الدكتور/ مصطفى عراقى - Dr.Mostafa Eraqi

أستاذ مساعد الميكروبيولوجى والمناعة بكلية العلوم

العينات



Protocols for Specimen Processing & Abbreviations

Protocols For Specimen Processing

Subsequent sections of this appendix are devoted to the basic protocols used in the diagnostic laboratory for processing specimens. These protocols describe the processing of specimens for isolation of bacteria, and pathogenic fungi where relevant. Detection of viruses from clinical specimens is usually accomplished by viral culture, antigen detection by immunofluorescence or ELISA, molecular methods, or serologic methods (see Chapter 32). The following types of specimens are considered:

  • Urine
  • Feces
  • Genital tract specimens
  • Skin and soft tissue specimens
  • Respiratory tract specimens (including nose, throat, eye and ear swabs, and sputum)
  • Cerebrospinal fluid
  • Pus
  • Other fluids such as pleural, pericardial fluids and joint aspirates
  • Blood
  • Bone marrow
  • Biopsy samples
  • Autopsy samples
  • Forensic samples

Abbreviations

Culture media

BA

Blood agar (different species of blood may be specified)

MAC

MacConkey agar

HE

Hektoen enteric agar

XLD

Xylose-lysine desoxycholate agar

CAMPY-BA

Campylobacter blood agar

CLED

Cysteine lactose electrolyte deficient agar

EMB

Eosin methylene blue agar

TM

Thayer-Martin agar

CA

Chocolate agar

TCBS

Thiosulfate citrate bit salts sucrose agar

SDA

Sabouraud agar

AnaBA

Anaerobic blood agar

AnaCNA

Anaerobic Colistin Nalidixic acid agar

AnaLVK

Laked horse blood, agar with vancomycin and kanamycin

 

 

 

  Incubation conditions

CO2

Air enriched with 5% carbon dioxide.

AnO2

Anaerobic environment; one in which oxygen has been removed and replaced by carbon dioxide and nitrogen. These conditions can be achieved in ‘gas jars’ with commercially available gas-generating sachets.Duration and temperature of incubation are also stated.

 

Other abbreviations

 

RBC

Red blood cells

WBC

White blood cells

NaOH

Sodium hydroxide

KOH

Potassium hydroxide

HCl

Hydrogen chloride

 

1- Urine

 

Specimen type

Mid-stream urine (MSU).

 

Catheter urine (CSU).

 

Suprapubic aspirate (SPA).

 

Early morning urine (EMU).

Examination of specimens

Macroscopically: note appearance (e.g. cloudy, bloodstained).

 

Microscopically: examine drop of urine (wet preparation; unstained).

 

Note presence and numbers of WBC (normal <10-50/cm3).

 

Note presence of RBCs, epithelial cells (a poorly-collected specimen), bacteria, crystals.

 

Commercial test kits available to detect WBCs and RBCs.

Culture

Quantity of bacteria important so quantitative and semiquantitive methods used.

 

Media: BA, Mac.

 

Incubation: air for 24-48 h at 35-37°C.

Likely pathogens (basic method)

Escherichia coli, Proteus, Staphylococcus saprophyticus, Enterococcus, Klebsiella, Pseudomonas, Salmonella, Candida (note that growth of yeasts is improved on SDA).

 

Generally a colony count of >= 105 organisms/ml of potential pathogens from an MSU is considered significant, however lower counts may be considered significant in symptomatic patients; presence of any number of bacteria in CSU or SPA specimens may be significant; any number of mycobacteria in EMU specimens may be significant.

Important pathogens (not isolated by above technique)

Mycobacteria:

 

·  EMU specimens best for isolation of mycobacteria; before culture, decontaminate with NaOH or HCl, centrifuge, neutralize deposit.

 

·  Microscopy: not recommended; other non-pathologic mycobacteria from water or environment may be detected.

 

·  Culture: inoculate centrifuged deposit on to Löwenstein-Jensen or Middlebrook agar (in screw-capped bottles to prevent desiccation and reduce hazard).

 

·  Incubation: air for 4-12 weeks at 35-37°C

 

Leptospira:

 

·  Can be demonstrated in urine in weeks 2-3 of infection by darkfield microscopy; urine must be very fresh (<15 minutes); culture possible, but difficult; diagnosis usually serologic.

 

Schistosoma:

 

·  Ova of S. haematobium can be seen by microscopy; urine usually contains RBCs.


2- Feces

 

Specimen type

Samples of feces preferable to rectal swabs; swabs from soiled diapers acceptable.

Examination of specimens

Macroscopically: note appearance, gross blood, mucus parasites.

