Skip to Main Content
Table 1.
Laboratory instructions for students.
StepsLaboratory Instructions
1 Obtain 10 prepared test tubes for your group. One of these is a control; do not add anything further to this tube. To the 3 tubes marked “1.5×” add 10 mL of your wastewater sample. To 3 other tubes add 0.1 mL of wastewater, and to the remaining 3 tubes add 1 mL of wastewater. Before you add your samples, make sure to label your tubes so that you remember the dilution of each tube. Replace the caps on your test tubes once finished, to help create an anaerobic environment. 
2 Incubate your tubes at ~35°C for 24–48 hours. 
3 Observe your test tubes. Those demonstrating both yellow liquid and gas in the Durham tube are positive results. Record your data in a table that you construct. 
4 You will now begin the process of inoculating Petri dishes with bacteria from only the test tubes that had a double-positive (gas plus color change) result in the presumptive test. Make sure that the dishes are properly labeled to keep track of your samples (note: it is possible to divide each Petri dish into 3 or 4 subsections to conduct multiple tests, if needed).
Flame sterilize an inoculation loop. Once cooled, dip it into the test tube to acquire a sample. Gently streak the sample onto the Petri dish. Repeat this step as necessary until all positive test tubes have been sampled and streaked. Make sure to flame sterilize, and then cool, your loop before obtaining each sample. 
5 Place the cover on your Petri dish and store it upside down. These will now be incubated for ~24 hours at 35°C. 
6 Observe your Petri dishes. Look for E. coli colonies, which will turn metallic green or blue-black. If a sample contains at least one E. coli colony, you count that as a positive result. This means that the test tube from which the sample originated contains E. coli. Of the 9 experimental test tubes that you originally started with, count which were positive for E. coli.
You will use these data and the “most probable number” table to calculate the number of bacteria in your sample. Your instructor will ask each group to share their results so that data can be collated for the entire class. 
StepsLaboratory Instructions
1 Obtain 10 prepared test tubes for your group. One of these is a control; do not add anything further to this tube. To the 3 tubes marked “1.5×” add 10 mL of your wastewater sample. To 3 other tubes add 0.1 mL of wastewater, and to the remaining 3 tubes add 1 mL of wastewater. Before you add your samples, make sure to label your tubes so that you remember the dilution of each tube. Replace the caps on your test tubes once finished, to help create an anaerobic environment. 
2 Incubate your tubes at ~35°C for 24–48 hours. 
3 Observe your test tubes. Those demonstrating both yellow liquid and gas in the Durham tube are positive results. Record your data in a table that you construct. 
4 You will now begin the process of inoculating Petri dishes with bacteria from only the test tubes that had a double-positive (gas plus color change) result in the presumptive test. Make sure that the dishes are properly labeled to keep track of your samples (note: it is possible to divide each Petri dish into 3 or 4 subsections to conduct multiple tests, if needed).
Flame sterilize an inoculation loop. Once cooled, dip it into the test tube to acquire a sample. Gently streak the sample onto the Petri dish. Repeat this step as necessary until all positive test tubes have been sampled and streaked. Make sure to flame sterilize, and then cool, your loop before obtaining each sample. 
5 Place the cover on your Petri dish and store it upside down. These will now be incubated for ~24 hours at 35°C. 
6 Observe your Petri dishes. Look for E. coli colonies, which will turn metallic green or blue-black. If a sample contains at least one E. coli colony, you count that as a positive result. This means that the test tube from which the sample originated contains E. coli. Of the 9 experimental test tubes that you originally started with, count which were positive for E. coli.
You will use these data and the “most probable number” table to calculate the number of bacteria in your sample. Your instructor will ask each group to share their results so that data can be collated for the entire class. 
Close Modal

or Create an Account

Close Modal
Close Modal