Fundamentals of microbiology
Geoffrey W. Hanlon
Chapter contents
Microscopy and staining of bacteria
Growth and reproduction of bacteria
Handling and storage of microorganisms
Isolation of pure bacterial cultures
Classification and identification
Key points
Introduction
Microorganisms are ubiquitous in nature and are vital components in the cycle of life. The majority are free-living organisms growing on dead or decaying matter whose prime function is the turnover of organic materials in the environment. Pharmaceutical microbiology, however, is concerned with the relatively small group of biological agents that cause human disease, spoil prepared medicines or can be used to produce compounds of medical interest.
In order to understand microorganisms more fully, living organisms of similar characteristics have been grouped together into taxonomic units. The most fundamental division is between prokaryotic and eukaryotic cells, which differ in a number of respects (Table 13.1) but particularly in the arrangement of their nuclear material. Eukaryotic cells contain chromosomes, which are separate from the cytoplasm and contained within a limiting nuclear membrane, i.e. they possess a true nucleus. Prokaryotic cells do not possess a true nucleus and their nuclear material is free within the cytoplasm, although it may be aggregated into discrete areas called nuclear bodies. Prokaryotic organisms make up the lower forms of life and include Eubacteria and Archaeobacteria. Eukaryotic cell types embrace all the higher forms of life, of which only the fungi will be dealt with in this chapter.
Table 13.1
Differences between prokaryotic and eukaryotic organisms
| Structure | Prokaryotes | Eukaryotes |
| Cell wall structure | Usually contains peptidoglycan | Peptidoglycan absent |
| Nuclear membrane | Absent | Present. Possess a true nucleus |
| Nucleolus | Absent | Present |
| Number of chromosomes | One | More than one |
| Mitochondria | Absent | Present |
| Mesosomes | Present | Absent |
| Ribosomes | 70S | 80S |
One characteristic shared by all microorganisms is the fact that they are small; however, it is a philosophical argument whether all infectious agents can be regarded as living. Some are little more than simple chemical entities incapable of any free-living existence. Viroids, for example, are small circular, single-stranded RNA molecules not complexed with protein. One particularly well-studied viroid has only 359 nucleotides (one-10th the size of the smallest known virus) and yet causes a disease in potatoes. Prions are small, self-replicating proteins devoid of any nucleic acid. The prion associated with Creutzfeld–Jakob disease in humans, scrapie in sheep and bovine spongiform encephalitis in cattle has only 250 amino acids and is highly resistant to inactivation by normal sterilization procedures.
Viruses are more complex than viroids or prions, possessing both protein and nucleic acid. Despite being among the most dangerous infectious agents known, they are still not regarded as living. Table 13.2 shows the major groups of viruses infecting humans.
Viruses
Viruses are obligate intracellular parasites with no intrinsic metabolic activity, being devoid of ribosomes and energy-producing enzyme systems. They are thus incapable of leading an independent existence and cannot be cultivated on cell-free media, no matter how nutritious. The size of human viruses ranges from the largest poxviruses, measuring about 300 nm, to the picornaviruses, such as the poliovirus which is approximately 20 nm. When one considers that a bacterial coccus measures 1000 nm in diameter, it can be appreciated that only the very largest virus particles may be seen under the light microscope, and electron microscopy is required for visualizing the majority. It will also be apparent that few of these viruses are large enough to be retained on the 200 nm (0.2 µm) membrane filters used to sterilize thermolabile liquids.
Viruses consist of a core of nucleic acid (either DNA as in vaccinia virus or RNA as in poliovirus) surrounded by a protein shell or capsid. Most DNA viruses have linear, double-stranded DNA but in the case of the parvovirus it is single stranded. The majority of RNA-containing viruses contain one molecule of single-stranded RNA, although in reoviruses it is double stranded. The protein capsid comprises 50–90% of the weight of the virus and, as nucleic acid can only synthesize approximately 10% its own weight of protein, the capsid must be made up of a number of identical protein molecules. These individual protein units are called capsomeres and are not in themselves symmetrical but are arranged around the nucleic acid core in characteristic symmetrical patterns. Additionally, many of the larger viruses possess a lipoprotein envelope surrounding the capsid arising from the membranes within the host cell. In many instances the membranes are virus modified to produce projections outwards from the envelope, such as haemagglutinins or neuraminidase. The enveloped viruses are often called ether sensitive, as ether and other organic solvents may dissolve the membrane.
The arrangement of the capsomeres can be of a number of types.
Reproduction of viruses
Because viruses have no intrinsic metabolic capability, they require the functioning of the host cell machinery in order to manufacture and assemble new virus particles. It is this intimate association between the virus and its host that makes the treatment of viral infections so complex. Any chemotherapeutic approach which damages the virus will almost inevitably cause injury to the host cells and hence lead to side-effects. An understanding of the life cycle of the virus is vital in determining suitable target sites for antiviral chemotherapy. The replication of viruses within host cells can be broken down into a number of stages.
Adsorption to host cell
The first step in the infection process involves virus adsorption on to the host cell. This usually occurs via an interaction between protein or glycoprotein moieties on the virus surface with specific receptors on the host cell outer membrane. Different cells possess receptors for different viruses. For example, the human immunodeficiency virus (HIV) possesses two proteins involved in adsorption to T lymphocytes and these are known as gp41 and gp120. There are receptors on the lymphocyte surface to which HIV will bind. The main receptor is CD4 to which the gp120 protein attaches. Other receptors are CXCR4 or CCR5 to which the gp41 protein binds. Both attachments are necessary for infection and lead to conformational changes in the HIV envelope proteins, resulting in membrane fusion.
Penetration
Enveloped viruses fuse the viral membrane with the host cell membrane and release the nucleocapsid directly into the cytoplasm. Naked virions generally penetrate the cell by phagocytosis. Bacteriophages are viruses which specifically attack bacteria and these inject their DNA into the host cell while the rest of the virus remains on the outside.
Uncoating
In this stage the capsid is removed as a result of attack by cellular proteases and this releases the nucleic acid into the cytoplasm. These first three stages are similar for both DNA and RNA viruses.
Nucleic acid and protein synthesis
The detailed mechanisms by which DNA- and RNA-containing viruses replicate inside the cell are outside the scope of this chapter and the reader is referred to the bibliography for further information. After nucleic acid replication, early viral proteins are produced, the function of which is to switch off host cell metabolic activity and direct the activities of the cell towards the synthesis of proteins necessary for the assembly of new virus particles.
Assembly of new virions
Again, there are differences in the detail of how the viruses are assembled within the host cell, but construction of new virions occurs at this stage and up to 100 new virus particles may be produced per cell.
Release of virus progeny
The newly formed virus particles may be liberated from the cell as a burst, in which case the host cell ruptures and dies. Infection with influenza virus results in a lytic response. Alternatively, the virions may be released gradually from the cell by budding of the host cell plasma membrane. These are often called ‘persistent’ infection; an example being hepatitis B.
Latent infections
In some instances a virus may enter a cell but not go through the replicative cycle outlined above and the host cell may be unharmed. The genome of the virus is conserved and may become integrated into the host cell genome where it may be replicated along with the host DNA during cell division. At some later stage the latent virus may become reactivated and progress through a lytic phase, causing cell damage/death and the release of new virions. Examples of this type of infection are those which occur with the herpes simplex viruses associated with cold sores, genital herpes and also chickenpox where the dormant virus may reactivate to give shingles later in life.
Oncogenic viruses
Oncogenic viruses have the capacity to transform the host cell into a cancer cell. In some cases this may lead to relatively harmless, benign growths, such as warts caused by papovavirus, but in other cases more severe, malignant tumours may arise. Cellular transformation may result from viral activation or mutation of normal host genes, called protooncogenes, or the insertion of viral oncogenes.
Bacteriophages
Bacteriophages (phages) are viruses that attack bacteria but not animal cells. It is generally accepted that the interaction between phage and bacterium is highly specific, and there is probably at least one phage for each species of bacterium. In many cases the infection of a bacterial cell by a phage results in lysis of the bacterium; such phages are termed virulent. Some phages, however, can infect a bacterium without causing lysis. In this case the phage DNA becomes incorporated within the bacterial genome. The phage DNA can then be replicated along with the bacterial cell DNA; this is then termed a prophage. Bacterial cells carrying a prophage are called lysogenic and phages capable of inducing lysogeny are called temperate. Occasionally some of the prophage genes may be expressed and this will confer on the bacterial cell the ability to produce new proteins. The ability to produce additional proteins as a result of prophage DNA is termed lysogenic conversion.
The discovery of bacteriophages in the early 20th century is attributed to two workers, Frederick Twort and Felix d’Herelle. In 1896 Ernest Hankin had made an observation that the waters of the Ganges River possessed antibacterial properties which may have led to a reduction in cases of dysentery and cholera in the areas surrounding the river. Twort and d’Herelle independently came to the conclusion that this effect must be due to a virus. Twort did not carry on with his research but d’Herelle quickly established the potential of bacteriophages in antibacterial therapy 10 years before the advent of antibiotics. It was the discovery of penicillin by Alexander Fleming in 1928 that led to the demise of bacteriophage therapy but interest is now increasing again due to the emergence of antibiotic-resistant strains of bacteria.
Archaeobacteria
Archaeobacteria are a fascinating group of prokaryotic microorganisms that are frequently found living in hostile environments. They differ in a number of respects from Eubacteria, particularly in the composition of their cell walls. They comprise methane producers, sulfate reducers, halophiles and extreme thermophiles. However, they are of little significance from a pharmaceutical or clinical standpoint and so will not be considered further.
Eubacteria
Eubacteria constitute the major group of prokaryotic cells that have pharmaceutical and clinical significance. They include a diverse range of microorganisms, from the primitive parasitic rickettsias that share some of the characteristics of viruses, through the more typical free-living bacteria to the branching, filamentous actinomycetes, which at first sight resemble fungi rather than bacteria.
Atypical bacteria
Rickettsiae
The family Rickettsiaceae includes three clinically important genera, Rickettsia, Coxiella and Bartonella. Although these are prokaryotic cells, they differ from most other bacteria both in their structure and in the fact that the majority of species lead an obligate intracellular existence. This means that, with a few exceptions, they cannot be grown on cell-free media, although unlike many viruses they do possess some independent enzymes. They have a pleomorphic appearance, ranging from coccoid through to rod-shaped cells; multiplication is by binary fission. Their cell wall composition bears similarities to that of Gram-negative bacteria (see later in this chapter) and in general they stain this way. The genus Rickettsia has a number of species that give rise to human diseases, in particular epidemic typhus (R. prowazekii), murine typhus (R. typhi) and spotted fevers (various species). These are characterized by transmission via insect vectors, particularly mites, ticks, fleas and lice.
The mode of transmission by these vectors varies depending upon the insect concerned. In the case of lice and fleas, the microorganisms multiply within the insect and get into the faeces. These insects then colonize humans and transmit the microorganism when the faeces or the insect itself is crushed onto the skin. No bite is necessary and the faeces may also be inhaled. Mites and ticks pick up the microorganism when they take a blood meal from an infected animal. They then pass on the infection to humans when they accidentally bite us.
Coxiella burnetii is the only species in the genus Coxiella and this gives rise to a disease called Q fever. Although the source of the disease is infected animals, usually no insect vector is involved and the most common route of transmission is by inhalation of infected dust. Bartonella quintana is the causative agent of trench fever which, as the name suggests, occurs typically under conditions of war and deprivation. Each of the infections described here can be treated with doxycycline, although the duration of therapy may vary depending upon the nature of the disease and its severity.
