Question

When your instructor adds \(1.0 \mathrm{~mL}\) of the \(E\). coli culture to the heated flask, he/she will start the stopwatch. This is \(\mathrm{T}_{0}\). Immediately swirl the flask to disperse the sample and remove \(1.5 \mathrm{~mL}\) with your pipette. If possible, do this step without removing the flask from the water bath. Begin the following serial dilution: \- Add the \(1.5 \mathrm{~mL}\) of broth from the flask to tube \(1 .\) \- Transfer \(1.0 \mathrm{~mL}\) from tube 1 to tube \(2 ;\) mix. Discard the pipette. Use a clean pipette for each transfer from here forward. \- Transfer \(1.0 \mathrm{~mL}\) from tube 2 to tube \(3 ;\) mix. \- Transfer \(1.0 \mathrm{~mL}\) from tube 3 to tube \(4 ;\) mix. \- Transfer \(1.0 \mathrm{~mL}\) from tube 4 to tube \(5 ;\) mix. \- Transfer \(1.0 \mathrm{~mL}\) from tube 5 to tube \(6 ; \mathrm{mix}\). \- Transfer \(1.0 \mathrm{~mL}\) from tube 6 to tube \(7 ;\) mix. \- Transfer \(0.1 \mathrm{~mL}\) from tube 1 to plate 1 and spread according to the spread-plate procedure. \- Transfer \(0.1 \mathrm{~mL}\) from tube 2 to plate 2 and spread. \- Transfer \(0.1 \mathrm{~mL}\) from tube 3 to plate 3 and spread. \- Transfer \(0.1 \mathrm{~mL}\) from tube 4 to plate 4 and spread. \- Transfer \(0.1 \mathrm{~mL}\) from tube 5 to plate 5 and spread. \- Transfer \(0.1 \mathrm{~mL}\) from tube 6 to plate 6 and spread. \- Transfer \(0.1 \mathrm{~mL}\) from tube 7 to plate 7 and spread.

Short answer

Follow the steps of serial dilution, transferring specified volumes from tube to tube, and plate 0.1 mL from each diluted tube onto separate plates.

Step-by-step solution

Prepare Initial Culture

Add 1.0 mL of the E. coli culture to the heated flask, start the stopwatch (T0), and immediately swirl the flask to disperse the sample.

Remove Sample and Begin Dilution

Remove 1.5 mL from the flask using your pipette, preferably without taking the flask out of the water bath. Add this 1.5 mL of broth to tube 1.

Serial Dilution Step 1

Using a clean pipette, transfer 1.0 mL from tube 1 to tube 2 and mix well.

Serial Dilution Step 2

With another clean pipette, transfer 1.0 mL from tube 2 to tube 3 and mix thoroughly.

Serial Dilution Step 3

Using a fresh pipette, transfer 1.0 mL from tube 3 to tube 4 and ensure proper mixing.

Serial Dilution Step 4

Transfer 1.0 mL from tube 4 to tube 5 with a clean pipette and mix the contents in tube 5.

Serial Dilution Step 5

Use a new pipette to transfer 1.0 mL from tube 5 to tube 6 and mix well.

Serial Dilution Step 6

Transfer 1.0 mL from tube 6 to tube 7 using a clean pipette and mix thoroughly.

Plate the Samples Step 1

Transfer 0.1 mL from tube 1 to plate 1 and spread it across the plate following the spread-plate procedure.

Plate the Samples Step 2

Using a separate pipette, transfer 0.1 mL from tube 2 to plate 2 and spread.

Plate the Samples Step 3

Transfer 0.1 mL from tube 3 to plate 3 using a clean pipette and then spread it.

Plate the Samples Step 4

Using a new pipette, transfer 0.1 mL from tube 4 to plate 4 and spread the sample.

Plate the Samples Step 5

Transfer 0.1 mL from tube 5 to plate 5 with a clean pipette and spread it over the plate.

Plate the Samples Step 6

Using a separate pipette, transfer 0.1 mL from tube 6 to plate 6 and spread.

Plate the Samples Step 7

Finally, transfer 0.1 mL from tube 7 to plate 7 using a new pipette and spread it as well.

Key concepts

E. coli Culture

E. coli is a commonly used bacteria in microbiology laboratories. Its genetic and structural simplicity makes it ideal for various experiments. Growing an E. coli culture involves incubating it in a nutrient-rich medium, allowing the bacteria to multiply rapidly. While handling E. coli, it is crucial to maintain a sterile environment to avoid contamination. Always use clean lab equipment and follow proper aseptic techniques to ensure the integrity of your cultures.

Spread-Plate Procedure

The spread-plate technique is used to evenly distribute a bacterial sample across an agar plate. Start by pipetting a small volume of your diluted sample onto the center of the plate. Using a sterile, L-shaped spreader, gently spread the sample over the entire surface of the agar. Rotating the plate while spreading helps in achieving even distribution. After spreading, cover the plate with its lid and incubate it in an appropriate environment for bacterial growth. This technique helps in isolating individual colonies, which can be further used for identification or quantification.

Microbiology Laboratory Techniques

Microbiology laboratories employ various techniques to study microorganisms. These include:
- Aseptic techniques: Ensures sterile conditions to avoid contamination.
- Culture methods: Growing microorganisms in controlled environments.
- Staining procedures: Microscopic examinations of bacteria.
- Molecular methods: DNA analysis for identifying and studying genetic material.
Consistently practicing these techniques is vital for obtaining accurate and reproducible results in microbiology experiments.

Pipetting Techniques

Proper pipetting techniques are essential for accuracy in experiments. Here are some tips:
- Always use a pipette appropriate for the volume you are measuring.
- Pre-wet the pipette tip by drawing and expelling the sample liquid a few times.
- Hold the pipette vertically when drawing the sample to ensure the correct volume.
- Never touch the pipette tip to avoid contamination.
Using the correct technique ensures consistent and reliable measurements, crucial for the success of your experiments.

Water Bath Usage

A water bath is used to maintain a constant temperature for samples. To use a water bath effectively:
- Set the desired temperature on the water bath and allow it to reach stability.
- Place your samples in a container that fits well within the water bath chamber.
- Ensure the water level covers the sample containers but does not overflow.
- Always monitor the temperature to maintain consistency.
Water baths are instrumental in processes like heating bacterial cultures or maintaining enzyme activity.