MIC 210

INTRODUCTION

Microbiology is the study of living organisms of microscopic size, which include bacteria, fungi, algae, protozoa, and the infectious agents at the borderline of life that are called viruses. It is concerned with their form, structure, reproduction, their relationship to each other and to other living organisms, their effects on human beings and on other animals and plants, their abilities to make physical and chemical changes in our environment, and their reactions to physical and chemical agents. These organisms are commonly called “microbes” or more exactly, as microorganisms. If an object has a diameter of less than 0.1 mm, the human eye cannot perceive it at all, and very little detail can be perceived in an object with a diameter of 1 mm. Generally speaking, therefore, organisms with a diameter of 1 mm or less are microorganisms and fall into the broad domain of microbiology.

SCOPE OF MICROBIOLOGY

Microbiology as a science deals with the study of microorganisms such as bacteria, protozoans, fungi, algae and other minute forms of life, and the benefits of such organisms. It extends to the study of the role of these organisms in Medicine (medical microbiology), Industry (industrial microbiology). Mycology (medical mycology), Agriculture (agricultural microbiology), Soil (soil microbiology), etc.

HISTORICAL DEVELOPMENT OF MICROBIOLOGY

The science of microbiology, like other sciences, had its origins in curiosity. However, the discovery of microorganisms had to await the invention of the microscope. The first microscope was discovered by a Dutch spectacle maker, Zaccharias Janssen (1590) and forms the basis of the modern compound microscope that is used by every microbiologist today. Later, Galileo (1610), invented an improved microscope, though with limited resolving power compared to later ones.

Robert Hook (1665) made and used a compound microscope and described the fascinating explorations of the newly discovered universe of microorganisms and published a book, Micrographia, containing descriptions and illustrations based upon his microscopic examination of higher organisms and filamentous fungi including molds and rusts.

The Dutch investigator, Antony van Leeuwenhoek (1632 – 1723) invented and made magnificent observations on the microscopic structure of seeds and embryos and on small invertebrate animals. His greatest contribution rests on his discovery of the microbial world: the world of “animalcules” or little animals as he and his contemporaries called them. A new dimension was thus added to biology. Leeuwenhoek described the diversity and abundance of different kinds of unicellular microorganisms as we know them today – protozoa, algae, yeasts, and bacteria.

For over 150 years after the discoveries of Janssen, Galileo, Hook, and Leeuwenhoek, knowledge of microscopy and microorganisms developed but slowly. There were relatively few microbiologists, and great technical problems in the science of optics and instrumental mechanics had to be solved.

Spontaneous generation controversy

After Leeuwenhoek had revealed the vast numbers of microorganisms present in nature, scientists began the debate of their origin. Initially there were two schools of thought:

1.      that microorganisms arose spontaneously from decomposing organic materials – a belief that became known as the doctrine of spontaneous generation or abiogenesis.

2.      that each organism was the progeny of an identical, preexisting organism.

Needham (1748) experimentally proved that bacteria arose spontaneously where no living organisms existed before. He placed boiled mutton broth in flasks; tightly stoppered them with corks, and at intervals examined them microscopically; bacteria appeared in large numbers. He reasoned that since boiling destroyed all living cells, therefore those that appeared later must have arisen spontaneously. Needham postulated the existence of a vegetative force that was necessary to confer life upon the nonliving ingredients of the liquid broth. It was also believed that maggots in decaying meat were derived spontaneously from transformations of the putrid meat itself. The doctrine of spontaneous generation was accepted without question until the renaissance.

In 1665, an Italian physician, Franscesco Redi questioned this hypothesis. He placed meat and fish in jars covered with very fine gauze and saw flies approach the jars and crawl on the gauze. He saw the eggs of the flies caught on the gauze and observed that the meat then putrefied without maggot formation. Maggots developed only when the flies’ eggs were deposited on the meat. Obviously the meat itself did not turn into maggots. Thus, he showed that the maggots that develop in putrefying meat are the larval stages of flies and will never appear if the meat is protected by placing it in a vessel closed with fine gauze so that flies are unable to deposit their eggs on it. Consequently, Redi absolutely weakened the doctrine that maggots develop spontaneously from meat (the doctrine of abiogenesis). However, for technical reasons, it was far more difficult to show that microorganisms are not generated spontaneously, and as time went on the proponents of the doctrine of spontaneous generation came to centre their claims more and more on the mysterious appearance of microorganisms in organic infusions. The opponents of this theory were therefore in a difficult position of having to prove otherwise.

Lazzaro Spallanzani (1765), an Italian naturalist, repeated the experiments of Needham, but with modifications. Instead of closing the flasks with corks, Spallanzani resorted to hermetic sealing (in aflame), before heating the contents. He found that if he boiled the broth long enough, no bacteria ever appeared. The broth remained clear   when observed with unaided eye and portions examined microscopically showed no sign of life. He therefore reasoned that Needham might not have boiled his broth long enough. Moreover, closing the flasks with corks was not enough mechanical plug to completely exclude air. He concluded that to render infusion permanently barren, it must be sealed hermetically and properly boiled. Animalcules could never appear unless new air somehow entered the flask and came in contact with the infusion.  

In 1775 Lavoisier discovered oxygen and the relation between air and life. This renewed controversy about abiogenesis, the objection to Spallanzani’s results now being that the exclusion of air (oxygen) from the flasks prevented the development of life. The proponents of spontaneous generation further maintained that such drastic treatment (heating) destroyed the life-supporting property of air so that even though spontaneous generation might occur, the organisms generated would be unable to multiply.

This argument was countered by Schroeder and von Dusch (1854) when they introduced the use of cotton into microbiologic practice. They drew air into flasks of broth after passing it through cotton filters that had previously been baked in an oven. The fact that the broth remained clear, while that in flasks that were not protected by cotton promptly became cloudy with a heavy bacterial population, clearly demonstrated that the source of microbial life was the outside air. Schroeder and von Dosch further showed that flasks were protected by a ward or plug of cotton wool in the mouth of the flask. If the flask was then boiled sufficiently, its contents remained sterile indefinitely. This is the origin of the familiar use of cotton plug for bacteriological culture tubes and flasks in the laboratory.

In spite of these demonstrations, long and bitter controversies still raged. Moreover, Schroeder and von Dusch were still not convinced by their own experiments and admitted the possibility that abiogenesis might occur under natural conditions.

By 1860, some scientists were of the hypothesis that microorganisms are agents that bring about chemical changes. A French scientist, Louis Pasteur (1822 – 1895), was a great proponent of this theory. However, he knew quiet well the acceptance of this concept was conditional on the demonstration that spontaneous generation does not occur. Stung by the continued claims of adherents to the doctrine of spontaneous generation, Pasteur finally turned his attention to this problem.

Pasteur first demonstrated that air does not contain microscopically observable “organized bodies”. He aspirated large quantities of air through a tube that contained a plug of guncotton as a filter. The guncotton was then removed and dissolved in a mixture of alcohol and ether, and the sediment was examined microscopically. The filtrate contained in addition to inorganic matter, considerable numbers of small round or oval bodies, indistinguishable from microorganisms. Pasteur next confirmed the fat that heated air can be supplied to a boiled infusion without giving rise to microbial development. Having established this point, he went on to show that in a closed system the addition of a piece of germ-laden guncotton to a sterile infusion invariably provoked microbial growth. These experiments showed Pasteur how germs can enter infusions and led him o demonstrate that infusions will remain sterile indefinitely in open flasks, provided that the neck of the flask is drawn out and bent down in such away that the germs from the air cannot ascend it (swan-necked flask). If the neck of such a flask was broken off, the infusion rapidly became populated with microbes. The same happened if the sterile liquid in the flask was poured into the exposed portion of the bent neck and then poured back. Pasteur rounded his study by demonstrating that the microorganisms are evenly distributed through the atmosphere.

Despite all these assertions, the proponents of spontaneous generation maintained a stubborn rear-guard action for some years.

The final blow to the spontaneous generation doctrine was dealt by an English physicist, John Tyndall (1877), who was an ardent partisan of Pasteur. In a series of experiments with infusions prepared from meat and fresh vegetables Tyndall obtained satisfactory sterilization by placing tubes of these infusions for 5 minutes in a bath of boiling brine. However, similar treatments of infusions prepared from dried hay proved completely inadequate. Worse still, his attempts to repeat the same experiments with other types of infusions failed to sterilize them with boiling brine, even after exposure for long periods of up to an hour. Tyndall finally realized what was happening. Dried hay contained highly resistant bacterial structures, later called spores. Prolonged boiling was necessary or a process of intermittent sterilization became known as tyndallization. Tynallization as developed by Tyndall is thus a method of sterilization by discontinuous heating, which could be used to kill all bacteria in infusions. Since growing bacteria are easily killed by brief boiling, all that is necessary is to allow the infusion to stand for a certain period before applying heat to permit germination of the spores with a consequent loss of their heat resistance. A brief period of boiling can then be used, and repeated, if need be, several times at intervals to trap any spores late in germination. Tyndall established that discontinuous boiling for 1 minute of five successive occasions would make infusion sterile, as opposed to a single continuous boiling for 1 hour.      

The works of Pasteur and Tyndall thus “disproved” the possibility of spontaneous generation, and their experimental findings have been used against the theory of spontaneous generation.

Germ theory of Fermentation

During the long controversy over spontaneous generation, a correlation between the growth of microorganisms in organic infusions and the onset of chemical changes in the infusion itself was frequently observed. These chemical changes were designated as “fermentation” and “putrefaction”, a process of decomposition that results in the formation of ill-smelling products, occurs characteristically and is a consequence of the breakdown of proteins, the principal organic constituents in such natural materials. Fermentation, a process that results in the formation of alcohols or organic acids, occurs characteristically in plant materials as a consequence of the breakdown of carbohydrates, the predominant organic compounds in plant tissues. 

In 1837, Cagniard de Latour, Schwann and Kutzing independently proposed that yeasts actively transform sugar into alcohol and carbon dioxide during fermentation. They postulated that in other fermentations, various specific microorganisms formed characteristic end products during their growth. This theory was bitterly attacked by chemists, who held the view that fermentation and putrefaction are purely chemical processes. However, Pasteur, a chemist by training, eventually convinced the scientific world that all fermentative processes are the results of microbial activity.

The germ theory of fermentation, stating that microorganisms bring about specific changes in their substrates, laid the foundation for important industrial development.

Germ theory of Disease

From the earliest times, disease was regarded as mysterious or even supernatural phenomenon. Ancient Greek and Roman physicians suspected that invisible, minute particulate agents caused certain diseases and that they could be transmitted in some way or other from one individual to the next.

The fact that certain bacteria produce disease was first clearly demonstrated by Robert Koch in 1876. He showed that spore-forming organism, Bacillus anthracis, was the cause of anthrax, which was then epidemic in sheep, cattle, and other domestic animals, and also occurred in man. To prove his assertion, Koch followed four experimental steps, which have since been known as Koch’s postulates:

1.      Find the suspected microorganism in every case of the disease.

2.      The microorganism must be isolated from the diseased host and grown in pure culture.

3.      The specific disease must be reproduced when a pure culture of the microorganism is inoculated into a healthy susceptible host.

4.      The microorganism must be recoverable once again from the experimentally infected host.

If carefully followed, these simple rules provide a logical basis for concluding that an organism is responsible for a given disease (or any other characteristic change, for that matter). There are conditions under which all the four rules cannot be observed. For example, apparently healthy individuals may be “carriers” of pathogenic microorganisms. Moreover, some infectious agents, such as certain viruses, have in the past been extremely difficult to isolate and cultivate outside the natural host. In these cases indirect evidence is sometimes necessary to establish the cause of an infectious process. Koch’s work proved unequivocally the biological specificity of disease agents.

DISTRIBUTION OF MICROORGANISMS

Microbes occur nearly everywhere in nature. They are carried by air currents from the earth’s surface to the upper atmosphere. They are found in oceans to many miles away on mountain tops. Fertile soils teem with microbes.

Microbes are carried by streams and rivers into lakes and other water bodies. They are spread by human wastes.

CLASSIFICATION OF MICROORGANISMS

·         Taxonomy is the science of classification of living forms. Organisms are arranged into taxonomic categories or taxa to facilitate research, scholarship and communication.

·         Systematics, or phylogeny, is the study of the evolutionary history of a group of organisms. The hierarchy of a taxa reveals evolutionary or phylogenetic relationships.

·         From the time of Aristotle, livings were categorized in two ways;

-          Plants or

-          Animals.

However, as biological sciences developed, biologists began looking for a natural classification system (one that grouped organisms based on ancestral relationships).

·         In 1857, Carl von Nageli, placed bacteria and Fungi in the Plant Kingdom.

·         In 1886, Ernst Haeckel proposed the Kingdom Protista to include bacteria, Protozoa, algae and Fungi. Fungi were later placed in their own Kingdom in 1959.

·         With the advent of electron microscopy, the physical differences between cells became apparent. The term prokaryotae was introduced in 1937 by Edward Chalton to distinguish cells having no nucleus from the nucleated cells of plants and animals.

·         In 1968, Robert Murray, proposed the Kingdom Prokaryotae.

·         In 1969, Whittaker founded the five Kingdom system in which prokaryotes were placed in the Kingdom Prokaryotae or Monera; and eukaryotes comprised the other four kingdoms: 

1.      Fungi

2.      Plantae

3.      Protista

4.      Animalia

·         The Kingdom Prokaryotae was based on microscopic observations. New techniques in molecular biology revealed that there are actually two types of prokaryotic cells and one type of eukaryotic cell. These cells differ in rRNA nucleotide sequences. Thus, the cell types are- the eukaryotes and two different types of prokaryotes (the bacteria and the archaea).

·         In 1978, Carl Woese proposed elevating the three cell types above kingdom, called domain. The archaea and bacteria are placed into their own separate separate domains. The animals, plants, fungi and protests are Kingdoms in the Domain Eukarya.

·         Thus, there are three domains;

1.      Domain Bacteria (contain peptidoglycan) – this includes all the        pathogenic prokaryotes as well as many of the non-pathogenic prokaryotes found in soil and water

2.      Domain Archaea - includes prokaryotes that do not have peptidoglycan in their cell walls. They often live in extreme environments, and carry out usual metabolic processes. Archaea include three major groups;

                                                             i.      The Methanogens, strict anaerobes that produce methane (CH₄) from carbon dioxide and hydrogen

                                                           ii.      Extreme halophiles, which require high concentrations of survival

                                                        iii.      Hyperthermophiles, which normally grow in hot, acidic environments    

2.      Domain Eukarya – which include animals, plants, fungi and protists.

3.       

MICROBIAL CELL TYPES

Prokaryotes and eukaryotes are chemically similar, since they both contain nucleic acids, proteins, lipids, and carbohydrates. They use the same kinds of chemical reactions to metabolize food, build proteins, and store energy. They differ in the structure of their cell walls and membranes, and the presence or absence of organelles (specialized cellular structures with specific functions) that tell them apart.

The major distinguishing characters of prokaryotes (Gk. for prenucleus) are:

1.      their genetic material (DNA) is not enclosed with a membrane

2.      they lack other membrane-bounded organelles

3.      their DNA is not associated with histone proteins (special chromosomal proteins found in eukaryotes).

