Bohomolets Microbiology Lecture #3


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By Ms. Kostiuk from Microbiology department

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  • Growth takes place in two levels. On one level, a cell synthesizes new cell components and increases its size, in the other level, the number of cells in the population increases. In most bacteria, growth involves increase in cell mass and number of ribosomes, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, forming of the two chromosomes, septum formation, and cell division.
  • Bacterial cells reproduce asexually by binary fission or transverse fission . b/ During this process there is an orderly increase in cellular structures and components, replication and segregation of the bacterial DNA, and formation of a septum cross wall which divides the cell into two progeny cells.  The process is coordinated by the bacterial membrane perhaps by means of mesosomes.  The DNA molecule is believed to be attached to a point on the membrane where it is replicated. c/ The 2 DNA molecules remain attached at points side-by-side on the membrane while new membrane material is synthesized between the two points. Other cytoplasmic components (ribosomes) are distributed to the 2 developing cells. d/ When septum formation is completed the cell splits into two progeny cells.  In many bacteria, the septum is degraded after cell division by autolysis, which leads in two independent daughter cells. In other bacteria, such as streptococci, septum separation is usually incomplete and the cell remain attached to one another to form chains.
  • You can see, that mesosome, which is formed by cytoplasmic membrane, takes part in binary fission of cell
  • The life cycle of a single bacterial cell may be taken as the time of division of a mother cell into two daughter cells, and then when one of the daughter cells divides into two more daughter cells. The time required to achieve a doubling of the population size is known as the doubling time or generation time . Generation time of bacteria influence to development of disease. So, if gas gangrene (agent is Clostridium perfringens) develop very rapidly, syphilis (agent is Treponema pallidum) is a chronic disease which can occur a lot of years.
  • 1. When a culture of bacteria is diluted and then transferred into fresh medium, the number of viable cells does not increase immediately. Before cells can multiply, they increase in length and synthesize macromolecules such as enzymes, proteins, RNA. This phase is the lag phase of cell division. The duration of the lag phase is apparently dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium. 2. During the exponential, or log phase , the cells divide at their maximum rate, and are most susceptible to the action of antibiotics and other deleterious agents. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. However, the division time of any species also depends on its genetic constitution. 3. Bacteria stop growing and enter the stationary phase once they have reached a population of about 5 billions cells per milliliter of medium. This occurs for one of two reasons: either a nutrient is used up or toxic products accumulate from the cells’ metabolism. In most cases, every cell in the population stops multiplying but none die. In other cases, some cells divide and an equal number die. Some organisms remain in the stationary phase for only a few hours, others remain for several days. 4. The total number of viable cells in the population begins to decrease at the onset of the death phase . As the limited factors intensify, cells begin to die in exponential numbers, and they are unable to multiply. The speed with which death occurs depends on the relative resistance of the species and how toxic the conditions are, but it is usually slower than the exponential growth phase. Viable cells often remain many weeks and months after this phase has begun.
  • We have discussed the growth of bacteria in the laboratory, in tube or flasks of liquid or on agar plates. In these containers, nutrients are not renewed after they have been used up, nor are waste products removed. This system is termed a closed system or batch culture . In batch culture growth, nutrients are expended, and metabolic products accumulate in the closed environment. Under these conditions, bacteria grow at an exponential rate for only a relatively short time. In nature, however, nutrients may be continuously replaced, waste materials are removed, and cells are washed away. In this system, termed an open system , the bacteria may remain in the exponential phase of growth for a long period. The open system found in nature can be simulated to some degree in the laboratory by using a device called a chemostat. In such continuously culture system , fresh medium replaces some of the spent medium, thus permitting continuous growth of a culture. Because end products do not accumulate and nutrients are not completely expended, the bacteria never reach stationary phase in a chemostat. Continuous culture use when we want to take large volume of microorganisms or their products.
  • Liquid. These media, termed broths, milks, or infusions, are made by dissolving various solutes in distilled water. A common laboratory medium, nutrient broth , contains beef extract and peptone dissolved in water. 2. Semisolid. At ordinary room temperature, semisolid media exhibit a clotlike consistency because they contain an amount of solidifying agent (agar or gelatin) that thickens them but does not produce a firm substrate. Semilolid media are used (for instance) to determine the motility of bacteria. 3. Solid media provide a firm surface on which cells can form discrete colonies and advantageous for isolating bacteria. It contain a solidifying agent that as thermoplastic: it is solid at room temperature and most incubation temperatures, and it melts at the boiling temperature of water (100 0 C). This agent is agar , a complex polysaccharide isolated from the red alga Gelidium.
