Teacher Preparation:

 

1 - 5 days before lab day:     

 

1. Prepare/Obtain sterile LB (Luria-Bertani) media for liquid culture

 

2. Prepare/Obtain 20 sterile LB agar plates for solid culture (this can be done by the students themselves)

 

1 day before lab day:

 

1. Prepare an "overnight culture" of E. coli TG1

 

a. Label a 15 mL sterile conical test tube "E. coli TG1 overnight"

 

b. Using a sterile inoculating loop, transfer one colony from the E. coli TG1 starter plate to the E. coli TG1 overnight test tube. 

 

1.) Agitate the inoculating loop in the media to ensure the bacteria are transferred. 

2.) Quickly recap the E. coli TG1 overnight test tube and discard the inoculating loop. 

3.) Incubate the E. coli TG1 overnight test tube at 37 °C with agitation.  Agitation of the test tube is important since the E. coli TG1 require oxygen to survive and replicate.  Agitation is not necessary yet desired.

4.) Incubation of the E. coli TG1 overnight test tube should last 18-24 h. 

 

*** You can store the E. coli TG1 starter plate in a refrigerator for future uses. 

 

2. Prepare solutions for chemistry

 

a. Heat a water bath to a gentle boil

 

b. Label 6 (six) 15 mL vessels (vial or test tube)

 

"A1," "A2," "A3," "B1," "B2," and "B3"

 

 

c. Add the appropriate mass of each chemical to each test tube according to the table below:

 

ID

Compound Name

Molecular Weight (MW)

mass to add (mg)

A1

2-nitrobenzaldehyde

151.12

54

A2

5-nitro-2-furaldehyde

141.09

51

A3

3-nitrobenzaldehyde

151.12

54

B1

4-bromophenylhydrazine hydrochloride

223.52

80

B2

4-cyanophenylhydrazine hydrochloride

169.62

61

B3

Aminoguanidine bicarbonate

136.11

49

 

d. To each test tube, add 12 mL of deionized water. 

 

(These solutions are now 0.03 M each.  Morality refers to the concentration in a liter of water. It is important the concentrations of the solutions be correct. If, in weighing out the chemical, you accidentally add too much or too little, adjust the volume of water so as to obtain the correct concentration.  For instance, if you weighed out 60 mg of A1, you will want to add 60/54 * 12 = 13.3 mL of water.)

 

e. Cap the tubes and place them in the boiling water bath.  Most of the chemicals will not dissolve in water at room temperature.  After about 3-5 min of heating, the solids should dissolve when you shake the tubes.

 

f. Store the solutions at room temperature until the lab day.

 

Lab day:         

 

Prepare 1/100 dilutions of "overnight culture"

 

1. Label a 15 mL sterile conical test tube "E. coli TG1 1/10"

 

2. Label 6 15 mL sterile conical test tubes "E. coli TG1 1/100"

 

3. Using the gradations on the sides of the tubes to measure, pour ~9 mL of sterile LB media into each of the seven test tubes. 

4. Pour 1 mL of the E. coli TG1 overnight culture into the tube marked E. coli TG1 1/10 using the gradations on the side of the tube to measure.  Cap this tube and invert it several times to mix.

 

5. Pour 1 mL of the E. coli TG1 1/10 dilution into each of the tubes marked E. coli TG1 1/100 Using the gradations on the sides of the tubes to measure. 

 

6. Cap the 1/100 dilution and invert them several times to mix.  Keep the 1/100 dilution at room temperature until they are used in the experiment.

 

Preparing student group materials

 

1. Each student group will need the following materials:

      

       6        - 1.5 mL Eppendorf tubes

       1        - Test Tube rack

       7        - transfer pipettes

       1        - Thin-tipped marker (for labeling)

       3       - Sterile LB agar plates

       1       - Glass Pasteur pipette

  1     - 1/100 dilution of E. coli TG1 overnight culture