الدكتور/ مصطفى عراقى - Dr.Mostafa Eraqi

أستاذ مساعد الميكروبيولوجى والمناعة بكلية العلوم

عملى ميكرو


UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECHv14        

Page 1 of 3

Section: Technical Manual

Subject Title: Table of Contents

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director

Revision Date:April 29, 2008

Annual Review Date:April 29, 2008

TECHNICAL MANUAL

 

TABLE OF CONTENTS


TOC o "1-3" h z u ALA (RAPID PORPHYRIN TEST) PAGEREF _Toc215137784 h 4

ANAEROBE IDENTIFICATION USING SPECIAL-POTENCY DISKS. PAGEREF _Toc215137785 h 6

SPS Disk for Differentiation of Anaerobic Cocci PAGEREF _Toc215137786 h 9

ANAEROBIC/CAMPYLOBACTER JAR SET UP. PAGEREF _Toc215137787 h 11

Anaerobic Jar PAGEREF _Toc215137788 h 11

Campylobacter Jar PAGEREF _Toc215137789 h 12

API Test Strips. PAGEREF _Toc215137790 h 13

IDENTIFICATION OF CORYNEBACTERIUM (API CORYNE) PAGEREF _Toc215137791 h 13

IDENTIFICATION OF ENTEROBACTERIACEAE (API 20E) PAGEREF _Toc215137792 h 17

IDENTIFICATION OF NON-ENTERIC GRAM-NEGATIVE RODS (API 20NE) PAGEREF _Toc215137793 h 24

SYSTEM FOR IDENTIFICATION OF NEISSERIA & HAEMOPHILUS (API NH) PAGEREF _Toc215137794 h 29

IDENTIFICATION OF STREPTOCOCCACEAE (API 20 Strep) PAGEREF _Toc215137795 h 33

API WEBSITE for Identification Profile. PAGEREF _Toc215137796 h 37

BACITRACIN DISK TEST. PAGEREF _Toc215137797 h 38

BILE ESCULIN TEST. PAGEREF _Toc215137798 h 40

BILE SOLUBILITY TEST. PAGEREF _Toc215137799 h 41

CATALASE TEST. PAGEREF _Toc215137800 h 42

CETRIMIDE PSEUDOMONAS SELECTIVE AGAR.. PAGEREF _Toc215137801 h 44

CRYPTOCOCCAL ANTIGEN.. PAGEREF _Toc215137802 h 45

E. coli O157 LATEX TEST (OXOID.. PAGEREF _Toc215137803 h 51

GENEPROBE-ACCUPROBE Staphylococcus aureus CULTURE IDENTIFICATION KIT. PAGEREF _Toc215137804 h 54

GERM TUBE TEST. PAGEREF _Toc215137805 h 61

GONOGEN (GC COAGGLUTINATION) TEST. PAGEREF _Toc215137806 h 63

HIPPURATE TEST. PAGEREF _Toc215137807 h 65



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECHv14

Page 2 of 3

Section: Technical Manual

Subject Title: Table of Contents (Cont'd)

 

INDOLE TEST. PAGEREF _Toc215137808 h 67

KOEHLER ILLUMINATION.. PAGEREF _Toc215137809 h 69

KOH STRING TEST. PAGEREF _Toc215137810 h 70

LAP TEST. PAGEREF _Toc215137811 h 72

MOTILITY TEST MEDIUM... PAGEREF _Toc215137812 h 74

MUG TEST (PGUA) PAGEREF _Toc215137813 h 76

NEISSERIA IDENTIFICATION SUGARS. PAGEREF _Toc215137814 h 78

ONPG TEST. PAGEREF _Toc215137815 h 80

ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM) PAGEREF _Toc215137816 h 82

OPTOCHIN SENSITIVITY TEST. PAGEREF _Toc215137817 h 84

ORNITHINE DECARBOXYLASE.. PAGEREF _Toc215137818 h 86

OXIDASE (API STRIP) PAGEREF _Toc215137819 h 88

OXIDASE (SPOT TEST DROPPER) PAGEREF _Toc215137820 h 90

PASTOREX STAPH PLUS TEST. PAGEREF _Toc215137821 h 91

PLATE STREAKING METHODS. PAGEREF _Toc215137822 h 93

PRO-AMP GLU-AMP TESTS. PAGEREF _Toc215137823 h 95

PYR TEST. PAGEREF _Toc215137824 h 97

QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE.. PAGEREF _Toc215137825 h 99

RapID ANA II SYSTEM... PAGEREF _Toc215137826 h 100

RapID MGP TEST. PAGEREF _Toc215137827 h 101

RapID VP TEST. PAGEREF _Toc215137828 h 103

RapID YEAST PLUS TEST. PAGEREF _Toc215137829 h 105

SIM (SULFIDE-INDOLE-MOTILITY) PAGEREF _Toc215137830 h 107

Staining Methods. PAGEREF _Toc215137831 h 109

ACID FAST STAIN FOR MYCOBACTERIA (KINYOUN) PAGEREF _Toc215137832 h 109

ACID FAST STAIN FOR NOCARDIA (MODIFED KINYOUN) PAGEREF _Toc215137833 h 111

ACRIDINE ORANGE STAIN.. PAGEREF _Toc215137834 h 113

BACTO 3-STEP GRAM STAIN PROCEDURE.. PAGEREF _Toc215137835 h 115



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECHv14

Page 3 of 3

Section: Technical Manual

Subject Title: Table of Contents (Cont'd)

 

EOSINOPHIL STAIN.. PAGEREF _Toc215137836 h 117

FLUOROCHROME STAIN for MYCOBACTERIUM... PAGEREF _Toc215137837 h 118

FUNGI-FLUORä STAIN.. PAGEREF _Toc215137838 h 120

GRAM STAIN.. PAGEREF _Toc215137839 h 122

GRAM STAINING MACHINE-MIDAS III OPERATION.. PAGEREF _Toc215137840 h 124

STAPHAUREX TEST. PAGEREF _Toc215137841 h 126

STREPTOCOCCAL GROUPING.. PAGEREF _Toc215137842 h 128

TETRAZOLIUM REDUCTION TEST (TTC) PAGEREF _Toc215137843 h 130

THERMONUCLEASE TEST. PAGEREF _Toc215137844 h 131

TRIBUTYRIN TEST. PAGEREF _Toc215137845 h 133

TSI (TRIPLE SUGAR IRON) PAGEREF _Toc215137846 h 134

TUBE COAGULASE TEST. PAGEREF _Toc215137847 h 137

UREA SLANT. PAGEREF _Toc215137848 h 139

XYLOSE FERMENTATION.. PAGEREF _Toc215137849 h 141

Record of Edited Revisions. PAGEREF _Toc215137850 h 142

BETA-LACTAMASE TESTING      Susceptibility Manual

DENKA MRSA SCREEN..................................................................................... Susceptibility Manual

HIGH LEVEL AMINOGLYCOSIDE TESTING................................................. Susceptibility Manual





UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH1v02

Page 1 of 2

Section: Technical Manual

Subject Title:ALA(Rapid Porphyrin                                     Test)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

ALA (RAPID PORPHYRIN TEST)

 

Principle

 

This test is used for rapidly detecting porphyrin as a means of speciating Haemophilus species.

Enzymes which convertALA(delta - aminolevulinic acid) to porphyrins in the biosynthesis of hemin (X factor) are produced by Haemophilus parainfluenzae but not by H. influenzae.  The production of porphyrins is detected by examination with an ultra-violet (UV) light.

 

Reagents

 

BBL  TAXO   Differentiation Disks ALA.  (Store refrigerated in the dark.  Allow 10-15 minutes for the container to reach room temperature before opening). 

Sterile distilled water

 

Other Materials

 

Petri dish

Inoculating loop

Gauze

Long-wave UV lamp

Forceps

 

Procedure

 

1.         Place oneALAdisk for each organism to be tested on the inside of a Petri dish using forceps.

2.         Moisten each disk with one drop of sterile water.

3.         Rub a loopful of the test organism onto the moistened disk holding it in place with sterile forceps.

4.         Saturate gauze with water, squeeze out any excess and place it in the petri dish as far away from the disks as possible.

5.         Incubate at 35oC.

6.         Examine at hourly intervals for 6 hours by removing the top of the petri dish and exposing the disks to UV light in a darkened room.  NB:  Wear UV safety goggles when using the UV light.



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Policy & Procedure Manual

Policy # MITECH1v02

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Technical Manual

 


 

Interpretation

 

A.        Positive:  Orange-red fluorescence

 

B.        Negative:  No fluorescence observed

 

Precautions

 

1.         Use for differentiating Haemophilus spp. only.

 

2.         Best results are obtained when a heavy inoculum is used.

 

3.        ALAis light sensitive.  Disks must be protected from light.

 

 

Quality Control

 

Test the following positive and negative controls each time an unknown is tested:

 

            Positive:          H. parainfluenza  (ATCC 7901)

            Negative:         H. influenzae      (ATCC 35056)

 

Reference

 

BBL  TAXO  Differentiation Disks ALA package insert, 1999.  Becton Dickinson Microbiology Systems.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH2v02

Page 1 of  5

Section: Technical Manual

Subject Title: Anaerobic Identification Using Special-Potency Disks

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

ANAEROBE IDENTIFICATION USING SPECIAL-POTENCY DISKS

 

Principle

Special potency antimicrobial disks of vancomycin (5 mg), kanamycin (1,000 mg) and colistin (10 mg) are used as an aid in determining the Gram reaction of anaerobes as well as in preliminary categorization of some anaerobic genera and species (Table 1).  In general, gram positive organisms are resistant to colistin and susceptible to vancomycin, while most gram negative organisms are resistant to vancomycin.  This difference is especially useful with some Clostridia that consistently stain gram negative.


Table 1 - Anaerobic Identification by Means of Special Potency Disks


 


Type of Organism

Response1 to Disk:

Vancomycin

(5 mg)

Kanamycin

(1,000 mg)

Colistin

(10 mg)

 

Gram negative

Gram positive

B. fragilis group

B. ureolyticus group

Fusobacterium spp.

Porphyromonas spp.

Veillonella spp.

R

S

R

R

R

S

R

V

V

R

S

S

R

S

V

R

R

S

S

R

S

1 R - resistant;  S - susceptible;  V - variable



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Policy & Procedure Manual

Policy # MITECH2v02

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Technical Manual

 


 


Materials

 

A.                Reagents


1.                  Special potency antibiotic disks:


            Vancomycin                5 mg

            Kanamycin                  1,000 mg

            Colistin                        10 mg


Store a small supply of disks (one carton each) in a tight container with desiccants at 40C.


2.                  Brucella or other anaerobic blood agar plate.


B.                 Supplies


1.                  Single disk dispenser or forceps

2.                  Ruler (divided into millimeters)


Procedure

 

1.                  Allow the container with disks to reach room temperature before opening it.

2.                  Subculture the isolate on a BAP.  To ensure an even, heavy lawn of growth, streak the first quadrant back and forth several times.  Streak the other quadrants to yield isolated colonies.

3.                  Place the three antibiotic disks on the first quadrant will apart from each other. 

4.                  If you have several organisms to test, first streak all the plates and then add the disks to them at the same time.

5.                  Incubate the plate(s) anaerobically for 48-72 hours at 35-370C.

6.                  Examine for zones of inhibition of growth around the disks.


Interpretation

 

A.        Susceptible:     Zone of inhibition of ³ 10 mm

B.        Resistant:        Zone of inhibition of < 10 mm



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Policy & Procedure Manual

Policy # MITECH2v02

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Technical Manual

 


 

Quality Control

 

A.                Test special potency antibiotic disks by lot when initially received and weekly thereafter.


B.                 Test Bacteroides fragilis (ATCC 25285), Clostridium perfringens (ATCC 13124), and Fusobacterium necrophorum (ATCC 25286) as described below under Procedure.  The results should show the following:



1.                  B. fragilis: resistant to all three antibiotics

2.                  F. necrophorum: resistant to vancomycin; susceptible to colistin and kanamycin

3.                  C. perfringens: susceptible to vancomycin and kanamycin and resistant to colistin


C.                 Record the results on a QC log.





























UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH2v02

Page 4 of 5

Technical Manual

 


 

 

SPS Disk for Differentiation of Anaerobic Cocci

 

Principle

Sodium polyanethol sulfonate (SPS), a commonly used anticoagulant, inhibits certain bacteria such as Peptostreptococcus anaerobius and the aerobe Gardnerella vaginalis.  Paper disks impregnated with 5% SPS can be used as a tool for differentiating P. anaerobius from other anaerobic cocci.


Materials


A.                Reagents


1.                  SPS Disks


a.                   Combine the following in a flask.

                                                SPS                             5 g

                                                Distilled water                        100 ml

b.                  After dissolving SPS, sterilize the mixture by filtration (0.22 mm pore size

                                    filter).

c.                   Dispense 20 ml onto sterile 1/4-inch diameter filter paper disks that are

spread inside empty, sterile petri dishes.  Allow these to dry for 72 hours at room temperature.

d.                  Store the disks at room temperature, and label with an expiration date of 6

                                    months.


2.                  SPS disks are also commercially available (Anaerobe Systems, Difco, Oxoid, Remel).  Store as indicated by the manufacturers. 

3.                  Brucella or other anaerobic blood agar plate.


B.                 Supplies


1.                  Single-disk dispenser or forceps

2.                  Ruler (divided into millimeters)


Procedure

 

1.                              Allow the container with disks to reach room temperature before use.

2.                  Subculture the isolates on a BAP.  To ensure an even, heavy lawn of growth, streak the first quadrant back and forth several times.  Streak the other quadrants to yield isolated colonies.



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Policy & Procedure Manual

Policy # MITECH2v02

Page 5 of 5

Technical Manual

 



3.                              Place the SPS disk on the first quadrant.

4.                  If you have several organisms to test, first streak all the plates and then add the disks to them at the same time.  You can use one plate for up to four tests.

5.                              Incubate the plate(s) anaerobically for 48-72 hours at 35-370C.

6.                              Examine for a zone of inhibition of growth around the disk. 


Interpretation

 

A.                Susceptible: Zone of inhibition of ³ 12mm


P. anaerobius usually gives a very large zone of inhibition (³ 16 mm), whereas other anaerobic cocci that appear susceptible to SPS give smaller zones.  To presumptively identify P. anaerobius, you must also consider the Gram stain, typical colonial morphology, and odor.  Some strains of P. micros may be susceptible to SPS.  Examine the Gram stain for the small cell size of P. micros and chaining characteristic of P. anaerobius.


B.                 Resistant: Zone of inhibition of <12 mm.


Quality Control

 

A.                Test each lot upon receipt and monthly thereafter.

B.                 Test P. anaerobius ATCC 27337 and Peptostreptococcus asaccharolyticus ATCC 29745 as described below under Procedure.  The results should show the following:

1.         P. anaerobius:             susceptible to SPS

2.         P. asaccharolyticus:    resistant to SPS

C.                 Record the results on a QC log.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH3v04

Page 1 of  2

Section: Technical Manual

Subject Title: Anaerobic/Campylobacter                             Jar Set Up

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:April 29, 2008

Annual Review Date:April 29, 2008

 

ANAEROBIC/CAMPYLOBACTER JAR SET UP

 

Anaerobic Jar


1.                  Anaerobic plates are kept in the nitrogen holding box until there is enough for a jar or until the end of the day.


2.                  Place the inoculated plates (max 14), biological indicators and anaerobic indicator strip into an empty anaerobic jar.


3.                  Tear open an AnaeroGen foil sachet at the tear-nick indicated and remove the Anaero Gen paper sachet from within.


4.                  Immediately place the paper sachet in the jar down the side of the plates.


5.                  Place the lid on the jar (no catalyst required) and seal with the clamp.

Note:   The time between opening the foil sachet and sealing the jar should not exceed      one minute.

Note:   Jar and lid must be labelled with the same number.


6.                  Label jar with date and place in walk in incubator.


Control Testing


An anaerobic indicator is added to each jar as it is set up to visually check that anaerobic conditions have been achieved and maintained.  Check the jar after 2 hours incubation to make sure the indicator does not indicate oxygen present.


Biological Indicator

Inoculate a quarter anaerobic plate with the following test organisms:

            Bacteroides fragilis ATCC 25285: growth

            Clostridium novyii ATCC 27606: growth

            Clostridium difficile ATCC 9089: growth

            Pseudomonas aeruginosa ATCC 27853: no growth


Record results on the anaerobic jar QC sheet.


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Policy & Procedure Manual

Policy # MITECH3v04

Page 2 of 2

Technical Manual

 


 

Campylobacter Jar

1.                  Campylobacter plates are kept in the CO2 incubator until there is enough for a jar or until4 p.m.(Any late cultures will be set up at the end of the shift).


2.                  Place a dampened paper towel into the bottom of an anaerobic jar.


3.                  Place the inoculated plates (max 14) into the jar.  Include a plate freshly inoculated with the control organism.


4.                  Tear open a CampyGen foil sachet at the tear-nick indicated and remove the CampyGen paper sachet from within.


5.                  Immediately place the paper sachet in the jar down the side of the plates.


6.                  Place the lid on the jar (no catalyst required) and seal with the clamp.

Note:   The time between opening the foil sachet and sealing the jar should not exceed      one minute.

Note:   Jar and lid must be labelled with the same number.


7.                  Label jar with date and place in walk in 42oC incubator.


Biological Indicator

Inoculate a campylobacter agar plate with the following test organism:


            Campylobacter jejuni ATCC 29428: growth

           

Record results on the anaerobic jar QC sheet.


Note:   The technologist on the enteric bench is responsible for the daily subculturing of the          control organism (3 new plates).  One newly subcultured plate will be incubated with the          reincubate culture jar.  The old control plate and the remaining 2 newly subcultured             plates will be kept in the CO2 incubator until the end of the day.


            The 2 new subcultured plates are for setting up new jars.  If more are needed, the technicians will subculture new plates from the old control plate.


UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH41v03

Page 1 of  4

Section: Technical Manual

Subject Title: API Test Strips - API                         CORYNE

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:March 03, 2006

Annual Review Date:April 29, 2008

API Test Strips

IDENTIFICATION OF CORYNEBACTERIUM (API CORYNE)


Principle

The API CORYNE system facilitates the 24 hour identification of C. jeikeium (CDC Group JK), other medically important Corynebacteria, Rhodococcus equi, Listeria species, Erysipelothrix rhusiopathiae, Actinomyces pyogenes, Arcanobacterium haemolyticum, Brevibacterium species and Gardnerella vaginalis.


The API CORYNE strip consists of 20 microtubes containing dehydrated substrates for the demonstration of enzymatic activity or the fermentation of carbohydrates (CHO).  The addition of a dense test suspension of bacteria rehydrates the enzymatic substrates.  The metabolic end products produced during incubation are detected through spontaneous coloured reactions or by the addition of reagents.


The fermentation tests are inoculated with an enrichment medium (containing pH indicator) which reconstitutes the CHO substrates.  Fermentation of CHO is detected by colour change in the pH indicator.


Materials

 

API Coryne strips - store at 2 - 80C

GP medium - store at 2 - 80C

McFarland Standard #6

Nitrate A - store at Room Temperature

Nitrate B - store at 2 - 80C

Zym A - store at 2 - 80C in the dark

Zym B - store at 2 - 80C in the dark

PYZ - store at 2 - 80C in the dark

H2O2 - store at 2 - 80C

Mineral oil

Sterile saline 3 ml



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH41v03

Page 2 of 4

Technical Manual

 


 


Procedure

1.               Preparation of Inoculum

a)                  Only pure cultures of a single organism should be used (heavily inoculated sheep BAP x 3; incubate for 24 hours at 350C in 5% CO2).


b)                  Using a sterile swab, harvest all the culture from 3 BAP and inoculate into 3 ml.

                        sterile saline to give a turbidity of at least McFarland #6.


2.               Preparation of the Strip

a)                  An incubation tray is supplied for each strip.


b)                  Dispense 5 ml of water into the wells of the tray.


3.               Inoculation of the Strip

a)                  Inoculate tests 1 ® 11 of the strip (NIT to GEL).


b)                  Avoid bubbles by tilting the strip slightly forward while placing the pipette tip on the side of the cupule.


c)                  Add 3 drops into each cupule for tests NIT to ES.


d)                 For the URE test fill the tube portion only.


e)                  For the GEL test, fill both the tube and cupule.  Then:         


f)                   For the last nine tests of the strip (O to GLYG transfer the rest of the bacterial suspension to an ampoule of GP medium.  Mix well.


g)                  Distribute the new suspension into the tubes only of tests O to GLYG


h)                  Overlay cupules URE and O to GLYG with mineral oil, forming a slight convex meniscus.


i)                    Cover with incubation lid and incubate the strip for 24 hours at 350C (non-CO2).


