Unfortunate News

[Ray]

I do not have that info, but I agree wholeheartedly that there will be generational degradation.

[neophyte]

Originally Posted by Subrosa

Ray, would you have any information about the effectiveness of homebrewed Innocucor after several generations? I know from culturing mixed cultures of microalgae that some species tend to predominate over time, while others disappear entirely.

I was thinking along the same lines of some species outcompeting others. That got me wondering about interspecific competition among the species in Innocuor. After applying Innocuor, some species would surely outperform others in terms of survival, and survival rates of each species would vary based on the orchid medium. It might be worthwhile to investigate the environmental requirements of each species of bacterium in Innocuor and figuring out which ones best thrive in specific orchid media (e.g. sphagnum moss, bark, etc.).

Considering that culturing all the species together would lead to some species outcompeting others, a good solution might be to simply figure out a) which species are best for one's own orchids and only using those ones and b) figuring out what cultural conditions that specific species of bacterium needs in vitro. Otherwise, one might end up with a culture predominated by one species of bacterium that ends up dying quickly in the orchid medium. I think that because Innocuor contains the correct proportions of all these bacteria, at least one will end up doing its job in the orchid medium (which is why Inncouor can be used for orchids in various media). But eventually, homebrewed Innocuor might become ineffective because it becomes predominated by the wrong species.

It's probably also a good idea to keep several jars of homebrewed probiotics because one of the jars might get contaminated with a virulent species or horizontal gene transfer might confer one population with toxin genes that would harm orchids.

[DirtyCoconuts]

I think it kind of comes down to the law of limiting reagents. If there is abundant food for all the strains then thy would ever compete and there would not be a need for any of them to die off

If in the replicating process there is a shortage of the fuel then you’d have the competition you discuss. But with sufficient sugar they should all make it

[neophyte]

DC, you are right that more abundant sugar means less competition, but there are other factors that affect growth besides the availability of food (and some affect the rate at which the bacteria can acquire food). The species that benefit from these factors (which vary by culture conditions) would crowd out the rest. So even with more sugar, the resources would be split unevenly.

[DirtyCoconuts]

I hear you. I wonder, and this is a reckless speculation, if the developers intended that with their careful selection of strains. Perhaps they keep each other in check the way a healthy biological system does.
This often happens with fungal colonies where as one starts to thrive, the waste it produces then fuels the competitiveness of another fungus which reduces the population of the first back to stasis.

?? Maybe but doubtful lol

[Ray]

They developed a way to maintain cooperating consortia of microbes.

I know their charge includes molasses, kelp, pure water, salt and clay - some of the species prefer being anchored, rather than swimming, I suppose. Maple syrup was substituted for the molasses when they made some for their own consumption.

When I’ve brewed my own, I’ve used 1:1:1:20+ Inocucor, molasses, KelpMax, RO water.

[DirtyCoconuts]

That’s the recipe I’m going with ray.
My garage insulated and air conditioned at stays at 75
I was going to use my new bottle and divide it into four parts and but three in the fridge as future seed bottles. The last quarter will be used for a two week brew and then I’ll jar that whole batch up and put in the garage fridge.

In the fridge how long do you think it will maintain viability?

[Ray]

Originally Posted by DirtyCoconuts

In the fridge how long do you think it will maintain viability?

You got me...

[thefish1337]

Originally Posted by Ray

I do not have that info, but I agree wholeheartedly that there will be generational degradation.

In the lab we avoid continually sub-culturing strains for this reason. Typically a strain can be stored at -80 for many years. That -80 stock is "streaked" onto a plate that we store in the fridge for *months*. We then use individual colonies from the fridge to start up *daily* cultures.

Applying the best practices to Inocucor it would be best to save a bottle of inocucor for as long as possible in the fridge or cool storage and use that bottle to start up all your amplifications. Ray suggests adding some molasses every once in a while (its probably best to add kelp and molasses). Once your original bottle is getting close to running out, amplify a batch and make a "stock" that you will use for all subsequent "generations". I put generations in quotation marks because in bulk culture the number of successive generations per fermentation is many orders of magnitude higher.

This reduces the number of "generations" on the microbes greatly. For example, if you fermented 5 batches per year using the last ferment to produce the next your inocucor would go through 5 subcultures per year. If you make stocks and store them for around a year and draw from that for fermentation the culture would only ever be 1 subculture away from the stock. I'm using a year here but nobody knows how long the stock is good for. After 1 year you would be 2 subcultures away. Once the original bottle is gone after 1 year you would have only subcultured twice from the original culture. In contrast, successive culturing would be 10 subcultures away from the original stock by the end of year 2.

Changes in the populations over time are inevitable but they can be slowed down with good culture practices.

[DirtyCoconuts]

Sweet. And thanks for that info