4.      their cell walls almost always contain the complex polysaccharide peptidoglycan

5.      they usually divide by binary fission. A process in which the DNA is copied and the cell splits into two cells. Binary fission involves fewer structures and processes than eukaryotic cell division. e.g. Bacteria.

Eukaryotes (Gk. for true nucleus) have nuclear membrane-bound linear structures of DNA called chromosomes which is separated from the cytoplasm by a nuclear membrane.

The DNA of eukaryotic chromosomes is consistently associated with chromosomal proteins called histones and with non-histones.

Eukaryotes also have a mitotic apparatus (various cellular structures that participate in a type of nuclear division called mitosis).

Eukaryotes have a number of organelles including mitochondria, golgi bodies, membrane-bound vacuoles, etc. Examples include Protozoa, Fungi, and Algae.

Figure 2.0 Internal structures of microbial cells. (a) Diagram of prokaryotic cell. (b). Diagram of eukaryotic cell. After: Madigan and Matinkho, 2006.

VIRUSES

Viruses are acellular (not cellular) organisms. They are structurally very simple; a virus particle contains a core made of only one type of nucleic acid, either DNA, or RNA. This core is surrounded by a protein coat. Sometimes the coat is encased by an additional layer, a lipid membrane called an envelope. Viruses can reproduce only using the cellular machinery of other organisms.

MICROSCOPY

A microscope is a tool or machine with the ability to increase the visual size of an object so that it is easier to see. A microscope must perform two functions:

·         Magnify (enlarge) the specimen to a size that can be seen by the human eye. Magnification is obtained by multiplying the objective lenses, i.e. 10X (low power), 40X (high power), and 100X (oil immersion). Most oculars magnify specimens by a factor of 10. Multiplying the magnification of a specific objective lens with that of the ocular; gives the total magnification. Most laboratory microscopes are equipped with three objectives, each capable of a different degree of magnification. These are referred to as the;

             a. Low power   - X10 X10  = X100

             b. High –dry      -X40 X10  = X400

             c. Oil immersion    X100 X10  = X1000.

·         Resolution (also called resolving power) – Is the smallest distance between two objects at which they may be seen as separate objects. It is also defined as the ability to distinguish two adjacent points as distinct and separate. 

Types of Microscopes

Microscopes are of two types;  (a). Light (or optical) and (b). Electron, depending upon the principle on which magnification is based. Light microscopy in which magnification is obtained by a system of optical lenses using light waves include;

i.                   Bright-field

ii.                 Dark-field

iii.              Fluorescence

iv.               Phase-contrast

Figure 3.0 The compound light microscope. Source: Tortora et al., 1993.

The electron microscope uses a beam of electrons in place of light waves to produce the image. Specimens can be examined by either;

                                         (v). Transmission, or

                                         (vi). Scanning electron microscopy

1.      Bright-field Microscopy – in this case the microscopic field (the area observed) is brightly lighted and the specimens appear dark because they absorb some of the light. Ordinarily, microorganisms do not absorb much light, but staining them with a dye greatly increases their light-absorbing ability, resulting in greater contrast and colour differentiation.

2.      Dark-field Microscopy – this produces a dark background against which objects are brilliantly illuminated. This is accomplished by equipping the light microscope with a special kind of condenser that transmits a hollow cone of light from the source of illumination. Most of the light directed through the condenser does not enter the objective; the field is essentially dark. However, some of the light rays will be scattered (diffracted) if the transparent medium contains objects such as microbial cells. This diffracted light will enter the objective and reach the eye; thus the object or microbial cell will appear bright in an otherwise dark microscopic field.

3.      Fluorescence Microscopy – Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wavelength and lesser energy content. Such substances are called fluorescent and the phenomenon is termed fluorescence. Application of this phenomenon is the basis of fluorescence microscopy. In practice, microbes are stained with a fluorescent dye and then illuminated with blue light; the blue light is absorbed and green light emitted by the dye.

4.      Phase-contrast Microscopy – this uses a conventional light microscope fitted with a phase-contrast objective and a phase contrast condenser. This special optical system makes it possible to distinguish unstained structures within a cell which differ only slightly in their refractive indices or thickness. In principle, this technique is based on the fact that light passing through one material and into another material of a slightly different refractive index and/or thickness will undergo a change in phase. These differences in phase, or wave-front irregularities, are translated into variations in brightness of the structures and hence are detectable by the eye.

5.      Electron Microscopy – the electron microscope, as the name suggests, uses a beam of electrons in place of light waves to produce the image. They are of two types;

                  (i). Transmission electron microscopy – this uses a beam of electrons

                        instead of light (because of shorter wavelength of electrons, structures

                        smaller than 0.2 µm can be resolved).

                   (ii). Scanning electron Microscopy – this also uses a beam of electrons

                         instead of light. It is used to examine surface features of cells and

                         viruses

                         (usually mg 1000X – 10, 000X). The specimen is subjected to a

                          narrow

                         electron beam which rapidly moves over (scans) the surface of the

                         specimen. 

PREPARATION OF SPECIMENS FOR LIGHT MICROSCOPY

Two general techniques are used to prepare specimens for light microscope examination;

a.      to suspend organisms in a liquid (the wet-mount or the hanging drop technique)

b.      to dry, fix, and stain films or smears of the specimen.

a.      The Wet-Mount and Hanging-Drop techniques

A wet mount is made by placing a drop of fluid containing the organisms onto a glass slide and covering the drop with a cover slip. To reduce the rate of evaporation and exclude the effect of air currents, the drop may be ringed with petroleum jelly or a similar material to provide a seal between the slide and the cover slip.

A special slide with a circular concave depression is sometimes used for examination of wet preparations. A suspension of microbial specimen is placed on a cover slip, and then inverted over the concave depression to produce a “hanging drop” of the specimen.

Advantages of wet preparation include;

·        It avoids the distortion of the morphology of microorganisms, which is common with dried and stained specimens.

·        Enables the determination of whether or not the organisms are motile.

·        Enables the observation of cytological changes occurring during cell division and to determine the rate at which the division occurs.

·        Some cell inclusion bodies, e.g. vacuoles and lipid material, can be observed readily in wet preparation.

b.      Fixed, stained smears

The essential steps in the preparation of a fixed, stained smear are;

1.      Preparation of the film or smear – this involves spreading the material containing the microorganisms over the surface of the slide.

2.      Fixation – this is when the film or smear is allowed to air dry. In order to prevent the cells from being washed off during the staining procedure, the slide is quickly passed through a Bunsen burner flame to heat-fix the cells to the slide.

3.      Application of one or more staining solutions – stains are added to the smear before microscopic examination. Fixed, stained smears are most frequently used for the observation of the morphological characteristics of bacteria. The advantages of this procedure are;

-  the cells are made more clearly visible after they are coloured, and

-  differences between cells of different species and within the same species can be demonstrated by use of appropriate staining solution.

STAINING METHODS

Staining is the colouring of microorganisms with a dye that emphasizes certain

structures. To apply acidic or basic dyes, three kinds of staining techniques are

used;

1.      Simple Stains – this is an aqueous or alcohol solution of a single basic dye. The primary purpose of a single stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible. The stain is applied to the fixed smear for a certain length of time and then washed off, and the slide is dried and examined. Occasionally, a chemical is added to the solution to intensify the stain; such an additive is called a mordant. The mordant increases the affinity of a stain for a biological specimen; and also to coat a structure (e.g., flagellum) to make it thicker and easier to see. Examples include Methylene blue, Carbolfuchsin, Crystal Violet, and Safranin.

2.      Differential Stains – Differential stains makes use of more than one stain to highlight the differences between cell parts or between cell types. The differential stains most frequently used for bacteria are the Gram stain and acid-fast stain.

·        Gram Stain – this was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It is one of the most useful staining procedures since it divides bacteria into two large groups; gram –positive and gram-negative. The gram staining procedure is as follows;

1.      A heat fixed smear is covered with a basic purple dye, usually crystal violet. Because the purple stain imparts its colour to all cells, it is referred to as a primary stain.

2.      After a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant. When the iodine is washed off, both gram positive and gram negative bacteria appear dark violet or purple.

3.      Next, the slide is washed with ethanol or an ethanol-acetone solution. This solution is a decolourizing agent, which removes the purple from the cells of some species but not from others.

4.      The alcohol is rinsed off, and the slide is then stained with safranin (counterstain), a basic red dye. The smear is washed again, blotted dry, and examined microscopically. 

The purple dye and the iodine combine with each bacterium and colour it

dark violet or purple. Bacteria that retain this colour after the alcohol has attempted to decolourize them are classified as gram-positive; bacteria that lose the dark the dark-violet or purple colour after decolourization are classified as gram-negative. Because gram-negative bacteria are colourless after the alcohol wash, they are nolonger visible. This is why the basic dye safranin is applied; it turns the gram-negative bacteria pink. Because gram positive bacteria retain the original purple stain, they are not affected by the safranin counterstain.

·         Acid-Fast Stain -This is a differential stain (divides bacteria into distinctive groups) where the acid-fast stain binds strongly only to bacteria that have a waxy material in their walls. The acid-fast bacteria (AFB) are not easily decolourized with acid-alcohol (HCl + 95% ethyl alcohol) after staining with hot carbolfuchsin dye since these bacteria have a thick, waxy lipid material that prevents the dye from being removed through the cell membrane. This staining method is used in the clinical laboratory for the identification of bacteria such as Mycobacterium tuberculosis (cause of tuberculosis) and M. leprae (cause of leprosy).

In the acid-fast staining procedure, the red dye carbolfuchsin is applied to a fixed smear, and the slide is gently heated for several minutes (heating enhances penetration and retention of the dye). The slide is cooled and washed with water. The smear is next treated with acid-alcohol, a decolourizer, which removes the red stain from bacteria that are not acid-fast. The acid-fast microorganisms retain the red colour because the carbolfuchsin is more soluble in the cell wall waxes than in the acid-alcohol. In non acid –fast bacteria, whose cell walls lack the waxy components, the carbolfuchsin is rapidly removed during decolourization, leaving the cells colourless. The smear is then stained with a methylene blue counterstain. Non acid-fast cells appear blue after application of the counterstain.

3.      Special Stains – special stains are used to colour and isolate specific parts of microorganisms, e.g., endospores, flagella, and to reveal the presence of capsules.

·         Negative staining for capsules – Many bacteria have a gelatinous covering called a capsule which is responsible for its virulence, the degree to which a pathogen can cause disease. To demonstrate the presence of capsules, the bacteria are mixed with a solution containing a fine colloidal suspension of coloured particles (usually India ink or Nigrosin) to provide a dark background and then the bacteria can be stained with a simple stain, such as safranin. Because of their chemical composition, capsules do not accept most biological dyes, such as safranin, and thus appear as halos surrounding each stained bacterial cell. The use of India ink illustrates a negative-staining technique; negative stains do not penetrate the capsule, and thus they provide a contrast between the capsule and the surrounding dark medium.

·         Endospore (spore) Staining- An endospore is a special resistant, dormant structure formed within a cell that protects a microorganism from adverse environmental conditions. Endospores, whenever they occur, cannot be stained by ordinary methods, such as simple staining and Gram staining, because the dyes do not penetrate the wall of the endospore. The most commonly used endospore stain is the Schaeffer-Futton endospore stain.

In this procedure, Malachite green, the primary stain, is applied to a heat-fixed smear and heated to steaming for about 5 minutes. The heat helps the stain to penetrate the endospore wall. Then the preparation is washed for about 30 seconds with water to remove the malachite green from all the cells’ parts except the endospores. Next, safranin, a counterstain, is applied to the smear to stain portions of the cell other than endospores.

·         Flagella Staining – Bacterial flagella are structures of locomotion too small to be seen with light microscopes without staining. A tedious and delicate staining procedure uses a mordant and the stain carbolfuchsin to build up the diameters of the flagella until they become visible under the light microscope.

BACTERIA

Bacteria are very small unicellular prokaryotic organisms, approximately 0.5 – 1.0 µm in diameter.

Shape and cellular arrangement

The shape of a bacterium is governed by its rigid cell wall. Typically bacterial cells are;

A.   Spherical; also called coccus (pl. cocci) – the cocci appear in several characteristic arrangements, depending on the plane of cellular division and whether the daughter cells stay together following division, they are divided into;

                           (i). Diplococci – where cells divide in one plane and remain attached predominantly in pairs

(ii). Streptococci – where cells divide in one plane and remain attached to form chains.

(iii). Tetracocci – where cells divide in two planes and characteristically form groups of four cells.

(iv). Staphylococci – where cells divide in three planes, in an irregular pattern, producing “bunches” of cocci.

(v). Sarcinae – where cells divide in three planes, in irregular pattern, producing a cuboidal arrangement of cells.

 

B.    Straight rods; also called bacillus (pl. bacilli) – the bacilli are not arranged in patterns as complex as those of cocci. They mostly occur singly or in pairs (diplobacilli). But some species form chains (streptobacilli).

C.    Helically curved rods; also called Spirillum (pl. spirilla) – the curved bacteria with less than one complete twist or turn, are said to have vibrioid shape, whereas this with one or more complete turns have a helical shape. Spirilla are rigid helical bacteria, whereas spirochetes are highly flexible.   

Although most bacterial species have cells that are of a fairly constant and characteristic shape, some have cells that are pleomorphic, i.e., that can exhibit a variety of shapes.

Bacterial fine structure

A typical bacterial cell is a prokaryotic cell with several structures external to the cell wall, the cell wall itself, and several structures internal to the cell wall.

Structures external to the cell wall

1.      Glycocalyx – glycocalyx is the general term used for substances that surround cells. The bacterial glycocalyx is a viscous (sticky), gelatinous polymer that is external to the cell wall and is composed of polysaccharide, polypeptide, or both. Its chemical composition varies widely with the species. For most part, it is made inside the cell and excreted to the cell surface.

If the glycocalyx is organized and firmly attached to the cell wall, it is called a capsule; but when unorganized and only loosely attached to the cell wall, the glycocalyx is called a slime.

The presence of a capsule can be determined by using negative staining, e.g. India Ink method.

Functions of the capsules

·         They may provide protection against temporary drying by binding

            water molecules.

·         They may block attachment of bacteriophages

·         They may be antiphagocytic, i.e., they inhibit engulfment of pathogenic bacteria by white blood cells and thus contribute to invasive or infective ability (virulence).

·         They may promote attachment of bacteria to surfaces; e.g., Streptococcus mutans (which causes dental caries), firmly adheres to the smooth surfaces of teeth because of its secretion of a water-insoluble capsular glucan.

·         If capsules are composed of compounds having an electrical charge, such as sugar-uronic acids, they may promote the stability of bacterial suspension by preventing the cells from aggregating and settling out, because cells bearing similarly charged surfaces tend to repel one another.

2.      Flagella – these are hair-like, helical appendages that protrude through the cell wall and are responsible for swimming motility. Their location on the cell varies and may be used in bacterial classification.

·         Monotrichous – with a single flagellum.

