There are a number of folks who do not have easy access to a pressure cooker or autoclave, or who are unwilling to learn to operate one. If it were not for this, these people might otherwise enjoy a hobby involving aseptic agar culture. This section was written with those people in mind. Understand please that the methods detailed here are not superior to the regular sterilization methods involving pressure cookers. In many respects they are inferior. If cost is a factor in obtaining a pressure cooker, you would do well to check out discount stores, second hand stores, and estate auctions. If fear of the pressure cooker is a factor, you should work on over coming this fear. For those people still in need of methods of sterilization not involving a pressure cooker, read on.
The methods detailed here were once in common usage amongst microbiologists, although some of them were used so long ago that these people were not known as microbiologists! In many cases these methods are still employeed in very specialized circumstances by modern microbiologists. In the mid 1800's J. Tyndall was working with beef broth infusions of bacteria and decided to try a hay infusion. In the meat broths he was always able to achieve sterile solutions with a 5 minute boil of the broth. Wonder of wonders, the hay infusion did not sterilize after 5 minutes of boiling. In fact, it was not sterilized after 5 hours of boiling! Only after 5 1/2 hours of boiling did he stand a decent chance of achieving a sterile infusion! He eventually went back to meat broths only to find that he now needed to boil these for 5 1/2 hours as well! He had discovered endospore forming bacteria. Meat broths tend to be invaded by bacteria which do not form endospores and are thus easily killed by a short boil. Hay however is in close contact with the soil throughout the summer as it grows. A whole host of Bacillus bacteria live in the soil, and many of them form endospores. Thus his hay infusions were rife with heat tolerant Bacillus endospores which would not die until after at least 5 1/2 hours, and often more, exposure to temperatures around 100 degrees Celcius. Fortunately today we have autoclaves and pressure cookers which achieve higher temperatures and greatly reduce the needed heating time. Unfortunately for Tyndall, the autoclave was not then available. He experimented and eventually came up with a more effective method for sterilizing his broth media.
Tyndallization is a process by which relatively short boils at regular atmospheric pressure may render a media sterile. The process is relatively simple and straight forward, and revolves around the fact that most endospore forming micro-organisms germinate from their endospores in 12 to 24 hours in optimal conditions. A solution to be sterilized is placed in a suitable container and heated to boiling. Remember no tightly closed glass containers! If you must use a bottle of some sorts, remember to plug the bottle with something along the lines of cotton, capped with foil. This will allow air to escape while preventing contaminants from entering the container as it cools. Boil the fluid for 10-15 minutes, then allow it to cool to room temperature naturally. Allow this to sit at room temperature for about 24 hours, +/- only 2 hours. This will allow the endospores to recognize the media as an optimal enviroment and begin growing. Much less time will not allow the slower spores to germinate, much more time will allow too much spoilage to begin, and may actually increase the number of endospores present! After the 24 hours of incubation at room temperature, bring the fluid to a boil and allow it to boil for 10-15 minutes. This will not kill all the endospores however. Allow the solution to incubate at room temperature for another 24 hours and then repeat the boil again. After the 4th such boil there is a good chance that proper sterilization has occurred, but you may wish to attempt a 5th boil just to be certain. The reason for the multiple boils is that not all of the endospores will germinate at once, some will hold over only to begin spoilage at a later date. By givingthem several chances to germinate, there is a greater chance of achieving sterilization by the Tyndallization method. Thus sterilization can be achieved at boiling water temperatures in 1 to 1 1/4 hours rather than 5 1/2 hours, which would destroy most agar medias.
Benefits of this method include passable sterilization without a pressure cooker or autoclave, without an excessively long boil, and without resorting to a higher temperature which might damage certain exotic media formulations. The disadvantages include the fact that the media has a much greater chance of becoming dehydrated than if it were pressure cooked, a slightly greater chance of incomplete sterilization, and the fact that it takes nearly a week before the media is available for culture.
A method by which glassware and even paper can be sterilized is by use of an electric oven. If the oven is set to between 320 and 340 degrees Farenheit (160-170 Celcius) sterilization of glass and paper will occur in approximately 2 to 3 hours. Paper chars at 356 F (180 C) and this is excessively high for sterilization, so these temperatures should be avoided. A meat thermometer placed on the same rack as the materials to be sterilizaed will allow the accuracy of the oven thermostat to be double checked. It is relatively common for modern ovens to be off by 10 to 20 degrees and more.
The advantage to this method of sterilizing equipment is that an autoclave is not needed, paper can be sterilized without getting wet, and glass utenzils and petri dishes are not at risk to be etched by steam. The disadvantages involve the longer length of time for sterilization, a slightly greater chance of incomplete sterilization, and the necessity of maintaining the oven within a narrow temperature range.
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