application of stains in clinical microbiology
TRANSCRIPT
SYMPOSIUM
Application of stains in clinical microbiology
BM Madison
Public Health Program Practice Of® ce, Division of Laboratory Systems, Centers for Disease Control andPrevention, Atlanta, Georgia, USA
Submitted November 9, 2000; accepted November 21, 2000
Abstract
Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stainremains the most commonly used stain because it detects and differentiates a wide range ofpathogens. The next most commonly used diagnostic technique is acid-fast staining that is usedprimarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agentsgrow slowly on culture media or may not grow at all; stains may be the only method to detectthese organisms in clinical specimens. In the hands of experienced clinical microscopists, stainsprovide rapid and cost-effective information for preliminary diagnosis of infectious diseases. Areview of the most common staining methods used in the clinical microbiology laboratory ispresented here.
Key words: acid-fast stain, ¯uorochrome stain, Gram stain
Gram stain in diagnostic microbiology
After more than 100 years, the Gram stain remainsthe most rapid and cost-effective diagnostic tool inthe clinical microbiology laboratory (Popescu andDoyle 1996). It is used to detect bacteria and toclassify them as either Gram positive or Gramnegative based on cell wall structure and content. Inconjunction with patient history and other labora-tory tests, the Gram stain is the preliminarydeterminant for diagnosing many serious infectiousdisease processes. The presence or absence ofbacteria, Gram stain reaction, and other microscopiccharacteristics are used to select the procedures thatwill be used for de®nitive identi®cation of organ-isms and speci®c antibiotic test panels. For manyinfectious diseases, the Gram stain is the basisfor surveillance and infection control decisions
(Washington 1986). Because of the impact that theGram stain has made on diagnosis, treatmentregimens and infection control issues, it is criticalthat the clinical microbiology laboratory provideaccurate and relevant results. Specimen quality canbe evaluated from Gram stain results by differen-tiating the number of epithelial and in¯ammatorycells in clinical specimens (Bartlett et al. 1987, Morinet al. 1992). For example, a sputum specimen withnumerous epithelial cells and few or no white bloodcells is judged to be saliva and generally should notbe cultured. The Gram stain is a cost-effective toolused to screen clinical specimens and aids inselecting those that will provide the most clinicallyrelevant culture results.
The Gram stain: a simple procedure and achallenge to interpret
The Gram stain procedure is straightforward,consisting of a primary stain (crystal violet), amordant (iodine), a decolorizer (alcohol or acetone)and a counterstain, usually safranin. Clinicallyrelevant smear results depend on collecting aquality specimen and selecting a portion of the
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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 ofLaboratory Systems, 4770 Buford Highway, NE, MS-G-25,Atlanta, GA 30341-3724, USA. Phone: ‡1 770-488-8133ã Biological Stain Commission.Biotechnic & Histochemistry 2001, 76(3): 119± 125.
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clinical material that is most representative of thedisease process. If the smear is too thick or too thin,organisms may not be seen. Adequate ®xation ofthe clinical specimen on microscope slides isanother important aspect for quality smears. Ex-cessive heat ®xation may cause subsequent distor-tion of organism morphology. Decolorization is themost critical step in the Gram stain procedure; over-or underdecolorization causes errors in interpretingGram stain results (Kruszak-Filipov and Shively1992). Careful adherence to the staining procedureis required for accurate results. Accuracy in inter-preting results is highly dependent on the trainingskill of the clinical microbiologists.
Old bugs: new faces and new concerns
New genera of bacteria continue to be identi®edfrom clinical specimens in immunocompetent andimmunocompromised hosts. Many bacteria aredamaged after exposure to antibiotic therapy andhostile environments; thus, their morphology andstaining characteristics often are atypical. Theclinical microbiologist now faces increasing chal-lenges in interpreting Gram stain results. Manyorganisms that commonly have been seen in clinicalspecimens tend to display atypical microscopicmorphological characteristics.