Chlamydiae
These are obligate intracellular parasitic bacteria that possess some independent enzymes but lack the ability to generate ATP. Two cellular forms are identified: a small (0.3 µm) highly infectious elementary body which, after infection, enlarges to give rise to the replicative form called the initial or reticulate body (0.8–1.2 µm). These divide by binary fission within membrane-bound vesicles in the cytoplasm of infected cells. Insect vectors are not required for the transmission of infection. Chlamydiae lack peptidoglycan in their cell walls and have weak Gram-negative characteristics.
Chlamydia trachomatis is the most important member of the group, being responsible for the disease trachoma, characterized by inflammation of the eyelids, which can lead to scarring of the cornea. This is the most common cause of infectious blindness worldwide. It is estimated that 400 million people are infected, with at least 6 million totally blind. The same species is also recognized as one of the major causes of sexually transmitted disease. C. psittaci and C. pneumoniae are responsible for respiratory tract infections. Chlamydial infections are responsive to treatment by tetracyclines, either topical or systemic as appropriate.
Mycoplasmas
The mycoplasmas are a group of very small (0.3–0.8 µm) prokaryotic microorganisms that are capable of growing on cell-free media but which lack cell walls. The cells are surrounded by a double-layered plasma membrane that contains substantial amounts of phospholipids and sterols. This structure has no rigidity owing to the absence of peptidoglycan, and so the cells are susceptible to osmotic lysis. The lack of peptidoglycan is also the reason for these bacteria being resistant to the effects of cell wall-acting antibiotics such as the penicillins, and also the enzyme lysozyme. Members of this group are pleomorphic, varying in shape from coccoid to filamentous. Most are facultative anaerobes capable of growth at 35 °C, and on solid media produce colonies with a characteristic ‘fried egg’ appearance. They contain a number of genera, of which the most important from a clinical point of view are Mycoplasma and Ureaplasma. M. pneumoniae is a major cause of respiratory tract infections in children and young adults, whereas U. urealyticum has been implicated in non-specific genital infections. Despite being resistant to the β-lactam antibiotics, these infections can be effectively treated using either tetracyclines or erythromycin.
Actinomycetes
Many of the macroscopic features of the actinomycetes are those that are more commonly found among the filamentous fungi but they are indeed prokaryotic cells. They are a diverse group of Gram-positive bacteria morphologically distinguishable from other bacteria because they have a tendency to produce branching filaments and reproductive spores. Nocardia contain a number of species that have been shown to be pathogenic to humans, but they occur principally in tropical climates. Reproduction in this genus is by fragmentation of the hyphal strands into individual cells, each of which can form a new mycelium. The genus Streptomyces contains no human pathogens but most species are saprophytic bacteria found in the soil. They are aerobic microorganisms producing a non-fragmenting, branching mycelium that may bear spores. The reason for their pharmaceutical importance is their ability to produce a wide range of therapeutically useful antibiotics, including streptomycin, chloramphenicol, oxytetracycline, erythromycin and neomycin.
Typical bacteria
Shape, size and aggregation
Bacteria occur in a variety of shapes and sizes, determined not only by the nature of the organisms themselves but also by the way in which they are grown (Fig. 13.1). In general, bacterial dimensions lie in the range 0.75–5 µm. The most common shapes are the sphere (coccus) and the rod (bacillus).
Some bacteria grow in the form of rods with a distinct curvature, e.g. vibrios are rod-shaped cells with a single curve resembling a comma, whereas a spirillum possesses a partial rigid spiral; spirochaetes are longer and thinner, exhibit a number of turns and are also more flexible. Rod-shaped cells occasionally grow in the form of chains but this is dependent upon growth conditions rather than being a characteristic of the species.
Cocci, however, show considerable variation in aggregation, which is characteristic of the species. The plane of cell division and the strength of adhesion of the cells determine the extent to which they aggregate after division. Cocci growing in pairs are called diplococci, those in four are tetrads and groups of eight are sarcina. If a chain of cells is produced resembling a string of beads this is termed a streptococcus, whereas an irregular cluster similar in appearance to a bunch of grapes is called a staphylococcus. In many cases this is sufficiently characteristic to give rise to the name of the bacterial genus, e.g. Staphylococcus aureus, Streptococcus pneumoniae.
