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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
close this folderPurulent exudates, wounds, and abscesses
View the documentIntroduction
View the documentCommonly encountered clinical conditions and the most frequent etiological agents
View the documentCollection and transportation of specimens
View the documentMacroscopic evaluation
View the documentMicroscopic examination
View the documentCulture
View the documentIdentification
View the documentSusceptibility testing
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover

Microscopic examination

A smear for Gram-staining and examination should be made for every specimen. In particular cases, or at the request of the clinician, a direct wet mount may be prepared and Ziehl-Neelsen staining carried out.

Gram-stained smear

Using a bacteriological loop, make an even smear of the most purulent part of the specimen on a clean slide. If only a swab is available, the slide should first be sterilized by being passed through the flame of a Bunsen burner and allowed to cool. The cotton swab should then be gently rolled over the glass surface, without rubbing or excessive pressure. Allow the slide to air-dry, protected from insects, or in the incubator. Fix by heat, stain and examine the smear under the oil-immersion objective (X 100). Inspect carefully and note the presence and the quantity (use + signs) of:


• polymorphonuclear granulocytes (pus cells);

• Gram-positive cocci arranged in clusters, suggestive of staphylococci;

• Gram-positive cocci in chains, suggestive of streptococci;

• Gram-negative rods resembling coliforms (Escherichia coli, Klebsiella, etc), other Enterobacteriaceae (Proteus, Serratia, etc.), nonfermentative rods (Pseudomonas spp), or obligate anaerobes (Bacteroides spp);

• large straight Gram-positive rods with square ends suggestive of Clostridium perfringens, the principal agent of gas gangrene, or Bacillus anthracis, the agent of anthrax;

• an extremely heavy and pleomorphic mixture of bacteria, including streptococci, Gram-positive and Gram-negative rods of various sizes including fusiform rods; such a picture is suggestive of an “anaerobic mixed flora” and should be reported as such;

Candida or other yeast cells, which are seen as ovoid Gram-positive budding spheres, often forming branched pseudomycelia;

Sulfur granules from actinomycosis or granules from a mycetoma should be crushed on a slide, Gram-stained and inspected for thin branched and fragmented Gram-positive filaments.

Direct microscopy

When requested, or when a fungal or parasitic infection is suspected, a wet preparation should be examined. If the pus is thick, a loopful should be mixed in a drop of saline. When looking for fungi, a drop of 10% potassium hydroxide should be used to clear the specimen. Apply a cover-slip and, using the X 10 and X 40 objectives, look especially for:


• actively motile amoebae in aspirate from a liver abscess;

• yeast cells of Histoplasma capsulatum var. duboisii, Blastomyces dermatitidis (in endemic areas), Candida spp;

• fungal hyphae and bacterial filaments in crushed granules from mycetoma,

• parasites (in endemic countries), such as microfilariae, scolices or hooks of Echinococcus, eggs of Schistosoma, Fasciola, or Paragonimus.

Acid-fast staining (Ziehl-Neelsen)

Ziehl-Neelsen staining should be performed when requested by the clinician. It is also advisable to make an acid-fast stained preparation when the pus shows no bacteria or when only faintly stained Gram-positive “coryneform” rods are seen on the Gram-stained smear. Tubercle bacilli should be suspected, in particular, in pus or purulent exudate from the pleura, joints, bone abscesses, or lymph nodes. Nontuberculous (so-called “atypical”) acid-fast bacilli are sometimes found in gluteal abscesses at the site of deep intramuscular injections. Such abscesses are often caused by rapidly growing mycobacteria belonging to the Mycobacterium fortuitum-chelonei group. In the tropics, discharge scraped from the base of a necrotic skin ulcer situated on a leg or an arm may be due to slow-growing acid-fast rods called M. ulcerans (Buruli ulcer). M. marinum is another slow-growing non-tuberculous acid-fast rod, which may be found in chronic, ulcerative, nodular lesions on the hands, arms, and other exposed skin surfaces in swimmers and fishermen.

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