Specimen Management

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Specimen Management

In the late 1800s, the first clinical microbiology laboratories were organized to diagnose infectious diseases such as tuberculosis, typhoid fever, malaria, intestinal parasites, syphilis, gonorrhea, and diphtheria. Between 1860 and 1900, microbiologists such as Pasteur, Koch, and Gram developed the techniques for staining and the use of solid media for isolation of microorganisms that are still used in clinical laboratories today. Microbiologists continue to look for the same organisms that these laboratorians did, as well as a whole range of others that have been discovered, for example, Legionella, viral infections, nontuberculosis acid-fast bacteria, and fungal infections. Microbiologists work in public health laboratories, hospital laboratories, reference or independent laboratories, and physician office laboratories (POLs). Depending on the level of service and type of testing of each facility, in general a microbiologist will perform one or more of the following functions:

This chapter presents an overview of issues involved in infectious disease diagnostic testing. Many of these issues are covered in detail in separate chapters.

General Concepts for Specimen Collection and Handling

Specimen collection and transportation are critical considerations, because results generated by the laboratory are limited by the quality and condition of the specimen upon arrival in the laboratory. Specimens should be obtained to preclude or minimize the possibility of introducing contaminating microorganisms that are not involved in the infectious process. This is a particular problem, for example, in specimens collected from mucous membranes that are already colonized with an individual’s endogenous or “normal” flora; these organisms are usually contaminants but may also be opportunistic pathogens. For example, the throats of hospitalized patients on ventilators may frequently be colonized with Klebsiella pneumoniae; although K. pneumoniae is not usually involved in cases of community-acquired pneumonia, it can cause a hospital-acquired respiratory infection in this subset of patients. Use of special techniques that bypass areas containing normal flora when feasible (e.g., covered brush bronchoscopy in critically ill patients with pneumonia) prevents many problems associated with false-positive results. Likewise, careful skin preparation before procedures such as blood cultures and spinal taps decreases the chance that organisms normally present on the skin will contaminate the specimen.

Appropriate Collection Techniques

Specimens should be collected during the acute (early) phase of an illness (or within 2 to 3 days for viral infections) and before antibiotics are administered, if possible. Swabs generally are poor specimens if tissue or needle aspirates can be obtained. It is the microbiologist’s responsibility to provide clinicians with a collection manual or instruction cards listing optimal specimen collection techniques and transport information. Information for the nursing staff and clinicians should include the following:

Instructions should be written so that specimens collected by the patient (e.g., urine, sputum, or stool) are handled properly. Most urine or stool collection kits contain instructions in several languages, but nothing substitutes for a concise set of verbal instructions. Similarly, when distributing kits for sputum collection, the microbiologist should be able to explain to the patient the difference between spitting in a cup (saliva) and producing good lower respiratory secretions from a deep cough (sputum). General collection information is shown in Table 5-1. An in-depth discussion of each type of specimen is found in Part VII.


Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory*

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Specimen Container Patient Preparation Special Instructions Transportation to Laboratory Storage before Processing Primary Plating Media Direct Examination Comments
Abscess (also Lesion, Wound, Pustule, Ulcer)                
Superficial Aerobic swab moistened with Stuart’s or Amie’s medium Wipe area with sterile saline or 70% alcohol Swab along leading edge of wound < 2 hrs 24 hrs/RT BA, CA, Mac, CNA optional Gram Add CNA if smear suggests mixed gram- positive and gram-negative flora
Deep Anaerobic transporter Wipe area with sterile saline or 70% alcohol Aspirate material from wall or excise tissue < 2 hrs 24 hrs/RT BA, CA, Mac, CNA Anaerobic
Gram Wash any granules and “emulsify” in saline
Blood or Bone Marrow Aspirate                
  Blood culture media set (aerobic and anaerobic bottle) or Vacutainer tube with SPS Disinfect venipuncture site with 70% alcohol and disinfectant such as Betadine Draw blood at time of febrile episode; draw two sets from right and left arms; do not draw more than three sets in a 24-hr period; draw ≥20 ml/set (adults) or 1-20 ml/set (pediatric) depending on patient’s weight Within 2 hrs/RT Must be incubated at 37° C on receipt in laboratory Blood culture bottles may be used. BA, CA BBA-anaerobic Direct gram Stain from positive blood culture bottles Other considerations: brucellosis, tularemia, cell wall–deficient bacteria, leptospirosis, or AFB
Body Fluids                
Amniotic, abdominal, ascites (peritoneal), bile, joint (synovial), pericardial, pleural Sterile, screw-cap tube or anaerobic transporter or direct inoculation into blood culture bottles Disinfect skin before aspirating specimen Needle aspiration < 15 min Plate as soon as received
Blood culture bottles incubate at 37° C on receipt in laboratory
May use an aerobic and anaerobic blood culture bottle set for body fluids
BA, CA, thio CNA, Mac (Peritoneal)
BBA, BBE, LKV anaerobic
Gram (vaginal fluid is recommended) May need to concentrate by centrifugation or filtration —stain and culture sediment
  Sterile, screw-cap container Disinfect skin before surgical procedure Take sample from affected area for biopsy Immediately/RT Plate as soon as received BA, CA, Mac, thio Gram May need to homogenize
Cerebrospinal Fluid                
  Sterile, screw-cap tube Disinfect skin before aspirating specimen Consider rapid testing (e.g., Gram stain; cryptococcal antigen) < 15 min < 24 hrs Routine Incubate at 37° C except for viruses, which can be held at 4° C for up to 3 days BA, CA (Routine)
BA, CA, thio (shunt)
Gram—best sensitivity by cytocentrifugation (may also want to do AO if cytocentrifuge not available) Add thio for CSF collected from shunt
Inner Sterile, screw-cap tube or anaerobic transporter Clean ear canal with mild soap solution before myringotomy (puncture of the ear drum) Aspirate material behind drum with syringe if ear drum intact; use swab to collect material from ruptured ear drum < 2 hrs 24 hrs/RT BA, CA, Mac (add thio if prior antimicrobial therapy)
Gram Add anaerobic culture plates for tympanocentesis specimens
Outer Aerobic swab moistened with Stuart’s or Amie’s medium Wipe away crust with sterile saline Firmly rotate swab in outer canal < 2 hrs/RT 24 hrs/RT BA, CA, Mac Gram  
Conjunctiva Aerobic swab moistened with Stuart’s or Amie’s medium   Sample both eyes; use swab premoistened with sterile saline < 2 hrs/RT 24 hrs/RT BA, CA, Mac Gram, AO, histologic stains (e.g., Giemsa) Other considerations: Chlamydia trachomatis, viruses, and fungi
Aqueous/vitreous fluid Sterile, screw cap tube     < 15 min/RT Set up immediately on receipt BA, Mac, 7H10, Ana Gram/AO  
Corneal scrapings Bedside inoculation of BA, CA, SDA, 7H10, thio Clinician should instill local anesthetic before collection   < 15 min/RT Must be incubated at 28° C (SDA) or 37° C (everything else) on receipt in laboratory BA, CA, SDA, 7H10, Ana, thio Gram/AO
The use of 10-mm frosted ring slides assists with location of specimen due to the size of the specimen
Other considerations: Acanthamoeba spp., herpes simplex virus and other viruses, Chlamydia trachomatis, and fungi
Foreign Bodies                
IUD Sterile, screw-cap container Disinfect skin before removal   < 15 min/RT Plate as soon as received Thio    
IV catheters, pins, Sterile, screw-cap container Disinfect skin before removal Do not culture Foley catheters; IV catheters are cultured quantitatively by rolling the segment back and forth across agar with sterile forceps four times; ≥15 colonies are associated with clinical significance < 15 min/RT Plate as soon as received if possible store < 2 hrs 4° C BA, Thio prosthetic valves    
GI Tract                
Gastric aspirate Sterile, screw-cap tube Collect in early AM before patient eats or gets out of bed Most gastric aspirates are on infants or for AFB < 15 min/RT Must be neutralized with sodium bicarbonate within 1 hr of collection BA, CA, Mac, HE, CNA, EB Gram/AO Other considerations: AFB
Gastric biopsy Sterile, screw-cap tube (normal saline < 2 hrs transport medium recomended)   Rapid urease test or culture for Helicobacter pylori < 1 hr/RT 24 hrs/4° C Skirrow’s, BA, BBA H&E stain optional: Immunostaining Other considerations: urea breath test
Antigen test (H. pylori )
Rectal swab Swab placed in enteric transport medium   Insert swab ~ 2.5 cm past anal sphincter; feces should be visible on swab Within 24 hrs/RT < 48 hrs/RT or store 4° C BA, Mac, XLD HE, Campy, EB Methylene blue for fecal leukocytes Other considerations: Vibrio, Yersinia enterocolitica, Escherichia coli O157:H7
Stool culture Clean, leak-proof container; transfer feces to enteric transport medium (Cary-Blair) if transport will exceed 1 hr   Routine culture should include Salmonella, Shigella, and Campylobacter; specify Vibrio, Aeromonas, Plesiomonas, Yersinia, Escherichia coli O157:H7, if needed
Follow-up may include Shiga toxin assay as recommened by CDC
Within 24 hrs/RT
Unpreserved < 1 hr/RT
72 hrs/4° C BA, Mac, XLD, HE, Campy, EB, optional: Mac-S; Chromogenic agar Methylene blue for fecal leukocytes
Optional: Shiga toxin testing
See considerations in previous rectal swabs
Do not perform routine stool cultures for patients whose length of stay in the hospital exceeds 3 days and whose admitting diagnosis was not diarrhea; these patients should be tested for Clostridium difficile
O&P O&P transporters (e.g., 10% formalin and PVA) Collect three specimens every other day at a minimum for outpatients; hospitalized patients (inpatients) should have a daily specimen collected for 3 days; specimens from inpatients hospitalized more than 3 days should be discouraged Wait 7-10 days if patient has received antiparasitic compounds, barium, iron, Kaopectate, metronidazole, Milk of Magnesia, Pepto-Bismol, or tetracycline Within 24 hrs/RT Indefinitely/RT   Liquid specimen should be examined for the presence of motile organisms  
Genital Tract                
Bartholin cyst Anaerobic transporter Disinfect skin before collection Aspirate fluid; consider chlamydia and GC culture < 2 hrs 24 hrs/RT BA, CA, Mac, TM, Ana Gram  
Cervix Swab moistened with Stuart’s or Amie’s medium Remove mucus before collection of specimen Do not use lubricant on speculum; use viral/chlamydial transport medium, if necessary; swab deeply into endocervical canal < 2 hrs/RT 24 hrs/RT BA, CA, Mac, TM Gram