Specimen Management

Chapter 5


Specimen Management



Objectives



1. State four critical parameters that should be monitored in the laboratory from specimen collection to set up and describe the effects each may have on the quality of the laboratory results (e.g., false negatives or positives, inadequate specimen type, incorrect sample).


2. Identify the proper or improper labeling of a specimen, and determine adequacy of a specimen given a patient scenario.


3. Define and differentiate backup broth, nutritive media, and differential and selective media.


4. Describe the oxygenation states (atmospheric conditions) associated with anaerobic, facultative anaerobic, capnophilic, aerobic, and microaerophilic organisms. Provide an example for each.


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.



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 Anaerobic
BBA, 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 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
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/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                
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, TM
Culture 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 shaft
Nails: send clippings of affected area
Skin: 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, Mac
PC OFPBL-cystic fibrosis		 Gram and other special stains as requested (e.g., Legionella DFA, acid-fast stain) Other considerations: AFB, Nocardia
UPPER                
Nasopharynx
Nose		 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, CA
BA, 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, Thio
Anaerobic: BBA, LKV, BBE		 Gram May need to homogenize
Urine                
Clean-voided midstream (CVS) Sterile, screw-cap container
Containers 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 midstream
Males: 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, Mac
Optional: 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/RT
preserved < 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

Only gold members can continue reading. Log In or Register to continue

Related posts:

  1. Chryseobacterium, Sphingobacterium, and Similar Organisms
  2. Liver and Lung Trematodes
  3. Infections of the Lower Respiratory System
  4. Gastrointestinal Tract Infections

Stay updated, free articles. Join our Telegram channel
Tags:
Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Specimen Management

Full access? Get Clinical Tree
Get Clinical Tree app for offline access