Often a heat-killed version of the bacteria is used, to contrast with live bacteria in infection assays. There are various different conditions being used in the literature, ranging from 50°C to 95°C, and various time duration of heating. What conditions do you think are reasonable and why is this the case? (Any good references?) thank you for comments.
if you use too high temperature you'll denature the tertiary structure of the epitopes and your sample will be useless as a vaccine. The temperature to be used will depend on the organism, but I would be careful with going above 65 Centigrade. I'm not saying not to do it, just to be careful.
10-15 min incubation at about 60-65 Centigrate should do the trick, but you'll need to test for viability and immune response and optimise the conditions to your particular needs.
I agree with Adam, about not boiling the sample, unless you want the cytoplasmic components for the seological analysis. If you are planning to work with pathogens (Bio Safety level 2 and 3) and would like to use the inactivated samples under Bio Safety level 1 conditions, then make sure the organism is inactivated/killed by plating. This is very important as time for inactivation varies dependent upon the organism, and the concentration of the organism in the solution. This will become even more important if it is a spore former. So unless you tell us what do you mean by infection assay? this is difficult to answer.
Thank you both very much for the comments. I agree that different bacteria and the nature of subsequent experimental design should be taken into account, in deciding on what heat killing conditions should be chosen (e.g. safety versus preserving important components).
>So unless you tell us what do you mean by infection assay?
I perform mammalian cell line based infection assays, in which a known cell density of host cells (mouse macrophages) are matched with a certain concentration of bacteria (live or heat killed) -- at pre-determined MOIs.
Then after some time, I measure the amounts of ROS/RNS (reactive oxygen/nitrogen species) induced by the bacteria. Of course, for heat-killed bacteria, it is merely a challenge, and not exactly infection.
Thank you!
Then I would not boil the samples, but heat for 65C for a length of time that kills the organism. You could use Alamar Blue if you want to use inactivated cells but still metabolically active. indicated by No growth on plating, but turns Alamar blue to red or pink with incubation of inactivated cells.
Thank you very much for your valuable inputs, Prof. Nammalwar Sriranganathan! It's very helpful.
What is the justification of using a heat-killed bacteria in any experiment?
This response may come a bit late but the purpose to heat kill any cell type / bacteria is to generate a positive control for dead-cell staining or on the other hand, negative control for any experiments requiring live, actively-respiring cells. Heat treatment of bacteria is also used to create bioparticles for endocytosis studies.
In some cases we need to use the inactivated bacteria. For example, when the purpose of test is to assay the anti-cancer or cancer-promoting effects of bacteria in MTT assay, we can not add the active bacteria solution to cancer cell line. Because active bacteria can definitely kill all cancer cells, like what contamination do. So before doing the test, it is necessary to inactive the bacteria.
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