 

·         Lophotrichous –with two or more flagella at one or both poles of the cell

               

                 

·         Amphitrichous – with one or more flagella at each end of the cell

          

·         Peritrichous – with flagella distributed over the entire cell.

 

The flagellum has three parts;

        a. the helical filament,

        b. the hook

        c. the basal body associated with the cytoplasmic membrane and the cell

            wall; and anchors the flagellum to the cell.

The filament of bacterial flagellum is compost of subunits of a protein called flagellin. The shape and wavelength of the flagellum are in part determined by the structure the flagellin protein and also to some extent by the direction of rotation of the flagellum.

The hook attaches to the basal body. The basal body is rather a complex structure; it connects the rest of the flagellum to the cell envelope (cell wall and cell membrane). The basal body consists of a small central rod that passes through a system of rings (L, P and S-M). In gram negative bacteria, an outer ring is anchored in the outer membrane and another in the peptidogylcan layer of the cell wall, and an inner ring is located within the cytoplasmic membrane. In gram positive bacteria, which lack the outer membrane, only the inner pair of rings is present.   

Flagella function in motility.

3.      Pili (Fimbriae) – these are hollow, non-helical, filamentous appendages that are thinner, shorter, and more numerous than flagella. They do not function in motility, since they are found on non-motile as well as motile species. 

Functions of fimbriae/pili

·         One type, called F pilus (or sex pilus), serves as the port of entry of genetic material during bacterial mating

·         Some pili, play a major role in human infection by allowing pathogenic bacteria to attach to epithelial cells lining the respiratory, intestinal or genitourinary tracts. This prevents the bacteria from being washed away by the flow of mucous or body fluids and permits the infection to be established. 

4. Bacterial cell wall

The cell wall of bacteria is a complex semi-rigid structure that is responsible for the characteristic shape of the cell. The cell wall surrounds the underlying, fragile plasma (cytoplasmic) membrane and protects it and the internal parts of the cell from adverse changes in the surrounding environment. Almost all prokaryotes have cell walls.  

Cell wall structure and composition

The bacterial cell wall is composed of a macromolecular network called peptidoglycan (sometimes called murein), which is present either alone or in combination with other substances. The peptidoglycan is composed of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are related to glucose.

Bacteria can be divided into two major groups, called gram -positive and gram -negative, on the basis of their reaction to Gram stain as developed in 1884 by a Danish Microbiologist, Christian Gram.

After Gram staining, gram -positive bacteria appear purple and gram- negative bacteria appear red. This difference in reaction to the gram stain arises because of differences in the cell wall structure of gram –positive and gram –negative cells.

 

Gram –positve                                                     Gram -negative

- Has a single thickened wall layer, composed            - Has thin murein layer, periplasm and the

Largely of murein together with techoic acid                 outer membrane layer.

- Wall not bounded by outer membrane and

there is no periplasmic space.

The gram- positive bacteria retain the CI complex formed by the reaction of Crystal Violet and Iodine when treated with organic solvents (i.e. alcohol or acetone). However, the CI complex is removed in gram negative bacteria.

The explanation of these differences is thought to be associated with difference in structure of the cell walls. When alcohol is added during the staining in gram- positive bacteria, thick walls shrink and close the pores so that the CI complex dissolved cannot come out. Gram- negative bacteria have a very thin rigid layer. The alcohol digests out easily the lipids of the outer membrane, the thin rigid layer and extracts out the CI complex from the cell walls. The cell then stain red when the counter-stain (Safranin) is added.

Therefore the cell walls of gram-positive and gram-negative bacteria differ in the following respects;

      Gram– positive                                                           Gram- negative

1. The cell walls of gram-positive  bacteria consists of many layers of peptidoglycan, forming a thick, rigid structure   2. Contain techoic acids, which consist primarily of an alcohol and phosphate 3. Have little or no lipopolysaccharides 4. Have small amounts of lipids (0 – 2%) 5. Appear violet blue after gram-staining 6. More resistant to physical disruption 7. Inhibited by basic dyes, e.g., crystal      Violet

1. Cell walls of gram-negative bacteria contain only a thin layer of peptidoglycan 2. Do not contain techoic acids 3. Lipopolysaccharides present 4. Larger amounts of lipids (10 – 20%) 5. Appear red after gram-staining 6. Less resistant 7. Not inhibited.

Functions of the cell wall

The cell wall prevents cells from rupturing when the osmotic pressure inside the cell is greater than outside the cell. It also helps maintain the shape of a bacterium and serves as a point of anchorage of flagella.

Structures internal to the cell wall

1.      Plasma (cytoplasmic) membrane – this is a thin, structure lying inside the cell wall and enclosing the cytoplasm of the cell. It is only 8nm thick, and serves as a critical barrier separating the inside of the cell (the cytoplasm) from its environment. If the membrane is broken, the integrity of the cell is destroyed, the internal contents leak into the environment, and the cell dies. The cytoplasmic membrane is also a highly selective barrier, enabling a cell to concentrate specific metabolites and excrete waste materials.

Structure of the membrane

The general structure of the bacterial plasma membrane is a phospholipid bilayer just like in eukaryotic cells.

Diagram showing structure of the cytoplasmic membrane. Pl (phospholipids), PP (peripheral proteins), IP ( Integral proteins)

 The two layers are composed of phospholipid molecules arranged in such a way that the polar, hydrophilic groups are to the outside of the bilayer. The fatty acid hydrocarbon (hydrophobic) groups are oriented towards the centre.

The structure of the bilayer is not rigid; the protein can move freely sideways and cannot flip off. This helps to maintain the bilayer structure. This dynamic arrangement of proteins and phospholipids is referred to as fluid mosaic model.           

Functions of the cytoplasmic membrane

a. It acts as a means of transportation system between the cell and the environment. It is through the cytoplasmic membrane that all nutrients pass, and it is also through the         cytoplasmic membrane that all waste products products leave the cell. The cytoplasmic

membrane is selectively permeable.

b. Respiration – the membrane contains enzymes that help in the breakdown of nutrients, e.g., glucose is broken down to release energy.

c. Photosynthesis – pigments and enzymes involved in photosynthesis are found in the infoldings of the cytoplasmic membrane that extend into the cytoplasm. These layers of membranes are called chromatophores.

d. Reproduction – Large infoldings of cell membrane called mesosomes found in gram- negative bacteria are thought to be involved in cell division. They are thought to initiate formation of septum during binary fission.

1.      Cytoplasm – this is the internal matrix of the cell contained inside the plasma membrane. It is composed of 80% water and is translucent, elastic and granular in appearance. Within the cytoplasm are nucleic acids, proteins, carbohydrates, inorganic ions, and other low molecular weight compounds. It is the site for many chemical reactions, such as protein synthesis, DNA synthesis and other cellular components.

2.      Cytoplasmic inclusions – These include food reserve materials and other granules such as those for storage of cellular metabolic products. The inclusions include volutin granules for the storage of phosphorus. They are referred to as metachromatic granules because they stain red when stained with methylene blue dye. They are found in cells growing in phosphate rich environments e.g., Corynebacterium diptheriae.

Polysaccharide granules are for storage of glycogen and starch. Lipid inclusions are for storage of fats.

3.      Ribosomes – These are sites for protein synthesis. They are composed of 60% RNA and 40% protein. They occur freely in the cytoplasm. Bacterial ribosomes are smaller (70S) compared to eukaryotic ribosomes (80S).

 

4.       Nuclear Area – this has a single long circular molecule of double stranded DNA, the bacterial chromosome. This is the cell’s genetic information, which carries all the information required for the cell’s structures and functions. They lack histones and are not surrounded by a nuclear envelope (membrane). In addition to the bacterial chromosome, bacteria often contain smaller circular, double stranded DNA molecules called plasmids.

5.      Plasmids - Plasmids are extra-chromosomal genetic elements; i.e., they are not connected to the main bacterial chromosome, and they replicate independently of chromosomal DNA. Plasmids contain genes which are not essential for bacterial growth and they can be lost by cell without affecting their growth. Plasmids may carry genes for such activities as antibiotic resistance, tolerance to toxic metals, production of toxins, and synthesis of enzymes. Plasmids can be transferred from one bacterium to another. Plasmid DNA is used for gene manipulation in biotechnology.

Bacterial Endospores

These are resistant bodies formed within the cells of endospore forming bacteria. They are formed in response to adverse environmental conditions (unfavourable growth conditions), such as desiccation and starvation. They are also resistant to high temperature, extreme pH, and chemical disinfectants. Endospores are therefore a means of survival against adverse conditions.

Each cell in endospore forming bacteria produces only one endospore and when favourable growth conditions return, each germinates into a single vegetative cell.

Endospores are resistant to normal staining procedures and can only be seen clearly with the endospore staining method. Endospores occur in the genera Bacillus, Clostridium, Desulfotomaculum and Streptolactobacillus.

In position the endospores may be;

a.      Central

b.      Terminal

c.      Subterminal.

Various endospore positions

Endospore Structure

The endospore is formed within the cell of endospore forming bacterium. The spore mother cell in which the endospore is formed is called a sporangium.

The exosporium is a thin protein coat occurring in some but not all species of endospore formers such as Bacillus cereus. The spore coat (inner and outer) is a thick layer (80% protein). This layer confers resistance of endospores to toxic chemicals. It also prevents uptake of stains.

The cortex layer contains wall like material, i.e., peptidoglycan. It absorbs water from the core.

The core is the part that develops to the future vegetative cell. It contains a cell wall, plasma membrane and cytoplasm as in the mother cell. During dormancy the core has a high concentration of dipicolinic acid and Ca⁺⁺ ions which associate to form calcium dipicolinate complex. This complex is thought to confer heat resistance to endospores.

The core is dehydrated and contains minimal cellular components; i.e., only those necessary for resumption of metabolism when endospore germinate.

Bacterial reproduction

When bacteria are inoculated into a suitable medium and incubated under optimum conditions for growth, a tremendous increase in the number of cells occur within relatively short time. Several methods of reproduction occur in bacteria;

                                   i. Binary fission

                                   ii. Budding

                                   iii. Fragmentation

                                   iv. Formation of conidiospores and sporangiospores.

i. Binary fission – this is the most common method of reproduction in bacteria. A cell divides into two identical daughter cells after development of a transverse wall (cross-wall) or septum. This is an asexual reproductive process.

Summary

1.      DNA gets attached to cytoplasmic membrane

2.      Cell enlarges and DNA duplicates

3.      Crosswalls form

4.      Cells separate

5.      daughter cells form.

ii. Budding – this is a reproduction method where a small protuberance (bud) develops at one end of the cell; this enlarges and eventually develops into a new cell which separates from the parent. An example of a bacterium that reproduces this way is Hyphomicrobium sp.

ii. Fragmentation – this is found in filamentous bacteria which reproduce by fragmentation of the filaments into small bacillary or coccoid cells, each of which gives rise to new growth.

iv. Formation of conidiospores or sporangiospores – this is found in members of the genus Streptomyces and related bacteria which produce many spores per organism by developing crosswalls (septation) at the hyphal tips; each spore gives rise to a new organism.

                          

                          A drawing of a typical streptomyces showing filamentous,

                     branching  growth   and  asexual spores (conidia) at the filament tips

                     (Tortora et al., 1993).

1.      Sexual reproduction in bacteria is is exemplified as conjugation where exchange of genetic material from a cell containing a sex or fertility factor (F) is transferred from an F⁺ strain (male) to a F^- strain (female). The sex factor is transferred through the sex pilus as seen in E. coli. (Fig 2.13).

                                    

The Process of conjugation in E. coli.  The donor bacterial cell has many type 1 pili (which play no role in conjugation) is connected by a sex pilus to the recipient cell (without appendages) (Stainer et al., 1986).)                          

                         

BACTERIAL GROWTH

Growth is an increase in the number of cells in a population, which can also be measured as an increase in microbial mass. In bacteria and other unicellular microbes growth continues until the cell divides into two in a process called binary fission.

In the most common bacterial reproduction, like binary fission, one cell divides, producing two cells, two producing four, etc. 

                                                                                        

                                   i.e.          1             2              2²            2³           2⁴             2ⁿ

                                   when n = the number of generations

Each succeeding generation, assuming no cell death, doubles the population. The total population N at the end of a given time period would be expresses as;

                                   (i)              N = 1 X 2ⁿ      

However, under practical conditions, the number of bacteria N_o inoculated at time zero is not 1 but more likely several thousand, so the formula becomes

                                   (ii)            N = N_o X 2ⁿ

Solving equation (ii) for n, we have

                                           Log₁₀N = log₁₀No + nlog₁₀2

                                                      n = log₁₀N – log₁₀N₀

                                                                     log₁₀2

By substituting the value of log₁₀2, which is 0.301 in the above equation, we get;

                                                      n = log₁₀N – log₁₀N₀

                                                                         0.301

This is same as;                            n =       1      (log₁₀N – log₁₀N₀)

                                                                 0.301                    

Hence                                             n = 3.3 (log₁₀N – log₁₀N₀)

Thus, by use of the above equation, one can calculate the number of generations that have taken place, providing he/she knows the initial population and the population after growth has occurred.

    

Normal growth curve of bacteria

When a bacterium is inoculated into a culture, it will undergo binary fission and the population increases regularly, doubling at regular time intervals (the generation time) during incubation. However, in real life bacterial populations do not increase regularly as expected. Instead they are subjected to various environmental pressures and stresses such as changes in temperature, pH, limited space, and even depletion of nutrients.

Thus when bacteria are grown in a batch culture, i.e., growth in limited volume of media where the nutrients are not removed, the culture does not increase steadily throughout. Instead a characteristic curve called the normal growth curve can be used to explain what happens to the numbers of viable cells during the incubation period; 

A.   The lag phase – this is the initial phase which follows immediately the inoculum is added to a new medium. The population remains temporarily unchanged. The individual cells increase in size, they synthesize new protoplasm. They synthesize enzymes or coenzymes in amounts required for optimal operation of the chemical machinery of the cell.

This is the time for adjustments in the physical environment around each cell. At the end of lag phase, each organism divides. However, since not all organisms complete the lag period simultaneously, there is a gradual increase in the population until the end of this period, when all cells are capable of dividing at regular intervals.

   

B.     Logarithmic or Exponential phase - during this period the cells divide steadily at a constant rate. The population is most nearly uniform in terms of chemical composition of cells, metabolic activity, and other physiological characteristics.

The generation, g (the time require for the population to double) can be determined from the number of generations n that occur in a particular time interval t. From the above equation for n, the generation time can be calculated by the following formula;

                             g = t/_n  =                       t           .

                                                        3.3 (log₁₀N – log₁₀N₀)

During exponential growth, the growth rate (i.e. the number of generations per hour), termed R, is the reciprocal of the generation time g.

                                   R   =          3.3 (log₁₀N – log₁₀N₀)

                                                                        t

Because, in general, cells in the logarithmic phase of growth are the most uniform and are in a more clearly defined condition than in any other phase, log phase cultures are commonly used for studies of microbial metabolism.

C.    The stationary phase – this is when the logarithmic phase of growth begins to taper off after several hours in gradual fashion, the stationary phase. This trend toward cessation of growth can be attributed to;

·        the exhaustion of some nutrients

·        the production of toxic products during growth

      The population remains constant for a time, perhaps due to complete cessation of 

        division or perhaps because the reproduction rate is balanced by an equivalent

        death rate.

