i am working with microbial suspension in broth and to determine the concentration of bacterial culture i have done serial dilution till 10^9 where 10^8 gives 38 colonies and 10^7 gives 179 colonies which plate should i consider to calculate the CFU/mL ?
I would not take any of them; there was something wrong with the dilution or the plating procedure. If the former, there is no guarantee the same mistake was not made also at more concentrated points of the dilution series, and if the latter, you have no way of spotting the faulty data.
I would start by ensuring my pipetting technique was correct (serial dilutions of a fluorophore are great for evaluating this). This includes changing tips between dilution steps and proper homogenization of the samples. Also, how are you plating the inocula? In my experience, using glass beads introduces less variation than using a spreading spatula. And of course, make sure all plates belong to the same production batch.
Hope it helps!
Dear Patel
You need to consider the two dilutions
There is a formula tu use the two dilutions
Dear Divyesh,
first of all, you need to have repetitions (from 3 to 6) of your plates, so that you can do some statistics. This will show you, which series (in range 30-300) is better to be used. Perhaps if you repeated the 10^7 sample, it would have been closer to 300 colonies? You can't know that until you have more data. Technically you could use plates from different dilutions, but it is not advisable because it may cause an estimation error ( Article Estimating the precision of serial dilutions and viable bact...
). The counts from these two dilutions should be similar if you multiply the lower one by 10, although it is not always the case since some colonies can grow together or sometimes even inhibit each other. Also remember that cells may not be evenly distributed in your diluted sample, therefore it must be pipetted or shaken.Good luck!
dear, compare your diluted culture with 0.5McFarland standard.
You can use both of dilutions, by calculating the count using each of them, and calculate the average of the two counts.
The late Dr. Scott Sutton discusses this in the linked document below. Various approaches mentioned above are used - averaging all within the countable range, using only the larger colony count, 3 or more rep's trusting that these will better show erroneous individual plate counts. Pick one - document and justify. http://www.microbiologynetwork.com/counting-colonies.asp
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