MadSci Network: Microbiology

Re: growing colonies of bacteria

Date: Fri Mar 30 06:33:36 2001
Posted By: Jim Caryl, Grad student, PhD Biochemistry & Molecular Biology, University of Leeds
Area of science: Microbiology
ID: 985905835.Mi

Hi Helen

Sounds like and interesting project. Certainly there is a very simple but effective method you can use to enumerate the bacteria. What you need to do is a 10-fold serial dilution of your 1:1 saliva:mouthwash sample.

I will Assume you have 1 ml saliva + 1 ml mouthwash, i.e. 2 ml :o) I will also assume that you are using standard petri-dishes and a rich media such as LB agar. You must also make sure you do not 'flood' the plate, i.e. you need to identify the a volume of sample that you can apply to the plate, which following 20 minutes 'soak-up' time, can be inverted and incubated without any fluid (re: your sample) swilling around (or running off!) the plate.

If you decide to inoculate with only a fraction of your sample (see above), remember to factor it up to a meaningful unit, i.e. cfu/ml (cfu = colony forming units)

Also, don't forget a control, i.e. saliva without mouthwash, as you need to determine the efficacy of the mouthwash.

Enough of that....SERIAL DILUTIONS:

1. Assuming you have that 2 ml of saliva/mouthwash, remove a fraction of it and dilute it by 10. eg. say you remove 0.5 ml, you will add this to a solution (such phosphate buffered saline - PBS, or any other diluant) of volume 4.5 ml. Your sample (bacteria) are now diluted by 10 (Dilution 10). You can now plate out a fraction of this (the same volume as you have been plating out with the dilution 0).

2. We don't stop there however, from your 10-fold dilution remove another 0.5 ml and add this to another 4.5 ml of fresh diluant. Your sample (bacteria) are now diluted by 100! (Dilution 100) Again, you plate the appropriate amount.

3. You can go further and take 0.5 ml of this 100-fold dilution and add to another 4.5 ml diluant to get a 1000-fold dilution (Dilution 1000) get the idea :o)

You will then find that starting with dilution 0 you have your lawn; at dilution 10 you many see visible colonies, but they'll be too numerous to count; dilution 100 you may get your countable colonies, if not then with dil-1000. Once you have counted your colonies on the most appropriate dilution, you need to factor back up to your original sample, i.e. 10 colonies on a 100-fold dilution plate equals 1000 colonies in your original sample.

*Remember that by adding an equal volume of mouthwash you are effectively diluting the saliva sample, so factor this into your control, i.e. dilute your control by an equal amount, just not using mouthwash. Don't forget that you'll need to do serial dilutions of your control also.

Hope that helps...once you're confident you can enumerate the bacteria you can play with other variables such as temperature etc.

Have fun

Jim Caryl
MAD Scientist.

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