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.
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.
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.