Gram Staining Problems

[Jonathan Bartlett]

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:

https://drive.google.com/file/d/0B-zu5qfA3muGdmU5b0NnWk5GVW8/view?usp=sharing

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

[John Griessen]

On 08/23/2017 10:19 PM, Jonathan Bartlett wrote:
> # 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).

I have not done this procedure ever, but...

If your crystal violet dries out because of heat in 30 seconds, it will be at a much higher concentration and that could be what
results in all violet/magenta looking color or black that we see in your photo. You probably need to wait until your
heat from fixing is dissipated, then add the few drops of crystal violet. that is how I would interpret your procedure list.

[Jonathan Bartlett]

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

[Jonathan Bartlett]

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

https://drive.google.com/file/d/0B-zu5qfA3muGUG1zcGxyM1AzNHM/view?usp=sharing

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 <jona...@bartlettpublishing.com> wrote: