Department of Energy Argonne National Laboratory Office of Science NEWTON's Homepage NEWTON's Homepage
NEWTON, Ask A Scientist!
NEWTON Home Page NEWTON Teachers Visit Our Archives Ask A Question How To Ask A Question Question of the Week Our Expert Scientists Volunteer at NEWTON! Frequently Asked Questions Referencing NEWTON About NEWTON About Ask A Scientist Education At Argonne Gram Stain on Unknown Culture
Name: Mike
Status: Other
Grade:  Other
Location: NJ
Country: United States
Date: January 2008


Question:
I performed a gram stain on an unknown culture and I followed the steps according to the book. crystal violet for 1 minute and rinse iodine for 1 minute and rinse decolorizer 15 seconds and rinse saffranin 1 min 30 sec and rinse........the only problem was that I had purple and pink staining. the decolorizer was on for exactly 15 seconds. i repeated the steps and got the same result. under the microscope I saw gram negative bacilli but no gram positive . Do you know why I had both pink and purple stains?



Replies:
If the amount of bacteria on the slide wasn't evenly spread on the surface you probably had portions that were more heavily smeared with bacteria than others. The decolorizing step is the most sensitive and tricky to get right. The purple portions could be under-decolorized, in other words the alcohol may not have penetrated all they way through the smear in the time you gave it. It could also be over-decolorized if the smear was too thin. You will have pink and purple areas on the same slide especially if the edges are thinner than the middle. I usually try to observe the outer edges of a slide where the smear is thinner. It's probably more important that you saw bacilli vs. whether they are gram pos or gram neg. Very few bacteria are actually gram pos. Can you separate them onto selective media such as PEA (for gram pos) or EMB or MacConkey for gram negative?

vanhoeck



Click here to return to the Molecular Biology Archives

NEWTON is an electronic community for Science, Math, and Computer Science K-12 Educators, sponsored and operated by Argonne National Laboratory's Educational Programs, Andrew Skipor, Ph.D., Head of Educational Programs.

For assistance with NEWTON contact a System Operator (help@newton.dep.anl.gov), or at Argonne's Educational Programs

NEWTON AND ASK A SCIENTIST
Educational Programs
Building 360
9700 S. Cass Ave.
Argonne, Illinois
60439-4845, USA
Update: June 2012
Weclome To Newton

Argonne National Laboratory