Bacteriology Handout PDF

Basic Microbiology Bacteriology Lecture notes prepared by Prof. C. Agyare Office: Room C103 Course content History/Development of Microbiology, classification and nomenclature Structure and function Culture media, growth requirements, dynamics of growt...

Basic Microbiology Bacteriology Lecture notes prepared by Prof. C. Agyare Office: Room C103 Course content History/Development of Microbiology, classification and nomenclature Structure and function Culture media, growth requirements, dynamics of growth Mode of reproduction Simple identification procedures, Gram staining and Important biochemical diagnostic methods Reference books Medical Microbiology by Kayser, F.H., Bienz, K.A., Eckert, J., Zinkernagel, R.M. (2005), Thieme Publication. Pharmaceutical Microbiology edited by W.B. Hugo and A.D. Russel, 6th Edition, Blackwell Sciences Ltd. Medical Microbiology, A guide to microbial infections: pathogenesis, immunity, laboratory diagnosis and control. Edited by Greenwood, D., Slack, R.C.B., Peuttherer, J.F. (2003), Churchill Livingstone Publication. Foundations in Microbiology by Talaro, K. and Talaro, A. (1996). Wm. C. Publishers. Summary of history and development of Microbiology Microbiology is the study of living organisms of microscopic size The term was introduced by French Chemist, Louis Pasteur who demonstrated that fermentation was caused by growth of bacteria and yeasts (1857-60). Anthony van Leuwenhoek first saw microorganism with aid of microscope in 1675 and termed them "animalcules “ Controversy of origin of microorganisms: Louis Joblot (1718) and John Needlam (1749) - Spontaneous generation. Lazzaro Spallanzani (1765 and 1776) and Louis Pasteur (1860-64) settled the controversy with the specific conditions required for sterility (115-120oC). Ferdinand Cohn (1876) discovered heat resistant spores Robert Koch in 1877 examined bacteria in dried film stained with aniline dyes and developed a method for isolation of pure cultures from mixed cultures on nutrient agar. 2 Classification of Bacteria The main groups of bacteria are distinguished by the microscopic observation of their morphology and staining reactions. Most medically important bacteria fall are classified on the basis of their shape. Details of structure provide a basis for a separate division into: 1. Filamentous bacteria (Actinomycetes), most of which are capable of true branching and which may produce a type of mycelium. 2. 'True' bacteria, which multiply by simple binary fission. 3. Spirochaetes, which divide by transverse binary fission. 4. Mycoplasmas, which lack a rigid cell wall. 5. Rickettsiae and Chlamydiae, which are strict intracellular parasites. Filamentous bacteria These are sometimes referred to as 'higher bacteria'. A few are of medical interest as pathogens, and some produce antibiotics. 1. Actinomyces. Gram-positive, non-acid-fast, tend to fragment into short coccal and bacillary forms and not to form conidia; anaerobic (e.g. Actinomyces israelii). 2. Nocardia. Similar to Actinomyces, but aerobic and mostly acid-fast (e.g. Nocardia asteroides). 3. Streptomyces. Vegetative mycelium does not fragment into short forms; conidia form in chains from aerial hypbae (e.g. Streptomyces griseus). 4. Mycobacterium. Acid-fast; Gram-positive, but does not readily stain by the Gram method; usually bacillary, rarely branching; aerobic (e.g. Mycobacterium tuberculosis) True bacteria Most medically important bacteria fall into this group. They are classified on the basis of their shape. 1. Cocci - spherical, or nearly spherical, cells 3 2. Bacilli - relatively straight, rod-shaped (cylindrical) cells 3. Vibrios and Spirilla - curved or twisted rod-shaped cells. Cocci The main groups of cocci are distinguished by their predominant mode of cell grouping and their reaction to the Gram stain. The different cocci are relatively uniform in size (usually about 1 μm in diameter). Some species are capsulate and a very few are motile. 1. Streptococcus. Gram-positive; cells mainly adherent in chains due to successive cell divisions occurring in the same axis (e.g. Streptococcus pyogenes); sometimes predominantly diplococcal (e.g. Streptococcus pneumoniae). Fig. 1. Streptococci 2. Staphylococcus and Micrococcus. Gram-positive; cells mainly adherent in irregular clusters due to successive divisions occurring irregularly in different planes (e.g. Staphylococcus aureus). Fig. 2. Staphylococci 4  3. Sarcina. Gram-positive cells mainly adherent in cubical arrays of eight, or multiples thereof, due to division occurring successively in three planes at right angles (e.g. Sarcina lutea). 4. Neisseria. Gram-negative; cells mainly adherent in pairs and slightly elongated at right angles to axis of pairs (e.g. Neisseria meningitidis). 5. Veillonella. Gram-negative; generaJly very small cocci arranged mainly in clusters and pairs; an aerobic (e.g. Veillonella parvulay. Bacilli The primary subdivision of the rod-shaped bacteria is made according to their staining reaction by the Gram method and the presence or absence of endospores. 1. Gram-positive spore-forming bacilli. Apart from some rare saprophytic varieties, the only bacteria to form endospores are those of the genera Bacillus, Paenibacillus (aerobic) and Clostridium (anaerobic). They are Gram-positive, but liable to become Gram negative in ageing cultures. The size, shape and position of the spore may assist recognition of the species, e.g. the bulging, spherical, terminal spore ('drumstick' form) of Clostridium tetani. 2. Gram-positive non-sporing bacilli. These include several genera. Corynebacterium is distinguished by a tendency to slight curving, a club-shaped or ovoid swelling of the bacilli, and their arrangement in parallel or angular clusters caused by the snapping mode of cell division. Erysipelothrix and Lactobacillus are distinguished by a tendency to grow in chains and filaments, and Listeria by flagella that confer motility. 3. Gram-negative bacilli. This large grouping includes numerous genera such as the pseudomonads and the family Enterobacteriaceae ('coliform bacilli') as well as smalI, often pleomorphic, bacilli represented by Haemophilus, Brucella, etc. ('parvobacteria'), and anaerobes such as Bacteroides and Prevotella. 