15 September 2016

Lecture #3: BACTERIAL GROWTH AND REPRODUCTION


  

Transverse binary fission is the most common asexual reproductive process whereby a single cell divides into two after developing a transverse cell wall.

Species of the genus Streptomyces produce many reproductive spores; each spore gives rise to a new organism. Related bacteria (genus Nocardia) produce extensive filamentous growth which is followed by fragmentation of the filaments into bacillary or coccoid cells.

Multiplication leads to an increase in the number of individual cell making up a population or culture. Growth denotes the increase in number beyond that present in the original inoculum. Thus, if we start with a single bacterium, the increase in population is by geometric progression.

Generation is the doubling of cell in number. The time interval required for the cell to divide is known as generation time. Not all bacteria have the same generation time. The generation time is dependent upon the nutrients in the medium and the physical conditions. Growth rate is expressed in terms of generation per hour.

When a given number of cells are inoculated into a fresh medium, the bacterial population is determined intermittently during an incubation period of 24 hours (more or less) and the logarithms of the cells versus time are a representation of population changes in the growth of a culture.
           


Section of curve                   Phase                         Growth rate

            A                                 Lag                             Zero
                                                Acceleration              Increasing
            B                                  Exponential               Constant
                                                Retardation               Decreasing
            C                                 Maximum                  Zero
                                                Stationary
            D                                 Decline                       Negative (death)


Four major phases of bacterial growth curve

1.      Lag phase – adaptation period

Enzymes and intermediates are formed and accumulate until they are present in concentrations that permit their growth to resume. The cells are metabolizing but there is a lag in cell division.

2.      Logarithmic or Exponential phase

The cells divide steadily at a constant rate. Growth rate is maximal during this phase. The population is most nearly uniform in terms of chemical composition of cells, metabolic activity and other physiological characteristics.

3.      Maximum stationary phase

There is a stopping of growth completely attributed to the exhaustion of nutrients and production of toxic products during growth. The population remains constant for a time as a result of complete stopping of division or the balancing of reproduction rate by an equivalent death rate.

4.      Death phase or period of decline

After stationary phase, bacteria may die faster than new ones produced, if some cells are still reproducing. Depletion of nutrients and accumulation of inhibitory products contributes to bacterial death. Bacteria die at different rates, just as they grow at different rates.

An appreciation of the normal growth curve is important; it must be understood that the cell are young and actively metabolizing during some phases of growth while in other phases they are dying, so there may be a great structural and physiological differences between cells taken at different times. In general, cells in the logarithmic phase of growth are commonly used for studies of metabolism because they are the most uniform and in a more clearly defined condition than any other bacterial cells.

There are many aspects of research relating to cell growth, organization and differentiation, with these aspects; it is desirable to have an entire population of cells in the same stage of growth cycle. The growth pattern in which all the cells will divide at the same time is known as synchronous growth. One can synchronize a population by manipulating the physical environment or the chemical composition of the medium.

Steady state or balanced growth is a condition wherein the bacterial population is maintained in the exponential or log phase. A bacterial population in this condition is used both in experimental research and in industrial processes. Devices for this are the turbidostat and the chemostat. In both devices, fresh medium is allowed to enter the culture vessel at the same rate as medium spent is removed to keep the culture at a constant volume.

Growth can be measured quantitatively by:

a.      Cell count – directly by microscopy or an electronic particle counter or indirectly by a colony count to determine viable bacteria.

b.      Cell mass – directly by weighing or by measuring nitrogen content or indirectly by turbidity.

c.       Cell activity – indirectly by relating the degree of biochemical activity to the size of the population.

