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**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.

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**Measurement of Bacterial Growth**

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**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.

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Generation time Time taken for a cell population to double in numbers and thus equivalent to the average length of the cell cycle

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**Growth in Batch Culture**

Typically, a batch culture passes through four distinct stages: Lag stage Logarithmic (exponential) growth Stationary stage Death stage

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**Growth in Batch Culture**

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**The Bacterial Growth Curve**

Four characteristic phases of the growth cycle are recognized. 1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.

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**The Bacterial Growth Curve**

The length of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

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**The Bacterial Growth Curve**

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population. Generation time (G) is defined as the time (t) per generation (n = number of generations).

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**The Bacterial Growth Curve**

3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space".

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**The Bacterial Growth Curve**

4. Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase

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**Generation Time (minutes) Escherichia coli Glucose-salts 17 **

Table 2. Generation times for some common bacteria under optimal conditions of growth. Bacterium Medium Generation Time (minutes) Escherichia coli Glucose-salts 17 Bacillus megaterium Sucrose-salts 25 Streptococcus lactis Milk 26 Lactose broth 48 Staphylococcus aureus Heart infusion broth 27-30

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**Lactobacillus acidophilus Milk 66-87**

Table 2. Generation times for some common bacteria under optimal conditions of growth. Lactobacillus acidophilus Milk 66-87 Rhizobium japonicum Mannitol-salts-yeast extract Mycobacterium tuberculosis Synthetic Treponema pallidum Rabbit testes 1980

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**The Bacterial Growth Curve**

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**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.

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**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

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**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

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**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

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**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

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**Table 1. Some Methods used to measure bacterial growth**

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

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**Table 1. Some Methods used to measure bacterial growth**

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

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**Calculation of Generation Time**

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).

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**Calculation of Generation Time**

the log plot

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**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.

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**Calculation of Generation Time**

The graph of the results reveals FOUR distinct phases which occur during the growth of a bacterial culture.

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**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

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**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

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**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 of minutes. Growth rate (min-1) = Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation

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**Mean Generation Time and Growth Rate**

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