I am determining MICs for M. tuberculosis using the alamar blue method described by Franzblau et al. I have had good success up until now. I am experiencing some variation in my MICs.
I grow my H37Ra research strain on 7H10 OADC agar until a sufficient lawn of bacteria is present. This takes about three weeks and I believe the cells are in the log phase of growth. I prepare a suspension of the cells (~ mcfarland 3) in 1 x PBS + 0.05% v/v tween 80 and I store the suspension in 1 ml aliquots containing 20% v/v glycerol at - 80 degrees.
When I do my MIC determinations, I swab new 7H10 OADC plates with my stored 1 ml aliquots and grow them until a sufficient lawn of bacteria is present (normally 2 weeks). From these plates I prepare a mcfarland 1 working solution to do my MIC determinations, further diluting them 1:10 to give ~ 2.5 x 10^5 CFU/ml.
My questions is, if I swab 7H10 OADC agar with a 1 week old stored aliquot vs a three month stored aliquot, could the cultures grown on 7H10 agar after two weeks have different physiologies and thus growth rates?
I have been experiencing a difference in growth rates when determining MICs and I am concerned it is due to my culture method. Some cultures have taken 7 days to read a result while others have taken 5 days. This results in a two fold difference in MICs.
yes the storage conditioned you have mentioned affect the MIC. Because the number of colony may decrease or increase in the storage condition.
Have you try with fresh drug stocks using the same stocks of MTB? Sometimes the drugs lose eficacy and that affects the MIC.
I always use fresh drugs that I prepare on the day. I understand that when you store cells at - 80, CFU/ml change, that is why I grow a new culture on 7H10 agar, using my stored culture and thus use the newly grown cells for my MIC determinations.
Bacterial count in McFarlad 1 for a bacterium such as E. coli is considerably different from that of Mycobacterium. Since the cells of the latter have lipophilic nature and tend to form clumps, even thorough mixing of the mycobacterial suspension can not guarantee equal cell numbers are seeding to the plates following each pipetting. This can affect growth rate. I think it would be better you define an end point time for your test regardless of the growth rate. For example in MODS test, some wells show mycobacterial growth within 5 days and some do it later, but the results should be read and report after 21 days.
The most important aspect is at what part of the growth phase of the microorganisms are that you are using in your assay. Microorganisms typically go through four partially mixed growth phases. The initial growth phase is the lag phase where the organisms have to adapt to the growth conditions and there is only a low increase in number of cells. This then goes over to the logarithmic growth phase where cells do not have to adapt to the growth conditions and the cells double exponentially with a straight line on a logarithmic scale. This eventually, when nutrients become limiting or byproducts that limit growth is excreted in the growth medium, leads to the stationary phase where the cell numbers remain constant and finally to the death phase when the cells start die and numbers decrease.
Cells collected during the exponential growth phase have the same physiological and biochemical parameters and if you use cells in this growth phase your results should be reproducable. A large inoculum of cells in the exponential growth phase decreases the lag phase in a new culture to a very high degree and this is an approach we followed in a paper with > 900 citations when determining MICs of bacteria (Eloff, 1998, Planta Medica 64, 711-714).
i think you should cultured bacteria in broth mediumfirst , then transfer them to agar medium and thus do MIC.
i think you should cultured bacteria in broth medium first , then transfer them to agar medium and then do MIC.
Yes, storage and culture method effect MIC value, If you are storing prepared media for more than two days, you may have background bacterial infection mainly no-turbidity type. when you seed the colony for MIC than this strain start growing much faster than the clinical isotype which you are going to find antimicrobial susceptibility. In another case for example if you are trying to find MIC in suspension culture you will find different result in comparison to disk diffusion test, because in solid media action become very slow, but in fluid media solubility of the compound and membrane functions may increase that provide both earlier better and much accurate results.
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