I am working with a strain which I have identified by 16S rRNA sequencing and it is identified as a Bacillus sp. But its showing a mixed reaction of Gram's staining. I am very confused. The culture is pure but after Gram's staining, some of the cells show violet color and some are showing pink color in the same slide. I have checked it again and again by applying different decolourizing time of decolourizer, but the results are still confusing.
Hi,
could you send us some picture? It could help us to better understand your problem.
Which kind of protocol are you using? It could be that something in the protocol is not working with the strain of bacteria you are interested in.
Have you tried to use a different purification protocol?
Please have a read at the following links (troubleshooting):
http://www.arrowscientific.com.au/index.php?option=com_content&view=article&id=48%3Adifferentiating-gram-negative-and-gram-positive-bacteria&catid=25&Itemid=40
https://www.atsu.edu/faculty/chamberlain/mosdoh/gramstainingrules.htm
https://www.microscopemaster.com/gram-stain.html
Best regards
Yes old culture of your strain can give you both reactions (Gram+ve and Gram-ve. Try to repeat the test on24h old culture. I think it will perform well.
With best wishes
Magda El-bendary thank you for your reply. Sir I have used fresh (24 h) old culture everytime for staining.
Francesco Desiderio Thankyou for your reply Sir. The links you provided is of great help. I will check it by other methods also. Here is the attached picture of my gram stained culture.
Hi,
Please check your procedures critically. While it is not impossible to have a novel occurrence in nature, the occurrence you cited is unlikely since Bacillus is a well studied bacterial Genus. It is almost impossible to have a strain test +Ve and -Ve for Gram staining at the same time. This is because of the role of peptidoglycan in the results of Gram staining. And the composition of peptidoglycan differ in most bacterial cells.
I suggest you try other measures as I feel something could be wrong with the procedure or steps. Take appropriate troubleshooting measures; cross-examine your procedures with different methodologies in the literature. Carry out other confirmatory tests to ascertain whether a bacterium is Gram +ve or -ve.
The bacterial strain should tilt towards one direction. But if you still have the same result, after doing every thing right, which I doubt based on current findings, then you have to perform in-depth study to understand the cellular make up of the Bacillus sp. The results of Gram stain is based on the composition of the bacterial cell wall. If something new happens, then there may have been a change in composition.
Pascal Chukwuebuka Okoye Thankyou sir for replying. I will try some other methods also to further confirm and clarify. I was wondering if is it possible that there could be some problem in the cells to retain the crystal violet-iodine complex ?
If the cell is same as other known bacterial cells, there shouldn't be any problems with retaining the crystal violet provided the procedures are followed explicitly.
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