I am performing some MIC tests on Streptococcus equi subsp. equi and am having difficulty with growth. There is no growth in the control wells where there is no antibiotic present. Does anyone have any suggestions as to why this might be?
Many thanks in advance.
Hi, is there any growth in the other wells or not? In the sense, are you able to see MICs with antibiotics used in the test?
My questions for you to understand the problem;
1. What is the media used?
2. How are you preparing the inoculum?
3. How are you culturing the organism for the MIC experiment? and most importantly, this organism is very slow growing and it requires at least to my knowledge > 36 hours to grow. This means, you might have to incubate the 96 well plates for more than 24 hours or even for 2 days.
The biggest disadvantage with this long incubation is that the media gets evaporated sometimes giving you false results. To over come this, you might have to use 100 uL of media per well rather than 50 uL of media. As well place a bowl with some water in the incubator.
4. Are you able to see growth on the plate after you prepare the inoculum for the test. In the sense, you prepare the inoculum, then the inoculum is dispensed into the wells. But, the same inoculum is plated on agar plates to determine the log CFUs. Are you doing that, if so, what is the count that you observe there?
5. Did you check the control wells for any bacteria, in the sense, when you did not see the growth in the control wells, did you take the broth from that wells and plate them on Blood agar ? to see if there is any viable bacteria..
6. What is the volume of media used in the wells? total volume is it 100 uL?
7. Are the plates placed in CO2 incubator through out or not?
If you can provide these details I can help you. You can contact me without hesitation any time.
Hope my answers are helpful for your work.
regards
Amarnath Pisipati
Please refer to the Clinical Laboratory Standard Institute(CLSI) Protocols. The media and methodology are in the protocols.
Due to the fastidious nature of the bacteria, the type of media used in the procedure is very critical.
Kindly get more information on your methodology by use of CLSI protocols and know the best media that can be used for your pathogen of interest
Maybe the problem is, that the antibiotics are in the control well too, I think it is the most common failure.
But maybe the problem is the media it should contain 5% horse blood and 20 mg/L ß-NAD(EUCAST) or the problem is the atmospaere. It should be 5% CO2.
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