D.   The decline or death phase - during this phase, the cells die faster than new cells are produced. The bacterial death is due to depletion of essential nutrients and the accumulation of inhibitory products, such as acids.

      During this phase, the number of viable cells decreases exponentially,

      essentially the inverse of the growth during the log phase.

      Bacteria die at different rates, just as they grow at different rates.

Transitional periods between growth phases

A culture proceeds gradually from one phase of growth to the next. This means that not all the cells are in an identical physiological condition toward the end of a given phase of growth. Time is required for some to catch up with others.

Synchronous growth – this involves the manipulation of the growth of cultures so that all cells will be in the same stage of their growth cycle, i.e., growing synchronously. However, the synchrony often lasts only a few generations, since even the daughters of a single cell soon get out of phase with one another.

A population can be synchronized by manipulating the physical environment or the chemical composition of the medium. For example, the cells may be inoculated into a medium at suboptimal temperature; if they are kept in this condition for some time, they will metabolize slowly but will not divide. When the temperature is subsequently raised, the cells will undergo a synchronized division.

The other method involves the separation of the smallest cells in a log phase culture by filtration or by differential centrifugation and is synchronized with each other.  

Continuous culture – this is useful in experimental research and in industrial processes, where it is often desirable to maintain a bacterial population growing at a particular rate in the exponential or log phase. This condition is known as steady state growth. The culture volume and the cell concentration are both kept constant by allowing fresh sterile medium to enter the culture vessel at the same rate that “spent” medium, containing cells, is removed from the growing culture. Under these conditions, the rate at which new cells are produced in the culture vessel is exactly balanced by the rate at which cells are being lost through the overflow from the culture vessel. This can be achieved by a chemostat or a turbidostat.  

 Measurement of Bacterial growth

Growth is measured by following changes in the number of cells or weight of cell mass. There are several methods for counting cell numbers or estimating cell mass, suited to different organisms or different situations;

A. Total cell count – The total number of cells in a population can be measured by counting a sample under the microscope, a method called the direct microscopic count. The direct microscopic count uses Petroff-Hausser counting chambers, which is a special slide accurately ruled into squares that are ¹/_4oo mm² in area. A glass cover slip rests ¹/₅₀ mm above the slide, so that the volume over a square is ¹/_20,000 mm³. A suspension of unstained bacteria can be counted in the chamber under a microscope, giving a measure of the number of cell per small chamber volume. Converting this value to the number of cells per milliliter of suspension is easily done by multiplying by a conversion factor based on the volume of the chamber sample. Direct microscopic counts can be made rapidly and simply with a minimum of equipment. The morphology of bacteria can also be observed as they are counted. However, it has several limitations;

·         Dead cells are not distinguished from living cells

·         Small cells are difficult to see under the microscope, and some cells are probably missed

·         Precision is difficult to achieve

·         A phase contrast microscope is required when the sample is not stained

·         The method is not usually suitable for cell suspensions of low density.

B. Viable count – This is used to count viable cells. A viable cell is defined as one that is

able to divide and form offspring. The usual way to perform viable count is to determine         

the number of cells in the sample capable of forming colonies on a suitable agar medium.   For this reason, the viable count is often called the plate count or colony count. The assumption made in this type of counting procedure is that each viable cell can yield one colony. There are two ways of performing a plate count: the spread plate and the pour plate methods.

          (i). The spread plate method - in this method a volume of an appropriately diluted

                 culture (not >0.1ml) is spread over the surface of an agar plate using a sterile glass

                 spreader. The plate is then incubated until colonies appear, and the number of

                 colonies is counted.

 

            (ii). The pour plate method – this is also referred to as the standard plate count

                  method. In this method a known volume of culture is pipetted into a sterile Petri-

                  plate; melted agar medium is the added and mixed well by gently swirling the plate

                  on the table top. 

Dilutions for spread and pour plate counts

With the spread and pour plate methods, the samples must always be diluted for one to obtain appropriate colony number (30 – 300). Since one rarely knows the approximate viable count ahead of time, it is usually necessary to make more than one dilution. Several 10-fold dilutions of the sample are commonly used.

The Actual procedure

1.      Culture of bacteria put in a suspension

2.      1 ml transferred to 99 ml dilution blank; 1 ml transferred to 2^nd 99 ml dilution blank; 1 ml transferred to 3^rd 99 ml dilution blank, etc.

3.      After the serial dilution as above;

a.      a sample from each dilution is pipetted on the surface of agar plate, and spread evenly over the surface of agar using a sterile glass spreader

b.      A sample from each dilution is pipetted into a sterile plate and 15 – 20 ml of sterile agar medium is poured into each plate and the plates gently rotated for thorough distribution of inoculum throughout the medium.

4.      Plates are placed, inverted, in an incubator for 24 hours or longer.

5.      A plate is selected which contains 30 – 300 colonies

6.      The Number of colonies counted on plate X dilution sample = Number of bacteria per ml.; e.g., 159 (Colonies on plate) X 10³  = 1.59 X 10⁵ Cells (colony forming units per ml of original sample).

7.      The viable counts are often expressed as colony forming units (CFUs) per mililiter.

These methods are routinely used for estimation of bacterial populations in milk, water, food, and many other materials.

A.    Turbidimetric methods – this uses a spectrophotometer or colorimeter. Turbidimetry is a simple, rapid method for following growth; however, the culture to be measured must be dense enough to register some turbidity on the instrument. This depends on the principle that a bacterial suspension absorbs and scatter light passing through them, so that a culture of more than 10⁷ – 10⁸ cells per ml appears turbid to the naked eye.

B.     Determination of Nitrogen content – the major constituent of cell material is protein, and since nitrogen is a characteristic part of proteins, one can measure a bacterial population or cell crop in terms of bacterial nitrogen.

C.    Determination of the dry weight of cells

D.   Measurement of a specific chemical change produced on a constituent of the medium. 

FUNGI

The fungi (sing; fungus) are a group of eukaryotic non-photosynthetic organisms that generally reproduce both sexually and asexually

General characteristics of fungi

Fungi are eukaryotic chemo-organotrophic organisms that have no chlorophyll. The vegetative body of a fungus, called a thallus (pl. thalli) may consist of a single cell as in yeasts, or more typically consists of filaments, 5 – 10µm across, which are commonly branched.

The yeast cell or mold filament is surrounded by a true cell wall. Some fungi are dimorphic; that is they exist in two forms. Some pathogenic fungi of humans and other animals have unicellular and yeast-like form in their host, but when growing saprophytically in soil or on a laboratory medium they have a filamentous mold form. However, the opposite dimorphic phenomenon occurs in some plant pathogens. In Taphrina deformans (which causes peach leaf curl) or in smuts (which causes cereal smuts), the mycelial form occurs in the host and the unicellular yeast-like form occurs in laboratory culture. Thus a fungal colony may be a mass of yeast cells, or it may be a filamentous mat of mold.

Morphology

In general, yeast cells are larger than most bacteria. Yeasts vary considerably in size, ranging from 1 – 5µm in width and from 5 – 30µm or more in length. They are commonly egg-shaped, but some are elongated and some spherical. Yeasts have no flagella or other organelles of locomotion.

The thallus of a mold consists essentially of two parts;

·         The mycelium (pl. mycelia), and

·         The spores (resistant, resting, or dormant cells).

The mycelium is a complex of several filaments called hyphae (sing. hypha). New hyphae generally arise from a spore which on germination puts out a germ tube or tubes. The germ tubes elongate and branch to form hyphae. Hyphae are composed of an outer tube-like wall surrounding a cavity, the lumen, which is filled or lined by protoplasm. Between the protoplasm and the wall is the plasmalemma, a double-layered membrane which surrounds the protoplasm. The hyphal wall consists of microfibrils composed for the most part of hemicellulose or chitin; true cellulose occurs only in the walls of lower fungi. Wall matrix material in which the microfibrils are embedded consists of proteins, lipids, and other substances.

Growth of a hypha is distal, near the tip. The major region of elongation takes place in the region just behind the tip. The young hypha may become divided into cells by crosswalls which are formed by centripetal invagination (inward growth) from the existing cell wall. These crosswalls constrict the plasmalemma and grow inward to form generally an incomplete septum (pl. septa) that has a central pore which allows for protoplasmic streaming. Even nuclei may migrate from cell to cell in the hypha. Hypae occur in three forms;

1.      Nonseptate, or coenocytic hyphae – such hyphae have no septa.

2.      Septate with uninucleate cells

3.      Septate with multinucleate cells. Each cell has more than one nucleus in each compartment.

Mycelia can be either vegetative or reproductive. Some vegetative mycelium penetrates into the medium in order to obtain nutrients; soluble nutrients are absorbed through walls. Reproductive mycelia are responsible for spore production and usually extend from the medium into the air.

Reproduction

Fungi reproduce both sexually and asexually. Asexual reproduction (also called somatic or vegetative reproduction) does not involve the union of nuclei, sex cells, or sex organs. It may be accomplished by;

1.      fission of somatic cells yielding two similar daughter cells;

2.      budding of somatic cells or spores, each bud a small outgrowth of the parent cell developing into a new individual;

3.      fragmentation or disjointing of the hyphal cells, each fragment becoming a new organism; or

4.      spore formation.

Asexual spores, whose function is to disseminate the species, are produced in large numbers. There are many kinds of asexual spores;

a.      Sporangiospores – these are single-celled spores formed within sacs called sporangia at the end of special hyphae called sporangiophores.

Aplanospores are non-motile sporangiospores. Zoospores are motile sporangiospores, their motility being due to the presence of flagella.

b.      Conidiospores or Conidia –small, single-celled conidia are called microconidia. Large multicelled conidia are called macroconidia. Conidia are formed at the tip or side of a hypha.

c.      Oidia or arthrospores – these are single-celled spores formed by disjointing of hyphal cells.

d.     Chlamydospores – these are thick walled, single-celled spores which are highly resistant to adverse conditions. They are formed from cells of the vegetative hypha..

e.      Blastospores – these are asexual spores formed by budding.

Sexual Reproduction

Sexual reproduction is carried out by fusion of the compatible nuclei of two parent cells. The process of reproduction begins with the joining of two cells and fusion of their protoplasts (plasmogamy), thus enabling the two haploid nuclei of two mating types to fuse together (karyogamy) to form a diploid nucleus. This is followed by meiosis, which again reduces the number of chromosomes to the haploid number.

The sex organelles of fungi, if they are present, are called gametangia. They may form differentiated sex cells (gametes) or may contain instead one or more gamete nuclei. . If the male and female gametangia are morphologically different, the male gametangium is called the antheridium and the female gametangium called the oogonium. The various methods of sexual reproduction (by which compatible nuclei are brought together in plasmogamy) include;

i.                   Gametic copulation – this is the fusion of naked gametes, one or both of which are motile.

ii.                 Gamete-gametangial copulation – this is where gametangia come into contact but do not fuse; the male nucleus migrates through a pore or fertilization tube into the female gametangium.

iii.              Gametangial copulation – two gametangia or their protoplasts fuse and give rise to a zygote that develops into a resting spore.

iv.               Somatic copulation – fusion of somatic or vegetative cells.

v.                  Spermatization – this is the union of a special male structure called a spermatium with a female receptive structure. The spermatium empties its contents into the latter during plasmogamy.

Sexual spores, which are produced by the fusion of two nuclei include;

a.      Oospores – which are formed within a special female structure, the oogonium. Fertilization of the eggs, or oospheres, by male gametes formed in antheridium gives rise to oospores.

b.      Zygospores – these are large, thickwalled spores formed when the tips of two sexually compatible hyphae, or gametangia, of certain fungi fuse together.

c.       Ascospores – these are single-celled spores produced in sac-like structures called asci (sing. Ascus). There are usually eight ascospores in each ascus.

d.      Basidiospores – these are single-celled spores borne on a club-shaped structure called a basidium.

Classification

Most fungi of domestic, industrial, or medical significance are members of the classes Phycomycetes, Acomycetes, Basidiomycetes and Fungi Imperfecti (Deuteromycetes). Basidiomycetes are important principally in agriculture.

Class 1: Phycomycetes

These are called the lower fungi. Majority of these are aquatic, some are amphibious, whereas others are terrestrial. Their somatic phase is either a unicellular thallus or a non-septate (coenocytic) mycelium. Asexual reproduction is either by sporangiospores or sometimes by conidia.

The sporangispores are formed within a sac-like structure called sporangium, borne on specialized structure called sporangiophore. In aquatic species, the sporangiospores are motile, hence called the zoospores.  

The zoospores are provided with one or two flagella for motility. The conidia are formed externally on morphologically differentiated hyphae called conidiophores.

Class 2: Ascomycetes

This class comprises both saprophytic and parasitic forms. The distinguishing characters include;

·         Septate mycelium with chitinous cell walls

·         Production of sexual spores called ascospores inside sac-like bodies called asci (sing. Ascus). Generally, there are eight ascospores in each ascus

The asci are formed as a result of sexual reproduction and may be contained within fruiting bodies called ascocarps.      

There is complete absence of motile cell in this class. Ascomycetes include yeasts, some common green and black molds, powdery mildews, cup fungi, morels, and truffles.

Class 3: Basidiomycetes

This is a class of fungi comprising both saprophytic and parasitic forms. Some of the serious plant parasites like rusts and smuts belong to basidiomycetes. The distinguishing characters of this class include;

·         Septate mycelium

·         Production of sexual spores called basidiospores outside a club-shaped structure called a basidium.

Usually, there are four basidiospores on each basidium. There is also a complete absence of motile cells.

Class 4: Deuteromycetes

This class comprises fungi reproducing exclusively by asexual means, usually by conidia. They are also referred to as “imperfect fungi” because of the lack of perfect or the sexual stage.

PROTOZOA

Protozoa are heterogeneous group of unicellular, non-photosynthetic, typically motile protists. Protozoans are the most highly specialized, and their cell structures and modes of life and reproduction the most complex, of all the protists. They are microscopic animals, each consisting of a single cell or group of cells to form a colony. They range in size upward from less than 7.5 µm, and are invisible to the naked eye. 

Apart from their role as parasites and cause of diseases in animals of interest to man, protozoa are of concern as nuisances and as helpful organisms of decomposition. One or more species of protozoa may be found in almost every conceivable habitat on earth. They are abundant in the soil, water, pond mud, dust, and oceans. They live, in part, upon other minute living things, including other protozoans and bacteria. 

Structure of Protozoa

Protozoa vary greatly in size according to species and physiological state. They are eukaryotic but no cell walls.

Pellicle or Periplast – this is the outer covering of protozoans and is composed of lipoprotein and takes the place of a cell wall. In some groups, such as amoebas, the pellicle is either absent or present as a thin, elastic covering that seldom infers with amoeboid movement and phagocytosis, both of which are motivated by cytoplasmic streaming. In other groups, especially the large and active flagellated or ciliated species, the pellicle is thicker and less flexible (giving a definite shape to the organism) and may be ridged or grooved or show other surface patterns. In addition to the pellicle, some species also possess a chitinous, cellulose or siliceous exoskeleton for additional protection.

