5

Specimen Management
OBJECTIVES General Concepts for Specimen Collection
1. State four critical parameters that should be monitored and Handling
in the laboratory from specimen collection to set up and
describe the effects each may have on the quality of Specimen collection and transportation are critical con-
the laboratory results (e.g., false negatives or positives, siderations because the results generated by the laboratory
inadequate specimen type, incorrect sample). are limited by the quality and condition of the speci-
2. Identify the proper or improper labeling of a specimen, men upon arrival in the laboratory. Specimens should
and determine the adequacy of a specimen given a patient be obtained to preclude or minimize the possibility of
scenario. introducing contaminating microorganisms that are not
3. Define and differentiate backup broth, nutritive media, and involved in the infectious process and can either inter-
differential and selective media.
fere with the growth of or outgrow the pathogen. This
4. Describe the oxygenation states (atmospheric conditions)
is a particular problem, for example, in specimens col-
associated with anaerobic and aerotolerant, facultative
anaerobic, aerobic, and microaerobic (microaerophilic and lected from mucous membranes that are already colonized
capnophilic) organisms. Provide an example for each. with an individual’s endogenous or “normal” microbiota;
5. Determine specimen acceptability and the proper these organisms are usually contaminants but may also
procedure for rejection or recollection. be opportunistic pathogens. For example, the throats of
6. List the critical parameters associated with the reporting of hospitalized patients on ventilators may be colonized with
direct and indirect organism detection. Klebsiella pneumoniae; although K. pneumoniae is not usu-
ally involved in cases of community-acquired pneumo-
nia, it can cause a hospital-acquired respiratory infection
in this subset of patients. Using special techniques that

M
icrobiologists work in public health laboratories, bypass areas containing normal microbiota when feasible
hospital laboratories, reference or independent (e.g., covered-brush bronchoscopy in critically ill patients
laboratories, and physician office laboratories with pneumonia) prevents many problems associated with
(POLs). The current trend in the diagnostic setting is chang- false-positive results. Likewise, careful skin preparation
ing the landscape of laboratory services. Many health care before procedures, such as blood cultures and spinal taps,
systems are consolidating microbiology to a single labo- decreases the chance that organisms normally present on
ratory. This creates a potential for an increase in the time the skin will contaminate the specimen.
between specimen collection and processing. The result may
be a delay in reporting critical results and compromised Appropriate Collection Techniques
integrity of the specimen.
Depending on the level of service and type of testing at Specimens should be collected during the acute (early)
each facility, in general, a microbiologist will perform one or phase of an illness (or within 2 to 3 days for viral infections)
more of the following functions: and before antimicrobials, antifungals, or antiviral medica-
• Cultivation (growth), identification, and antimicrobial tions are administered, if possible. Swabs generally are poor
susceptibility testing of microorganisms specimens if tissue or needle aspirates can be obtained. It
• Direct detection of infecting organisms by microscopy is the microbiologist’s responsibility to provide clinicians
• Direct detection of specific products of infecting organ- with a collection manual or instruction cards listing opti-
isms using chemical, immunologic, or molecular mal specimen collection techniques and transport informa-
techniques tion. Information for the nursing staff and clinicians should
• Detection of antibodies produced by the patient in include the following:
response to an infecting organism (serology) • Safety considerations
This chapter presents an overview of the issues involved • Selection of the appropriate anatomic site and specimen
with infectious disease diagnostic testing. Many of these • Collection instructions, including the type of swab or
issues are covered in detail in separate chapters. transport medium

58
 CHAPTER 5 Specimen Management 59

• T  ransportation instructions, including time and temper- Anticoagulants are used to prevent clotting of specimens
ature constraints such as blood, bone marrow, and synovial fluid because
• Labeling instructions, including patient demographic microorganisms will otherwise be bound up in the clot. The
information (minimum of two patient identifiers) type and concentration of anticoagulant are very important
• Special instructions, such as patient preparation because many organisms are inhibited by some of these
• Sterile versus nonsterile collection devices chemicals. Sodium polyanethol sulfonate (SPS) at a con-
• Minimal acceptable quality and recommended quantity centration of 0.025% (w/v) is usually used because Neisseria
Instructions should be written so that specimens col- spp. and some anaerobic bacteria are particularly sensitive
lected by the patient (e.g., urine, sputum, or stool) are to higher concentrations. Because the ratio of specimen to
handled properly. Most urine or stool collection kits con- SPS is so important, it is necessary to have both large (adult-
tain instructions in several languages, but nothing sub- size) and small (pediatric-size) tubes available, so organisms
stitutes for a concise set of verbal instructions. Similarly, in small amounts of bone marrow or synovial fluid are
when distributing kits for sputum collection, the microbi- not overwhelmed by the concentration of SPS. SPS is also
ologist should be able to explain to the patient the differ- included in blood culture collection systems. Heparin is also
ence between spitting in a cup (saliva) and producing good a commonly used anticoagulant, especially for viral cultures,
lower respiratory secretions from a deep cough (sputum). although it may inhibit the growth of gram-positive bacteria
General collection information is shown in Table 5.1. An and yeast. Citrate, ethylenediaminetetraacetic acid (EDTA),
in-depth discussion of each type of specimen is found in or other anticoagulants should not be used for microbiol-
Part VII. ogy because their efficacy has not been demonstrated for
most organisms. It is the microbiologist’s job to make sure
Specimen Transport media containing the appropriate anticoagulant is used for
each procedure. The laboratory generally should not specify
Ideally, most specimens should be transported to the lab- a color (“yellow-top”) tube for collection without specify-
oratory within 2 hours of collection. There are instances ing the anticoagulant (SPS) because at least one popular
where the time from collection to laboratory processing brand of collection tube (Vacutainer, Becton, Dickinson
should not exceed 15 minutes if not refrigerated or placed and Company) has a yellow-top tube with either SPS or
in specific transport media (Table 5.2). All specimen con- trisodium citrate/citric acid/dextrose (ACD); ACD is not
tainers should be leak-proof, and the specimens should be appropriate for use in microbiology.
transported within sealable, leak-proof plastic bags with
a separate section for paperwork; resealable bags or bags Specimen Storage
with a permanent seal are common for this purpose. Bags
should be marked with a biohazard label (Fig. 5.1). Many If specimens cannot be processed as soon as they are received,
microorganisms are susceptible to environmental condi- they must be stored (Table 5.1). Several storage methods
tions such as the presence of oxygen (anaerobic bacteria), are used (refrigerator temperature [4°C], ambient [room]
changes in temperature (Neisseria meningitidis), or changes temperature [22–25°C], body temperature [35–37°C], and
in pH (Shigella). Thus the use of special preservatives or freezer temperature [either −20°C or −70°C]), depending
temperature-controlled or holding media for the transpor- on the type of transport media (if applicable) and the etio-
tation of specimens is important to ensure organism viabil- logic (infectious) agents suspected. Urine, stool, viral speci-
ity (survival). mens, sputa, swabs, and foreign devices such as catheters
should be stored at 4°C. Serum for serologic studies may be
Specimen Preservation frozen for up to 1 week at −20°C, and tissues or specimens
for long-term storage should be frozen at −70°C.
Preservatives, such as boric acid for urine or polyvinyl
alcohol (PVA) and buffered formalin for stool for ova and Specimen Labeling
parasite (O&P) examination, are designed to maintain the
appropriate colony counts (urines) or the integrity of tro- Specimens should be labeled with the patient’s name, iden-
phozoites and cysts (O&P), respectively. Other transport tifying number (hospital or sample number), birth date,
or holding media maintain the viability of microorganisms date and time of collection, source, and the initials of the
present in a specimen without supporting the growth of the individual that collected the sample. Enough information
organisms. This maintains the organisms in a state of sus- must be provided on the specimen label so that the speci-
pended animation so that no organism overgrows another men can be matched with the test requisition when it is
or dies out. Stuart’s medium and Amie’s medium are two received in the laboratory.
common holding media. Sometimes charcoal is added to
these media to absorb fatty acids present in the specimen Specimen Requisition
that could result in pH changes in the media and the kill-
ing of fastidious (fragile) organisms such as Neisseria gonor- The specimen (or test) requisition is an order form that is
rhoeae or Bordetella pertussis. sent to the laboratory along with a specimen. Often the
 60 PA RT I I
TABLE a
5.1   Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory

General Principles in Clinical Microbiology

Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Abscess (Also Lesion, Wound, Pustule, Ulcer)
Superficial Recommend Wipe area Aspirate or tissue ≤2 h 24 h/RT BA, CA, BBA, LKV, Gram Contamination of
E-swab with sterile are preferred if Mac, CNA BBE surface material may
transport saline possible, pass optional introduce normal
system or or 70% swab deeply microbiota. Add CNA
aerobic swab alcohol. into the lesion if smear suggests
moistened along leading mixed gram-positive
with Stuart’s edge of wound. and gram-negative
or Amie’s flora.
medium
Deep Anaerobic Wipe area Aspirate material ≤2 h 24 h/RT BA, CA, Mac, BBA, LKV, Gram Wash any granules and
transporter; with sterile from wall or CNA BBE “emulsify” in saline.
≥1 mL if saline excise tissue.
sample or 70%
alcohol.
Blood
Blood culture Disinfect Draw blood at time Within 2 h/RT ≤2 h/RT Blood culture Blood Direct Gram C or BCYE if F. tularen-
media set veni- of febrile epi- Must be bottles, culture stain from sis is suspected.
(aerobic and puncture sode; draw two incubated aerobic; bottles, positive Other considerations:
anaerobic site with sets from right at 37°C consider anaero- blood cul- brucellosis, tularemia,
bottle) chlorhex- and left arms; on receipt isolator bic. ture bottles. cell wall–deficient
Disinfect the idine- do not draw in labora- tubes fungi bacteria, leptospi-
container with alcohol. more than four tory. and other rosis, or AFB; blood
70% isopro- sets in a 24-h intracellular cultures should be
pyl alcohol or period; draw agents. collected before
chlorhexidine, ≥20 mL/set administration of
wait 30 s (adults) or 1–20 antibiotics when
mL/set (pediat- possible.
ric) depending
on patient’s
weight; or per
manufacturer’s
instructions.
 Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Bone Marrow Aspirate
Bone Blood culture Puncture site ≤24 h, RT if in 24 h, RT BA, CA BBA Isolator tubes for the
marrow bottles or 1.5 prepared culture bottle May use blood detection of Brucella
mL lysis- by primary or tube culture and intracellular
centrifugation care bottles if bacteria.
tube provide for volume is
surgical sufficient.
incision.
Body Fluids
Amniotic Sterile, screw- Disinfect skin Needle aspiration ≤15 min <24 h/RT: May use an BBA, BBE, Gram May need to con-
abdomi- cap tube or with iodine Pericardial aerobic and LKV centrate by cen-
nal, anaerobic prepara- fluid and anaerobic trifugation or
ascites transporter or tion before other fluids blood culture filtration—stain and
(perito- direct inocu- aspirating for fungal bottle set for culture sediment.
neal), lation into specimen. cultures. body fluids.
bile, blood culture <25 h/4°C: BA, CA, thio,
joint bottles, or Incubate CNA, Mac
(syno- capped blood cul-
vial), syringe ture bottles
peri- at 37°C on
cardial, receipt in
pleural laboratory.
<24 h/4°C:
Pericardial
fluid and
fluids for
fungal
cultures.
Bone Sterile, screw- Disinfect skin Take sample from Immediately/RT Plate as soon BA, CA, Mac, BBA, BBE, Gram May need to homog-
cap container before affected area for as received. thio LKV enize.
surgical biopsy.
procedure.

CHAPTER 5
Cerebrospinal Fluid (CSF)
Sterile, screw- Disinfect Consider rapid ≤15 min RT <24 h/RT BA, CA (Rou- Gram—best If only 1 tube, submit to
cap tube skin with testing (e.g., Never Except for tine) sensitivity by microbiology first to
iodine or Gram stain; refrigerate for viruses, BA, CA, Mac, cytocentrifu- avoid contamination;
chlorhexi- cryptococcal bacteriology. which can thio (shunt) gation (may otherwise tube 2.

Specimen Management
dine antigen). be held at also want Add thio for CSF col-
before 4°C for up to do AO lected from shunt.
aspirating to 3 days. if cytocen- Recommended to also
specimen. trifuge not collect blood culture.
available).
Continued

61
 62 PA RT I I
TABLE
5.1    Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory—cont’d

Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments

General Principles in Clinical Microbiology

Ear
Inner Sterile, screw- Clean ear Aspirate material ≤2h 24 h/RT BA, CA (add BBA Gram Add anaerobic culture
cap tube or canal with behind drum thio if prior plates for tympano-
anaerobic mild soap with syringe if antimicrobial centesis specimens.
transporter solution. eardrum intact; therapy)
use flexible
shaft swab to
collect material
from ruptured
eardrum.
External Wound or Wipe away Firmly rotate swab ≤2 h/RT 24 h/4°C BA, CA, Mac Gram
E-swab trans- crust with in outer canal.
port system or sterile
aerobic swab saline.
moistened
with Stuart’s
or Amie’s
medium
Eye
Conjunc- Direct culture Sample both eyes; Plates ≤15 min, 24 h/RT BA, CA Gram, AO, Other considerations:
tiva inoculation to use separate RT histologic Chlamydia trachoma-
BA and Choc; swabs pre- Swabs stains (e.g., tis, viruses, and fungi.
or E-swab moistened with ≤2 h/RT Giemsa)
transport sterile saline.
system
Aqueous/ Sterile, screw- Prepare eye ≤15 min/RT <24 h/RT BA, CA Gram/AO Other considerations:
vitreous cap tube for needle Set up fungal media; some
fluid aspiration. immedi- anesthetics may be
ately on inhibitory to some
receipt. organisms.
Corneal Bedside inocu- Clinician ≤15 min/RT <24 h/RT BHI 10% Gram/AO Other considerations:
scrap- lation of BHI should Must be incu- Sheep The use of Acanthamoeba spp.,
ings 10% instill local bated at blood, C, 10-mm herpes simplex virus
anesthetic 28°C (SDA) SDA with frosted ring and other viruses,
before col- or 37°C antibiotics slides assists Chlamydia trachoma-
lection. (everything with location tis, and fungi.
else) on of specimen
receipt in because of
laboratory. the size of the
specimen.
 Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Foreign Bodies
IUD Sterile, screw- Disinfect skin ≤15 min/RT Plate as Thio
cap container before soon as
removal. received.
IV cath- Sterile, screw- Disinfect skin Do not culture ≤15 min/RT Plate as BA, Thio, pros-
eters, cap container with alco- Foley catheters; soon as thetic valves
pins hol before IV catheters are received,
removal. cultured quanti- if possible;
tatively by rolling store <2
the segment h/4°C.
back and forth
across agar
with sterile for-
ceps four times;
≥15 colonies
are associated
with clinical
significance.
GI Tract
Gastric Sterile, screw- Collect in Most gastric ≤15 min/RT <24 h/4°C BA, CA, Mac, Gram/AO Other considerations:
wash or cap tube early AM aspirates are Must be HE, CNA, AFB.
lavage before on infants or for neutral- EB
patient AFB. ized with
eats or sodium
gets out of bicarbon-
bed. ate within
1 h of col-
lection.
Gastric Sterile, screw- Rapid urease test <1 h/RT 24 h/4°C Skirrow, BA BBA H&E stain Other considerations:
biopsy cap tube with or culture for optional: urea breath test;
transport Helicobacter immunos- antigen test (H.
media pylori. taining. pylori).

CHAPTER 5
Continued

Specimen Management
63
 64 PA RT I I
TABLE
5.1    Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory—cont’d

Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments

General Principles in Clinical Microbiology

Rectal Swab placed in Insert swab, 1–1.5 ≤2 h/RT <24 h/RT BA, Mac, HE, Methylene Optional: Mac-sorbitol,
swab enteric trans- in past anal Campy, EB blue for chromogenic agar,
port medium sphincter; feces fecal leuko- Shiga toxin testing.
should be vis- cytes. Other considerations:
ible on swab. Vibrio (TCBS), Yer-
sinia enterocolitica
(CIN), Escherichia coli
O157:H7, N. gonor-
rhoeae, Shigella,
Campylobacter,
herpes simplex virus
and carriage of group
B streptococci.
Stool Clean, leak- Routine culture Within 24 h/ 24 h/4°C BA, Mac,, HE, Methylene Optional: Mac-sorbitol,
(feces) proof should include RT in holding <48 h/RT or Campy, EB; blue for chromogenic agar,
routine container; Salmonella, media 4°C fecal Shiga-toxin testing.
culture transfer feces Shigella, and Unpreserved ≤1 leukocytes; See considerations in
to enteric Campylobacter; h/RT optional: previous rectal swabs.
transport specify Vibrio, Shiga toxin Do not perform routine
medium Aeromonas, testing. stool cultures for
(Cary-Blair- Plesiomo- patients whose
holding nas, Yersinia, length of stay in the
medium) Escherichia coli hospital exceeds
O157:H7, if 3 days and whose
needed. admitting diagnosis
was not diarrhea;
these patients should
be tested for Clos-
tridiodes difficile.
Clostrid- Sterile, leak- ≤1 h, RT 2 days, 4°C CCFA Nucleic acid testing is
iodes proof con- 1–24 h, 4°C for culture more sensitive than
difficile tainer or nucleic culture.
acid Patients should be
detection passing ≥3 liquid or
soft stools per 24
hours without having
received laxatives.
 Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Esch- Sterile leak- ≤1 h, RT unpre- <24 h, 4°C Mac-sorbitol Shiga toxin EIA and
erichia coli proof served unpre- nucleic acid testing
O157-H7 container, ≤24 h, RT or served are more sensitive
or other or Cary- 4°C in swab than culture. Bloody
Shiga- Blair holding transport or liquid stools
toxin medium system collected following
producing 6 days of onset of
serotypes symptoms yield more
positives.
Serotyping for O and H
antigens.
O&P O&P transport- Collect three Wait 5–10 days Fresh nonpre- Indefinitely/ Fresh liquid
ers (e.g., 10% specimens minimum (up served liquid RT specimen
formalin and every to 2 weeks) specimens should be
PVA) other if patient should be examined
day at a has received examined for the
minimum antiparasitic within 30 min presence
for out- compounds, of passage; of motile
patients; barium, iron, semiformed organisms.
hospi- Kaopectate, within 1 hour
talized metronida- of passage.
patients zole, Milk of Specimen in
(inpatients) Magnesia, fixatives, 24
should Pepto-Bismol, h/RT.
have or tetracycline.
a daily
specimen
collected
for 3 days;
specimens
from inpa-
tients hos-
pitalized
more than

CHAPTER 5
3 days
should be
discour-
aged.
Genital Tract

Specimen Management
Female
Bartholin Anaerobic Disinfect skin ≤2 h/RT 24 h/RT BA, CA, Mac, BBA, LKV, Gram
cyst transporter with iodine TM, CNA BBE
prepara-
tion before
collection.
Continued

65
 66 PA RT I I
TABLE
5.1    Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory—cont’d

Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments

General Principles in Clinical Microbiology

Cervix Swab moist- Remove Do not use ≤2 h/RT 24 h/RT BA, CA, TM Gram
ened with mucus lubricant on
Stuart’s before col- speculum;
or Amie’s lection of use viral/chla-
medium specimen. mydial trans-
port medium,
if necessary;
swab deeply
into endocervi-
cal canal.
Cul-de- Anaerobic Submit aspirate. <2 h/RT 24 h/RT BA, CA, Mac, BBA, LKV, Gram
sac fluid transporter TM, CNA BBE
Endome- Anaerobic Surgical biopsy ≤2 h/RT 24 h/RT BA, CA, Mac, BBA, LKV, Gram
trium transporter or transcervi- TM, CNA BBE
cal aspirate
via sheathed
catheter.
Products Sterile tube or If no discharge ≤2 h/RT 24 h/RT BA, CA, Mac, BBA, LKV,
of con- anaerobic can be TM, CNA BBE
ception transport obtained, wash
system periurethral area
with povidone-
iodine soap;
rinse with water.
Insert swab 2–4
cm into urethra,
rotate for 2 s
Urethra Swab moist- Collect 1 Collect discharge ≤2 h/RT 24 h/RT TM Gram Other considerations:
ened with hour after by massaging chlamydia, myco-
Stuart’s patient’s urethra against plasma.
or Amie’s last urina- pubic symphy-
medium tion. sis or insert flex-
Remove exu- ible swab 2–4
date from cm into urethra
urethral and rotate swab
opening. for 2 s; collect
at least 1 h
after patient has
urinated.
 Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Vagina Swab moist- Remove Swab secretions ≤2 h/RT 24 h/RT BA, TM Gram Examine Gram stain
ened with exudate. and mucous Culture is not for bacterial vagino-
Stuart’s membrane of recom- sis, especially white
or Amie’s vagina. mended blood cells, clue
medium If a smear is also for the cells, gram-positive
required, use a diagnosis rods indicative of
second swab. of bacterial Lactobacillus, and
vaginosis; curved, gram-nega-
inoculate tive rods indicative of
selective Mobiluncus spp.
medium Subculture Group
for group B B streptococcus
streptococ- enrichment broth to
cus (LIM chromogenic screen-
broth) if ing agar, or use
indicated broth for nucleic acid
for vaginal/ testing.
rectal screen
in pregnant
women.
Male or Swab transport Remove surface of ≤2 h/RT 24 h/RT
female the lesion with
genital sterile scalpel;
lesion rub base of
lesion with a
sterile swab.
H. ducreyi CA with vanco- Gram Gram stain resembling
mycin (3μg/ a “school of fish.”
Ml)
Male
Prostate Swab moist- Clean urethral Collect secretions ≤2 h/RT for 24 h/RT for BA, CA, Mac, BBA, LKV, Gram
ened with meatus with on swab or in swab; imme- swab; plate TM, CNA BBE

CHAPTER 5
Stuart’s soap and tube. diately if in secretions
or Amie’s water and tube/RT immediately
medium or massage if in tube
sterile, screw- the prostate
cap tube through the
rectum.

Specimen Management
Continued

67
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TABLE
5.1    Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory—cont’d

Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments

General Principles in Clinical Microbiology

Urethra Swab moist- Insert flexible ≤2 h/RT 24 h/RT TM Gram Other considerations:
ened with swab 2–4 cm chlamydia, myco-
Stuart’s into urethra plasma.
or Amie’s and rotate for
medium 2 s or collect
discharge.
Hair, Nails, or Skin Scrapings (for Fungal Culture)
Clean, screw- Nails or Hair: collect hairs Within 72 h/RT Indefinitely/ SDA, IMAcg, CW
top tube skin: wipe with intact shaft. RT SDAcg
with 70% Nails: send
alcohol clippings of
affected area.
Skin: scrape skin
at leading edge
of lesion.
Respiratory Tract
Lower
BAL, BB, Sterile, screw- Anaerobic culture ≤2 h/RT 24 h/4°C BA, CA, Mac, BBA, LKV (only Gram and other Other considerations:
BW or top container appropriate only CNA acce­ptable special stains quantitative cul-
endo- if sheathed (pro- for protected as requested ture for BAL, AFB,
tracheal tected) catheter broncho- (e.g., Legio- Legionella (BCYE),
aspirate used. scopic nella DFA, Nocardia, Myco-
brushing in acid-fast plasma, Pneumocys-
anaerobic stain). tis, Cytomegalovirus.
transport).
Sputum Sterile, screw- Have patient Have patient collect ≤2 h/RT <24 h/4°C BA, CA, Mac, Gram and other Other considerations:
top container brush from deep cough; Cystic fibrosis special stains AFB, Nocardia,
teeth and specimen should patients, add as requested Legionella (BCYE).
then rinse be examined BCSA/, (e.g., Legio-
or gargle for suitability for OFPBL, nella DFA,
with water culture by Gram Mannitol salt acid-fast
before col- stain; induced and IMA stain)
lection. sputa on pedi-
atric or uncoop-
erative patients
may be watery
because of saline
nebulization.
 Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Upper
Nasophar- Swab moist- Insert flexible ≤15 min, RT 24 h/RT BA, CA Other considerations:
ynx ened with swab through without trans- add special media
Stuart’s nose into pos- port media for Corynebacterium
or Amie’s terior nasophar- ≤2 h/RT using diphtheria (cystine-
medium ynx and rotate transport tellurite or Loeffler’s
for 5 s; speci- media medium), pertus-
men of choice sis, Chlamydia, and
for Bordetella Mycoplasma.
pertussis.
Nasal Swab transport Premoisten swab ≤2 h/RT 24 h/RT B, chromo-
with sterile genic agar
saline; insert for MRSA
approximately screening
1–2 cm into
nares; rotate
against nasal
mucosa.
Pharynx Swab moistened Swab posterior ≤2 h/RT 24 h/RT BA or SSA Other considerations:
(throat) with Stuart’s or pharynx and add special media
Amie’s medium tonsils for C. diphtheria
Swab dry with or (cystine-
without silica tellurite or Loeffler’s
gel for S. pyo- medium), Neisseria
genes and C. gonorrhoeae, and
diphtheriae epiglottis (Haemophi-
lus influenzae)
Tissue
Collected Anaerobic Disinfect Do not allow ≤15 min/RT 24 h/RT BA, CA, Mac, BBA, LKV, Gram May need to homog-
during transporter or skin. specimen to dry CNA, Thio; BBE enize.
surgery sterile, screw- out; moisten
or biopsy cap tube with sterile,

CHAPTER 5
procedure saline if not
bloody.
Urine
Male and Sterile tube or Unpreserved Specified by Chlamydia and N. gon-
female transport ≤2 h, RT; manufac- orrhoeae detection.

Specimen Management
voided for medium ≤24 h, 4°C turer
nucleic provided by
acid manufacturer
detection
Continued

69
TABLE
5.1    Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory—cont’d

70 PA RT I I
Storage Primary
Patient Special Transportation Before Plating Anaerobic Direct
Specimen Container Preparation Instructions to Laboratory Processing Media Media Examination Comments
Clean- Sterile, screw- Females: clean Preserved ≤24 24 h/4°C BA, Mac Check for Plate quantitatively at
voided cap container area with h/RT Optional: pyuria, 1:1000; consider

General Principles in Clinical Microbiology

midstream Containers that soap and Unpreserved chromogenic Gram stain plating quantitatively
(CVS) include a vari- water, then ≤1/2 h/RT agar not recom- at 1:100 if patient is a
ety of chemi- rinse with mended female of childbear-
cal urinalysis water; hold ing age with white
preservatives labia apart blood cells and pos-
may also be and begin sible acute urethral
used. voiding in syndrome.
commode;
after several
mL have pas­
sed, collect
midstream.
Males: clean
glans with
soap and
water, then
rinse with
water; retract
foreskin;
begin voiding
in commode;
after several
mL have
passed, col-
lect mid-
stream.
Straight Sterile, screw-cap Clean urethral Insert catheter into Unpreserved 24 h/4°C BA, Mac Gram or Plate quantitatively at
catheter container or area (soap bladder; allow ≤1/2 h/RT Optional: check for 1:100 and 1:1000.
(in and urine transport and water) first 15 mL to Preserved ≤ 24 chromogenic pyuria Culture of Foley cath-
out) tube with boric and rinse pass; then col- h/RT agar eters is not recom-
acid preservative (water). lect remainder. mended.
Supra- Sterile, screw- Disinfect Needle aspiration Immediately/RT Plate as BA, Mac, CNA BBA, LKV, Gram or Plate quantitatively at
pubic cap container skin. above the sym­ soon as Thio BBE check for 1:100 and 1:1000.
aspirate or anaerobic physis pubis received pyuria
transporter through the
abdom­inal wall into
the full bladder.