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Policy & Procedure Manual

Policy # MITECH41v03

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Technical Manual

 


 

 

Interpretation

REACTIONS TABLE

TESTS

REACTONS

NEGATIVE RESULTS

POSITIVE RESULTS

NIT


Addition of NIT A + NIT B

(10 min)

NIT

NITrate reduction

Colourless

Very pale pink

Dark pink

Red

PYZ


PYZ (10 min)

PYZ

PYraZinamidase

Colourless

Very pale brown

Very pale orange

Brown

Orange

PyrA ® BNAG

Addition of ZYM A + ZYM B (10 min)

PyrA

Pyrrolidonyl Arylamidase

Colourless

Pale orange

Orange

PAL

Alkaline Phosphatase

Colourless

Beige-pale purple

Pale orange


Purple

bGUR

Beta GlucURonidase

Colourless

Pale grey

Pale beige


Blue

bGAL

Beta GALactosidase

Colourless

Beige-pale purple

Purple


µ GLU

Alpha GLUcosidase

Colourless

Beige-pale purple

Pale green


Purple

BNAG

N-Acetyl-B Glucosaminidase

Colourless

Beige-pale purple

Pale brown

Pale grey



Brown


 



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REACTIONS TABLE (Cont'd)

TESTS

REACTONS

NEGATIVE RESULTS

POSITIVE RESULTS

ESC

ESCulin (b Glucosidase)

Colourless

Grey

Black

URE

UREase

Yellow

Orange

Red

Pink

[GEL]

GELatine (hydrolysis)

No diffusion of black pigment

Diffusion of black pigment

O

GLU

RIB

XYL

MAN

MAL

LAC

SAC

GLYG

Control (Fermentation)

GLUcose         }

RIBose            }

XYLOSE        }

MANnitol       }          Fermentation

MALtose         }

LACtose         }

Sucrose            }

GLYcoGen     }






Red


Orange






Yellow


Yellow-orange

CAT


Addition of H2O2   3% (1 min)

CAT

CATalase (ESC or GEL test)

No bubbles

Bubbles


a)      Record results on a API CORYNE profile sheet.


b)      Refer to the API WEBSITE for Identification Profile



References

1.                  Coyle, Marie B., Benjamin Lipsky.  1990.  Coryneform Bacteria in Infectious Diseases: Clinical and Laboratory Aspects.  Clinical Microbiology Reviews.  3:227-246.


2.                  Freney, J.M.T.  Duperron, C. Couturier, W. Hansen, F. Allard, J.M. Boueufgras, D. Monget, J. Fleurette.  Evaluation of API Coryne in Comparison with Conventional Methods for Identifying Coryneform Bacteria, Journal of Clinical Microbiology, January 1991, Vol. 29, p. 38-41.


3.                  Murray P.A., et al.  Manual of Clinical Microbiology, 7th ed., 1999.




UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH42v03

Page 1 of  7

Section: Technical Manual

Subject Title: API Test Strips - API 20E

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:March 03, 2006

Annual Review Date:April 29, 2008

 

IDENTIFICATION OF ENTEROBACTERIACEAE (API 20E)

Principle

The API 20E system facilitates the 24-hour identification of Enterobacteriaceae as well as 24 or 48-hour identification of other Gram negative bacteria.


The API 20E strip consists of microtubes containing dehydrated substrates for the demonstration of enzymatic activity and carbohydrate (CHO) fermentation.  The substrates are reconstituted by adding a bacterial suspension.  After incubation, the metabolic end products are detected by indicator systems or the addition of reagents.  CHO fermentation is detected by colour change in the pH indicator.


Materials

API 20E strips - store at 2-80C

0.85% sterile saline

Nitrate A - store at 2-80C

Nitrate B - store at 2-80C

Mineral oil

Zinc dust

Kovacs Reagent                                  ý

Voges - Proskauer Reagents               ý

Ferric Chloride                                    ý          Store at 2-80C            

H2O2                                                                     ý

Oxidase Reagent                                


OF Dextrose                                       ý          ID of non-

Motility Medium                                 ý          Enterobacteriaceae


Procedure

1.                  Preparation of Inoculum

a)                  Add 5 ml. of 0.85% saline to a sterile test tube.

b)                  Using a sterile inoculating loop, carefully touch the centre of a well isolated colony (2-3 mm. Diameter) and thoroughly emulsify in the saline.



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2.                  Preparation of the Strip

a)                  An incubation tray and lid is supplied for each strip.

b)                  Dispense 5 ml of water in to the tray.


3.                  Inoculation of the Strip

a)                  Remove the cap from the tube containing the bacterial suspension and insert a 5 ml.  Pasteur pipette.

b)                  Tilt the API 20E incubation tray and fill the tube section of the microtubes by placing the pipette tip against the side of the cupule.


Note:   The ADH, LDC, ODC, H2S, AND URE reactions can be interpreted best if these microtubes are slightly underfilled.

c)                  Fill both the TUBE and CUPULE section of [CIT], [VP] and [GEL] tubes.

d)                 After inoculation, completely fill the cupule section of the ADH, LDC, ODC,

                        H2S and URE tubes with mineral oil.

e)                  Using the excess bacterial suspension, inoculate an agar slant or plate (non-selective media such as nutrient agar, blood agar or tryptic (trypticase) soy agar is suggested) as a purity check and for oxidase testing, serology, and/or additional biochemical testing.  Incubate the slant or plate for 18-24 hours at 350C.


4.                  Incubation of the Strip


a)                  After inoculation, place the plastic lid on the tray and incubate the strip for 18-24 hours at 350C in a  non-CO2 incubator.


b)                  Weekend incubation:  The biochemical reactions of the API 20E should be read after 18-24 hours incubation.  If the strips cannot be read after 24 hours incubation at 350C, the strips should be removed from the incubator and stored at 2-80C (refrigerator) until the reactions can be read.


5.                  Reading the Strip

a)                  After 18 hours of incubation and before 24 hours incubation, record all reactions not requiring the addition of reagents.


b)                  If the GLU tube is negative (blue or green), do not add reagents.  Reincubate a further 18-24 hours.



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c)                  If the GLU is positive (yellow):


i.                    Perform the oxidase test.


A portion of the growth from the agar slate or plate, inoculated from the 20E bacterial suspension, should be rubbed onto filter paper to which a drop of oxidase reagent (1% tetramethyl-p-phenylenediamine dihydrochloride) has been added.  The area where the growth has been added will turn dark purple within 10 seconds if the reaction is positive and will be colourless or light purple if negative.


Note:   (a)        Nichrome wire loops should NOT be used in performing

            the oxidase test.  Nichrome wire can cause a false positive reaction.


            (b)        The oxidase test should NOT be performed using bacterial

            growth from selective media such as MacConkey, EMB, etc.


Note:   (a)        Before addition of reagents, observe GLU tube (positive or

                        negative) for bubbles.


(b)               The nitrate reduction and indole tests must be performed last since these reactions release gaseous products which interfere with the interpretation of other tests on the strip.  The plastic incubation lid should not be replaced after the addition of these reagents.


ii.                  Add the reagents to TDA and VP tubes.  If positive, the TDA reactions will be immediate, whereas the VP reaction may be delayed up to 10 minutes.


iii.                The Kovacs' reagent should then be added to theINDtube.           


iv.                The Nitrate Reduction test should be performed on all oxidase positive organisms.  The reagents should be added to the GLU tube after the Kovacs Reagent has been added to theINDtube.



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Interpretation

a)                  Record results on a API 20E analytical profile sheet.


b)                  The tests are separated into groups of three.  The following numerical value is assigned to each reaction recorded:


1-                  positive reaction in the first test of the group

2-                  positive reaction in the second test of the group

            4-         positive reaction in any test

0-                  negative reaction in any test


c)                  Refer to the API WEBSITE for Identification Profile



Reference

1.         Murray P.A., et al.  Manual of Clinical Microbiology, 7th ed., 1999.



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SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE

 

TUBE

INCUBATION

POSITIVE

NEGATIVE

COMMENTS

ONPG

 

Yellow

Colourless

(1)     Any shade of yellow is a positive reaction.

(2)     VP tube, before the addition of reagents, can be used a negative control.

 

ADH

18-24 hr

36-48 hr

Red orOrange

Red

Yellow

Yellow or Orange

Orange reactions occurring at 36-48 hours should be interpreted as negative.

 

LDC

18-24 hr

36-48 hr

Red orOrange

Red

Yellow

Yellow or Orange

Any shade of orange within 18-24 hours is a positive reaction.

At 36-48 hours, orange decarboxylase reactions should be interpreted as negative.

 

ODC

18-34 hr

36-48 hr

Red orOrange

Red

Yellow

Yellow or Orange

Orange reactions occurring at 36-48 hours should be interpreted as negative.

 

CIT

 

Turquoise or

Dark Blue

Light Green

Or Yellow

(1)     Both the tube and cupule should be filled.

(2)     Reaction is read in the aerobic (cupule) area.

 

H2S

 

Black Deposit

No Black Deposit

(1)     H2S production may range from a heavy black deposit to a very thin black line around the tube bottom.  Carefully examine the bottom of the tube before considering the reaction negative.

(2)     A "browning" of the medium is a negative reaction unless a black deposit is present.  "Browning" occurs with TDA positive organisms.

 

URE

18-24 hr

36-48 hr

Red orOrange

Red

Yellow

Yellow or Orange

A method of lower sensitivity has been chosen.  Klebsiella, Proteus and Yersinia routinely give positive reactions.

 

TDA

Add 1 drop 10% Ferric chloride.

 

Brown-Red

 

 

Yellow

(1)     Immediate reaction.

(2)     Indole positive organisms may produce a golden orange colour due to indole production.  This is a negative reaction.

 

IND

Add 1 drop Kovacs Reagent

 

Red Ring

 

 

Yellow

(1)     The reaction should read within 2 minutes after the addition of the Kovacs reagents and the results recorded.

(2)     After several minutes, the HCl present in Kovacs reagent may react with the plastic of the cupule resulting in a change from a negative (yellow) colour to a brownish-red.  This is a negative reaction.

 



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SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE (cont'd)

 

TUBE

INCUBATION

POSITIVE

NEGATIVE

COMMENTS

VP

Add 1 drop of 40% Potassium Hydroxide, then 1 drop of alpha-napthol.

(1)     Wait 10 minutes before considering the reaction negative.

(2)     A pale pink colour which appears immediately after the addition of reagents but which turns dark `pink or red after 10 minutes should be interpreted as positive.

Motility may be observed by hanging drop or wet mount preparation.

 

 

Red

Colourless

GEL

 

Diffusion of the pigment

No diffusion

(1)     The solid gelatin particles may spread throughout the tube after inoculation.  Unless diffusion occurs, the reaction is negative.

(2)     Any degree of diffusion is a positive reaction.

 

 

 

 

GLU

 

 

 

 

 

 

 

 

 

 

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Yellow

Or Gray

 

 

 

 

 

 

 

 

 

Yellow

 

 

 

Blue or

Blue-Green

 

 

 

 

 

 

 

 

 

Blue or

Blue-Green

 

COMMENTS FOR ALL CARBOHYDRATES

 

Fermentation

(Enterobacteriaceae, Aeromonas, Vibrio)

(1)     Fermentation of the carbohydrates begins in the most anaerobic portion (bottom) of the tube.  Therefore, these reactions should be read from the bottom of the tube to the top.

(2)     A yellow colour at the bottom of the tube only indicates a weak or delayed positive reaction.

 

Oxidation (Other Gram-negatives)

(1)     Oxidative utilization of the carbohydrates begins in the most aerobic portion (top) of the tube.  Therefore, these reactions should be read from the top to the bottom of the tube. 

(2)     A yellow colour in the upper portion of the tube and blue in the bottom of the tube indicate oxidative utilization of the sugar.  This reaction should be considered positive only for non-Enterobacteriaceae gram negative rods.  This is a negative reaction for fermentative organisms such as Enterobacteriaceae.




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SUMMARY OF RESULTS - 18- 24 HOUR PROCEDURE (cont'd)

 

TUBE

INCUBATION

POSITIVE

NEGATIVE

COMMENTS

 

GLU

 

After reading GLU reaction, add 2 drops 0.8% sulfanilic acid and 2 drops 0.5% N. N-dimethyl-alpha-naphthylamine

 

(1)     Before addition of reagents, observe GLU tube (positive or negative) for bubbles.  Bubbles are indicative of reduction of nitrate to the nitrogenous (N2) state.

(2)     A positive reaction may take 2-3 minutes for the red colour to appear.

(3)     Confirm a negative test by adding zinc dust or 20 mesh granular zinc.  A pink-orange colour after 10 minutes confirms a negative reaction.  A yellow colour indicates reduction of nitrates to the nitrogenous (N2) state.

 

NO2

N2 gas

Red

 

Bubbles: Yellow after reagents and zinc

Yellow

 

Orange after reagents and zinc

 

MAN

INO

SOR

Catalase

 

After reading carbohydrate reaction, add 1 drop 1.5% H2O2

 

(1)     Bubbles may take 1-2 minutes to appear. 

(2)     Best results will be obtained if the test is run in tubes which have no gas from fermentation.

 

 

 

Bubbles

 

 

No bubbles



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Policy & Procedure Manual

Policy # MITECH43v03

Page 1 of 5


Section: Technical Manual

Subject Title: API Test Strips - API 20NE

Issued by: LABORATORY MANAGER

Original Date:August 3, 2003

Approved by: Laboratory Director


Revision Date:March 03, 2006

Annual Review Date:April 29, 2008

 

IDENTIFICATION OF NON-ENTERIC GRAM-NEGATIVE RODS (API 20NE)


Principle

The API 20NE system facilitates the identification of non-fastidious Gram-negative rods not belonging to the Enterobacteriaceae within 48 hours.


The API 20NE strip consists of microtubes containing dehydrated media and substrates. The media microtubes containing conventional tests are inoculated with a bacterial suspension which reconstitutes the media. After incubation, the metabolic end products are detected by indicator systems or the addition of reagents. The substrate microtubes contain assimilation tests and are inoculated with a minimal medium. If the bacteria are capable of utilizing the corresponding substrate, then they will grow.


Materials

API 20NE strips - store at 2-80C

0.85% sterile saline

Mineral oil

Zinc dust

AUX Medium                                  }

James Reagent                                 }

Nitrate 1 - store at 2-80C                }                Store at 2-80C

Nitrate 2 - store at 2-80C                }

      Oxidase Reagent

 

Procedure

1.      Preparation of Inoculum

a)                  Add 2 ml. of 0.85% saline to a sterile test tube.

b)                  Using a sterile inoculating loop, carefully touch the centre of a well isolated colony (2-3 mm. Diameter) and thoroughly emulsify in the saline. The suspension turbidity should be equal to a 0.5 McFarland standard.


2.                  Preparation of the Strip

a)                  An incubation tray and lid are supplied for each strip.

b)                  Dispense 5 ml of distilled water in to the tray.


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4.                  Inoculation of the Strip

a)                  Remove the cap from the tube containing the bacterial suspension and insert a sterile pipette.

b)                  Tilt the API 20NE incubation tray and fill the TUBE section of the NO3 to PNPG microtubes by placing the pipette tip against the side of the cupule.

c)                  Open an ampule of AUX Medium and add 200 uL of the bacterial suspension to the ampule. Mix well with a pipette while avoiding the formation of air bubbles.

d)                 Using the AUX Medium bacterial suspension, fill both the TUBE and CUPULE section of [GLU] to [PAC]. Do not overfill the cupules. Fill to a flat or slightly convex meniscus.

e)                  After inoculation, completely fill the CUPULE section of the 3 underlined tests,      

                        GLU, ADH and URE tubes with mineral oil.

f)                   Using the excess bacterial suspension, inoculate an agar slant or plate (non-selective media such as nutrient agar, blood agar or tryptic (trypticase) soy agar is suggested) as a purity check and for oxidase testing, and/or additional biochemical testing.  Incubate the slant or plate with the API 20NE strip.


4.                  Incubation of the Strip


a)                  After inoculation, place the plastic lid on the tray and incubate the strip for 24 hours at 300C in a non-CO2 incubator.


5.                  Reading the Strip

a)                  After 24 hours incubation, record all reactions not requiring the addition of reagents.

b)                  Perform the oxidase test.


A portion of the growth from the agar slate or plate, inoculated from the 20NE bacterial suspension, should be rubbed onto filter paper to which a drop of oxidase reagent (1% tetramethyl-p-phenylenediamine dihydrochloride) has been added.  The area where the growth has been added will turn dark purple within 10 seconds if the reaction is positive and will be colourless or light purple if negative.

Note:  

(a)        Nichrome wire loops should NOT be used in performing the oxidase              test. Nichrome wire can cause a false positivereaction.


(b)        The oxidase test should NOT be performed using bacterial growth from selective media such as MacConkey, EMB, etc.




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c)                  Assimilation tests are observed for bacterial growth. An opaque cupule indicates a positive reaction.

d)                 Protect the assimilation tests with the incubation tray lid during the reading of the Nitrate and TRP tests.

e)                  Perform the Nitrate test.


i.                    Add one drop of Nitrate 1 and one drop of Nitrate 2 reagents to NO3 cupule.

ii.                  After 5 minutes a red color indicates a positive reaction.

iii.                A negative reaction may be due to the production of nitrogen. Add Zinc dust to the NO3 cupule. After 5 minutes a colorless cupule indicates a positive reaction. A pink-red cupule indicates a negative reaction.


f)                   Perform the TRP test.


i.                    Add one drop of JAMES Reagent.

ii.                  The reaction takes place immediately, producing a pink color in the entire cupule if the reaction is positive.



Interpretation

1.                  Use the API 20NE analytical profile index.

 

2.                  The tests are separated into groups of three.  The following numerical value is assigned to each positive reaction recorded:


1 - positive reaction in the first test of the group

2 - positive reaction in the second test of the group

4 - positive reaction in the third test of the group


3.                  Refer to the API WEBSITE for Identification Profile


4.                  The strip must be reincubated in the following cases:


i.                    If the profile cannot be found in the API web site.

ii.                  If the following note is indicated for the profile obtained:


IDENTIFICATION NOT VALID

BEFORE 48-HR INCUBATION


iii.                If the strip is to be reincubated, remove the reagents from the NO3 and TRP cupules and then cover these tests with mineral oil.



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iv.                Reincubate the strip for another 24 hours at 30oC in a non-CO2 incubator.

v.                  Read all the tests again, except for NO3, TRP and GLU.


READING TABLE

TESTS

SUBSTRATES

REACTONS/ENZYMES

NEGATIVE RESULTS

POSITIVE RESULTS

N03

Potassium

nitrate

NITrate reduction to nitrites

NIT 1 + NIT 2 / 5 min

        colourless                   pink-red

NITrates to nitrogen

Zn / 5 min

       pink                      colourless

TRP

tryptophane

indole production

JAMES / immediate

       colourless /                    pink

pale green / yellow

GLU

glucose

Acidification

blue to green

yellow

ADH

arginine

arginine dihydrolase

yellow

orange/pink/red

ure

urea

Urease

yellow

orange/pink/red

ESC

esculin

hydrolysis (b-glucosidase)

yellow

grey/brown/black

GEL

gelatine

(with India ink)

hydrolysis (protease)

no pigment diffusion

diffusion of black pigment

PNPG

p-nitrophenyl-b-D-galactopyranoside

b-galactosidase

colourless


yellow

[GLU]

glucose

Assimilation

transparent

opaque

[ARA]

arabinose

Assimilation

transparent

opaque

[MNE]

mannose

Assimilation

transparent

opaque

[MAN]

mannitol

Assimilation

transparent

opaque

[NAG]

N-acetyl-glucosamine

Assimilation

transparent

opaque

[MAL]

maltose

Assimilation

transparent

opaque

[GNT]

gluconate

Assimilation

transparent

opaque

[CAP]

caprate

Assimilation

transparent

opaque

[ADI]

adipate

Assimilation

transparent

opaque

[MLT]

malate

Assimilation

transparent

opaque

[CIT]

citrate

Assimilation

transparent

opaque

[PAC]

phenyl-acetate

Assimilation

transparent

opaque

OX

see oxidase test

cytochrome oxidase

colorless/

light purple

dark purple




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Quality Control

To be performed on receipt of every new lot of strip by the QC bench technologist.

Reference


Reference Package Insert - api 20NE system for the identification, bioMerieux Inc., Missouri USA.

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH44v05

Page 1 of  4

Section: Technical Manual

Subject Title:API Test Strips -APINH

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:March 03, 2006

Annual Review Date:April 29, 2008

 

SYSTEM FOR IDENTIFICATION OF NEISSERIA & HAEMOPHILUS (APINH)

 

Principle

The API NH strip consists of 10 microtubes containing dehydrated substrates, which enable the performance of 12 identification tests (enzymatic reactions or sugar fermentations), as well as the detection of a penicillinase (particular interest in Haemophilus influenzae, Haemophilus parainfluenzae, Branhamella catarrhalis (Moraxella catarrhalis) and Neisseria gonorrhoeae).