Plasma membrane – located just beneath the pellicle is the membrane which encloses the cytoplasm.

Endoplasm – this is the inner, very fluid zone of cytoplasm and contains many of the common microbial organelles (food vacuoles, contractile vacuoles, nucleus, mitochondria, etc.).

Ectoplasm – this is the outer, less fluid, peripheral zone of cytoplasm. This layer gives rise to a number of specialized structures used for locomotion, defense, and feeding (flagella, cilia, trichocysts, cytostome, cytopharynx (gullet), anal pore, and contractile fibers).

Contractile vacuoles – these are conspicuous in species of Protozoa that reside in fresh water but are also found in some marine and parasitic species. The vacuoles appear to be regulators of intracellular water and hence intracellular osmotic pressure. Due to low osmotic tension of fresh water and the much higher tension of intracellular fluids, cell in fresh water tend to take in water. Such cells would burst unless the excess water taken in is excreted. 

Flagella and Cilia – the protozoan flagella act as swimming appendages. The cilia are much like flagella in structure and function but are shorter. Cilia, like flagella, produce movement by sending out undulations and also appear to originate in basal granules in the ectoplasm.

Reproduction in Protozoa

Asexual reproduction – Asexual reproduction by binary fission is the most common method of reproduction among protozoa. In this process the nucleus and the remainder of the cell divide into two equal parts. Division occurs longitudinally in flagellate (Sarcomastigophora) forms such as Euglena, transversely in Ciliates (Ciliophora) like Paramecium. The daughter cells separate and continue to perform their own life processes. The rate of binary fission depends upon the species and upon various environmental factors. It may take place several times a day or may require a few days. 

A second form of asexual reproduction is budding: a small part of the parent separates and develops into a new individual.

A third method of asexual reproduction, sporulation, is found in members of the class Sporozoa. Sometimes, as in Plasmodium (Malaria parasite), there is a complicated life cycle involving more than one host. Certain stages are performed in one host (Mosquito), the other stages only in a second host (Human).

Sexual reproduction – A sexual type of reproduction occurs in certain protozoa such as Paramecium. These organisms occasionally undergo conjugation. Each cell behaves as though it were both male and female, each fertilizing the other by nuclear exchange during temporary union. Genetic variation may occur, binary fission after conjugation yields progeny that may differ in some way from the two parent cells before conjugation

Encystment – During the life cycle of some protozoans some cells may produce a thick cell wall, lose water, and become dormant. During this stage metabolism is reduced to a minimum or ceases entirely, and the dormant cell resists unfavourable environmental conditions such as prolonged drought, heat, increased salinity, or unfavourable pH. Such a stage may be formed by mature, growing cells during asexual cycles of development or just after conjugation of gametes during sexual reproductive cycles. In protozoa such cells are called cysts. Cells going into the cyst stage are said to be encysting.

After a physiologically and genetically determined period or, depending on species and cyst type, when growth conditions become favourable, the cell inside the cyst wall takes in water, resumes activity, bursts the cell wall, and emerges in the actively growing trophozoite stage. The encysted cell is said to have excysted.

Nutrition of Protozoa

Protozoa are chemoorganotrophic. Ingestion of foods by protozoa is accomplished by three methods:

·         by phagocytosis

·         by means of cytostome, and

·         by pinocytosis

i. By phagocytosis – In phagocytosis, like in amoeba, two or more pseudopodia are extruded like fingers around the food particle. The fingers merge into one, with the particle trapped within. The section of plasma membrane surrounding the food particle is pinched off and becomes the food vacuole within which the food undergoes digestion.

ii. By means of cytostome – In some protozoans, like in ciliates, food particles are wafted by cilia into a deep pouch or invagination of the cell coating called a cytopharynx. The food particle passes through the cell coating at the inner end of the cytopharynx into a cytoplasmic digestive food vacuole.

iii. By pinocytosis – Pinocytosis is a somewhat similar process for ingesting fluid rather than particulate matter, though not dependent on cilia; the pinocytic vesicles are numerous and very tiny.

Classification of Protozoa

The protozoans are divided into three phyla based on their methods of locomotion, morphology, mode of reproduction, and nutrition.

1. Phylum Sarcomastigophora – these are protozoa that always bear one or more flagella as the locomotor organelles. Their cell division is always longitudinal. Examples include Trypanosomes, Trichomonas, Leishmania, Euglena, and Trichonympha.

Trypanosome cell is slender and leaf-shaped, with posteriorly directed single flagellum which is attached through part of its length to the body of the cell, to form undulating membrane. They are parasitic in vertebrates, where they develop in the blood stream, being transmitted from host to host by biting insects. They cause disease such as African sleeping sickness, transmitted by tsetse fly. Trypanosomes are osmotrophic and absorb their nutrients from blood of the host. The Trichomonads have 4 – 6 flagella and are harmless inhabitants of the gut of vertebrates.

Trichomonas species inhabit the human body and are pathogenic e.g., T. vaginalis, T. buccalis (or T. tenax?), and T. hominis.

The amoebas – these are protozoa in which amoeboid locomotion is the predominant mode of cell movement; although some of them are able to produce flagella as well. The simplest members of this group are amoebas, which have characteristically amorphous cells due to continuous shape changes brought about by the extension of pseudopodia (Gr. pseudo = false; pod = foot), which are protoplasmic extrusions that flow or creep forward from the main body of the cell. The rest of the organism flows into the pseudopodium, and the animal moves slowly from place to place. This sort of motility is called amoeboid movement. They are phagotrophic, where the pseudopodia surround and engulf food particles, which then become enclosed in a vacuole, where digestion occurs. Waste products are excreted through the plasma membrane and pellicle to the outside. Most amoebas are free-living soil or water organisms which phagocytize smaller prey. A few inhabit the animal gut, including the forms that cause disease (amoebiasis), like Entamoeba histolytica.

• Entamoeba histolytica – this is the best known species of amoeba pathogenic to man. It attacks the walls of the intestine. It feeds characteristically but not exclusively, upon red blood cell. They penetrate the intestinal lining and cause intense inflammation and ulcers (amoebic dysentery). By means of proteolytic enzymes they can burrow through the lining and penetrate deep into the intestinal wall so that, occasionally, rupture of the intestine occurs. The patient may then die of peritonitis caused by escape of the bacteria of the feaces into the abdominal cavity.

Entamoeba histolytica may also get into the lymph and blood vessels and then be carried to the liver, lungs, brain, and other organs where they become localized and cause the formation of large obscesses.

Reproduction – E. histolytica possess the power of asexual or vegetative

multiplication. In this process the cell divides itself into two cells, a

phenomenon called cell fission.  Cell fission is said to be binary when

the cell divides into two equal parts, each part having (theoretically) all

the physiological and genetic potential of the parent cell. During

binary fission the cells divide by a constriction somewhere near the

physiological middle of the cell, i.e., by transverse fission. The nucleus

undergoes mitosis, typical of eukaryons.

Transmission of Amoebiasis –E. histolytica is passed in feaces, most

commonly in the dormant, thick-walled, encysted form. Amoebiasis is

often chronic and may be present with little definite symptomatology

for a long time. Thus amoebic infections, especially of the intestine,

like many other infections is often unknowingly disseminated widely

by mild cases or carriers. Carriers who handle food may transmit cysts

to food via their soiled hands. These cysts, after being swallowed,

rapidly excyst in the intestine. Feaces-soiled hands and flies appear to

be the major vectors of most intestinal pathogens: protozoan, bacterial,

and viral. 

Anything recently contaminated with feaces from a chronic case or

carrier of amoebiasis may transmit the cysts. Transmission on fruits

and vegetables can occur when human sewage and feaces are used for

fertilizer. Under such circumstances fresh vegetables (lettuce and

celery, for instance) may have viable E. histolytica cysts upon them

when eaten.

2. Phylum Apicomplexa – this is a very diverse group of parasitic protozoa. They possess no means of independent locomotion and show gliding movement. They are distinguished by the production of minute resistant spores and reproduce by multiple fission. All of them are parasitic and many pathogens are included in this group, e.g., Plasmodium species which cause malaria.
• Malaria – Malaria is one of the most important of the arthropod-borne (Gr. Arthron = joint; pod = foot) human parasites. Hundreds of thousands of persons die annually from this disease and millions are made chronically ill by it.
• Malarial parasite – Malaria in man is caused by a protozoan parasite of the genus Plasmodium. There are four species of Plasmodium that cause human malaria:  

a.       Plasmodium vivax (from the vivacious activity of its trophozoite stage), is most widely distributed in temperate zones. It requires about 48 hours to complete its development within the red cells. The chills, therefore, commonly occur at intervals of 48 hours, or every third day, and hence this type of malaria is called the tertian (third) fever.

b.       Plasmodium malariae, which mature approximately every fourth day. This species causes quartan (fourth) fever.

c.        Plasmodium falciparum (the word falciparum is derived from the curved or sickle-shaped falciform – gametes). It requires from 24 – 48 hours or more for development. This type of malaria is called estivo-autumnal fever because in temperate climates it typically occurs in late summer and autumn. It is most prevalent in tropical zones. It is more severe than the other forms of malaria and is less easily controlled by antimalarial drugs.

d.       Plasmodium ovale – this resembles P. vivax, and causes a disease much like tertian malaria but milder.

The life history of Plasmodium involves two stages of development:

            (i). Asexual, passed in the human body, and

            (ii). Sexual, passed in the female species of Anopheles mosquito.

Mosquitoes are two-winged insects (Diptera) which pass through four stages of development; the egg, the larva (wiggler), the pupa, and the fully developed insect (imago). The three stages develop in water.

The female Anopheles mosquito may be recognized by her stance as she bites: a “head-on” position, with hind legs in the air. She usually has spots of silver or gray on her wings and often gray bands on her legs. The non-malaria bearing varieties are usually brownish or brown-gray and bite with body nearly parallel to the skin.

• Cycle in man – An infected mosquito introduces the parasites into the blood of its victim with its saliva when it bites. The female mosquito bites only to obtain blood proteins for egg production. The males live offensively on plant juices. The parasites undergo a short period of multiplication in certain tissues, particularly in the liver. This is called the exoerythrocytic (or pre-erythrocytic) cycle. Very soon their asexual progeny enter red cells and grow within them. This stage of the parasite is the trophozoite stage. The parasite multiplies asexually within the red cells, forming a number of small bodies or segments. Finally the affected erythrocyte breaks up, and the segments escape into the circulating blood. Each segment is a new, active parasite called a merozoite. It no sooner gets out of one red cell than it attacks another erythrocyte and in turn multiplies. In this way the blood is soon teeming with the parasites and the infection becomes clinically manifest; the intrinsic incubation period (bite to first symptom) is completed. The patient becomes anaemic and weakened by loss of so many red cells, and possibly also suffers from poisonous products formed by the parasites.

The parasites appear in the blood in successive generations, all the individuals of which divide and burst out of the erythrocytes at about the same time. Each such process causes the chills and fever so characteristic of malaria. A chill indicates that a fresh crop of parasites has matured and entered the circulation.

After passing through several cycles of asexual development as described above, round, distinctive gametocytes appear in the blood of the patient. These are larger than the asexual forms and are easily recognized microscopically by trained persons in the blood smears of the infected patients. Gametocytes undergo no further development in human erythrocytes. They die if not taken up by a mosquito.

• Cycle in mosquito – When an Anopheles mosquito bites a person who has mature gametocytes in his/her blood, the sexual stage of the parasite begins.

After fertilization of the female by the male gamete in the stomach of the mosquito, the motile zygotes invade the cell lining the mosquito’s stomach and multiply there, forming a sac (oocyst) wherein the parasites undergo further development by fission. The sac ruptures, liberating numerous new young parasites. After moving about for some days inside the mosquito, they reach the mosquito’s salivary glands and from there are injected into man when the insect bites. The life cycle is thus complete. Because of the necessary period of sexual reproduction of the parasite, a mosquito that has just bitten a malaria patient cannot transmit the disease to another person until the end of 12 days – the extrinsic incubation period. A mosquito, once infected, remains so for the rest of its life, may be two months or more.

3. Phylum Ciliophora – this is a large and very varied group of aquatic, phagotrophic organisms. They are motile by means of numerous short, hair-like projections, termed cilia. The cell has two nuclei (macronucleus and micronucleus), differing in structure and function. The cell division is always transverse. Examples include Tetrahymena, Paramecium, Nassula, Balantidium, etc. 

• Paramecium – this s ovoid or pear-shaped (pyriform) and is called the “slipper animalcule” because of its slipper-like outline. It is commonly found in pond water. It maintains its form by means of a flexible but tough external pellicle that has protruding through it symmetrically arranged rows of cilia. Its cilia move in a highly coordinated rhythmic manner which permits the organism to swim in a spiral manner gracefully as a seal. Near one end of the cell is a depression (sometimes called the oral groove or peristome) which leads to the mouth or cytostome. Food (bacteria, smaller protozoans) is wafted by cilia in this area through the cytostome into the cytopharynx. The food is then taken into food vacuoles which form at the en of the cytopharynx. Digestion and absorption occur, and indigestible material is extruded through a posterior anal opening or cytopyge. Contractile vacuoles in the cytoplasm help maintain ion and water balance.

Paramecium can presumably defend or position itself by means of trichocysts (small organelles beneath the pellicle). Upon activation, the trichocysts secrete a substance which (outside the pellicle) forms long, sticky filaments. However, the major function of trichocysts is still obscure; defense, achoring, and ion regulation have been suggested.   

Reproduction – Paramecium multiplies asexually by binary fission and sexually by conjugation. Some cells have only one nucleus (macronucleus), and multiply only vegetatively, i.e., by binary fission; whereas other cells have two differing nuclei: a large macronucleus or “vegetative” polyploidy nucleus and a minute, diploid micronucleus. Only cells containing micronuclei can conjugate. Most conjugating cells are morphologically indistinguishable sexually.

In conjugation, the cells fuse and each diploid micronucleus undergoes meiotic divisions, producing four haploid nuclei in each cell. Of these, three degenerate, one divides meiotically. Thus there are two haploid (gamete-like) nuclei. One haploid nucleus from each cell of the conjugal pair is exchanged with the counterpart in the other cell. The residual and the exchange haploid nuclei in each cell then combine, thus forming a diploid nucleus and achieving genetic recombination.

In each cell the new nucleus, now haploid zygote, twice divides by mitosis, yielding four diploid nuclei, two of which become macronuclei, the other two micronuclei. The older macronucleus disintegrates. The conjugants now separate, each with two macro- and two micronuclei. In each cell one of the micronuclei disintegrates; the remaining one divides by mitosis. Each of the conjugants now again has two of each kind of nucleus. Each of these tetranucleate cells divides by binary fission. Each daughter cell normally has one macronucleus and one micronucleus. In some instances a micronucleus may be “lost in the shuffle”; such a cell has lost its sex factor and multiplies only asexually.

VIRUSES

Viruses are infectious agents so small that they can only be seen at magnifications provided by the electron microscope. Viruses are incapable of independent growth in artificial media. They can only grow in animal or plant cells or in microorganisms. They reproduce in these cells by replication. They lack metabolic machinery of their own and depend on the host cells to carry out their vital functions. However, like the host cells, viruses have the genetic information for replication; information in their genes for usurping the host cell’s energy-generating and protein-synthesizing systems.

