Bacterial Growth Growth of bacteria refers to increase

Bacterial Growth • Growth of bacteria refers to increase in the number of cells. • Bacterial population increase in number by division of cells. • When the bacterial cells have the right amount of nutrition, temperature, p. H, osmotic pressure, trace elements, oxygen and space, the cells continue to divide. • As the growth continues, population size increases • Different bacteria take different time for cell division.

Binary fission

Mechanisms of Growth • Binary fission is the most common mechanism. • Budding, fragmentation and spore formation are rare. • In binary fission, there are several stages called DNA replication, cell elongation, septum formation, septum completion and cell separation. DNA replication: the cell accumulates enough nutrition for duplicating its DNA. The chromosomal DNA replicates itself within few minutes and two circular copies of DNA are formed, which are attached to the cell membrane.

Mechanisms of Growth Cell elongation: Protein, RNA, carbohydrate, lipid and all components increase in amount; so the cell elongates. Septum formation: The cell membrane begins to grow inwards, forming a septum, separating the newly replicated DNA into two new cells. Septum completion: when the septum completes itself the DNA and cytoplasm are divided into two equal-sized cells. Cell separation: The new cells form cell wall and binary fission is complete. A typical E coli needs 20 minutes to divide in its optimal condition.

Mechanisms of Cell Division Septum formation Septum completion Cell separation

Other mechanisms of cell growth Budding, Fragmentation Sporangiospore formation • Budding: When certain bacteria(e. g. Rhodopseudomonas acidophila gets optimal condition, it grows a small bud at one end of the cell wall. The bud grows bigger with replicated DNA and other cellular material. When the bud receives enough material for independent existence, it separates itself from its parent cell.

• Fragmentation: In optimal condition, Nocardia cells produces 3 -4 fragments from the existing cell. All the fragments grow bigger and function as new cell. • Sporangiospore formation: Streptomyces replicates its DNA into many copies and develops cross walls at its tip to produce sporangiospore, each of which develop into new cells. • The difference between endospore and sporangiospore is - One endospore grows into one cell, so endospore formation is not cell growth. - Many sporangiospores are produced from one cell and each Sporangiospore grows into new cell. So this is growth.

Measurement of Growth • Microscopic method Take cells on a graded microscopic slide and count the number of cells per microscopic field. Since the slide has a known amount of volume, number of cells per ml can be calculated. • Cultural method Add a known volume of microbial suspension on nutrient agar medium, spread/pour, incubate overnight and count and calculate CFU/ml in the inoculum.

Measurement of growth • Electronic method Bacterial suspension is passed through an electronic particle counter. The fine orifice inside the counter allows one bacteria to pass at a time. Since cells have greater resistance than media, each passing cell generates a signal due to difference in resistance. • Turbidimetric method Cells have greater optical density than media. Therefore, the more cells in the suspension, the more turbidity the suspension has. The turbidity is measured as amount of scattered light in a spectrophotometer or a photocell.

Terms about Bacterial Growth • Generation means the event of formation of daughter cells from mother cells. • Generation time means the time needed to produce daughter cells from mother cell. Each bacteria has its specific generation time under specific conditions. • Growth rate is the increase of cell number per unit time. The number of cells increased in one hour is the growth rate/hour. • Death rate is the decrease in the number of living cells per unit time.

Mathematical expression of bacterial growth • If we have N 0 cells at the beginning and N cells at the end of time t, and if the cells divide n number of times: then in an optimal condition, the number of n cells will be N= N 02 • Number of generations, n = 3. 3 (log N- log N 0) • Generation time, g =t/n Example: If we find 4096 cells after 5 hours of growth and the bacteria takes 50 minutes to double, then number of generation n=6; and there were 128 cells in the inoculum.

Phases of Bacterial Growth in Culture • Lag phase • Exponential or logarithmic (log) phase • Stationary phase • Death phase (decline phase)

Lag Phase • Lag phase • occurs immediately after inoculation • It is the stage in the growth curve when the cells are alive, adapting and synthesizing macromolecules, but not yet dividing • cells do not grow; cells per volume media do not increase • Cells adapt to the new media and incubation parameters. The more time taken in adaptation, the longer the lag phase.