4. Vibrios and Spirilla. Vibrios and the related campylobacters are recognized as short, non-f1exuous, comma-shaped bacilli (e.g. Vibrio cholerae) and spirilla as non-f1exuous 5 spiral filaments (e.g. Anaerobiospirillum, 'Spirillum minus'). They are Gram-negative and mostly motile, having polar flagella and showing very active 'darting' motility. Fig. 3: Spirilla Spirochaetes These organisms differ from the 'true' bacteria in being slender flexuous spiral filaments which, unlike the spirilla, are motile without possession of flagella. The staining reaction, when demonstrable, is Gram-negative. The different varieties are recognized by their size, shape, wave form and refractility, observed in the natural state in unstained wet films by dark-ground microscopy. Fig. 4: Spirochaetes 1. Borrelia. Larger and more refractile than the other pathogenic spirochaetes and more readily stained by ordinary methods; coils large and open, with a wavelength of 2-3 μm; by electron microscopy a leash of between eight and twelve fibrils, each about 0.02 μrn thick, is seen twisted round the whole length of the protoplast (e.g. Borrelia recurrentis). 2. Treponema. Thinner filaments in coils of shorter wavelength (e.g. 1.0-1.5μrn), typically presenting a regular 'corkscrew' form; feebly refractile and difficult to stain except by silver impregnation methods; by electron microscopy, a leash of four fibrils is seen wound round the protoplast within the cell wall (e.g. Treponema pallidum). 6  3. Leptospira. The coils are so fine and close (wavelength about 0.5 μm) that they are barely discernible by darkground microscopy, though can clearly be seen winding round two axial filaments by electron microscopy. One or both extrernities of the organism are hooked, or recurved, so that it may take the shape of a walking-stick, an 'S' or a 'C' (e.g. Leptospira interrogans). Mycoplasmas These are prokaryotes that differ from 'true' bacteria in their smaller size and their lack of a rigid cell wall, which leads to extreme pleomorphism and sensitivity to external osmotic pressure. The viable elements range from 0.15 to over 1 μm in diameter, the smallest being capable of passing through filters that retain conventional bacteria. Mycoplasmas can be cultivated on cell-free nutrient media, and are the smallest and simplest organisms capable of autonomous growth. E.g. M. genitalium, M. pneumoniae, M. Pulmoni. Fig. 5: Mycoplasmas Rickettsiae and chlamydiae The rickettsiae are rod-shaped, spherical or pleomorphic Gram-negative organisms. They are generally smaller than 'true' bacteria, but are still resolvable in the light rnicroscope. Most are strict parasites that can grow only in the living tissues of a suitable animal host, usually intracellularly (e.g. Rickettsia prowazekii). Chlamydiae are similar to rickettsiae, but have a more complex intracellular cyc1e (e.g. Chlamydia trachomatis). 7 STRUCTURE OF BACTERIA Fig. 6: Bacterial cell In spite of their small size bacteria have a well organized cellular structure (Fig. 6). Like the cells of higher organisms the bacterial cell possesses a nucleus, a cytoplasm and a cytoplasmic membrane. The true bacteria and the actinomycetes resemble plant cells in possessing, in addition, a cell wall which is distinct from the cytoplasm and cytoplasmic membrane. 8 Table 1: Composition and function(s) of the various structures of the bacterial cell Structure Function(s) Predominant chemical composition Flagella Swimming movement Protein Stabilizes mating bacteria during Sex pilus Protein DNA transfer by conjugation Common pili or Attachment to surfaces; protection Protein fimbriae against phagotrophic engulfment Attachment to surfaces; protection Capsules (includes against phagocytic engulfment, Usually polysaccharide; "slime layers" and occasionally killing or digestion; occasionally polypeptide glycocalyx) reserve of nutrients or protection against desiccation Cell wall Prevents osmotic lysis of cell Gram-positive Peptidoglycan (murein) protoplast and confers rigidity and bacteria complexed with teichoic acids shape on cells Peptidoglycan prevents osmotic lysis Peptidoglycan (murein) and confers rigidity and shape; outer Gram-negative surrounded by phospholipid membrane is permeability barrier; bacteria protein-lipopolysaccharide associated LPS and proteins have "outer membrane" various functions Permeability barrier; transport of Plasma membrane solutes; energy generation; location of Phospholipid and protein numerous enzyme systems Ribosomes Sites of translation (protein synthesis) RNA and protein Highly variable; Often reserves of nutrients; additional Inclusions carbohydrate, lipid, protein or specialized functions inorganic 9 Chromosome Genetic material of cell DNA Plasmid Extrachromosomal genetic material DNA The cell wall The cell wall is a complex rigid structure which gives bacteria their definite shape. It must possess considerable strength so as to enable it to withstand the osmotic pressure (in some cases this is as much as 20 atmospheres) of the intracellular constituent. When the cell wall is absent, bacteria would burst, since the delicate cytoplasmic membrane would by itself be unable to withstand this high internal pressure. The thickness of most bacterial cell walls under normal conditions of growth is in the range of from 10 to 20 nm, the cell walls of Gram-positive bacteria being in general thicker than those of Gram-negative bacteria. The rigidity of the bacterial cell wall is due to the presence of a basal three-dimensional enveloping structure known as peptidoglycan or murein. The cell wall peptidoglycan is composed chemically of a backbone consisting of the amino sugars N-acetyl glucosamine and N-acetyl muramic acid, to the muramic acid residues of which are attached peptide side-chains comprising a limited number of amino acids, D- and L-alanine, D-glutamic acid and either L-lysine, diaminopimelic acid, diaminobutyric acid or ornithine. In some species either glycine or aspartic acid is also present. In the intact cell the three-dimensional structure of the peptidoglycan is completed by extensive cross-linking between neighbouring peptide chains. In Staphylococcus aureus this appears to occur through a glycine pentapeptide, linking lysine and terminal D- alanine residues. In certain bacteria e.g. Bacillus spp, Micrococcus lysodeikticus and Enterococcus faecalis, the wall mucopeptide of the intact cell is susceptible to the action of lysozyme. As a result of this the wall is broken down leaving a structure known as protoplast, which is bounded only by the cytoplasmic membrane. Protoplasts are osmotically sensitive and undergo rapid lysis in isotonic or hypotonic solutions. They can, therefore, only be maintained in intact form in hypertonic