Gram-positive cocci
Staphylococci and Streptococci are the most commonGram-positive cocci seen on Gram stained smears inthe clinical microbiology laboratory. Typically,Staphylococci are arranged in grape-like clusterswith some single cells and pairs. Streptococci usuallyare arranged in pairs and chains. Streptococci aremore typical in microscopic morphology, but tendto lose their ability to retain the crystal violet-iodinecomplex and appear Gram-negative. The differencein the structure and cell wall content of these twogenera frequently is apparent in Gram stains ofclinical specimens. The morphological variabilityin staphylococcal cells on Gram stained smears ofclinical specimens seems to reveal reactions tochemotherapeutic agents and colonization pres-sures in their environment. More recently, cells ofstaphylococci in smears prepared from bloodcultures reveal large cocci with the appearance ofadditional deposits of cell wall material resemblingyeast cells (personal observation). Several newgenera of Gram-positive cocci such as Stomatococcus,Aerococcus and Abiotrophia spp. are frequently foundas opportunistic agents of infection in immunocom-promised hosts (Ruoff 1999). Identi®cation of these
organisms is dif®cult and tedious, and the Gramstain result is a key to the identi®cation process.
Gram-positive rods
When Gram-positive rods were seen on Gramstained smears in the past, they were discountedas skin or environmental contaminants. Smallpleomorphic cells were typically Corynebacteriumspp. or diptheroids. Large Gram-positive rods werelikely to be a member of Bacillus spp. or Clostridiumspp. Small Gram-positive rods are observed morefrequently in specimens from sterile body sites inimmunocompromised patients. Gram-positive rodsin Gram stain smears often vary in size and shape,making it unclear whether one or more genera oforganisms are present. Some rods enlarge to the sizeof fungal hyphae. Gram-positive rods from sterilebody sites can no longer be discounted as skincontaminants or diphtheroids. These organismseasily lose cell wall material and stain Gram-variable or Gram-negative. Although spores rarelyare seen in clinical specimens, their presenceprovides some degree of assurance that the organ-ism is Gram-positive rather than Gram-negative. Asshown in Fig. 1, the spore-forming Bacillus circulanshas lost cell wall material and appears as a Gram-negative rod.
Attempts to identify isolates of Gram-positiverods biochemically are tedious and time-consum-ing. The clinical microbiologist, however, mustprocess each case on an individual basis inconsultation with the clinician to determine therelevance of the isolate. The term ``diphtheriod’’rarely is used in the clinical setting since theseorganisms may present as frank pathogens in theimmunocompromised host. Aside from the varia-tion in morphology and Gram stain reactions,several new genera of Gram-positive rods havebeen classi®ed as opportunistic human pathogens(Clarridge and Spiegel 1995). Some examples areBrevibacterium spp. and Exigubacterium spp. that arepart of the indigenous ¯ora of humans. Clinicalmicrobiologists must determine the true Gramreaction of bacilli and give careful attention toboth Gram-positive and Gram-negative organismsbecause of bioterrorism concerns. Gram-negativeorganisms such as Francisella tularensis and Brucellaspp. must not be mistaken for overdecolorizedsmall Gram-positive rods.
Gram-negative cocci
Gram-negative cocci are seen most often in smearsof respiratory and genital specimens. These organ-
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isms must be observed carefully to determinewhether they represent aged or overdecolorizedGram-positive cocci. Close attention to the arrange-ment, size and shape of the organisms often willprovide suf®cient information to differentiate theseorganisms from Gram-positive cocci. Althoughmost Gram-negative cocci seen in respiratoryspecimens are endogeneous microbiota, Branham-mella catarrhalis, previously Neisseria catarrhalis, isisolated frequently from infectious processes (Doern1986). N. gonorrhoeae is the most common patho-genic Gram-negative coccus identi®ed in genitalspecimens. N. meningiditis frequently colonizes thenasopharynx and may disseminate and causemeningitis in children.
Gram-negative rods
Gram-negative rods are among the most commonorganisms seen in Gram stained smears becausethey are so common in nature. They are agents of awide range of infections including bacteremia,septicemia, and urinary tract, upper and lowerrespiratory tract, and wound infections. Somebacteria such as Pseudomonas aeruginosa, Klebsiellapeumoniae, Fusobacterium spp., Campylobacter spp.and others have unique Gram stain morphologythat may be used to provide presumptive diagnosticclues to clinicians. Experienced microscopists,however, can only presumptively differentiateP. aeruginosa from members of the Enterobacteria-ceae on direct Gram stained smears approximately80% of the time (Sewell 1987, Bartlett et al. 1985).Interestingly, a Gram-negative coccobacillaryorganism Acinetobacter anitratus, has been shownto stain Gram-positive and mimic Staphylococci inclinical smears (personal observations). Acineto-bacter is distinguished easily from Staphylococci onroutine culture since it grows well on MacConkey’sagar, and Staphylococci are inhibited. These observa-tions support the fact that the Gram stain should beused as an adjunct to culture in clinical situationsand should not be used alone to identify bacteria.
