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Table 2.
Lab materials and instructions for preparation.
Materials 
  • Phenol red lactose broth

  • Levine eosin-methylene blue (EMB) agar

  • Petri dishes

  • Pipettes

  • Test tubes

  • Test-tube caps

  • Test-tube racks

  • Durham tubes

  • Safety gloves

  • Incubator

  • Inoculator loops

  • Bunsen burner

  • Biohazard bag

  • 10% bleach solution

 
Instructions 
Nine experimental test tubes are needed per group of students, plus one control. To reduce the amount of materials needed, 4 students per group is recommended. 
Create the phenol red lactose broth using the easy-to-follow instructions on the package. Add 5 mL of the broth to 6 of the 9 experimental test tubes, plus the control. For the remaining 3, however, you want to use 5 mL of broth that is 1.5× strength. This is done to provide enough lactose for the tubes receiving 10 mL of the wastewater sample. Label these tubes as “1.5×”. Thus, each group should have 7 tubes of normal strength broth and 3 tubes of 1.5× strength. An inverted Durham tube should also be placed in each test tube so that the open end of the tube is face down and the tube is at least halfway submerged in the broth. Then the test tubes should be autoclaved to sterilize them before use. The autoclave process will completely fill the Durham tubes with solution. Remove the test tubes from the autoclave and cover them with sterile test-tube caps. These caps will later be used to create an anoxic environment for anaerobic metabolism. 
Lastly, using the instructions on the package, prepare enough Levine EMB agar for 3 Petri dishes per group, plus a few backups. During the lab, ensure that students follow proper safety guidelines, including use of gloves when handling wastewater samples, proper disposal or sterilization of biohazardous waste (which includes anything that has touched wastewater; consult your institution's specific safety protocols), and disinfection of lab tables using a bleach solution. 
Materials 
  • Phenol red lactose broth

  • Levine eosin-methylene blue (EMB) agar

  • Petri dishes

  • Pipettes

  • Test tubes

  • Test-tube caps

  • Test-tube racks

  • Durham tubes

  • Safety gloves

  • Incubator

  • Inoculator loops

  • Bunsen burner

  • Biohazard bag

  • 10% bleach solution

 
Instructions 
Nine experimental test tubes are needed per group of students, plus one control. To reduce the amount of materials needed, 4 students per group is recommended. 
Create the phenol red lactose broth using the easy-to-follow instructions on the package. Add 5 mL of the broth to 6 of the 9 experimental test tubes, plus the control. For the remaining 3, however, you want to use 5 mL of broth that is 1.5× strength. This is done to provide enough lactose for the tubes receiving 10 mL of the wastewater sample. Label these tubes as “1.5×”. Thus, each group should have 7 tubes of normal strength broth and 3 tubes of 1.5× strength. An inverted Durham tube should also be placed in each test tube so that the open end of the tube is face down and the tube is at least halfway submerged in the broth. Then the test tubes should be autoclaved to sterilize them before use. The autoclave process will completely fill the Durham tubes with solution. Remove the test tubes from the autoclave and cover them with sterile test-tube caps. These caps will later be used to create an anoxic environment for anaerobic metabolism. 
Lastly, using the instructions on the package, prepare enough Levine EMB agar for 3 Petri dishes per group, plus a few backups. During the lab, ensure that students follow proper safety guidelines, including use of gloves when handling wastewater samples, proper disposal or sterilization of biohazardous waste (which includes anything that has touched wastewater; consult your institution's specific safety protocols), and disinfection of lab tables using a bleach solution. 
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