Measurement of Bacterial

Growth

Measuring Growth
Direct Method

Plate Count
Filtration
Direct Microscopic Count
Indirect Method

Turbidity
Metabolic Activity
Dry weight

Direct Method

1.Plate count: (viable count)

Most frequently used method of measuring bacterial
populations.
Inoculate plate with a sample and count number of colonies.
Advantages:
Measures viable cells

Disadvantages:
Takes 24 hours or more for visible colonies to appear.
Only counts between 25 and 250 colonies are accurate.
Must perform serial dilutions to get appropriate numbers/plate.

Plate Counts: Perform serial dilutions of a sample

Example:
32 colonies , 1/10000
Plate Count : 32x10000/ml = 320,000/ml

Filtration

2. Filtration:
Used to measure small quantities of bacteria.
Example: Fecal bacteria in a lake or in ocean water.

Procedure:
A large sample (100 ml or more) is filtered to
retain bacteria.
Filter is transferred onto a Petri dish.
Incubate and count colonies.

3.Direct Microscopic Count:

Petroff-Hausser Counting Chamber

Direct Microscopic Count:

Procedure:
A specific volume of a bacterial suspension (0.01 ml) is placed
on a microscope slide with a special grid.
Stain is added to visualize bacteria.
Cells are counted and multiplied by a factor to obtain
concentration.
Advantage:
No incubation time required.
Disadvantages:
Cannot always distinguish between live and dead bacteria.
Motile bacteria are difficult to count.
Requires a high concentration of bacteria (10 million/ml).

Example: 12 bacteria in one square

12 X 25 = 300bacteria
Total volume held is 0.02 mm3
300/.02 = 15000 or 1.5 X 104Bacteria per mm3
1cm3 = 1000mm3 (10 x 10 x 10)
1.5 X 104 x 1000 = 1.5 X 107/cm3 or mL

Sample added to the

surface of
the grid, the whole grid
has 25
large squares, the total
volume
that can be added is
0.02mm3

Indirect Method

1.Turbidity

Turbidity:
As bacteria multiply in media, it becomes turbid.
Use a spectrophotometer to determine % transmission or absorbance.
Multiply by a factor to determine concentration.

Advantages:
No incubation time required.
Disadvantages:
Cannot distinguish between live and dead bacteria.
Requires a high concentration of bacteria (10 to 100 million cells/ml).

2. Metabolic Activity:
-measure product as a function of time
As bacteria multiply in media, they produce certain products:
Carbon dioxide
Acids

Measure metabolic products.

Expensive

Metabolic Conversion/Enzyme Assay

Example:
1 bacterium produces 4.6 x 1012 NADH/sec/cell under idea growth

conditions.
In a 1 ml sample of growing cells, 5.2 x 10 23 NADH/sec/ml are

produced per second (as revealed by a color-based assay of NADH on

the sample)
Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023 NADH/sec/cell) =

2.3 x 1024 cells/ml

*Nicotinamide adenine dinucleotide, abbreviated NAD+

3. Dry Weight
Procedure:
1.Bacteria or fungi in liquid media are
centrifuged.
2.Resulting cell pellet is weighed.
Doesnt distinguish live and dead cells.

Determining dry mass of a fixed volume

Example:
An E. coli cell has a dry mass of
about 7.0 x 10-19 mg.
A 1 ml sample with a dry mass of
2 mg therefore has:
2 mg/ml x 1 cell/7 x 10-19 mg
= 2.8 x 1020 cells/ml