 

Microscopically: note WBCs and RBCs in wet preparation; ova, cysts and parasites in concentrated suspension.

 

Electron microscopy can be useful for rapid detection of some viruses.

Culture

Media/inoculum: Mac or EMB, HE or XLD

 

Incubation: air for 18-48h at 35-37°C.

 

Campy-BA

 

Incubation: microaerophilic (10% O2) for 24-48h at 43°C

Likely pathogens(basic method)

Salmonella, Shigella Campylobacter, Escherichia coli (see below for verotoxin-producers).

Important pathogens (not isolated by above technique)

History and presentation of patient dictates culture type.

Pathogens demonstrated by special culture techniques

Verotoxin producing E. coli (EHEC)

 

·  Medium: sorbitol Mac (verotoxin producers do not usually ferment sorbitol; other E. coli do).

 

·  Incubation: as basic method above.

 

Yersinia enterocolitica:

 

·  Medium: CIN (cefsulodin, irgasan, novobiocin) agar.

 

Incubation: air for 48-96h at 30°C.

 

Vibrio cholerae:

 

·  Medium: TCBS.

 

·  Incubation: air for 18h at 35°C.

 

·  Medium for enrichment: alkaline peptone water (pH 9).

 

·  Incubation: air for 3-6h at 35°C.

 

·  Subculture to TCBS and incubate as above

 

Pathogens demonstrated microscopically:

 

·  Ova.

 

·  Cysts.

 

·  Helminth parasites.

 

·  Entamoeba histolytica/dispar (mobile form visible if specimen examined early on).

 

·  Giardia lamblia trophozoites (acute diarrhea) or cysts.

 

·  Rotaviruses (and certain other viruses) visible by electron microscopy.


3- Genital tract specimens

 

Specimen type

Swabs transported in buffered medium essential due to fastidiousness and multiplicity of species causing genital infections.

Examination of specimens

Microscopically:

 

Wet preparation (for Trichomonas vaginalis, Candida).

 

·  Gram stain: note WBCs, Gram-negative intracellular diplococci (Neisseria gonorrhoeae); presence of WBCs in absence of any of above species, consider Chlamydia trachomatis; clue cells characteristic of bacterial vaginosis.

Culture

Media: BA, TM (or other medium selective for gonococci), Group B streptococcus selective broth

 

Incubation:

 

BA (CO2) for 18-72h at 35-37°C.

 

TM (CO2) for 18-72h at 35-37°C.

 

Group B streptococcus selective broth (CO2) 18-24h at 35-37°C then subculture to BA

Likely pathogens(basic method)

N. gonorrhoeae, Candida albicans, Streptococcus agalactiae (group B streptococcus), Staphylococcus aureus, Listeria monocytogenes.

Important pathogens (not isolated by above technique)

Chlamydia trachomatis:

 

·  Culture: inoculate specimen onto monolayer of irradiated McCoy cells; examine after 48h for characteristic inclusions.

 

·  Alternatively direct examination of specimen by immunofluorescent staining techniques.

 

Haemophilus ducreyi:

 

·  Medium: culture on CA &plus; isovitalex 1%.

 

·  Incubation: CO2 for 2-9 days at 30-34°C.

 

Treponema pallidum:

 

·  Cannot be cultured in vitro.

 

·  Demonstrated in primary and secondary lesions by dark ground microscopy of exudate; otherwise diagnosis depends upon serology.

 

Mycoplasma and Ureaplasma

 

·  specialized media - SP-4 broth or agar


4- Skin and soft tissue specimens

 

Specimen type

Tissue from biopsy, scrapings, or debridement is optimal. Swab samples from skin lesions of limited value. Needle aspirate of deep abscess also acceptable. Vesicular fluid or swab samples from lesion base optimal for viral culture and microscopic examination (by EM or DFA)

Examination of specimens

·  Fungal Infections

 

Microscopically (skin scrapings): for fungal infections stain portion of tissue scrapings with Calcofluor white in KOH and examine in wet preparation for fungal hyphae using fluorescence microscopy.

 

·  Bacterial Infections

 

Microscopically: Gram stain

Culture

·  Fungal Infections

 

Use untreated portion of sample.

 

Medium: SDA.

 

Incubation: air for 1-10 days at ambient temp (approximately 25°C).

 

·  Bacterial Infections

 

Media: BA, MAC orEMB,CA; if anaerobes suspected anaBAP, anaCNA and/or anaLKV or equivalent.

 

Incubation: air or 5% CO2 (BA, CA, MAC/EMB), or AnO2 (anaerobic media) for 18-48h at 35-37°C.

Likely pathogens(basic method)

Fungal Infections

 

Trichophyton, Epidermophyton and Microsporum Candida and other skin yeasts (important in immunocompromised, e.g. Trichosporon beigelii, Cryptococcus neoformans; also Sporothrix schenkii).

 

Bacterial Infections

 

·  Frequent: Staphylococcus aureus, Streptococcus pyogenes, enterobacteria and pseudomonads Bacteroides, Candida.

 

·  Infrequent: Propionibacterium acnes, Corynebacterium diphtheriae (from cutaneous diphtheria), Corynebacterium ulcerans, Erysipelothrix rhusiopathiae, Bacillus anthracis.

 

·  Other Infections: Mycobacterium particularly M. marinum (see section on Sputum below).

Important pathogens (not isolated by above technique)

Treponema pallidum: cannot be cultivated in vitro; examine fluid from primary and secondary lesions by dark ground microscopy.

Pathogens demonstrated by special culture techniques

Microscopically:

 

·  Leishmania tropica, L. mexicana (amastigotes in smear from skin lesion).

 

·  Dracunculus medinensis (adult, or in fluid from vesicle).

 

·  Onchocerca volvulus (microfilariae in skin snips from nodule).

 

·  Sarcoptes scabiei (mites in skin scrapings or hooked out from burrow).


5- Respiratory tract samples

 

Pharyngitis

 

Specimen type

Throat swabs

Examination of specimens

Microscopy of no value.

Culture

Media: BA, (tellurite BA if Corynebacterium diphtheriae suspected).

Incubation

BA (AnO2) (anaerobic incubation enhances streptococcal beta-hemolysis) 18-48h at 35-37°C.

Likely pathogens(basic method)

Streptococcus pyogenes (also beta-hemolytic streptococci of groups C and G), C. diphtheriae, Candida (thrush - important in immunocompromised).


6- Colonization

 

Specimen type

Nasal swabs (usually to detect carriage of pathogens and not to sample site of infection)

Culture

BA, TM

Incubation

Media: BA, TM (5% CO2)

Likely pathogens(basic method)

Staphylococcus aureus (including MRSA) Streptococcus pyogenes, Neisseria meningitidis, C. diphtheriae.


7- Pertussis

 

Specimen type

Nasopharyngeal swabs

Culture

BA, CA, and Bordet-Gengou or Regan-Lowe, for Bordetella pertussis.

Incubation

BA, CA (5% CO2), Regan-Lowe (air), 18-48h at 35-37°C.

Likely pathogens(basic method)

Bordetella pertussis


8- Otitis media

 

Specimen type

Tympanocentesis fluid or exudate; outer ear swabs of limited value

Examination of specimens

Microscopically: Gram stain

Culture

Media: BA, CA

Incubation

BA, CA (5% CO2), 18-48h at 35-37°C

Likely pathogens(basic method)

Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Moraxella catarrhalis.






9- Otitis externa

 

Specimen type

Exudate or aspirate; outer ear swabs of limited value

Examination of specimens

Microscopically: Gram stain

Culture

Media: BA, CA, EMB/MAC

Incubation

BA, CA, EMB/MAC (5% CO2), 18-48h at 35-37°C

Likely pathogens(basic method)

Pseudomonas aeruginosa, Staphylococcus aureus.


10- Eye infections

 

Specimen type

Eye swabs

Examination of specimens

Microscopically: Gram-stained smears: note WBCs and bacteria specimens.

 

Direct immunofluorescence (DFA; direct fluorescent antibody stain) for Chlamydiatrachomatis if clinical history suggestive.

Culture

Media: BA, CA.

Incubation

CO2 for 18-48 h at 35-37°C.

Likely pathogens(basic method)

S. pneumoniae, S. aureus, H. influenzae, N. gonorrhoeae.

Important pathogens (special techniques)

C. trachomatis (for methods see Genital Tract Specimens, above).


11- Pneumonia

 

Specimen type

Sputum

Examination of specimens

Macroscopically: note appearance (purulent, mucoid, salivary), volume.

 

Microscopically: Gram stain - note polymorphs, bacteria (numbers, different types, predominant morphotype), squamous epithelial cells (presence indicates contamination with mouth flora - criteria for rejection).

Culture

Media: BA, CA. EMB/MAC

Incubation

CO2 for 24-48h at 35-37°C.

Likely pathogens(basic method)

S. pneumoniae, H. influenzae, S. aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis

Important pathogens (not isolated by the above technique)

(Note that laboratory needs to be warned if these pathogens are suspected.)

 

Anaerobes: If lung abscess suspected, follow basic method and inoculate anaBA and incubate anaerobically for 48h.

 

Legionella pneumophila:

 

·  Medium: buffered charcoal yeast extract agar.

 

·  Incubation: CO2 for 3-14 days at 35-37°C.

 

Fungi

 

·  Microscopy: Calcofluor white/KOH, Gomori-Methenamine Silver (GMS)

 

·  Media SDA, Mycosel, Inhibitory Mold Agar, and/or BHI-BA

 

·  Incubation - air, 4-6 weeks, 30°C

 

·  Pathogens: Aspergillus, Zygomycetes, Candida, Histoplasma, Coccidioides immitis

 

Mycobacteria:

 

·  If mycobacteria suspected, handle sputum in safety cabinet.

 

·  Before culture: decontaminate sputum with an equal volume of 4% NaOH to kill other bacteria. Neutralize with HCl or dilute.

 

·  Microscopy: Ziehl-Neelsen, Kinyoun&apos;s or auramine O stain; observe for acid-fast bacilli.

 

·  Culture Löwenstein-Jensen or Middlebrook agar (in screw capped bottles to prevent desiccation) and Middlebrook 7H9 broth or similar.

 

·  Incubation: air for up to 8-12 weeks at 35°C

 

·  Alternatively use automated growth detection system. Add sputum homogenate and antibiotic cocktail to suppress other respiratory organisms to commercial medium. Incubation: air for 6 weeks at 35°C. Growth detected by automated detection of CO2.

Other important pathogens

Viruses such as respiratory syncytial virus and influenza virus detected by EIA, immunofluorescence or cell culture of nasopharyngeal washings; Pneumocystis carinii - microscopy using immunofluorescence staining, GMS or Calcofluor white-KOH; Paragonimus westermanii (eggs in sputum).


12- Cerebrospinal fluid

 

Specimen type

This specimen is irreplaceable and must be processed as soon as possible after collection, with utmost care.

Examination of specimens

Macroscopically:

 

·  Note volume, color (presence of xanthochromia); presence of clot (will invalidate attempts at accurate cell count).

 

Microscopically:

 

·  Gram stain.

 

Chemically & Hematologically:

 

·  Analysis of protein and glucose content.

 

·  RBC & WBC count with differential

 

·  Antigen detection for Cryptococcus if indicated

Culture

Media: BA, CA SDA (if Cryptococcus suspected from microscopy or history); Löwenstein-Jensen, Middlebrook or other suitable for mycobacteria (if suspected from microscopy or history).

 

·  Incubation: BA & CA (CO2) for 18-48h at 35-37°C; SDA (air) for 18-48h at 30°C; Löwenstein-Jensen (or equivalent): air for up to six weeks at 37°C.

Likely pathogens(basic method)

S. pneumoniae, H influenzae, N. meningitidis, S. agalactiae, E. coli, Listeria monocytogenes, Cryptococcus neoformans, M. tuberculosis

Important pathogens (not isolated by above technique)

Leptospira interrogans; viruses - cell culture or PCR enteroviruses, mumps virus, herpes simplex virus; Naegleria (amebae plus WBCs and RBCs may be visible).


13- Pus

 

Specimen type

Aspirate of exudate. Swabs sample of limited value.

Examination of specimens

Microscopically: Gram stain: note WBCs (polymorphs), bacteria and fungi (numbers, different types, predominant type).

 

·  If actinomycetes are suspected, examine pus for sulphur granules.

Culture

Media: BA, CA, Mac/EMB, if anaerobes suspected add anaBA, LVK, anaCNA or equivalent, anaTHIO if Actinomyces suspected

 

Incubation:

 

·  BA (air) 35-37°C for 1-5 days.

 

·  Mac/EMB (air) 35-37°C for 1-5 days.

 

·  CA (CO2) 35-37°C for 1-5 days.

 

·  anaerobic media (AnO2) 35-37°C for 2-5 days.

 

Note that many fastidious anaerobes are particularly susceptible to oxygen in early stages of colony formation, so anaerobic plates should be incubated undisturbed for 48 h unless examined in an anaerobic jar or chamber.

Likely pathogens(basic method)

Staphylococcus aureus, Streptococcus pyogenes, enterococci, Pasteurella spp., Yersinia pestis, Enterobacteriaceae and pseudomonads, anaerobes including Clostridium, Bacteroides, and Fusobacterium, Nocardia, and Actinomyces (may require 1-2 weeks of incubation).

Important pathogens (not isolated by above technique)

Rapidly growing Mycobacterium spp.:

 

Pus from some lesions (e.g. injection abscesses) from which no organisms are recovered should be examined microscopically and cultured for mycobacteria (using methods for Sputum, above).


14- Other fluids

 

Specimen type

Pleural, pericardial, ascitic and joint fluids.

Examination of specimens

Macroscopically: note appearance and volume. Centrifuge fluid and examine deposit.

 

Microscopically: Gram stain: note polymorphs, lymphocytes bacteria (numbers, different types, predominant type). Cytologic stain.

Culture

Use sediment.

 

Media: BA, CA, MAC/EMB, if anaerobes suspected add anaBA, LVK, anaCNA or equivalent

 

Incubation:

 

·  BA (air) 35-37°C for 1-5 days.

 

·  Mac/EMB (air) 35-37°C for 1-5 days.

 

·  CA (CO2) 35-37°C for 1-5 days.

 

·  anaerobic media (AnO2) 35-37°C for 2-5 days.

Likely pathogens(basic method)

S. aureus, Streptococcus, Enterococcus, Neisseria, Haeomphilus, Enterobacteriaceae, Pseudomonas aeruginosa, others.

Important pathogens (not isolated by above technique)

Examine microscopically by Gram stain and culture for mycobacteria if indicated (see Sputum above).


15- Blood

 

Specimen type

Volume of blood collected, ratio of blood to culture medium and presence of antibiotics all affect this important diagnostic process; scrupulous adherence to aseptic collection of samples is essential; inoculate blood directly into culture medium at bedside; use two bottles of culture medium (aerobic and anaerobic); distribute at least 20ml of blood between the two culture bottles. Collect 2-3 separate sets of blood cultures from

 

separate venipuncture sites. Most commercial systems do not require subculture, but use automated techniques to detect gas produced as a result of bacterial growth. Manual systems include a biphasic (agar & broth) medium (Septi-Chek®), lysis-centrifugation of blood followed by agar plating of lysed blood concentrate (Isolator®), and an atmospheric pressure signal device (Signal®).

Examination of specimens

Transport blood cultures rapidly to the laboratory, Incubate at 35-37°C. Incubate in automated commercial system for 5-7 days. When growth detected, Gram stain and subculture as appropriate. For manual system, examine daily and Gram stain and/or subculture as appropriate. Prolonged incubation (up to 21days) may be required for growth of some organisms (e.g. Brucella).

Subculture

Media: BA, CA, MAC/EMC. Anaerobic media as appropriate and as indicated by Gram stain results

 

Incubation:

 

·  BA, CA (CO2) for 24-48h at 35-37°C.

 

·  Mac/EMB (air) for 24-48h at 35-37°C.

 

·  anaerobic media (AnO2) for 24-48h at 35-37°C.

Likely pathogens(basic method)

S. aureus, S. pneumoniae, E. coli, P. aeruginosa, K. pneumoniae, Enterococcus, other Enterobacteriaceae, other Streptococcaceae. Bacteroides, Candida. Any isolate from more than one blood culture considered significant; polymicrobial infections may be found and isolation of one species does not preclude possibility of a second; normal skin commensals such as coagulase-negative staphylococci are common contaminants.


16- Bone marrow

 

Specimen type

Usually only available for microbiologic evaluation when collected for hematologic reasons; if collected and manipulated aseptically, bone marrow can be processed as for blood culture.

Likely pathogens(basic method)

Histoplasma, Salmonella typhi, Brucella, mycobacteria, Leishmania donovani can be demonstrated in stained smears.


17- Biopsy samples

As with cerebrospinal fluid, process biopsy samples with care as these are irreplaceable; processing protocol dictated by patient history; divide sample into three parts for histology, bacteriology and virology (note that specimens for bacteriology and virology must not be placed in histologic fixatives). Lymph nodes and other tissues may require maceration; this process may produce aerosols and should be carried out with appropriate precautions.


18- Autopsy samples

Collection of samples requires aseptic technique; superficial contaminants may be eliminated by searing outer surface with hot iron before cutting or by washing the sample several times in sterile broth or saline. Useful for single infectious etiologies in overwhelming infections and in well established disease entities, e.g. tuberculosis, histoplasmosis.


19- Forensic samples

All samples must be retained for legal purposes, precluding any destructive processing; it is imperative that all samples and slides are clearly labelled; staff should sign for custody of samples


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جوال: المملكة العربية السعودية:   00966565709849
جمهورية مصر العربية:               00201005111249
هاتف المكتب  :                         0096664044088
تحويلة داخلية:                            4088

البريد الالكترونى: m.eraqi@mu.edu.sa

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