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:

• Cultivation (growth), identification, and antimicrobial susceptibility testing of microorganisms

• Direct detection of infecting organisms by microscopy

• Direct detection of specific products of infecting organisms using chemical, immunologic, or molecular techniques

• Detection of antibodies produced by the patient in response to an infecting organism (serology)

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:

• Safety considerations

• Selection of appropriate anatomic site and specimen

• Collection instructions including type of swab or transport medium

• Transportation instructions including time and temperature

• Labeling instructions including patient demographic information (minimum of two patient identifiers)

• Special instructions such as patient preparation

• Sterile versus nonsterile collection devices

• Minimal acceptable quality

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.

TABLE 5-1

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

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 AnaerobicBBA, LKV, BBE	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 receivedBlood culture bottles incubate at 37° C on receipt in laboratory	May use an aerobic and anaerobic blood culture bottle set for body fluidsBA, 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
Bone
	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
Ear
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)BBA-(anaerobic)	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
Eye
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/AOThe 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 testAntigen 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 neededFollow-up may include Shiga toxin assay as recommened by CDC	Within 24 hrs/RTUnpreserved < 1 hr/RT	72 hrs/4° C	BA, Mac, XLD, HE, Campy, EB, optional: Mac-S; Chromogenic agar	Methylene blue for fecal leukocytesOptional: Shiga toxin testing	See considerations in previous rectal swabsDo 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
FEMALE
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
Cul-de-sac	Anaerobic transporter		Submit aspirate	< 2 hrs/RT	24 hrs/RT	BA, CA, Mac, TM, Ana	Gram
Endometrium	Anaerobic transporter		Surgical biopsy or transcervical aspirate via sheathed catheter	< 2 hrs/RT	24 hrs/RT	BA, CA, Mac, TM, Ana	Gram
Urethra	Swab moistened with Stuart’s or Amie’s medium	Remove exudate from urethral opening	Collect discharge by massaging urethra against pubic symphysis or insert flexible swab 2-4 cm into urethra and rotate swab for 2 seconds; collect at least 1 hr after patient has urinated	< 2 hrs/RT	24 hrs/RT	BA, CA, TM	Gram	Other considerations: Chlamydia, Mycoplasma
Vagina	Swab moistened with Stuart’s or Amie’s medium or JEMBEC transport system	Remove exudate	Swab secretions and mucous membrane of vagina	< 2 hrs/RT	24 hrs/RT	BA, TMCulture is not recommended for the diagnosis of bacterial vaginosis; inoculate selective medium for group B Streptococcus (LIM broth) if indicated on pregnant women	Gram	Examine Gram stain for bacterial vaginosis, especially white blood cells, clue cells, gram-positive rods indicative of Lactobacillus, and curved, gram-negative rods indicative of Mobiluncus spp.
MALE
Prostate	Swab moistened with Stuart’s or Amie’s medium or sterile, screw-cap tube	Clean glans with soap and water	Collect secretions on swab or in tube	< 2 hrs/RT for swab; immediately if in tube/RT	Swab: 24 hrs/RT; tube: plate secretions immediately	BA, CA, Mac, TM, CNA	Gram
Urethra	Swab moistened with Stuart’s or Amie’s medium or JEMBEC transport system		Insert flexible swab 2-4 cm into urethra and rotate for 2 seconds or collect discharge on JEMBEC transport system	< 2 hrs/RT for swab; within 2 hrs for JEMBEC system	24 hrs/RT for swab; put JEMBEC at 37° C immediately on receipt in laboratory	BA, CA, TM	Gram	Other considerations: Chlamydia, Mycoplasma