AFB, Acid-fast bacilli; AM, morning; AO, acridine orange stain; BA, blood agar; BAL, bronchial alveolar lavage; BB, bronchial brush; BBA, brucella blood agar; BBE, Bacteroides bile esculin agar; BCSA, B. cepacia selective agar;
BCYE, buffered charcoal-yeast extract agar; BHI, brain heart infusion agar; BW, bronchial wash; CA, chocolate agar; CCFA, cycloserine-cefoxitin-fructose agar; Campy, selective Campylobacter agar; CIN, cefsulodin-Igrasan-
novobiocin agar, CNA, Columbia agar with colistin and nalidixic acid; CW, calcofluor white stain; DFA, direct fluorescent antibody stain; EB, enrichment broth; GC, Neisseria gonorrhoeae; GI, gastrointestinal; Gram, Gram stain;
H&E, hematoxylin and eosin; HE, Hektoen enteric agar; IMA, inhibitory mold agar; IMAcg, inhibitory mold agar with chloramphenicol and gentamicin; IUD, intrauterine device; LKV, laked blood agar with kanamycin and vancomy-
cin; Mac, MacConkey agar; Mac-S, MacConkey-sorbitol; OFPBL, oxidative-fermentative polymyxin B-bacitracin-lactose-agar; O&P, ova and parasite examination; PVA, polyvinyl alcohol; RT, room temperature; SDA, Sabouraud
dextrose agar; SDAcg, Sabouraud dextrose agar with cycloheximide and gentamicin; SPS, sodium polyanethol sulfonate; SSA, group A streptococcus selective agar; thio, thioglycollate broth; TM, Thayer-Martin agar.
aSpecimens for viruses, chlamydia, and mycoplasma are usually submitted in appropriate transport media at 4°C to stabilize respective microorganisms.
 CHAPTER 5 Specimen Management 71

• Th e specimen was received in a fixative (formalin),

which, in essence, kills any microorganism present.
• The specimen has been received for anaerobic culture
from a site known to have anaerobes as part of the nor-
mal microbiota (vagina, mouth).
• The specimen is dried.
• Processing the specimen would produce information of
questionable medical value (e.g., Foley catheter tip).
It is important to always talk to the requesting physi-
cian or another member of the health care team before
discarding unacceptable specimens. This is particularly
important if the specimen was collected using an invasive
technique such as a surgical biopsy and collection of a new
• Fig. 5.1 Specimen bag with biohazard label, separate pouch for specimen would be difficult or impossible. In these cases,
paperwork, and self-seal. (Courtesy Allegiance Healthcare Corp., mislabeling of a specimen or requisition may be corrected
McGaw Park, IL.) by having the person who collected the specimen and filled
out the paperwork come to the laboratory and correct the
requisition is a hard (paper) copy of the physician’s orders problem; a mislabeled specimen or requisition should not
and the patient’s demographic information (e.g., name and be identified over the telephone. However, correction of
hospital number). Sometimes, however, if a hospital infor- mislabeled specimens must be completed at the discre-
mation system offers computerized order entry, the requi- tion of the laboratory’s standard operating procedures. It
sition is transported to the laboratory electronically. The is often necessary to do the best possible job on a less than
requisition should contain as much information as possible optimal specimen if it would be impossible to collect the
regarding the patient history and diagnosis. This informa- specimen again because the patient is taking antibiotics,
tion helps the microbiologist work up the specimen and the tissue was collected at surgery, or the patient would
determine which organisms are significant in the culture. A have to undergo a second invasive procedure (bone mar-
complete requisition should include the following: row or spinal tap). A notation regarding improper collec-
• The patient’s name tion should be added to the final report in this instance
• Hospital identification number because only the primary caregiver is able to determine the
• Age and date of birth validity of the results.
• Sex
• Collection date and time Specimen Processing
• Ordering physician
• Exact nature and source of the specimen Depending on the site of testing (hospital, independent
• Diagnosis (may be ICD-10-CM code) laboratory, physician’s office laboratory) and how the speci-
• Current antimicrobial therapy mens are transported to the laboratory (in-house, courier, or
driver), microbiology samples may arrive in the laboratory in
Rejection of Unacceptable Specimens large numbers or as single tests. Although batch processing
may be possible in large independent laboratories, hospital
Criteria for specimen rejection should be set up and distrib- testing is typically performed as specimens arrive. Surgical
uted to all clinical practitioners. In general, specimens are specimens or specimens from a patient in the emergency
unacceptable if any of the following conditions apply: room should be processed immediately for any direct testing
• The information on the label does not match the infor- orders such as Gram staining or nucleic acid testing, prior to
mation on the requisition or the specimen is not labeled the administration of antibiotics. When multiple specimens
at all (the patient’s name or the source of the specimen is arrive at the same time, priority should be given to those
different). that are most critical, such as cerebrospinal fluid (CSF),
• The specimen has been transported at the improper tissue, blood, and sterile fluids. Urine, throat, sputa, stool,
temperature. or wound drainage specimens can be saved for later. On
• The specimen has not been transported in the proper arrival in the laboratory, the time and date received should
medium (e.g., specimens for anaerobic bacteria submit- be recorded. Acid-fast, viral, and fungal specimens are usu-
ted in aerobic transports). ally batched for processing. When a specimen is received
• The quantity of specimen is insufficient for testing (the with multiple requests but the amount of specimen is insuf-
specimen is considered quantity-not-sufficient [QNS]). ficient to do all of them, the microbiologist should call the
• The specimen is leaking. clinician to prioritize the testing. Any time a laboratory staff
• The specimen transport time exceeds the recommended member contacts the physician or nurse, the conversation
postcollection to set-up time or the specimen is not and agreed-upon information should be documented to
preserved. ensure proper follow-up.
72 PA RT I I General Principles in Clinical Microbiology