solutions. Protoplasts may also be obtained from certain bacteria by growing them in the presence of penicillin or other antibiotics which are capable of inhibiting cell wall synthesis. Protoplasts are much more readily prepared from Gram-positive than from Gram- negative bacteria. This is not surprising, as the peptidoglycan is a relatively minor component of the Gram-negative cell wall. Gram-negative organisms appear to retain a good deal of the cell wall lipopolysaccharide and lipoprotein. Peptidoglycan is the major constituent of the cell walls of Gram-positive bacteria constituting from 50% to as much as 90% of the wall. In Gram-negative species, 10 however, mucopeptide (= peptidoglycan) constitutes only from 5 to 10% of the wall, but even under these circumstances it is responsible for the osmotic stability of the cell This is because the osmotic pressure of the Gram-negative cell appears to be considerably less than that of the Gram-positive cell. In addition to mucopeptide, Gram-positive cell walls contain polysaccharides and teichoic acids. The latter are polymeric complexes of ribitol phosphate or glycerol phosphate with simple sugar or amino sugar residues, together with alanine in the unnatural D-configuration. Gram-positive cell walls do not appear to contain protein as an internal component of the wall but in some cases protein may be present in a microcapsular or capsular layer, e.g. the M proteins of the streptococci, and the glutamyl polypeptides of Bacillus species Gram-negative cell walls are considerably more complex biochemically than Gram- positive cell walls, and unlike the latter contain a high concentration of lipid and protein. From the marked difference in cell wall composition shown between the Gram-positive and Gram-negative bacteria it might be expected that the chemical composition of the cell wall could be useful as a taxonomic criterion. Cytoplasmic membrane Beneath the cell wall as an anatomically distinct structure is the cytoplasmic membrane. The membrane is semi-permeable, allowing the passage of water but impermeable to large molecular weight compounds and also many small molecular weight compounds, e.g. simple sugars and amino acids. Because of this it constitutes the osmotic barrier of the cell, controlling the passage of nutrients into the cytoplasm and of end products of metabolism out of it. Compared with the cell wall the cytoplasmic membrane is very thin, from 6 to 10 nm in cross-section. The membrane has a bimolecular leaflet structure consisting of a sandwich of two layers of lipid, the hydrophobic side-chains of which are on the inside and the hydrophilic residues on the outside, in contact on the one hand with the external environment and on the other with the cytoplasm. Interspersed in the lipid bilayers are molecules of protein with their hydrophilic residues projecting on the outside and inside of the membrane. Morphologically, the bacterial cytoplasm is much simpler than the cytoplasm of higher organisms. However, certain bacteria, particularly Gram-positive bacteria, contain in- vaginations of the cytoplasmic membrane. These structures known as mesosomes can sometimes be seen attached to the bacterial chromosome; they are often closely related to the septum of dividing cells. It is thought that they may have a regulatory role in cell 11 division, ensuring that each daughter cell receives a nucleus. Some may be involved in the secretion of extracellular products. The Cytoplasm The main structural components of the bacterial cytoplasm would appear to be the ribonucleoprotein granules, known as ribosomes They measure from 10 to 20 nm in diameter, and are the sites of protein synthesis. It has been estimated that a single bacterium may contain upwards of 10,000 ribosomes. The ribosomes have been known to occur in groups called polysomes linked together like beads on a chain by messenger RNA. The individual ribosomes have a sedimentation coefficient of 70 Svedberg units. Each 70S ribosome is a complex of two smaller units with sedimentation coefficient of 50S and 30S. In the presence of magnesium these form stable complexes to yield a 70S ribosome. In low magnesium concentrations, however, they dissociate and in the absence of magnesium further degradation occurs. The nucleus The nucleus carries the genetic blueprint of the cell coded in the nucleotide sequence of its desoxyribonucleic acid (DNA). The bacterial nucleus differs from the nucleus of higher cells. 1. It does not possess a nuclear membrane, and is therefore in direct contact with the cytoplasm of the cell. This may well facilitate rapid transfer of messenger RNA from the nucleus to the ribosomes. Cells lacking a nuclear membrane are sometimes referred to as procaryotic in contrast to eucaryotic cells which possess a nuclear membrane. 2. The bacterial DNA does not appear to be associated with a basic protein, though the presence of some protein in the nucleus has not been excluded. Many bacteria possess in addition to the above structure flagella, fimbriae or pili, capsules, spores and various types of granules. Since these structures are found only in certain species their demonstration is of importance in bacterial identification Flagella Flagella are the locomotor organs of bacteria. They are long, contractile hair-like filaments measuring from 12 to 19 nm in diameter, and usually several micrometres in length. In some bacterial species, e.g. the enterobacteriaceae, they are arranged along the sides of the organism. This is known as peritrichous flagellation. In others, such as Pseudomonas, flagella occur singly or in tufts at one or both ends - polar flagellation. Individually, flagella are too thin to be seen by ordinary light microscopy but they can be stained. They are best demonstrated by electron microscopy when they appear, 12 particularly those from peritrichously flagellated bacteria, as helical or wavelike structures. In electron micrographs the flagella can be seen to pass through the cell wall and to originate either from the cytoplasm or from the cytoplasmic membrane. Though they are not attached to the cell wall, flagella can apparently initiate movement only when the cell wall is present. Thus protoplasts prepared from Bacillus species by lysozyme treatment and possessing apparently normal flagella, are non-motile. Relative to their size, the speed with which the bacteria can be propelled is considerable; actively motile species are capable of travelling at up to 50 μm/sec. Chemically, flagella