Stepping up or maintaining a culture

[Idontknow]

I am in Japan and don't have easy or cheap access to cultures.
It requires having a friend who is travelling overseas to pick up and bring back in their suitcase. After the long wait for the cultures, I of course must repay the person with gifts etc so it ends up adding another cost. Considering the high cost of quality meat here, I do want to try to be thrifty where I can while making it more convenient for myself to have cultures on hand when I need them.

I was thinking about propagating and making some slants, plates, or dried cultures.
Has anyone done this before? I have done this with beer yeasts, washing a yeast cake and keeping it in the fridge. Adding glycerin can help keep a yeast viable for freezing.
I also have a dehydrator so perhaps I can dry a culture before freezing.

What would you recommend if you were to try any methods of increasing your supply of a starter culture? What medium would you use to propagate the culture?

I was thinking about gelatin with some sugar added. I can use a pressure cooker to autoclave that first. Then I would streak the plate or slants with freeze dried culture straight from the packets that I have. Just to be absolutely clear: I would not be using a culture derived from anything that was in contact with my meat projects (backslopping). The culture would be straight from the manufacturer's packaging using aseptic handling.

I also have Erlenmeyer flasks and a magnetic stirplate. I use that to increase my starter culture for brewing beer. What medium would you suggest for that? I could heat it passively by running the entire thing inside my curing chamber, as my stir plate doesn't have heating functionality.

After stepping-up a culture, I could either freeze it or dry it. Which do you think would be the best or most reliable method? Dry and then freeze, freeze, add glycerin and freeze?

I have access to Agar, Glycerin, Gelatin, Various Starches and Sugars.
Are there any people here that have done this or similar culturing methods?
I've done some basic microbiology courses but have not done anything with meat cultures so I am open to suggestions from any bacteria wranglers here.
Thanks and cheers!

 

[mike243]

Sorry,I aint no help, like my beer, food and women germ free

 

[daveomak]

Cultures are grown using the strain of bacteria native to the area folks are trying to replicate the results of that specific area.. You will need a CDC clean room environment, so as to not contaminate your cultures...

 

[fishwrestler]

In my opinion no started is needed I took the advice of Robert Goodrick for Vancouver BC I started with whole muscle an my chamber is developing it on mold. He also does not use any starter in his salami. Dave you would need a CDC environment. You can reactivate a culture using distilled water and spray or dredge your cased sausage in it. This was the first piece of meat I put in my chamber, the mold has spread to all my other product I have added except smoked items

 

[Jonok]

A common method I used in my Infectious Disease research days was to isolate the desired organism on an appropriate agar plate, and then, on a second pass (streaked from a .5 McFarland suspension of colonies you are sure represent your organism), heavily inoculate a vial of sterile (autoclaved or 0.5u filtered), skim milk with the pure strain, freeze the vial in liquid Nitrogen, then into the lyophilizer or -70c freezer as appropriate. No need for a clean room, just good aseptic bench technique. Pouring plates is likely when you would contaminate something, so incubating your plates for 24 hrs at 40c and tossing any that show growth is advisable even if you do have a hood in which to pour them in is a good idea.

Kept several hundred strains of lyophilized strep pneumo pure, happy, and safe in storage for upwards of a year, and for transport halfway across the world with no particular environmental requirements.

 

[Idontknow]

Jim, thank you for the detailed response! I hope you dont mind my follow-up questions.

For rapid freezing, I can get my hands on dry ice which can drop samples to -70C temporarily. Good enough?
I might be able to get a McFarland reference but if I can't, would that be a big problem? Its a visual inspection right?
I can see some references in google image search and that should get me in the ballpark? Any advice there?

I also don't have a lyophilizer, but I have a vacuum sealer with a port for attachments.
It isn't a very strong vacuum, but with a very thin substrate it might desiccate the sample.
I haven't done the math yet to see if I can get it to sublimate ice under that weak vacuum.
(funny side-story, I once reversed the valve on a bicycle pump and vacuum distilled a bottle of wine for shits and giggles)

My Autoclave is an instant pot with sterilize function that I would run twice with a 2 hour break between runs.
Good enough?

I have cooking agar which is just no-additive agar.
Should I just add a small amount of sugar and milk to that agar before sterilizing?
Do you have any suggestions on making the growth media more selective?
I currently have Lactobacillus sakei, Staphylococcus carnosus, Staphylococcus xylosus, Pediococcus pentosaceus, and Penicillium Nalgiovense
Would it help to have the same nitrate and salt levels in the medium to be more selective to these strains, or should I not bother with that?

I think my last question would be, what volume of vial do you think is sufficient?
I'm thinking that it would be convenient to shoot for 1kg meat inoculated per vial, and to buy a 500 pack of 2ml vials with locking snap lids that can be autoclaved. Would you suggest I use a larger volume vial?

Cheers and thanks a lot!

 

[Jonok]

Hmm
If you’re going to freeze them using dry ice, I’d probably do glass vials with (ideally) rubber stoppers and crimp on lids. 5 or 10 cc should be adequate (like the bottles that medicine for injection comes in). In order to cool them optimally, I’d get something like a yeti coffee cup and fill it part way with acetone, and incrementally add dry ice until it largely quits bubbling (which should be somewhere around -80c). Then I would dip a vial (with something other than your bare hands, of course,) about half full of inoculated milk in and swirl it around until the milk is completely frozen to the sides.

Asepically place the sterilized cap.

If you can keep it on dry ice for the trip, you’re good to go.

Drying it, other than with a lyophilizer is going to be tricky.

To sub your cultures, The Lactobacillus and Pediococcus would be happy in just about any common medium, the penicillium will just sporulate if you leave it alone and let it dry out over a day or so, the staphylococci.won’t do well on plain agar, but if you were to mix in 25% by volume meat broth of some sort, ideally without a ton of salt, they’ll probably grow ok.
If you can’t take the stuff on dry ice, you could also either pour slants, and surface inoculate or just half fill a vial with nutrient agar as above and do a stab inoculation. These will likely remain viable for a few days to a week or so at room temperature after overnight incubation at around 40c, as none of the bugs you’re working with are super fastidious.
Lemme know if you have any other questions.
Jim

 

[Idontknow]

Great! Thanks for sharing your knowledge! I will take it all in consideration and plan this out before I give it a try.

Just clarifying I am not travelling with these. A friend brought a couple freeze dried packages from the US to me in Japan and I want to keep a stock of these cultures on hand without having to try to arrange another friend to bring back some in their suitcase.

 

[daveomak]

Your chamber may be developing mold, but it is continually being diluted with the bacteria from your locale... Local strains are more hardy than strains introduced from an outside environment.. That's all I was saying in reference to a CDC clean room..

In my opinion no started is needed I took the advice of Robert Goodrick for Vancouver BC I started with whole muscle an my chamber is developing it on mold. He also does not use any starter in his salami. Dave you would need a CDC environment. You can reactivate a culture using distilled water and spray or dredge your cased sausage in it. This was the first piece of meat I put in my chamber, the mold has spread to all my other product I have added except smoked items
View attachment 390939

 

[Idontknow]

I appreciate the input but where I am I don't trust the local flora and fauna to give a good product. I am able to get lacto pickles going pretty easily with wild strains, but there is other stuff around. My house is prone to mildew (classic crappy Japanese rabbit hutch post war construction) and the air quality around here is questionable. Im also pretty new to the fermenting of sausages so I approach it like I am dealing with ebola :-) I have had to throw out a bad batch of beer once and that was terrible. I can't even imagine the hell a bad batch of sausage might be.

 

[Jonok]

[QUOTE="Idontknow, post: 1940035
Just clarifying I am not travelling with these. A friend brought a couple freeze dried packages from the US to me in Japan and I want to keep a stock of these cultures on hand without having to try to arrange another friend to bring back some in their suitcase.[/QUOTE]

Ah, then I would suggest the freezing in skim milk, quick freezing as above, and storage in the coldest, most stable (no defrost cycles) freezer you have available.
Jjk

 

[Idontknow]

Ok, thanks!

 

[BGKYSmoker]

Man

 

[Fueling Around]

This isn't rocket science.
I'm a propulsion engineer (token rocket scientist).
I followed the thread until the biologists went beyond my comprehension level