Reproduction of microorganisms • Bacteria multiply by transverse division. • Yeasts multiply by budding. • Actinomycetes by fragmentation of filaments.
Bacterial replication is a coordinated process in which two equivalent daugther cells are produced. • For growth to occur, there must be sufficient metabolites to support the synthesis of the bacterial components and especially the nucleotides for DNA synthesis.
The production of two daughter bacteria requires the growth and extension of the cell wall components followed by the production of a septum (cross wall) to divide the daughter bacteria into two cells. • Septum formation is initiated at the cell membrane. The septum grows from opposite sides toward the center of the cell, causing cleavage of the daughter cells.
Chromosome replication is initiated at the membrane, and each daughter chromosome is anchored to a different portion of membrane. • In some bacterial cells, the DNA associates with mesosomes. • As the bacterial membrane grows, the daughter chromosomes are pulled apart. • Commencement of chromosome replication also initiates the process of cell division, which can be visualized by the start of septum formation between the two daughter cells.
Generative (ordoubling) time • It is the time, covering the beginning of division of the mother cell up to the formation of two new cells. • The average generative time is about 20 – 30 minutes in a majority of medically important bacteria. • They are some exceptions among pathogenic bacteria: • Mycobacterium tuberculosis has the generative time about 18 hours, • Mycobacterium leprae has even much longer generative time than otherspecies (10 – 20 days).
The length of the generative time is in a direct dependance on the length of an incubation or prodromed period of infections. • In a certain extent, the same rule is applied for antibiotic therapy.
Populationdynamics • When bacteria are added to a medium, they require time to adept to the new environment before they begin dividing. This hiatus is known as lag phase of growth. • The bacteria will grow and divide at a doubling time characteristic of the strains and determined by the conditions during the exponencial phase. During this phase, the number of bacteria will increase to 2n , in which n is the number of generations. • The cultures runs out of metabolites, or a toxic substances builds up in the medium. The bacteria then stop growing and enter the stationary phase.
Themultiplicationofbacteria in a closedsystemis limited by exhaustionofnutrientsandincreaseofhydrogenionsandtoxicmetabolites • Growth cycle in a closed system: • After inoculation of a fresh medium of the closed system, bacteria grow and multiply in successive phases: • lag phase (or phase of adaptation) • phase of acceleration (or phase of physiological youth) • phase of exponential growth • stationary growth phase • phase of decline
The majority of bacterial and fungal species of medical importance grow in artificial media in the laboratory. • Some bacteria cannot be cultivated on solid nutrient media surfaces and can only be grown in cell cultures (e.g. chlamydia, rickettsia). • Some bacterial species cannot be grown at all except in experimental animals (e.g. Treponema pallidum). • Viruses must be grown in cell or tissue cultures as they are incapable of free-living existence. • Some parasites (e.g. Trichomonas vaginalis) can be cultivated in liquid media but it is easier to detect them by microscopic examination (Giemsa staining).
There are some basic conditions for cultivation of bacteria: • Optimum environmental moisture. It is possible to cultivate bacteria in liquid media or in solid media with a gelling agent (agar) binding about 90 % of water. • Optimum temperature for cultivation of bacteria of medical importance is about 37 °C. Saprophytic bacteria are able to grow at lower temperatures. • Optimum pH of culture media is usually 7.2-7.4. Lactobacillus sp. needs acid pH and Vibrio cholerae alkaline pH reaction for the growth. • Optimum constituents of bacteriological culture media. All culture media share a number of common constituents necessary to enable bacteria to grow in vitro.
Optimum quantity of oxygen in cultivation environment • Bacteria obtain energy either by oxidation or by fermentation, i.e. oxidation-reduction procedure without oxygen. Bacteria are classified into four basic groups according to their relation to atmospheric oxygen: • Obligate aerobes – reproduce only in the presence of oxygen. • Facultative anaerobes – reproduce in both aerobic and anaerobic environments. Their complete enzymatic equipment allows them to live and grow in the presence or absence of oxygen. • Obligate anaerobes – grow only in the absence of free oxygen (i.e. unable to grow and reproduce in the presence of oxygen). Some species are so sensitive that they die if exposed to oxygen. • Anaerobic aerotolerant microbes do not need oxygen for their growth and it is not fatal for them. • Some aerobic and anaerobic bacteria need 5-10 % CO2 in the environment (microaerophilic).
SPORES(endospores)the spore isformedinsidetheparentvegetative cell – hence thename „endospores“
The spore is a dehydrated, multishelledstructurethatprotectsandallowsthebacteria to exist in „suspendedanimation“. • Itcontains a complete copy ofthe chromosome, the bare minimum concentrationsofessentialproteinsandribosomes, and a highconcentrationofcalciumbound to dipicolinicacid.