are composed mainly, if not entirely, of protein. They consist of strands wound round each other in helical forms which in certain species, e.g. Vibrio, are surrounded by sheaths. The strands are made up of a globular protein called flagellin, disposed helically around a hollow core. Under acid conditions the subunits dissociate, but on reversal of these conditions they re-aggregate to form intact flagella. Fimbriae or pili Fimbriae or pili are hair-like processes arising like flagella from granules in the cytoplasmic membrane and are only found in certain bacteria, particularly in Gram- negative bacilli. They differ from flagella in being shorter and thinner, straight, and play no part in the motility of the organism. Fimbriae cannot be demonstrated with the light microscope but can be seen in suitably prepared electron micrographs. They are best developed when the bacteria are grown in fluid media. They are probably structures by which bacteria can attach themselves to cells and to other particulate material. Certain bacteria possess specialized fimbriae or pili which are longer and thinner than the common type. These appear to be hollow and to constitute conjugation tubes through which DNA is transferred from one organism to another during conjugation; they are consequently referred to as sex pili Spores Bacteria of the genera Bacillus and Clostridium produce highly resistant dormant forms known as spores. These appear first as round or oval bodies which form within the cytoplasm of the organism. They are highly refractile (cellular structures that refract light) but stain with great difficulty. In simple Gram-stained preparations they appear as clear unstained areas. When stained, however, they hold the stain firmly and are not readily decolorized. The spore may be situated in the centre of the bacillus - equatorial, near one end – sub- terminal, or protruding from one end - terminal, the precise location being constant within a single species. In the Bacillus genus the diameter of the spore is normally less 13 than that of the bacterium but in the genus Clostridium the spore is usually wider than the bacterium. The first stage in the formation of the spore is a condensation of chromatin (the substance that forms chromosomes and contains DNA, RNA, and various proteins), apparently representing half the cellular DNA, in an area at one end of the bacterium known as the spore field which is destined to be the site of formation of the new spore. At the same time a transverse septum derived from the cytoplasmic membrane forms by a process of invagination, and divides the developing spore, now known as the forespore, from the rest of the bacillus, known as the sporangium. From this stage on, the two areas of the cell are functionally distinct, the sporangium continuing to produce components characteristic of the vegetative form, and the forespore producing components characteristic of the spore. The dividing septum eventually completely encircles the forespore as a double-layered membrane, the inner layer of which becomes the spore wall. The spore cortex is then laid down between the inner and outer layers and the process completed by transformation of the outer layer into the spore coats and exosporium. Finally, when the spore is fully formed, the body of the organism degenerates and the spore is set free, the entire process culminating in the release of the mature spore taking from four to eight hours to complete. Spores are dormant bacterial forms; they are non-metabolizing and non-reproducing. Spore DNA is not replicated, messenger RNA is not produced- and the spore protein synthesizing system is defective as judged by in vitro incorporation experiments. Their biological importance derives from their high resistance to a variety of agencies to which the vegetative form is susceptible-heat, ultra-violet irradiation, mechanical disruption and most chemical disinfectants. They can, as a result, survive for much longer periods than the vegetative form in environments which would be completely inimical to the latter. Spores have, in fact, been recovered from dried plants which have been preserved for some 300 years. Of particular importance to medicine is their high resistance to heat; some will resist boiling for several hours. This necessitates the employment, as in the autoclave, of temperatures of over 100°C for the sterilization of sporulating species. Spores show marked differences from vegetative organisms in morphology, chemical composition and antigenic structure. In the centre is the cytoplasm of the spore which appears to be much more homogeneous than the cytoplasm of the vegetative form. The cytoplasm is bounded by a delicate membrane or spore wall; outside the spore wall is a thick cortex of low density and low affinity for dyes. The cortex appears to contain most of the mucopeptide of the spore. This is surrounded by the coats of the spore of which up 14 to three may be present. Finally, outside the spore coat there is in many cases a loosely attached outer layer or exosporium. The most striking chemical differences between spores and vegetative cells are their lower free water content, and the presence of a large amount (on average about 10% of the mass of the spore) of an unusual compound, dipicolinic acid (DPA), occurring as its calcium salt. Spores have been shown to possess a number of spore-specific antigens and to produce a variety of enzymes differing both in antigenic and physical properties from the corresponding enzymes of the vegetative form. The spores are unusually rich in sulphur containing amino acids, notably cysteine, which appears to be located mainly in the spore coat. The disulphide linkages derived from these are responsible for the increased radiation resistance of the spore. The heat resistance of the spore is in some way related to the presence of a calcium dipicolinic acid chelate. In general, sporulation is initiated in conditions where, as a result of nutrient limitation, rapid growth is terminated. It would appear, however, that sporing bacteria possess in effect two genomes, one for the vegetative cell and one for the spore. During vegetative growth transcription of the spore genome is inhibited, and as sporulation proceeds the transcription of the vegetative genome is inhibited. Under suitable conditions the spore regenerates the vegetative form. This process, known as germination, is initiated apparently by the stimulation of certain so-called trigger nutrients, usually