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

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

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

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 35^oC.

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.

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.

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

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

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.

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.

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.

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.

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 42^oC 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.

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

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).

Interpretation

REACTIONS TABLE

REACTIONS TABLE (Cont'd)

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, 7^th ed., 1999.

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.

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.

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.

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, 7^th ed., 1999.

SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE

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

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

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.

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.

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.

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

v.                  Read all the tests again, except for NO_3, TRP and GLU.

READING TABLE

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.

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 35-37^oC, 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.

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 35-37^oC in aerobic conditions.

5.            Incubation

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

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

Reactions Table (Cont'd)

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.

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.

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

                        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

Interpretation

SUMMARY OF RESULTS – 4 to 24 HOUR PROCEDURE

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

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.

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 35^oC 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.

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.

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 35^oC 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.

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, 7^th ed., 1999; p. 1665.

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

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.

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.

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.          

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

            -20^oC 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 56^oC for 30 minutes.

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

            7.         Pronase - Rehydrate with 2.5 mL dH₂O, can be stored at 2^oC-8^oC 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.

            Other Materials

            Boiling water bath

            56^oC 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 56^oC 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.

Performing the tests:

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

            1.         Set up and label the slide as follows:

					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.

            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: