Chapter 26 Specimen Collection and Processing for Microbiology

Chapter 26

Specimen Collection and Processing

for Microbiology

Wang Hui

General specimen collection and processing issue

First part of this chapter

Specimen Collection

1. Successful laboratory diagnosis of a microbial infection depends on many factors beginning with a well-collected sample.

2. Proper specimen selection, collection, and transport are all essential to ensure that a specimen is representative of the disease process and minimally contaminated with microorganisms present in adjacent tissues.

Site and Timing

1. Collect the sample from the correct anatomic site . eg. a superficial sample of a lesion is not useful in identifying the cause of a deep wound infection.

2. The timing of sample collection is also important. eg, when submitting a specimen for bacterial culture, samples should be

collected before the administration of antibiotics

Collection Techniques

1. Sterile technique and equipment. 2. Sufficient volume

3. After collection, the specimen must be placed in an appropriately labeled leak-proof container.

Requisition slip

1. Each specimen must be accompanied by a requisition slip to evaluate the specimen appropriately and relay the test results back to health care provider without delay.

2. The requisition slip should contain these information :

patient name, age, gender, identification number, location, name of health- care provider, time and date of collection, specimen type, diagnosis, and test(s) requested.

Transport of Specimens

1. Rapid, optimally in less than 2 hours.

2. For delays in transport, most specimens should be refrigerated.

3. Exceptions: blood, cerebrospinal fluid (CSF), and specimens to be examined for anaerobes, fastidious organisms such as Neisseria gonorrboeae and Bordetella pertussis, and Trichomonas vaginalis, all of which should be maintained at room temperature.

Specimen rejection criteria(1)

1. Improper transport temperature 2. Improper transport container or medium

3. Prolonged transport time 4. Unlabeled or mislabeled specimen 5. Broken or cracked container

6. Leaking specimen


7. Dried-out specimen

8. Inappropriate specimen for test requested

9. Inadequate volume

10. Specimen in fixative (for culture)

11. Duplicate sample in 24-hr period (for urine, sputum, feces culture)


1. When specimens are rejected, the health care provider is notified so that another specimen may be properly submitted.

2. If information on the requisition is incomplete, laboratory personnel should ask a responsible person to provide the information before processing the specimen further.

3. If a specimen is mislabeled, the sample should be recollected. Relabeling of a specimen is acceptable only for difficult to collect specimens, such as tissue obtained during a surgical procedure or CSF.

Specimen not routinely accepted for anaerobic culture(1)

1. Throat, nasopharyngeal, or gingival swabs

2. Sputa

3. Bronchial wash, lavage, or brush (except when collected with a protected double lumen catheter)

4. Gastric and bowel contents

Specimen not routinely accepted for anaerobic culture(2)

5. Ileostomy and colostomy effluent

6. Voided or catheterized urine

7. Female genital tract specimens collected through the vagina

8. Surface swabs of ulcers, wound, and abscesses

Standard Precautions

1. All specimens should be presumed to contain transmissible agents and therefore should be collected and handled using standard precautions.

2. Use of gloves, gown, mask, and protective eyewear when there is a risk of coming in contact with the specimen

3. In most clinical laboratories, a special area is designed for processing clinical samples for culture.

Clinical Diagnosis by Microbiology Laboratory method(1)

1. Direct Examination Gram stain (general) acid-fast bacilli (AFB) (mycobacteria ) KOH and/or calcofluor white preparation

(fungi ) wet mount (parasites), etc. Other techniques for directly examining specimens :

direct fluorescent antibody stains (DFA),

enzyme immunoassays (EIAs),

DNA hybridization or amplification assays, etc.

Clinical Diagnosis by Microbiology Laboratory method(2)

2. Isolate, Culture and Identification A combination of media types is used to isolate

bacteria and fungi (include enriched, nonselective, selective, or differential media);

Viruses can only be cultured within mammalian cells, three main categories: primary, low-passage finite and continuous cell lines

Culture of parasites is generally not performed.

Clinical Dignosis by Microbiology Laboratory method(3)

3. Lengths of culture time Most routine bacterial cultures are incubated

for 2 to 3 days. Mycobacterial and fungal cultures are in-

cubated for as long as 6 weeks. Viral cell cultures are incubated for varying

lengths of time depending on the specimen source and the growth rate of the viruses that are typically recovered from that site

Clinical Dignosis by Microbiology Laboratory method(4)

4. The condition of incubation 35℃ for bacteria and viruses 30℃ for fungi Various atmospheric conditions may be

utilized including ambient, CO2 enriched, microaerophilic and anaerobic.

Specific recommendations for each specimen type

The second part of this chapter

Blood - specimen collection(1)

1. In general, blood for culture should not be obtained using an intravascular device.

2. When performing a venipuncture, the skin must be adequately disinfected to minimize contamination with normal skin flora.

3. Blood should be collected and incubated into the blood culture bottles using the same needle.


4. Blood specimens should be collected before administering antimicrobial agents.

5. Optimally, the specimen should be collected just before a fever spike; however, prac-tically, the specimen should be collected immediately after the spike.

6. For adults, 20 to 30mL of blood should be collected per venipuncture. Less blood is required for children .


7. For adult patient, two sets of cultures should be collected per febrile episode to help distinguish probable pathogens from pos-sible contaminants

8. No more than four sets should be submitted in a 24-hour period.

9. Inoculated blood culture vials should be held at room temperature until they reach the laboratory.

Blood culture(1)

1. Cultures for rapidly growing bacteria and yeast are usually incubated for 5 to 7 days.

2. Cultures for mycobacteria and slowly grow-ing fungi are held for as long as 42 days.

3. Many types of blood culture systems are a-vailable, including both manual and auto-mated. Each system utilizes a noninvasive method (i.e., colorimetric, fluorescent, or manometric methods for detecting CO2 or other gases) to monitor growth.

Blood culture(2)

4. As soon as growth is detected from the blood specimen, a stain is performed (Gram, acid-fast, or Giemsa stain) to determine the type of microorganism present.

5. Positive stain results are considered a critical value and called directly to the patient’s health care provider .

6. Then the specimen should be subcultured to solid media.

Culture of catheter tips

1. Performed to determine the source of a bacteremia.

2. Semiquantitative catheter tip culture method :

The segment is rolled across a blood agar plate four times

Cultures yielding organisms present in more than 15 CFU are considered to be significant, potentially indicating a catheter-related in-fection.

Cerebrospinal fluid (CSF)(1)

1. Cerebrospinal fluid (CSF) is submitted for microbiological analysis when meningitis or encephalitis is suspected.

2. For meningitis, the likely infection agent differs depending on the duration of symp-toms. The most likely bacterial agent of acute meningitis will also vary with the age of the individual and whether the disease is comunity or nosocomially acquired.

Cerebrospinal fluid (CSF)(2)

3. Most infectious cases of encephalitis are a result of viral infection, both arthropod and nonarthropod borne.

4. Parasitic infections of the central nervous system also occur, with varying clinical presentations.

Probable infectious cause of meningitis

Duration of symptoms Probable Pathogen

< 24 hr Pyogenic bacteria

1-7 days Enteroviruses , Pyogenic bacteria

≥4 wk Mycobacterium tuberculosis

Treponema pallidum

Brucella spp , Candida spp

Leptospira interrogans

Borrelia burgdorferi

Cryptococcus neoformam

Coccidioides immitis

Histoplasma capsulatum

CSF - specimen collection

1. Obtained by lumbar spinal puncture

2. Generally at least 0.5mL of CSF (smear, culture, antigen tests )

For mycobacterial culture, at least 3mL (greater volumes increase recovery)

CSF – transportation

1. Transported to the laboratory promptly and processed as soon as possible.

2. If a delay in processing is unavoidable, the specimen should be held at room temperature.

3. If greater than 1.0mL of CSF is received for a given test the fluid is centrifuged to allow the test to be performed on the concentrate sediment

CSF-laboratory diagnosis

1. Gram stain

2. antigen tests

3. India ink test (Cryptococcus neoformans )

4. dark-field microscopy of a concentrated specimen (Leptospires)

5. acid-fast bacilli (AFB) (mycobacteria )

6. bacterial culture

7. yeast and fungi culture

8. viral culture

Gastrointestinal Tract(1)

1. Feces, and in some cases rectal swabs, are submitted to the laboratory primarily to determine the etiologic agent infections diarrhea or food poisoning.

2. Feces should be collected in a clean container with a tight lid and should not be contaminated with urine, barium, or toilet paper. Optimally be examined within 2 hours of collection.


3. Rectal swabs should be placed in a tube transport system containing modified Stuart-’s medium.

4. Unpreserved stool specimens should be maintained at refrigerator temperature dur-ing storage and transport.


5. It is becoming standard practice to reject stool specimens for bacterial culture and parasite examination from patients who have been hospitalized longer than 3 days . For such patients, examination for the toxins produced by Clostridium difficile is recom-mended.

Gastrointestinal Tract -laboratory diagnosis

1. Bacterial culture

selective and differential medium (Mac Conkey agar, Hektoen enteric or xylose-lysine-desoxycholate agar,etc)

2. Fungal culture of stool is not recommended.

3. Viral culture

4. To detect parasites, stool is examined microscopically for the presence of protozoa, helminth eggs, and larvae.

Genital Tract(1)

1. Genital tract specimens are sent to the laboratory for determining the cause of vari-ous clinical syndromes, including vulvova-ginitis, bacterial vaginosis, etc.

2. Many specimens will be contaminated with the normal microbiota of the genital tract or skin; therefore, the microbiologist must differentiate the normal flora from potential pathogens.

Genital Tract(2)

3. Organisms such as N. gonorrhoeae, C. trachomatis, and Haemophilus ducreyi are always pathogenic, whereas organisms such as the Enterobacteriaceae, S. aureus, and group B streptococci are pathogenic only in some clinical situations.

Genital Tract -laboratory diagnosis(1)

1. direct Gram stain (only a few situations )

eg., gram-negative diplococci within poly-morphonuclear leukocytes

2. wet mount preparation of vaginal secretions

clue cells :epithelial cells covered with small coccobacillary bacteria

3. vaginal pH normal≤4.5

4. whiff test positive :generation of a pungent, fishy odor on addition of 10% KOH to the specimen


5. bacterial culture: depend on the source and the organisms likely to cause disease at that site

Tissue and aspirates should be plated to media capable of recovering fastidious organisms.

Specimens from the cervix, vagina, and urethra should at a minimum be evaluated for N. gonorrhoeae and C. trachomatis by culture or a direct detection method.


6. Fungal culture of female genital tract specimens is not productive.

7. Viral culture remains the gold standard for detection of HSV.

Lower Respiratory Tract

1. primarily to determine the etiologic agent of pneumonia

2. Specimen types:

sputum (expectorated or induced), tracheal aspirates, transtracheal aspirates, bronchial washes, bronchial brushings, and bronchoalveolar lavage fluids.

3. delivered promptly to the laboratory. if delays are unavoidable , refrigerated .

Lower Respiratory Tract -laboratory diagnosis(1)

1. Gram-stained smear low-power magnification to determine the

number of squamous epithelial cells and/or neutrophils present

oil immersion to determine the relative amounts of organisms present

Intracellular organisms should be specifical-ly noted.

2. culture ,selective and nonselective media, In addition, a medium capable of recovering fastidious organisms

Lower Respiratory Tract -laboratory diagnosis(2)

3. bronchial brush specimens (0.01 to 0.001mL)

a smear for Gram staining is prepared by cytocentrifugation, and 0.01mL of the speci-men is plated to appropriate media using a pipette or calibrated loop.

4. bronchoalveolar lavage (10 to 100mL )

a smear is prepared by cytocentrifugation and Gram stained (presence or absence of intracellular organisms), a 0.001-mL aliquot of the specimen is inoculated onto agar media

Upper Respiratory Tract

1. Nasopharyngeal aspirates, washings, and swab specimens are primarily used for the diagnosis of viral respiratory infections but may also be submitted to diagnose pertussis, diphtheria, chlamydia infections, and candidiasis, as well as identify carriers of N. meningitidis or S. aureus.

2. Throat swab specimens are generally collected to diagnose group A streptococcal pharyngitis or to detect shedding of viruses such as enteroviruses, HSV, or CMV.

Tissues

1. procured at great expense and considerable risk to the patient; therefore, for optimal evaluation enough material should be collected to allow both histopathologic and microbiologic examination.

2. After collection, tissues should be placed in a sterile container and transported rapidly to the laboratory to prevent drying.

Tissues -laboratory diagnosis

1. homogenized by mincing with a sterile scalpel, grinding with a mortar and pestle or tissue grinder

2. Gram stain or other stains examined for presence of microorganisms,

leukocytes, and squamous epithelial cells 3. routine culture, liquid medium and enriched agar

medium 4. bone marrow aspirates, in collection tubes for the

lysis centrifugation blood culture system or in a sterile container

Urine

1. Acceptable methods of urine collection in-clude midstream clean catch, catheterization, and suprapubic aspiration. Foley catheter tips should not be accepted for culture.

2. promptly to the laboratory and processed within 2 hours of collection . If delays are unavoidable, refrigerated.

Urine -laboratory diagnosis

1. Screening urine specimens Gram stain dipstick tests that combine nitrate reductase

and leukocyte esterase

2. Quantitative bacterial culture

0.001-mLplastic or wire calibrated loop

blood and Mac-Conkey agars

Skin and Subcutaneous Lesions(1)

1. Ideally, the infected material is aspirated with a needle and syringe. For transport, the material is expelled into sterile container that is tightly capped and promptly delivered to the laboratory.

2. If an aspirate cannot be obtained, swab specimens of exudate collected from the deep portion of the lesion are acceptable.

3. For bacterial and fungal cultures, swabs may be placed in tube transport system contain-ing modified Stuart’s medium.

Skin and Subcutaneous Lesions(2)

4. To recover anaerobes, an additional swab specimen must be collected and placed in an anaerobic transport system.

5. For viral culture, the specimen (aspirate or swab) should be placed in viral transport medium and kept on ice.

6. If a delay in processing is unavoidable, specimens may be stored in the refrigerator, except those for recovery of anaerobes (room temperature )

Skin and Subcutaneous Lesions -laboratory diagnosis

1. Gram-stain

2. appropriate media for culture

3. If detection of mycobacteria is requested, specimens should be decontaminated and concentrated. The sediment is used to prepare a smear for staining for AFB and to inoculate mycobacterial media.

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Chapter 26 Specimen Collection and Processing for Microbiology