Summary of methods for measuring bacterial growth

            Method                                              Applications

Microscopic count                           Enumeration of bacteria in milk and vaccine

Plate count                                        Enumeration of bacteria in milk, water, foods,
                                                            soil, cultures

Membrane or molecular filter        same as plate count

Nitrogen determination                  measurement of cell crop from heavy culture
                                                            suspensions to be used for research in metabolism

Weight determination                     same as for nitrogen determination

Turbidimeteric measurement        microbiological assays, estimation of cell crop in
                                                            broth, culture aqueous suspension

Measurement of biochemical         microbiological assays
                        activity


ENZYMES AND THEIR REGULATION

To live, grow and reproduce, a cell must be capable of performing an immensely of chemical changes, and these chemical changes lies in the activity of the enzymes. In a sense, the enzymes may be regarded as the working part of the cell.

There are two types of enzymes based on the site of action

1.      Intracellular or endoenzymes – functioning in the cell. It synthesizes cellular material and also performs catabolic reactions which provide the energy requirements of the cell.

2.      Extracellular or exoenzymes – functioning outside the cell. It performs whatever changes are necessary on the nutrients in the medium to allow the foods to enter the cell.

Based on the presence of substrate and enzyme formation, bacterial enzymes are divided:

1.      Constitutive – these are always produced by the cell, independently of the composition of the medium in which it grows.

2.      Adaptive (induced) – these are produced by the cell only in response to the presence of a particular substrate; they are produced only when needed.

The growing culture technique is used routinely for the characterization of the enzymatic activities of microorganisms. Results of such test provide information necessary for their identification. The resting cell technique and cell free preparation are principally used in research work, the purpose of which is to determine how the organisms accomplishes each specific change.

Procedure for growing culture technique:

1.      Inoculation of the bacteria into a medium containing the substrate.
2.      Incubation of the medium containing the bacteria for 1 or more days.
3.      Examination for a change or disappearance of the substrate and presence of end products.

The living cell neither synthesizes nor catabolizes more material than is required for normal metabolism and growth, all this cells for precise control mechanisms of cellular metabolism. The control of cellular metabolism eventually involves the regulation of enzyme activity. Generally speaking, enzymes can be controlled or regulated in two ways:

1.      Genetic control – inducing and suppressing enzyme synthesis is at the genetic level.

2.      Direct control of catalysis – by altering the concentrations of substrate or reactants or by coupling with other processes which usually implies regulation of the ligands which do not participate in the catalytic process itself.

Feedback inhibition – is one type of this control by which the end product of a series of metabolic reactions inhibits the activity of an earlier enzyme of the sequence.


BACTERIAL METABOLISM

The term metabolism denotes all the organized chemical activities performed by a cell, which comprises two general types: (1) energy production (2) energy utilization

Energy production

Most bacteria obtain energy by carrying out chemical reactions which liberate energy. Some forms of life such as green plants, can utilize radiant energy and are designated as phototrophs. Others rely upon oxidation of chemical compounds and they are called chemotrophs.

The systems in bacteria that transform chemical and radiant energy into a biologically useful form include:

1.      Respiration – in which the molecular oxygen is the ultimate electron acceptor. The pathway of aerobic dissimilation is exceedingly complex. The most important respiratory mechanism for final oxidation is the tricarboxylic acid cycle of Krebs, which together with the known reactions of glycolysis, can account for the complete oxidation of glucose.

Kreb cycle – an enzyme system which converts pyruvic acid to CO2 in the presence of O2 accompanying release of energy which is trapped in the form of ATP (adenosine triphosphate) molecules.


2.      Fermentation – the foodstuff molecule is usually broken down into two fragments, one which is then oxidized by the center.

The major route of glucose catabolism in most cells is glycolysis or the Embden–Meyerhoff pathway, an anaerobic process of glucose dissemination, which may occur by sequence of enzyme catalyzed reactions, to pyruvic acid.
Although the basic pathway is the same for all types of cells, the properties of certain enzymes are not uniform in all species or cell types. Such variations are introduced for purpose of cellular distinction and control of specific steps in the pathway.

Respiration in the presence of oxygen, glucose is changed into CO2 and water. It is much more efficient process than fermentation because many molecules of ATP are generated. Fermentation in the absence of oxygen, glucose is changed into different end products and only a few molecules of ATP is generated

3.      Photosynthesis – a process whereby microorganisms with chlorophyll obtain energy from the environment in the form of light.