The viral genetic material is either DNA or RNA, but never both. The nucleic acid is enclosed in a highly specialized protein coat of varying design. The coat protects the genetic material when the virus is outside the host cell.   

The structurally complete mature and infectious virus is called the virion.

Therefore, viruses can be defined as noncellular infectious entities whose genomes are a nucleic acid (either DNA or RNA); which reproduce only in living cells; and which use the cells’ biosynthetic machinery to direct the synthesis of specialized particles (virions), which contain the viral genomes and transfer them efficiently to other cells.

Viruses are generally divided into three major groups based on the kind of host they infect:

1.      Bacterial viruses (bacteriophage or phage) – are viruses that are able to infect bacterial cells.

2.      Plant viruses – which are parasites of flowering plants; ferns, fungi, algae, etc.

3.      Animal viruses –are obligate intracellular parasites of insects, fish, reptiles, birds, and many mammals.

General characteristics of viruses

All virions are composed of protein and nucleic acid core. The coat, or capsid, is composed of separate protein segments known as capsomeres. Capsomeres chemically bond with one another to surround the core, forming either a helical or polyhedral (many sided) shape.

The virions’ genetic material contains information necessary to synthesize the capsid proteins and enzymes required for completing its life cycle. The capsid aids in viral attachment to the host cell, determines the antigenic nature of the virus, and protects the enclosed nucleic acid. The core of a virion contains genetic material which is either DNA or RNA. These could be;

·         dsDNA

·         ssDNA

·         dsRNA

·         ssRNA.

The viral genes are only expressed after the virion has penetrated a suitable host cell and enter the process of viral replication. This reproductive process differs greatly from that carried out by cellular forms. Viruses do not “grow” inside the host as do other obligate intracellular parasites. Instead of simply “feeding” off of the host, viruses take command of their host’s metabolic pathways and direct them in the production of essential enzymes and products required for the production of more virions.

The five stages of virus infection and replication are:

·         Adsorption

·         Penetration and Uncoating

·         Component replication and biosynthesis

·         Assembly

·         Release

BACTERIOPHAGES

These are viruses that infect bacteria.

General characteristics

·         Bacteriophages are widely distributed in nature and exist for most, if not all bacteria.

·         They are composed of a nucleic acid core surrounded by a protein coat.

·         Bacteriophages occur in different shapes, although many have a tail through which they inoculate the host cell with viral nucleic acid. 

There are two main types of bacteriophages;

1.      Lytic or virulent phages,

2.      Temperate or avirulent phages.

When lytic phages infect cells, the cells respond by producing large numbers of new viruses. That is, at the end of the incubation period the host cell bursts or lyses, releasing new phages to infect other host cells. This is called lytic cycle.

In temperate type of infection, the result is not so readily apparent. The nucleic acid is carried and replicated in the host bacterial cells from one generation to another without any cell lysis. However, temperate phages may spontaneously become virulent at some subsequent generation and lyse the host cells. 

Phage morphology and structure

All phages have a nucleic acid core covered by a protein coat, or capsid. The capsid is made up of morphological subunits, called capsomeres. The capsomeres consist of a number of protein subunits or molecules called protomers.

Most phages occur in one of the two structural forms;

a.      Cubic or helical symmetry – these have regular solids or, more specifically polyhedral. These are rod shaped.

b.      Polyhedral, which are icosahedral in shape (icosahedron is a regular polyhedron with 20 triangular facets, each of which is an equilateral triangle).

Some bacteriophages, e.g., T-even (T₂, T₄ and T₆), have very complex structures, including a head and a tail.

General morphology of viruses

Viruses may be classified into several morphological types on the basis of their capsid architecture;

Polyhedral viruses

Many animal, plant, and bacterial viruses are polyhedral viruses, i.e., they are many sided. The capsid of most polyhedral viruses is in the shape of an icosahedron, a regular polyhedron with 20 triangular faces and 12 corners. The capsomere of each face form equilateral triangle; e.g., the poliovirus.

Helical viruses

These resemble long rods that may be rigid or flexible. The viral nucleic acid is found within a hollow, cylindrical capsid that has a helical structure; e.g., TMV.

Complex viruses

Some viruses, particularly bacterial viruses, have very complicated structures and are called complex viruses; e.g., bacteriophage.

Taxonomy of viruses

The International Committee on Taxonomy of Viruses (ICTV) has not yet established higher taxa (order through kingdom) for viruses. However, viruses are grouped by nucleic acid type, morphology, and the presence or absence of an envelope. The ICTV has organized the 2000 known species of viruses into 73 families, each of which shares morphologic characteristics and reproductive strategies. Family names end in –viridae. Likewise, genera of viruses share certain characteristics.

A viral species is defined as a group of viruses sharing the same genetic information and ecological niche. 

VIRAL MULTIPLICATION

For a virus to multiply, it must invade a host cell and take over the host’s metabolic machinery. A single virus can give rise to several or even thousands of similar viruses in a single host cell. This process can drastically change host cell and can even cause its death.

Multiplication of bacteriophages

The best understood viral life cycles are those of phages. Phages can multiply by two alternative methods;

·         The lytic cycle or

·         Lysogenic cycle

The lytic cycle

This is exemplified by T-even bacteriophages (T₂, T₄, and T₆) in their host, E. coli. The virions of T-even bacteriophages are large, complex, with a characteristic head and tail structure.

The multiplication cycle of these viruses can be divided into five distinct stages;

1.      Attachment

2.      Penetration

3.      Biosynthesis

4.      Maturation, and

5.      Release.

1.      Attachment – After a chance collision between phage particles and bacteria, attachment, or adsorption occurs. During this process, an attachment site on the virus attaches to a complementary receptor site on the bacterial cell. This attachment is a chemical interaction in which weak bonds are formed between the attachment and receptor sites. T-even bacteriophages use tail fibres as attachment sites. The complementary receptor sites are on the bacterial cell wall.

2.      Penetration – After attachment, the T-even phage injects its DNA into the bacterium. To do this, the phage’s tail releases an enzyme, phage lysozyme, which breaks down a portion of the bacterial cell wall. During penetration, the tail sheath of the phage contracts, and the tail core reaches the plasma membrane, the DNA from the phage’s head passes through the tail core and through the plasma membrane and enters the bacterial cell. The capsid remains outside the bacterial cell. Therefore, the phage particle functions like a hypodermic syringe to inject its DNA into the bacterial cell.

3.      Biosynthesis – Once the phage DNA has reached the cytoplasm of the host cell, biosynthesis of viral nucleic acid and protein occurs. Host protein synthesis is stopped by;

·         Virus-induced degradation of host DNA,

·         Virus proteins that interfere with transcription, or

·         Repression of translation.

             Initially, the phage uses the host cell’s nucleotides and several of its enzymes

             to synthesize many copies of phage DNA. Soon after, biosynthesis of viral

             proteins begins.

             Thus soon after infection, complete phages cannot be found in the host cell.

             Only separate components – DNA and protein – can be detected. This period

             during viral multiplication, when complete, infective virions are not yet

             present is called the eclipse period.

  

4.      Maturation – The next sequence of events is called maturation. In this process, phage DNA and capsids are assembled into complete virions. The phage heads and tails are separately assembled from protein subunits, and the head is filled with phage DNA and attached to the tail.

5.      Release –this is the final stage of viral multiplication and the term lysis is generally used for this stage in T-even phages because in this case the plasma membrane actually breaks open (lyses). Lysozyme, whose code is provided by a phage gene; is synthesized within the cell. This enzyme causes the bacterial cell to break down, and the newly produced phages are released from the host cell. The released phages infect other susceptible cells in the vicinity, and the viral multiplication cycle is repeated within these cells.

The time that elapses from phage attachment to release is called burst time and averages 20 – 40 minutes. The number of newly synthesized phage particles released from a single cell is referred to as burst size and usually ranges from about 50 – 200

The Lysogenic cycle

In contrast to T-even phages, some viruses do not cause lysis and death of host cell when they multiply. They proceed through a lysogenic cycle by incorporating their DNA into the host cell’s DNA. This is called lysogeny, and the phage remains latent. These phages are called lysogenic or temperate phages. The participating bacterial host cells are called lysogenic cell. Examples include the bacteriophage lambda (      ).

The lysogenic cycle include;

1.      Upon penetration, the linear phage DNA forms a circle

2.      This circle can multiply and be transcribed,

3.      Leading to the production of new phage and to lysis (lytic cycle).

4.      Alternatively, the circle can recombine with and become part of the circular bacterial DNA (lysogenic cycle). The inserted phage DNA is now called a prophage. The phage genes that would otherwise

5.      t the synthesis and release of new virions are repressed (turned off). Every time the host cell’s machinery replicates the bacterial chromosome,

6.      It also replicates the prophage DNA. The prophage remains latent within the progeny cells.     

7.      However, a rare spontaneous event can lead to the excision (popping of the 

      phage DNA, and the initiation of the lytic cycle.

Importance of Lysogeny

1.      The lysogenic cells are immune to re-infection by the same phage

2.      The host cell may exhibit new properties

3.      It makes specialized transduction possible.

CULTIVATION OF VIRUSES

Bacteriophages can be grown in pure cultures of host cells that have been plated on the surface of nutrient agar. As lytic phage complete their life cycles, they destroy their host cells and produce a clear spot, or plaque, on the plate surface.

The plaque counts have also been used in the enumeration of viruses because the number of plaques is proportional to the number of infectious virus particles present. Each virion gives rise to a single plaque, just as a bacterium gives rise to a single colony.

Embryonated chicken eggs – Since viruses can grow only in living cells, one of the most economical and convenient methods for cultivating a number of viruses is the chicken embryo technique.

Fertile chicken eggs incubated for 5 – 12 days can be incubated through the shell aseptically, and the opening sealed with paraffin wax and the egg incubated at 36^oC for the time required for virus growth. Chick embryos contain several different types of cells in which various viruses will replicate.

Tissue cultures – Cell cultures are choice of propagation of viruses because they are;

1.      convenient

2.      economical to maintain

3.      Show cytopathic effects.

In this case, the tissue structure deteriorates as the virus multiplies. This deterioration is called the cytopathic effect (CPE).

Animals – Some viruses cannot be cultivated in cell culture or in embryonated chicken eggs and must be propagated in living animals. Mice, guinea pigs, rabbits and primates are used for this purpose. Animal inoculation is also a good diagnostic tool because the animal can show typical disease symptoms and histological (tissue) sections of infected tissue can be examined microscopically.

Cultivation of plant viruses – Plant viruses can be cultivated by direct mechanical inoculation of virus suspensions by rubbing on leaves of living plants. Rubbing is accomplished with the aid of an abrasive such as carborundum. This can lead to the formation of local lesions as well as general infection. Some plant viruses can replicate to large numbers in infected plants.

MICROBIAL GROWTH

Microbial growth refers to the number of cells, not the size of cells. Microbial growth has;

i.        Nutritional requirements

ii.       Physical requirements.   

Nutritional Requirements

All forms of life share certain nutritional requirements for growth and normal functioning;

1.      All organisms require a source of energy. Some rely on chemical compounds for their energy and are called chemotrophs. Other can utilize radiant energy (light) and are called phototrophs. Both chemotrophs and phototrophs exist among bacteria.

2.      All organisms require a source of electrons for their metabolism. Some organisms can use reduced inorganic compounds as electron donors and are termed lithotrophs (some may be chemolithotrophs, others photolithotrophs. Other organisms use organic compounds as electron donors and are called organotrophs (some are chemoorganotrophs, others photoorganotrophs).

3.      All organisms require carbon in some form for use in synthesizing cell components. All organisms require at least small amounts of CO₂; However, some can use CO₂ as their major,or even sole, source of carbon, such organisms are called autotrophs. Others require organic compounds as their carbon source and are called heterotrophs.

Microbial nutritional groups

category	Principle energy source	Principle carbon source	Example
Photoautotrphs	Sunlight	CO2	Green sulfur and purple sulfur bacteria Cyanobacteria and algae
Photoheterotroph	Sunlight	Organic compounds	Purple and green sulfur bacteria
Chemoautotroph	Inorganic chemical compounds	CO2	Chemolithotrophic bacteria
Chemoheterotrophs	Organic compounds	Organic compounds	Most bacteria, Fungi and Protozoans

(i) Photoautotroph :  These use light as principle energy source and carbon dioxide as the principle  carbon source. These include photosynthetic organisms, higher plants, algae and photosynthetic bacteria ie. green sulfur bacteria and purple sulphur bacteria.

(ii) Photoheterotrophs: these use light as principle energy source and organic compounds as the principle carbon sources. This category include the green and purple non sulfur bacteria.

(iii) Chemoautotrophs:  these bacteria use inorganic compounds principle energy source and carbon dioxide as the principle carbon source. Energy is obtained through oxidation of reduced inorganic compounds eg.

NH₄ + 3/2 O₂ →→→ NO^-₂ + 2H⁺ + H₂O + 62.1 Kcal, as in Nitrosomonas,

Fe ⁺² + 2H ⁺+ 1/2 O₂ →→→ Fe ⁺² + H₂O +e , 17.0 Kcal, as in Thiobacillus ferroxidans

S + 1/2O₂ →→→→ SO₄  ^-2 + 2H⁺ + 48.5 Kcal,  as in  T. thiooxidans

(iv) Chemoheterotrophs

These microorganisms utilize the organic compounds as the principle source of carbon and energy. This group constitutes most microorganisms. Most bacteria, all fungi and protozoa belong to this group. In this category, there is no distinction between the carbon and the energy sources. The same organic compound is also used as the energy and carbon source.

4.      All organisms require nitrogen in some form for the cell components. Bacteria are extremely versatile in this respect. They can use atmospheric nitrogen. Others thrive on inorganic nitrogen compounds such as nitrates, nitrites, or ammonium salts, and still others derive nitrogen from organic compounds such as amino acids.

5.      All organisms require oxygen, sulphur and phosphorus. Oxygen is provided in various forms such as water, components of various nutrients; or molecular oxygen. Microbes that use molecular oxygen, are called aerobes; and produce more energy from nutrients than do microbes that do not use oxygen. Organisms that require oxygen to live are called obligate aerobes.

The organisms that can use oxygen when it is present but are able to continue growth in the absence of oxygen are called facultative anaerobes. Obligate anaerobes are organisms that are unable to use molecular oxygen for energy-yielding reactions. They are in fact harmed by it, e.g. Clostridium.

- Sulphur is needed for synthesis of certain amino acids (Cysteine, Cystine, and   

  Methionine).

- Phosphorus, usually supplied in the form of phosphate, is an essential 

  component of nucleotides, nucleic acids, phospholipids, techoic acids, and

  other compounds.

6.      All organisms require metal ions, such as K⁺, Ca²⁺, Mg²⁺, and Fe²⁺ for normal growth. Other metal ions are also needed but usually only at very low concentrations, such as Zn²⁺, Cu²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, B³⁺ and Co²⁺; these  are often termed trace elements and often occur as contaminants of other compounds of culture media in amounts sufficient to support bacterial growth.

