If bacteria or fungi are seen on microscopic examination, appropriate culture media should be inoculated. Independently from the results of microscopy, all specimens of pus or exudate should preferably be inoculated on to a minimum of three culture media:
• a blood agar plate for the isolation of staphylococci and streptococci;
• a MacConkey agar plate for the isolation of Gram-negative rods; and
• a tube of broth that can serve as enrichment medium for both aerobes and anaerobes, e.g., thioglycollate broth or cooked meat medium.
The size of the inoculum should be determined according to the result of the microscopic examination, and may vary from one loopful to a few drops. If massive numbers of organisms are seen on the Gram-stained smear, the specimen may even have to be diluted in a small amount of sterile broth before plating out. If a swab is used for the inoculation, it should be applied to a small area of the plate and the rest of the surface streaked out with a loop. If the swab is dry, it should first be moistened in a small quantity of sterile broth or saline. In all cases, the technique of inoculation should provide single colonies for identification and susceptibility tests.
Prior to inoculation, the blood agar plate should be dried for 20 minutes in an incubator, to minimize the risk of overgrowth by spreading Proteus spp. The inoculated plate should be incubated at 35 °C in a candle jar. Routinely, all media should be incubated for two days and inspected daily for growth. If culture for fastidious organisms is requested, longer incubation (1-2 weeks or more) will be necessary. If growth appears in the broth, it should be Gram-stained and subcultured on appropriate culture media. Additional culture media should be used if specially requested, or if indicated by the results of the microscopic examination, as in the following examples.
• If staphylococci have been seen, an additional mannitol salt agar is helpful in obtaining pure growth and in making a preliminary distinction between S. aureus and other cocci.
• If streptococci have been observed, their identification may be hastened by placing a differential bacitracin disc on the initial streaking area.
• If yeasts or fungi have been observed, the specimen should also be inoculated on to two tubes of Sabouraud dextrose agar, one to be incubated at 35 °C, the other at room temperature, both to be observed during one month. (Blood agar is sufficient for the isolation of Candida spp.)
• If acid-fast rods have been seen in the Ziehl-Neelsen stained smear, up to 3 tubes of Löwenstein-Jensen medium should be inoculated. If the specimen also contains non-acid-fast bacteria, it should first be decontaminated. Rapidly growing mycobacteria, such as M. fortuitum, may be killed by the decontamination process; they produce growth within 3 - 7 days on blood agar and MacConkey agar. Branched, filamentous, partially acid-fast rods in pus from the pleura or from a brain abscess will probably be Nocardia asteroides, which grows on blood agar within a few days.
• Pus from patients with arthritis, pleuritis, osteitis, or cellulitis, particularly from children under 5 years of age, should also be inoculated on to a chocolate agar plate for the recovery of H. influenzae.
• Culture in a strictly anaerobic atmosphere is essential when the Gram-stained smear shows “anaerobic mixed flora” and also when the specimen produces a typical foul odour. Anaerobic blood agar is also necessary for the growth of Actinomyces. Anaerobic culture will be requested by the clinician when he or she suspects clostridial gas gangrene. Methods for anaerobic bacteriology are described on” Anaerobic bacteriology”.