Gram stain modi� cations
Several modi®cations of the Gram stain procedurehave been made since Gram developed the test inthe late 1800s. Hucker’s modi®cation (Hucker 1921)currently is used in most clinical laboratories andprovides greater reagent stability and better differ-entiation of organisms. The choice of the decolorizermay 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 lessexperienced personnel. Acetone is a more rapiddecolorizer with a shorter range of reproducibilityand is recommended only for experienced person-nel. Faintly staining Gram-negative rods may beenhanced by counterstaining with safranin for1±2 min rather than the usual 30 sec, by adding0.05% basic fuchsin to the safranin or by substitut-ing carbol fuchsin (Kruczak-Filipov and Shively1992).
Despite its many modi®cations and uses, theGram stain has limitations. Organisms that lack anintact cell wall will not stain. The integrity of the cellwall of Gram-positive bacteria that are old, dead ordamaged by antibiotics or other chemotherapeuticagents often are disrupted and do not retain thecrystal violet-iodine complex during decolorization,and appear Gram-variable or Gram-negative.Although some yeast, fungal elements and someparasites may be seen, the Gram stain is not theoptimal method for determining the presence ofthese organisms in direct smears of clinical speci-mens (Chapin and Murray 1999). The stain isunable to penetrate easily the cell wall of somebacteria (Beveridge et al. 1991).
Acridine orange stain
Acridine orange is another stain that has provedvaluable 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 clinicalmaterial stain yellow-green. Acridine orange is anexcellent stain for detecting spirochetes such asTreponema pallidium in touch preps from genitallesions and other body ¯uids.
Coupled with cytocentrifugation, this stain canbe 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. Forcon®rming the Gram reaction of organisms seen onacridine orange stained smears, slides may beoverstained with the Gram stain (Lauer et al. 1981).
Stains for detecting acid-fastorganisms
Acid-fast microscopy of sputum smears is the mostcost-effective procedure to screen for infectiouscases of presumed tuberculosis. The causative agentof tuberculosis, Mycobacterium tuberculosis, has awaxy lipid component in its cell wall that prevents
Clinical microbiology stains 121
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the organism from being stained easily with mostbiological stains. Once stained with a phenol dyemixture, however, acid-fast organisms are notdecolorized easily with an acid-alcohol mixture.Acid-fast microscopy provides information to de-termine the presence of acid-fast organisms inclinical specimens and to support infection controlmeasures that prevent transmission of tuberculosis.It is useful for helping clinicians initiate treatmentand monitor progress of antituberculous drugtherapy. Acid-fast microscopy also serves as adeterminant for performing other laboratory testssuch as the nucleic acid ampli®cation tests foridentifying M. tuberculosis in clinical specimens.Other nontuberculous Mycobacterium spp. also stainacid-fast.
Three acid-fast staining techniques are com-monly used: Kinyoun, Ziehl-Nielseen and ¯uoro-chrome stains (Kent and Kubica 1985). Kinyoun andZiehl-Nielseen stained smears are examined by lightmicroscopy under oil immersion. Acid-fast organ-isms appear as red bacilli, and epithelial cells andother material stain blue when methylene blue isused as a counterstain (Fig. 2). Malachite green orbrilliant green also may be used as counterstains.Ziehl-Nielsen stain is heated to enable carbolfuchsin to penetrate the cell wall and is known asa hot stain. Kinyoun stain has a higher concentra-tion of fuchsin and phenol and does not requireheat; thus, it is called a cold acid-fast stain(Smithwick 1976). Tergitol, a wetting agent, hasbeen used to enhance the penetration of carbolfuchsin across the lipid cell wall of acid-fastorganisms.
Modi®cations of basic fuchsin stains are used todetect other microorganisms and cell structures. Aweaker acid, sulfuric rather than hydrochloric,is used to detect nontuberculous mycobacteria,Nocardia spp., Rhodococcus spp., Tsukamurella spp.,Gordona spp. and Legionella micdadei, which areweakly or partially acid-fast. Some organismsincluding rapid growing mycobacteria tend to losetheir acid-fast properties with age and require aweaker acid alcohol decolorization to detect acid-fast properties on repeated subculture. Some rapidgrowing mycobacteria and Nocardia spp. havesimilar colonial and microscopic characteristics suchas Corynebacterium spp. Modi®ed acid-fast stainingis a key test used in the identi®cation scheme ofthese bacteria.
