Question

Enumerating the viable and total cell concentration of a population of a microbial isolate can be a really laborious task for a microbiologist. Especially when a microbiologist studies the same isolate for several years, it often becomes practical to determine the relationship between optical density (OD) and cell concentration. Once this relationship (determined by a standard curve) is determined, the OD of an isolate in a broth can be routinely used to determine the population's concentration. Why must a standard curve be prepared for each isolate when using OD measurements to determine cell concentration? Also describe an experiment that would generate this type of standard curve.

Answer 1

For determination of the concentration of the bacterial cells in any given sample, it is essential that the O.D of the sample is compared with the standard curve to know the concentration of bacterial cells in the given sample .

A standard curve is prepared by using various known dilution of the single bacterial strain . The optical density is plotted on the y-axis against the concentration on the log x axis. Any point along the straight portion of the line can be used to calculate the viable cell count from that dilution factor.This curve is then used to know the bacterial cell concentration of a unknown sample of the same bacterial strain .

Each isolate differs with the other with respect to the cell morphology and its ability to aggregate or produce some secretions . Which might interfere with the O.D (optical density) measurement , hence altering the results therefore it is essential that the different standard curves are prepared for different isolates .

Experiment to generate standard curve

1. Grow the bacterial cells in suitable growth medium

2. After growth is completed . Centrifuge and seperate bacterial cells from the growth medium .

3. Decant the supernant and place the cells in suitable suspension medium .

4 . Prepare serial dilution .

5 . Using spectrophotometer take optical densities of all dlutions by putting samples in the special cuvettes provided with the instrument .

6. Do not forget to baseline the blank or suspension solution before taking O.D of dilutions .

7. Plot a graph -optical density is plotted on the y-axis against the concentration on the log x axis