Growth and Multiplication of Bacteria
Bacterial growth can be defined as an orderly increase of all the chemical components of the cell. Cell multiplication is a con-sequence of growth that leads to an increase in the number of bacteria making up a population or culture. Most bacteria divide by binary fission in which the bacteria undergo cell division to produce two daughter cells identical to the parent cell. Bacterial growth can be equated to cell number: one bacterium divides into two, these two produce four, and then eight, and so on (Fig. 2-15). The growth rate of a bacterium is therefore measured by measuring the change in bacterial number per unit time.
Generation time is the time required for a bacterium to give rise to two daughter cells under optimum conditions. The generation time for most of the pathogenic bacteria, such as E. coli, is about 20 minutes. The generation time is longer (i.e.,20 hours) for M. tuberculosis and longest (i.e., 20 days) for M.leprae. A bacterium replicates and multiplies rapidly producingmillions of cells within 24 hours. For example, E. coli in about 7 hours can undergo 20 generations and produce 1 million cells, in about 10 hours undergo 30 generations and produce 1 billion cells, and in 24 hours produces 1021 cells (Fig. 2-16). However, in actual practice, the multiplication of bacteria is arrested after a few cell divisions due to exhaustion of nutrients and accumulation of toxic products.
Microbial concentrations can be measured in terms of (i) cell concentration (the number of viable cells per unit volume of culture) or (ii) biomass concentration (dry weight of cells per unit volume of culture). The number of bacteria at a given time can be estimated by performing a total count or viable count.
Total count: This denotes the total number of bacteria in thesample, irrespective of whether they are living or dead. This is done by counting the bacteria under the microscope using counting chamber or by comparing the growth with standard opacity tubes.
Viable count: This usually indicates the number of livingor viable bacteria. This count can be obtained by dilution or plating method.
In dilution method, several tubes with liquid culture media are incubated with varying dilutions of sample and the viable count is calculated from the number of tubes showing bacterial growth. This method is widely used in microbiological testing of water for presumptive coliform count in drinking water.
In the plate method, a sample is diluted and small volume of it is
spread on the surface of an agar plate. The number of colonies that grow after
a suitable incubation time indicates viable count of the bacteria.
When a broth culture is inoculated with a small bacterial inocu-lum, the population size of the bacteria increases showing a clas-sical pattern. The bacterial growth curve shows the following four distinct phases (Fig. 2-17):
1. Lag phase: After a liquid culture broth is inoculated, themultiplication of bacteria does not start immediately. It takes some time to multiply. The time between inoculation and beginning of multiplication is known as lag phase. In this phase, the inoculated bacteria become acclimatized to the environment, switch on various enzymes, and adjust to the environmental temperature and atmospheric conditions. During this phase, there is an increase in size of bacteria but no appreciable increase in number of bacterial cells. The cells are active metabolically. The duration of the lag phase varies with the bacterial species, nature of culture medium, incubation temperature, etc. It may vary from 1 hour to several days.
2. Log phase: This phase is characterized by rapid exponentialcell growth (i.e., 1 to 2 to 4 to 8 and so on). The bacterial
population doubles during every generation. They multiply at their maximum rate. The bacterial cells are small and uniformly stained. The microbes are sensitive to adverse conditions, such as antibiotics and other antimicrobial agents.
3. Stationary phase: After log phase, the bacterial growth almoststops completely due to lack of essential nutrients, lack of water oxygen, change in pH of the medium, etc. and accumulation of their own toxic metabolic wastes. Death rate of bacteria exceeds the rate of replication of bacteria. Endospores start forming dur-ing this stage. Bacteria become Gram variable and show irregular staining. Many bacteria start producing exotoxins.
4. Decline phase: During this phase, the bacterial populationdeclines due to death of cells. The decline phase starts due to (a) accumulation of toxic products and autolytic enzymes and (b) exhaustion of nutrients. Involution forms are common in this stage. Growth rate during different phases of bacterial growth curve is summarized in Table 2-5.
The continuous culture is a method of culture useful for industrial and research purpose. This is achieved by using a special device for replenishing nutrients and removing bacte-rial population continuously so that bacteria growth is not inhibited due to lack of nutrients or due to accumulation of toxic bacterial metabolites.
A variety of factors affect growth of bacteria. These are discussed below:
Bacteria on the basis of their oxygen requirements can be classi-fied broadly into aerobic and anaerobic bacteria.
Aerobic bacteria: They require oxygen for their growth. Theymay be:
o Obligate aerobes—which can grow only in the presence of oxygen (e.g., P. aeruginosa).
o Facultative aerobes—which are ordinary aerobes but can also grow without oxygen (e.g., E. coli). Most of the pathogenic bacteria are facultative aerobes.
o Microaerophilic bacteria—those bacteria that can grow in the presence of low oxygen and in the presence of low (4%) concentration of carbon dioxide (e.g., Campylobacter jejuni).
Some fermentative organisms (e.g., Lactobacillus plantarum) are aerotolerant but do not contain the enzyme catalase or superoxide dismutase. Oxygen is not reduced, and therefore hydrogen peroxide (H2O2) and nascent oxygen (O22) are not produced.
Anaerobic bacteria: Obligate anaerobes are the bacteria thatcan grow only in the absence of oxygen (e.g., Clostridium botulinumClostridium tetani, etc.). These bacteria lack superoxide dismutaseand catalase; hence oxygen is lethal to these organisms.
◗ Carbon dioxide
The organisms that require higher amounts of carbon dioxide (CO2) for their growth are called capnophilic bacteria. They grow well in the presence of 5–10% CO2 and 15% O2. In candle jar, 3% CO2 can be achieved. Examples of such bacteria include H. influenzae, Brucellaabortus, etc.
The optimum temperature for most of the pathogenic bacteria is 378C. The optimal temperature, however, is variable; depend-ing on their temperature range, growth of bacteria is grouped as follows:
o Psychrophiles:These bacteria are cold loving microbes thatgrow within a temperature range of 0–208C. Most of soil and water saprophytes belong to this group.
o Mesophiles:These are moderate temperature loving microbesthat grow between 258C and 408C. Most of pathogenic bac-teriabelong to this group.
o Thermophiles:These are heat loving microbes. They can growat a high temperature range of 55–808C. B. stearothermophilus is an example.
Most pathogenic bacteria grow between pH 7.2 and 7.6. Very few bacteria, such as lactobacilli, can grow at acidic pH below 4.0. Many food items, such as pickles and cheese, are prevented from spoilage by acids produced during fermentation.
V. choleraeis an example of the bacteria that can grow at analkaline (8.2–8.9) pH.
Depending on the source of energy bacteria make use of, they may be classified as phototrophs (bacteria deriving energy from sunlight) or chemotrophs (bacteria deriving energy from chem-ical sources).
◗ Osmotic pressure
Microbes obtain almost all their nutrients in solution from surrounding water. Hence factors such as osmotic pressure and salt concentration of the solution affect the growth of bacteria. Bacteria by virtue of mechanical strength of their cell wall are able to withstand a wide range of external osmotic variations. Organisms requiring high osmotic pressures are called osmo-philic bacteria. Sudden exposure of bacteria to hypertonic solu-tion may cause osmotic withdrawal of water, leading to osmotic shrinkage of the protoplasm ( plasmolysis). On the other hand, sudden transfer of bacteria from concentrated solution to dis-tilled water may cause excessive imbibition of water leading to swelling and bursting of cell ( plasmoptysis).