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