Isolating Microbes from Wine
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Just curious, why would you want to?
There's a lot of literature out there and a boatload of microbes in wine. And the populations of the little creatures change during the wine making process. I've read there's roughly 56 yeast strains and 36 bacteria strains in a batch of wine. Probably more. And lately there's the idea that there is an influential microbial terroir in wines, also. Wine making is a straightforward process but the microscopic activity and chemical reactions are so incredibly and beautifully complex.
I don't know how to do it but I'm guessing a Petri dish and electron microscope would be helpful.
There's a lot of literature out there and a boatload of microbes in wine. And the populations of the little creatures change during the wine making process. I've read there's roughly 56 yeast strains and 36 bacteria strains in a batch of wine. Probably more. And lately there's the idea that there is an influential microbial terroir in wines, also. Wine making is a straightforward process but the microscopic activity and chemical reactions are so incredibly and beautifully complex.
I don't know how to do it but I'm guessing a Petri dish and electron microscope would be helpful.
BernardSmith
Senior Member
Microbes .. or yeast cells? You can easily observe yeast cells using an inexpensive microscope . Most yeast cells are between 3 and 4 micometers (1/1000 of a mm). There are relatively simple ways to grow yeast cells in a petri dish and then isolate the specific cells you are interested in and grow those cells and not others. You might check out Yeast: The Practical Guide to Beer Fermentation by Chris White and Jamil Zainasheff (Brewers Publications, 2010) pps 173 - 261. But you really need to have some experience working with cultures to make good use of the processes and techniques the authors suggest.
What is the goal here - just curiosity, or are you looking for a method to identify and potentially treat problems? It's also possible to isolate microorganisms without identifying them, and vice versa.
At the most basic (and inexpensive) level, you could get some culture plates with various culture media and plate out samples of your wine. It's then relatively straightforward, as @BernardSmith notes, to identify yeast vs bacterial using a microscope. But it sounds like you're looking for more granularity than that.
ETS labs runs a 'Scorpions' panel, which uses QPCR (quantitative polymerase chain reaction) to identify and quantify various yeasts and bacteria in your wine sample. The focus is on microorganisms that can cause problems (eg brettanomyces and acetic acid bacteria). The method works on a liquid wine sample, which has the added advantage that some organisms that may grow in wine are resistant to culturing on solid media. Cost is $126 at time of posting, so it's not cheap but not ridiculously expensive either. Certainly if you're a commercial winery and you suspect a problem, it's well worth it.
If you want to do this at home, you're looking at at least a few thousand dollars for a QPCR machine and associated molecular biology tools, plus reagents and consumables (some of which in themselves are quite expensive). You could do a cheaper, non-quantitative version with a regular PCR machine and agarose gel, but it's still going to be a tough and costly project unless you have access to a microbiology lab and the knowledge of how to use it.
At the most basic (and inexpensive) level, you could get some culture plates with various culture media and plate out samples of your wine. It's then relatively straightforward, as @BernardSmith notes, to identify yeast vs bacterial using a microscope. But it sounds like you're looking for more granularity than that.
ETS labs runs a 'Scorpions' panel, which uses QPCR (quantitative polymerase chain reaction) to identify and quantify various yeasts and bacteria in your wine sample. The focus is on microorganisms that can cause problems (eg brettanomyces and acetic acid bacteria). The method works on a liquid wine sample, which has the added advantage that some organisms that may grow in wine are resistant to culturing on solid media. Cost is $126 at time of posting, so it's not cheap but not ridiculously expensive either. Certainly if you're a commercial winery and you suspect a problem, it's well worth it.
If you want to do this at home, you're looking at at least a few thousand dollars for a QPCR machine and associated molecular biology tools, plus reagents and consumables (some of which in themselves are quite expensive). You could do a cheaper, non-quantitative version with a regular PCR machine and agarose gel, but it's still going to be a tough and costly project unless you have access to a microbiology lab and the knowledge of how to use it.
The traditional way to isolate microbes/ yeast lines is with agar plates.
1 a drop of culture or fermenting fruit or sewage sample or etc is put on the edge of an agar plate. A single line is drawn through which thins out the cell density.
2 the platinum inoculation loop is sterilized in a Bunsen burner flame then cooled. Next a Z pattern is run through the first line dragging cells and creating more space between them.,,, approx half of the plate is now used.
3 the platinum loop is again sterilized in fire and cooled. The plate is rotated 90 degrees and a second Z is created with one line of the last Z. The purpose again is to create space between cells so that individual colonies are isolated.
4 the plates are grown out at a temp suitable for what you are looking for. ex. 37C for staph or E. coli. for five days. Again depends on what you are looking for.