Anatomy
Figure 13.2 is a diagrammatic representation of a typical bacterial cell. The various components are described below.
Capsule.
Many bacteria produce extracellular polysaccharides, which may take the form of either a discrete capsule firmly adhered to the cell or a more diffuse layer of slime. Not all bacteria produce a capsule and even those that can will only do so under certain circumstances. Many encapsulated pathogens, when first isolated, give rise to colonies on agar which are smooth (S) but subculturing leads to the formation of rough colonies (R). This S to R transition is due to loss in capsule production. Reinoculation of the R cells into an animal results in the resumption of capsule formation, indicating that the capacity has not been lost irrevocably.
The function of the capsule is generally regarded as protective, as encapsulated cells are more resistant to disinfectants, desiccation and phagocytic attack. In some organisms, however, it serves as an adhesive mechanism; for example, Streptococcus mutans is an inhabitant of the mouth that metabolizes sucrose to produce a polysaccharide capsule enabling the cell to adhere firmly to the teeth. This is the initial step in the formation of dental plaque, which is a complex array of microorganisms and organic matrix that adheres to the teeth and ultimately leads to decay. The substitution of sucrose by glucose prevents capsule formation and hence eliminates plaque.
A similar picture emerges with Staph. epidermidis. This bacterium forms part of the normal microflora of the skin and was originally thought of as non-pathogenic. With the increased usage of indwelling medical devices coagulase-negative staphylococci, in particular Staph. epidermidis, have emerged as the major cause of device-related infections. The normal microbial flora has developed the ability to produce extracellular polysaccharide, which enables the cells to form resistant biofilms attached to the devices. These biofilms are very difficult to eradicate and have profound resistance to antibiotics and disinfectants. It is now apparent that the dominant mode of growth for aquatic bacteria is not planktonic (free swimming) but sessile, i.e. attached to surfaces and covered with protective extracellular polysaccharide or glycocalyx.
Cell wall.
Bacteria can be divided into two broad groups by the use of the Gram-staining procedure (see later in this chapter for details), which reflects differences in cell wall structure. The classification is based upon the ability of the cells to retain the dye methyl violet after washing with a decolourizing agent such as absolute alcohol. Gram-positive cells retain the stain whereas Gram-negative cells do not. As a very rough guide, the majority of small rod-shaped cells are Gram negative. Most large rods, such as the Bacillaceae, lactobacilli and actinomycetes, are Gram positive. Similarly, most cocci are Gram positive, although there are notable exceptions, such as the Neisseriaceae.
Bacteria are unique in that they possess peptidoglycan in their cell walls. This is a complex molecule with repeating units of N-acetylmuramic acid and N-acetylglucosamine (Fig. 13.3). This extremely long molecule is wound around the cell and crosslinked by polypeptide bridges to form a structure of great rigidity. The degree and nature of crosslinking vary between bacterial species. Crosslinking imparts to the cell its characteristic shape and has principally a protective function. Peptidoglycan (also called murein or mucopeptide) is the site of action of a number of antibiotics, such as penicillin, bacitracin, vancomycin and cycloserine. The enzyme lysozyme is also capable of hydrolysing the β–1–4 linkages between N-acetylmuramic acid and N-acelylglucosamine.
Figure 13.4 shows simplified diagrams of a Gram-positive and a Gram-negative cell wall. The Gram-positive cell wall is much simpler in layout, containing peptidoglycan interspersed with teichoic acid polymers. These latter are highly antigenic but do not provide structural support. Functions attributed to teichoic acids include the regulation of enzyme activity in cell wall synthesis, sequestration of essential cations, cellular adhesion and mediation of the inflammatory response in disease. In general, proteins are not found in Gram-positive cell walls. Gram-negative cell walls are more complex, comprising a much thinner layer of peptidoglycan surrounded by an outer bilayered membrane. This outer membrane acts as a diffusional barrier and is the main reason why many Gram-negative cells are much less susceptible to antimicrobial agents than are Gram-positive cells.