Membersofseveralbacterialgenera are capableofformingendospores: • Bacillus anthracis • Clostridium tetani • Clostridium botulinum • Clostridium perfringens • and other, but never gram-negative microbes
Spore formation is a means by which some bacteria are able to survive extremly harsh environmental conditions. • The genetic material of the bacterial cells is concentrated and than surrounded by a protective coat, rendering the cell impervious to desiccation, heat and many chemical agents.
The bacteria in the stage of spore is metabolically inert and can remain stable for months to years. When exposed to favorable conditions, germination can occur, with the production of single cell that subsenquently can undergo normal replication. • It should be obvious that the complete eradication of disease caused by spore-forming microorganisms is difficult or impossible. • The two major groups of bacteria that form spores are the aerobic genus Bacillus (e.g. disease anthrax) and the anaerobic genus Clostridium (e.g. disease tetanus, botulinismus).
Sporulation • The sporulation process begins when nutritional conditions become unfavorable, depletion of the nitrogen or carbon source (or both) being the most significant factor. • Sporulation occurs massively in cultures that have terminated exponential growth as a result of such depletion.
Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components. • These changes represent a true process of differentiation. A series of genes whose products determine the formation and final composition of the spore are actived, while another series of genes involved in vegetative cell function are inactivated. • These changes involve alterations in the transcriptional specifity of RNA polymerase, which is determined by the association of the polymerase core protein with one or another promoter-specific protein called a sigma factor. Different sigma factors are produced during vegetative growth and sporulation.
Sporulation • Morphologically, sporulation begins with the isolation of a terminal nucleus by the inward growth of the cell membrane. • The growth process involves an infolding of the membrane so as to produce a double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore.
Sporulation • The two spore membranes now engage in the activity synthesis of special layer that will form the cell envelope: • the spore wall and cortex, lying between the facing membranes, and the coat and exosporium lying outside the facing membrane. • In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of unique spore constituents.
Core • The core is the spore protoplast. • It contains a complete nucleus (chromosome), all of the components of the proteins-synthetizing apparatus, and an energy-generating system based on glycolysis. Cytochromes are lacking even in aerobic species, the spores of which rely on shorted electron transport pathway involving flavoproteins. A number of vegetative cell enzymes are increased in amount (eg. alanine racemase), and a number of unique enzymes are formed (eg. dipicolinic acid synthetase). • The energy for germination is stored as 3-phosphoglycerate rather than as ATP.
Core • The heat resistance of spores is due in part to their dehydrated state and in part to the presence in the core of large amounts (5 – 15% of the spore dry weight) of calcium dipicolinate, which is formed from an intermediate of the the lysine biosynthetic pathway. • In some way not yet understood, these properties result in the stabilization of the spore enzymes, most of which exhibit normal heat lability when isolated soluble form.
Spore wall • The innermost layer surrounding the inner spore membrane is called the spore wall. • It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.
Cortex • The cortex is the thickest layer of the spore envelope. • It contains an unusual type of peptidoglycan, with many fewer cross-links than are found in cell wall peptidoglycan. • Cortex peptidoglycan is extremly senstitive to lysozyme, and its autolysis plays a key role in spore germination.
Coat • The coat is composed of a keratin-like protein containing many intramolecular disulfide bonds. • The impermeability of this layer confers on spores their relative resistance to antibacteral chemical agents.
Exosporium • The exosporium is a lipoprotein membrane containing some carbohydrate.
Germination • The germination process occurs in three stages: • activation, • initiation, • outgrowth.
Activation • Even when placed in an environment that favors germination (eg. nutritionally rich medium) bacterial spores will not germinate unless first activated by one or another agent that damages the spore coat. • Among the agents that can overcome spore dormancy are heat, abrasion, acidity, and componds containing free sulfhydryl groups.
Initiation • Once activated, a spore will initiate germination if the environmental conditions are favorable. • Different species have evolved receptors recognise different effectors as signaling a rich medium. • Binding of the effector activates an autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes.
Outgrowth • Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. • A period of active biosynthesis follows. This period, which terminates in cell division, is called outgrowth. • Outgrowh requires a supply of all nutrients essenial for cell growth.
The spore stain • Spores are most simply observed as intracellular refractile bodies in unstained cell suspensions or as colorless areas in cell stained by conventional methods. • The spore wall is relatively impermeable, but dyes can be made to penetrate it by haeting the preparation. • The same inpermeability then serves to prevent decolorization of the spore by a period of alcohol treatment sufficient to decolorize vegetative cells. The latter can finnaly be counterstained.Spores are commonly stained with malachite green or carbolfuchsin.