I am entirely self-trained. I am trying to do a gram stain from a vegetable fermentation (I have done sauerkraut and cucumber). From what I understand, the bacteria from a vegetable ferment should be almost all lactobacilli, which are mostly gram-positive. However, every time I do a gram staining procedure against either one, it comes out gram-negative. Does anyone have any tips to share?
-- The photo is below. I am assuming that this is actually bacteria that is in the photo:
My procedure (I am doing this on a table with a plastic container for a drain/sink):
- Put on 2-3 drops of liquid from fermentation
- Let it dry
- Heat fix with butane lighter (3 passes)
- Apply a few drops of crystal violet and wait 1 minute (NOTE - I don't know if this is unusual, but my crystal violet dries out about halfway through the minute).
- Wash off crystal violet with water.
- Apply several drops of iodine and wait 1 minute
- Wash off iodine with water
- Apply a few drops of ethyl alcohol and immediately rinse off
- Apply a few drops of Safranin O and wait 1 minute
- Wash off Safranin O with water
- Dry reverse side of slide, add a drop to the top, and add a cover slip
- View using 40x objective
Originally I thought that the alcohol was staying on too long. That is still entirely possible but I am getting it washed off as quickly as I can. Then I thought that maybe my sauerkraut just happened to have a gram-negative bacteria (maybe Pseudomonas fluorescens or something). But when it was also happening with my cucumbers I figured it is probably not that.
Anyway, I am very frustrated, and any help is appreciated.
Jon
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