simple sugars, amino acids and ribosides present in the environment. It can be also be induced in some cases by unnatural means, e.g. by mechanical abrasion or by exposure to certain surface active compounds. In general, the readiness with which generation may be induced under such conditions is greatly increased by prior mild heat treatment The capsule The capsule is an outer covering of jelly-like material surrounding the cell wall. It is not essential to the life of the cell since it may be lost spontaneously by mutation and, in some cases, removed enzymically without loss of viability. Occasionally, the outer limit of the capsule may be poorly defined; it is then usually referred to as a slime layer. The capsules of most of the pathogenic bacteria are viscous polysaccharide gums which give the colony of the organism a mucoid appearance. Capsules have a low affinity for aniline dyes, and are most reliably demonstrated by 'negative staining' procedures. Polysaccharide capsules are readily washed off the cell with water and water must therefore be avoided in capsule stains. Organisms of the Bacillus genus are unusual in that their capsules are composed largely of polypeptide. In pathogenic species the 15 capsules inhibit the engulfment of the bacteria by the phagocytes, and play an important part in determining virulence; if the capsule is lost by either mutation or enzymic degradation the organism becomes avirulent. Microcapsule Certain bacteria are known to possess surface antigens, which exist in a distinct layer external to the cell wall, though insufficiently thick to be seen by ordinary microscopic methods. This layer is sometimes referred to as a microcapsule. Important examples of microcapsular antigens are the M antigens of the streptococci and the Vi antigens of Salm. typhi. Of these, the M antigens of the streptococci have probably most claims to be described as microcapsular, since they can be removed enzymically without affecting the viability of the organism or the integrity of the cell wall. In the intact cell they are normally covered by a true capsule. Metachromatic or volutin granules Intracellular granules may be observed in many species of bacteria. The most important of these are the metachromatic or volutin granules which are highly characteristic of the corynebacteria and which are of practical importance in the identification of these organisms. Metachromatic granules stain very intensely with basic dyes. They are granules of highly polymerized metaphosphate. Metachromatic granules accumulate in the bacterial cell only when the latter is grown on relatively rich media. Certain bacterial species show the presence of granules of glycogen like polysaccharide and of lipid. Many bacteria when grown under unsuitable environmental conditions assume an unusual and frequently bizarre morphology; this may take the form of spherical, lemon- shaped, boat-shaped or irregular coarse spiral structures usually known as involution forms. Most involution forms appear to be non-viable under ordinary conditions of cultivation. GROWTH OF BACTERIA Reproduction in bacteria The majority of bacteria reproduce by simple division or binary fission. Other bacteria undergo reproduction involving genetic exchange Binary fission Fission occurs when the cell reaches a critical size, but the immediate stimulus of the division process is unknown. The first stage in fission consists of a division of the nucleus, followed by division of the cell. The circular chromosome divides into two identical circles which segregate at opposite ends of the cell. At the same time, the cell 16 wall is laid down in the middle of the cell. Nuclear division generally occurs in phase with cell division but when cells are growing rapidly it tends to outpace cell division (Fig. 7). Consequently during rapid growth the cells usually show two or more nuclei, and one or more transverse septa prior to division. Fig. 7: Binary fission Two main types of cell division have been described. During the first, characteristic of Gram-negative bacteria, the cell appears to divide by a constrictive or pinching process. In the second, there is an in-growth of the cytoplasmic membrane to form a transverse septum, the new transverse cell wall being laid down between adjacent layers of membrane. Reproduction involving genetic exchange For many years, it was thought that binary fission was the only method of reproduction in bacteria, but it is now known that there are three methods of reproduction in which genetic exchange can occur between pairs of cells, and thus a form of sexual reproduction is exhibited. These processes are transformation, conjugation and transduction. Transformation A culture of Streptococcus pneumoniae deficient in capsular material could be made to produce normal capsulated cells by the addition of cell-free filtrate from a culture in which a normal capsulated strain had been growing. The material in the culture filtrate responsible for the re-establishment of capsulated cells is DNA. Conjugation Conjugation is a natural process found in certain bacterial genera and involves the active passage of genetic material from one cell to another by means of the sex pili. However, despite the resemblance of this process to the complete genetic exchange found in eukaryotes, it is not possible to designate male and female bacteria. Bacteria which are 17 able to effect transfer contain in their genetic make-up a fertility factor and are designated F+ strains. These are able to transfer part, and in some cases, all of their genetic material to F- strains. Transduction There are certain groups of viruses, called bacteriophages (phages), which can attack bacteria. This attack involves the injection of viral DNA into bacterial cells which then proceed to make new virus particles and destroy cells. Some viruses, known as temperate viruses, do not cause this catastrophic event when they infect their host, but can pass genetic material from one cell to another. Types of bacterial growth In the lab bacterial growth can be seen in three main forms: Developemt of colonies Transformation of a clear broth medium to a turbid suspension Biofilm formation, in which growth is spread thinly (300-400μm) over an inert surface and nutrition obtained from bathing fluid Mean Generation Time The rate at which fission occurs varies with different species. Many species, e.g. E. coli and many Gram-negative bacilli, multiply very rapidly, dividing, in their most rapid