Hey there,

You want to work with spores of which organism?

Hi, to start we will work with B. atrophaeus and B. sterothermophilus, and in a second stage will continue with C. diffile or C. perfringes

Excellent!

At first, I will try to give you general explanations, and then go a little bit more in details as an example of how I would proceed for C. difficile, since it is the bacterium I am usually working with.

First of all, I would mention that you will surely have to prepare specific culture media for every different organisms.For each organism, you would have to prepare media with specific purposes:

1) A medium for growing vegetative cells (prior to make them sporulate)

2) A medium suited for the sporulation (where you deposit you vegetative cells)

3) A medium for germination of the spores into observable vegetative cells (so you can visually count the spores after you completed your sporicidal activity test)

Now, i will tell you a bit more in details what I would do I I want to test the sporicidal activity of a given product on C. difficile spores. Before giving you those recommendations, I would assume that you have an anaerobic chamber where you could grow and manipulate C. difficile!

1) First step, I would grow C. difficile vegetative cells on solid BHI-Agar petris.

2) Second step, spread those vegetative cells on TY-Agar petris, where they will sporulate.

3) Recuperate as much spores as possible (by adding PBS and scraping the petris).

4) Do a purification of the spores by making an Ethanol shock: Incubation of the collected spores and residual bacteria from step 3. For that, you add to your collected cells a mix of 50% Ethanol/H20, for 1 hour. (this is because we know that this mix kills all vegetative cells, but let the spores intact). When the Ethanol shock is over, you resuspend them in PBS.

5) Count the amount of spores you have. (Spores/mL). You have to plate different dilutions of the cleaned preparation you just got, on a a medium that allows spores to germinate back into vegetative cells, and allows you to count colonies (CFU). For C. difficile, this medium is called BHI-TAG-Agar.

6) When you know your quantity of spores/mL in you preparation, I would proceed with the sporicidal test, with a precise amount of spores to incubate for a given amount of time. I would include a negative control where you incubate you spores with PBS, and a positive control where you would incubate your spores with a solution of Hydrogen Peroxyde (know to kill C. difficile spores), for example.

7) After the incubations of your spores with your product are done, you would count the surviving spores in each samples, using the same medium for spores germination (BHI-TAG-Agar).

8) Do the ratio calculations : Amount of spores that survived the incubation/ amount of spores counted before the incubation.

So this is how I would proceed with C. difficile, I would use same general steps for other organisms, taking into consideration that other organisms have their specific media for vegetative cell growth, for spore formation, and for spore germination. If working with other organisms, we surely would have to verify if spores are stable in a PBS solution for conservation, and if the 50% ethanol/H2O used to kill vegetative cells in the purifying step is also working properly with other bacteria.

If you are wondering what is the exact composition of each media I mentionned here, you can go in our article to get more details.In this article, we also explain some of the stuff I just told you about spore production, spore recovery and count, vegetative cell production and count, etc.

https://www.researchgate.net/publication/259723569_Prevention_of_Clostridium_difficile_spore_formation_by_sub-inhibitory_concentrations_of_tigecycline_and_piperacillintazobactam

Hope this can help you, if you have any other question, just let me know!

Article Prevention of Clostridium difficile spore formation by sub-i...

Dear Juan

This manuscript may help you

Validation of peracetic acid as a sporicide for sterilization of working

surfaces in biological safety cabinets

http://www.jmidonline.org/upload/sayi/9/JMID-00604.pdf