Log Phase • Log phase or exponential phase – It starts when lag phase ends – During this phase the microbe is growing by binary fission at the maximum rate possible – Cells per volume increases and generation time reaches a constant maximum – The cells are metabolically the most active; so rate of utilization of nutritional media is proportional to number of cells in the media – Cells are constantly dividing, DNA is constantly being replicated. Therefore the cells are sensitive to excess heat, radiation, drugs, anti-microbial chemicals.

Stationary phase • As soon as cells growing in the log phase reaches a point of saturation in terms of cell number/volume, the cell growth rate slows down for limitation of nutrient concentration • Waste materials from active cells accumulate • Cells start to die from lack of food and toxicity of wastes • Cell growth = cell death, there is an equilibrium • Surviving cells metabolize slowly but produce secondary metabolites • p. H, gas, critical growth requirements become unfavorable • Cells often become resistant to changes in growth parameters

Death Phase • Nutrient depletion and waste accumulation becomes intolerable for the cells • Number of dead cells overcome number of live cells • The number of cells decrease logarithmically • Either the whole population dies or a number of resistant cells with low metabolic activity survives (endospore/VBNC)

Exception of Growth Phases • exponentially growing culture inoculated into same media, same growth conditions – no lag phase • old culture, same media & conditions – require lag phase because cells need to recover, regain metabolic abilities. • Cells damaged (heat, radiation, toxic chemicals) – require lag phase as cells repair damage • Cells transferred from rich medium to poor culture medium, require lag phase as cells have to synthesize more enzymes etc to enable synthesis of macromolecules not present in poor culture medium

Significance of Bacterial Growth • The shorter the lag phase, the quicker the growth, the more profit for the industry • Log phase is the most desired stage for industries that produce cell itself (eg. Yeast) or primary metabolites (eg. Enzyme, protein) • Stationary phase is good for harvesting secondary metabolites , eg, antibiotics • The knowledge of microbial growth and its manipulation allows us to make industrial profit by maintaining log phase or stationary phase when necessary. • The knowledge of how to stop growth/metabolism is the key to preserve food, materials and to cure infectious disease.

Synchronous Growth • It means a situation in a cell culture when all the cells divide at the same time. • Important in the study of genetics and metabolism • The easiest way to synchronize bacterial growth is to add some cytostatic agent so that cells don’t divide and they all maintain the same state of metabolism and cell cycle. • When the cytostatic agent is removed, all cells start to divide at the same time.

Control of Bacterial Growth • Physical method • • • Heat Pasteurization Filtration Osmotic pressure radiation

Physical Method • Dry heat (170 -180º C for 1 hr) denatures the cells • Moist heat = heat + vapour pressure Vegetative cells and spores destroyed At 121º C (15 psi) for 10 -15 min in autoclave • Ionozing radiation X-rays, electron beams cause destruction of DNA Nonionizing radiation UV ray Most effective wave length ~ 260 nm cause

Physical Method • Physical separation by filtration; Passage of liquid through a filter pore size of 0. 2µm or less and collection in a sterile container. Examples. HEPA and membrane filters. • Osmotic pressure Use of high concentration of salts and sugars for preserving foods Create hypertonic environment that cause water to leave the cell • Low temperature: Water in the cells crystallize below 0 C

Chemical method • Factors affecting efficiency of chemicals – Concentration of agent – Initial load of microbes – Environmental: Temperature, p. H, salt – Presence of organic matter – Time or exposure

Microbial Preservation • Microbial preservation means maintaining cells in a viable condition without growth • It is most conveniently done by adding microbes in a minimal growth medium where they will survive but not grow • For short-term, microbes can be stored in a minimal media at 4º C • For long term, microbes should be added to a cryopreservative and stored at -80º C in a cryo-vial.