09-17-2012, 07:15 PM (This post was last modified: 09-17-2012, 09:53 PM by Administrator.)
I am currently performing a salmonella mutagenicity assay and experiencing some difficulty.
I am culturing a frozen stock into nutrient broth and growing a 16 hour culture and there is growth in my broth. When culture is transferred to top agar and plated on minimal glucose base there is no growth of culture.
10-17-2012, 11:01 PM (This post was last modified: 04-15-2015, 04:46 PM by SunilNagpal.)
Ames test is used to assess the mutagenic potential of chemical compounds. The test is therefore a confirmation of mutagenicity. Many a times it has been observed that the number of false positive and false negative in this case is more due to mutation at different locations than as desired. Salmonella is preferred for quick estimation as compared to traditional method in which rodents are used. A strain of Salmonella typhimurium is used in which mutation in genes those are involved in synthesis of histidine is carried out. They are auxotrophic mutants and therefore need histidine for their growth. The test involved evaluation of cells those can grow in histidine free media due to mutagenic action.
In your case, if growth is observed in broth and not in second passage onto agar then kindly review /investigate following things to arrive at the root cause of your issue.
1) Review if there is any difference in these two types of media with respect to pH, incubation conditions, and nutrient contents –histidine concentration if any. If the difference is found then that may be the cause of no growth onto the agar.
2) Since you are transferring growth from liquid to agar, this is second passage and many a times mutants are very unstable with respect to number of passages. If possible, try inoculation of lyophilized suspension directly onto agar that is single passage.
3) Check the growth of positive control and negative control. If there is no growth on positive controls then the issue is with respect to media itself. Positive control-results are very important to conclude & investigate further in your case.
4) Try incubation in liquid for longer time period than what you had done (12hours). At least incubate for 24 hours. And then transfer onto the agar media.
Also investigate other parameters like autoclave time temperature of agar media and incubation condition like temperature, aeration, Rh etc.