Gross Examination of Specimen Selection of Culture Media

All processing should begin with a gross examination of the Primary culture media are divided into several categories. The
specimen. Areas with blood or mucus should be located and first is nutritive media, such as blood agar. Nutritive media
sampled for culture and direct examination. Stool should support the growth of a wide range of nonfastidious microor-
be examined for evidence of barium (i.e., chalky white ganisms and are considered nonselective because, theoretically,
color), which would preclude O&P examination. Nota- the growth of most organisms is supported. Nutritive media
tions should be made on the handwritten or electronic can also be differential, in that microorganisms can be distin-
work card regarding the status of the specimen (e.g., bloody, guished on the basis of certain growth characteristics evident on
cloudy, clotted) so that if more than one person works on the medium. Blood agar is considered both a nutritive and dif-
the sample, the results of the gross examination are available ferential medium because it differentiates organisms based on
for consultation. whether they are alpha (α)-, beta (β)-, or gamma (γ)-hemolytic
(Fig. 5.2). Selective media support the growth of one group
Direct Microscopic Examination of organisms but not another by adding antimicrobials, dyes,
alcohol, or other inhibitory chemicals to a particular medium.
All appropriate specimens should have a direct micro- MacConkey agar, for example, contains the dye crystal violet,
scopic examination (smear of the primary specimen). which inhibits gram-positive organisms. Columbia agar with
The direct examination serves several purposes. First, colistin and nalidixic acid (CNA) is a selective medium for
the quality of the specimen can be assessed; for example, gram-positive organisms because the antimicrobials colistin and
sputa can be rejected that represents saliva and not lower nalidixic acid inhibit gram-negative organisms. Selective media
respiratory tract secretions by quantitation of white blood can also be differential media if, in addition to their inhibitory
cells or squamous epithelial cells present in the specimen. activity, they differentiate between groups of organisms. Mac-
Second, the microbiologist and clinician can be given an Conkey agar, for example, differentiates between lactose-fer-
early indication of what may be wrong with the patient menting and nonfermenting gram-negative rods by the color
(e.g., 4+ gram-positive cocci in clusters in an exudate). of the colonial growth (pink or clear, respectively); this is shown
Third, the work-up of the specimen can be guided by in Fig. 5.3. Fastidious organisms are organisms that require spe-
comparing what grows in culture to what was seen on cific nutritional or environmental conditions to enhance their
the original smear. A situation in which three different growth. Chocolate agar is considered an enrichment media
morphotypes (cellular types) are seen on direct Gram that is used in most routine cultures for the enhancement of
stain but only two grow in culture, for example, alerts the growth for commonly isolated fastidious organisms such as
microbiologist to the fact that the third organism may be Haemophilus spp. In some cases (sterile body fluids, tissues, or
an anaerobic bacterium. If there were more than three deep abscesses in a patient receiving antimicrobial therapy),
organisms on the culture plate that were not visible on backup broth (also called supplemental or enrichment broth)
Gram stain, this would indicate possible contamination. medium is inoculated, along with primary solid (agar) media,
Gram stains are often also layered with cells and debris. so small numbers of organisms present may be detected; this
Organisms that appear on the surface of white blood allows detection of anaerobes in aerobic cultures and organ-
cells actually may be ingested organisms that are no lon- isms that may be damaged by either previous or concurrent
ger viable or capable of growth. It is imperative that the antimicrobial therapy. Thioglycollate (thio) broth, brain-heart
Gram stain results and specimen culture correlate to the infusion broth (BHIB), and tryptic soy broth (TSB) are com-
type of specimen to ensure that accurate information is mon backup broths.
provided to the clinician. Selection of media to inoculate for any given specimen
Direct examinations are usually not performed on throat, is usually based on the organisms most likely to be involved
nasopharyngeal, or stool specimens because of the presence in the disease process at the particular site of infection. For
of abundant normal microbiota but are indicated from most example, in determining what to set up for a CSF specimen,
other sources. one considers the most likely pathogens that cause meningitis
The most common stain in bacteriology is the Gram (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria
stain, which helps the clinician visualize rods, cocci, meningitidis, Escherichia coli, group B streptococcus) and selects
white blood cells, red blood cells, or squamous epithe- media that will support the growth of these organisms (blood
lial cells present in the sample. The most common direct and chocolate agar at a minimum). Likewise, if a specimen is
fungal stains are KOH (potassium hydroxide), PAS collected from a source likely to be contaminated with normal
(periodic-acid Schiff ), GMS (Grocott’s methenamine microbiota—for example, an anal fistula (an opening of the
silver stain), and calcofluor white. Although rarely used surface of the skin near the anus that may communicate with
in the clinical laboratory, the most common direct acid- the rectum)—the laboratorian might want to add a selective
fast stain is the Kinyoun modification (cold method) of medium, such as CNA, to suppress gram-negative bacteria and
the Ziehl-Neelsen (hot method) procedure. Chapter 6 allow gram-positive bacteria and yeast to be recovered.
describes the use of microscopy in clinical diagnosis in In addition to primary plating media, chromogenic
more detail. agars are now available for a variety of organisms that
 CHAPTER 5 Specimen Management 73

A B

• Fig. 5.2 Examples of various types of hemolysis on

blood agar. (A) Streptococcus pneumoniae showing
alpha (α)-hemolysis (i.e., greening around colony). (B)
Staphylococcus aureus showing beta (β)-hemolysis
(i.e., clearing around colony). (C) Enterococcus faeca-
lis showing gamma (γ)-hemolysis (i.e., no hemolysis
C
around colony).

A B
• Fig. 5.3 MacConkey agar. (A) Escherichia coli, a lactose fermenter. (B) Pseudomonas aeruginosa, a
nonlactose fermenter.
74 PA RT I I General Principles in Clinical Microbiology