 

The reactions produced during incubation result in spontaneous color changes or are revealed by the addition of reagents.


After a 2-hour incubation period at a temperature of 35-37oC, the reading of the reactions is performed visually and identification is obtained by consulting the profile list.


Reagents

APINHstrips

NaCl 0.85% Medium (2 ml)

JAMES reagent

ZYM B reagent

Swab

Incubation box

Result sheet

1 package insert

McFarland Standard, point 4 on the scale

Mineral oil

Pipettes

Ampule rack

Ampule protector


Procedure

1.                  Specimen Processing

The microorganisms to be identified must first be isolated as separate colonies by streaking the specimen onto Blood agar, Chocolate agar or Martin-Lewis agar according to standard microbial techniques.



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Technical Manual

Subject Title:API Test Strips -APINH



2.                  Preparation of Strip

Each strip is composed of 10 cupules.  Each cupule has an open and closed area (cupule and tube).  An incubation tray is supplied for each strip.  It serves as a support and individual chamber while both protecting the strip from contaminants in the air and assuring the humid atmosphere necessary to avoid dehydration during incubation.


·  Remove the strip from its individual packaging

·  Place the strip in the incubation box

·  Discard the desiccant sachet


Record the specimen number on the flat portion of the tray (do not record the number on the lid as it may be misplaced during handling).


3.                  Preparation of the Inoculum

            ·  Open an ampule of NaCl 0.85% Medium (2 ml) with the ampule protector.

            ·  Using a swab, pick up a few well-isolated colonies and prepare a suspension with a

                turbidity equivalent to 4 McFarland, ensuring it is well mixed.

            ·  The suspension should be used immediately after preparation.


4.                  Inoculation of the Strip

            ·  Distribute the prepared bacterial suspension into the cupules, avoiding the formation of

               bubbles (tilt the strip slightly forwards and place the tip of the pipette or PSIpette against

               the side of the cupule):

-          Only fill the tube part of the first 7 microtubes (PEN to URE): about 50 ml.

-          Fill tube and cupule of the last 3 microtubes LIP/ProA, PAL/GGT, bGAL/IND: about 150 ml, avoiding the formation of a convex meniscus.

            ·  Cover the first 7 tests (PEN to URE) with mineral oil (underlined tests).


   NOTE: The quality of the filling is very important: tubes which are insufficiently or excessively full may cause false positive or false negative results.

                             ·  Close the incubation box.

                             ·  Incubate for 2 hours at 35-37oC in aerobic conditions.


5.            Incubation

         Incubate for 2 hours at 35-37oC in aerobic conditions.



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Technical Manual

Subject Title:API Test Strips -APINH



6.            Reading the Strip


         Refer to the Reactions Table for a description of how to read the reactions.


         Note all spontaneous reactions (PEN to bGAL) and record them as + or -.

         · Add 1 drop of ZYM B reagent to microtubes 8 and 9: LIP/ProA and PAL/GGT.

         · Add 1 drop of JAMES reagent to microtube 10: bGAL/IND.

         · Wait 2 minutes then read the reactions by referring to the Reading Table in this package

            insert and record them on the result sheet.

-          If the LIP reaction is positive (blue pigment), interpret the ProA reaction as negative, whether the ZYM B reagent has been added or not.

-          If, after a 2-hour incubation period, several reactions (fermentation, penicillinase) are doubtful, re-incubate the strip for another 2 hours and read the reactions again (the enzymatic tests should not be re-read in this case).


Reactions Table

 

TESTS

REACTIONS

SUBSTRATES

QTY

(mg)

RESULTS

NEGATIVE

POSITIVE

 

1) PEN

 

PENicillinase

 

Penicillin G

 

1.36

 

Blue

(penicillinase absent)

 

Yellow

Yellow-green

Yellow-blue

(penicillinase present

 

2) GLU

3) FRU

4) MAL

5) SAC

 

GLUcose (Acidification)

FRUctose (Acidification)

MALtose (Acidification)

SACcharose/Sucrose

(Acidification)

 

Glucose

Fructose

Maltose

Sucrose

 

0.5

0.1

0.1

0.5

 

 

Red

Red-orange

 

 

Yellow

Orange

 

 

6) ODC

 

Ornithine DeCarboxylase

 

Ornithine

 

0.55

 

Yellow-green

Grey-green

 

Blue

 

7) URE

 

UREease

 

Urea

 

0.41

 

Yellow

 

Pink-violet

 

8a) LIP

 

LIPase

 

5-bromo-3-indoxyl-caprate

 

0.033

 

Colorless

Pale grey

 

Blue

(+precipitate)

 

 

9a) PAL

 

Alkaline Phosphatase

 

Para-Nitrophenyl-phosphate 2CHA

 

0.038

 

Colorless

Pale yellow

 

Yellow

 

 

10a) bGAL

 

Beta GALactosidaase

 

Para-Nitrophenyl-BD

galactopyranoside

 

0.04

 

Colorless

 

Yellow



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Technical Manual

Subject Title:API Test Strips -APINH



 

Reactions Table (Cont'd)


TESTS

REACTIONS

SUBSTRATES

QTY

(mg)

RESULTS

NEGATIVE

POSITIVE

 

 

 

8b) ProA

 

 

 

Proline Arylamidase

If LIP is +. ProA is always -

 

 

 

Proline-4-methoxy-

b naphthylamide

 

 

 

0.056

 

 

ZYM B / 3 min

 

Yellow

Pale orange

(brown if LIP +)

 

 

Orange

 

 

 

9b) GGT

 

 

 

Gamma Glutamyl Transferase

 

 

 

Gamma glutamyl

4-methoxy-

b naphthylamide

 

 

 

 

0.049

 

ZYM B / 3 min

 

Yellow

Pale orange

(yellow-orange if PAL +)

 

 

Orange

 

 

 

 

 

10b)IND

 

 

 

INDole

 

 

 

Tryptophane

 

 

 

0.036

 

JAMES / 3 min

 

Colorless

 

 

Pink


Interpretation


a)      Record results on a API NH profile sheet.

b)      Refer to the API WEBSITE for Identification Profile


Quality Control

To be performed on receipt of every new lot of strip by the QC bench technologist.


QC organisms to be used:

            Neisseria gonorrhoea              ATCC 31426

            Haemophilus influenzae                      ATCC 10211

            Branhamella catarrhalis                     ATCC 23246

            Haemophilus paraphrophilus             ATCC 49917

Reference


Reference Package Insert - api NH system for the identification of Neisseria and Haemophilus bioMerieux Inc., Missouri USA.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH45v02

Page 1 of  4

Section: Technical Manual

Subject Title:API Test Strips -API20 Strep

Issued by: LABORATORY MANAGER

Original Date:April 29, 2008

Approved by: Laboratory Director


Revision Date:

Annual Review Date:

 

IDENTIFICATION OF STREPTOCOCCACEAE (API 20 Strep)

Principle

API20 Strep facilitates the group or species identification of most streptococci and enterococci, and those most common related organisms.


TheAPI20 Strep consists of 20 microtubes containing dehydrated substrates for the demonstration of enzymatic activity or the fermentation of sugars. The enzymatic tests are inoculated with a dense suspension of organisms, made from a pure culture, which is used to reconstitute the enzymatic substrates. During incubation, metabolism produces colour changes that are either spontaneous or revealed by the addition of reagents. The fermentation tests are inoculated with an enriched medium which rehydrates the sugar substrates. Fermentation of carbohydrates is detected by a shift in the PH indicator.


Materials

API20 Strep strips                             ý

APIGP medium, 2 ml                        ý Store at 2-80C

Sterile distilled water,  2 ml                ý

Mineral oil


NIN – Ninhydrin                                ý

ZYM A Reagent                                 ý Store at 2-80C

ZYM B Reagent                                 ý

Voges - Proskauer Reagents               ý

           


Procedure

6.                  Preparation of Inoculum

a)                  Add 2 ml of sterile distilled water without additives to a sterile test tube.

b)                  Using a sterile swab,  make a dense suspension with a turbidity greater than

4 McFarland standard from a fresh, pure culture; two plates may be necessary for an adequate inoculum. This suspension must be used immediately after preparation.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH45v02

Page 2 of  4

Section: Technical Manual

Subject Title:API Test Strips -API20 Strep

 

7.                  Preparation of the Strip

a)                  An incubation tray and lid is supplied for each strip.

b)                  Dispense 5 ml of water in to the tray.

c)                  Label the tray with the patient name and order number.


8.                  Inoculation of the Strip

a)                  Remove the cap from the tube containing the bacterial suspension.

b)                  When inoculating theAPI20 Strep strip, avoid the formation of bubbles (tilt the strip slightly forwards and place the tip of a Pasteur pipette against the side of the cupule):


The first half of the strip (tests VP to ADH) will be inoculated with this suspension

a)                  For tests VP to LAP, distribute approximately 100µl into each capule.

b)                  For the ADH test: fill the tube only.


For the second half of the strip (RIB to GLYG):

a)                  Carefully open an ampule ofAPIGP Medium and transfer the rest of the suspension (approximately 0.5 ml) into it. Mix well.

b)                  Distribute this new suspension into the tubes only.

c)                  Fill the capule of the underlined tests (ADH to GLYG) with mineral oil to form a convex meniscus.

d)                 Place the lid on the tray.

e)                  Using the excess bacterial suspension, inoculate a blood agar plate  as a purity check. Incubate at in CO2 overnight.


9.                  Incubation of the Strip


a)                  Incubate the strip at 360C in aerobic conditions for 4 to 4½ hours to obtain a first reading and for 24 hours to obtain a second reading if required.


10.              Reading the Strip

a)                  After 4 hours of incubation: add the reagents:

                        VP test: 1 drop each of VP 1 and VP 2

                        HIP test: 2 drops of NIN

                                    PYRA, aGAL, ßGUR, ßGAL,PALand LAP tests:

                                    1 drop each of ZYM A and ZYM B

                        Wait 10 minutes; record the reactions on theAPI20 Strep analytical profile sheet



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH45v02

Page 3 of  4

Section: Technical Manual

Subject Title:API Test Strips -API20 Strep

 

                        Reincubation is necessary in the following cases:

-          low discrimination;

-          unacceptable or doubtful profile;

-          or if the following comment is given for the profile:

            IDENTIFICATION NOT VALID

         BEFORE 24 HOURS INCUBATION

In this case, after 24 hours, reread the reactionsESC, ADH and RIB to GLYG. Do not reread the enzymatic reactions (HIP, PYRA, aGAL, ßGUR, ßGAL,PAL, LAP) and VP


                        Record the reactions on theAPI20 Strep analytical profile sheet



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH45v02

Page 4 of 4

Section: Technical Manual

Subject Title:API Test Strips -API20 Strep

Interpretation

SUMMARY OF RESULTS – 4 to 24 HOUR PROCEDURE

TESTS

RESULTS

NEGATIVE

POSITIVE

 

VP

Add VP 1 + VP2/ wait 10 minutes

Colourless

Pink-Red

 

HIP

Add NIN/wait 10 minutes

Colourless/Pale blue

Bluish-grey

Dark blue/Violet

 

ESC

4 hours

24 hours

4 hours

24 hours

Colourless

Pale yellow

Colourless

Pale yellow

Light grey

Black

Grey

Black

 

PYRA

Add ZYM A + ZYM B/ wait 10 minutes (PYRA to LAP)

Colourless or very pale orange

Orange

aGAL

Colourless

Violet

ßGUR

Colourless

Blue

ßGAL

Colourless or very pale violet

Violet

PAL

Colourless or very pale violet

Violet

LAP

Colourless

Orange

ADH

Yellow

Red

 

RIB

4 hours

24 hours

4 hours

24 hours

Red

Orange/Red

Orange/Yellow

Yellow

ARA

Red

Orange/Red

Orange/Yellow

Yellow

MAN

Red

Orange/Red

Orange/Yellow

Yellow

SOR

Red

Orange/Red

Orange/Yellow

Yellow

LAC

Red

Orange/Red

Orange/Yellow

Yellow

TRE

Red

Orange/Red

Orange/Yellow

Yellow

INU

Red

Orange/Red

Orange/Yellow

Yellow

RAF

Red

Orange/Red

Orange/Yellow

Yellow

AMD

Red

Orange/Red

Orange/Yellow

Yellow

GLYG

Red orOrange

Bright Yellow


d)                 The tests are separated into groups of three.  The following numerical value is assigned to each reaction recorded:

3-                  positive reaction in the first test of the group

4-                  positive reaction in the second test of the group

            4-         positive reaction in any test

1-                  negative reaction in any test


e)                  Refer to the API WEBSITE for Identification Profile



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH46v03

Page 1 of  1

Section: Technical Manual

Subject Title: API WEBSITE for Identification Profile

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:March 14, 2007

Annual Review Date:April 29, 2008

 

API WEBSITE for Identification Profile

 

 

  1. Read API biochemical strip as per reading procedure.

 

  1. Go to API website:


https://apiweb.biomerieux.com/servlet/Authenticate?action=prepareLogin

 

3.      Login: gsmall

Password: micro2

 

4.      Enter reactions or profile number

 

5.      Obtain identification

 

6.      Copy the identification and percent probability to the LIS workcard.

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH5v02

Page 1 of  2

Section: Technical Manual

Subject Title: Bacitracin Disk Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director

Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

BACITRACIN DISK TEST

Principle

 

This is a screening test for the presumptive identification of Group A Streptococci which are

susceptible to 0.04U bacitracin.  Other beta-haemolytic streptococci are usually resistant to this concentration of bacitracin.

 

Reagents

 

Bacto Differentiation Disks Bacitracin (0.04U). Store refrigerated.

5% Sheep Blood Agar (BA)

 

Other Materials

 

Culture loop

Cotton swabs

Forceps

 

Procedure

 

1.         Inoculate the surface of the BA with the suspect beta haemolytic Streptococcus.  Streak for confluent growth.

2.         Using aseptic technique, place a bacitracin disk onto the inoculated surface. 

3.         Incubate in O2 at 35oC X 18-24 hr.

 

Interpretation

 

Susceptible:  any zone of inhibition around the disk (Presumptive Group A Streptococcus).

Resistant:    growth up to the edge of the disk

 

Precautions

 

1.         Other beta-haemolytic streptococci may be susceptible to bacitracin.  Therefore this test can be used only for the presumptive identification of Gp. A Strep.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH5v02

Page 2 of  2

Technical Manual

 


 

 

 

Quality Control

 

Test with known susceptible and resistant control strains weekly.

            Susceptible:     Gp.A Strep. (ATCC 19615)

            Resistant:        Gp.B Strep. (ATCC 13813)

 

Reference

1.         Difco Differentiation Disks Bacitracin package insert.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH7v02

Page 1 of  1

Section: Technical Manual

Subject Title: Bile Esculin Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

BILE ESCULIN TEST

Principle

 

This test determines the ability of an organism to grow in the presence of bile and to hydrolyze the glycoside esculin to esculetin and glucose.  The test is used to presumptively identify Group D Streptococci.

 

Materials

 

Bile esculin agar slant / plate

Culture loop

 

Procedure

 

1.         Heavily inoculate a bile esculin slant / plate with the suspect organism.

2.         Incubate in O2 at 35oC for 18-24 hr.

 

Interpretation

 

Positive:          Presence of a dark brown to black colour on the slant.

 

Negative:         No blackening of the medium.  Growth may occur, but this does not indicate esculin

                        splitting.

 

Quality Control

 

Each new lot of media should be tested with known control strains.

 

            Positive:          E. faecalis        (ATCC 29212)           

            Negative:         Gp.B Strep.     (ATCC 13813)

            No Growth:     Gp.A Strep.     (ATCC 19615)

 

References

 

1.                  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams

            and Wilkins,BaltimoreMD, 1980, p4-12.

 



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH8v02

Page 1 of 1

Section: Technical Manual

Subject Title: Bile Solubility Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

BILE SOLUBILITY TEST

Principle

 

Tests the ability of alpha haemolytic streptococci to lyse in the presence of bile salts.  This test is used for the identification of Streptococcus pneumoniae.

 

Reagents

 

BBL Spot Test dropper (10% sodium desoxycholate).

 

Procedure

 

1.         Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.

2.         Place 1 drop of the reagent directly on isolated colonies of suspected S. pneumoniae.

3.         Keep the plates very level to prevent the reagent from running and washing a non- pneumucoccal colony away, producing a false positive result.

4.         Incubate at room temperature on the bench for 15-30 minutes until the reagent drys.  Do not invert the plate; leave the lid ajar.

5.         Examine the colonies for lysis.

 

Interpretation

 

            Positive (bile soluble):             Lysis of the colonies.

 

            Negative (bile insoluble):        No lysis of colonies.

 

Quality Control

 

Test with known positive and negative control strains weekly.

 

            Positive:          S. pneumoniae             (ATCC 6303)

            Negative:         Viridans Streptococcus (LPTP 8610)

 

References

 

1.        MurrayPA, et al.  Manual of Clinical Microbiology, 7th ed., 1999; p. 1665.

 

2.         BBL Desoxycholate Reagent Droppers package insert, April 1991.

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH9v02

Page 1 of  2

Section: Technical Manual

Subject Title: Catalase Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

CATALASE TEST

Principle

 

Detects the presence of the enzyme catalase which hydrolyzes H2O2 to produce H2O and O2.  This test is used to differentiate Staphylococci (catalase positive) from Streptococci (catalase negative).

 

Reagents

 

Hydrogen peroxide (H2O2), 3%

            Store in a dark bottle and avoid any undue exposure to light.

            Keep refrigerated at all times when not in use.

 

Other Materials

 

Clean glass microscope slides

Plastic culture loop or wooden applicator stick

 

Procedure

 

1.         Pick a colony from an 18-24 hr culture and place it on a clean glass slide.  Avoid carry over of blood agar which can cause false positives.

2.         Put one drop of 3% H2O2 over the organism on the slide.  Do not reverse the order of the procedure as false positive results may occur.  Do not mix.

3.         Observe for immediate bubbling (gas liberation) and record the result.

4.         Discard the slide into a discard container.

 

Interpretation

 

Positive test:    Immediate bubbling, easily observed (02 formed)

 

Negative test:  No bubbling


Precautions

 

1.         Carry over of blood agar must be avoided.

2.         Growth for testing must be from an 18-24 hr culture.

3.         3% H2O2 is caustic - avoid exposure to skin.  If H2O2 does get on the skin, immediately flood the area with 70% ethyl alcohol, not water.



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Policy & Procedure Manual

Policy # MITECH9v02

Page 2 of  2

Technical Manual

 


 

4.         Aerosols may be released by the bubbling of the O2.

5.         H2O2 is unstable and breaks down easily on exposure to light.  The solution must be kept refrigerated in the dark.

 

Quality Control

 

H2O2 is very unstable and should be tested daily or immediately prior to its use.

 

            Positive:          S. aureus  (ATCC 25923)

            Negative:         Gp. A. Strep.  (ATCC 19615)

 

References

 

1.         MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore MD., 1980, p51-58.

 

2.        MurrayPA, et al.  Manual of Clinical Microbiology, 7th ed., 1999; pp 426-427.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH10v02

Page 1 of  1

Section: Technical Manual

Subject Title: Cetrimide Pseudomonas Selective Agar

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

CETRIMIDE PSEUDOMONAS SELECTIVE AGAR

 

Principle

 

Cetrimide Selective Agar is used for the identification of Pseudomonas aeruginosa.  Cetrimide is a compound that has germicidal activity against most organisms except Pseudomonas aeruginosa.  Also pigment production is enhanced on this media. 

 

Procedure

 

1.                  Divide the plate into approximately 8 pie shaped divisions.

2.                  Streak the test organism (pure culture) onto one of the pie shaped divisions.

3.                  Incubate at 350C for 18 - 24 hours.

 

Interpretation

 

Pseudomonas aeruginosa will grow on this media and will be pigmented a pale green to dark blue-green colour.  All other organisms will not grow or will be non-pigmented.

 

Quality Control

 

Test with positive and negative controls each time the test is set up.

 

            Positive:          Pseudomonas aeruginosa   (ATCC 27853)  

            Negative:         Escherichia coli   (ATCC 25922)                  

 

Reference

 

1.         PML Technical Manual,  1990.          


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH11v02

Page 1 of 6

Section: Technical Manual

Subject Title: Cryptococcal Antigen

Issued by: LABORATORY MANAGER

Original Date:March 20, 2000

Approved by: Laboratory Director


Revision Date:November 10, 2008

Annual Review Date:April 29, 2008

 

CRYPTOCOCCAL ANTIGEN

 

Latex particles coated with anti-cryptococcal globulin (ACGR) reacts with cryptococcal polysaccharide antigen (in CSF or serum) causing a visible agglutination.

