I am working MDR-TB and performing a time-kill assay. I use 1 x 10^5 CFU/ml cells from 7H10 OADC agar (~14 days incubation) and inoculate them into liquid culture (Sauton media). I then monitor CFU/ml on treatment days of 0,1,2,3 and 6 using 7H10 OADC. I noticed that my control MDR-TB have a gradual decrease over the days i.e, with regards to log10 CFU/ml, 5.09, 5.27, 5.20, 5.05 and 4.64. Comparing day 0 to day 6, there is difference of 0.45. Statistically, there is no difference between the control and RIF+INH treatment, but a difference for the peptide I am testing.
I would like to know if I can still use these results and if the decrease of my MDR-TB control is something natural.
Before I got to the time-kill assay, I was using the microtiter Alamar Blue assay and by 6, the MDR-TB control was sufficiently changing Alamar Blue from blue to pink indicating sufficient growth.
I also performed the assay using H37Ra and there was an increase in growth over the six days.
I really hope someone can shed light on this issue?
All experiments were conducted in triplicate and independent of each other.
Can you clarify why you are going from 7H10/OADC to Sauton's to 710/OADC? What detergents are you using in each medium?
The decrease you are seeing may be due to the fact that Sauton's is a defined medium that is very, very minimal compared to 7H9/OADC. In our hands, cultures grown in Sauton's grow more slowly, seem to aggregate more and do not reach comparable ODs when compared to 7H9/OADC. You should also be aware that growth in Sauton's changes the transcriptome of Mtb, can alter its susceptibility to anti-mycobacterial compounds and its susceptibility to other metabolites. If you don't need a defined medium at this step, which you may since you are testing peptides and not conventional compounds, I'd suggest using 7H9/OADC and determining if you still observe this effect.
If there is a bona fide reason that a defined medium must be used, you have a few options: you could pre-culture your strains in 7H9/OADC to mid-log, wash and re-innoculate into Sauton's, and then allow this culture to reach mid log before restarting new cultures for your time-course assay. This acclimates your bacteria to the minimal media and would ensure that the you aren't seeing 'growing pains' in your time-kill assay. Alternately, you could try HdB, another minimal media, and go about acclimating it as above. Some strains grow better in HdB vs Sauton's or vice-versa. Just be sure to use tyloxapol, not tween-80, as your detergent in any minimal media as we have observed that tween without BSA is toxic to the culture. Good luck!
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Thanks Kathryn, my Sauton media contains tween 80 (0.05 % v/v) and glucose (2 % w/v). The reason for going from 7H10 OADC to Sauton is that I require the assay to be conducted in liquid culture. The other reason is that my peptide is antagonised by the components of OADC. As mentioned, I determined MICs and Synergy MICs for H37Ra and MDR using alamar blue and after 5 - 7 days there is positive growth (blue to pink). I also determined the growth of the controls after 7 days when doing this and for H37Ra it was ~ 1 x 10^7 CFU/ml and for the MDR strain it was ~ 5 x 10^6 CFU/ml. So there is positive growth in the media without a decrease. I of course include the necessary controls to make sure it is H37Ra and MDR growth that I am observing. Now, all of a sudden when I conducted the time-kill, I observe this change. It could be due to any of the reasons that you mentioned. Its just so confusing for me, because I never changed any parameters for the MDR strain moving from MIC, to MBC, to Synergy MIC.
Do you know what components of OADC are antagonistic? You could supplement 7H9 with ADS instead of OADC but my guess is that it is the BSA, not the oleic acid, that is your peptide antagonist (but you'd have to determine that empirically if you haven't already).
Oleic acid binds to the peptide, so does albumin and catalase might inhibit a Fenton reaction type mechanism observed for the peptide. So its a bit tricky, hence the need for Sauton media. I also need to use physiological Ca++ and Mg++ as this also has an effect on the peptides MICs.
Interesting. Sounds like a complex issue but one that you have a handle on.
If you haven't already, I would try pre-conditioning your cultures as described above and seeing if that makes a difference. As always, good luck!
Dear Shane,
I have done a lot of TKK and what is very important performing reproducible TKK is working with frozen work stocks. We make work stocks in 7H9 media with a density of about 2x10^7 cfu/mL, these cultures should be of actively growing Mtb. We made the mistakes once to increase the density of the workstocks to 1x10^8, however the Mtb was in a more static phase and was not growing like it should be.
Good luck and enjoy working on TB
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