SYMPOSIUM

Application of stains in clinical microbiology

BM Madison
Public Health Program Practice Of® ce, Division of Laboratory Systems, Centers for Disease Control and
Prevention, Atlanta, Georgia, USA

Submitted November 9, 2000; accepted November 21, 2000

Abstract
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Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain
remains the most commonly used stain because it detects and differentiates a wide range of
pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used
primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents
grow slowly on culture media or may not grow at all; stains may be the only method to detect
these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains
provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A
review of the most common staining methods used in the clinical microbiology laboratory is
presented here.
For personal use only.

Key words: acid-fast stain, ¯uorochrome stain, Gram stain

Gram stain in diagnostic microbiology
After more than 100 years, the Gram stain remains
the most rapid and cost-effective diagnostic tool in
the clinical microbiology laboratory (Popescu and
Doyle 1996). It is used to detect bacteria and to
classify them as either Gram positive or Gram
negative based on cell wall structure and content. In
conjunction with patient history and other labora-
tory tests, the Gram stain is the preliminary
determinant for diagnosing many serious infectious
disease processes. The presence or absence of
bacteria, Gram stain reaction, and other microscopic
characteristics are used to select the procedures that
will be used for de®nitive identi®cation of organ-
isms and speci®c antibiotic test panels. For many
infectious diseases, the Gram stain is the basis
for surveillance and infection control decisions
(Washington 1986). Because of the impact that the
Gram stain has made on diagnosis, treatment
regimens and infection control issues, it is critical
that the clinical microbiology laboratory provide
accurate and relevant results. Specimen quality can
be evaluated from Gram stain results by differen-
tiating the number of epithelial and in¯ammatory
cells in clinical specimens (Bartlett et al. 1987, Morin
et al. 1992). For example, a sputum specimen with
numerous epithelial cells and few or no white blood
cells is judged to be saliva and generally should not
be cultured. The Gram stain is a cost-effective tool
used to screen clinical specimens and aids in
selecting those that will provide the most clinically
relevant culture results.
The Gram stain: a simple procedure and a
challenge to interpret

Address for correspondence: Bereneice M. Madison, Ph.D.,
Senior Staff Fellow, Centers for Disease Control and Preven-
tion, Public Health Program Practice Of® ce, Division of
Laboratory Systems, 4770 Buford Highway, NE, MS-G-25,
Atlanta, GA 30341-3724, USA. Phone: ‡1 770-488-8133
ã Biological Stain Commission.
Biotechnic & Histochemistry 2001, 76(3): 119± 125.
The Gram stain procedure is straightforward,
consisting of a primary stain (crystal violet), a
mordant (iodine), a decolorizer (alcohol or acetone)
and a counterstain, usually safranin. Clinically
relevant smear results depend on collecting a
quality specimen and selecting a portion of the

119
 clinical material that is most representative of the
disease process. If the smear is too thick or too thin,
organisms may not be seen. Adequate ®xation of
the clinical specimen on microscope slides is
another important aspect for quality smears. Ex-
cessive heat ®xation may cause subsequent distor-
tion of organism morphology. Decolorization is the
most critical step in the Gram stain procedure; over-
or underdecolorization causes errors in interpreting
Gram stain results (Kruszak-Filipov and Shively
1992). Careful adherence to the staining procedure
is required for accurate results. Accuracy in inter-
preting results is highly dependent on the training
skill of the clinical microbiologists.
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Old bugs: new faces and new concerns
New genera of bacteria continue to be identi®ed
from clinical specimens in immunocompetent and
immunocompromised hosts. Many bacteria are
damaged after exposure to antibiotic therapy and
hostile environments; thus, their morphology and
staining characteristics often are atypical. The
clinical microbiologist now faces increasing chal-
lenges in interpreting Gram stain results. Many
For personal use only.
organisms that commonly have been seen in clinical
specimens tend to display atypical microscopic
morphological characteristics.