growth phase and under optimal conditions, once in every 20 to 30 minutes. Some organisms, however, e.g. tubercle bacilli, grow very slowly, dividing only once or twice in 24 hours. The time interval between one cell division and the next is called the generation time. When considering a growing culture containing many thousands of cells, a mean generation time is usually calculated. If a single cell reproduces by binary fission, then the number of bacteria n in any generation will be as follows: 1st generation n = 1 x 2 = 21 2nd generation n = 1 x 2 x 2 =22 3rd generation n = l x 2 x 2 x 2 = 23 yth generation n= 1x2y =2y For an initial inoculum of n0 cells, as distinct from one cell, at the yth generation the cell population will be: n = n0 x 2y This equation may be rewritten thus: log n = log n0 + y log 2 18 y = (log n – log n0) / log 2 = (log n – log n0) / 0.301 where y is the number of generations that have elapsed in the time interval between determining the viable count n0 and the population reaching n. If this time interval is t, then the mean generation time G is given by the expression: G = t / y = 0.301t / (log n – log n0) Growth curves When bacteria are inoculated into a fluid medium there is a delay of some hours before they begin to multiply. This period of delay is known as the lag phase (A). Although there is no obvious multiplication during the lag phase, bacteria are nevertheless highly active in it. From the beginning they increase in size and synthesize cellular material. This synthetic activity is clearly shown if the nitrogen concentration of a culture, which is a measure of the amount of bacterial protein present, is plotted against time The lag phase is followed by the logarithmic phase (B), during which the bacteria are multiplying at their maximum rate. The time required for one bacterial division during the logarithmic phase is known as the generation time. In the log phase the number of organisms present in each generation period is virtually twice that in the previous period. Consequently if the logarithm of the number of organisms is plotted against time the result is a straight line. The phase is of relatively short duration, lasting at most for some hours. In its termination three factors are of importance 19  1. Exhaustion of nutrients. This factor is probably of much greater importance to bacteria growing naturally than under the usual conditions of a laboratory culture 2. The accumulation of toxic metabolic end-products which inhibit bacterial growth 3. The achievement of a maximum population density. It would appear that even under conditions of optimal nutrition, and in the absence of significant inhibition by metabolic end-products, there is a minimal amount of living space in which a bacterium will grow Following the log phase there is a stationary phase (C), during which the bacterial population remains roughly constant. In this phase a balance is apparently struck between bacterial multiplication and bacterial death. The stationary phase is followed by the phase of decline (D), during which the number of organisms progressively decreases, the culture eventually becoming sterile. Complete sterility may not be achieved, however, for a considerable time NUTRITIONAL REQUIREMENTS During the growth of bacteria there occurs an active synthesis of the complex constituents of the cell. In order that this synthesis may take place, the elements which are ultimately incorporated in the cell protoplasm must be present in an assimilable form in the medium in which the organism is growing. Phosphorus, sulphur, sodium, potassium, iron and other elements required in small amounts are readily assimilated from simple salts present in the environment of the organism. Different types of bacteria, however, show considerable differences in the nature of the food material which they can accept as sources of the elements carbon and nitrogen. These differences correspond in general with differences in the synthetic power of the bacteria. Autotrophs Some organisms – autotrophs - are endowed with considerable synthetic power and can utilize very simple inorganic compounds, such as carbon dioxide and nitrates or ammonium salts, as sources of carbon and nitrogen respectively. From these they can build up their complex carbohydrates, proteins, fats and other essential constituents. Heterotrophs Heterotrophs differ from the autotrophs in requiring organic sources of carbon such as are found in carbohydrates, proteins, amino acids and fatty acids, for growth. 20 Almost all types of organic compounds can be utilized by some species of bacteria. These compounds serve not only as sources of energy but also as sources of the carbon required for the synthesis of cell material. Among the heterotrophic bacteria, however, there are marked differences in nutritional requirements. Some, such as E. coli, are relatively non-exacting in their demands, being capable of growing on fairly simple media containing glucose or other carbohydrate as a source of energy and common inorganic salts as a source of nitrogen, phosphorous, sulphur and other mineral requirements. Growth factors Many of the parasitic bacteria, however, are much more fastidious than E. coli and will not grow on a simple glucose salt medium such as described above, but require the addition of complex organic compounds, which they are unable to synthesize for themselves. Such compounds are known as growth factors. In general, the nutritional demands of bacteria (i.e. the range of growth factors they require) can be correlated with their degree of parasitism. As a result of long habituation, the more highly parasitic bacteria have become adapted to the well-stocked nutritional environment of the animal body and, finding many of their complex organic requirements pre-formed, do not need the elaborate synthetic powers of the less exacting free-living bacteria. Bacterial growth factors can be conveniently classified in three large groups: 1. Amino acids 2. Bacterial vitamins 3. A miscellaneous group of compounds of unrelated chemical composition Amino acids Amino acid requirement may vary from that of comparatively non-exacting organisms, such as Salm. typhi, which needs only a single amino acid, tryptophane, to that of highly exacting organisms such as Leuconostoc mesenteroides, which requires 17 amino acids, none of which it can synthesize. In general, the requirement for amino acids is most frequently for the aromatic type, since these are the most difficult to