 

I.          Specimen Collection and Processing

 

            5 mL of blood is collected in a serum separator tube and separated by centrifugation. The serum is removed to a vial and refrigerated until testing. Specimens are stored a

            -20oC after testing.

 

            Spinal fluid is collected in clean, sterile, centrifuge tubes.  Specimens are stored refrigerated after testing.

            Note:  Fungus culture should also be set up.

 

II.        Procedure

 

            Reagents

 

            Meridian CALAS (Cryptococcal Antigen Latex Agglutination System)

            1.         GBDA - Glycine buffered diluent with albumin.

            2.         Detect Latex (ACGR) - Anti-cryptococcal globulin reagent.

            3.         Control Latex (NGR) -Normalglobulin reagent.

            4.        AGC- Antiglobulin control.  Rehydrate with 1.5 mL dH2O.

            5.         NC - Negative control.  Rehydrate with 2.5 mL dH2O and inactivate the negative at 56oC for 30 minutes.

            6.        CAC- Cryptococcal antigen control (Positive control).

            7.         Pronase - Rehydrate with 2.5 mL dH2O, can be stored at 2oC-8oC for approximately 1 month.

 

            Note:   Ensure that all reconstituted vials are thoroughly dissolved before use

 

            All reagents are stored refrigerated.  Do not interchange reagents with a kit having a different lot number.  Allow reagents to warm to room temperature before use.  Mix gently before use.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH11v02

Page 2 of  6

Section: Technical Manual

Subject Title: Cryptococcal Antigen


 

            Other Materials

            Boiling water bath

            56oC heating block

            1.0 x 0.1 mL pipettes

            Rotator

            Small serologic test tubes

            Test tube rack

            Marking pen

            Applicator sticks

 

            The following are provided byMeridian:

            Capillary pipettes

            Rubber bulb

            Ring slide

 

            Method

 

            Specimen preparation:

 

            1.         Store refrigerated if testing is not done immediately.

 

                        (a)        Inactivate serum by mixing 500 µL of serum and 500 µL of pronase solution in a 12 x 75 mm tube and incubate at 56oC for 15 minutes.  Further inactivate in a boiling water bath for 5 minutes.  This constitutes a 1:2 dilution.

 

                        (b)        Centrifuge CSF at 3500 rpm for 15 mins.  Inactivate the supernatant in a boiling water bath for 5 minutes.

 

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH11v02

Page 3 of 6

Section: Technical Manual

Subject Title: Cryptococcal Antigen


           

Performing the tests:

 

            Note:   Controls must be run each time a patient specimen is tested.

 

            1.         Set up and label the slide as follows:

 


Control           

Latex

(NGR)




Detect

Latex

(ACGR)


           CAC                            NC                 AGC                           TEST

            (POS                           (NEG              (Anti-

            Control)                       Control)           globulin

                                                                        Control)

           

 

                       

         

           





Not

Used

 
















            2.         Gently resuspend the latex particles in the Detect Latex (ACGR) and Control Latex(NGR) reagents.  Rock each reagent just prior to use.

                        Place one drop of Detect Latex (ACGR) or Control Latex (NGR) into the designated      

                         rings.

 

3.                  Place 25 μL (one drop) of the cryptococcal antigen control (CAC) into the designated rings.  Repeat with the negative control (NC) and anti-globulin control (AGC)

 

            4.         Place 25 μL of specimen in the designated rings.

           

            5.         Using a separate applicator stick, mix the contents of each ring thoroughly, spreading the contents over the entire surface area.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH11v02

Page 4 of 6

Section: Technical Manual

Subject Title: Cryptococcal Antigen



            6.         Place the slide on the rotator and rotate at 125 rpm for 5 minutes.

 

            7.         Read the reactions immediately.

 

            8.         Rate the agglutination as follows:

                        Positive = any evidence of agglutination (granulation or clumping)

                        Negative = a homogenous suspension of particles with no visible clumping.

                         Detect Latex (ACGR) + Positive Control = Positive

                         Detect Latex (ACGR) + Negative Control = Negative

                         Control Latex (AGC) + Anti-globulin Control = Positive

                         Control Latex (AGC) + Positive Control = Negative

                         Control Latex (AGC) + Negative Control = Negative

                        

            9.         Patient specimens showing any agglutination in Detect Latex (ACGR) should be titrated against both Detect Latex (ACGR) and Control Latex (AGC) reagents.

 

                        (a)        Prepare two-fold serial dilutions of the specimen using 200 µL of GBDA in each of 8 test tubes labelled as follows:

 

 

            Tube    1        2      3         4        5          6           7        8

 

            Serum  1:4   1:8  1:16  1:32   1:64    1:128   1:256 1:512

 

            CSF    1:2   1:4    1:8    1:16    1:32   1:64    1:128    1:256

 

                        (b)        Transfer one drop of each dilution into 2 rings.

                        (c)        Add one drop of Detect Latex (ACGR) to one ring of each dilution.

                        (d)       Add one drop of Control Latex (NGR) to each of the other rings.

                        (e)        Mix using separate applicator sticks.

(f)                Place the slide on the rotator and rotate at 125 rpm for 5 minutes.

                        (g)        Read the results as follows:

 

                                    1+ = fine granulation against a milky background

                                    2+ = small but definite clumps against a slightly cloudy background

                                    3+ = large and small clumps against a clear background

                                    4+ = large clumps against a clear background

 

                        (h)        If tube #8 gives an agglutination of 2+ or

                                    greater, the specimen must be further serially

                                    diluted until a titre may be obtained.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH11v02

Page 5 of 6

Section: Technical Manual

Subject Title: Cryptococcal Antigen


 

Interpretation of results

 

            Negative:         Negative result in initial screening tests against Detect  Latex(ACGR).

 

            Positive:          The titre is reported as the highest dilution showing a 2+ or greater reaction with Detect Latex (ACGR) and negative with Control Latex(NGR).

 

            Nonspecific Interference:       The titre with Detect Latex (ACGR) is at least 4-fold higher than the titre with Control Latex(NGR).

 

Uninterpretable test:   The titre with Detect Latex (ACGR) is less than 4-fold greater than the titre with Control Latex(NGR).

 

III.       Reporting

 

            Telephone all positive reports.

 

            Negative Report:         "Cryptococcal antigen not detected by latex agglutination."

 

            Positive Report:          "Cryptococcal antigen detected at a titre of         by latex agglutination."

 

            Non-specific or Uninterpretable Report:

                                                "Cryptococcal antigen uninterpretable by latex agglutination."

 

IV.       Precautions

 

            The ring slide must be thoroughly cleaned after each use as follows:

 

            (a)        Soak in hypochlorite overnight.

            (b)        Scrub using detergent.

            (c)        Rinse well with tap water.

            (d)       Rinse 3 times with distilled water.

            (e)        Dry thoroughly using paper towels.

            (f)        Wipe clean with lint-free tissue.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH11v02

Page 6 of 6

Section: Technical Manual

Subject Title: Cryptococcal Antigen


 

V.        Quality Control

 

            The pattern of reactions for the controls must be as follows.

 

             Control Latex(NGR)             -                       -                       +

             Detect Latex(ACGR)            +                      -                       N/A

                                                        CAC                   NC                 AGC

 

            Failure to obtain this pattern indicates that the test must be repeated and the patient test results cannot be reported.

 

VI.       References

 

Product Insert, 1986 Rev 7/01 Meridian Diagnostics Inc.,3471 River Hills Dr.,Cincinnati,Ohio45244.  (513)-271-3700.

 

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH15v02

Page 1 of  3

Section: Technical Manual

Subject Title: E. coli O157 Latex Test (Oxoid)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

E. coli O157 LATEX TEST (OXOID)


Principle

The Latex test will demonstrate by slide agglutination, E. coli strains possessing the O157 antigen.  Sorbitol MacConkey Agar (SMAC) should be used as the primary screen.  Non-sorbital fermenting colonies (NSF) are tested with the latex reagents, to determine if the isolate belongs to the O157 serogroup, and is therefore a potential vero-cytotoxin (VT) producing strain.


Reagents

DR621 Test Latex -                consists of latex particles sensitized with specific rabbit antibody reactive with the O157 antigen.


DR622 Control Latex -           consists of latex particles sensitized with pre-immune rabbit globulins.


Storage

Do not freeze.  Store at 20C - 80C.  Do not use kit beyond the expiry date.


Procedure

NSF colonies may be taken from SMAC plates or alternatively NSF isolates may be inoculated onto non-selective agar media for testing.


It is necessary to test up to 10 separate NSF colonies to ensure a high probability of detection from mixed cultures.


1)                  Bring the latex reagents to room temperature. Make sure the latex suspensions are mixed by vigorous shaking.  Expel any latex from the dropper pipette for complete mixing.


2)                  Dispense 1 drop of the Test latex onto a circle of the black slide.  Place it close to the edge of the circle.



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Policy & Procedure Manual

Policy # MITECH15v02

Page 2 of  3

Technical Manual

 


 


3)                  Add some loopfuls or a pasteur pipette drop of saline to the circle.  Ensure that the latex and saline do not mix at this stage.


4)                  Using a loop, pick off a portion of the colony to be tested and carefully emulsify in the saline drop.


5)                  Mix the Test latex and suspension together and spread to cover most of the reaction area using the loop.  Flame the loop.


6)                  Rock the slide in a circular motion, observing for agglutination.  Do not rock the card for more than 1 minute and do not use a magnifying glass.


7)                  If no agglutination occurs, then proceed to test other NSF colonies if these are present.


8)                  If agglutination with the test reagent does occur, then it is necessary to test a further portion of the colony with the control reagent to ensure that the isolate is not an autoagglutinating strain.


9)                  When finished, dispose of the reaction slide into disinfectant.


Interpretation

a)         Positive result -                       Agglutination of the Test latex occurs within 1 minute.

                                                            No agglutination of the Control latex.  *Perform

biochemical tests to confirm that the organism is an E. coli strain.


b)         Negative result -                      no agglutination of the Test latex.


c)         Non-interpretable result -        clumping of the Control latex.


References

1.                  Borczyk A., Lior H., Crebin B. 1987.  Int. J. Food. Microbiol. 4, 347-349.


2.                  Konowalchuk J., Speirs J. and Stavric S.  1977.  Infect. Immune. 18, 775-779.


3.                  ScotlandS., Day N. and Rowe B.  1980.  FEMS Microbiol. Lett. 7, 15-17.


4.                  Centers for Disease Control.  1982.  Morbid Mortal Wkly. 31, 580-585.



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Policy & Procedure Manual

Policy # MITECH15v02

Page 3 of  3

Technical Manual

 



5.                  Karmali M., Steel B., Petric M. and Lim C.  1983.  Lancet 8, 619-620.


6.                  Johnson W., Lior H. and Bezanson.  1983.  Lancet 8, 76.


7.                  March S. and Ratnam.  1986.  J. Clin. Microbiol.  23, 869-872.


8.                  Krishnan C., Fitzgerald V., Dakin S. and Behme R.  1987.  J. Clin. Microbiol. 25, 1043-1047.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH53v04

Page 1 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification

Issued by: LABORATORY MANAGER

Original Date:January 29, 2002

Approved by: Laboratory Director

Revision Date:July 30, 2007

Annual Review Date:April 29, 2008


GENEPROBE-ACCUPROBE Staphylococcus aureus CULTURE IDENTIFICATION KIT


Principle

 

Within the genus Staphylococcus, S. aureus is the most clinically significant species due to the incidence and severity of the infections it can cause.  Because problems with identification of clinical isolates exist, ACCUPROBE offers a rapid, objective method for the identification of S. aureus based on the detection of specific ribosomal RNA sequences that are unique to S. aureus.

This test is to be used for the identification of S. aureus isolated from culture.

 

Materials

 

The ACCUPROBE STAPHYLOCOCCUS AUREUS CULTURE IDENTIFICATION TEST

 

The ACCUPROBE CULTURE IDENTIFICATION REAGENTKIT– Reagents 1, 2, 3

 

Materials required but not provided

1 µl plastic sterile inoculating loops, wire loops, plastic needles or applicator sticks for selecting colonies

Control culture strains

Incubator or water bath (35-37oC)

Water bath or heating block (60 +/-1oC)

Micropipettes (50ul, 300 µl)

Re-pipettes (50ul, 300µl)

Vortex mixer

 

 

Sample Collection and Preparation

 

The ACCUPROBE  Staphylococcus aureus Culture Identification Test is designed to determine the identity of S .aureus from culture.

 

1.      Solid Media Method

Growth from appropriate solid media, with morphology suggestive of staphylococci may be tested.  The culture should be less than 48 hours old.  It can be tested as soon as the growth is visible.

a)      Growth can be removed with a 1 µl disposable plastic loop, a wire loop, a disposable plastic needle, or an applicator stick.  Swabs should not be used due to the small volume of liquid in which the cells are subsequently re-suspended.


 

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Policy # MITECH53v04

Page 2 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification

 

b)      If a single colony is to be tested, it should be at least 1mm in diameter.  A 1 µL loopful of cells or several (3-4) smaller colonies can be tested.

c)      Avoid taking any of the solid media with the cells.

d)     The operator may elect to inoculate another culture plate at this time to confirm the purity of the isolate. 

 

2.      Broth Culture Method

Appropriate broth cultures, such as Trypticase Soy or Brain Heart Infusion with turbidity equivalent to or greater than a McFarland 1 Nephelometer Standard may be tested.  Broth cultures, incubated for up to 72 hours at 37C may be used.  Pipette a 50 µl sample from the well mixed broth suspension into the Probe Reagent Tubes, as described below.

 

Procedure

 

1.         Equipment Preparation

Adjust the incubator or water bath to 35 –37oC.

Adjust the water bath or heating block to 60 + 1oC.

Prepare the Gen-Probe luminometer for operation.

 

2.                  Controls

Staph aureus ATCC29213 – Positive

Staph epidermidis ATCC12228 – Negative

 

3.         Sample Preparation

a)      Open the foil pouch by cutting evenly across the top of the pouch.  Remove enough Probe Reagent Tubes to test the culture and/or controls.  Reseal the pouch by folding the opened edge over several times and securing with adhesive tape or a clip.  Leave the desiccant pillow in the pouch.

b)      Label a sufficient number of Probe Reagent Tubes to test the culture isolates and/or controls.  Remove and retain the caps.

c)      Pipette 50 µl of Reagent 1 (Lysis Reagent) into all Probe Reagent Tubes.  If broth cultures are to be tested, do not add Reagent 1 to the Probe Reagent Tubes.

d)     Transfer the sample from the solid media or 50 µl of a well mixed broth culture into the labeled Probe Reagent Tubes as described in the Sample Collection and Preparation section.  Twirl the loop, needle or stick in Reagent 1 (Lysis Reagent) to remove the cells if testing growth from solid media and mix thoroughly.

e)      Recap the Probe Reagent Tubes and incubate at 35-37oC for 5 minutes in a water bath or 10 minutes at 35-37oC in an incubator.


 

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Policy # MITECH53v04

Page 3 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification

 

4.           Hybridization

a)   Remove the Probe Reagent Tubes from the water bath or incubator.  Remove and                             retain the caps.  Pipette 50 µl of Reagent 2 (Hybridization Buffer) into all Probe Reagent tubes.

b)   Recap the Probe Reagent Tubes and incubate for 15 minutes at 60 + 1oC in a water bath or heating block.

 

5.           Selection

a)      Remove the Probe Reagent Tubes from the water bath or heating block.  Remove and retain the caps.  Pipette 300 µl of Reagent 3 (Selection Reagent) into each tube.  Recap the tubes and VORTEX them to mix completely.

b)      Incubate the Probe Reagent Tubes for 5 minutes at 60 + 1oC in a water bath or heating block.

c)      Remove the Probe Reagent Tubes from the water bath or heating block and leave them at room temperature for at least 5 minutes.  Remove and discard the caps.

Read the results in the luminometer within 1 hour after removing from the water bath or heating block.

 

6.          Detection

a)      Select the appropriate protocol from the menu of the luminometer software.

b)      Using a damp tissue or paper towel, wipe each tube to ensure that no residue is present on the outside of the tube and insert the tube into the luminometer according to the instrument directions.

c)      When the analysis is complete, remove the tube(s) from the luminometer.

 

 

Procedural Notes

 

1.      Reagents:  Reagent 2 (Hybridization Buffer) may precipitate.  Warming and mixing the solution at 35-60oC will dissolve the precipitate.

 

2.      Temperature:  The Sample Preparation, Hybridization and Selection reactions are temperature dependent. Therefore, it is imperative that the incubator, water bath or heat block is maintained within the specified temperature range.

 

3.      Time:  The Hybridization Reaction should be started within 1 hour of adding the cells and Reagent 1 to the Probe Reagent Tubes.

The Hybridization and Selection reactions are time dependent.  Hybridize at least 15 minutes but no more than 20 minutes.  Incubate the Probe Reagent Tubes during the Selection Step for at least 5 minutes but no more than 6 minutes.


 

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Policy # MITECH53v04

Page 4 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification

4.      WaterBath:  The level of water in the water bath should be maintained to ensure that the entire liquid reaction volume in the Probe Reagent Tubes is submerged.

 

5.      Vortexing:  It is critical to have a homogeneous mixture during the Selection Step, specifically after the addition of Reagent 3.

 

6.      Trouble-shooting: a) Elevated negative control values can be caused by insufficient mixing after adding Reagent 3 (Selection Reagent) or by testing mixed cultures.  Because mixed cultures can occur, a portion of the growth may be streaked onto the appropriate agar medium and incubated to check for multiple colony types.

b)  Low positive control values can be caused by insufficient cell numbers or by testing mixed or aged cultures.  Because mixed cultures can occur, a portion of the growth may be streaked onto the appropriate agar medium and incubated to check for multiple colony types.

 

7.      ACCUPROBE also has identification kits for Streptococcus pneumoniae and Enterococcus.  The method for Enterococcus is identical as for Staph aureus, but the tubes used would be from the Enterococcus Probe Kit.  The method for Streptococcus pneumoniae differs from the Staph aureus method, by omitting the incubation at 35-37C in sample preparation (Step ‘e’). 

 

Results

 

1.      Interpretation of Results

The results of the ACCUPROBE Staphylococcus aureus Culture Identification Test are based on the following cut-off values.  Samples producing signals greater than or equal to these cut-off values are considered positive.  Signals less than these cut-off values are considered negative.  Results in repeat ranges should be repeated.

 

2.      Quality Control and Acceptability of Results

Negative control (Staph epidermidis,ATCC12228) and positive control (Staph aureus,ATCC29213) should satisfy the following results.

 

 ACCULDR                                         LEADER

Cut-off Value                         1, 500 PLU                                          50,000 RLU

Repeat Range                         1,200-1,499 PLU                                40,000- 49,999 RLU

Negative Control                     <600 PLU                                           <20,000 RLU

Positive Control                       >1,500 PLU                                        >50,000 RLU

 

Reference

 

Gen-Probe Incorporated,San Diego,CA, ACCUPROBE STAPH AUREUS CULTURE IDENTIFICATION TEST package insert http://www.gen-probe.com/pdfs/pi/102944RevG.pdf


 

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Policy & Procedure Manual

Policy # MITECH53v04

Page 5 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification

 

OPERATION OFGEN-PROBE LUMINOMETER

 

A.        Instrument Startup

 

Verify that the instrument is properly connected to the power source.  Verify that there is sufficient printer paper and that the Detection Reagent Reservoirs contain sufficient reagent to completely analyze the samples to be tested.  See SectionVII-A for instructions to replace printer paper and SectionIII-I to replace Detection Reagents.

 

Operation of the LEADER 50i luminometer is controlled by the operator through the CONTROL PANEL.  After the POWER SWITCH is placed in the “ON” position theMAINMENU appears on the display.

     LEADER 50                  SELECT:

 

START   PROGRAM   LIST   UTIL  WASH

 








Software functions are accessed through the eight function keys on the right.


“START” allows the operator to access the MEASURE MENU.  Proceeding through this menu allows the operator to measure and analyze samples.


“PROGRAM” allows the operator to edit or delete protocols and to add new protocols to the memory.  A maximum of 30 protocols may be stored in the software, and all are identified and accessed by a number.


LIST” allows the operator to print out the parameters contained in a selected protocol.


“UTIL” allows the operator to view or change instrument parameters.  These include date and time, passcode assignment, calibration factor, and background limit.


“WASH” allows the operator to prime the reagent injection system.


“STOP” allows the operator to interrupt the protocol in progress and exit to run a stat procedure.


“RESUME” allows the operator to return to the protocol that was in process when a stat run was begun.


“ENTER” allows the operator to accept the value on the display.