Gram-positive cocci
Staphylococci and Streptococci are the most common
Gram-positive cocci seen on Gram stained smears in
the clinical microbiology laboratory. Typically,
Staphylococci are arranged in grape-like clusters
with some single cells and pairs. Streptococci usually
are arranged in pairs and chains. Streptococci are
more typical in microscopic morphology, but tend
to lose their ability to retain the crystal violet-iodine
complex and appear Gram-negative. The difference
in the structure and cell wall content of these two
genera frequently is apparent in Gram stains of
clinical specimens. The morphological variability
in staphylococcal cells on Gram stained smears of
clinical specimens seems to reveal reactions to
chemotherapeutic agents and colonization pres-
sures in their environment. More recently, cells of
staphylococci in smears prepared from blood
cultures reveal large cocci with the appearance of
additional deposits of cell wall material resembling
yeast cells (personal observation). Several new
genera of Gram-positive cocci such as Stomatococcus,
Aerococcus and Abiotrophia spp. are frequently found
as opportunistic agents of infection in immunocom-
promised hosts (Ruoff 1999). Identi®cation of these
120 Biotechnic & Histochemistry 2001, 76(3): 119± 125
organisms is dif®cult and tedious, and the Gram
stain result is a key to the identi®cation process.
Gram-positive rods
When Gram-positive rods were seen on Gram
stained smears in the past, they were discounted
as skin or environmental contaminants. Small
pleomorphic cells were typically Corynebacterium
spp. or diptheroids. Large Gram-positive rods were
likely to be a member of Bacillus spp. or Clostridium
spp. Small Gram-positive rods are observed more
frequently in specimens from sterile body sites in
immunocompromised patients. Gram-positive rods
in Gram stain smears often vary in size and shape,
making it unclear whether one or more genera of
organisms are present. Some rods enlarge to the size
of fungal hyphae. Gram-positive rods from sterile
body sites can no longer be discounted as skin
contaminants or diphtheroids. These organisms
easily lose cell wall material and stain Gram-
variable or Gram-negative. Although spores rarely
are seen in clinical specimens, their presence
provides some degree of assurance that the organ-
ism is Gram-positive rather than Gram-negative. As
shown in Fig. 1, the spore-forming Bacillus circulans
has lost cell wall material and appears as a Gram-
negative rod.
Attempts to identify isolates of Gram-positive
rods biochemically are tedious and time-consum-
ing. The clinical microbiologist, however, must
process each case on an individual basis in
consultation with the clinician to determine the
relevance of the isolate. The term ``diphtheriod’’
rarely is used in the clinical setting since these
organisms may present as frank pathogens in the
immunocompromised host. Aside from the varia-
tion in morphology and Gram stain reactions,
several new genera of Gram-positive rods have
been classi®ed as opportunistic human pathogens
(Clarridge and Spiegel 1995). Some examples are
Brevibacterium spp. and Exigubacterium spp. that are
part of the indigenous ¯ora of humans. Clinical
microbiologists must determine the true Gram
reaction of bacilli and give careful attention to
both Gram-positive and Gram-negative organisms
because of bioterrorism concerns. Gram-negative
organisms such as Francisella tularensis and Brucella
spp. must not be mistaken for overdecolorized
small Gram-positive rods.
Gram-negative cocci
Gram-negative cocci are seen most often in smears
of respiratory and genital specimens. These organ-
 isms must be observed carefully to determine
whether they represent aged or overdecolorized
Gram-positive cocci. Close attention to the arrange-
ment, size and shape of the organisms often will
provide suf®cient information to differentiate these
organisms from Gram-positive cocci. Although
most Gram-negative cocci seen in respiratory
specimens are endogeneous microbiota, Branham-
mella catarrhalis, previously Neisseria catarrhalis, is
isolated frequently from infectious processes (Doern
1986). N. gonorrhoeae is the most common patho-
genic Gram-negative coccus identi®ed in genital
specimens. N. meningiditis frequently colonizes the
nasopharynx and may disseminate and cause
meningitis in children.
