Transcript Growth in Batch Culture

Microbial Growth
A.Growth in Batch Culture
B.Mean Generation Time and Growth Rate
C.Measurement of Microbial Growth
D.Continuous Culture
E.Factors Influencing Growth
F.Growth in Natural Environments
Growth in Batch Culture
• “Growth” is generally used to refer to the
acquisition of biomass leading to cell
division, or reproduction
• A “batch culture” is a closed system in
broth medium in which no additional
nutrient is added after inoculation of the
broth.
Growth in Batch Culture
• Typically, a batch culture passes through
four distinct stages:
–
–
–
–
Lag stage
Logarithmic (exponential) growth
Stationary stage
Death stage
Growth in Batch Culture
Mean Generation Time
and Growth Rate
• The mean generation time (doubling time) is
the amount of time required for the
concentration of cells to double during the
log stage. It is expressed in units
1 of minutes.
mean generation time
• Growth rate (min-1) =
• Mean generation time can be determined
Mean Generation Time
and Growth Rate
Mean Generation Time
and Growth Rate
Growth of E. coli 23716,
9-20-01 batch culture
y = 0.0187e
0.0069x
2
R = 0.9928
10
A425
1
0.1
0.01
0
200
400
600
800
time, min
1000
1200
1400
1600
Measurement of
Microbial Growth
• Microscopic cell counts
– Calibrated “Petroff-Hausser counting chamber,”
similar to hemacytometer, can be used
– Generally very difficult for bacteria since cells tend
to move in and out of counting field
– Can be useful for organisms that can’t be cultured
– Special stains (e.g. serological stains or stains for
viable cells) can be used for specific purposes
• Serial dilution and colony counting
– Also know as “viable cell counts”
Measurement of
Microbial Growth
• Serial dilution and colony counting
– Also know as “viable cell counts”
– Concentrated samples are diluted by serial
dilution
– The diluted samples can be either plated by
spread plating or by pour plating
Measurement of
Microbial Growth
• Serial dilution (cont.)
– Diluted samples are spread onto media in petri dishes
and incubated
– Colonies are counted. The concentration of bacteria in
the original sample is calculated (from plates with 25 –
250 colonies, from the FDA Bacteriological Analytical
Manual).
– A simple calculation, with a single plate falling
into the statistically valid range, is given below:
CFU
# colonies counted
in original sample 
ml
(dilution factor)(volume plated, in ml)
Measurement of
Microbial Growth
• Serial dilution (cont.)
– So you’ve seen all this before, huh?
– What if there is more than one plate in the
statistically valid range of 25 – 250 colonies?
– For details on this standard method, see:
FDA Bacteriological Analytical Manual
– and specifically the section on:
Aerobic Plate Counts
– Really.
– heh, heh.
Measurement of
Microbial Growth
CFU
C


ml [(1* n1)  (0.1* n 2)  ...] * d1 * V
• Where:
C = Sum of all colonies on all plates between 25
- 250
n1= number of plates counted at dilution 1
(least diluted plate counted)
n2= number of plates counted at dilution 2
(dilution 2 = 0.1 of dilution 1)
Measurement of
Microbial Growth
● Membrane filtration
Used for samples with low microbial concentration
– A measured volume (usually 1 to 100 ml) of sample
is filtered through a membrane filter (typically with a
0.45 μm pore size)
– The filter is placed on a nutrient agar medium and
incubated
– Colonies grow on the filter and can be counted
–
Measurement of
Microbial Growth
• Turbidity
– Based on the diffraction or “scattering” of light
by bacteria in a broth culture
– Light scattering is measured as optical
absorbance in a spectrophotometer
– Optical absorbance is directly proportional to
the concentration of bacteria in the suspension
Measurement of
Microbial Growth
• Mass determination
– Cells are removed from a broth culture by
centrifugation and weighed to determine the
“wet mass.”
– The cells can be dried out and weighed to
determine the “dry mass.”
• Measurement of enzymatic activity or other
cell components
Growth in Continuous Culture
• A “continuous culture” is an open system in which fresh
media is continuously added to the culture at a constant
rate, and old broth is removed at the same rate.
• This method is accomplished in a device called a
chemostat.
• Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as the
nutrient feed is maintained.
Lab Ex. 19: Enumeration of Bacteria – Pour Plate Technique
Bacterial growth may be determined based on increase in the
number of cells.
Reproduction and thus growth leads to increase in cell number and
thus the population of the bacterial culture.
Bacterial growth follows specific patterns as seen in the growth
curve patterns for bacteria.
The time it takes for a population of bacterial cells to double in
number is called the generation time.
Bacterial growth may be determined by using either
Cell Mass methods or Cell Number methods.
Cell Mass methods:
Turbidity measurements by Spectrophotometry
Total Protein
Total DNA
Cell Number methods:
Direct Microscopic Counts (hemocytometer)
Pour Plate
Most Probable Number
Membrane Filter
Enumeration methods may yield either total counts or viable counts.
Total count is a count of cells including dead and live cells.
Viable count is a count of only those cells that are alive in the sample.
Pour Plate method:
This is the most commonly used method for enumeration of
bacteria in a wide variety of samples including milk, food,
meat, soil etc.
Pour plate methods yield a count of only the living cells in the
sample and thus are a viable count.
There are two steps to the process: dilution of the sample so
that various dilutions of the sample may be inoculated
onto plates and a count of the colonies that grow made;
the second step is the plating of the dilutions so that each
cell in the diluted sample may then grow and form colonies
that will in turn become visible to the naked eye and can be
counted.
Dilution is important since the colonies will have to be counted and
with a concentrated sample there may be too many colonies
than can be accurately counted.
Plating is important since a count of only the living cells is
required in this procedure (only living cells will be able to
multiply and form colonies)
Samples of milk, meat and soil will be used in the exercise.
Rules to keep in mind:
- only living cells are counted. Why?
- only plates with colony numbers between 30-300 are useable
(<30 or >300 are not statistically valid)
- use aseptic technique
- dilutions to be done accurately
Procedure:
- aseptically dilute sample with sterile water blanks
- make 1:10 dilutions as instructed, based on sample
- aseptically transfer 1ml volume of dilutions that are to be
plated onto sterile Petri plates
- pour molten agar onto the sample in Petri dish and mix
thoroughly
- incubate as instructed for growth of colonies
- during next lab period, count colonies on plates
- use the formula:
cfu/ml =
# of colonies x
1
dilution