On the first day, the vegetative bacteria would die and those spores that germinate by next day are then killed the following day. The process depends on germination of spores in between inspissation. If the spores fail to germinate then this technique cannot be considered sterilization. At temperature 100o C: ƒ Boiling: Boiling water (100o C) kills most vegetative bacteria and viruses immediately. Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not required, certain metal articles and glasswares can be disinfected by placing them in boiling water for 10-20 minutes. The lid of the boiler must not be opened during the period. ƒ Steam at 100o C: Instead of keeping the articles in boiling water, they are subjected to free steam at 100o C. Traditionally Arnold’s and Koch’s steamers were used.
An autoclave (with discharge tap open) can also serve the same purpose. A steamer is a metal cabinet with perforated trays to hold the articles and a conical lid. The bottom of steamer is filled with water and heated. The steam that is generated sterilizes the articles when exposed for a period of 90 minutes. Media such as TCBS, DCA and selenite broth are sterilized by steaming. Sugar and gelatin in medium may get decomposed on autoclaving, hence they are exposed to free steaming for 20 minutes for three successive days. This process is known as tyndallisation (after John Tyndall) or fractional sterilization or intermittent sterilization. The vegetative bacteria are killed in the first exposure and the spores that germinate by next day are killed in subsequent days.