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Diagnostic Microbiology 170-1 Provides an Overview of the Most Commonly Used Stains

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Diagnostic Microbiology 170-1 Provides an Overview of the Most Commonly Used Stains PART Infectious Diseases XVII Section cells is used to judge the suitability of a specimen for culture. For 1 example, the presence of more than 10 epithelial cells per low-power field in a sputum specimen is highly suggestive of a specimen contami- General Considerations nated with oral secretions. In addition, a preliminary assessment of the etiologic agent can be made based upon the morphology (e.g., cocci vs rods) and stain reaction (e.g., Gram-positive isolates are purple; Gram- negative are red) of the microorganisms. However, a negative Gram stain does not rule out infection as 104 to 105 microorganisms per mL Chapter 170 in the specimen are required for detection by this method. In addition to the Gram stain, many other stains are used in micro- biology, both to detect organisms and to help infer their identity. Table Diagnostic Microbiology 170-1 provides an overview of the most commonly used stains. Carey-Ann D. Burnham and Isolation and Identification Gregory A. Storch The approach to isolation of microorganisms in a clinical specimen will vary depending on the body site and pathogen suspected. For body sites that are usually sterile, such as cerebrospinal fluid, nutrient-rich media such as sheep blood agar and chocolate agar are used to aid in Laboratory evidence to support the diagnosis of an infectious disease the recovery of fastidious pathogens. In contrast, stool specimens may be based on 1 or more of the following: direct examination of contain abundant amounts of commensal bacteria and thus to isolate specimens using microscopic or antigen detection techniques, isola- pathogens, selective and differential media must be used. Selective tion of microorganisms in culture, serologic testing, host gene expres- media will inhibit the growth of some organisms to aid in isolation of sion patterns, and molecular detection of an organism, resistance suspect pathogens; differential media rely on growth characteristics determinant, or virulence factor. Additional important roles of the or carbohydrate assimilation characteristics to impart a growth pattern diagnostic microbiology lab are antimicrobial susceptibility testing to that differentiates organisms. MacConkey agar supports growth of guide in the selection of the most appropriate antimicrobial therapy Gram-negative rods while suppressing Gram-positive organisms, and and supporting hospital infection prevention in the detection and a color change in the media from clear to pink distinguishes lactose- characterization of pathogens associated with nosocomial infections. fermenting organisms from other Gram-negative rods. Special media, such as Sabouraud dextrose agar and inhibitory mold agar, are used to SPECIMEN COLLECTION recover fungi in clinical specimens. Many pathogens, including Bar- The success of microbiology cultures, that is, isolation of a pathogen if tonella, Bordetella pertussis, Legionella, Mycoplasma, and certain fungal present, is directly linked to specimen collection techniques. In pathogens such as Malassezia furfur, require specialized growth media general, this means collecting the correct specimen type for the or incubation conditions. Consultation with the laboratory is advised disease or condition in question and promptly transporting the speci- when these pathogens are suspected. men to the laboratory for analysis. Although for some conditions Once an organism is recovered in culture, additional testing will be swab specimens may be necessary, in general, a swab is a suboptimal performed to identify the isolate. Confirmation of microbial identity specimen. A swab is only able to hold a very small amount of speci- has classically been performed using phenotypic tests that rely on the men (approximately 100 µL), and using a traditional swab, only a phenotypic properties of an isolate. Some examples include carbohy- small fraction of organisms that are absorbed onto a swab will be drate assimilation patterns, indole production, and motility. However, released back into the culture. Flocked swabs, coupled with transport these methods are not able to resolve all organisms to species level and medium, improve organism recovery. However, when possible, fluid require incubation time. In some instances, sequence based identifica- or tissue should be submitted to the laboratory for analysis. If anaero- tion, for bacteria usually based on sequence analysis of the bacterial bic infection is suspected, the sample should be transported in appro- 16S rRNA gene, is used for organism identification (particularly priate medium to preserve viability of anaerobic bacteria. For the organisms that are