 

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Policy & Procedure Manual

Policy # MITECH53v04

Page 6 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification

B.        ANALYZING SAMPLES

The following guide outlines the general procedure for analyzing samples using the LEADER 50i luminometer.  Prior to reading samples, the LEADER 50i luminometer should be allowed a 20 minute warm-up period. Press the “ON” switch at the back of the machine above the electrical cord. To maintain instrument life, the instrument power should be turned “OFF” if it is not to be used within a 24 hour period.


The following sections are a guide for measuring samples.  Refer to the display screens and follow the instrument prompts.  Key in the instrument commands as indicated.


Note:   Before analyzing samples in the LEADER 50i luminometer, perform 2 wash cycles to prime reagent lines and pumps. PressWASH: Insert empty tube: Close cover.  Repeat a second time.  (See Step #3)


DISPLAY

COMMANDS

EXPLANATION

     LEADER 50                  SELECT:

START  PROGRAM  LIST  UTIL WASH

 

1.

 

Press START key.

 

This command accesses the MEASURE Function.

CHECK REAGENT LEVELS

PRESS START TO PROCEED

 

2.


 

Check reagent bottles for sufficient volume to run assays.  Press START key.

 

This display is a reminder to the operator to monitor reagent volume.

WASH CYCLE

0=NO          1=YES

 

3.



 

Press 1, ENTER. Place an empty tube in the chamber and close the cover.

 

Pressing 1, ENTER initiates the wash cycle. Chamber lid opens. When cover is closed, instrument will prime injectors. At completion, cover will open. Selecting 0 will bypass wash cycle.

OPERATOR:

PRESS ENTER TO ACCEPT, 1 FORNEWNAME

 

4.


 

Press ENTER.

 

Up to 24 characters may be entered. If ID is not desired, press ENTER to advance to the next screen.


ENTERKITLOT NUMBER?

0=NO      1=YES

 

5.


 

Key in 0. Press ENTER.

 

Up to 10 characters may be entered. Press 0 to bypass kit lot number entry.

START PROTOCOL NUMBER ________

PRESS ENTER

 

6.


 

Key in 3. Press ENTER.

 

Optical chamber cover will open. Assay header will print.


PROTOCOL 3 APBACFUNG

SELECTDATAMODE: 0=NEW   1=OLD   2=EDIT

 

7.


 

Key in 0. Press ENTER.

 

Choosing 1 allows use of references from most recent run. Can be used to retrieve references in case of power failure during run.

 

Choosing 2 allows manual entry of data as tubes are run.

 

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Policy # MITECH53v04

Page 7 of 7

Section: Technical Manual

Subject Title: ACCUPROBE S. aureus Culture Identification


DISPLAY

COMMANDS

EXPLANATION

SAMPLE 1 REPLICATE 1

INSERT TUBE, CLOSE COVER

 

8.


 

Insert first sample after removing cap. Close chamber cover.

 

Sample is processed as above.


SAMPLE 2 REPLICATE 1

INSERT TUBE, CLOSE COVER

 

9.



 

Insert second sample after removing cap. Close chamber cover.

 

Continue to process samples in same manner until all samples are processed.

SAMPLE N REPLICATE 1

INSERT TUBE, CLOSE COVER

 

10.


 

 

Press STOP.

 

After all tubes are processed, press STOP.

PROTOCOL 3 APBACFUNG

SELECTDATAMODE: 0=NEW   1=OLD   2=EDIT

 

11.



 

Press 2, ENTER. Place a clean empty tube in chamber and close cover.

 

Run will terminate. Display will return toMAINMENU. Choosing 0 allows operator to continue running samples. Choosing 1 allows operator to process aSTATsample.

PROTOCOL 3 APBACFUNG

SELECTDATAMODE: 0=NEW   1=OLD   2=EDIT

 

12.


 

 

MAIN MENU display.


The LEADER 50i luminometer can be left “ON” to continue analysis using different protocols.  Alternatively, the operator may cease operation for the day.  If the instrument is not to be used within a 24 hour period, turn the instrument power switch to “OFF.”


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH16v02

Page1 of  2

Section: Technical Manual

Subject Title: Germ Tube Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

GERM TUBE TEST

Principle

 

This is a rapid test for the presumptive identification of C. albicans.

 

Reagents

 

Bovine serum

            A small volume to be used as a working solution may be stored

            at 2 to 8oC.  Stock solution can be dispensed into small

            tubes and stored at -20oC.

 

Other Materials

 

Clean glass microscope slides

Glass coverslips

Vitek tubes (13 x 100 mm)

Pasteur pipettes

 

Procedure

 

1.         Put 3 drops of serum into a small Vitek tube.

2.         Using a Pasteur pipette, touch a colony of yeast and gently emulsify it in the serum.  The pipette can be left in the tube.

3.         Incubate at 37oC for 2-4 hours but no longer.

4.         Transfer a drop of the serum to a slide for examination.

5.         Coverslip and examine microscopically using x 40 objective.

 

Interpretation

 

Germ tubes are appendages half the width and 3 to 4 times the length of the yeast cell from which they arise.  There is no constriction between the yeast cell and the germination tube.

 

Positive test:    presence of short lateral filaments (germ tubes)

 

Negative test: yeast cells only



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Technical Manual

 


 

 

Precaution

 

C. tropicalis may form pseudohyphae which may be falsely interpreted as germ tubes.

 

 

Quality Control

 

Set up known controls each time a test is run.

 

            Positive:          C. albicans      (ATCC 10231)

            Negative:         C. tropicalis     (ATCC 13803)

 

 

Reference

 

1.        MurrayPA, et al.  Manual of Clinical Microbiology, 7th ed., 1999; pp. 1189-1191.


 

 

 



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH17v02

Page 1 of  2

Section: Technical Manual

Subject Title: Gonogen

            (GC Coagglutination) Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

GONOGEN (GC COAGGLUTINATION) TEST

Principle

 

The Gonogen II test is a coagglutination test for the confirmatory identification of N. gonorrhoeae.

 

Reagents

 

            I:  Buffer

            II: Gonogen reagent (antibodies)

            Positive control reagent

            Negative control reagent

 

Other Materials

 

            Test tray: consists of wells with special

            matrix and absorbent material

            Glass tubes (12 x 75mm) (not provided)

            Glass dropper rod assembly

            Plastic transfer pipets

 

Procedure

 

1.         Preparation of sample

 

            a)         In a 12x75 mm tube dispense 500 μL of reagent I (buffer) using the glass dropper rod assembly provided (demarcation line).

 

            b)         Using a swab, make a suspension of approximately 30 colonies to match a McFarland 1 turbidity standard.

 

            c)         Press swab against side of tube to express as much liquid as possible.

 

            d)         Vortex reagent II and add 1 drop to the tube.

 

            e)         Mix and set sit for at least 5 minutes.


 

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Technical Manual

 


 

 

2.         Test

 

            a)         With a plastic transfer pipet, add 2 drops of each test suspension into a well of the test tray using a separate well for each test.

 

            b)         using a clean plastic transfer pipet, add 1 drop of reagent I (buffer) to each completed test well.

Interpretation

 

            Positive:          Pink to red dot in well of test tray.

 

            Negative:         White to pale pink dot in well of test tray.

 

                        Note:   1.         A colour reaction more intense than the negative control should be interpreted as positive.

 

                                    2.         If color reaction is questionable, reincubate tube at RT for 3 minutes and repeat test.

 

                                    3.         If specimen suspension is made too turbid a faint background colour will occur.  This should NOT be interpreted as a positive result.

 

 

Quality Control

 

The positive and negative controls must be tested whenever a test is run.  The test is performed in the same manner except 1 drop of the control reagent is added to 500 μL of buffer rather than a suspension of the test organism.

 

References

 

1.         Gonogen II package insert, October 1993.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH19v02

Page 1 of  2

Section: Technical Manual

Subject Title: Hippurate Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

HIPPURATE TEST

Principle

 

This test determines the ability of bacteria to hydrolyse sodium hippurate. One of the end products, glycine is detected by the addition of ninhydrin reagent.

 

Reagents

 

Hippurate disks (store refrigerated)

Ninhydrin reagent

Sterile distilled water

 

Other Materials

 

Sterile tube (13 x 100mm)

Bacteriology loop

Sterile graduated pasteur pipette

 

Procedure

 

1.         Place a Hippurate disk into a sterile tube and add 0.4 mL sterile water.

 

2.         Heavily inoculate the tube with a loopful of the test organism.

 

3.         Incubate at 350C for 2 hours.

 

4.         Following incubation add 5 drops of ninhydrin reagent to the tube and shake gently.

 

5.         Reincubate tube for 10 minutes and read reaction.

 

Interpretation

 

Positive:                      Deep purple-blue colour

 

Negative:                     No colour change or light purple



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Technical Manual

 


 

 

Precautions

 

1.         A heavy inoculum is necessary to obtain a high concentration of enzyme.

 

2.         Do not incubate longer than 30 minutes after addition of ninhydrin reagent because a false positive reaction could result.

 

 

Quality Control

 

Test with known positive and negative controls each time the test is preformed.

 

Positive:          Campylobacter jejuni (ATCC 29428)

 

Negative:         Campylobacter coli     (CPI B7080)

 

 

Reference

 

1.         Difco package insert.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH20v02

Page 1 of  2

Section: Technical Manual

Subject Title: Indole Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

INDOLE TEST

 

Principle

Bacteria that produce the enzyme tryptophanase will deaminate tryptophan to indole, pyruvic acid and ammonia in the presence of a co-enzyme pyridoxal phosphate.


Indole combines with Ehrlich's / Kovac's reagent to form a red-coloured complex.


Materials

Test A:                        Filter paper strips impregnated with Ehrlich's reagent.


Test B:             Kovac's reagent

                        2% Tryptone broth (Difco, Oxoid)


Method

A:        Filter paper strips are suspended over tubes of ONPG / PAM media, and incubated at 350C overnight.


Interpretation

Positive test - development of red colour on the strip.

Negative test - white-yellow colour.


B:        1.         Inoculate the tryptone broth, and incubate at 350C overnight.

2.                  Add a few drops of Kovac's reagent to the broth.


Interpretation

Positive test - red colour in the upper layer.

Negative test - light-yellow colour in the upper layer.



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Technical Manual

 


 


Quality Control

Test the following positive and negative controls weekly:


Positive:          Proteus vulgaris (ATCC 13315)

Negative:         Klebsiella pneumoniae (ATCC 13883)

Reference

1.        Murray,PA, et al.  Manual of Clinical Microbiology 7th ed. 1999.

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH21v02

Page 1 of  1

Section: Technical Manual

Subject Title: Koehler Illumination

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

KOEHLER ILLUMINATION


The microscope should be set up using Koehler illumination for all parasitology examinations.  This ensures that all the light from the lamp is being focused onto the specimen and that the field to be examined is evenly illuminated.


Procedure


1.                  Turn the lamp on.


2.                  Bring the condenser up to the top position, with the top lens swung in.


3.                  Open the condenser diaphragm.


4.                  Place a specimen on the stage and focus with the 10x objective.


5.                  Close the field diaphragm.


6.                  Lower the condenser until the edge of the field diaphragm is in sharp focus.


7.                  Center the field diaphragm image with condenser centering screws.


8.                  Open the field diaphragm until the edge just disappears from view.


9.                  Remove one eyepiece.


10.              Looking down the eyepiece tube, close the condenser diaphragm until the illumination is approximately 2/3 full.


11.              Replace the eyepiece.


12.              Repeat for each objective lens when changed.


Reference

1.                  Baron E., Finegold S.M., Bailey & Scott's Diagnostic Microbiology, 8th ed., The C.V.

Mosby Company, p64.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH22v02

Page 1 of  2

Section: Technical Manual

Subject Title: KOH String Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

KOH STRING TEST

Principle

 

The formation of a string (DNA) in 3% KOH indicates that the isolate is a gram negative organism.

 

Reagents

 

3% KOH

 

Other Materials

 

Glass slides

Culture loop

 

Procedure

 

1.         Place a drop of 3% KOH onto a  glass slide.

 

2.         Emulsify in KOH a loopful of the culture from a BA incubated for 18-24 hr.

 

3.         Continue to mix the suspension for 60 sec and by slowly lifting the loop, observe for the formation of a string.

 

Interpretation

 

Positive:          formation of a string within 60 seconds

 

Negative:         failure to form a string

 

Precautions

 

1.         False positive and false negative results may occur.

 

Quality Control

 

Known controls should be tested each time the test is performed.

            Positive:          P. aeruginosa  (ATCC 27853)

            Negative:         S. aureus         (ATCC 25923)

 

 

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Technical Manual

 


 

 

References

 

1.        MurrayPAet al.  Manual of Clinical Microbiology, 7th ed., 1999; p. 1671.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH23v02

Page 1 of  2

Section: Technical Manual

Subject Title: Lap Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

LAP TEST

Principle

 

LAP (Leucine-β-naphthylamide) impregnated disks serve as a substrate for the detection of Leucine aminopeptidase.  Following the hydrolysis of the substrate by the enzyme the resulting β-naphthylamine produces a red colour upon the addition of cinnamaldehyde reagent.  This test is usually used, in conjunction with other tests, for the identification of streptococci and other catalase negative gram positive cocci.

 

Reagents

 

LAP discs

Cinnamaldehyde reagent (0.01% p-dimethylamino-cinnamaldehyde)

(disks and reagents are both in LAP kit)

Glass slide

Innoculating loop

Forceps

Sterile distilled water

 

Procedure

 

1.         Place a LAP disk onto a glass slide and moisten it with one drop of sterile distilled water.

2.         Rub a loopful of the culture onto the moistened disk holding it in place with sterile forceps.

3.         Leave at room temperature for 5 minutes.

4.         After 5 minutes, add 1 drop of cinnamaldehyde reagent.

 

Interpretation

 

Positive:          red colour within one minute

 

Negative:         no colour change or slight yellow colour

 

Quality Control

 

Test known positive and negative controls each time an unknown is run.

 

            Positive:          E. faecalis        (ATCC 29212)

            Negative:         Leuconostoc    (ATCC 8923)


 

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Policy # MITECH23v02

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Technical Manual

 


 

 

Reference

 

1.         Carr-Scarborough Microbiologicals package insert 1991.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH24v02

Page 1 of  2

Section: Technical Manual

Subject Title: Motility Test Medium

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

MOTILITY TEST MEDIUM

Principle

 

Motility Test Medium is a semi-solid agar designed to demonstrate motility by diffusion.

 

Motility Test Medium is a modification of the formula of Tittsler and Sandhoizer.  The medium contains small amounts of agar and gelatin, as well as triphenyltetrazolium chloride (TTC).  TTC is a soluble compound which is taken up by the bacterial cells.  Once the substance has been absorbed by the bacteria, it is reduced, releasing the acid formazan, a highly pigmented red, insoluble compound.

 

Organisms are stabbed into the medium with an inoculating wire.  If the organisms are motile, they will diffuse into the soft medium laterally from the line of inoculation, resulting in a diffuse, pink colour throughout the medium.  Non-motile organisms grow along the line of inoculation only, producing a pinkish-red line with no diffusion.

 

Storage

 

Upon receipt store at 2 - 8oC, away from direct light.  Media should not be used if there are signs of contamination, deterioration (shrinking or discoloration), or if the expiration date has passed.

 

Limitations

 

Motility tests often show a false-negative reaction.  The organism may be weakly motile, or the flagella may be damaged due to heating, shaking, or other trauma. Hanging drop motility may be performed from an inoculated tryptone broth incubated for 2-4 hours to confirm motility results.  Consult appropriate microbiological texts for procedure.

 

TTC may be inhibitory to some fastidious bacteria.

 

Most motility of bacteria should be interpreted at 35oC: however, certain bacteria such as Yersinia enterocolitica demonstrate the best motility at 25oC. 

 

Organisms that require oxygen for growth, such as Pseudomonas aeruginosa, will produce a spreading film on the surface of the medium, and will not fan out from the inoculation line where oxygen is depleted.

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Policy & Procedure Manual

Policy # MITECH24v02

Page 2 of  2

Technical Manual

 


 

Procedure

 

1.         Tube Method

 

Prior to inoculation, the medium should be brought to room temperature.  Inoculate selected colonies of a pure 18 to 24 hour culture, or from a turbid broth culture 4-8 hours old.  Using a straight needle, stab the centre of the medium about 1/4" from the top.  Incubate the tubes with the caps loose at 35oC (see "Limitations") for 18 - 24 hours.  Observe for motility.

 

2.         Plate Method

 

If using a multipoint inoculation system, make a pour plate form the 18 ml tube by gently melting the agar in a boiling water bath and dispensing the liquid medium into a sterile petri dish.  Prepare the inoculum by touching the top of one  or two well isolated colonies and inoculating into a broth.  Stab the inoculum into the medium using the modified pins of a replicator or by using a straight needle.  Incubate aerobically at 350C (see "Limitations") for 16-18 hours.  Examine for the presence of a pink diffusion from the point of inoculation.  

 

Interpretation

 

Positive:          A diffuse pink colour occurring throughout the medium.

Negative:         A pinkish red line at the stab site with no diffusion.

 

Quality Control

 

Test the following positive and negative control organisms on receipt of each shipment:

 

Positive:          Escherichia coli  (ATCC 25922)

Negative:         Klebsiella pneumoniae  (ATCC 13883)

 

References

 

1.                  Finegold, S.M., and E. J. Baron, Bailey and Scott's Diagnostic Microbiology, 7th ed., C.V. Mosby,St. Louis, 1986.  Koneman, E.W., et al., Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott,Philadelphia, 1979.  Lennette, E.H., et al., Manual of Clinical Microbiology, 4th ed., American Society for Microbiology,Washington,D.C., 1985.


2.                  MacFaddin, J.F., Biochemical Tests for Identification of Medical Bacteria, Williams and Wilkins,Baltimore, 1980.  Tittsler R.P., andL.A.Sandhoizer, J. Bacteriol., 31:575, 1936.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH25v02

Page 1 of  2

Section: Technical Manual

Subject Title: Mug Test (PGUA)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

MUG TEST (PGUA)

Principle

 

If an organism produces the enzyme glucuronidase it will break down the substrate ortho-nitrophenyl-beta-glucuronide liberating the ortho-nitrophenyl producing a yellow colour.  This test is used, in conjunction with others, for the identification of E. coli.

 

Reagents and Materials

 

            PGUA tablets

            13x100mm tubes

            Tryptone water

            Kovac's reagent

 

Procedure

 

1.         Prepare a dense suspension of the test organism (lactose-fermenter only) in 0.25 mL of the tryptone water.

2.         Add 1 PGUA tablet to the tube.

3.         Incubate at 36oC for 4 hours.

4.         Examine the tube for development of a yellow colour.

5.         Add 1 drop of Kovac's Indole reagent and observe for the development of a red colour.

 

Interpretation

 

            MUG positive:                        Yellow colour

            MUG negative:           Colourless

 

            Indole positive:           Red colour after addition of Kovac's

            Indole negative:          Kovac's remains yellow

 

            MUG               INDOLE                     INTERPRETATION / ACTOIN

 

             +                        +                               report as E. coli

             -                         +                               set up VITEK  Identification

             +                        -                                set up VITEK  Identification

             -                         -                                set up VITEK  Identification



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Policy & Procedure Manual

Policy #MITECH25v02

Page 2 of  2

Technical Manual

 


 

Precautions

 

1.         E. coli O157:H7 and non-motile strains which produce verotoxin are MUG test negative.

 

 

Quality Control

 

The following controls are tested weekly:

                                                                                    MUG               INDOLE

 

                        E. coli (ATCC 25922)                         +                        +

 

                        P. mirabilis (ATCC 12453)                -                         -

 

 

Reference

 

1.         Prolab package insert



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH26v02

Page 1 of  2

Section: Technical Manual

Subject Title: Neisseria Identification

                        Sugars

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

NEISSERIA IDENTIFICATION SUGARS

Principle

 

The test determines the ability of bacteria to produce acid products from carbohydrates.  Used as a method to identify Neisseria species and other fastidious organisms.

 

Materials

 

Cystine Proteose Peptone Agar (CPPA) media: - glucose, maltose, lactose, sucrose, control (no sugar).

Inoculating loop.

 

Procedure

 

1.         For each tube, scrape a full 3 mm loopful of growth from the surface of a 24 hour chocolate agar subculture plate.

2.         Deposit this inoculum a few millimetres below the surface of the medium.

3.         Incubate at 35oC.

4.         Examine tubes after 1, 4 and 24 hours incubation.

 

Interpretation

 

            Positive:          Yellow colour at top of tube

            Negative:         Red (alkaline) to orange (neutral) colour.

