Measurement of Bacterial Growth
● Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosome, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission.
Measurement of Bacterial Growth
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.
Generation time
Time taken for a cell population to double in numbers and thus equivalent to the average length of the cell cycle
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
Table 2. Generation times for some common bacteria under optimal conditions of growth.
BacteriumMedium
Generation Time (minutes)
Escherichia coli
Glucose-salts 17
Bacillus megaterium
Sucrose-salts 25
Streptococcus lactis
Milk 26
Streptococcus lactis
Lactose broth 48
Staphylococcus aureus
Heart infusion broth
27-30
Lactobacillus acidophilus
Milk 66-87
Rhizobium japonicum
Mannitol-salts-yeast extract
344-461
Mycobacterium tuberculosis
Synthetic 792-932
Treponema pallidum
Rabbit testes 1980
Table 2. Generation times for some common bacteria under optimal conditions of growth.
The Bacterial Growth Curve
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.
Factors that Influence Growth
● Temperature– Most bacteria grow throughout a range of approximately
20 Celsius degrees, with the maximum growth rate at a certain “optimum temperature”
– Psychrophiles: Grows well at 0ºC; optimally between 0ºC – 15ºC
– Psychrotrophs: Can grow at 0 – 10ºC; optimum between 20 – 30ºC and maximum around 35ºC
– Mesophiles: Optimum around 20 – 45ºC– Moderate thermophiles: Optimum around 55 – 65 ºC– Extreme thermophiles (Hyperthermophiles):
Optimum around 80 – 113 ºC
Factors that Influence Growth
● pH– Acidophiles:
● Grow optimally between ~pH 0 and 5.5– Neutrophiles
● Growoptimally between pH 5.5 and 8– Alkalophiles
● Grow optimally between pH 8 – 11.5
Factors that Influence Growth
● Salt concentration– Halophiles require elevated salt concentrations to
grow; often require 0.2 M ionic strength or greater and may some may grow at 1 M or greater; example, Halobacterium
– Osmotolerant (halotolerant) organisms grow over a wide range of salt concentrations or ionic strengths; for example, Staphylococcus aureus
Factors that Influence Growth
● Oxygen concentration– Strict aerobes: Require oxygen for growth (~20%)– Strict anaerobes: Grow in the absence of oxygen; cannot
grow in the presence of oxygen– Facultative anaerobes: Grow best in the presence of
oxygen, but are able to grow (at reduced rates) in the absence of oxygen
– Aerotolerant anaerobes: Can grow equally well in the presence or absence of oxygen
– Microaerophiles: Require reduced concentrations of oxygen (~2 – 10%) for growth
Table 1. Some Methods used to measure bacterial growth
Method Application Comments
Direct microscopic count
Enumeration of bacteria in milk or cellular vaccines
Cannot distinguish living from nonliving cells
Viable cell count (colony counts)
Enumeration of bacteria in milk, foods, soil, water, laboratory cultures, etc.
Very sensitive if plating conditions are optimal
Turbidity measurement
Estimations of large numbers of bacteria in clear liquid media and broths
Fast and nondestructive, but cannot detect cell densities less than 107 cells per ml
Measurement of total N or protein
Measurement of total cell yield from very dense cultures
only practical application is in the research laboratory
Measurement of Biochemical activity e.g. O2 uptake CO2 production, ATP production, etc.
Microbiological assays
Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers
Measurement of dry weight or wet weight of cells or volume of cells after centrifugation
Measurement of total cell yield in cultures
probably more sensitive than total N or total protein measurements
Table 1. Some Methods used to measure bacterial growth
Because of the very large differences in the number of cells present at the peak and at the start/end of the experiment, it's hard to see what's going on from this graph. It's much easier to see the whole experiment if you plot the number of viable cells on a logarithmic scale (or more simply, plot the log of cell number).
Calculation of Generation Time
the log plot
Calculation of Generation Time
As you can see, the indirect method of counting (optical density) closely parallels the direct method (viable count). (At later time points, you can see that the number of viable cells declines faster than the optical density of the culture. It will be even easier to see the results if we concentrate on the first 12 hours of the experiment.
Calculation of Generation Time
The graph of the results reveals FOUR distinct phases which occur during the growth of a bacterial culture.
Calculation of Generation Time
When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide.
B = number of bacteria at the beginning of a time interval b = number of bacteria at the end of the time interval
G = t 3.3 log b/B
Calculation of Generation Time
Example: What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000 cells in four hours of growth?
G = t_____ 3.3 log b/B G = 240 minutes 3.3 log 107/104 G = 240 minutes 3.3 x 3 G = 24 minutes
Calculation of Generation Time
Mean Generation Timeand 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 of minutes.
● Growth rate (min-1) =
● Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation
timegeneration mean1
Mean Generation Timeand Growth Rate