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Freeze-Thaw Cell Disruption of Bacillus spp.

Repeated freezing and thawing of bacterial cells disrupts them because of the repeated formation of sharp ice crystals - many beginning to grow in the inside of the cells. Sort of like burst in balloon by using needles from the inside!

1. THE important thing is to apply this technique to GROWING (a present progressive word!) bacteria. "Growing" means more than being merely "alive." Spores and seeds can be alive, but not growing. For a bacterial culture, this means its turbidity is in the process of increasing - it is more than it was a minute ago, and less than it will be a minute from now. An overnight culture that is now turbid is probably NOT what you want as it has probably grown and reached stationary phase. You MUST have actively growing cells. Otherwise, your Bacillus will be in a toughed or sporulated state. How to easily do this:
    
   1. For each final culture you want, make up two flasks of sterile growth medium. Inoculate one of them, but incubate both - so that the sterile one is at the same temperature.
   2. When you see turbidity in the first flask, transfer a drop to the sterile flask, and continue incubating that second flask. At first you will not see any turbidity in it. (Yes, I know that students like macho, highly turbid cultures, but even a million cells per milliliter is far more than you need. Lower numbers mean you need make less dilution later.
   3. As soon as you can first detect turbidity (about a million cells per milliliter), add ice to quickly chill the culture and instantly stop its growth. To chill a 37°c culture to 4°c add ice that weighs 40% as much as the culture liquid. (E.g.: if you had a 100 ml culture, add ice until the flask becomes 40 grams heavier.) / Obviously, warmer cultures will need more ice, and cooler ones less. BUT extra ice won't hurt. Once the culture is at 4°c, scoop out the extra ice.
   
    
2. Now you have a chilled culture of bacteria "stopped" in the midst of rapid growth. None should be sporulated. How do you prepare this for freezing and thawing?
    
   1. Distribute one milliliter aliquotes into small PLASTIC tubes (pre-labelled 0 through 10) such as microfuge tubes (glass will crack under the expansive pressures of water's freezing).
   2. Put tube "0" into an ice bath, and put that into the refrigerator to slow the ice's melting. This tube is your control - it will never be frozen.
   3. Put all the other tubes in the freezer and allow them to freeze.
   4. Once all the tubes are frozen, take out 2 through 10 and allow them to thaw (but not stand around all day, and don't thaw in hot water!
   5. Once thawed, put 2-10 back in the freezer to freeze.
   6. Once all are frozen, thaw 3-10
   7. Keep going around and around until you have only #10 thawed and put that back in the freezer. (Up to this point #10 has been frozen ten times, but only thawed nine times.)
   
    
3. You are now ready to count the numbers of surviving - ahem! "living" - bacteria in each tube. This is done by a method called "plate counts." Any live bacterium that gets to the agar surface will start growing and proliferating and make a colony. Any dead bacterium does nothing. Thus a living bacterium has another name - a colony-forming unit, or a "cfu." In short, you want to determine the concentrations of cfu's in each of the tubes sitting in your freezer.
    
4. How to do plate counts: see "Plate Counts" (Or go the home page and click on the Site Index logo to the left of the title. Then look up "plate counts.")

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