The lipopolysaccharide component of the outer membrane can be shed from the wall upon cell death. It is a highly heat-resistant molecule known as endotoxin, which has a number of toxic effects on the human body, including fever, shock and even death. For this reason it is important that solutions for injection or infusion are not just sterile but are also free from endotoxins.
Cytoplasmic membrane.
The cytoplasmic membranes of most bacteria are very similar and are composed of protein, lipids, phospholipids and a small amount of carbohydrate. The components are arranged into a bilayer structure with a hydrophobic interior and a hydrophilic exterior. The cytoplasmic membrane has a variety of functions.
• It serves as an osmotic barrier.
• It is selectively permeable and is the site of carrier-mediated transport.
• It is the site of ATP generation and cytochrome activity.
• It is the site of cell wall synthesis.
The cytoplasmic membrane has very little tensile strength and the internal hydrostatic pressure of up to 20 bar forces it firmly against the inside of the cell wall. Treatment of bacterial cells with lysozyme may remove the cell wall and, as long as the conditions are isotonic, the resulting cell will survive. These cells are called protoplasts and, as the cytoplasmic membrane is now the limiting structure, the cell assumes a spherical shape. Protoplasts of Gram-negative bacteria are difficult to obtain because the layer of lipopolysaccharide protects the peptidoglycan from attack. In these cases mixtures of EDTA and lysozyme are used and the resulting cells, which still retain fragments of cell envelope, are termed spheroplasts.
Nuclear material.
The genetic information necessary for the functioning of the cell is contained within a single circular molecule of double-stranded DNA. When unfolded, this would be about 1000 times as long as the cell itself and so exists within the cytoplasm in a considerably compacted state. It is condensed into discrete areas called chromatin bodies that are not surrounded by a nuclear membrane. Rapidly dividing cells may contain more than one area of nuclear material but these are copies of the same chromosome, not different chromosomes, and arise because DNA replication proceeds ahead of cell division.
In addition to the main chromosome, cells may contain extra pieces of circular double-stranded DNA which are called plasmids. These can encode a variety of products which are not necessary for the normal functioning of the cell but confer some sort of selective advantage. For example, the plasmids may contain genes conferring antibiotic resistance or the ability to synthesize toxins or virulence factors. Plasmids replicate autonomously (i.e. independent of the main chromosome) and in some cases are able to be transferred from one cell to another (maybe of a different species).
Mesosomes.
These are irregular invaginations of the cytoplasmic membrane which are quite prominent in Gram-positive bacteria but less so in Gram-negative bacteria. It has been proposed that they have a variety of functions, including cross-wall synthesis during cell division and furnishing an attachment site for nuclear material, facilitating the separation of segregating chromosomes during cell division. They have also been implicated in enzyme secretions and may act as a site for cell respiration. However, it has also been suggested that they are simply artefacts which arise as a result of preparation for electron microscopy.
Ribosomes.
The cytoplasm of bacteria is densely populated with ribosomes, which are complexes of RNA and protein in discrete particles 20 nm in diameter. They are the sites of protein synthesis within the cell and the numbers present reflect the degree of metabolic activity of the cell. They are frequently found organized in clusters called polyribosomes or polysomes. Prokaryotic ribosomes have a sedimentation coefficient of 70S, compared to 80S ribosomes of eukaryotic cells. This distinction aids the selective toxicity of a number of antibiotics. The 70S ribosome is made up of RNA and protein and can dissociate into one 30S and one 50S subunit.
Inclusion granules.
Certain bacteria tend to accumulate reserves of materials after active growth has ceased, and these become incorporated within the cytoplasm in the form of granules. The most common are glycogen granules, volutin granules (containing polymetaphosphate) and lipid granules (containing poly β-hydroxybutyric acid). Other granules, such as sulphur and iron, may also be found in the more primitive bacteria.