  • The tube on the left shows no motility, the tube in the right shows motility.
  • Depending upon what is added, a microbiologist can make a medium for nearly any purpose. As a result, only a few species of bacteria or fungi cannot yet be cultivated artificially. Media are used for primary isolation, to maintain cultures in the lab, to determine biochemical and growth characteristics, and other functions. General purpose media are designed to grow as broad a spectrum of microbes as possible. As a rule, they contain a mixture of nutrients that could support the growth of pathogens and nonpathogens alike. Examples include nutrient agar and broth. Meat-peptone agar consist of meat extract, peptone, agar-agar and distilled water. 2. An enriched medium contains complex organic substances such as blood, serum, or special growth factors (vitamins, amino acids) that certain species must have in order to grow. Pathogenic Neisseria (one species causes gonorrhea) are grown on chocolate agar, which is made by heating blood agar. 3. A selective medium contains one or more agents that inhibit the growth of a certain microbe or microbes (A, B, C) but not others (D) and selects, microbe D and allows it to grow. Selective media are very important in primary isolation of a specific type of microorganism from samples containing dozens of different species. For example, Mannitol salt agar, which is used to isolate members of the genus Staphylococcus. It is selective for this genus because its members can grow in the presence of 7.5% chloride, whereas many other species are inhibited by this high concentration. 4. Differential media grow several types of microorganisms and are designed to display visible differences among those microorganisms. Differentiation shows up as variations in colony size and color, in media color changes. 5. Transport media are used to maintain and preserve specimens that have to be held for a period of time before clinical analysis or to sustain delicate species that die rapidly if not held under stable conditions.
  • It is often possible to determine in which class organisms belong by growing them in a nutrient agar medium in shake tubes. The tube with agar not yet solidified is inverted several times, which disperses the bacteria throughout the tube. The agar is then allowed to harden. The top of the tube is highly aerobic and the bottom is anaerobic, since heating it drives off oxygen from the medium and solidified agar inhibits diffusion of oxygen. Therefore, the location in the tube of bacterial growth indicates their requirement for oxygen.
  • Two general methods of cultivation are the most simple and available. One involves incubating culture in anaerobe jars, from which the oxygen has been removed. A common type of anaerobe jar contains two main elements: a disposable packet that produces hydrogen, and a catalyst that combines the hydrogen with any free oxygen to form water. A second method involves adding chemicals to the medium that react with oxygen, such as sodium thioglycollate, cysteine, glucose, etc. the medium is then often boiled to drive out the air before the bacteria are inoculated.
  • The main goal for doctors is to make accurate diagnosis of infectious disease. For it we should to identify of agent which caused this disease. In clinical laboratories, it is critical to identify quickly the agents that are isolated from patients to the best possible treatment can be given. To do this, an organism is described in as many ways as is practically possible. The investigator identifies the organism as to genus and perhaps species by comparing the properties of the unknown organism with organisms that have already been identified. The numbers and types of tests used depend upon the type of infectious agent one is trying to detect or identify, and the type of specimen being examined. Another important part of identifying an organism depends on disease symptoms.
  • Colonial appearance is one of important feature for identification of microorganisms. Different groups of microbes have different types of colonies. It is cultivating properties.
  • Bacteria can form S and R forms of colonies. It is quite stable characteristic of culture. S means smooth, R means rough. For the most of pathogenic bacteria S-form is usual. But under unfavourable condition bacterial culture can change to other form (transformation S form to R and R to S – very rarely). When it virulence and antigen features of bacteria can be changed. This phenomenon is termed dissociation.
  • Pigments can protect bacterium from UV lights.
  • Certain isolation techniques are based on the concept that if an individual bacterial cell is separated from other cells and provided adequate space on a nutrient surface, it will grow into a discrete mound of cells called a colony . Because it was formed from a single cell, a colony consists of just that single species and no other.
  • The species is the basic taxonomic unit. Several similar species of bacteria are grouped into a genus , and many similar genera are included within a family . Families, in turn, are grouped into orders , many orders that have similar characteristics constitute a class , and several classes constitute a division . Similar division, in turn, are grouped into kingdoms . At the present time, all bacteria are officially classified in a single kingdom Prokaryotae because of their common prokaryotic cell structure.