Not all the biological functions of metal ions are known, but Fe²⁺, Mg²⁺, Zn²⁺ , Mo⁶⁺, Mn²⁺, and Cu²⁺  are known to be cofactors for various enzymes.

7.      All organisms also require organic growth factors which are essential organic compounds that an organism is unable to synthesize; they must be directly obtained from the environment. The growth factors include vitamins and vitamin-like compounds. These function either as coenzymes for several enzymes or as the building blocks for coenzymes. Some bacteria are capable of synthesizing their entire requirement of vitamins from other compounds in the culture medium, but others cannot do so and will not grow unless the required vitamins are supplied preformed to them in the medium.

8.      All living organisms require water, and in the case of bacteria all nutrients must be in aqueous solution before they can enter the cells. Water is a highly polar compound that is unequaled in its ability to dissolve or disperse cellular components and to provide a suitable milieu for the various metabolic reactions of a cell. Moreover, the high specific heat of water provides resistance to sudden, transient temperature changes in the environment. Water is also a chemical reactant, being required for the many hydrolytic reactions carried out by the cell.

Physical conditions required for microbial growth

Just as bacteria vary greatly in their nutritional requirements, so do they exhibit diverse responses to physical conditions such as temperature, gaseous conditions, and pH.

1.      Temperature – since all processes of growth are dependent on chemical reactions and since the rates of these reactions are influenced by temperature, the pattern of bacterial growth can be profoundly influenced by this condition. The temperature that allows for most rapid growth during a short period of time (12 – 24 hrs) is known as the optimum growth temperature.

On the basis of their temperature relationships, bacteria are divided into three main groups;

·         Psychrophiles – these are able to grow in cold temperatures (at 0^oC or lower), though they grow best at high temperatures. They are called “cold lovers”. The physiological factors responsible for the low temperature maxima for strict psychrophiles are not entirely clear, but some factors like instability of their ribosomes and various enzymes, increased leakage of cell components, and impaired transport of nutrients have been implicated.

·         Mesophiles – grow best within a temperature range of approximately 25 -40^oC. For example, all bacteria that are pathogenic for humans and warm-blooded animals are mesophiles, most growing at about body temperature (37^oC).

·         Thermophiles – these grow best at temperatures above 45^oC. The growth range of many thermophiles extends into the mesophilic region; these species are called facultative thermophiles.

Other thermophiles cannot grow in the mesophilic range and are called true thermophiles, obligate thermophiles or stenothermophiles. Factors that have been implicated in the ability to grow at high temperatures are an increased thermal stability of ribosomes, membranes, and various enzymes. Loss of the fluidity that exists within the lipid bilayer of the cytoplasmic membrane may be a factor governing the minimum temperature.

  

2.      Gaseous requirements – The principle gases that affect growth are oxygen and carbon dioxide. Bacteria display a wide variety of responses to free oxygen that it is convenient to divide them into four groups on the following bases

·         Aerobic bacteria - which require oxygen for growth and can grow when incubated in an air atmosphere.

·         Anaerobic bacteria – these do not use oxygen to obtain energy; oxygen is toxic for them and they cannot grow when incubated in an air atmosphere. Some can tolerate low levels of oxygen (non-stringent or tolerant anaerobes), but others (stringent or strict anaerobes) cannot tolerate even low levels and may die upon brief exposure to air.

·         Facultatively anaerobic bacteria – these do not require oxygen for growth, although they may use it for energy production if it is available. They are not inhibited by oxygen and usually grow as well under an air atmosphere as they do in the absence of oxygen.

·         Microaerophilic bacteria these require low levels of oxygen for growth but cannot tolerate the level of oxygen present in an air atmosphere

3.      Acidity or Alkalinity (pH) – microbes that grow well in acid environments are known as acidophiles, those that favour more neutral conditions may be called neutrophiles, and those that favour more alkaline environments are alkalophiles. A last group that favours environments that are high in salt concentrations are called halophiles. Radical shifts in pH can be prevented by incorporating a buffer (i.e. a substance that resists change in pH) into the medium. A buffer is a mixture of a weak acid and its conjugate base (e.g. acetic acid (CH₃COOH) and an acetate (CH₃COO ^-).Such mixtures have maximum buffering capacity at the pH where the concentration of the acid equals that of its conjugate base

.

4.      Light – some phototrophic bacteria must be exposed to a source of light for illumination, since light is their source of energy.

5.      Hydrostatic pressure – bacterial growth may be influenced by hydrostatic pressure. Microbes obtain almost all their nutrients in solution from the surrounding water. They therefore require water for growth and are actually about 80 – 90% water. High osmotic pressures have the effect of removing necessary water from a cell.

CULTURE MEDIA

A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture medium. Some bacteria can grow well on just about any culture medium; others require special media, and others cannot grow on any non-living medium yet developed. The microbes that grow and multiply in or on a culture medium are referred to as a culture. 

Requirements of a culture medium

1.      It must contain the right nutrients for the particular microorganism we want to grow

2.      It should contain sufficient moisture, a properly adjusted pH, and a suitable level of oxygen, or perhaps none at all.

3.      The medium must initially be sterile – i.e., it must initially contain no living microorganisms – so that the culture will contain only the microorganisms (and their offspring) we add to the medium.

4.      Finally, the growing culture should be incubated at the proper temperature.

A wide variety of media are available for the growth of microorganisms in the laboratory. Most of these media, which are available from commercial sources, have pre-mixed components and require only the addition of water and sterilization.

When it is desirable to grow bacteria on a solid medium, a solidifying agent such as agar is added to the medium. Agar is a complex polysaccharide derived from a marine alga, and it has long been used as a thickener in foods such as jellies, soups, and ice cream. 

Important properties of agar that make it valuable in microbiology

·         Few microbes can degrade agar, so it remains solid.

·         Agar melts at about the boiling point of water but remains liquid until the temperature drops to about 40^oC. For laboratory use, agar is held in water baths at about 50^oC. At this temperature, it does not injure most bacteria when it is poured over a bacterial inoculum.

·         Once the agar has solidified, it can be incubated at temperature approaching 100^oC before it liquefies; this property is useful when thermophilic bacteria are being grown.

·         Agar media are usually contained in test-tubes or Petri-dishes. The test tubes are called slants when they are allowed to solidify with the tube held at an angle so that a large surface for growth is available. When the agar solidifies in a vertical tube, it is called a deep. Petri-plates named for their inventor, are shallow dishes with a lid that nests over the bottom to prevent contamination. 

Types of culture media

To be appropriately prepared for microbial growth, a medium must provide an 

energy source as well as sources of carbon, nitrogen, sulfur, phosphorus, and any

necessary growth factors that the organism is unable to synthesize.

1.      Chemically defined Medium – this is a medium whose exact chemical composition is known. Organisms that require many growth factors are described as fastidious. Chemically defined media are usually reserved for laboratory experimental work or for the growth of autotrophic bacteria.

2.      Complex Media – these are media whose exact chemical composition varies slightly from batch to batch. These complex media are made up of nutrients such as extracts from yeasts, meat, or plants, or digests of proteins from these and other sources. Most heterotrophs are routinely grown on complex media. In complex media, the energy, carbon, nitrogen, and sulfur requirements of the growing microorganism are met largely by protein. Vitamins and other organic growth factors are provided by meat extracts or yeast extracts. If a complex medium is in liquid form, it is called nutrient broth. When agar is added, it is called nutrient agar.

3.       Selective and Differential Media – selective media are designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes. For example, dyes such as brilliant green selectively inhibit gram-positive bacteria, and this dye is the basis of a medium called Brilliant Green agar that is used to isolate the gram-negative Salmonella.

Differential media make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. Similarly, pure cultures of microorganisms have identifiable reactions with differential media in tubes or plates. Sometimes, selective and differential media are used together; e.g., MacConkey agar.

4.      Enrichment Culture – Because bacteria present in small numbers can be missed, especially if other bacteria are present in much larger numbers, it is sometimes necessary to use an enrichment culture. This is often the case for soil or feacal samples.

The medium for an enrichment culture is usually liquid and provides nutrients and environmental conditions that favour the growth of a particular microbe but are not suitable for the growth of other types of microbes. In this sense, it is designed to increase very small numbers of the desired type of microbe to detectable levels.  

PURE CULTURES

A pure culture, sometimes called axenic culture refers to the growth of a single type of microbe in an environment free of any other kind of living thing. Many microbiologists prefer to use the term “axenic” in order to avoid the misunderstanding that pure cultures are genetically pure. It is a population of microbes of the same species but may contain some individuals with mutations.

Aseptic transfer technique

The aseptic technique (aseptic means free from sepsis and not liable to microbial putrefaction) is required to transfer the pure culture from one vessel to another.

Besides preventing contamination of pure cultures with unwanted microorganisms, proper aseptic technique also protects the microbiologist from contamination with the culture, which should always be treated as a potential pathogen.

Aseptic technique involves avoiding any contact of the pure culture, sterile medium, and sterile surfaces of the growth vessel with contaminating microorganisms. To accomplish this task;

1.      the work area is cleansed with a disinfectant to reduce the number of potential contaminants;

2.      the transfer instruments are sterilized, e.g., by heating a transfer loop in a Bunsen burner flame before and after transferring;

3.      the work is accomplished quickly and efficiently to minimize the time of exposure during which contamination of the culture or laboratory worker can occur.

The normal steps for transferring a culture from one vessel to another are as follows;

            a .flame the transfer loop

            b. open and flame the mouths of the culture tubes

            c. pick up some of the culture growth and transfer it to the fresh medium

            d. flame the mouths of the culture vessels and reseal them

            e. reflame the inoculating loop.

METHODS FOR ISOLATING PURE CULTURES

Several different methods are used for the establishment of pure cultures of microorganisms;

1.      The streak plate method – In this procedure a loopful of bacterial cells is diluted by drawing it across a medium until only single cells are deposited at a given location. The growth of each isolated cell results in the formation of a discrete colony.

The key principle of this method is that, by streaking a dilution gradient is established across the face of the plate as bacterial cells are deposited on the agar surface. The streaking can be done in different patterns.

2.      The spread plate method – this method involves;

·        Aseptically applying a known volume of suspension to a suitable solid medium,

·        Sterilize a spreading rod by dipping in alcohol and flaming,

·        Use a sterile rod to spread suspension over the surface of the medium,

·        Incubate,

·        Observe the colonies.

3.      The pour plate technique – In this method, a known volume of a microbial suspension is mixed with liquefied agar medium and poured into a Petri-plate; after incubation the numbers of colonies that develop are noted.  

MAINTENANCE OF PURE CULTURES

Once a microorganism has been isolated and grown in pure culture, it is necessary to maintain the viable culture, free of contamination, for some period of time. There are several methods;

1.      Periodic subculturing.

2.      Refrigeration at 0 – 5^oC for short storage times.

3.      Freezing in liquid nitrogen at -196^oC for prolonged storage.

4.      Lyophilization (also called freeze-drying) to dehydrate the cells.

By sufficiently lowering the temperature or by removing water microbial growth is precluded but viability in a dormant state is maintained, permitting preservation of microorganisms for extended periods.

MICROBIAL CONTROL

Control refers to the reduction in numbers and/or activity of the total microbial flora. The principal reasons for controlling microorganisms are;

(i). to prevent transmission of disease and infection,

(ii). to prevent contamination by or growth of undesirable microorganisms, and

(iii). to prevent deterioration and spoilage of materials by microorganisms.

TERMINOLOGY OF MICROBIAL CONTROL

·         Terms related to destruction of organisms

1.      Antimicrobial agent – Anything that kills or interferes with the multiplication, growth, or activity of microorganisms. The term usually refers to chemical agents such as disinfectants and antibiotics but may also be used to describe such physical agents of control as ultraviolet (UV) or intense heat.

2.      Disinfection – the process of destroying vegetative pathogens but not necessarily endospores or viruses. Usually, a disinfectant is a chemical applied to an object or a material. Disinfectants tend to reduce or inhibit growth; they usually do not sterilize. This term is usually applied to use of liquid chemical solution on surfaces, or to elimination of pathogens in water; e.g. chlorination.

3.      Sterilization – the process of destroying all forms of microbial life on an object or in a material. This includes the destruction of endospores – the most resistant form of microbial life. Sterilization is absolute; there are no degrees of sterilization

-          Moist heat, 121^oC for 15 minutes

-          Dry heat, 170^oC for 120 minutes.

4.      Antiseptics – is the chemical disinfection of the skin, mucous membranes, or other living things. This term is applied to treatment of wounds. Antisepsis is a specific kind of disinfection.

5.      Germicide – (cide= kill) is a chemical agent that rapidly kills microbes but not necessarily their endospores. A bactericide kills bacteria; a sporicide kills endospores; a fungicide kills fungi; a virucide kills viruses, and an amoebicide kills amoeba.

6.      Antibiotic – a chemical produced by a microbe that is able to kill or inhibit the growth or activity of another microbe. These chemicals should be nontoxic to the host, work at very low concentrations, and be nonantigenic.

·         Terms related to suppression of organisms

1.      Bacteriostasis – a condition in which bacterial growth and multiplication are inhibited, but the bacteria are not killed. If the bacteriostatic agent is removed, bacterial growth and multiplication may resume. Fungistasis refers to the inhibition of fungal growth. Refrigeration is bacteriostatic; many chemicals such as dyes are bacteriostatic rather than bactericidal.

2.      Asepsis – (asepsis= without infection) is the absence of pathogens from an object or area. Aseptic techniques are designed to prevent the entry of pathogens into the body. Whereas surgical asepsis is designed to exclude all microbes, medical asepsis is designed to exclude microbes associated with communicable diseases.

Air infiltration; ultraviolet lights; personnel masks, gloves and gowns; and instrument sterilization are all factors in achieving asepsis.

3.      Degerming – is the removal of transient microbes from the skin by mechanical cleansing or by the use of antiseptic. For routine injections, alcohol swabs are often used; before surgery, iodine-containing products are often used.

4.      Sanitization – the reduction of pathogens on eating utensils to safe public health levels by mechanical cleansing or chemicals. Any chemicals used must be compatible with safety and palatability of foods.

Conditions influencing microbial control

          Many biological characteristics influence the rate at which microorganisms are

          killed or inactivated by various agents. These include;

1.      Environment – the physical or chemical properties of the medium or substance carrying the organisms, i.e., the environment, has profound influence on the rate as well as the efficacy of microbial destruction. For example, the effectiveness of heat is much greater in acid than in alkaline material. The consistency of the material (aqueous or viscous) will markedly influence the penetration of the agent, and high concentrations of carbohydrates generally increase the thermal resistance in organisms.

The presence of extraneous organic matter can significantly reduce the efficacy of an antimicrobial agent by inactivating it or protecting the microorganisms from it.

2.      Types of microbe – species of microbes differ in their susceptibility to physical and chemical agents. In spore-forming species, the growing vegetative cells are much more susceptible than the spore forms; bacterial spores are extremely resistant. In fact bacterial spores are the most resistant of all living organisms in their capacity to survive under adverse physical and chemical conditions.

