Summer Science Academy Experiment:
Estimating the Number of Bacteria on a Solid Surface
TIME NEEDED: ½-1 hour(day 1); ½hour (day 2)
Because of their very small size, counting the number of bacteria in a sample can be difficult at best. Although direct counts are possible with a microscope, they require a lot of time and expertise. An easier method is to spread bacteria over a wide area (i.e. nutrient agar plate) and count the number of colonies that grow. If the bacteria are spread out enough, each bacterial cell in the original sample should produce a single colony. Usually, bacterial samples must be diluted considerably to obtain reasonable counts.
When one intends to determine the number of cells in a bacterial culture one way of doing this is by carrying out serial dilution. Since bacterial cell numbers are usually very high in your original sample, plating out this sample in an undiluted fashion would just lead to the creation of a bacterial lawn (a smear of many, many individual bacteria colonies that are all growing next to or on top of one another).
Bacterial cell numbers need to be reduced, which is done by repeatedly diluting the amount of bacteria you have in your sample. A small amount of bacteria sample is mixed with a diluent solution (such sterile broth), and then successive dilutions are made. A small amount of each of the diluted bacteria samples is then spread onto an agar plate. The numbers of bacteria colonies that grow on each plate are counted. By working backwards using multiplication with the "dilution factor" (the number of times that you have diluted the bacteria sample with the diluent solution), you will be able to make a determination of the numbers of bacteria in your original sample.
This method has some drawbacks, however. Injured bacteria may not always form colonies. Also, since there is no single diluent solution that supports the growth of all types of bacteria, some bacteria may be left out of any given counting procedure.
Tubes containing 1 ml of sterile 0.9% NaCl (sterile saline)
Tubes of sterile broth (2 per sample)
Agar plates (3 per sample)
Follow this procedure for each of the surfaces you want to test:
For each surface you will test, label 3 agar plate as follows: "direct count", "1:10" and "1:100". Also, label 2 tubes of sterile broth for each sample: one will be "1:10" and the other will be "1:100". Make sure you also label the plates and tubes with the type of sample you will be collecting.
- Unwrap a sterile swab. Dip it into the tube of sterile saline solution. Gently press the wet swab against the inside of the saline solution tube to get rid of any excess saline. Take the wet swab out of the tube of saline. Be careful not to let the wet cotton tip touch anything.
- Gently rub the wet swab against one half of the surface you want to test bacteria from. (such as on half of the phone receiver, or one side of your locker handle). Now take this swab and gently rub it onto the entire surface of one of your agar plate. Be careful not to dig into the agar.
- Unwrap another sterile swab. Again, dip it into the sterile saline solution and squeeze out any excess saline against the side of the tube. Use this 2nd wet swab to remove a sample from the 2nd half of your solid surface. You will now be diluting this sample with sterile broth in the next steps.
- Take this 2nd swab and dip it into one of your tubes of sterile broth labeled "1:10". (this will become a 1:10 dilution of your sample) Swish the swab around on the broth to mix in any bacteria. Remove the swab and discard it. Screw the cap on the broth tube and shake it upside down a bunch of times to evenly mix the bacteria.
- Unwrap another sterile swab and dip it into the 1:10 dilution tube that you just prepared. Squeeze out the excess liquid against the side of the tube as before. Gently rub this swab on the surface of the agar plate labeled "1:10". Discard this swab when you are done.
- Unwrap another sterile swab. Dip it into the tube labeled 1:10. This time you should not squeeze out any of the excess liquid. Instead, take the very wet swab out of the 1:10 tube and quickly put it into the tube labeled "1:100". Swish the swab around to mix in the bacteria, then remove the swab and discard it. Cap the tube and shake the tube upside down a bunch of time to mix the bacteria. You have now made a 1:100 dilution of your original sample.
- Unwrap another sterile swab and dip it into the 1:100 dilution liquid. Squeeze out the excess liquid and then gently rub the swab all over a plate labeled "1:100". Discard this swab when you are done.
- Repeat all these steps for each of the surfaces you want to test. You will need 3 agar plates for each (a "direct" count and a "1:10" and "1:100" count plate). When you are all through, put the agar plates in a 37 degree incubator or another suitable warm place (such as on top of the fridge). You should see bacteria in 24-48 hours.
- Count the number of bacteria colonies that appear on each of the plates that has between 30 and 200 colonies. Any plate which has more than 200 colonies is designated as "too many to count" (TMTC). Plates with fewer than 30 colonies do not have enough individuals to be statistically acceptable.
- To compute the estimated number of bacteria on the surface that you tested, use the following formula:
B = N/D
B = number of bacteria
N = number of colonies counted on a plate
D = dilution factor (either 1, 10 or 100)