PCR is a genetic technique to say what is in a soup like sewage from a neighborhood. PCR does not isolate cell lines. At best it can give a rough percentage of organisms. Any organism detected has been isolated for identification with a Z plate as above.
Selective plating is a common technique as use PDA agar for identifying yeast and mold or bromicresol for isolating gram negative or penecillin for isolating resistant organisms. ,,,, Families are described by what they grow in as anaerobes need CO2 or psychrophiles will grow at a refrigerator temp or acetobacter will use ethyl alcohol as the only carbon source etc etc.
Grouping into a micro family count is done with dilution plates ex if the 1/1000 dilution on PDA has 96 colonies the food is said to have 96,000 yeast and mold per gram. (Sounds like a off flavor milk)
With sterile media isolation is a basic intro lab skill,,, not hard. ID takes skill.
1 a drop of culture or fermenting fruit or sewage sample or etc is put on the edge of an agar plate. A single line is drawn through which thins out the cell density.
2 the platinum inoculation loop is sterilized in a Bunsen burner flame then cooled. Next a Z pattern is run through the first line dragging cells and creating more space between them.,,, approx half of the plate is now used.
3 the platinum loop is again sterilized in fire and cooled. The plate is rotated 90 degrees and a second Z is created with one line of the last Z. The purpose again is to create space between cells so that individual colonies are isolated.
4 the plates are grown out at a temp suitable for what you are looking for. ex. 37C for staph or E. coli. for five days. Again depends on what you are looking for.
PCR is a genetic technique to say what is in a soup like sewage from a neighborhood. PCR does not isolate cell lines. At best it can give a rough percentage of organisms. Any organism detected has been isolated for identification with a Z plate as above.
Selective plating is a common technique as use PDA agar for identifying yeast and mold or bromicresol for isolating gram negative or penecillin for isolating resistant organisms. ,,,, Families are described by what they grow in as anaerobes need CO2 or psychrophiles will grow at a refrigerator temp or acetobacter will use ethyl alcohol as the only carbon source etc etc.
Grouping into a micro family count is done with dilution plates ex if the 1/1000 dilution on PDA has 96 colonies the food is said to have 96,000 yeast and mold per gram. (Sounds like a off flavor milk)
With sterile media isolation is a basic intro lab skill,,, not hard. ID takes skill.
Last edited:
The first thing that came to mind when I read this.
The second...
I'm leaving this alone, this one is for @Rice_Guy
. And I wasn't wrong. Look at him go! 
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Thanks for all the information. To answer the question of "why", I simply just wanted to for intellectual scientific betterment. (I'm a nerd like that). So, I did it!
Here is my method and what I think I found:
1.) Sterile filter wine using a Buchner Funnel to capture any possible microbes
2.) Blot the filter pad onto selective media growth agar in a petri dish
3.) Incubate the petri dishes at 37C for 48-72 hours
4.) Certain selective agar will only grow specific microbes (In this case Saccharomyces & Brettanomyces yeast), but to identify the bacteria (which used a different selective agar) I had to do a KOH test
5.) Dilute Potassium Hydroxide (KOH) in water and then submerge a bacterial colony in the KOH solution. If the bacteria becomes viscous/stringy, then it's Gram-negative. If the bacteria dissolves evenly, then it's Gram-positive. (All my bacteria came back Gram-positive)
6.) Have fun!
Here are some photos of what I got.
Thanks,
Tin_Man
Here is my method and what I think I found:
1.) Sterile filter wine using a Buchner Funnel to capture any possible microbes
2.) Blot the filter pad onto selective media growth agar in a petri dish
3.) Incubate the petri dishes at 37C for 48-72 hours
4.) Certain selective agar will only grow specific microbes (In this case Saccharomyces & Brettanomyces yeast), but to identify the bacteria (which used a different selective agar) I had to do a KOH test
5.) Dilute Potassium Hydroxide (KOH) in water and then submerge a bacterial colony in the KOH solution. If the bacteria becomes viscous/stringy, then it's Gram-negative. If the bacteria dissolves evenly, then it's Gram-positive. (All my bacteria came back Gram-positive)
6.) Have fun!
Here are some photos of what I got.
Thanks,
Tin_Man
Attachments
Nicely done! I'm assuming that nice laminar flow hood isn't just something you have kicking around in your basement
Any plans for further ID of your bugs?
Fantastic!
I completely understand. I've spent a lot of time and a bit of money just to satisfy my curiosity.
This hobby has hit me hard but this is one aspect I'll watch from the sidelines.
I completely understand. I've spent a lot of time and a bit of money just to satisfy my curiosity.
This hobby has hit me hard but this is one aspect I'll watch from the sidelines.