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Gram-negative rods
Gram-negative rods are among the most common
organisms seen in Gram stained smears because
they are so common in nature. They are agents of a
wide range of infections including bacteremia,
septicemia, and urinary tract, upper and lower
respiratory tract, and wound infections. Some
bacteria such as Pseudomonas aeruginosa, Klebsiella
For personal use only.
peumoniae, Fusobacterium spp., Campylobacter spp.
and others have unique Gram stain morphology
that may be used to provide presumptive diagnostic
clues to clinicians. Experienced microscopists,
however, can only presumptively differentiate
P. aeruginosa from members of the Enterobacteria-
ceae on direct Gram stained smears approximately
80% of the time (Sewell 1987, Bartlett et al. 1985).
Interestingly, a Gram-negative coccobacillary
organism Acinetobacter anitratus, has been shown
to stain Gram-positive and mimic Staphylococci in
clinical smears (personal observations). Acineto-
bacter is distinguished easily from Staphylococci on
routine culture since it grows well on MacConkey’s
agar, and Staphylococci are inhibited. These observa-
tions support the fact that the Gram stain should be
used as an adjunct to culture in clinical situations
and should not be used alone to identify bacteria.
Gram stain modiŽ cations
Several modi®cations of the Gram stain procedure
have been made since Gram developed the test in
the late 1800s. Hucker’s modi®cation (Hucker 1921)
currently is used in most clinical laboratories and
provides greater reagent stability and better differ-
entiation of organisms. The choice of the decolorizer
may vary. When 95% ethanol is used, decoloriza-
tion time will be long, intermediate with acetone-
alcohol, fastest with acetone. Use of 95% ethanol is
the preferred decolorizer for students and less
experienced personnel. Acetone is a more rapid
decolorizer with a shorter range of reproducibility
and is recommended only for experienced person-
nel. Faintly staining Gram-negative rods may be
enhanced by counterstaining with safranin for
1±2 min rather than the usual 30 sec, by adding
0.05% basic fuchsin to the safranin or by substitut-
ing carbol fuchsin (Kruczak-Filipov and Shively
1992).
Despite its many modi®cations and uses, the
Gram stain has limitations. Organisms that lack an
intact cell wall will not stain. The integrity of the cell
wall of Gram-positive bacteria that are old, dead or
damaged by antibiotics or other chemotherapeutic
agents often are disrupted and do not retain the
crystal violet-iodine complex during decolorization,
and appear Gram-variable or Gram-negative.
Although some yeast, fungal elements and some
parasites may be seen, the Gram stain is not the
optimal method for determining the presence of
these organisms in direct smears of clinical speci-
mens (Chapin and Murray 1999). The stain is
unable to penetrate easily the cell wall of some
bacteria (Beveridge et al. 1991).
Acridine orange stain
Acridine orange is another stain that has proved
valuable for detecting bacteria in clinical specimens.
At pH 4.0, bacteria stain red-orange while non-
bacterial cells such as yeasts, epithelial cells,
polymorphonuclear leukocytes and other clinical
material stain yellow-green. Acridine orange is an
excellent stain for detecting spirochetes such as
Treponema pallidium in touch preps from genital
lesions and other body ¯uids.
Coupled with cytocentrifugation, this stain can
be sensitive for detecting bacteria in cerebrospinal
¯uid. Acridine orange is a ¯uorochrome, and a
¯uorescence microscope equipped with appropriate
®lters must be used to observe stained smears. For
con®rming the Gram reaction of organisms seen on
acridine orange stained smears, slides may be
overstained with the Gram stain (Lauer et al. 1981).
