[DIYbio] Re: Gram Staining Problems

I *think* I managed to get this done right.  Below is my image:


I think the problem was that I did not have enough crystal violet and iodine.  I switched from adding a few drops to flooding it in both steps, and it now seems to work.  It is showing gram positive bacilli and both gram positive and gram negative cocci (if I am looking at it correctly).

Jon


On Wed, Aug 23, 2017 at 10:19 PM, Jonathan Bartlett <jonathan@bartlettpublishing.com> wrote:
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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