It would be great to have your opinion in that: what is the valid approach to prepare a standard the inoculum with known cfu/.ml? I prepared an overnight broth culture (18hrs) at 37 C, afterwards the turbidity of culture is adjusted to match that of a 0.5 McFarland standard (108 CFU/ml). How accurate is this method when we are using different strains for same antibacterial testing? Standard methods indicate the use of growth method (2-6 hrs broth culture until turbid) or direct suspension method (from an overnight colonies on agar plate) when preparing the inoculum, but in some publications, a 24 hrs overnight culture is being used then later its turbidity is adjusted to Mcfarland standard. So is the later method valid? I also tried to plot the serially diluted cfu/ml with the OD readings after 24 hours incubation of an overnight culture at 37C, but it didn't give convincing results, there were variations.
Hi Dima,
Why don't you test the growth of each of your strain (ie do it three times for each strain). For example, at each OD measurement check the CFU by plating. Also record the time each measurement is taken. Plot on graph to determine the the growth characteristics of your bacteria, ie lag, log, stationary phase. You would want to use each of your strain at the same phase. If you have good methodology (good dilution and plating techniques, keeping conditions similar for all strains [ie. not leaving some cultures sitting around while testing others])and always use culture at the same determined OD, then you should get similar numbers of bacteria in your standard inoculum (though always check inoculum bacterial numbers by plating). Many strains can be in the declining phase by 24 hours (depending on many factors) and hence will give variable results. What phase you want to use depends on your aim but often mid-log phase growth is used. So, if you know your growth characteristics, you can adjust the bacterial numbers to 108CFU/ml safely by diluting. Once diluted you can check whether the density is similar to your McFarland but don't forget, visual checking is subjective especially as the colour of the culture broth can influence your perception. It takes a bit of work initially but then later you save time and you can be sure that you have a solid system.
Many many thanks Aniko... it seems i did things differently.. But didnt get an explainable match of counts to OD..so i did something wrong. for ex..initially am aiming at 108 cfu/ml hence i used to incubate broth culture for 124hrs then adjust its dilution to read 0.1 on spectro.but then i thought better to validate this by plate counting and OD. I did tenfold serial dilutions of the 24 hrs broth culture followed by plating and reading each dilution.however unexplainae results for instance dilution of 1/10-4 gave toonumerous to count on plate and below 0.001 OD!
Once again..thx
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