Isolates shows different activity in the same medium after three transfer. What is the reason?
It depends upon the bacteria. Like some strains of P. aeruginosa exhibit hypermutation at very high frequencies while there are others which are quite stable. Its advisable to maintain a stock culture and carrying out your experiments with subcultures from your stock.
Naveen Sathyan,
I am thankful to you, but my problem is that as i mentioned in discussion part of the question organisms showing different activity after three sequential transfer , study related to morphology remain same like colony characteristic etc . but only difference observed in metabolic activity i want to know what is the reason behind it and one thing i want to mention here is sequential transfer is done from master culture similarly i also tried with different master culture slant
If you are doing serial transfers of cultures then it may be very difficult to generate a homogeneous culture. Remember that you are dealing with very very large population sizes with bacterial cultures. The standard approach is to streak for single colonies. Each isolated colony arose from a single cell and therefore should be genetically homogeneous.
Hello Sagar,
Typically USP guidelines suggests that you do not subculture your bacterial cultures for more than six passages.
Theoretically speaking, spontaneous mutations arise at a frequency of 1/100,000,000 so there might be mutations arising in your culture during successive rounds of transfers.
If you keep your sample as a broth cultre, re-isolate and purify it by streak plating on a solid culture medium and then obtain a well-isolated colony. the well isolated colony is assumed to have arisen from a single cell, and therefore assumed to be pure.
transfer this to a fresh sterile medium (preferably an agar slant) and use this as your master stock culture.
Broth cultures can easily be contaminated because of their fluid state. In addition, you will not be able to monitor contamination since you cannot see the cells growing as colonies.
Another possibility is that perhaps the gene coding for the enzymatic activity you are monitoring might probably be plasmid-borne? If that is the case, there might be loss of plasmid if you are using this trait as a marker.
I hope these thoughts can help :)
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