Hi everyone
I am trying to determine MIC of some beta-lactam antibiotics against A. baumannii and E. coli by broth dilution methods following Andrew's 2001 protocol (Determination of minimum inhibitory concentrations,2001). So, firstly, I need to maintain the inoculum concentration 5 x 10^6 per ml (Mc Farland standard) for the test set-up. I have done an absorbance index for both the bacteria with a particular spectrophotometer. However, when maintaining the concentration it seems too diluted; since for instance, to make 5 ml of working concentration of inoculum I need 1 μl or less amount of overnight culture according to the index. Nevertheless the following morning I found no growth neither in the different antibiotics concentration wells nor in the growth control even. I know I could confirm the McFarland standard by using McFarland standard kit, however, its not possible for me at this moment to one. Can you please suggest me how I could maintain McFarland standard of A. baumannii and E. coli?
Thanks in advance.
Dear Mahbulbul, you can use McFarland densitometer (Biosan Den-1B) or you can prepare your own turbidity standart as in the attached file.
Best regards.
mert
You need to compare absorbance readings with McF. then put CFU for same or if if you already know what OD corresponds to cell number. You may calculate accordingly. Generally 1 OD for E coli turns out to be 3-5x10^9 cell/ml. But it various from instruments. There was about ten times difference for E coli when we used it. I mean 1 OD corresponds to 10 McF in our laboratory.
Above comment by Mert Sudagidan explained well already.
When you are preparing your inoculum from your liquid culture using 1 ul or less, the error is very high.
Prepare home made0.5 McFarland standard as indicated by Mert. (your preparation must be fresh and prepared every 2 weeks)d
- From your fresh overnight culture (max 16 Hrs), or from your solid culture, it 's important to avoid clumping, let your supsension stand for 5 minutes, from the supernatant, prepare a suppension equivalent to 0,5 McFarland.
the approximate cell density of 0.5 McfFarland is 1 to 2.5 X 10"8 cfu / ml
- dilute 250 ul in 5 ml (to obtain 5.10'"6 cfu/ml)
A periodic check of your colony count from the positive control well should be done.
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