A rapid and more sensitive method for detectingacid-fast bacilli in clinical specimens is the ¯uor-ochrome staining method. Two staining methods(Blair 1969, Truant 1962) are used in ¯uorochromeacid-fast microscopy, Blair’s stain with auramine O
as the primary stain, and Truant’s, which uses acombination of both auramine O and rhodamine B.Acid-fast organisms stain green with auramine Oand yellow-gold when auramine O-rhodamine B isthe primary stain depending on the ®lter systemused with the ¯uorescence microscope. At leastthree counterstains can be used with ¯uorochromestaining. Potassium permanganate, a strong oxidiz-ing agent, can be used with both primary stains(Chapin and Murray 1999). It acts as a quenchingagent, decreasing the background ¯uorescence ofcellular debris, and the yellow or green acid-fastorganisms can be seen against a dark background.Acridine orange can be used as a counterstain withauramine O. Cellular debris and nonacid-fastorganisms stain yellow-orange in contrast to thegreen 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 TBFluorostain kit (Polysciences, Inc. 1993). Whereascounterstains enhance the background and providebetter recognition of acid-fast stained organisms,they are not necessary.
Truant’s stain has been reported to provideincreased sensitivity for detecting acid-fast organ-isms when smears are stained at 37 oC. With thismodi®cation as well as other techniques for acid-fast staining, cross-contamination and preventingthe stain from drying on the slides are importantconcerns (McCarter and Robinson 1994).
Fluorochrome acid-fast microscopy has manyadvantages over basic fuchsin staining. It providesmore sensitivity for detecting smaller numbers ofacid-fast organisms in clinical specimens. Thestained smears may be screened at £200 magni®ca-tion and con®rmed at £400 magni®cation. Theability to examine ¯uorochrome stained smears at£200 magni®cation rather than £1000 as requiredfor light microscopy decreases reading time andturnaround time for reporting acid-fast smearresults. Fluorochrome stained smears can be re-stained with a basic fuchsin staining method ifdesired (Ebersole 1992). Fluorochrome stainedsmears lose their ¯uorescence if not read within24 hr after staining and if not protected from light.
Fluorescent stains such as auramine-rhodamine,auramine-carbol fuschin, and acridine orange alsocan be used as screening techniques for intestinalprotozoa such as Cryptosprodium parvum, Cyclosporacayetanensis and Isopora belli. These organisms causesevere diarrhea in both immunocompromised andimmunocompetent hosts. Oocysts of these parasitesmay be dif®cult to detect without special staining. Amodi®ed acid-fast stain such as Zehl-Nielsen orKinyoun’s is used often to con®rm the presence of
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these organisms in clinical specimens (Garcia et al.1999, Long et al. 1991, Shimizu 1992a,b).
Stains for detecting fungi andparasites in clinical specimens
Potassium hydroxide (10±15% KOH) traditionallywas used to observe fungi in clinical specimens bysome experienced microscopists. It dissolves mucusand debris in clinical material, permitting easierobservation of fungi that may be present in clinicalspecimens. Enhanced clearing of the clinicalmaterial has been achieved by heating the KOH
prepared smear before microscopic examination.KOH is not highly recommended today for detect-ing fungi in clinical specimens because of theexpertise required to read wet mount preparationson the light microscope.
Calco¯uor white is recommended for detectingfungi in clinical specimens and can be used as a wetmount in combination with KOH (Pasarell andSchell 1992). Calco¯uor white is a ¯uorescent opticalbrightening agent that binds nonspeci®cally to beta-linked polysaccharide polymers found in fungi andother organisms. When observed with a ¯uores-cence microscope equipped with the appropriateexcitation ®lters, fungi ¯uoresce bright green on acalco¯uor white stain as shown with the yeastCandida albicans in Fig. 3. Another advantage of thecalco¯uor white stain over the KOH prep is thatdried smears can be prepared, stained and exam-ined later without the problem of drying that is aconcern with KOH preparations. The calco¯uorwhite stained slides may even be restained afterseveral days when ¯uorescence has been quenched.