difficult to culture). recovery of some organism types, such as viruses and Neisseria gonor- Matrix-assisted laser desorption-ionization time-of-flight mass spec- rhoeae, specific transport media may be required. Considerations spe- trometry (MALDI-TOF MS) is a rapid and accurate technique that is cific to the collection of blood cultures will be addressed in the blood based on generating a protein fingerprint of an organism and compar- culture section. ing that fingerprint to a library of known organisms to produce an identification. This method can identify bacteria or yeast growing in LABORATORY DIAGNOSIS OF BACTERIAL culture in a matter of minutes, and the consumable costs for these AND FUNGAL INFECTIONS analyses are minimal. Although the scope and availability of molecular methods for detection of bacterial and fungal pathogens is increasing at a rapid pace, the BloodCulture state-of-the art for the diagnosis of many of these infections is depen- The performance of blood cultures is one of the most important func- dent upon microscopic detection of organisms or cultivation of organ- tions of the clinical microbiology laboratory. Most blood cultures are isms on culture media. performed by collecting blood into bottles of nutrient-rich broth to facilitate the growth of bacteria and yeast. Some blood culture media Microscopy contain resins or other agents to help neutralize antibiotics that may The Gram stain is an extremely valuable diagnostic technique to be present in the patients’ blood. Blood culture bottles are then incu- provide rapid and inexpensive information regarding the absence or bated on an automated blood culture incubator that will monitor the presence of inflammatory cells and organisms in clinical specimens. blood culture bottle at regular intervals for evidence of growth. Once For some specimen types, the presence of inflammatory and epithelial the instrument detects evidence of microbial growth, it will alarm to 1231 Downloaded for dr.Rahmat Dani Satria, M.Sc, Sp.PK ([email protected]) at Universitas Gadjah Mada from ClinicalKey.com by Elsevier on July 28, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. PartXVII ◆ Infectious Diseases 1232 Table170-1 Stains Used for Microscopic Examination TYPE OF STAIN CLINICAL USE Gram stain Stains bacteria (with differentiation of Gram-positive and Gram-negative organisms), fungi, leukocytes, and epithelial cells Potassium hydroxide (KOH) A 10% solution dissolves cellular and organic debris and facilitates detection of fungal elements in clinical specimens Calcofluor white stain Nonspecific fluorochrome that binds to cellulose and chitin in fungal cell walls, can be combined with 10% KOH to dissolve cellular material Ziehl-Neelsen and Kinyoun stains Acid-fast stains, using basic carbolfuchsin, followed by acid–alcohol decolorization and methylene blue counterstaining Acid-fast organisms (e.g., Mycobacterium) resist decolorization and stain pink A weaker decolorizing agent is used for partially acid-fast organisms (e.g., Nocardia, Cryptosporidium, Cyclospora, Isospora) Auramine-rhodamine stain Acid-fast stain using fluorochromes that bind to mycolic acid in mycobacterial cell walls and resist acid–alcohol decolorization; usually performed directly on clinical specimens Acid-fast organisms stain orange-yellow against a black background Acridine orange stain Fluorescent dye that intercalates into DNA, used to aid in differentiation of organisms from debris during direct specimen examination, and also for detection of organisms that are not visible with Gram stain Bacteria and fungi stain orange, and background cellular material stains green Lugol iodine stain Added to wet preparations of fecal specimens for ova and parasites to enhance contrast of the internal structures (nuclei, glycogen vacuoles) Wright and Giemsa stains Primarily for detecting blood parasites (Plasmodium, Babesia, and Leishmania), detection of amoeba in preparations of cerebral spinal fluid, and fungi in tissues (yeasts, Histoplasma) Trichrome stain Stains stool specimens for identification of protozoa Direct fluorescent-antibody stain Used for direct detection of a variety of organisms in clinical specimens by using specific fluorescein- labeled antibodies (e.g., Pneumocystis jiroveci, many viruses) alert the laboratory. Approximately 80% of blood cultures that will There are a number of rapid diagnostic assays that can be used ultimately be positive are identified within the 1st 24 hr of incubation. directly on positive blood culture broth to identify pathogens com- A portion of broth from a blood culture bottle that has signaled posi- monly associated with bacteremia and some antimicrobial resistance tive is then Gram-stained and cultured onto appropriate growth media determinants. Most of these rapid diagnostic assays are based on so that the organism
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