The purpose of this laboratory activity is to determine the number of microorganisms in a 1 ml sample. The sample could be soil, water, sewage, food, blood, urine, or almost anything (see chapter 3 in your text or one of the lab manuals). A sample is diluted and then rediluted several times with known volumes of sterile water (see the figure and discussion on serial dilution in your text). The object of this serial dilution is to reduce the number of organisms per ml so they can be counted (between 30 and 300 colonies) when a 1 ml sample is plated. The number of viable cells in the original sample can be estimated by multiplying the number of colonies in a plate by the dilution factor for the sample plated. For example, if 150 colonies are counted for 1 ml of a 1:10,000 dilution, there were150 x 10,000 = (1.5 x 10 to the 2nd power)(10 to the 4th power) = 1.5 x 10 to the 6th power = 1,500,000 viable cells in 1 ml of the original undiluted sample.
1. Practice your pipetting technique using a 1 ml pipette and a 10 ml pipette. Determine how to obtain and deliver 1 ml and 9 ml quantities of water accurately and without spills.
2. Set up a series of 9 sterile tubes and use a sterile 10 ml pipette to deliver 9 ml of sterile water to each.
3. Add exactly 1 ml of broth culture to the first tube using a sterile 1 ml pipette. Flush the pipette and mix the bacteria throughout the water by alternately drawing about 1 ml of the suspension into the pipette and squirting it back into the tube.
4. After 3 flushes draw up exactly 1 ml of dilution #1 and add it to tube #2. Flush and mix as before.
5. Draw up exactly 1 ml of dilution #2 and add it to tube #3. Continue making successive dilutions until all 9 tubes have been used.
6. Put 1 ml of the #9 dilution into a sterile plate. Repeat for the #8, #7, and #6 dilutions.
7. Add a tube of agar to each plate. Gently rotate the plate to mix the contents and then allow the agar to solidify.
8. After the agar has solidified, invert the plates and incubate at 37 degrees C for 24-48 hrs. Be sure to label the agar side of the plate.
9. After 48 hrs, remove the plates and observe the results. Choose a plate with more than 30 but less than 300 colonies for counting. It may help to place the plate over a sheet of graph paper and count the exact number of colonies.
10. Estimate the number of bacterial cells per 1 ml of the undiluted broth.
11. Obtain an agar slant and innoculate it with a small amount of cells from a colony on a plate.
12. Take an opportunity to make a Gram stain of a typical colony and any unusual colonies that are present on your plates.
Discuss in your notebook the implications of the results you observe.
We will form interest groups to investigate one of these projects.
Each group will spend time during lab developing an experimental protocol. Discuss your protocol with the instructor and prepare a short presentation to the class. The class will critiqie each protocol. It is important to identify all materials needed for next week and the source of each item.
Next week you will begin the experimental protocol.