produce pigmented colonies based on the genus or species different organisms present in the specimen; such semi-
of a particular group of bacteria or yeast. These media are quantitative information is usually sufficient for the physi-
often used for screening specimens for pathogens, such as cian to be able to treat the patient.
methicillin-resistant Staphylococcus aureus (MRSA), vanco-
mycin-resistant enterococci (VRE), and Candida species, Incubation Conditions
to name a few.
Routine primary plating media and direct examinations Inoculated media are incubated under various temperatures
for specimens commonly submitted to the microbiology and environmental conditions, depending on the organ-
laboratory are shown in Table 5.1. Samples received on swab isms suspected—for example, 25° to 30°C for fungi and
should be plated to the least inhibitory media first, followed 35° to 37°C for most bacteria, viruses, and acid-fast bacil-
by additional media prior to making a smear for Gram lus. A number of different environmental conditions exist.
staining. Chapter 7 on bacterial cultivation reemphasizes Aerobes grow in ambient air, which contains 21% oxygen
the strategies for selection and the use of bacterial media. (O2) and a small amount (0.03%) of carbon dioxide (CO2).
Anaerobes usually cannot grow in the presence of O2, and
Specimen Preparation the atmosphere in anaerobe jars, bags, or chambers is com-
posed of 5% to 10% hydrogen (H2), 5% to 10% CO2, 80%
Many specimens require some form of initial treatment to 90% nitrogen (N2), and 0% O2. Aerotolerant micro-
before inoculation onto primary plating media. Such proce- organisms are anaerobes that do not use oxygen but are
dures include homogenization, grinding of bone, or minc- not killed by a small amount. Microaerobic refers to both
ing of tissue; concentration by centrifugation or filtration capnophilic and microaerophilic organisms. Capnophiles,
of large volumes of sterile fluids, such as ascites (peritoneal) such as Haemophilus influenzae and Neisseria gonorrhoeae,
or pleural (lung) fluids; or decontamination of respiratory require increased concentrations of CO2 (5% to 10%) and
specimens, such as those for legionellae or mycobacteria. approximately 15% O2. This atmosphere can be achieved
Traditional fiber swab specimens have an internal mattress by a candle jar (3% CO2) or a CO2 incubator, chamber,
core that can trap organisms and may be vortexed (mixed) or bag. Microaerophiles (Campylobacter jejuni, Helicobacter
in 0.5 to 1 mL of saline or broth for 10 to 20 seconds to pylori) grow under reduced O2 (5% to 10%) and increased
dislodge material from the fibers. Flocked swabs contain no CO2 (8% to 10%). This environment can also be obtained
mattress core. The fibers are designed to ionically bind the in specially designed chambers, jars, or bags. Both anaero-
negative charges on the surface of cells (Copan Diagnostics, bic and microaerobic environments may be produced using
Murrieta, CA). Some flocked swabs come in liquid media; an automated microprocessor-controlled system, such as
the specimen can be mixed in the original container by vor- the Advanced Axonomat, to create the desired atmospheric
texing prior to inoculating media and preparing a direct balance of gases required for specific organismal growth
smear. (Advanced Instruments, Norwood, MA). More detailed
information is included in Chapter 40.
Inoculation on Solid Media
Specimen Work-Up
Specimens can be inoculated (plated) onto solid media
either quantitatively by a dilution procedure or by means One of the most important functions that a microbiologist
of a quantitative loop or semiquantitatively using an performs is deciding what is clinically relevant regarding
ordinary inoculating loop. Urine cultures and tissues from specimen work-up. Considerable judgment is required to
burn victims are plated quantitatively; everything else is decide what organisms to look for and report. It is essential
usually plated semiquantitatively. Plates inoculated for to recognize what constitutes indigenous (normal) micro-
quantitation are usually streaked with a 1:100 or 1:1000 biota and what constitutes a potential pathogen. Indiscrim-
loop. Plates inoculated for semiquantitation are usually inate identification, susceptibility testing, and reporting of
streaked out in four quadrants. A variety of automated normal microbiota can contribute to the unnecessary use of
medium inoculators are also available. Detailed methods antibiotics and the potential emergence of resistant organ-
for streaking solid media are provided in Chapter 7, Fig. isms. Because organisms that are clinically relevant to iden-
7.9. Semiquantitation is referred to as streaking for isola- tify and report vary by source, the microbiologist should
tion because the microorganisms present in the specimen know which organisms cause disease at various sites. Part
are successively diluted out as each quadrant is streaked VII contains a detailed discussion of these issues.
until finally each morphotype is present as a single colony.
Numbers of organisms present can subsequently be graded Extent of Identification Required
as 4+ (many, heavy growth) if growth is out to the fourth
quadrant, 3+ (moderate growth) if growth is out to the As health care continues to change, one of the most prob-
third quadrant, 2+ (few or light growth) if growth is in lematic issues for microbiologists is the extent of culture
the second quadrant, and 1+ (rare) if growth is in the first work-up. Microbiologists still rely heavily on definitive iden-
quadrant. This tells the clinician the relative numbers of tification, although shortcuts, including the use of limited
 CHAPTER 5 Specimen Management 75

TABLE a study in which a group of physicians was asked whether

5.2   Common Transport Media they would treat a patient with a sore throat given two
separate laboratory reports—that is, one that stated “many
Media Description
group A streptococci” and one that stated “few group A
Amies transport media Recovery of aerobic and streptococci.” Although group A streptococcus (Streptococ-
anaerobic bacteria cus pyogenes) is considered significant in any numbers in a
Amies transport media Recovery of aerobic and symptomatic individual, the physicians said that they would
with charcoal anaerobic bacteria; charcoal treat the patient with many organisms but not the one with
neutralized bacterial toxins few organisms. Thus, although a pathogen (group A strep-
and other inhibitory sub-
tococcus) was isolated in both cases, one word on the report
stances, maintains pH
(either many or few) made a difference in how the patient
Anaerobic transport Numerous commercial sys- would be treated.
media tems available; recovery of
In communicating with the physician, the microbiologist
anaerobes, and microaero-
bic bacteria can prevent confusion and misunderstanding by not using
jargon or abbreviations and by providing reports with clear-
Cary-Blair Recovery of enteric pathogens
cut conclusions. The microbiologist should not assume that
Formalin (5%–10%), PVA Recovery of gastrointestinal the clinician is fully familiar with laboratory procedures or
(poly-vinyl alcohol), parasites; some may be the latest microbial taxonomic schemes. Thus, when appro-
SAF (sodium acetate- acceptable for immunoas-
priate, interpretive statements should be included in the
acetic acid-formalin), says
Total-Fix, Eco-Fix written report along with the specific results. One example
would be the addition of a statement such as “suggests con-
Stuart’s transport media Recovery of bacteria
tamination at collection” when more than three organisms
Universal transport Recovery of chlamydia, myco- are isolated from a clean-voided midstream urine specimen.
media plasmas and ureaplasmas Laboratory newsletters should be used to provide phy-
and viruses sicians with material such as details of new procedures,
nomenclature changes, and changes in usual antimicrobial
susceptibility patterns of frequently isolated organisms. This
identification procedures in some cases, are becoming com- last information, discussed in more detail in Chapter 11,
monplace in most clinical laboratories. Careful application is very useful to clinicians when selecting empiric therapy.
of knowledge of the significance of various organisms in Empiric therapy is based on the physician determining the
specific situations and thoughtful use of limited approaches most likely organism causing a patient’s clinical symptoms
will keep microbiology testing cost-effective and the labora- and then selecting an antimicrobial that, in the past, has
tory’s workload manageable, while providing for optimum worked against that organism in a particular hospital or geo-
patient care. graphic area. Empiric therapy is used to initiate treatment
Complete identification of a blood culture isolate, such following a direct testing method such as a Gram stain or
as Clostridium septicum as opposed to a genus identification nucleic acid test before the results of the patient’s culture
of Clostridium spp., will alert the clinician to the possibility are known and may be critical to the patient’s well-being in
of malignancy or other disease. At the same time, a pre- cases of life-threatening illnesses.
sumptive identification of Escherichia coli if a gram-nega- Positive findings should be communicated to the clini-
tive, spot indole-positive rod is recovered with appropriate cian in a timely manner, and all verbal reports should be
colony morphology on MacConkey agar (flat, lactose-fer- followed by written confirmation of results. Results should
menting colony that is precipitating bile salts) is probably be generated electronically in the laboratory information
permissible from an uncomplicated urinary tract infection. system (LIS).
In the final analysis, culture results should always be com-
pared with the suspected diagnosis. The clinician should be Critical (Panic) Values
encouraged to supply the microbiologist with all pertinent
information (e.g., recent travel history, pet exposure, perti- Certain critical results must be communicated to the cli-
nent radiograph findings) so that the microbiologist can use nician immediately. Each clinical microbiology laboratory,
the information to interpret culture results and plan appro- in consultation with its medical staff, should prepare a list
priate strategies for work-up. of these so-called “panic values.” Common panic values
include the following:
• Positive blood cultures
Communication of Laboratory Findings
• Positive spinal fluid Gram stain or culture
To fulfill their professional obligation to the patient, micro- • S treptococcus pyogenes (group A streptococcus) in a surgi-
biologists must communicate their findings to those health cal wound
care professionals responsible for treating the patient. This • Gram stain suggestive of gas gangrene (large boxcar-
task is not as easy as it may seem. This is nicely illustrated in shaped gram-positive rods)
76 PA RT I I General Principles in Clinical Microbiology

