I am conducting microbiological testing on QC samples recently. I did a test to get Total bacterial count (TBC) on a sample. And I used pour plate method. I am encountering some errors recently.
My 1:10 plate does not show any count but my 1:100 plate has 1 or 2 counts. Similar case, sometimes my 1:100 plate shows higher count than my 1:10 plate.
I am aware that this is not right. I could not find out what would be the possible reasons. How should I minimize the errors?
I would be grateful if anyone advice me on this. I would like to improve my technical skills.
Thank You in advance.
The sample is so diluted that the difference between 1:10 and 1:100 dilutions is not statistically significant: '1 or 2 is not higher than 0.' (Ideal dilutions yield 20 to 200 colonies per plate.)
Hi Kavitha,
As per my knowledge, this is happen due to pipette out non-homogeneous sample preparation. If the contaminant bacteria are present in the sample and so that if we are pipette out fixed amount of aliquot to prepare next dilution, we didnt know in that aliquot contaminant bacteria present or not ?
To overcome from this problem,follow this steps:
1. Put NC along with test sample (Standard practice) ,also put Duplicate plates for each dilution to get accurate result.
2. Vortex is most important step. when you are doing dilutions and plating aliquot in the plates, before that each and every time do vortex about 1-2 min. and then do next step.
It will make homogeneous suspension of dilutions and if the contaminant bacteria ll present in the sample they ll get homogenize and you ll get accurate result.
3. If you are analyze new sample which is not validated,then validate the method first. May be your sample shows inhibition so that after certain dilution, you got counts on plate.
I hope this ll help in your routine analysis.
It sounds like you've a) over-diluted the sample, 2) have a non-homogenous sample, or 3) have not enough growth of your strain. I'd start troubleshooting by culturing from all dilution steps, including the undiluted sample.
The higher the dilution of sample the more error is introduced into your results. try a spread plate method with at neat, 1:2, 1:5, 1:10 or even smaller dilutions. You should aim to count the least diluted sample that it is possible to enumerate on a plate . Usually a plate with around 200 colonies Above this is perhaps to much to count and below the dilutions will have more error. Sources of possible error can be as simple as not changing pipette tips between dilutions.
All good answers, but there is another thing you should consider... Is the product you are testing at all inhibitory to bacterial growth? For cosmetics, FDA CoBAM goes out to -6 on dilution as most cosmetics have a bacterial inhibitor. Sometimes it takes dilution of the inhibitory effect to see growth.
Also, if there is a possibility of agar above 42C, you could try pre-pouring the plates, then putting 1 ml on top of the set agar and using a plate spreader to cover the whole top.
It may be due to the antimicrobial activity of the product so by diluting the sample you eliminate this activity.
What should be done is to perform suitability of the counting method.
It may be due to the container you had diluted in; so in this why you should run negative control.
I hope this will help you Kavitha Subramaniam .
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