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Name: Mike
Status: Other
Grade:  Other
Location: NJ
Country: United States
Date: January 2008

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?

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?


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