  • The best classification schemes group organisms that are related through evolution and separate those that are unrelated. Unfortunately, this is more difficult in the case of bacteria than in plants and animals. But, some of the modern techniques of bacterial taxonomy used to group organisms according to their evolutionary relationship. There are numerical and genetic taxonomy. Numerical taxonomy is based on calculating the percentage of characteristics that two groups have in common. In this approach, the investigator conducts a large number of tests to determine whether certain features are present or absent in an organism. These tests include such characteristics as the ability to degrade lactose, the ability to form endospores, and the presence of flagella. Some characteristics such as motility and spore-forming ability depend on many genes and therefore are more fundamental and important properties. The final result of classification by numerical taxonomy is expressed in terms of a similarity coefficient , defined as the percentage of the total number of characters tested that are common to 2 strains. The strains that have more than a 90% similarity coefficient are termed a single species. 2. Molecular approaches to taxonomy . Resent developments in methods of classification are based on comparing more directly the information contained in the DNA of different groups of microorganisms. Because the genetic information of a cell is encoded in its DNA, the relatedness of 2 organisms is directly connected to the similarity of base sequences in their DNA. So, if numerical classifications are based in phenetic characteristics, genetic taxonomy based on similarity of DNA sequences. Another product that has been sequenced recently to determine genetic relatedness between organisms is 16S ribosomal RNA, a component of the small subunit of prokaryotic ribosomes. Now, every new obtained species should be identified with this technique. Chemical and serological analysis are subsidiary and cannot be used for classification along.
  • At the present time, the taxonomic scheme for bacteria is practical and convenient, consisting of descriptions of organisms, including their morphology, staining characteristics, and nutritional and metabolic properties. The descriptions of bacteria are contained in the reference text, Bergey’s Manual of Systematic Bacteriology .
  • Organisms are normally named according to a binomial system in which the organism is identified by its genus and species. Bacteria are referred to by their unique binomial name, consisting of the genus and species names of each organism. The name of bacterium sometimes reflect the physiology or ecology of the organism. As examples, St. epidermidis, which live on skin. In other cases, the bacterial name may indicate the name of the person who discovered it (Shigella, Salmonella). Name of bacterium can reflect name of disease which it causes. Or presence of pigment in bacterial culture – St. aureus.
  • Bohomolets Microbiology Lecture #3

    1. 1. Growth, development and cultivation of microbes Evolution and classification of microorganisms
    2. 2. Possible mechanisms of propagation and surviving of procaryotic organisms Different bacteria Formation of L-forms Nonsporoforming parasite bacteria Change into nonculturable forms Spirochetes Formation of cyst Chlamidiae Intracellular and extracellular forms formation Mycoplasmas Budding Bacillus, Streptomycetes Formation of spores Actinomycetes, mycobacterium, mycoplasmas Fragmentation Organisms Mechanism
    3. 3. Inclusions of Chamidiae in infected cells
    4. 4. Growth is a steady increase in all of the chemical components of an bacterium and usually results in an increase in the size of a cell
    5. 5. Steps of binary fission a/ A parent cell prepares for division b/ Enlarging of bacterium cell wall, cell membrane, and overall volume. The wall form the transverse septum, and the duplicated chromosome becomes affixed to a special membrane site. c/ The wall septum grows inward, and the chromosomes are pulled toward opposite cell ends as the membrane enlarges. d/ The septum is synthesized completely through the cell center.
    6. 6. Division of bacterial cell Streptococcus pyogenes
    7. 7. Participation of mesosome in bacterium fission <ul><li>N – nucleoid </li></ul><ul><li>PC – cell wall </li></ul><ul><li>MC – cytoplasmic membrane </li></ul><ul><li>M – mesosome </li></ul><ul><li>S – septum </li></ul>
    8. 8. Doubling time of various bacteria under optimal conditions 23 28 Staphylococcus aureus in rabbit testes 1980 ( 33 hours) Treponema pallidum 336 (2 weeks) 800 ( >13 hours) Mycobacterium tuberculosis 23 28 Bacillus subtilis 16 20 Escherichia coli 8 10 Clostridium perfringens Time required for one cell to grow to visible colony (hr) Generation time (min) Species
    9. 9. The phase of bacterial growth in closed system 1. The lag phase , during which vigorous metabolic activity occurs but cells do not divide. 2. The log (logarithmic) phase is when rapid cell division occurs. 3. The stationary phase in which the total number of viable cells remains constant. 4. The death phase in which the total number of viable bacteria decreases exponentially.
