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Thread: Question for those using Wallerstein Differential

  1. #1
    Join Date
    Apr 2009
    Posts
    12

    Arrow Question for those using Wallerstein Differential

    I have been using Wallerstein Differential media for my aerobic/anaerobic baceterial count. Until now, I've always gone by the directions- counting dark green colonies as bacterial and any white/light green colonies as cyclohexamide resistant yeast. However, there have always been some ambiguities (colonies with a dark green center but otherwise white or filamentous colonies with dark green bacterial colonies on the inside).


    My pH indicator showed my pH was dropping on my plates where there was only white colonies. I decided to pick some colonies and examine them under the microscope. What I had assumed was wild yeast is most definitely lactobacillus (rod shaped, catalase negative).

    I did see some yeast in one of the very light colonies I picked but overall I am seeing lacto.

    Does anyone have any experience with this. I am trying to collect some precise data and this is certainly holding me back.

    For the record, I'm pour plating with 5 ml samples for aerobic plates, incubated for 3 days and 2.5 ml samples for anaerobic plates, incubated for 7 days. I'd definitely be open to switching to UBA if I thought it would help.

  2. #2
    Join Date
    May 2009
    Posts
    1
    In my experience it is not a good idea to go off of just the color of the colonies for determining the type of contaminant. Even in brewing yeast there can be variation in the way they take up/metabolize the bromocresol, generally they take up the dye and are green. In my case, I found Lacto to be small white colonies, pedio to be small green colonies, and Brett to be fairly variable. In the wide world of metabolism and the many bacteria/yeast that you can come across, it best to do some staining and look at it under a microscope so you can get a better idea of what it could be. When in doubt ferment it out

  3. #3
    Join Date
    Dec 2003
    Location
    Lebanon, OR
    Posts
    18

    Gram stain and have a peek under the scope

    Ditto on pc95. Use plates to grow out, isolate with differential media; but confirm by microscope.

    Gram staining helps highlight morphology, but not essential.

    Get the book- Illus. Guide to Microbes & Sediments in Wine, Beer & Juice to help identify what you see.
    https://interactive.asbcnet.org/sour...2206&SKU=52206

    The truth is, however, you need only discern whether it's yeast (and estimate the relative size and condition), or bacterial rods (most likely) or cocci.

    We just grow out on 3M Petrifilm Aerobic plates/anaerobic conditions, and then go from there- staining colonies we pick off the plates and then growing out if they interest us.

    Hope this helps.
    Scott

  4. #4
    Join Date
    Apr 2009
    Posts
    12
    Thank you for the replies. I've been putting in some serious time with a spectroscope and have started getting a feel for visual colony identification (something that was generally lacking in my previous cloning work). I also didn't think I could visualize bacterial cells with a standard biological microscope (dead wrong). I'll suck it up and file it under "progress"

  5. #5
    Join Date
    Jun 2005
    Location
    Lexington, KY, USA
    Posts
    79
    Scott has good advice here and we have a couple papers to recommend on use of media in the brewing industry. (I used to make the four media at the Siebel Institute and wrote about those and comparative media). And yes you can detect bacteria very well under x400 magnification. Remember that acid producers turn the bromocresol green around them yellow - part of the differential media properties.

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