Hair, Nails, or Skin Scrapings (for fungal culture)
	Clean, screw-top tube	Nails or skin: wipe with 70% alcohol	Hair: collect hairs with intact shaftNails: send clippings of affected areaSkin: scrape skin at leading edge of lesion	Within 24 hrs/RT	Indefinitely/RT	SDA, IMAcg, SDAcg	CW
Respiratory Tract
LOWER
BAL, BB, BW	Sterile, screw-top container		Anaerobic culture appropriate only if sheathed (protected) catheter used	< 2 hrs/RT	24 hrs/4° C	BA, CA, Mac, CNA	Gram and other special stains as requested (e.g., Legionella DFA, acid-fast stain)	Other considerations: quantitative culture for BAL, AFB, Legionella, Nocardia, Mycoplasma, Pneumocystis, cytomegalovirus
Sputum, tracheal aspirate (suction)	Sterile, screw-top container	Sputum: have patient brush teeth and then rinse or gargle with water before collection	Sputum: have patient collect from deep cough; specimen should be examined for suitability for culture by Gram stain; induced sputa on pediatric or uncooperative patients may be watery because of saline nebulization	< 2 hrs/RT	24 hrs/4° C	BA, CA, MacPC OFPBL-cystic fibrosis	Gram and other special stains as requested (e.g., Legionella DFA, acid-fast stain)	Other considerations: AFB, Nocardia
UPPER
NasopharynxNose	Swab moistened with Stuart’s or Amie’s medium		Insert flexible swab through nose into posterior nasopharynx and rotate for 5 seconds; specimen of choice for Bordetella pertussis	< 2 hrs/RT	24 hrs/RT	BA, CABA, chromogenic agar		Other considerations: add special media for Corynebacterium diphtheriae, pertussis, Chlamydia, and Mycoplasma
Pharynx (throat)	Swab moistened with Stuart’s or Amie’s medium		Swab posterior pharynx and tonsils; routine culture for group A Streptococcus (S. pyogenes) only	< 2 hrs/RT	24 hrs/RT	BA or SSA		Other considerations: add special media for C. diphtheriae, Neisseria gonorrhoeae, and epiglottis (Haemophilus influenzae)
Tissue
	Anaerobic transporter or sterile, screw-cap tube	Disinfect skin	Do not allow specimen to dry out; moisten with sterile, distilled water if not bloody	< 15 min/RT	24 hrs/RT	BA, CA, Mac, CNA, ThioAnaerobic: BBA, LKV, BBE	Gram	May need to homogenize
Urine
Clean-voided midstream (CVS)	Sterile, screw-cap containerContainers that include a variety of chemical urinalysis preservatives may also be used	Females: clean area with soap and water, then rinse with water; hold labia apart and begin voiding in commode; after several mL have passed, collect midstreamMales: clean glans with soap and water, then rinse with water; retract foreskin; after several mL have passed, collect midstream		Preserved within 24 hrs/RT unpreserved < 2 hrs/RT	24 hrs/4° C	BA, MacOptional: Chromogenic agar	Check for pyuria, Gram stain not recommended	Plate quantitatively at 1 : 1000; consider plating quantitatively at 1 : 100 if patient is female of childbearing age with white blood cells and possible acute urethral syndrome
Straight catheter (in and out)	Sterile, screw-cap container	Clean urethral area (soap and water) and rinse (water)	Insert catheter into bladder; allow first 15 mL to pass; then collect remainder	< 2 hrs/RTpreserved < 24 hrs/RT	24 hrs/4° C	BA, Mac	Gram or check for pyuria	Plate quantitatively at 1 : 100 and 1 : 1000
Indwelling catheter (Foley)	Sterile, screw-cap container	Disinfect catheter collection port	Aspirate 5-10 mL of urine with needle and syringe	< 2 hrs/4° C (preserved < 24 hrs/RT)	24 hrs/4° C	BA, Mac	Gram or check for pyuria	Plate quantitatively at 1 : 1000
Suprapubic aspirate	Sterile, screw-cap container or anaerobic transporter	Disinfect skin	Needle aspiration above the symphysis pubis through the abdominal wall into the full bladder	Immediately/RT	Plate as soon as received	BA, Mac, Ana, Thio	Gram or check for pyuria	Plate quantitatively at 1 : 100 and 1 : 1000