I have done a gram staining for an unknown bacteria. That was confirmed that the bacteria is gram positive. But some of the bacilli did not stained properly in the middle? What could be the reason for that?
my predictions,
1. Cells are in different cell cycle stage
2. that could be spores.
Hello Mohamed Atheef
How old is the culture?
Old cultures tend to lose the peptidoglycan cell wall, which predisposes gram-positive cells to be gram variable or gram-negative.
Cultures should undergo evaluation while they are fresh.
Best.
This is a fresh culture. Which was incubated for overnight at 37 degree celcius.
With special reference to the spore shapes in the stained bacteria, It seems that many spores are released from the cells and stained in pale red.
Hi Mohamed Atheef
If you have used a fresh culture, then there are other possibilities that you may have to consider.
a. Some bacteria, after staining with Gram Stain yield a pattern called gram-variable where a mix of pink and purple cells are seen.
b. There are certain genera, Actinomyces, Arthrobacter, Corynebacterium, Mycobacterium, and Propionibacterium who have cell walls particularly sensitive to breakage during cell division, resulting in gram-negative staining of these gram-positive cells.
c. In cultures of Bacillus, Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness during growth coincides with an increasing number of cells that stain gram-negative.
d. Some bacteria do not stain as expected with Gram Stain. For example, for Mycobacterium spp., the waxy nature of the coat renders the bacteria not readily stainable with dyes used in Gram Stain, though the bacteria are considered to be gram positive.
e. You may also have an unusual gram-positive cell wall structure that may cause bacteria to stain gram-negative or gram-variable.
Best.
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