10.5: Post-lab Questions

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Standard Plate Count

Using the formula V1D1=V2D2, find the final dilution of the following (remember that D1=1):
1. 10.0ml of a sample is transferred to 90.0ml of diluent
2. 4.0ml of a sample is transferred to 7.0ml of diluent
Using the formula determine the original cell density in the sample:\[\begin{align*} \text {Number of CFU:}\\ \text {OCD }&= \text { Amount plated } \times \text { dilution factor} \end{align*}\nonumber \]
1. You counted 72 colonies on a plate in your dilution series. The plate was inoculated with 1.0ml from a 10-4 test tube, what is the OCD?
2. You counted 235 colonies on a plate in your dilution series. The plate was inoculated with 0.1ml from a 10-7 test tube, what is the OCD?
1. You must make a 10-1 dilution using 5.0ml of your original sample. Show how you would do this. Hint: What volume of diluent do you need to add the 5.0ml to in order to perform and 10-1 dilution?
2. You must make a 10-2 dilution using 5.0ml of your original sample. Show how you would do this.
3. Show how you would make a 10-1 dilution using 4.0ml of your original sample.
4. Show how you would make a 10-2 dilution using 6.0ml of your original sample.
1. You are instructed to add 2.0ml of your original sample to 2.0ml of diluent. What is your dilution?
2. You are instructed to add 2.0ml of your original sample to 4.0 ml of diluent. What is your dilution?
3. In your own words, explain the difference between the above dilutions:
1.0ml of sample added to 99.0ml of diluent is a 10-2 dilution. What would happen if you added 0.1ml instead of 1.0ml? What would the final dilution be?
The colony count of a 10-8 plate in a dilution series is 162.
1. What is the bacterial count in the original sample?
2. What would the colony count be on the 10-7 dilution?
3. What would the colony count be on the 10-9 dilution?
Set up a dilution scheme, using the following materials, with a final plating of four plates with dilutions of 10-5 through 10-8. You must use exactly all 4 water blanks and plate four plates. Diagram the dilution scheme showing all tubes, plates, and dilutions.

Original sample

3 sterile water blanks (99mL)

1 sterile water blanks (9ml)

4 Petri plates

The Turbidimetric Method

Indirect Determination of Growth by Optical Density

Materials:

2 Groups per Table-
Your Dilution Series
Original E. coli
DI water (for blank)
TSB (for blank)
8 cuvettes and a cuvette rack (Dispose cuvettes with liquid in Biohazard trash can)
Kimwipes
5mL pipettes

Procedures:

You will measure the absorbance (OD) of each one of your dilutions, and the original E. coli broth, individually in a spectrophotometer. The spectrophotometer is a machine that shines a light through a liquid sample. A perfectly clear sample will transmit all the light. A sample that is cloudy (turbid) or colored will absorb some of the light and impede the transmission of the light. Therefore there is a direct relationship between turbidity and absorbance and an indirect relationship between turbidity and transmission. Do not write directly on cuvette tubes or cuvette rack. You may label the rack with tape.

Follow the directions on the Spectrophotometer for calibrating it. Ask your instructor if you do not know how to use the instrument, do not attempt to guess. It is important to blank the spectrophotometer with the sterile un-inoculated water to zero absorbance.
Follow the diagram below to set up the cuvettes.
1. Using a 5ml pipette, dispense 2ml of sterile water into 1 cuvette as your blank (for the dilution tubes).
2. Using a 5ml pipette, dispense 2ml of TSB into 1 cuvette as your blank (for the E.c. culture).
3. Using a 5ml pipette, dispense 2ml of the original E. coli culture into a cuvette.
4. Using a separate 5ml pipette for each dilution tube, dispense 2ml of each dilution into a corresponding cuvette.
5. Use the blank cuvette (water only) to blank the spectrophotometer. Place the cuvettes sequentially into the spectrophotometer and read the optical density of the original culture and each of your dilutions. Record the O.D values into the table, plot them on a graph, and answer the questions.

Results:

Fill out the following table:

Tube/cuvette	Dilution	O.D.
1. Original broth	1:1
2.
3.
4.
5.
6.

Graph the results on the attached graph. And answer the questions below. Be careful to set the axes correctly and space the dilutions correctly.

Turbidimetric Method Questions

Why is it important to have a plate count along with the absorbance measurement?
What is the relationship between the O.D. values graphed and the cell number from the SPC for your culture?

Contributors and Attributions

Kelly C. Burke (College of the Canyons)