Expert: Florence M Rollwagen - 9/10/2008

Question
a sample of pure culture that was gram stained - saw a field of red and purple cocci?  Adjacent cells were not the same?  What did I do or shoudl I conclude I contaminated?

Answer
Hi Cindy:  Thanks for your question.  

First let me ask if this is a culture given to you by your instructor to practice the Gram stain, or whether it is a colony you isolated from the environment.  If it came from the instructor, it's probably not contaminated.

Contamination is always a problem in micro.  If you saw a field of red and purple cocci there will be two possibilities.

First, and easiest to check is to determine if the Gram stain was done correctly.  The slide needs to be FLOODED with stain.  Sometimes we get variable staining if all the bacteria have not come in contact with the stain.  To correct this, make sure you don't have too many bacteria on the slide.  Too many bacteria will affect how thoroughly the stain gets to the bacteria.  Make several slides, stain for different periods of time (I know your instructions give you the exact number of minutes, but you can vary this by at least 30 sec to 1 min on either side).  Also be careful when you de-stain.  You don't want to take off too much color, so make sure you vary this as well.  If you are still getting variable results, you should check for contamination as described below.

Second, you should streak your culture for isolation.  If you don't know how to do this look it up on the Internet.  It should also be in your lab book, or ask your lab instructor.  I can't describe it in this forum, it has to be demonstrated.  You should get colonies of different description if you have a contaminated culture.  Different description means size, color, etc.

Repeat the Gram stain first, it's the easiest to do.  If you consistently get variable results, then streak for isolation.

I hope this answer has helped you.  Please write back if you have more questions.

FM Rollwagen, PhD