I use Rose bengal agar cloramfenicol for search yeast and molds for inclusion in petri plat. Which is the method correct for count? I'm not sure if i count everything or only the bigger one?
Yes, you have to count every individual colony.
However, in practice the question is more, what is deemed a countable plate and how important is it. In many laboratories you will find that technicians and scientists won't bother to count each and every colony on the plate if you have a visibly high number of colonies. They will for example simply say (with a bit of experience), the count for this plate is greater than 300 (write
Nevertheless, you will of course still need a count, so it is common practice to do one of the following:
- Split your plate into sectors of equal size (you can draw lines on the bottom of the plate), count all colonies in one sector and then multiply this number by the number of sectors you have divided in. So, say, you have split your plate into 4 sectors (quadrants) by drawing a big cross on it, you would count the colonies in one quadrant and then multiply by 4. This is obviously an extrapolation.
- The other option, is to dilute your sample until you get it within countable range. So you could do a serial dilution, put it onto plates and then once your organisms have grown you can count the entire plate and then multiply by the dilution factor. So, say, for example you have your sample in an enrichment broth and do two 1/10 dilutions (1/100) and then plate out 0.1mL (counts as 1/10), you have a final dilution factor of 1000. So you would need to multiply the count that you get on this imaginary plate by 1000.
In your sample you obviously have a variety of different organisms. It is probably advisable to check official methods for what they recommend. Here is an excerpt from the official BAM method for Yeasts, Molds and Mycotoxins:
Count plates after 5 days of incubation. If there is no growth at 5 days, re-incubate for another 48 h. Do not count colonies before the end of the incubation period because handling of plates could result in secondary growth from dislodged spores, making final counts invalid. Count plates containing 10-150 colonies. If mainly yeasts are present, plates with 150 colonies are usually countable. However, if substantial amounts of mold are present, depending on the type of mold, the upper countable limit may have to be lowered at the discretion of the analyst. Report results in colony forming units (CFU)/g or CFU/ml based on average count of triplicate set. Round off counts to two significant figures. If third digit is 6 or above, round off to digit above (e.g., 456 = 460); if 4 or below, round off to digit below (e.g., 454 = 450). If third digit is 5, round off to digit below if first 2 digits are an even number (e.g., 445 = 440); round off to digit above if first 2 digits are an odd number (e.g., 455 = 460). When plates from all dilutions have no colonies, report mold and yeast counts (MYC) as less than 1 times the lowest dilution used. Isolate individual colonies on PDA or MA, if further analysis and species identification is necessary.
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