D. Presumptive confirmation test
Select 10 typical C. perfringens colonies from TSC or TSC-egg yolk agar plates and
inoculate each into a tube of freshly deaerated and cooled fluid thioglycollate broth.Incubate in standard incubator 18-24 h at 35°C. Examine each culture by Gram stain and check for purity. C. perfringens is a short, thick, Gram-positive bacillus. If there is evidence of contamination, streak contaminated culture(s) on TSC agar containing egg yolk and incubate in anaerobic jar 24 h at 35°C. Surface colonies of C. perfringens are yellowish gray with 2-4 mm opaque zones caused by lecithinase activity. This procedure is also used for isolating C. perfringens from chopped liver broth whenever the organism is not detected by direct plating on TSC agar.
Iron-milk presumptive test. Inoculate modified iron-milk medium with 1 ml of actively growing fluid thioglycollate culture and incubate medium at 46°C in a water bath. After 2 h, check hourly for "stormy fermentation." This reaction is characterized by rapid coagulation of milk followed by fracturing of curd into spongy mass which usually rises above medium surface. Remove positive tubes to prevent spilling over into water bath. For this reason, do not use short tubes for the test. Cultures that fail to exhibit "stormy fermentation" within 5 h are unlikely to be C. perfringens. An occasional strain may require 6 h or more, but this is a questionable result that should be confirmed by further testing. Some strains of C. baratii react in this manner, but this species can be differentiated by its inability to liquefy gelatin in lactose-gelatin medium. The rapidity with which the "stormy fermentation" occurs depends on the strain and the initial population. Therefore, only actively growing cultures are appropriate for this test. The presumptive test in iron-milk medium may be sufficient for some purposes. However, the completed test must always be performed with isolates associated with food poisoning outbreaks. The following tests must be included for the completed test.