Remember Matt? He’s still lying in the hospital bed waiting to hear what caused his illness and the local oyster industry is keen to get back into business.
What counting method will you use?
Unfortunately, a direct microscopic count or a count using a spectrophotometer would not tell us what we need to know – how many live Vibrio are in the water. So we choose a viable plate count.
What dilution scheme will you use?
We need to get plate counts between 30 and 300, and we think that the concentration maybe as high as 1 million (106) per mL, but it could be much lower. So, we should go ahead and do the first plate without even diluting! After that we could do standard serial ten-fold (1/10) dilutions.
How many dilutions do you need?
You definitely need at least 1 dilution, in case the number is somewhere in the 300 or 600 per mL range. Without a dilution, that wouldn’t be countable. If there are >300 colonies in a 1/10 dilution, there are >3000 actual cells per mL. As the count may well be much higher than this, it would be sensible to make more dilutions and not waste much time or energy. That way you should end up with a plate that has between 30 and 300 colonies. It might be more work in the short term, but in the long run it may save you having to repeat the experiment.
So, how many dilutions? Let’s assume the count might be as high as 106 bacteria per mL. If you prepare a 10-4 (1/10,000) dilution of the water, and plate 1mL, there should be 100 colonies on the plate after incubation. As the count may be lower, it would be advisable to plate the undiluted water, as well as samples of the 10-1, 10-2, 10-3 and 10-4 dilutions.