Calco¯uor white has also been used in a rapidmethod to detect Pneumocystis carinii cysts inbronchoalveolar lavage samples. Smears forP. carinii may be scanned at £40 and con®rmed at£1000. These organisms are seen frequently inpatients with acquired immune de®ciency syn-drome (AIDS) and must be distinguished fromyeast cells of Histoplasma and Candida spp. Calco-¯uor white has been reported to have advantagesover the Gomori methenamine silver and toluidineblue stains because it is a more rapid and cost-effective method for detecting P. carinii (Baselski etal. 1990, Stratton et al. 1991). Experience inrecognizing the morphology of the highly charac-
Clinical microbiology stains 123
Fig. 3. Candida albicans ¯ uoresces bright green withcalco¯ uor white stain when observed by ¯ uorescencemicroscopy. £400.
Fig. 1. Bacillus circulans, a Gram-positive spore form-ing bacillus that has lost cell wall material and appearsGram-negative. £400.
Fig. 2. Acid-fast bacilli appear red with Kinyoun andZiehl-Nielsen’s basic fuchsin stains. Epithelial cells andother background material appear blue when methy-lene blue is used as the counterstain. £400 (Acid-fastorganisms are observed best at £1000 under oilimmersion).
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teristic double parenthesis structure of Peumocystiscarinni is very important.
India ink staining is another procedure that hasbeen used primarily for detecting the presence ofcapsule producing organisms, such as Cryptococcusneoformans, in cerebrospinal ¯uid (McGinnis andSchell 1992). The appropriate mixture of India inkand clinical specimen for optimal visualization offungi and the ability to distinguish red and whiteblood cells from nonbudding yeast cells in clinicalspecimens are challenges for many inexperiencedmicroscopists. The calco¯uor white stain worksespecially well for observation of fungi in cere-brospinal ¯uid and has replaced the use of India inkin many laboratory settings. In addition, the muchmore sensitive and speci®c antigen detection test forthe capsular polysaccharide of Cryptococcus neofor-mans is now commonly used because C. neoformansantigen is often found in spinal ¯uid and bloodspecimens of HIV infected patients.
Grocott-Gomori’s methenamine-silver stain oncewas used commonly in the microbiology laboratoryfor observing fungi in clinical specimens (Woodsand Walker 1996). Because of the expertise requiredfor maintaining optimal staining results of this time-consuming method, many microbiologists usecalco¯uor white for rapid detection of fungi.Gomori methenamine-silver is still used routinelyin histology laboratories for pathological examina-tion of tissue and other clinical specimens.
Wright-Giemsa stain is used for examiningclinical specimens for fungi, particularly the intra-cellular yeast form of Histoplasma capsulatum inblood and tissue specimens (Chapin and Murray1999, Woods and Walker 1996). Histoplasma andother fungi are seen more commonly in clinicalspecimens of patients with human immunode®-ciency viral infection. Specimens with Histoplasmaare seen in both the microbiology and hematologylaboratories.
Wright-Giemsa stain is the primary method fordetecting and identifying Plasmodium spp., thecausative agents of malaria as well as for otherblood and tissue parasites such as Leishmaniadonovani, Trypansoma spp., Babesia microti andmicro®lariae (Garcia and Bruckner 1988). Theseparasites are observed on both thick and thinsmears from whole blood or buffy coat smears.Such organisms are seen most often in pro®ciencypanels and only frequently enough in clinicalspecimens to encourage efforts to maintain compe-tency.
Examination for intestinal protozoa is requestedroutinely in the clinical microbiology laboratory.Con®rmatory identi®cation is performed by micro-
scopic examination of a permanent stained smear ofstool or other appropriate clinical specimen. Themost commonly used stain for detecting ova andparasites in fecal specimens is a modi®cation ofGormori’s original tissue stain trichrome techniqueof Wheatly. The trichrome stain is a rapid, simpleprocedure that produces uniformly well stainedsmears of intestinal protozoa, human cells, yeastcells, and artifactual material in approximately45 min or less (Chapin and Murray 1999, Wheatley1951).
Summary
Microscopic examination of stained smears in theclinical microbiology laboratory still provides themost cost-effective method of detecting manypathogens and judging specimen quality despitethe development of rapid detection systems andmolecular diagnostic probes. The stains used inclinical microbiology are relatively simple to per-form, but the complexity of the procedures residesin microscopic examination, differentiating speci®corganisms, evaluating the staining reactions, anddetermining the signi®cance of what is detected bythe microscopist. These issues are easier for theclinical microbiologist with microscopy experienceand knowledge of the various disease entities(Chapin 1995).
Acknowledgments
Special thanks to Drs. Ron Doyle and BeverlyMetchock for reviewing the manuscript. Photo-graphs from Public Health Image Library, Centersfor Disease Control and Prevention.
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