synthesize. Gram-positive bacteria are much more exacting in their demands for amino acids than are Gram-negative bacteria. Even where considerable amino acid requirement exists, bacteria are nevertheless able to meet some of their needs by synthesis from ammonium salts Nitrate can be utilized as an alternative source of nitrogen by some of the heterotrophic bacteria. The organism must, however, first reduce it to ammonia through the action of the enzyme nitratase. In some cases amino acid requirement depends on the vitamin composition of the medium. If the medium is deficient in a vitamin required for amino 21 acid synthesis, the vitamin being one that the organism cannot synthesize, the appropriate corresponding amino acid must be supplied preformed Vitamins Bacterial vitamins play a catalytic role in bacterial metabolism, generally as constituents of coenzymes, which, (in contrast to the amino acids), are required by bacteria in very low concentrations. The most important of the bacterial vitamins are those belonging to the B group of animal vitamins, - biotin, nicotinic acid, pantothenic acid, pyridoxine, para minobenzoic acid, riboflavine, thiamine and folic acid. Their role in bacterial metabolism is essentially similar to their role in tissue metabolism. Other important bacterial vitamins are lipoic acid, which is required in the oxidative decarboxylation of pyruvic acid, and haematin which is a component of bacterial cytochromes and of the enzymes peroxidase and catalase. Other factors The need for purines and pyrimidines is related to the growth factor composition of the medium. Oleic acid is required by the corynebacteria and Erysipelothrix, and the polyamines putrescine and spermidine by H. infiuenzae and H. parainfiuenzae. The power of bacteria to synthesize growth factors is of great importance in connection with animal nutrition, since animals can utilize vitamins produced by bacteria in the intestine Bacteria in the body have an ample supply of their required growth materials. In the laboratory, however, it is essential, when attempting to grow a particular organism, to ensure that the medium satisfies all its nutritional requirements. Blood or other body fluids can usually be relied on to supply a considerable variety of food materials such as amino acids and growth factors lacking in the simpler laboratory media. Carbon dioxide All bacteria require for the initiation of growth a low concentration of carbon dioxide; this is essential to heterotrophic organisms as a means of replenishing the dicarboxylic acids of the Krebs cycle. Sufficient carbon dioxide both to initiate and to maintain growth is usually obtained from endogenous metabolism. Large amounts of carbon dioxide are, however, indispensable to gonococci and meningococci, and to the bovine variety of Br. abortus. Oxygen Bacteria show considerable differences in their requirement for and tolerance of molecular oxygen. Some, the obligatory aerobes, grow only in its presence. Organisms of this type, e.g. gonococci, meningococci and V. cholerae, grow poorly in the depths of fluid media where the oxygen tension is insufficiently high for significant growth. Others, 22 the obligatory anaerobes, grow only when all traces of oxygen are absent from the medium. The majority of pathogenic bacteria are facultative anaerobes, i.e. organisms which will grow in both aerobic and anaerobic conditions. The inhibition of growth by oxygen is due to the production of hydrogen peroxide, a substance highly toxic to bacteria, coupled with a lack of the detoxicating enzyme, catalase, of aerobic organisms. Temperature Each type of bacterium multiplies best within a restricted temperature range. For most of the pathogenic bacteria optimum growth temperature (i.e. the temperature at which the organism multiplies at a maximum rate but which is not necessarily the temperature of maximum yield) is 37°C, with upper and lower growth limits of 40 to 50oC and 15 to 20oC respectively. In general, the more highly parasitic a bacterium the closer does its temperature range of growth approximate to 37°C, the temperature to which it has become accustomed by association with the animal body. Organisms with an optimal temperature of about 20 - 45°C are known as mesophiles Some bacteria, notably certain free-living members of the Pseudomonas genus, have an optimum temperature of around 20oC, and are capable of significant growth in temperatures below l0oC, a temperature at which mesophiles will not grow. Organisms of this type, which are known as psychrophiles, are widely distributed in nature; they are not of pathogenic importance though they may be responsible for food decomposition. Psychrotrophs, on the other hand, although they can also grow at 0 ◦C, have much higher temperature optima (20–30oC). Members of this group are often economically significant due to their ability to grow on refrigerated foodstuffs. At the other extreme are the thermophiles, many of which have optimum temperatures in the range of 40 to 80°C. Extreme thermophiles have optimum values in excess of this, and can tolerate temperatures in excess of 100oC. A member of the primitive bacterial group called the Archaea has been reported as growing at a temperature of 121oC. It is probable that the unusual behaviour of the thermophilic bacteria is due to the high heat resistance of their enzyme proteins. Thermophilic bacteria, though not pathogenic, are of importance since they may be responsible for spoilage in food that has been processed by heat. pH Microorganisms are strongly influenced by the prevailing pH of their surroundings. Organisms that grow best near neutral pH are called neutralophiles or neutrophiles. Most bacteria grow best at pH values of 7.4 - 7.6, on the alkaline side of neutrality. Some bacterial species to grow best at acidic pH and are referred to as acidophiles. Acidophiles grow best at pH less than 5.5. Some bacteria can grow at pH as low as 0.8. Alkalophiles are organisms that grow best at alkaline pH 23 The pH range (between minimum and maximum values) is greater in fungi than it is in bacteria. Salinity The concentration of NaCl affects the growth of bacteria. Organisms that tolerate high concentration of salt are called halophiles. Moderate halophiles which include marine bacteria grow best at salt (NaCl) concentrations of about 3%. Extreme halophiles exhibit maximal growth rates in saturated brine solutions. These organisms grow well in salt concentrations in excess of 15%. Pressure