Re: How can I count the number of microorganisms in a drop of river water?

Hi Taylor -

You can find some additional information to help you with a search for CFU agar on our search engine.

The best way to answer your question involves analyzing the number of "colony forming units" or CFU in a defined volume of the water, commonly 0.1 milliliters (mL). To do this spread 0.1mL of your sample on a plate, incubate it, and count the number of colonies the next day, and after 2 days of incubation. Each colony grows from a single cell, so the number of colonies is related to the number of cells you put on the plate.

This procedure will not pick up *all* bacteria in a sample. Your culture conditions will likely be performed in ambient atmosphere, and at ~25°C (room temperature) or 37°C (body temperature). Some bacteria will not grow well, or at all, in the concentration of oxygen gas present in the air, or at the given temperature(s) you select. However, these conditions should be adequate for most species commonly encountered in environmental water sources. Furthermore, if you use *selective* media, you may only be culturing for particular kinds of bacteria. We commonly use an agar media called MacConkey agar to grow coliform bacteria such as E. coli and related species. If you use a nutrient media such as Brain-Heart Infusion, Sheep's Blood Agar, or Luria-Bretani (LB) agar, you may pick up additional species.

If you have a lot of bacteria, you may get a "lawn" of microbial growth on the plate, something of little help, other than it tells you that you probably have >5,000 CFU in your sample, or a swarming species of bacterium that took over the plate. If this happens, perform a dilution series to determine how many bacteria are in your sample.

    Starting tube     10X dilution    100X dilution    1000X dilution
       0.1mL      -->    +0.9mL   
         |               =1.0mL
         V             take 0.1mL  -->     +0.9mL
   plate 0.1mL to          |               =1.0mL
      agar plate           V              take 0.1mL -->     +0.9mL
                     plate 0.1mL to           |              =1.0mL  
                        agar plate            V                 |
                                        plate 0.1mL to          V
                                           agar plate      plate 0.1mL to
                                                             agar plate


    `0.1 or 10-1          10-2                 10-3             10-4

                         FINAL DILUTIONS PER MILLILITER

1. Label each of your plates with the dilution plated (as in the figure above), the date and source of the water.
2. Incubate the plates for 18 hours.
3. Count the number of colonies on your plate - it's best to use plates in the dilution series that have tens or only a few hundred colonies, numbers you can easily counte.
4. Divide that number by the dilution of 10 that you made, so if you have 33 colonies on the 10^-2 plate, you divide 33 by 0.01. It turns out that 33/0.01, is the same as 33 X 100, so that means you started with 3300 colonies per mL of your original water source. You can look at this calculation as:

        # colonies X 10+dilution
   

5. Put the plates back in the incubator and let them grow for another day.
6. Count the colonies again to see if any new ones grew in the additional time.