Stains for detecting acid-fast
organisms
Acid-fast microscopy of sputum smears is the most
cost-effective procedure to screen for infectious
cases of presumed tuberculosis. The causative agent
of tuberculosis, Mycobacterium tuberculosis, has a
waxy lipid component in its cell wall that prevents
Clinical microbiology stains 121
 the organism from being stained easily with most
biological stains. Once stained with a phenol dye
mixture, however, acid-fast organisms are not
decolorized easily with an acid-alcohol mixture.
Acid-fast microscopy provides information to de-
termine the presence of acid-fast organisms in
clinical specimens and to support infection control
measures that prevent transmission of tuberculosis.
It is useful for helping clinicians initiate treatment
and monitor progress of antituberculous drug
therapy. Acid-fast microscopy also serves as a
determinant for performing other laboratory tests
such as the nucleic acid ampli®cation tests for
identifying M. tuberculosis in clinical specimens.
Other nontuberculous Mycobacterium spp. also stain
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acid-fast.
Three acid-fast staining techniques are com-
monly used: Kinyoun, Ziehl-Nielseen and ¯uoro-
chrome stains (Kent and Kubica 1985). Kinyoun and
Ziehl-Nielseen stained smears are examined by light
microscopy under oil immersion. Acid-fast organ-
isms appear as red bacilli, and epithelial cells and
other material stain blue when methylene blue is
used as a counterstain (Fig. 2). Malachite green or
brilliant green also may be used as counterstains.
For personal use only.
Ziehl-Nielsen stain is heated to enable carbol
fuchsin to penetrate the cell wall and is known as
a hot stain. Kinyoun stain has a higher concentra-
tion of fuchsin and phenol and does not require
heat; thus, it is called a cold acid-fast stain
(Smithwick 1976). Tergitol, a wetting agent, has
been used to enhance the penetration of carbol
fuchsin across the lipid cell wall of acid-fast
organisms.
Modi®cations of basic fuchsin stains are used to
detect other microorganisms and cell structures. A
weaker acid, sulfuric rather than hydrochloric,
is used to detect nontuberculous mycobacteria,
Nocardia spp., Rhodococcus spp., Tsukamurella spp.,
Gordona spp. and Legionella micdadei, which are
weakly or partially acid-fast. Some organisms
including rapid growing mycobacteria tend to lose
their acid-fast properties with age and require a
weaker acid alcohol decolorization to detect acid-
fast properties on repeated subculture. Some rapid
growing mycobacteria and Nocardia spp. have
similar colonial and microscopic characteristics such
as Corynebacterium spp. Modi®ed acid-fast staining
is a key test used in the identi®cation scheme of
these bacteria.
A rapid and more sensitive method for detecting
acid-fast bacilli in clinical specimens is the ¯uor-
ochrome staining method. Two staining methods
(Blair 1969, Truant 1962) are used in ¯uorochrome
acid-fast microscopy, Blair’s stain with auramine O
122 Biotechnic & Histochemistry 2001, 76(3): 119± 125
as the primary stain, and Truant’s, which uses a
combination of both auramine O and rhodamine B.
Acid-fast organisms stain green with auramine O
and yellow-gold when auramine O-rhodamine B is
the primary stain depending on the ®lter system
used with the ¯uorescence microscope. At least
three counterstains can be used with ¯uorochrome
staining. Potassium permanganate, a strong oxidiz-
ing agent, can be used with both primary stains
(Chapin and Murray 1999). It acts as a quenching
agent, decreasing the background ¯uorescence of
cellular debris, and the yellow or green acid-fast
organisms can be seen against a dark background.
Acridine orange can be used as a counterstain with
auramine O. Cellular debris and nonacid-fast
organisms stain yellow-orange in contrast to the
green acid-fast bacilli (Chapin and Murray 1999).
Erichrome black T also can be used as a counter-
stain with auramine O-rhodamine B in the TB
Fluorostain kit (Polysciences, Inc. 1993). Whereas
counterstains enhance the background and provide
better recognition of acid-fast stained organisms,
they are not necessary.
