We are examining some Bacillus species that are endophytic in plants. These bacteria are resistant to isolation. We think that they are resistant because the endospores are dormant? Are there cultural additives that are known to break dormancy? Are there shock treatments that may work to break endospore dormancy? Any ideas will be appreciated.
One of the recognized methods to break the dormancy of Bacillus endospores is through autoclaving them! simple and very effective!
ACTIVATION OF BACTERIAL ENDOSPORES
A. KEYNAN, Z. EVENCHIK, H. 0. HALVORSON, AND J. W. HASTINGS
J. Bacteriology 88: 313-318.
The classical method is to expose them to 65 C for 45 Min, 34 C for 48 h works as well. Or, suspend spores in reducing agent (see reference above)
Hi James, I think following paper will be useful to you!
http://jb.asm.org/content/184/5/1296.full
The requirements for germination are generally species-specific. For Bacillus cereus two of the known germinants are L-alanine and inosine. The combination of the two provides a faster germination. Another purine ribonucleoside, the adenosine, has also been described as a germinant for B. cereus. The ability of ribonucleosides to induce germination is most likely related to the structural properties of the sugar and not of the nucleoside. For strain ATCC14579, among the 20 amino-acids tested only L-cystein, L-alanine, L-threonin and L-glutamine were able to induce germination. Moreover, the germination rate depends not only on the nature and concentration of germinants but also of the temperature and pH and varies from one strain to another. In B. cereus germination can also be induced by chemical agents such as a mixture of dipicolinic acid and CaCl2 or by high pressure (100 to 200 MPa).
Thank you for the suggestions. It seems like heat treatments and chemical inducers (like amino acids) may be used. We'll experiment with some of these methods. Thank you very much for the suggestions. Jim White, Rutgers University
Pre-culture in a rich culture broth like BHI (brain heart infusion) broth.
You can incubate the spore suspension at 80 degree in a waterbath for 20min and then plate the suspension on isolation media. This will not get rid of other vegatative, nonsporulating isolates, but also break the spore dormancy and allow the spores to germinate in fresh media.
Just found this question - but here goes anyway for whoever might be doing something similar. [If anyone here works with endospores I would appreciate if they could also provide an opinion on my questions about the terminology of endospore vs spore, see url below]
The answers on heat treatment are all useful, and it the endospores will then germinate if put in/on a good nutrient source. But please do NOT autoclave, the purpose of the autoclave (121 degrees centigrade) is to kill endospores, as most vegetative bacteria are killed by boiling or even lower temperatures. The heat treatment also does also act as a selective factor because it kills the vegetative bacteria that could interfere with isolation of your bacillus.
https://www.researchgate.net/post/Should_the_term_Endospore_always_be_used_preferentially_to_spore
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