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Standard Curve To Get A Known Number of Cells Christine L. Case, Ed.D. Biology Professor Skyline College My home page

A standard curve is a graph relating a measured quantity (e.g., radioactivity, fluorescence, or optical density) to concentration of the substance of interest in "known" samples. You prepare and assay "known" samples containing the substance in amounts chosen to span the range of concentrations that you expect to find in the "unknown" samples. You then draw the standard curve by plotting assayed quantity (on the Y axis) vs. concentration (on the X axis). Such a curve can be used to determine concentrations of the substance in "unknown" samples

Preparation of a standard curve-to get a suspension containing a known number of bacteria:

1. Obtain a 24 - 48-hr culture of bacteria.

2. Standardize a spectrophotometer with sterile nutrient broth.

3. Determine the abs. of the culture.

4. Transfer 1.0 ml of the culture to a bottle containing 99 ml of nutrient broth to make a 1:100 dilution. Determine the abs. of the 1:100 dilution.

5. Transfer 1.0 ml of the 1:100 dilution to another 99-ml bottle of nutrient broth to make a 1:10⁴ dilution. Determine the abs. of the 1:10⁴ dilution.

6. Transfer 1.0 ml of the 1:10⁴ dilution to another 99-ml bottle of nutrient broth to make a 1:10⁶ dilution. Determine the abs. of the 1:10⁶ dilution.

7. Complete the following table:

	Abs. (Y axis)
1:100
1:104
1:106

A. Use the original undiluted culture.

1. Using a sterile 1-ml pipette, aspecially transfer 1.0 ml of the bacteria into a 99-ml dilution blank, label the tube "1:10²" and discard the pipette.

2. Using a different sterile 1-ml pipette, transfer 1.0 ml of the 1:100 dilution to a 99-ml dilution blank, label the tube "1:10⁴.

3. Mark the bottoms of 4 sterile nutrient agar plates with the dilution: 1:10⁵, 1:10⁴, 1:10³, 1:10².

4. Using a sterile 1-ml pipette, aspetically transfer 0.1 ml of the 1:10⁴ to the 10^-5 plate and 1.0 ml to the 1:10⁴ plate.

5. Aseptically transfer 0.1 ml of the 1:10² dilution to the 10³ plate and 1.0 ml to the 10² plate.

6. Sterilize a spreading rod by dipping it in alcohol and burning off the alcohol. Spread the bacteria over the surface of the agar using the sterile spreading rod. Start with the highest dilution. Why?

7. Incubate the plates inverted at 37°C for 24-48 hr.

8. Select the plate with between 25-250 colonies. Count the colonies and determine the number colony forming units per milliliter (cfu/ml) in the original sample. See actual plates: Sample 1 | Sample 2

B. Repeat steps 1-8 with each dilution prepared above to complete the following table:

	Abs.	cfu/ml (Y axis)
1:100
1:104
1:106

Plot abs. (Y axis) and cfu/ml (X axis).

How can you quickly use this graph to prepare a culture containing 1000 cells/ml from a turbid 24-hr culture of bacteria?