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Issue with Simple Stain Procedure

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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Issue with Simple Stain Procedure

Postby meay7c » Fri Feb 05, 2010 3:19 am

Hello:

I am a new micro bio student. This week's lab was a nightmare (me -->lab-a-phobia :roll: ). My partner and I were to prepare bacterial smears first and use the same slides for a simple staining. The problem was the bacterial smears were very faint. We collected bacteria from a solid and a broth and used the needle and loop to determine which was better suitable for this procedure. The smear was very faint and out of 7 slides maybe 2-3 were descent.

We used the best 3 slides for staining. My partner flooded the slides with the meth blue, crys violet, and fuschin. The stains were left on for the recommended time. After rinsing, our smears disappeared. We did not have the opportunity to repeat the procedure. I wanted to know for future staining procedures, what went wrong. I was wondering what could have led to our smears disappearing. Were the bacteria applied correctly? Did we use too much stain? Were we holding the slide incorrectly during staining? Is there anyone who can advise me on this procedure? I would be grateful for any feedback.

Thanks.
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Re: Issue with Simple Stain Procedure

Postby billw » Fri Feb 05, 2010 2:27 pm

Sounds like insufficient drying and/or over-rinsing. Over-rinsing is a very common problem with new students. Just a few drops from an eye-dropper is usually sufficient for rinse - much more and you end up washing away much of the bacteria. Plus, if the smear was insufficiently dried or you waited too long before rinsing, the bacteria will not stick to the glass and will be washed off with the rinse.

So:

1. Dry the smear thoroughly (but slowly, so as not to cook the bacteria).
2. Apply the stains for only the required amount of time (a full gram-stain process should take less than three minutes).
3. Rinse very gently with a small amount of water.
http://microbiology101.com/
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