Freezing bacteria
- Thread starter light-G
- Start date
Well depends, if not protected e.g. by adding glycerine ice crystals may form and the membrane can be disrupted (esp. by repreated thawing and freezing). To determine the reduced viability simply count the cfus (colony forming units) before and after freezing.
To see reduction in membrane fluidity however needs a full blown lab
But before toying around with bacteria, be sure you follow appropriate safety procedures.
To see reduction in membrane fluidity however needs a full blown lab
But before toying around with bacteria, be sure you follow appropriate safety procedures.
light-G said:
The effects are the same as for freezing any other type of cell – the ice crystals form inside the cells that can rupture the cell membrane and kill the cells. A number of chemicals act as cryopreservants that minimize this damage, such as glycerol, DMSO or sucrose. But for bacteria this is not nearly as important an issue as it is for eukaryotic cells for a number of reasons…..
- bacteria have a strong cell wall
- bacteria don’t have numerous internal organelles that can be damaged
- bacteria are unicellular organisms and, as such, are much better equipped to deal with extremes of temperature than, for instance, a weak differentiated specialized animal cell.
- many species of bacteria can form spores that are extremely resistant to temperature extremes
- bacteria have the advantage of huge colony numbers and fast cell division rates, so even if, say, 90% of all the cells die during a freeze-thaw cycle, the colony needs only a single survivor to re-establish itself once optimal conditions return.<P>
light-G said:
Apart from performing the actual experiment, you can point to the fact that the freezing of bacterial stocks at -80<sup>O</sup>C is standard laboratory practice. With some Googling you will be able to find no end of simple freezing protocols.<P>
light-G said:
Are you in a position to perform such experiments?
A simple freezing solution of Tris, MgCl<sub>2</sub>, NaCl and glycerol can be used. You merely mix a 1:1 ratio of bacterial culture with the freezing solution (500ul total) and put the eppindorfs into the freezer. Using log-phase cells works best. Unfreezing and plating out serial dilutions of the frozen stocks will determine the survival rate.
Proper methodology will require you to control for numerous different variables.....
- bacterial species (Gram + versus Gram –)
- different cryopreservants
- different temps
- different salt concentrations
etc etc <P>
If you are going to perform an experiment on freezing bacteria, then the species of that bacteria is probably the most influential variable. There are some species of bacteria that only live in extreme environments, such as boiling water and ice. So, freezing some bacteria may not have an effect, possibly only putting them into a dormant state--which has been shown many times. hope this helps.
a_nabacus said:
Yes, that’s true. But let’s face it – the poster gives the distinct impression of a high school student. Even professional microbiologists in fully equipped laboratories can find it difficult to culture ‘extremophile’ bacteria. A high school science laboratory has no chance in hell of doing it, let alone obtaining such bacteria in the first place.
If this person is going to actually perform this experiment, then we’re talking about lab strains of <I>E.coli</I> that can be grown in simple LB medium and can be frozen at -80<sup>o</sup>C, or even in a standard -20<sup>o</sup>C freezer.<P>