Hello
I really hope somebody can help me with that, because I have no clue.
Basically I am about to create a growth curve of Corallococcus corallodies, as I don't know how long is exponential phase in that organisms and nobody knows.
I will prepare 10 tubes of cells growing in liquid media with 1 day interval in incubator on shaker.
From each sample on day 10 I will take 1 mL for dilution series.
Spectrophotometry is, as I suppose, not a problem as I am just going to measure each sample against blank, which is medium. (should I repeat measurements or once is enough?)
But now I have to create dilution series, and my problem is: I don't know how much should I dilute each of these samples, and how many dilutions per sample should I actually plate?
I assume that samples 1-3 days old will need less dilution than samples 7-10, basically because if I use the same dilutions for each then on former samples I will hardly see anything, and on latter I will not be able to count colonies. But maybe that thinking is wrong ?
And I was thinking about how many plates should I create for each sample, i.e. sample 1 day could have 4 plates of 4 dilutions - 10 -1, 10 -2, 10 -3 and 10 -4, whereas sample 10 day - 10 -6, 10 -7, 10 -8 and 10 -9.
I hope this all make sense . Thank you very much for any help. :)
10/25/18
Dear Jacek,
Your approach is OK. Unfortunately, if you don't know how many CFU there are in a suspension, you need to make several dilutions as you described, and plate all of them to get plates with enough CFU for an accurate count. Preparing 3-4 plates of each dilution should be adequate. One suggestion: If you have a hemocytometer and the your cells are large enough, you can make a dilution of your cells and count them under a microscope. This will be total count (live + dead cells). Once you know this, you will know what dilutions to make for plating to get live counts.
Another suggestion: If you will be growing the organism for 10 days, I wouldn't wait until the 10th day to do all the dilutions & plating. Some of the cells sampled in the early phase of growth may die during storage before they're plated on day 10, resulting in an inaccurate count. Better to dilute and plate cells on a day to day basis for the entire growth cycle to avoid this problem.
I hope this information helps you.
Bill Colonna Center for Crops Utilization Research, Iowa State University, Ames, Iowa, USA [email protected]
Your approach is sound but labor intensive. What I would suggest is for you to do a pilot study first. Start with a few tubes (maybe 5) that you culture over 10 days. Every day, take 1 mL out from one of the tubes (alternate between the tubes so they have more or less the same volume over the culture time frame) and check the OD using a spectrophotometer. Make a growth curve based on the OD, so you have an idea of the growth kinetics. If the culture crashes in 5 days, there is no need to continue for 10 days.
Once you narrow down you culture time frame, you can go to the next phase. Start with enough tubes to last you the entire culture time frame (5 days or 10 days or longer). Every day, take 1 mL out from one of the tubes and check the OD using a spectrophotometer. You also do serial dilutions and plate 3 dilutions to get a rough bacterial count (to correlate with OD). In the beginning, do 101, 102 and 103 dilutions. As you see the OD go up and the cultures expand, you could start doing 104, 105 and 106. If you see that your cultures are growing slow, plate lower dilutions. You could do a single plate and then repeat the experiment or you could do multiple plates for each dilution.
These are very helpful responses! Thank you very much W.J. Colonna and Julie Joseph
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