•  lood smear positive for malaria

B (transfer) and store data regarding positive cultures or anti-
• Positive cryptococcal antigen test microbial susceptibility results. Results of individual organ-
• Positive acid-fast stain ism antibiograms (patterns) can then be retrieved monthly
• Detection of a select agent (e.g., Brucella) or other signif- so hospital-wide susceptibility patterns can be studied for
icant pathogen (e.g., Legionella, vancomycin-resistant S. the emergence of resistant organisms or other epidemiologic
aureus, or other antibiotic-resistant organisms as outlined information. LIS systems can be interfaced with printers
by the facility and infection control policies) or electronic facsimile machines (faxes) as well as accessed
through smartphones or tablets for quick and easy reporting
and information retrieval, further improving the quality of
Expediting Results Reporting: patient care.
Computerization
Visit the Evolve site for a complete list of procedures,
Laboratories process information using an LIS or laboratory review questions, and case studies.
information system. Many LIS systems are, in turn, inter-
faced with a hospital information system (HIS). Between Bibliography
the HIS and LIS, most functions involved in ordering and
reporting laboratory tests can be handled electronically. Bennett J, Dolin R, Blaser M: Principles and practice of infectious dis-
Order entry, patient identification, and specimen identifica- eases, ed 9, Philadelphia, PA, 2020, Elsevier Saunders.
tion can be handled using the same type of bar coding that is Carroll KC, Pfaller MA, Landry ML, et al.: Manual of clinical micro-
commonly used in supermarkets. The LIS also takes care of biology, ed 12, Washington, DC, 2019, ASM Press.
result reporting and supervisory verification of results, stores Daley P, Castricianao S, Chernesky M, Smiej M: Comparison of
flocked and rayon swabs for collection of respiratory epithelial
quality control data, allows easy test inquiries, and assists
cells from uninfected volunteers and symptomatic patients, J Clin
in test management reporting by storing, for example, the Microbiol 44:2265, 2006.
number of positive, negative, and unsatisfactory specimens. Leber AL: Collection, transport, and manipulation of clinical speci-
LIS systems are capable of interfacing (communicating) mens. In Leber AL, ed: Clinical microbiology procedures handbook,
with microbiology instruments to automatically download ed 4, Washington, DC, 2016, ASM Press.
Chapter Review
1. All of the following are differential media except: 8. The laboratory received a blood culture specimen and
a. Blood agar placed it directly in the incubator. At 18 hours, the cul-
b. Chocolate agar ture indicated positive growth. The technologist com-
c. MacConkey’s agar pleted a Gram stain and plated some of the culture for
d. Eosin methylene blue agar incubation and identification. Identify the discrepancy
2. Selective media: or next step in the process.
a. May be differential a. The technologist must wait at least 18 hours for
b. Allow growth of all organisms growth on the agar plates to identify the organism.
c. Are used for growth of fastidious organisms b. The technologist failed to call the doctor or nurse
d. Contain dyes or antibiotics to suppress the growth immediately upon completion of the Gram stain
of some organisms with the result.
3. A urine specimen was collected at 8:00 a.m. The nurse c. The technologist completed the process correctly.
labeled the sample and placed it next to the tube sta- d. The technologist must place the blood culture bot-
tion to be sent to the laboratory for testing. The speci- tle back into the culture system until there is evi-
men was received in the laboratory for culture at 1:00 dence of growth on the agar plates.
p.m. on the same day. The microbiologist should: 9. True or False
a. Set up a routine bacteriology culture _____
F Nutritive media supports the growth of all or-
b. Set up a routine bacteriology culture and note the time ganisms.
c. Reject the specimen _____
T A requisition should include the patient’s name,
d. Call the nurse to explain why the specimen was hospital ID, birth date, specimen source, and col-
rejected and request a new sample lection date/time.
4. Neisseria gonorrhoeae are capnophilic organisms and _____
F EDTA is commonly used to collect blood for
require: specimens in the microbiology laboratory.
a. 5% to 10% CO2, 15% O2 _____
T The majority of microbiologic specimens
b. 5% to 10% H2, 0% O2 should be set up within 2 hours of collection.
c. 0.3% CO2, 21% O2 _____
T Serum samples may be stored frozen for up to
d. 8% to 10% CO2, 5% to 10% O2 1 week.
5. Specimens may include all of the following except: 10. Matching: Match each term with the correct description.
a. Sputum
b. Tissue biopsy ____
D SPS a. selective
c. Vacutainer needle ____
E aerobic b. positive CSF
d. Rectal swab ____
I ambient c. differential
6. Enrichment broth is used to: temperature d. blood culture
a. Shorten incubation time by providing excess nutrients ____
G enrichment systems
b. Suppress normal flora to allow pathogens to grow ____
A Campy e. requires oxygen
c. Detect small numbers of anaerobes ____
F Helicobacter sp. f. microaerophilic
d. Increase the growth of fastidious and pathogenic ____
J chocolate agar g. gram-negative broth
organisms ____
H facultative h. with or without
7. The laboratory received a tissue biopsy collected in sur- anaerobe oxygen
gery 3 hours earlier from a pancreatic cyst transported ____
B critical value i. room temperature
in a gauze pad, covered in saline, and placed in a bio- ____
C blood agar j. fastidious organisms
hazard bag. The microbiologist should:
  
a. Set up the tissue specimen after processing to blood
agar (BA), MacConkey, and chocolate agars 11. Short Answer
b. Complete a Gram stain and determine whether the Explain why direct Gram stains are not typically per-
specimen is acceptable formed on throat, nasopharyngeal, or stool specimens.
  
c. Reject the specimen and cancel the order in the
Direct Gram stains are not typically performed on throat,
laboratory information system nasopharyngeal or stool specimens because of the large
d. Set up the specimen and make a note in the LIS amount of normal microbiota present. Identifying a
regarding transport pathogen within the Gram stain would be difficult or
potentially misleading due to the numbers of contaminating
bacteria.

76.e1