 

            Organism                               Glu      Mal      Lac      Suc      Cont

 

            N. gonorrhoeae                       +         -          -          -          -

            N. meningitides                       +         +         -          -          -

            M. catarrhalis                          -          -          -          -          -

 

Precautions

 

1.         Inoculum must be heavy.

2.         False positive results may occur if tubes are incubated in CO2.

3.         Tubes that appear bright yellow should be gram stained to check for contamination.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH26v02

Page 2 of  2

Technical Manual

 


 

 

Quality Control

 

The following controls are run each time the test is performed:

 

            N. gonorrhoeae   (ATCC 43069)

            N. meningitidis     (ATCC 13090)

            M. catarrhalis      (ATCC 8176)

 

Reference

 

1.         Murray PA, et al.  Manual of Clinical Microbiology, 7th ed., 1999; pp. 592-598.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH27v02

Page 1 of  2

Section: Technical Manual

Subject Title: ONPG Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

ONPG TEST

Principle

 

This test is used to demonstrate the presence or absence of the enzyme B-galactosidase using the substrate ortho-nitrophenyl-D-galactopyranoside in order to differentiate lactose-fermenting from non lactose-fermenting organisms and in the identification of B. cepacia.

 

Reagents

 

ONPG disks (Store refrigerated)

Sterile saline

 

Other materials

 

Sterile tube (13 x 100 mm)

Bacteriology loop

Sterile graduated Pasteur pipette

 

Procedure

 

1.         Place an ONPG disk into a sterile tube and add 0.2 mL saline.

2.         Heavily inoculate the tube with a loopful of the test isolate.

3.         Incubate at 35oC for up to 4 hours.

 

Interpretation

 

Positive:  yellow colour within 4 hours

 

Negative:  colourless at 4 hours

 

Precautions

 

1.         A heavy inoculum is necessary to obtain a high concentration of enzyme.

 

Quality Control

 

Test with known positive and negative controls each time the test is performed.

            Positive:          E. coli              (ATCC 25922)           

            Negative:         P. vulgaris       (ATCC 13315)

 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH27v02

Page 2 of  2

Technical Manual

 


 

 

References

 

1.                  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams

and Wilkins,BaltimoreMD., 1980, p120-128.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH28v02

Page 1 of  2

Section: Technical Manual

Subject Title: ONPG-Phenylalanine-

            Motility Medium (ONPG-PAM)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

ONPG-PHENYLALANINE-MOTILITY MEDIUM (ONPG-PAM)

Principle

 

This test is used to determine an organism's motility, its ability to ferment lactose and produce phenylalanine deaminase. The medium is primarily used as a screening procedure for the detection of enteric pathogens.

 

Reagents

 

ONPG-PAM tube

10% Ferric chloride

 

Other materials

 

Culture wire

 

Procedure

 

1.         Inoculate the ONPG-PAM tube by stabbing the centre of it to the bottom of the tube.

2.         Incubate the tube in O2 at 35oC X 18 hours.

3.         Read the tube for ONPG, motility and indole.

4.         Add 2 drops of 10% ferric chloride solution and read the phenylalanine result.

 

Interpretation

 

            ONPG:                                                positive:           yellow

                                                            negative:          no colour change

 

            Motility:                                  positive:           diffuse growth from line of inoculum

                                                            negative:          growth does not spread from line of inoculum

 

            Phenylalanine (PPA):              positive:           green

                                                            negative:          yellow/brown

 

           


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH28v02

Page 2 of  2

Technical Manual

 


 

 

Quality Control

 

Test with control organisms each time a new batch of meda is prepared.

 

 

ONPG

Motility

PPA

K. pneumoniae (ATCC 13883)

+

-

-

P. vulgaris (ATCC 13315)

-

+

+

 

           

References

 

1.                  Murray,PA, et al.  1999.  Manual of Clinical Microbiology, 7th ed., American Society for

            Microbiology,Washington,D.C.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH29v03

Page 1 of 2

Section: Technical Manual

Subject Title: Optochin Sensitivity Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director

Revision Date:July 30, 2007

Annual Review Date:April 29, 2008

 

OPTOCHIN SENSITIVITY TEST

Principle

 

This test is used to determine an organism's susceptibility to the chemical optochin (ethylhydrocupreine hydrochloride) for the presumptive identification of S. pneumoniae.

 

Reagents

 

Bacto Optochin Disks (5 µg disk) Store refrigerated

5% Sheep Blood Agar (BA)

 

Other Materials

 

Culture loop

Forceps

Cotton swabs

 

Procedure

 

1.         Inoculate the suspected alpha haemolytic colony onto a BA to obtain confluent growth.

 

2.         Using aseptic technique place an optochin disk onto the surface of the inoculated agar.  Press down with forceps.

 

3.         Incubate at 35oC in CO2 for 18-24 hours.

 

Interpretation

 

Susceptibile:    Zone of inhibition of at least 14 mm

Resistant:        Zone of inhibition less than 14 mm

 

Quality Control

 

Test with known susceptible and resistant control strains weekly:

 

            Susceptible:     S. pneumoniae   (ATCC 6303)

            Resistant:        Viridans Strep.              (LPTP 8610)


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH29v02

Page 2 of  2

Technical Manual

 


 

 

References

 

1.                  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore MD., 1980, p245-249.

 

2.         Difco package insert, July 1983.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH30v02

Page 1 of 2

Section: Technical Manual

Subject Title: Ornithine Decarboxylase

Issued by: LABORATORY MANAGER

Original Date:December 15, 2005

Approved by: Laboratory Director

Revision Date:

Annual Review Date:April 29, 2008

 

ORNITHINE DECARBOXYLASE

Principle

 

This test is used to determine an organism's ability to decarboxylate or hydrolyze the amino acid - ornithine, forming an amine - putrescine that produces an alkaline pH. It is primarily used for the differentiation of S. lugdunensis from other coagulase-negative staphylococci.

 

Reagents

 

Mǿller’s ornithine decarboxylase (1% ornithine) with bromcresol purple. OXOID. Store refrigerated.

Mineral Oil

 

Other Materials

 

Culture loop

 

Procedure

 

1.      Inoculate one or two colonies of a 18 to 24 hour culture of staphylococci into the ornithine decarboxylase tube.

2.      Overlay the inoculated tube with approximately 1 mL of mineral oil.

3.      Incubate at 35oC in O2 for 18-24 hours.

4.      Observe for purple colour.

 

Interpretation

 

Positive:          Turbid, purple colour (alkaline)

Negative:         Bright yellow colour (acidic)

 

Quality Control

 

Test with known positive and negative control strains weekly:

 

            Positive:          S. lugdunensis   (ATCC 700328)

            Negative:         S. aureus           (ATCC 25923)


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH30v02

Page 2 of  2

Technical Manual

Subject Title: Ornithine Decarboxylase

 

 

References

 

1.                  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins,BaltimoreMD., 1980.

 

2.         Izenberg H.D. 2003. Decarboxylase-Dihydrolase Tests 3.17.15, in Clinical Microbiology Procedures Handbook, 2nd ed. Vol.1 ASM Press,Washington,D.C.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH31v02

Page 1 of  2

Section: Technical Manual

Subject Title: Oxidase (API Strip)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

OXIDASE (API STRIP)

Principle

 

This test determines whether an isolate produces oxidase enzymes.  This test is mainly used, in conjunction with other tests, for the identification of gram negative organisms and Bacillus species.

 

Reagents

 

API Oxidase Reagent

1.         0.2% Aqueous ascorbic acid: Reconstitute ascorbic acid with 25 ml sterile distilled water.  This solution may be refrigerated for up to 28 days.  The expiry date must be written on the bottle.

 

2.         N,N,N,-Tetramethyl-p-phenylenediamine-dihydrochloride:

            Reconstitute with 5 ml of the 0.2% aqueous ascorbic acid.  It is recommended that this be re-constituted 4-5 hours before use.  This solution may be refrigerated for up to 7 days at 2 - 8oC.  The expiry date must be written on the bottle.

 

Other Materials

 

API filter paper

API oxidase tray

Wooden applicator stick

 

Procedure

 

1.         Place a filter paper in the oxidase tray and moisten entire paper with oxidase reagent. Allow to air dry. May be used for up to 1 week.                         

2.         Transfer a portion of the colony to the filter paper using a wooden applicator stick.

3.         Observe for 30 seconds.

 

Interpretation

 

Positive:          Development of a purple colour within 30 seconds

Negative:         No colour change

 

Precautions

 

Nichrome wire may cause false positive reactions.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH31v02

Page 2 of  2

Technical Manual

 


 

 

Quality Control

 

Test daily with known positive and negative controls.

 

            Positive:          P. aeruginosa     (ATCC 27853)

            Negative:         K. pneumoniae   (ATCC 13883)

 

References

 

1.         API Oxidase package insert 3/80.

 

2.                  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams

            and Wilkins,BaltimoreMD, 1980, p249-260.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH32v02

Page 1 of  1

Section: Technical Manual

Subject Title: Oxidase

                        (Spot Test Dropper)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

OXIDASE (SPOT TEST DROPPER)

Principle

 

This test determines whether an isolate produces oxidase enzymes and is used for the identification of Neisseria species isolated from primary plates.

 

Reagents

 

Spot Test dropper.  Store at room temperature.

 

Procedure

 

1.         Hold the dropper upright and squeeze gently to crush the glass ampule inside the dispenser.

 

2.         Add 2 - 3 drops directly to the colonies to be tested and observe for 30 seconds.

 

Interpretation

 

Positive:          Development of a purple colour within 30 seconds

 

Negative:         No colour change

 

Note:               Colonies which are positive must be subcultured immediately since prolonged exposure will result in death of the organisms.

 

Quality Control

 

Test daily with known positive and negative controls.

            P. aeruginosa (ATCC 27853) :  positive

            E. coli (ATCC 25922)             :  negative

 

References

 

1.         Difco Spot Text Oxidase reagent package insert 1985.

 

2.                  MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams

            and Wilkins,BaltimoreMD, 1980, p249-260.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH33v02

Page 1 of  2

Section: Technical Manual

Subject Title: Pastorex Staph Plus Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

PASTOREX STAPH PLUS TEST


Principle

A rapid slide latex agglutination test for the detection of clumping factor, capsular polysaccharide and protein A produced by most strains of S. aureus.


Reagents and Materials

Pastorex test latex suspension (store refrigerated)

Disposable reaction cards

Plastic stick


Procedure

1.                  Confirm the identification of a suspect Staphylococcus by Gram stain and catalase test.

2.                  Allow the latex reagent to warm to room temperature before use.

3.                  Shake the reagent so that all of the particles are resuspended.

4.                  Dispense one drop of latex test reagent in one of the circles on the reaction card.

5.                  Dispense one drop of negative control reagent in another circle on the card.

6.                  Emulsify 1 to 3 colonies into the test latex with a loop for 10 seconds.

7.                  Repeat step 6 for the negative control reagent.

8.                  Gently rock the card for 30 seconds and look for clumping.

9.                  Discard the card into a discard container.


Interpretation

Positive test:    Clumping within 20 seconds with the test latex particles only.


Negative test:  No clumping in either latex.


Uninterpretable test:   Clumping in the negative control.


Precautions

1.                  False positive results may occur after 40 seconds.

2.                  False positive agglutination can occur with organisms other than staphylococci.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH33v02

Page 2 of 2

Technical Manual

 



 

Quality Control

Test known positive and negative controls daily.


            Positive:          S. aureus  (ATCC 29213)

            Negative:         S. epidermidis  (ATCC 12228)

 

References

1.         Pastorex Staph Plus package insert Feb. 1999.

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH34v02

Page 1 of  2

Section: Technical Manual

Subject Title: Plate Streaking Methods

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

PLATE STREAKING METHODS

 

Blood Agar and MacConkey Agar for Urine Cultures

 

1 uL disposable loop

Inoculate in one continuous streak down the middle of the plate.  With the same loop, streak out the entire plate at 90o to the initial inoculum.  Streak a minimum of 15 lines.

 


                                                                                                                        1o inoculum

 


                                                                                                                       

 

 

 

 

Martin-Lewis Agar

 

Inoculate plate with specimen swab in a "Z" pattern across the plate (with continuous rotation of the swab while inoculating).  Streak out the entire plate with a sterile loop at 90o to the initial inoculum.  Streak a minimum of 15 lines.





                                                                                                                        1o inoculum








UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH34v02

Page 2 of 2

Technical Manual

 


 

Growth Quantitation:

 


  1 +    

  2 +

  3 +

 


Isoplater Streaking

 

Manual Streaking

 

Inoculate specimen with swab or loop onto the entire first quadrant of the agar plate.  Use a sterile loop and streak out the second, third and fourth quadrants as per diagram:

1                                                                                                                                                                                            2





                                                            Use a sterile loop








                                  3                                                                                          4

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH35v02

Page 1 of  2

Section: Technical Manual

Subject Title: Pro-Amp Glu-Amp Tests

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

PRO-AMP GLU-AMP TESTS

Principle

 

Rapid chromogenic tests for the identification of pathogenic Neisseria.

 

Reagents

 

Pro-Amp tablets

Glu-Amp tablets

Fast Blue BB solution

Sterile Saline

 

Other Materials

 

Sterile Tubes (13 x 100mm)

 

Procedure

 

1.         Suspend the growth from Choc media in 2 tubes of 0.25 ml saline to achieve the turbidity > #2 McFarland standard.

2.         Add 1 tablet to the respective tube.

3.         Incubate at 36oC x 4 hours.

4.         After incubation add 3 drops of Fast Blue BB solution to each tube and read results after 10 minutes.

 

Interpretation

 

            Positive:          Orange/salmon colour

            Negative:         Yellow colour

 

Organism                                                       Glu-Amp                                            Pro-Amp

 

           N. gonorrhoeae                                         -                                                            +

           N. meningitidis                                          +                                                           v

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH35v02

Page 2  of 2

Technical Manual

 


 

 

Quality Control

 

Test with control organisms when test is run:

 

            N. gonorrhoeae (ATCC 43069)

            N. menigitidis (ATCC 13090)

 

Reference

 

1.         Pro lab package insert, February 1985.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH36v02

Page 1 of 2

Section: Technical Manual

Subject Title: PYR Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

PYR TEST

Principle

 

PYR (L-pyrrolidonyl-β-naphthylamide) impregnated disks serve as a substrate for the detection of pyrrolidonyl peptidase.  Following the hydrolysis of the substrate by the enzyme the resulting β-naphthylamine produces a red colour upon the addition of cinnamaldehyde reagent.  This test is used, in conjunction with others, for the identification of catalase negative, gram positive cocci including Enterococci and Group A Streptococci.

 

Reagents

 

PYR discs

Cinnamaldehyde reagent (0.01% p-dimethylamino-cinnamaldehyde)

(disks and reagents are both in PYR kit)

Glass slide

Innoculating loop

Forceps

Sterile distilled water

 

Procedure

 

1.         Place a PYR disk onto a glass slide and moisten it with one drop of sterile distilled water.

2.         Rub a loopful of the culture onto the moistened disk holding it in place with sterile forceps.

3.         Leave at room temperature for 2 minutes.

4.         After 2 minutes, add 1 drop of cinnamaldehyde reagent.

 

Interpretation

 

Positive:          Pink or cherry red colour within one minute

 

Negative:         No colour change or slight yellow colour

 

Quality Control

 

Test knows positive and negative controls each time an unknown is run.

 

            Positive:          Group A streptococcus  (ATCC 19615)

            Negative:         Group B streptococcus  (ATCC 13813)


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH36v02

Page 2 of 2

Technical Manual

 


 

 

Reference

 

1.         Carr-Scarborough Microbiologicals package insert 1990.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH37v04

Page 1 of 1

Section: Technical Manual

Subject Title: Quantitation of Organisms & Cells on Smears & Culture

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:April 01, 2007

Annual Review Date:April 29, 2008

 

QUANTITATION OF ORGANISMS & CELLS ON SMEARS & CULTURE


Microscopic:

 

Gram Smear:                                                                                       Report as:

   ±  --- <1 per oil immersion field                                                       Few

   +  --- 1 - 5 per oil immersion field                                                   1+

  ++  --- 5 - 10 per oil immersion field                                     2+

 +++  --- >10 per oil immersion field                                                  3+

 

Acid Fast Bacilli Smear:                                                                     Report as:

3-9 per smear                                                                                       Few

1-9 per ten 250X fields                                                                       1+

1-9 per 250X field                                                                              2+

10-90 per 250X field                                                                          3+

>90 per 250X field                                                                             4+

 

Culture:

                                                                                                            Report as:

   ± --- few colonies in primary inoculum                                            Scant growth

   + --- confluent growth in primary inoculum                                    Light growth

  ++ --- growth up to 2nd quadrant                                                     Moderate growth

 +++ --- growth in or >3rd quadrant                                                   Heavy growth

 

Size of colonies:

            lg    -    large

            med   - medium

            sm    -  small

            tiny  -   tiny

            ppt   -   pinpoint

 

References

 

·         H.D. Izenberg. 2003. in Clinical Microbiology Procedures Handbook, 2nd ed. Vol.1 ASM Press,Washington, D.C

 

·         Acid-fast Bacilli (AFB): Change in Reporting the Enumeration of AFB Smears,Labstract,OntarioPublic Health Laboratories, January 2007

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH38v02

Page 1 of 1

Section: Technical Manual

Subject Title: RapID ANA II System

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

RapID ANA II SYSTEM


Principle

The RapID ANA II System is a qualitative micromethod employing conventional and chromogenic substrate for the identification of medically important anaerobic bacteria of human origin.


The tests used in it are based upon the microbial degradation of specific substrate detected by various indicator systems.  The reactions are a combination of conventional tests and single-substrate chromogenic tests.


Materials

1.                  RapID ANA II panels

2.                  Suspension fluid

3.                  Kovacs spot indole reagent

4.                  RapID ANA II reagent

5.                  RapID ANA ID forms


Procedure

Make an equivalent McFarland #3 turbidity suspension of 18-24 hours AnO2 culture (not more than 72 hours) in the supplied suspension fluid.  Mix it thoroughly - can be used up to 15 minutes.  Inoculate an agar (BA FAA) plate for purity and incubate for 24 hours anaerobically.  Peel the lid off the panel marked "peel to inoculate".  Using the Pasteur pipette, transfer the entire contents into the right upper corner of the panel. Seal the panel. Level the contents in the panel and slowly tilt the panel so that every chamber receives an equal amount of suspension.  Incubate the panel at least four hours (not more than six hours) in non-CO2 incubator at 35-370C.  After the incubation period, read the panel prior to adding the reagents and write results on the ID form.  Add the reagents as per instructions.  Allow 30 seconds but not more than two minutes.  Read it and score on the form.


Interpretation and Identification

Please follow the guidelines from the manufacturer and see RapidANAERIC.exe for identification code.


See RapID ANA II System Insert #iii08-1/94.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH39v02

Page 1 of 2

Section: Technical Manual

Subject Title: RapID MGP Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

RapID MGP TEST


Principle

Rapid MGP Medium is a 5 hour test for the differentiation of Enterococcus faecium and E. faecalis from Enterococcus gallinarum and E. casseliflavus based on the ability to acidify the carbohydrate methyl-glucopyranoside (MGP).


Reagents

Rapid MGP Medium (Hardy Diagnostics)

Bacteriology loop


Procedure

1.                  Using a sweep of colonies from an 18-24 hour pure culture of the organism to be tested, stab the MGP media with the loop.  There should be a visible cell paste on the loop as the media is inoculated.


2.                  Incubate aerobically at 350C for 5 hours.


3.                  Observe for the development of a yellow colour along the stab line indicating a positive test.


4.                  Reincubate weak reactions for 24 hours.


Interpretation

Positive:          yellow colour along stab line

Negative:         colour remains blue


E. casseliflavus            +

E. gallinarum              +

E. faecalis                    -

E. faecium                   -



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Quality Control

Positive and negative controls are run each time the test is set up.


Positive:          E. casseliflavus   (ATCC 12755)


Negative:         E. faecalis  (ATCC 19966)    


Reference

1.         Hardy Diagnostics package insert 1999.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH40v02

Page 1 of 2

Section: Technical Manual

Subject Title: RapID VP Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:March 2, 2006

Annual Review Date:April 29, 2008

 

RapID VP TEST

 

Purpose

To aid in the identification of S. milleri.

 

Media

MR - VP broth


Procedure

1.                  Transfer approximately 0.2 ml of VP broth into a sterile 13 x 100 test tube.


2.                  Using a sterile inoculating wire inoculate the test organism heavily into the broth.


3.                  Incubate the tube at 350C for 5 hours.


4.                  After incubation add 1 drop of alpha-naphthol and 1 drop of 40% KOH.


5.                  Shake the tube gently for one minute to expose the medium to air.  Allow 10-15 minutes for reaction to develop.


Interpretation

            Positive     -     Red colour

            Negative    -     No colour change within 10-15 minutes


Precautions

The order of adding reagents are important; alpha-naphthol followed by 40% KOH.


Quality Control

 

Quality control must be performed on each new lot of Rapid VP reagent before being put into general use and once weekly with the following organisms:

 

            S. pyogenes (ATCC 19615):   Negative

            Streptococcus species Group F (ATCC 12392):         Positive

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH40v02

Page 1 of 2

Section: Technical Manual

Subject Title: RapID VP Test


Reference

 

Ruoff, K.L., Ferraro, M.J. 1986.  Presumptive identification of S. milleri in 5h J Clin Microbiol 24:495-497.