Truant’s stain has been reported to provide
increased sensitivity for detecting acid-fast organ-
isms when smears are stained at 37 o C. With this
modi®cation as well as other techniques for acid-
fast staining, cross-contamination and preventing
the stain from drying on the slides are important
concerns (McCarter and Robinson 1994).
Fluorochrome acid-fast microscopy has many
advantages over basic fuchsin staining. It provides
more sensitivity for detecting smaller numbers of
acid-fast organisms in clinical specimens. The
stained smears may be screened at £200 magni®ca-
tion and con®rmed at £400 magni®cation. The
ability to examine ¯uorochrome stained smears at
£200 magni®cation rather than £1000 as required
for light microscopy decreases reading time and
turnaround time for reporting acid-fast smear
results. Fluorochrome stained smears can be re-
stained with a basic fuchsin staining method if
desired (Ebersole 1992). Fluorochrome stained
smears lose their ¯uorescence if not read within
24 hr after staining and if not protected from light.
Fluorescent stains such as auramine-rhodamine,
auramine-carbol fuschin, and acridine orange also
can be used as screening techniques for intestinal
protozoa such as Cryptosprodium parvum, Cyclospora
cayetanensis and Isopora belli. These organisms cause
severe diarrhea in both immunocompromised and
immunocompetent hosts. Oocysts of these parasites
may be dif®cult to detect without special staining. A
modi®ed acid-fast stain such as Zehl-Nielsen or
Kinyoun’s is used often to con®rm the presence of

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Fig. 1. Bacillus circulans, a Gram-positive spore form-
ing bacillus that has lost cell wall material and appears
Gram-negative. £400.
these organisms in clinical specimens (Garcia et al.
1999, Long et al. 1991, Shimizu 1992a,b).
Stains for detecting fungi and
parasites in clinical specimens
For personal use only.
Potassium hydroxide (10±15% KOH) traditionally
was used to observe fungi in clinical specimens by
some experienced microscopists. It dissolves mucus
and debris in clinical material, permitting easier
observation of fungi that may be present in clinical
specimens. Enhanced clearing of the clinical
material has been achieved by heating the KOH
Fig. 2. Acid-fast bacilli appear red with Kinyoun and
Ziehl-Nielsen’s basic fuchsin stains. Epithelial cells and
other background material appear blue when methy-
lene blue is used as the counterstain. £400 (Acid-fast
organisms are observed best at £1000 under oil
immersion).
prepared smear before microscopic examination.
KOH is not highly recommended today for detect-
ing fungi in clinical specimens because of the
expertise required to read wet mount preparations
on the light microscope.
Calco¯uor white is recommended for detecting
fungi in clinical specimens and can be used as a wet
mount in combination with KOH (Pasarell and
Schell 1992). Calco¯uor white is a ¯uorescent optical
brightening agent that binds nonspeci®cally to beta-
linked polysaccharide polymers found in fungi and
other organisms. When observed with a ¯uores-
cence microscope equipped with the appropriate
excitation ®lters, fungi ¯uoresce bright green on a
calco¯uor white stain as shown with the yeast
Candida albicans in Fig. 3. Another advantage of the
calco¯uor white stain over the KOH prep is that
dried smears can be prepared, stained and exam-
ined later without the problem of drying that is a
concern with KOH preparations. The calco¯uor
white stained slides may even be restained after
several days when ¯uorescence has been quenched.
Calco¯uor white has also been used in a rapid
method to detect Pneumocystis carinii cysts in
bronchoalveolar lavage samples. Smears for
P. carinii may be scanned at £40 and con®rmed at
£1000. These organisms are seen frequently in
patients with acquired immune de®ciency syn-
drome (AIDS) and must be distinguished from
yeast cells of Histoplasma and Candida spp. Calco-
¯uor white has been reported to have advantages
over the Gomori methenamine silver and toluidine
blue stains because it is a more rapid and cost-
effective method for detecting P. carinii (Baselski et
al. 1990, Stratton et al. 1991). Experience in
recognizing the morphology of the highly charac-
Fig. 3. Candida albicans ¯ uoresces bright green with
calco¯ uor white stain when observed by ¯ uorescence
microscopy. £400.