Energy utilization

Once energy is obtained, bacteria as well as other organisms utilized it in various ways:

1.      For the biosynthesis of new cell components
2.      For the maintenance of physical and chemical integrity of the cell.
3.      For the activity of the locomotor organelles
4.      For transporting solutes across membranes
5.      For heat production

Death – is the reversible loss of the ability of the cell to reproduce.

Empiric test of death – is determined by culturing the cells on solid media. A cell is considered dead if it fails to give rise to a colony.


MICROBIAL GENETICS

From the point of view of genetics, a characteristic of all forms of life is the general uniformity or “likeness” in characteristics of the progeny and parent. A particular organism, however, may not exhibit the same characteristics when grown under a variety of different environmental conditions. A deviation from the parent form in the bacteria of the same species growing under the different or identical condition is known as variation. It occurs as a result of:

1.      Genotypic changes – involves alteration of genes

a.      Mutation

Any gene is capable of changing or mutating to a different form, so that it causes an altered characteristic. The act of mutating is known as mutation or it is an alteration in form or qualities. In mutation, there is a permanent transmissible change in characters of offspring from those of parents. It occurs in two ways:

(1)   Spontaneously – under natural conditions due to tautomeric shift of electrons in purine or pyrimidine base

(2)   Induced – due to exposure of cells to some mutagenic agents such as base analogue, alkylating agents, ultraviolet radiations, etc.

b.     Recombination

It occurs when a portion of the genetic material from a donor is transferred to a recipient cell. There are three processes by which recombination takes place:

(1)   Transformation – soluble DNA from the donor cell is released into the surrounding medium and taken up by a recipient cell.

(2)   Transudation – fragment of the donor chromosome is carried to recipient cell by a temperate bacteriophage (virus–eating bacteria)

(3)   Conjugation–plasmid mediated – two bacteria actually join together to permit transfer of the genetic material through the pilus.

Plasmid – is a small, extrachromosomal genetic unit which cannot be integrated into the chromosome and not transferred by an infectious process (unless taken up by a generalized transducing bacteriophage).

They are clinically significant because:

(a)   They carry genes for resistance to therapeutic agents

(b)   They lead to emergence of strains to new combinations with antigenic and virulent factors.

Their presence is detectable only when the genes they carry confer new properties on the host.


2.      Phenotypic changes – changes are imposed by the environment without genetic change. It is less stable than genotypic change and is not inherited.

a.      Morphological modifications

(1)   Cellular variation

(a)   Large capsules are produced by certain species of bacteria when grown in milk and none at all when grown in nutrient broth.

(b)   Spores are formed when certain species of bacteria are placed in an environment that is unfavorable for their growth.

(c)    Flagella are removed from a certain bacteria when placed in a medium containing alcohol.

(2)   Colony variation

Colony is a group of bacteria formed from the reproduction of a single organism and generally visible to the naked eye.

Dissociation is a kind of bacterial variation in which there is a change in the observable characteristics of growth in colonies resulting in the production of a new one. A change from S (smooth) to R (rough) forms is an example of dissociation. Other types are:

(a)   M (mucoid) – viscous and slimy
(b)   H (spreading) to O (nonspreading)
(c)    D (dward or diptheroid)

Pleomorphism is variation in size, shape and appearance of bacteria of the same species growing under favorable conditions.

Involutions or degenerative forms – these are abnormal forms assumed by microorganisms growing under unfavorable conditions.

b.     Physiological variation

Adaptation – variations (changes in bacterial make–up) that represent physiologic adjustment to the environment

(1)   Ability to decompose sugars
(2)   Variation in nutritive requirement
(3)   Susceptibility to infection by viruses
(4)   Immunological characteristics
(5)   Virulence

Attenuation – an important form of adaptation which indicates a loss in disease–producing ability to a given organism.




  







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