1. Bacterial Growth Brock, Pelczar Tortora
Mic 101 : L 9 STT
Bacterial Growth
Brock, Pelczar Tortora

2. 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, pH, 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.

3. Binary fission

4. Mechanisms of Growth Binary fission is the most common.
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.
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.

5. Budding, Fragmentation and Sporangiospore formation
Budding: When the bacteria Rhodopseudomonas acidophila gets optimal condition, it grows a small bud at one end of the cell wall. The buds grows bigger with replicated DNA and other cellular material. When the bud receives enough material for independent existance, 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.

6. 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.
Electronic method
Bacterial suspension is passed through an electronic particle counter. The fine orifice inside the counter allows one bacteria to pass at 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.

7. 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.
Eg. 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.

8. Mathemetical expression of bacterial growth
If we have N0 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 cells will be N= N02
Number of generations, n = 3.3 (log N- log N0)
Generation time, g =t/n
Eg. 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. n

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

10. 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.

11. 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 at the rate of Log 2 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.

12. 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
pH, gas, critical growth requirements become unfavorable
Cells often become resistant to changes in growth parameters

13. 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)

14. 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

15. 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, cell inoculum) 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.

16. 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.

17. Control of Bacterial Growth
Rate of death
-The number of microbes
- Environmental influences
-Time of exposure
- Microbial characteristics
Physical method
Heat
Pasteurization
Filtration
Osmotic pressure
radiation
Chemical method
Factors affecting efficiency of chemicals
Concentration of agent
Initial load of microbes
Environmental: Temperature, pH, salt
Presence of organic matter
Time or exposure
Nature of organism

18. 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 min in autoclave
• Ionozing radiation X-rays, -rays, electron
beams cause destruction of DNA
Nonionizing radiation UV ray
Most effective wave length ~ 260 nm cause
destruction
• Physical separation by filtration;
Passage of liquid through a filter pore size
of 0.2mm or less and collection in a sterile
Container. Eg. 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

19. Chemical method Factors affecting efficiency of chemicals
Concentration of agent
Initial load of microbes
Environmental: Temperature, pH, salt
Presence of organic matter
Time or exposure
Nature of organism
Examples: halogens (Chlorine gas, Bromine and Iodine solution, alcohol, antiseptics, antibiotics

20. 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 cryo-preservative and stored at -80º C in a cryo-vial.

21. Questions
How can we measure and enumerate the growth of organisms dividing by binary fission?
Describe any particular phase of growth of microbes?
Why can not the bacteria grow infinitely in culture?
How are the bacterial growth phases interesting for industrial profit? How can growth phases be manipulated to make profit in the industry?
Which one is a reproductive method in bacteria and why: endospore or sporangiospore?