Clinical microbiology stains 123
 teristic double parenthesis structure of Peumocystis
carinni is very important.
India ink staining is another procedure that has
been used primarily for detecting the presence of
capsule producing organisms, such as Cryptococcus
neoformans, in cerebrospinal ¯uid (McGinnis and
Schell 1992). The appropriate mixture of India ink
and clinical specimen for optimal visualization of
fungi and the ability to distinguish red and white
blood cells from nonbudding yeast cells in clinical
specimens are challenges for many inexperienced
microscopists. The calco¯uor white stain works
especially well for observation of fungi in cere-
brospinal ¯uid and has replaced the use of India ink
in many laboratory settings. In addition, the much
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more sensitive and speci®c antigen detection test for
the capsular polysaccharide of Cryptococcus neofor-
mans is now commonly used because C. neoformans
antigen is often found in spinal ¯uid and blood
specimens of HIV infected patients.
Grocott-Gomori’s methenamine-silver stain once
was used commonly in the microbiology laboratory
for observing fungi in clinical specimens (Woods
and Walker 1996). Because of the expertise required
for maintaining optimal staining results of this time-
For personal use only.
consuming method, many microbiologists use
calco¯uor white for rapid detection of fungi.
Gomori methenamine-silver is still used routinely
in histology laboratories for pathological examina-
tion of tissue and other clinical specimens.
Wright-Giemsa stain is used for examining
clinical specimens for fungi, particularly the intra-
cellular yeast form of Histoplasma capsulatum in
blood and tissue specimens (Chapin and Murray
1999, Woods and Walker 1996). Histoplasma and
other fungi are seen more commonly in clinical
specimens of patients with human immunode®-
ciency viral infection. Specimens with Histoplasma
are seen in both the microbiology and hematology
laboratories.
Wright-Giemsa stain is the primary method for
detecting and identifying Plasmodium spp., the
causative agents of malaria as well as for other
blood and tissue parasites such as Leishmania
donovani, Trypansoma spp., Babesia microti and
micro®lariae (Garcia and Bruckner 1988). These
parasites are observed on both thick and thin
smears from whole blood or buffy coat smears.
Such organisms are seen most often in pro®ciency
panels and only frequently enough in clinical
specimens to encourage efforts to maintain compe-
tency.
Examination for intestinal protozoa is requested
routinely in the clinical microbiology laboratory.
Con®rmatory identi®cation is performed by micro-
124 Biotechnic & Histochemistry 2001, 76(3): 119± 125
scopic examination of a permanent stained smear of
stool or other appropriate clinical specimen. The
most commonly used stain for detecting ova and
parasites in fecal specimens is a modi®cation of
Gormori’s original tissue stain trichrome technique
of Wheatly. The trichrome stain is a rapid, simple
procedure that produces uniformly well stained
smears of intestinal protozoa, human cells, yeast
cells, and artifactual material in approximately
45 min or less (Chapin and Murray 1999, Wheatley
1951).
Summary
Microscopic examination of stained smears in the
clinical microbiology laboratory still provides the
most cost-effective method of detecting many
pathogens and judging specimen quality despite
the development of rapid detection systems and
molecular diagnostic probes. The stains used in
clinical microbiology are relatively simple to per-
form, but the complexity of the procedures resides
in microscopic examination, differentiating speci®c
organisms, evaluating the staining reactions, and
determining the signi®cance of what is detected by
the microscopist. These issues are easier for the
clinical microbiologist with microscopy experience
and knowledge of the various disease entities
(Chapin 1995).
Acknowledgments
Special thanks to Drs. Ron Doyle and Beverly
Metchock for reviewing the manuscript. Photo-
graphs from Public Health Image Library, Centers
for Disease Control and Prevention.
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