The solute concentration affects the osmotic pressure that is exerted across the plasma membrane. The cell walls of bacteria make them relatively resistant to changes in osmotic pressure, but extreme osmotic pressure can result death of bacteria cells. In hypertonic solutions bacteria cells may shrink and desiccated, in hypotonic solutions the cells may burst. Bacteria that can grow in solutions with high solute concentration are referred to as osmotolerant. Those that grow best at high osmotic pressures are referred to as osmophiles. Hydrostatic pressure (pressure exerted by a water column as a result of the weight of the water column) has influence on bacterial growth rate. Hydrostatic pressures greater than 200 atmospheres generally inactivate enzymes and disrupt membrane transport processes. Organisms that can grow at very high hydrostatic pressures are referred to as barotolerant. Those that grow best at high hydrostatic pressures are referred to as barophiles. Light radiation Exposure to visible light can cause death of bacteria. The exposure leads to formation of singlet oxygen which can result in death of bacteria cells. Some bacteria produce pigments that protect them against the lethal effects of light. Eg yellow, orange or red carotenoid pigments interfere with the formation and action of singlet oxygen. Bacteria possessing carotenoid pigments can tolerate much higher levels of exposure to sunlight than non-pigmented ones. Cultivation of Bacteria Bacteria may be cultivated in the laboratory by providing them with suitable nutrient material. This may be supplied either in fluid or in solid media. When inoculated into fluid media most species grow diffusely throughout the medium but some, particularly those which prefer a high oxygen tension, grow mainly on the surface of the medium while others grow mainly at the bottom. A fluid culture of a rapidly growing organism such as E. coli may contain after overnight incubation as many as 109 to 1010 organisms per ml. On solid media bacteria grow in the form of colonies, each colony consisting of the descendants of a single cell deposited on the surface of the medium. If a large number of 24 organisms is seeded onto the medium the colonies will fuse together and produce a confluent sheet of growth. Culture media Artificial media containing the required nutrients used for the cultivation of microorganisms A wide variety of consumable nutrients may be required by bacteria. Some bacteria can grow in simple aqueous solution containing an energy source, such as glucose, and a few inorganic ions. For the routine cultivation of bacteria, a cheap source of all likely nutrients is desirable, and it should also be remembered that even bacteria whose minimum requirements are very simple grow far better on more highly nutritious media. The media usually employed are prepared from protein by acid or enzymic digestion. Typical sources are muscle tissue (meat), casein (milk protein) and blood fibrin. Their digestion provides a supply of the natural amino acids and, because of their origin as living tissue, they will also contain vitamins or growth factors and mineral traces. Solutions of these digests, with the addition of sodium chloride to optimize the tonicity, comprise the common liquid culture media of the bacteriological laboratory. If it is required to study the characteristic colony appearance of cultures, the above media may be solidified by a natural carbohydrate gelling agent, agar, which is derived from seaweed. Agar found in the cell wall of several species of red algae, especially Oriental members of the genus Gelidium. Eg Palmaria polysiphonia It is used as a solidifying agent in the preparation of candies, creams and lotions, and canned fish and meat; as a texturizer or emulsifier in ice cream and frozen desserts; as a clarifying agent in winemaking and; and as a sizing material in fabrics. It is an excellent laboratory medium for growing bacteria, because it is not dissolved by salts or consumed by most microorganisms. Agar is extracted from seaweed by boiling, and is cooled and dried and sold as flakes or cakes. Originally called agar-agar, a Malay word for a local seaweed, it was produced in East Asia but is now made in other Pacific coastal regions such as California and Australia. In addition, a vast array of special culture media have been developed containing chemicals which by either their selective inhibitory properties or characteristic changes act as selective and diagnostic agents to pick out and identify bacterial species from specimens containing a mixture of microorganisms. Anaerobes may be grown by placing cultures in an oxygen free atmosphere, or adding a reducing agent such as cooked meat or sodium thioglycollate to the media. Staining of bacteria 25 When examined in the living condition bacteria are seen to be transparent, colourless and homogeneous or finely granular. In unstained wet preparations the contrast between bacteria and background is poor. They are therefore usually examined in stained preparations. Staining not only increases contrast and therefore facilitates the visualization of bacteria but also, when stains specific for these are employed, permits the recognition of certain structural features. For general purposes the stain most frequently employed is Gram's stain. It was discovered by a Danish Physician, Hans Christian Gram, in 1884. On the basis of their reactions in this stain bacteria may be classified into two large groups: Gram-positive and Gram-negative. This classification is of great importance in relation to bacterial identification. Gram stain procedure begins with primary staining with crystal violet, which stains all bacteria cells blue-purple. Gram iodine is then added as a mordant. A mordant increases the affinity of the primary stain for the bacteria cells. The cells are rinsed with acetone or alcohol to try to wash out the primary stain. A red counter stain (safranin) is then applied, which stains the bacteria that were decolourised in the previous step so that they can be seen easily. References I. Medical Microbiology by Kayser, F.H., Bienz, K.A., Eckert, J., Zinkernagel, R.M. (2005), Thieme Publication. II. Medical Microbiology, A guide to microbial infections: pathogenesis, immunity, laboratory diagnosis and control. Edited by Greenwood, D., Slack, R.C.B., Peuttherer, J.F. (2003), Churchill Livingstone Publication. III. Foundations in Microbiology by Talaro, K. and Talaro, A. (1996). Wm. C. Publishers. 26