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH41v02

Page 1 of 2

Section: Technical Manual

Subject Title: RapID Yeast Plus Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

RapID YEAST PLUS TEST

Purpose

Used for the identification of yeast and yeast like organisms.


Materials

Rapid Yeast Plus Panel

Rapid Yeast Plus Reagent A and Reagent B

Rapid ID Inoculating fluid 2ml

Pasteur Pipettes

Cotton swabs


Procedure

1.                  Use a cotton swab suspend sufficient growth of the yeast in the Inoculating fluid to give a suspension heavy enough to obliterate the black lines on the inoculation card.


2.                  Peel back the panel lid over the inoculation port by pulling the tab marked "Peel to inoculate".


3.                  Using a Pasteur pipette transfer the entire contents of the inoculation fluid into the upper right hand corner of the panel and then reseal the panel.


4.                  Tilt the panel back away from the biochemical wells at approx. a 45% angle.


5.                  While tilting back gently rock the panel from side to side to evenly distribute the inoculum along the rear baffles.


6.                  Slowly tilt the panel forward toward the reaction cavities until the inoculum flows along the baffles into the biochemical wells.


7.                  Incubate panel at 300C for 4 hours.


8.                  After incubation peel the label lid from over the reaction cavities.


9.                  Add 1 drop of reagent A to cavities 7 to 14 inclusive.


10.              Add 1 drop of reagent B to cavities 16-18 inclusive.



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11.              Read results after 30 seconds but no more than 1 minute after adding reagent.  Record results onto supplied report form, and look results up in the Rapid ID Yeast Plus code book for interpretation.


Interpretation

Well #

Positive

Negative

 

1 to 5


Yellow

Blue to green

6


Yellow

Red, pink, orange, gold

7 to 14


Any yellow

Clear or cream

15


Red or dark red-orange

Yellow, yellow-orange or orange

16-18


Purple, red or dark pink

Clear straw, orange, pale to medium pink


Quality Control

Control strains are set up for each new lot number of panels.


References

1.         Rapid ID yeast plus package insert issue #7/98.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH42v02

Page 1 of 2

Section: Technical Manual

Subject Title: SIM (Sulfide-Indole-Motility)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

SIM (SULFIDE-INDOLE-MOTILITY)

Principle

 

1.         To determine the ability of an organism to liberate hydrogen sulfide (H2S) from sulphur-bearing amino acids producing a visible, black colour reaction.

2.         To determine the ability of an organism to split indole from the tryptophan molecule.

3.                              To determine if the organism is motile or non-motile.

 

This test is used, in conjunction with others, for the identification of Enterobacteriaceae when unable to identify using VITEK or API system.

 

Reagents

 

Kovac's Reagent

 

Other Materials

 

SIM Medium.

Inoculating wire or sterile glass pasteur pipette.

 

Procedure

 

1.         With a pasteur pipette, draw up a small amount of previously inoculated TSB.

2.         Stab vertically into the medium to within 1/4 to 1/2 inch from bottom: withdraw inoculating needle following line of inoculation.

3.         Incubate O2, 35oC X 18-24 hours.

4.         Add a few drops of Kovac's reagent and observe for development of a red colour.

 

Interpretation

 

H2S production

            (a)        Positive:  any blackening of the medium                               

            (b)        Negative:  no blackening

           


 

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Motility

            (a)        Positive:          motile organisms migrate from the stab line and diffuse into the medium causing  turbidity. They may exhibit fuzzy streaks of growth.  (Compare with an uninoculated tube.)

            (b)        Negative:         bacterial growth accentuated along stab  line; surrounding medium remains clear.

Summary:

 

            Results are recorded as follows.  Remember that H2S is first, then indole and finally motility.

                        -/-/-      no H2S, indole neg, non motile

                        -/-/+     no H2S, indole neg, motile

                        -/+/-     no H2S, indole pos, non motile

                        -/+/+   no H2S, indole pos, motile

                        +/-/-     H2S, indole neg, non motile

                        +/-/+   H2S, indole neg, motile

                        +/+/-   H2S, indole pos, non motile

                        +/+/+   H2S, indole pos, motile

 

            Refer to Manual of Clinical Microbiology for specific organism reactions.

 

Precautions

 

1.         An H2S-producing organism may exhibit blackening on SIM medium, but none on TSI medium.

2.         Some H2S inhibition occurs when the temperature exceeds 34oC.

3.         Many bacteria are motile at one temperature and non-motile when at another.

4.         If a motility test is difficult to interpret, compare with an uninoculated motility tube.  If still in doubt, perform a wet prep or hanging drop preparation using a heavy loopful of an 18-24 hr culture.

 

Quality Control

 

Quality control must be performed on each new lot of SIM before being put into general use.

            K. pneumoniae (ATCC 13883):          -/-/-

            P. vulgaris (ATCC 13315):                 +/+/+

 

References

 

1.         MacFaddin JF, Biochemical Tests for identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore MD, 1980, p162-173, 173-183, 214-218.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH431v03

Page 1 of 2

Section: Technical Manual

Subject Title: Acid Fast Stain for             Mycobacteria (Kinyoun)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:March 13, 2006

Annual Review Date:April 29, 2008

Staining Methods

ACID FAST STAIN FOR MYCOBACTERIA (KINYOUN)

 

Principle

 

To stain Mycobacteria present in specimens and cultures.

 

Mycobacteria are different to stain with common aniline dyes.  However, they will stain with basic fuchsin.  Once stained, they retain the dye despite treatment with mineral acids i.e.  HCl H2SO4.  This property of acid fastness may be due to a lipd fraction called mycolic acid.  Mycobacteria also exhibit degrees of resistance to decolourization with alcohol.

 

Materials

 

Kinyoun Carbol fuchsin

3% HCl in 95% ethanol

Brilliant green

 

Procedure

 

1.                  Prepare smear over an area of 2-3 sq. cm.

 

2.                  Heat fix smear on heating block (560C/1 hr).  Then hold to incinerator for 10 secs.

 

3.                  Place slide on stain rack and allow to cool.  Flood with Kinyoun Carbol fuschsin for 5 min.

 

4.                  Rinse off stain with water.

 

5.                  Decolourize with 3% acid alcohol for 3 mins.

 

6.                  Rinse with water.

 

7.                  Repeat decolourization for 1-2 mins. or until no red appears.

 

8.                  Rinse with water.

 

9.                  Flood slide 3-4 mins. with Brilliant green.


 

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10.              Rinse with water.

 

11.              Air dry.  DO NOT BLOT.

 

 

Microscopy

 

Place a drop of oil between the specimen and coverslip and oil again on top.  Smears are examined with oil immersion lens.  The coverslip prevents cross contamination.  Spend an average of 15 mins. on each slide.  The total area of the specimen must be examined.

 

 

Interpretation

The filaments of Mycobacterium species appear red-stained against a blue background.

 

 

Quality Control

 

Mycobacterium Control Slide (MicroBioLogics Cat. No. QCSL41-10) set up simultaneously with the processing of clinical specimens.

Control Results:

Positive Control: Red-stained bacilli

Negative Control: Blue-green-stained bacilli

 

 

 

References

 

Baker, F.J., Breach, M.R.  1980.  Medical Microbiological Technique, p. 15

 

Murray,PA.et al.  Manual of Clinical Microbiology, 8th edition, 2003 ASM,Washington,D.C.

 

 

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH432v03

Page 1 of 2

Section: Technical Manual

Subject Title: Acid fast stain for Nocardia (Modifed Kinyoun)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2006

Annual Review Date:April 29, 2008

 

ACID FAST STAIN FOR NOCARDIA (MODIFED KINYOUN)


Principle

Nocardia species possess the unique characteristic of resisting decolorization with acid alcohol.


Reagents

1.                  Carbol-fuchsin

Basic fuchsin solution             (3 g basic fuchsin in 100 mL 95% ethyl alcohol)

10 mL

Phenol 5% aqueous                 90 mL


2.                  Decolourizer (1% sulfuric acid)

H2SO4 (concentrated)             1 mL

Distilled water                                    99 mL


3.                  Methylene blue

Methylene blue                       0.3 g

Distilled water                                    100 mL


Staining Procedure

1.                  Prepare 2 smears: one for the modified Kinyoun (procedure as follows), the other slide to be stained in parallel with non-modified KINYOUN method.

2.                  Fix the smear by gentle heating.

3.                  Flood the smear with Carbol fuchsin solution.

4.                  Allow the slide to stand for 5 minutes.

5.                  Wash the smear with tap water.

6.                  Decolorize the smear with 1% sulfuric acid until no more colour appears in the washing (approx. 1 min.).

7.                  Rinse with tap water.

8.                  Counterstain with methylene blue about 1 minute.

9.                  Rinse with tap water and air dry.



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Microscopy

 

Place a drop of oil between the specimen and coverslip and oil again on top.  Smears are examined with oil immersion lens.  The coverslip prevents cross contamination.

 

 

Interpretation

The filaments of Nocardia species and Rhodococcus appear red-stained against a blue background.

The smear stained with unmodified Kinyoun should not show red-stained bacilli.



Quality Control

Stain a positive control slide of Nocardia species simultaneously with the clinical specimens.

Nocardia species appear as red-stained bacilli against a blue background.



References

Murray,PA.et al.  Manual of Clinical Microbiology, 8th edition, 2003 ASM,Washington,D.C.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH433v02

Page 1 of 2

Section: Technical Manual

Subject Title: AcridineOrangeStain

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

ACRIDINE ORANGE STAIN

Principle

 

Acridine orange is a fluorescent dye which will bind to the nucleic acid of bacteria and other cells.  It is recommended for use for the detection of microorganisms in direct smears.  It is useful for the rapid screening of specimens from normally sterile sites (eg. CSF) and blood smears, or smears containing proteinaceous material where differentiation of organisms from background material may be difficult.   

 

Reagents

 

AcridineOrangespot test dropper.  Stored at room temperature.

Absolute Methanol

 

Procedure

 

1.         Prepare a smear of the specimen to be stained.

 

2.         Allow to air dry.

 

3.         Fix with methanol for 1 to 2 minutes.

 

4.         Hold the dropper upright and squeeze gently to crush the glass ampoule inside the dispenser.

 

5.         Flood the slide with the acridine orange and stain for 2 minutes.

 

6.         Rinse thoroughly with tap water and allow to air dry.

 

7.         Examine with a fluorescent microscope using low and oil immersion objectives.

 

Interpretation

 

Bacteria and fungus stain bright orange.  The background appears black to yellow green.  Leukocytes will stain yellow, orange and red.


 

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Quality Control

 

Stain a smear of Streptococcus pneumoniae (ATCC 6303) each time the test is performed.   

 

References

 

1.         Difco Spot Test Acridine Orange Stain package insert, 1984.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH434v02

Page 1 of 2

Section: Technical Manual

Subject Title: Bacto 3-Step Gram Stain                   Procedure

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008



BACTO 3-STEP GRAM STAIN PROCEDURE

Principle

 

To be used for problem smears to determine the Gram reaction of organisms.


Materials

3-Step Stabilized Iodine Technique

Bacto Gram Crystal Violet

Bacto Stabilized Gram Iodine

Bacto 3-Step Gram Safranin-S


3-Step Technical Iodine Technique

Bacto Gram Crystal Violet

Bacto Gram Iodine

Bacto 3-Step Gram Safranin-T


Microscope slides

Bunsen burner or methanol

Bacteriological loop

Swabs

Blotting paper

Microscope with oil immersion lens

Bactrolä Gram Slide

Bactrolä Disks



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Procedure


1.                  Flood the fixed smear with primary stain (Bacto Gram Crystal Violet) and stain for 1 minute.

2.                  Remove the primary stain by gently washing with cold tap water.

3.                  Flood the slide with mordant (Bacto Stabilized Gram Iodine or Bacto Gram Iodine (traditional formulation) and retain on the slide for 1 minute.  (Refer to LIMITATIONS OF THE PROCEDURE, #5)

4.                  Wash off the mordant with decolourizer / counterstain (Bacto 3-Step Gram Safranin-S or Bacto 3-Step Gram Safranin-T).  (NOTE: Do not wash off iodine with water).  Add more decolourizer / counterstain solution to the slide and stain 20-50 seconds.

5.                  Remove the decolourizer / counterstain solution by gently washing the slide with cold tap water.

6.                  Blot with blotting paper or paper towel or allow to air dry.

7.                  Examine the smear under an oil immersion lens.



Interpretation

REACTION



3-STEP TECHNIQUE

using either Bacto Gram Safranin-S or Bacto Gram Safranin-T


                       Gram-positive



                       Gram-negative



Purple-black

to purple cells


Red-pink to

fuchsia cells

Quality Control

Run controls daily using 18-24 hour cultures of known gram-positive and gram-negative microorganisms. 



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH435v02

Page 1 of  1

Section: Technical Manual

Subject Title: Eosinophil Stain

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

EOSINOPHIL STAIN

Principle

 

A stain for the detection of eosinophils in clinical specimens.

 

Reagents

 

AJP Scientific Eosinophil stain:

            Solution I - Eosin Y

            Solution II - Buffer Ph 6.5

            Solution III - Methylene Blue

            Stored at room temperature

 

Procedure

 

1.         Make a thin smear and spread evenly.

 

2.         Fix slide by air drying or with gentle heat.

 

3.         Cover slide with solution I and leave for 30 seconds.

 

4.         Add solution II to cover slide.  Mix gently and allow to stain for 3 to 5 minutes.

 

5.         Wash off with tap water and drain.

 

6.         Cover slide with solution III and immediately wash off with tap water.  Drain and air dry.

 

 

Interpretation

 

Eosinophils stain with red cytoplasm and bright red granules.

 

Reference

 

1.         A.J.P. Scientific INC package insert.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH436v02

Page 1 of 2

Section: Technical Manual

Subject Title: Fluorochrome Stain for Mycobacterium

Issued by: LABORATORY MANAGER

Original Date:March 13, 2006

Approved by: Laboratory Director


Revision Date:April 11, 2007

Annual Review Date:April 29, 2008

 

FLUOROCHROME STAIN for MYCOBACTERIUM

 

Principle

Mycobacteria possess the unique characteristic of resisting decolorization with acid alcohol as well as staining poorly, if at all with Gram stain.


Reagents

BD TB Fluorescent Stain Kit (REF 212521):

·   Auramine stain solution

·   Decolorizer solution (Hydrochloric acid/Isopropanol)

·   Potassium permanganate solution (0.5%)


Preparation of Smear

Staining Procedure

 

1.         Place heat fixed smear on a staining rack.


2.         Flood the smear with the thoroughly mixed auramine solution and let stand for 20 minutes.


3.         Rinse the smear with tap water.


4.         Decolorize the smear with decolorizer for 2-3 minutes.


5.         Rinse with tap water.


6.         Flood the smear with Potassium permanganate counterstain and let stand for 4-5 minutes. 

            Excessive treatment (>5 min.) should be avoided as it may reduce the fluorescence of       stained bacilli.


7.         Rinse with tap water and air dry.


8.         Examine slide under the UV microscope.



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Interpretation

Acid fast organisms: reddish-orange fluorescence.

Confirm all positive results with a non-modified KINYOUN  Stain.


 

Quality Control

 

Mycobacterium Control Slide (MicroBioLogics Cat. No. QCSL41-10) - stain simultaneously with the processing of clinical specimens.

 

Expected Control Results:

Positive Control: bacilli with reddish-orange fluorescence

Negative Control: no fluorescence

 

 

Reference


Murray,PA.et al.  Manual of Clinical Microbiology, 8th edition, 2003 ASM,Washington,D.C.


BD TB Fluorescent Stain Kit T (REF 212521), Difco-BBL package insert




UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH437v02

Page 1 of  2

Section: Technical Manual

Subject Title: Fungi-fluorä  Stain

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

FUNGI-FLUORä STAIN

 

Principle

The Fungi-Fluorä stain is used for the rapid identification of various fungal infections in fresh or frozen clinical specimens.


The active, fluorescing dye in the staining solution is Cellufluor which is the disodium salt of 4,4'-bis[4-anilino-6-bis-(2-hydroxyethel) amino-s-triazin-2-ylamino]-2,2'-stilbenedisulfonic acid.  Fungi-Fluorä staining solution is a 0.05% solution of this dye in deionized water with potassium hydroxide added as a clearing agent.  The Fungi-Fluorä counter staining solution B is an aqueous solution of Evans Blue dye used to reduce background fluorescence.  Cellufluor binds nonspecifically to beta-linked polysaccharides found in the cell walls of various organisms such as chitin and cellulose.


Materials

Staining Solution A

Counterstaining Solution B

Absolute alcohol

Water

Fluorescent Microscope (250-400 nm filter)

 

Precautions

1.         Store in a dark or opaque bottle, tightly sealed, at room temperature.


2.         Avoid eye or skin contact: use gloves and protective glasses.


Procedure

1.                  Prepare smear of specimen and allow to air dry.


2.                  Fix on the rack with absolute alcohol for 5 mins. until dry.  Fixed smears can be held indefinitely until ready to stain and examine.


3.                  Add few drops of Fungi-Fluor solution A (Cellufluor) for 1 minute.


4.                  Rinse gently with tapwater.



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5.                  Apply coverslip to wetted slide and examine with the fluorescent microscope using the designated filter.  If there is a delay, add distilled water to the coverslip just prior to examination.  Use a fresh tube of water daily.


6.                  Optional for thicker smears.  Add few drops of the counterstain Fungi-Fluor solution B.  Rinse gently with tap water and then proceed as in step 5 above.


Quality Control

Stain a smear of Candida albicans daily.


Interpretation

Use 20x or 40x objective.


Fungal elements will appear yellow-green against a red-orange background when counterstain is used.  Observe for characteristic morphology.


References

1.                  Manufacturers' Instructions (Data Sheet #316).  Fungi-Fluorä kit - Polysciences, Inc., July 1995


2.                  Clin. Micro. Newsletter 9:33-36,March 1, 1987.

K.L. McGowna.  "Practical Approaches to Diagnosing Fungal Infections in Immunocompromised Patients".


3.                  J. Clin. Micro. 28:393-394, Feb. 1990.  V.S. Baselski et al.  "Rapid Detection of Pneumocystis carinii in Bronchoalveolar Lavage Samples by Using Cellofluor Staining".

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH438v02

Page 1 of  2

Section: Technical Manual

Subject Title: Gram Stain

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

GRAM STAIN

 

Principle

Bacteria can be recognized as gram positive (blue-black/purple) if they retain the primary dye complex of crystal violet and iodine in the face of attempted decolourization, or as gram negative (pink) if decolourization occurs as shown by the cell accepting the counterstain safranin.


Generally the mechanism of the Gram stain is:  The fixed bacteria are stained with the triphenylmethane dye, crystal violet.  Next the smear is flooded with Grams solution which oxidatively forms an insoluble complex with the crystal violet.  The smear is then flooded with the organic solvent, acetone-alcohol.  Depending on cell permeability the crystal violet-iodine complex will be washed from Gram negative bacteria in solvent but not from Gram positive bacteria.  Upon counterstaining with safranin, organisms which had been discolorized by the ethanol (Gram negative) will stain pink.  Gram positive organisms which retained the crystal violet will appear blue-black/purple microscopically.


Materials

Crystal violet solution

Grams Iodine solution

Acetone alcohol

Safranin solution


Procedure

1.                  Prepare the film on the slide and allow to air dry.

DO NOT HEAT TO DRY FILM.


2.                  When film is dry, place slide on heating block for several minutes.  Slide should be just warm to your hand.

DO NOT OVERHEAT.

3.                  Allow slide to cool - this will happen quickly - in just a few seconds.

DO NOT ADD STAIN TO HOT SLIDE.



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4.                  Flood slide with crystal violet - leave 1 minute.


5.                  Wash gently with water.


6.                  Flood slide with Grams Iodine - leave 1 minute.


7.                  Wash iodine from slide with acetone-alcohol mixture.  Add a few more drops of acetone-alcohol until no more colour comes from film - usually 30 seconds.


8.                  Wash gently with water.


9.                  Flood slide with safranin - leave 1 minute.


10.              Wash gently with water.  Clean back of slide with tissue and place slide in tray.  


Precaution

1.                  At no time should the film (smear) be exposed to too much heat.  When the specimen is still wet, heat causes coagulation of the protein resulting in heavy overstaining which cannot be removed by the decolourizer.  A thick smear will also show more tendency to "lift off" during staining.


2.                  Rinsing the Grams Iodine off with the decolorizer gives more stability to the CV-GI complex and false over decolorizing will not take place.


3.                  Flooding a hot slide with crystal violet will cause the stain to precipitate and make decolourizing much more difficult.