    10. 10. Continuous culture growing in chemostat Multiplying bacteria Reservoir with medium <ul><li>Closed system – batch culture </li></ul><ul><li>Open system – continuous culture </li></ul>
    11. 11. Categories of media classification for bacteria cultivation Consistency Liquid Semisolid Solid
    12. 12. Bacteria growth in semisolid media
    13. 13. Categories of media classification for bacteria cultivation Functional type General purpose Enriched Selective Differential Specimen transport Anaerobic growth
    14. 14. Enriched medium Blood agar with Enterococcus faecalis
    15. 15. Differential media MacConkey agar differentiates between lactose-fermenting bacteria (indicated by a pink-red reaction in the center of the colony) and lactose-negative bacteria (indicated by an off-white colony with no dye reaction).
    16. 16. Growth of bacteria with different requirements in oxygen in shake tubes
    17. 17. General methods of cultivation of anaerobe microorganisms <ul><li>Anaerobe jars </li></ul><ul><li>Special medium for anaerobic growth </li></ul>
    18. 18. Methods of identifying bacteria <ul><li>Microscopic analysis: shape, cell arrangement, inclusions, endospores, capsules, motility, Gram staining. </li></ul><ul><li>Culturing: appearance of colonies, including texture, size, shape, pigment; speed of growth and patterns of growth in liquid medium </li></ul><ul><li>3. Biochemical tests: detection products of microbial metabolism. Examples include tests for fermentation of sugars, capacity to digest proteins, presence of enzymes such as catalase, oxidase. </li></ul><ul><li>4. Molecular biological techniques: analysis of DNA sequences with DNA technology </li></ul><ul><li>5. Serological techniques : analysis of antigen properties. </li></ul>
    19. 19. Colonies of bacteria Clostridium sporogenes Bacillus spp. Pseudomonas aeruginosa Bacillus anthracis Mycobacterium tuberculosis Mycoplasma pneumoniae
    20. 20. Colonies of fungi genus Microsporum
    21. 21. S - and R – forms of colonies
    22. 22. Bacterial pigments Nocardia
    23. 23. Method for isolating bacteria A pure culture is a population of microorganisms which are belonged to one species and grown on nutrient medium
    24. 25. Special terms Strain is a culture of microorganisms derived from a single parent (organism or place). Type is a culture of microorganisms which differs in structure or metabolism from other culture of that species. Clone is a population of microorganisms that are descendents of a single mother cell
    25. 26. Subspecies in bacteria <ul><li>♦ Subspecies </li></ul><ul><li>♦ Types: </li></ul><ul><li>Morphovar (differs in structure of cell) </li></ul><ul><li>Chemovar (differs in metabolism) </li></ul><ul><li>Biovar (differs in number of biological features) </li></ul><ul><li>Phagevar ( differs in susceptibility to bacterial viruses) </li></ul><ul><li>Serovar (differs in antigenic makeup) </li></ul>
    26. 27. Taxonomic ranks of the bacterium Leptospira interrogans interrogans Species Leptospira Genus Leptospira ceae Family Spirochaeta les Order Scotobacteria Class Gracilicutes Division (Phymlum) Prokaryotae Kingdom Example Formal rank
    27. 28. Taxonomy system of classification Properties A lot of different phenetic features Chemical features Antigenic features Genetic features Numerical taxonomy Genetic taxonomy Cheme- taxonomy Serological taxonomy
    28. 29. Bergey’s Manual of Systematic Bacteriology. First edition – 1923. Classification of bacteria based on one or number of the most characteristic features
    29. 30. Genera Species Author’s name Bacterial morphology Disease’s name Pigment forming Habitat Author’s name Escherichia,Salmonella,Shigella Vibrio, Staphylococcus, Streptococcus Vibrio cholerae, Salmonella enteritidis Staphylococcus aureus, Pseudomonas aeruginosa Escherichia coli, Staphylococcus epidermidis Shigella sonney, Borrelia burgdorferi