Part 1: Teacher Demonstration

1. Divide the class into teams of 4 students each.
2. Distribute Student Lab Sheet: Coliform Counts to each student.
3. Have students assist with the preparation of the VRBA and hamburger solution.

Preparation of the Violet Red Bile Agar (VRBA)

1. Prepare the VRBA as per instructions on the label.
2. In a flask, add 41.5 grams of agar to 1 L of water (use the best water available, e.g., distilled, etc.)
   • A general rule is that it takes about 20 ml per Petri dish. This will tell you about how many ml of agar to prepare.
3. Once you have the VRBA agar in the flask, bring it to a slow boil. Make sure the agar is at a rolling boil. Be very careful, as it can flash boil over the top very quickly.
   • The agar should be translucent with no undissolved granules of agar on the sides of the flask.
4. Cool the agar slightly. Caution: It will harden if cooled too long. Monitor the agar temperature as it cools. The best temperature for pouring is 111–115° F (44–46° C). A water bath set at this temperature would be ideal.

Preparation of the Hamburger Solution

1. Add 90 ml of sterile saline solution to the blender.
2. Weigh out 10 grams of hamburger on sterile aluminum foil. (Wear safety gloves.)
3. Add the hamburger to the sterile saline solution in the blender. Blend for about 1 minute on high. The concentration of the hamburger is 1 in 10.

Part 2: Student Activity

Have Each Team of Students:

1. Prepare Test Tubes and Petri Dishes
   • Label 5 Petri dishes on the bottom: “10,” “100,” “1,000,” “10,000,” and “control.”
   • Set up 3 test tubes and label them: “100,” “1,000,” and “10,000.”
   • Add 9 ml of sterile saline solution to each of the 3 test tubes.
2. Inoculate Petri Dishes (see Student Lab Sheet: Coliform Counts)
   • 1 in 10
     • Pipette 1 ml of the 1-in-10 hamburger solution directly into the Petri dish marked “10.”
     • Carefully swirl the dish to cover the surface. Cover the Petri dish.
   • 1 in 100
     • Pipette 1 ml of the 1-in-10 hamburger solution into the test tube marked “100.” Now the concentration of the hamburger is 1-in-100.
     • Thoroughly mix the solution by holding the test tube by the top and gently striking the bottom with the finger on the other hand for about 5 strikes.
     • Pipette 1 ml of this solution into the Petri dish marked “100.”
   • Repeat this procedure for the 1-in-1,000 and 1-in-10,000 dilutions.
   • Have students draw the dilution and plating scheme in their food safety portfolios.
3. Add the Agar to the Petri Dishes Containing the Hamburger Solutions
   • Help the students learn to properly pour the agar out of the flask into the Petri dishes. Show them how to flame the mouth of the flask.
   • Pour about 10 ml of agar into each Petri dish containing the hamburger solution and then swirl the dish to mix and evenly cover the bottom of the dish.
   • As soon as the agar is solidified, pour in another 4 to 6 ml of agar and swirl again to spread evenly.
   • Pour a control dish to make sure the agar is not contaminated.
   • Store the dishes upright until the agar is solid. Then invert the dishes, seal them with Parafilm, and place in the incubator at 95° F (35° C) or let the dishes sit at room temperature (away from the sun) overnight. Examine the Petri dishes for growth the next day and record observations.
4. Record Data
   • Students should examine the sealed Petri dishes for the presence of colonies. Remind them to be sure that when counting the colonies, they multiply by the dilution factor. This should give relative numbers of coliforms in the hamburger.
   • Students should report their findings to the class for analysis and discussion.