Quality Control

Run control slides concurrently with unknowns at least once daily using known smears containing Gram positive and Gram negative bacteria.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH439v02

Page 1 of  2

Section: Technical Manual

Subject Title: Gram Staining Machine Operation

Issued by: LABORATORY MANAGER

Original Date:December 15, 2005

Approved by: Laboratory Director


Revision Date:

Annual Review Date:April 29, 2008

 

GRAM STAINING MACHINE-MIDAS III OPERATION

 

Principle

Adapting the principle of Gram Stain, the MIDAS III will carry fixed smears through different staining solutions for a pre-set time to produced a Gram stained smear.

 

Materials

Staining Rack

Crystal violet solution

Gram’s Iodine solution

Acetone alcohol (50%/50%)

Safranin solution


Instrument Programming Procedure


1.      Press “Program”

2.      Follow the instructions on the screen and program the duration of each staining step as follow:


For Program 1 (thin smears)


Step

Station

Reagent

Time Set

1

1

Crystal Violet

1 minute

2

5

Water

1 minute; flow rate 1500

3

2

Gram’s Iodine

1 minute

4

5

Water

1 minute; flow rate 1500

5

3

Acetone Alcohol

20 seconds

6

5

Water

1 minute; flow rate 1500

7

4

Safranin

1 minute

8

5

Water

1 minute; flow rate 1500

9

6

Dry bath

2 minutes




UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH439v02

Page 2 of  2

Section: Technical Manual

Subject Title: Gram Staining Machine Operation

For Program 2 (thick smears):

Step

Station

Reagent

Time Set

1

1

Crystal Violet

1 minute

2

5

Water

1 minute; flow rate 1500

3

2

Gram’s Iodine

1 minute

4

5

Water

1 minute; flow rate 1500

5

3

Acetone Alcohol

45 seconds

6

5

Water

1 minute; flow rate 1500

7

4

Safranin

1.5 minutes

8

5

Water

1 minute; flow rate 1500

9

6

Dry bath

2 minutes


3.      Press “End” to save program.


Procedure


Daily Set-up:

1.      Remove staining station covers.

2.      Discard old stains.

3.      Wash containers with tap water.

4.      Fill containers with fresh stains/decolourizer.

5.      Record stain change in LIS micqc.


Staining Procedure:

1.      Heat fix smears

2.      Load fixed smears on MIDAS III slide holder

3.      Load holder onto the prongs of MIDAS III.

4.      Press “RUN”, then “1” for regular thin smears OR “2” for thicker sputum smears.

5.      Press “Enter”

6.      Remove smear holder when staining is complete.

Quality Control

Run control slides concurrently with unknowns at least once daily using known smears containing Gram positive and Gram negative bacteria.


Reference

MIDAS III operation manual.

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH44v02

Page 1 of 2

Section: Technical Manual

Subject Title: Staphaurex Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

STAPHAUREX TEST


Principle

 

A rapid slide latex test for the detection of clumping factor and protein A produced by most strains of S. aureus.

 

Reagents and Materials

 

Staphaureux latex suspension (store refrigerated)

Disposable reaction cards

Culture loop or wooden applicator stick

 

Procedure

 

1.         Confirm the identification of a suspect Staphylococcus by Gram stain and catalase test.

2.         Allow the latex reagent to warm to room temperature before use.

3.         Shake the reagent so that all of the particles are resuspended.

4.         Dispense one drop of latex reagent onto the reaction card.

5.         Add 1-3 colonies to the drop, mix well with a loop or wooden applicator stick.

6.         Rock the slide for 20 seconds and look for clumping.

7.         Discard the slide into a discard container.

 

Interpretation

 

Positive test:    Clumping within 20 seconds with the sensitized latex particles.

Negative test:  No clumping

 

Precautions

 

1.         False positive results may occur after 20 seconds.

2.         False positive agglutination can occur with  E. coli and 

            C.  albicans


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH44v02

Page 2 of 2

Technical Manual

 


 

 

Quality Control

 

Test known positive and negative controls daily:

 

            Positive:          S. aureus  (ATCC 25923)

            Negative:         S. epidermidis  (ATCC 12228)

 

References

 

1.         Staphaurex Package insert, July 1992.

 

 



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH44v03

Page 1 of  2

Section: Technical Manual

Subject Title: Streptococcal Grouping

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

STREPTOCOCCAL GROUPING

Principle

 

This test is used to determine the Lancefield group of an isolate.  Latex particles labelled with specific group antisera will agglutinate in the presence of the corresponding antigen after extraction with nitrous acid.

 

Reagents

 

Pro-lab Streptococcal grouping Latex kit.

 

Other Materials

 

Droppers

Disposable slides

Wooden stirring sticks

13x100 mm test tubes

 

Procedure

 

1.         Label one test tube for each isolate.

2.         Add 2 drops of Extraction Reagent 1 to each tube.

3.         Suspend 4 beta-haemolytic colonies in the Extraction Reagent 1.

4.         Shake tube to mix.

5.         Add 2 drops of Extraction Reagent 2 to each tube.

6.         Shake tube to mix.

7.         Add 2 drops of Extraction Reagent 3 to each tube.  Mix

8.         Dispense one drop of each latex suspension to be tested onto separate circles on the test card.

9.         Using a pasteur pipette, add one drop of extract to the latex suspension.

10.       Mix the latex and extract with the wooden stick using the complete area of the circle.

11.       Gently rock the card for 2 minutes and look for agglutination.



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH44v02

Page 2 of 2

Technical Manual

 


 

Interpretation

 

Positive:          Strong visible agglutination within 2 minutes.

 

Negative:         Milky appearance without visible agglutination.

 

 

Precautions

 

1.         False positive reactions have been known to occur with organisms from unrelated genera eg. E. coli, Klebsiella sp., Pseudomonas sp.

 

 

Quality Control

 

Test reagents are checked weekly.

 

Each test should be tested with at least one extra grouping latex suspension as a negative control.

 

 

Reference

 

1.         Pro-lab Streptococcal Grouping package insert.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH45v02

Page 1 of  1

Section: Technical Manual

Subject Title: Tetrazolium Reduction Test (TTC)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

TETRAZOLIUM REDUCTION TEST (TTC)

 

Principle

 

To differentiate between E. faecalis and E. faecium.

 

E. faecalis reduces the colorless compound (Tetrazolium-chloride) to an insoluble substance - formozan which is red.

 

Material

 

BHI broth/Tetrazolium-chloride (TTC)

 

Procedure

 

Inoculate a loopful of an overnight plate culture to 1 ml of TTC broth.  Incubate at 350C and observe the reaction at 2 hours.  If negative, reincubate up to 8 hours / overnight.

 

Interpretation

 

Positive - deep magenta

Negative - colourless or faint pink

 

Quality Control

 

Positive:          E. faecalis  (ATCC 29212)

Negative:         E. faecium  (ATCC 19434)

 

References

 

1.                  J. Gen. Microbiology (1965), 38, 279-287.

           


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH46v02

Page 1 of  2

Section: Technical Manual

Subject Title: Thermonuclease Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

THERMONUCLEASE TEST

Principle

 

Staphylococcus aureus contains a heat-stable thermonuclease and coagulase negative staphylococcus does not.  This is a rapid test to differentiate between the two organisms.

 

Materials

 

Toluidine blue-O DNA plate (Q-Lab)

13x100 mm tube with white cap

pasteur pipettes

 

Procedure

 

1.         Dispense 2 - 3 mL of blood broth from BacT/Alert bottle showing gram positive cocci in clusters in the direct Gram stain into a sterile capped 13x100 mm tube.

2.         Place tube in heating block, 1000C for 15 minutes.

3.         Let cool to room temperature.

4.         Centrifuge at approximately 2500 rpm for 3 minutes.

5.         Inoculate a pre-warmed (35oC for 1 hour) toluidine blue-O DNA plate by filling wells (cut well with the end of a pasteur pipette) with 2 drops of the supernatant.

6.         Incubate the plate at 35oC in the upright position (agar side down).

7.         Inspect the plate at, 1 hour, 2 hours and 4 hours and again after overnight incubation if negative at 4 hours.

8.         Always run negative and positive control wells with each plate each day.

 

Interpretation

 

Positive:          Pink zone of clearing at the edge of the well with a darker blue ring at the outer periphery of the zone;    indicates thermonuclease activity

 

Negative:         No zone or a small clear zone around the well


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH46v02

Page 2 of 2

Technical Manual

 


 


Quality Control

 

1.         Inoculate 5 day negative patient BacT/Alert bottles with 0.5 mL of a slightly turbid suspension of (a) S. aureus (ATCC 25923) and (b) S. epidermidis (ATCC 12228) in trypticase soy broth.

 

2.         Incubate the bottles overnight at 36oC on the shaker.

 

3.         Remove 3 - 6 mL of the broth-blood from the bottles and process in the same manner as the patient specimens (steps 1 to 4). Always QC new controls before use with patient specimen.

 

4.            Supernatants may be kept refrigerated for up to 1 month for use as controls.

 

Reference

 

1.         Rafner, H.B., & Stretton C.W. 1985.  Thermonuclease test for same day identification of S. aureus in blood cultures.  J. Clin. Microbiol. 21:995-996.

 

 


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH47v02

Page 1 of  1

Section: Technical Manual

Subject Title: Tributyrin Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

TRIBUTYRIN TEST

Principle

 

A rapid chromogenic test for the identification of M. catarrhalis.

 

Reagents

 

Prolab Tributyrin (TRIB) tablets.

            Store at room temperature.

Sterile saline.

 

Other Materials

 

Sterile tubes (13 x 100 mm)

 

Procedures

 

1.         Suspend the growth from CHOC in 0.25 mL (6 drops) saline to achieve the turbidity >#2 McFarland standard.

2.         Add 1 tablet to the tube.

3.         Incubate at 350C x 4 hours.

4.         Examine the tube for development of a yellow colour.

 

Interpretation

 

Positive:          Yellow/yellow orange colour

 

Negative:         Red

 

Quality Control

 

Test the following organism weekly:

 

            Positive:             M. catarrhalis  (ATCC 8176)         

            Negative:            N. gonorrhoeae (ATCC 43069)

 

References

 

1.         Prolab package insert, February 1985.

 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH48v02

Page 1 of  3

Section: Technical Manual

Subject Title: TSI (Triple Sugar Iron)

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

TSI (TRIPLE SUGAR IRON)

Principle

 

To determine the ability of an organism to attack a specific carbohydrate incorporated in a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulfide (H2S) production.  This test is used, in conjunction with others, for the identification of enteric pathogens.

 

Materials

 

TSI Slant

Inoculating wire or sterile glass pasteur pipette.

 

Procedure

 

1.         Using the an inoculating wire, dip into the previously inoculated TSB.

2.         Stab the butt of the TSI to within 1/4 inch from bottom, draw out and fishtail over slant.  Do not tighten cap.

3.         Incubate O2, 35oC X 18-24 hours.

 

Interpretation

 

Carbohydrate utilization:

            1.         Fermentation of glucose only

                        (a)        slant:  red colour (alkaline reaction)

                        (b)        butt:   yellow colour (acid reaction)

 

            2.         Fermentation of glucose and sucrose and/or lactose

                        (a)        slant:  yellow colour (acid reaction)

                        (b)        butt:   yellow colour (acid reaction)

 

            3.         Neither glucose nor lactose nor sucrose fermented

                        (a)        slant:  red colour (alkaline reaction)

                        (b)        butt: (i)  aerobic organism

                                                    (a)  No growth

                                                    (b)  No colour change

                                            (ii) facultative organism

                                                 red colour (alkaline reaction)



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH48v02

Page 2 of 3

Technical Manual

 


 

Gas production:

 

            1.         Aerogenic:

                        (a)        Gas production:  CO2 and H2

                        (b)        Evident by one of the following:

                                     (i)        a single gas bubble

                                     (ii)       bubbles in the medium

                                     (iii)      splitting of medium

                                     (iv)      complete displacement of the medium from bottom of the tube leaving a clear area

                                      (v)      slight indentation of medium from the side of the tube

 

            2.         Anaerogenic:

                                    No gas production

                                   

H2S production:

 

                                    The presence of a black precipitate (ferrous sulfide) is evident by:

(i)                     A black colour spread throughout the entire butt masking the

            acidity;  may even be a slight evidence  on the slant

                                          (ii)        A black ring near the top of the butt area

            (iii)      A black precipitate scattered throughout the butt  but not entirely 

                        masking the acidity present

Summary:

 

            The ways of recording the TSI reactions are listed below.  Remember that the slant is first, followed by the butt reaction.

 

                        acid/acid                                              +/+

                        acid/acid/gas                                       +/+ with gas

                        acid/acid/gas/H2S                                +/+ with H2S

                        alkaline/acid                                        -/+

                        alkaline/acid/gas                                  -/+ with gas

                        alkaline/acid/gas/H2S                          -/+ with gas and H2S

                        alkaline/acid/H2S                                 -/+ with H2S

                        alkaline/alkaline                                   -/-

                       

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH48v02

Page 3 of 3

Technical Manual

 


 

Precautions

 

1.         The TSI tube should be read within 18-24 hr.  If read earlier, a false +/+ reaction may occur; if after 24 hr, a false -/-reaction may occur.

 

2.                  An H2S organism may produce so much black precipitate that the acidity in the butt is completely masked.  If H2S is produced, an acid condition exists in the butt.

 

3.         There is no inhibitor in this medium, therefore any organism may grow.  Be certain that the organism tested is a catalase positive, gram negative bacillus.

 

4.         S. typhi usually produces a ring of H2S near the surface of the butt.  Occasionally the amount of H2S produced is so small that it will not be detected in TSI, but will show up in SIM media.

 

5.         Some organisms produce such an abundance of gas that the medium may be completely displaced by gas, resulting in the medium being blown up into the cap of the tube.  Use caution to avoid contamination.

 

6.         Do not tighten the cap of a TSI tube.  A free exchange of air is necessary to enhance the alkaline reaction of the slant.

 

 

Quality Control

 

Test the media each time it is prepared using the following organisms:

 

            E. coli:             (ATCC 25922)            :           +/+

            P. mirabilis:    (ATCC 12453)            :           -/+/H2S

            P. aeruginosa: (ATCC 27853)            :           -/-

 

References

 

1.         MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore MD, 1980, p183-194.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH49v02

Page 1 of  2

Section: Technical Manual

Subject Title: Tube Coagulase Test

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

TUBE COAGULASE TEST

Principle

 

This test is used to speciate staphylococci by determining the ability of an isolate to clot plasma by producing the enzyme coagulase.

 

Reagents

 

Rabbit plasma 

 

            1.         Reconstitute one vial at a time with sterile distilled water (volume determined by vial size).

 

            2.         Store refrigerated before and after reconstitution.  Use within 72 hours of reconstitution.

 

Other Materials

 

Sterile glass tubes (tube method)

Culture loop or wooden applicator stick

 

Procedure

 

            1.         Add 0.5 mL of plasma to a sterile glass tube.

 

            2.         Emulsify a large loopful of a pure colony of Staphylococcus into the plasma.

 

            3.         Incubate at 35oC for 4 hr, observing every 30 minutes for clot formation.

           

4.                  If there is no visible clot at the end of 4 hours, leave at room temperature overnight and observe for clot formation.

 

Interpretation

 

                        Positive:          Clot formation

 

                        Negative:         No clot formation


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH49v02

Page 2 of 2

Technical Manual

 


 

 

Precautions

 

1)         When observing the tube, do not shake or agitate the tube.

 

Quality Control

 

Each time a coagulase test is performed, known positive and negative cultures must be tested.

 

            Positive:          S. aureus  (ATCC 25923)

            Negative:         S. epidermidis  (ATCC 12228)

 

References

 

1.         MacFaddin, J.F., Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins,BaltimoreMD, 1980, pgs. 64-77.


 

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH50v02

Page1 of  2      

Section: Technical Manual

Subject Title: Urea Slant

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

UREA SLANT

 

Principle

To determine the ability of an organism to split urea by the action of the enzyme urease forming two molecules of ammonia with resulting alkalinity.


Materials

Urea Slant

Bacteriology loop


Procedure

1.                  From one isolated colony, heavily inoculate the urea slant.

2.                  Incubate O2, 350C.

3.                  Read at 3 hours and again at 18-24 hours.

 

Interpretation

 

Positive:          Intense pink-red colour.

                        Rapidly positive:         1 to 6 hours (Proteus spp.)

                        Delayed positive:        ³ 18 hours

 

Negative:         No colour change

 

Precautions

 

Urea test media rely on the demonstration of alkalinity, thus are not specific for urease.  The utilization of peptones or other proteins may cause an increase in pH.

 

Quality Control

 

Controls should be set up weekly.

 

            P. mirabilis (ATCC 12453):               Positive - 4 hours

            K. pneumoniae (ATCC 13883):          Weak positive - 18 hours

            E. coli (ATCC 25922):                        Negative


         

UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy # MITECH50v02

Page 2 of 2

Technical Manual

 


 


References

 

1.         MacFaddin JF, Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore MD, 1980, p298-308.

 



UHN/MSH Microbiology Department

Policy & Procedure Manual

Policy #MITECH52v02

Page 1 of  1

Section: Technical Manual

Subject Title: Xylose Fermentation

Issued by: LABORATORY MANAGER

Original Date:July 31, 2000

Approved by: Laboratory Director


Revision Date:February 15, 2002

Annual Review Date:April 29, 2008

 

XYLOSE FERMENTATION

Principle

A rapid chromogenic test for the identification of E. gallinarum.


Reagents

Prolab d-xylose tablets.

Sterile staline.


Other Materials

Sterile tubes (13 x 100 mm)


Procedures

1.                  Suspend the growth from BA in 0.25 mL saline to achieve the turbidity >#2 McFarland standard.

2.                  Add 1 tablet to the tube.

3.                  Incubate at 35 - 370C x 2 hours.

4.                  Examine the tube for development of a yellow colour.


Interpretation

Positive:          Yellow / yellow orange colour

Negative:         Red


Quality Control

The following organisms are tested weekly:


            Positive:          E. gallinarum (ATCC 35038)

            Negative:         E. faecalis (ATCC 29212)


References

1.         J. Clin. Microbiol. 12, 620-623, 1980.


Record of Edited Revisions


Manual Section Name: Technical Manual


Page Number / Item

Date of Revision

Signature of Approval

Annual Review

August 3, 2003

Dr. T. Mazzulli

Annual Review

May 16, 2004

Dr. T. Mazzulli

Annual Review

April 20, 2005

Dr. T. Mazzulli

Annual Review

April 10, 2006

Dr. T. Mazzulli

API website login change

March 14, 2007

Dr. T. Mazzulli

Annual Review

April 11, 2007

Dr. T. Mazzulli

Gen-Probe for S. aureus ID added

April 11, 2007

Dr. T. Mazzulli

Gram Staining machine MIDAS III added

April 11, 2007

Dr. T. Mazzulli

Fluorochrome stain for Mycobacterium – name of stains added to the procedure steps.

April 11, 2007

Dr. T. Mazzulli

Page number changes

April 11, 2007

Dr. T. Mazzulli

Added quantitation of AFB smear to Quantitation of Smear and Culture

April 11, 2007

Dr. T. Mazzulli

Annual Review

April 11, 2007

Dr. T. Mazzulli

Operation of Gen-Probe Luminometer Instructions changed

July 30, 2007

Dr. T. Mazzulli

Optochin Test – change from MHB to BA

July 30, 2007

Dr. T. Mazzulli

ACCUPROBE S. aureus Operation of GEN-PROBE luminometer change

Dec 4, 2007

Dr. T. Mazzulli

Change Gram quantitation for + to report as “Few”

April 1, 2008

Dr. T. Mazzulli

New procedure change for Streptococcus grouping

April 29, 2008

Dr. T. Mazzulli

Anaerobic jar organisms – wrong QC organism listed – changed

April 29, 2008

Dr. T. Mazzulli

Annual Review

April 29, 2008

Dr. T. Mazzulli

AddedAPIstrep strip

April 29, 2008

Dr. T. Mazzulli

Removed ACCUPROBE for S. aureus ID

April 29, 2008

Dr. T. Mazzulli

RemovedCRYSTALforMRSAID

April 29, 2008

Dr. T. Mazzulli

Cryptococcal Antigen revised

April 29, 2008

Dr. T. Mazzulli












































جامعة المجمعة

Medical laboratories Department

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جوال: المملكة العربية السعودية:   00966565709849
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Workshop registration

CLICK ON THE LINK BELOW FOR REGISTRATION


Seminar and Workshop in Blood Bank

 

Scan By Mobile


 

ِApplied Clinical Biocemistry

ِApplied Clinical Microbiology

Clinical Bacteriology

إحصائية الموقع

عدد الصفحات: 143

البحوث والمحاضرات: 124

الزيارات: 1054424