18.7.1: Procedure Microbal Resistance to Cheotherapeutic Agents

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MATERIALS

Plasmid DNA (pAMP) on ice,
calcium chloride solution on ice,
2 sterile culture tubes,
1 tube of LB broth,
2 plates of LB agar,
2 plates of LB agar with ampicillin (LB/amp),
sterile 1 ml transfer pipettes,
sterile plastic inoculating loops,
bent glass rod, turntable,
alcohol,
beaker of ice,
water bath at 42°C.

ORGANISM

LB agar culture of Escherichia coli

MICROBIAL RESISTANCE PROCEDURE: demonstration

1. Label one LB agar plate "Transformed Bacteria, Positive control" and the other LB agar plate "Wild-Type Bacteria, Positive Control."

Label one LB/amp agar plate "Transformed Bacteria, Experiment" and the other LB/amp agar plate "Wild-Type Bacteria, Negative Control."

2. Label one sterile culture tube "(+) AMP" and the other "(-) AMP." Using a sterile 1ml transfer pipette, add 250 µl of ice cold calcium chloride to each tube. Place both tubes on ice.

Using a sterile plastic inoculating loop, transfer 1-2 large colonies of E. coli into the (+) AMP tube and vigorously tap against the wall of the tube to dislodge all the bacteria. Immediately suspend the cells by repeatedly pipeting in and out with a sterile transfer pipette until no visible clumps of bacteria remain. Return tube to the ice.

3. Repeat step 3 this time using the (-) AMP tube and return to the ice.

4. Using a sterile plastic inoculating loop, add one loopful of pDNA (plasmid DNA) solution to the (+) AMP tube and swish loop to mix the DNA. Return to the ice.

5. Incubate both tubes on ice for 15 minutes.

6. After 15 minutes, "heat-shock" both tube of bacteria by immersing them in a 42°C water bath for 90 seconds. Return both tubes to the ice for 1 minute or more.

7. Using a sterile 1ml transfer pipette, add 250 µl of LB broth to each tube. Tap tubes with your fingers to mix. Set tubes in a test tube rack at room temperature.

8. Using a sterile 1ml transfer pipette, add 100 µl of E. coli suspension from the (-)AMP tube onto the LB/amp agar plate labeled "Wild-Type Bacteria, Negative Control." Add another 100 l of E. coli from the (-)AMP to the LB agar plate labeled "Wild-Type Bacteria, Positive Control."

9. Using a bent glass rod dipped in alcohol and flamed, spread the bacteria thoroughly over both agar plates. Make sure you reflame the glass rod between plates.

10. Using a sterile 1ml transfer pipette, add 100 µl of E. coli suspension from the (+) AMP tube onto the LB/amp agar plate labeled "Transformed Bacteria, Experiment." Add another 100 l of E. coli from the (+) AMP to the LB agar plate labeled "Transformed Bacteria, Positive Control."

11. Immediately spread as in step 10.

12. Incubate all plates at upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

The procedure is summarized in Fig. 3.

Figure \(\PageIndex{1}\): Transfer of Plasmid-Mediated Resistance to Ampicillin in *E. coli*. (Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

Contributors and Attributions

Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)