3.      Physiological state of the microbe – the physiological state of cells may influence the susceptibility of an antimicrobial agent. Young actively metabolizing cells are apt to be more easily destroyed than old, dormant cells in the case of an agent that causes damage through interference with metabolism; non-growing cells would not be affected.

Mode of action of antimicrobial agents

The manner in which antimicrobial agents inhibit or kill can be attributed to the following kinds of actions;

a.      Damage to the cell wall or inhibition of cell wall synthesis – the cell wall proper provides a protective covering to the cell in addition to participating in certain physiological processes. Damage at any of these areas may initiate a number of subsequent changes leading to cell death.

b.      Alteration of the permeability of the cytoplasmic membrane – the plasma membrane, located just beneath the cell wall, is the target of many microbial control agents. This membrane actively controls the passage of nutrients into the cell and the elimination of wastes from the cell. Damage to the lipids or proteins of the plasma membrane by antimicrobial agents, typically causes cellular contents to leak into the surrounding medium and interferes with the growth of the cell. Several types of chemical agents and antibiotics work at least in this manner.

c.      Damage to proteins and nucleic acids – microbial cells have enzymes, which are primarily protein and are vital to cellular activities. Functional proteins are due to their three-dimensional shape. This shape is maintained by chemical bonds that link adjoining portions of the amino acid chain as it folds back and forth upon itself. Some of those bonds are hydrogen bonds, which are susceptible to breakage by heat or certain chemicals; breakage results in denaturation of proteins.

Because DNA and RNA carry the genetic message, damage to these nucleic acids by radiation or chemicals is frequently lethal to the cell; this damage prevents both replication and normal functioning.

d.     Inhibition of enzyme action – this also acts by interference with metabolic pathways.

e.      Coagulation of cell contents – this destroys cells when internal contents separate into liquid portion and an insoluble mass. This process also causes denaturation of cell proteins and nucleic acids.

f.       Oxidation – this is the breakdown of large molecules into smaller ones by the loss of electrons.

METHODS OF MICROBIAL CONTROL

A.   Physical methods of microbial control

The major physical agents or processes used for the control of microbes are

·         Temperature (high or low)

·         Desiccation

·         Osmotic pressure

·         Radiation

·         Filtration

a.      High temperatures – Every type of organism has optimum, minimum, and

maximum growth temperature.  Temperature above the maximum generally kill, while those below the minimum usually produce stasis (inhibition of metabolism) and may even be considered preservative. High temperatures combined with high moisture are one of the most effective methods of killing microorganisms. There is dry heat and moist heat procedures of microbial control.

Moist heat kills microorganism by coagulating their proteins and is much more rapid and effective than dry heat, which destroys microorganisms by oxidizing their chemical constituents.

Thus, procedures by which heat is employed are divided into two categories;

a.             Moist heat

b.             Dry heat

Moist heat – these include:

i.                   Steam under pressure – this uses steam under pressure for sterilization. This provides temperatures above those obtainable by boiling. This has the advantages of rapid heating, penetration, and moisture in abundance, which facilitates the coagulation of proteins. In the laboratory, this is achieved by an autoclave.

ii.                 Fractional sterilization- For those microbiological media, solutions of chemicals, and biological materials that cannot be heated above 100^oC without being damaged, but can however withstand temperature of free-flowing steam (100^oC), it is possible to sterilize them by fractional sterilization (Tyndallization). This method involves heating the material at 100^oC on three successive days with incubation periods in between. Resistant spores germinate during the incubation periods; on subsequent exposure to heat, the vegetative cells will be destroyed.

iii.              Boiling water – The practice of exposing instruments for short periods of time in boiling water is more likely to bring about disinfection (destruction of vegetative cells of microbes) rather than sterilization. Boiling water cannot be (and is not) used in the laboratory as a method of sterilization.

iv.               Pasteurization – Milk, Cream, and certain alcoholic beverages (beer and wine) are subjected to a controlled heat treatment (called pasteurization) which kills microorganisms of certain types but does not destroy all organisms. Pasteurized milk is not sterile milk. 

Dry heat – these include:

i.                   Hot-air sterilization – Dry-heat, or hot-air sterilization is recommended where it is either undesirable or unlikely that steam under pressure will make direct and complete contact with the materials to be sterilized. This is used in laboratory items like glassware, such as Petri-dishes and pipettes, as well as oils, powders and similar substances.

This may be achieved by a special electric or gas oven or even the kitchen store oven. For laboratory glassware, a 2 –hour exposure to a temperature of 160^oC is sufficient for sterilization.

ii.               Incineration – Destruction of microorganisms by burning is practiced routinely in the laboratory when the transfer needle is introduced into the flame of the Bunsen burner.

Incineration is used for the destruction of carcasses, infected laboratory animals, and other infected materials to be disposed of.

b. Low temperatures – Temperatures below the optimum for growth depress the rate of metabolism, and if the temperature is sufficiently low, growth and metabolism cease. Low temperatures are useful for preservation of cultures, since microbes have a unique capacity for surviving extreme cold.

Agar-slant cultures of some bacteria, yeasts and molds are customarily stored for long periods of time at refrigeration temperatures of about 4 – 7^oC or liquid nitrogen at -196oC. Low temperatures are merely microbiostatic whereas high temperatures are microbicidal.

Drying – Since chemical reactions of microbes are basically carried out in water, drying cells by removing water from the environment will greatly inhibit their action. Many foods are dried to prevent spoilage by microbial action. Drying may be bacteriostatic or bactericidal.

In the process of lyophilization, organisms are subjected to extreme dehydration in the frozen state and then sealed in a vacuum. In this condition, desiccated (lyophilized) cultures of microbes remain viable for many years.

Radiation – Energy transmitted through space in a variety of forms is generally called radiation. The electromagnetic spectrum contains three ranges of antimicrobial radiation:

-          Ionizing radiation – which include the X-rays and gamma radiation. These have extremely short wavelengths and produce highly reactive units, called free radicals (H and OH). The free radicals quickly combine with proteins and DNA to cause death.

-          Ultraviolet radiation in the range of 265 – 280 nm is microbicidal. This wavelength does not penetrate glass or plastic but is absorbed by the proteins and nucleic acids of microbes. These molecules are easily destroyed when they absorb this energy.

Uv light is used to control microbes on the surfaces of transfer cabinets, hair brushes, and operating tables. However, Uv light must be carefully controlled since it may cause blindness and skin cancer.

Filtration – this uses a variety of filters that can remove microbes from liquids or gases. The mean pore diameter of filters ranges from one to several micrometers. They are available in several grades.

Filters do not act as mere mechanical sieves; porosity alone is not the only factor preventing the passage of organisms. Other factors, such as electric charge on the filter, the electric charge carried by organisms, and the nature of the fluid being filtered can influence the efficiency of filtration. 

Sound – Ultrasonic sound may be used to destroy microbes. Although sound at this frequency cannot be heard, it is able to coagulate cell proteins and disintegrate cell components. This method of control may be used in research to cut microbes to pieces and isolate their internal components. It may also be used in the hospital in the form of an ultrasonic “bath” for cleaning organic materials from instruments making them easier to disinfect or sterilize.

B.     Control by chemical agents –A large number of chemical compounds have the ability to inhibit the growth and metabolism of microorganisms or kill them. The major antimicrobial agents are:

i.             Alcohols

ii.           Halogens

iii.         Phenol and phenolic compounds

iv.        Heavy metals and their compounds

v.          Dyes

vi.        Aldehydes

vii.      Oxidizing agents

viii.    Detergents

ix.        Gaseous agents

Alcohol – Ethyl alcohol (CH₃CH₂OH), in concentrations between 50 – 90%, is effective against vegetative or non-spore forming cells. For practical application a 70% concentration of alcohol is generally used. Alcohols are protein denaturants, and this property may, to a large extent account for their antimicrobial activity. 

Alcohols are also lipid solvents, and hence they may damage lipid complexes in the cell membrane. They are also dehydrating agents.

Phenol – Phenol or carbolic acid is a powerful disinfectant when used in a 5% solution. It is virucidal, bactericidal, and fungicidal. It operates on cells to destroy proteins, cell membrane and other cell components. It also inactivates enzymes and causes leakage of amino acids from cells.

Heavy metals – Most of the heavy metals, either alone or in certain compounds, exert a detrimental effect upon microorganisms. Heavy metals and their compounds act antimicrobically by combining with cellular proteins and inactivating them.

Two of the most commonly used heavy metals as antimicrobials are silver and mercury. The metals may be bacteriostatic, bactericidal, and fungicidal. Mercury may be used in inorganic compounds (e.g. mercuric chloride, mercuric oxide) which are very corrosive, toxic to animals. 

In the case of mercuric chloride the inhibition is directed at enzymes which contain the sulfhydryl grouping.

High concentrations of salts of heavy metals like mercury, copper, and silver coagulate cytoplasmic proteins, resulting in damage or death to the cell. 

Salts of heavy metals are also precipitants and high concentrations of such salts could cause cell death.

Dyes – Many of the dyes used for staining are antimicrobial. Crystal violet is added to media as a bacteriostatic agent since it is able to inhibit Gram-positive bacteria. A chemical relative called gentian violet is also antifungal and has been used in the treatment of fungal infections of the mouth; e.g., thrush, caused by Candida albicans.

Oxidizing agents – Several agents control microbes by either releasing oxygen themselves, stimulating the release of oxygen from other molecules, or directly oxidizing cells and cell components.

These include iodine, iodophores, chlorine and its derivatives, hydrogen peroxide, and potassium permanganate.

Chlorine gas (Cl₂) is used regularly to kill microbes in water purification and sewage treatment plants. Sodium hypochlorite (NaOCl) is the form of chlorine found in household products. Sodium hypochlorite is bactericidal and active against most viruses.

Aldehydes – These are the class of chemicals with the general formula RCHO (aldehydes). Two of the most effective are formaldehyde and glutaraldehyde. Both are highly microbicidal, and both have the ability to kill spores (sporicidal). Formaldehyde is an extremely reactive chemical. It combines readily with vital organic compounds such as proteins and nucleic acids. It is likely that interaction of formaldehyde with these cellular substances accounts for its antimicrobial action.

Glutaraldehyde is effective against vegetative bacteria, fungi, bacterial and fungal spores, and viruses.

Detergents – Surface-tension depressants, or wetting agents, used primarily for cleansing surfaces are called detergents; e.g., soap. However, soap is a poor detergent in hard water; hence more efficient cleaning agents have been developed, called surfactants or synthetic detergents, many of which are superior to soap. These are extensively used in the laundry and dishwashing powders, shampoos and other washing preparations. Some are highly bactericidal.

Halogens – This uses iodine which has effective germicidal effect. It is an oxidizing agent and this may account for its antimicrobial action.

C.    Antimicrobial drugs – The treatment of a disease with a chemical substance is known as chemotherapy; the chemical substance is called a chemotherapeutic agent. A satisfactory chemotherapeutic agent must;

1.      Destroy or prevent the activity of a parasite without injuring the cells of the host or with only minor injury to its cells

2.      Be able to come into contact with the parasite by penetrating the cells and tissues of the host in effective concentrations

3.      Leave unaltered the host’s natural defense mechanisms, such as phagocytosis and the production of antibodies. 

Chemotherapeutic agents that are isolated from cultures of microbes are called antibiotics. To be useful as chemotherapeutic agents antibiotics must have the following qualities;

·        They should have the ability to destroy or inhibit many different species of pathogenic microorganisms. This is referred to as a “broad spectrum” antibiotic.

·        They should prevent the ready development of resistant forms of the parasite.

·        They should not produce undesirable side effects in the host, such as sensitivity or allergic reactions, nerve damage, or irritation of the kidneys and gastrointestinal tract.

·        They should not eliminate the normal microbial flora of the host, because doing so may upset the “balance of nature” and permit normally non-pathogenic microbes, or particularly pathogenic forms normally restrained by the usual flora, to establish a new infection.

Most antibiotics demonstrate some form of host damage if used at high levels or for prolonged periods. The best antibiotics are those that result in the fewest and mildest side effects. Examples of antibiotics are Penicillin produced by Penicillium notatum and chrysogenum (these are effective against gram positive bacteria like Staphylococcus, Streptococcus).

Antibiotics taken orally may have their effectiveness reduced or inhibited by many factors e.g., Penicillin G and Erythromycin are promptly inactivated by acid liquids such as ginger ale and lemon juice. Milk and dairy products interfere with absorption of some tetracycline antibiotics because they combine with or absorb the drugs and prevent them from entering the circulatory system.

Development of drug resistance

All microorganisms will experience a change in the gene frequency of their population over generations of time as a result of environmental change. Antibiotics act as agents of natural selection favouring those cells that have the genetic ability to withstand the effects of the drug. Those that survive become the parents of new populations of drug-resistant bacteria. The development of resistance can only be slowed, not stopped because evolution is characteristic of life.  

Microorganisms can become resistant to antibiotics in four ways;

·         They may stop producing the drug sensitive structure

·         They can modify the sensitive structure

·         They can become impermeable to the drug, or

·         They can release enzymes that inactivate the antibiotic.

Classification of antibiotics

Antibiotics can be classified in several ways, e.g., some are bactericidal and others are bacteriostatic. They may be classified on the basis of their mode of action, i.e., the manner in which they manifest their damage upon microbial cells.

On their mode of action, the major points of attack of antibiotics on microorganisms include;

·         Inhibition of cell-wall synthesis – Among the antibiotics whose antimicrobial activity is expressed by inhibition of the biosynthesis of the peptidoglycan cell-wall structure are the penicillins, cephalosporins, cycloserine, vancomycin, and bacitracin.

·         Damage to the cytoplasmic membrane – Several polypeptide antibiotics produced by Bacillus species have the ability to damage cell membrane structure. They adversely affect the normal permeability characteristics of the cell membrane. Included in this category are the polymyxins, gramicidins, and tyrocidines.

·         Inhibition of nucleic acid and protein synthesis – These antibiotics interfere with the synthesis of nucleic acids and proteins. These include Streptomycin, Tetracyclines, Chloramphenicol, and Erythromycin.

Biological control agents

Biological control of pathogens, i.e., total or partial destruction of pathogen population by other organisms, occurs routinely in nature. For example, there are several diseases in which the pathogen cannot develop in certain areas either because the soil, called suppressive soil, contains microorganisms antagonistic to the pathogen, or the antagonist may be avirulent strains of the same pathogen e.g., in cross protection.. The antagonist may

·         reduce  the inoculum density (by destroying propagules or preventing their formation),

·         directly kill them,

·         displace the pathogen from the host,

·         suppress germination and growth,

·         produce antibiotics or other toxic chemicals,

·         stimulate host resistance,

·         offer competition.

Examples include

i.             Trichoderma harzianum – control soilborne pathogens

ii.         Agrobacterium radiobacter – control crown gall, caused by A. tumefasciens

iii.       Pseudomonas fluorescens – against Rhizoctonia and Pythium

iv.        Bacillus subtilis – control damping off

ECONOMIC IMPORTANCE OF MICROORGANISMS

Food and Beverage Production - A variety of important foods in our diets are produced with the aid of microbial activity. In the dairy industry, fermented milks are produced by inoculating pasteurized milk with a known culture of microorganisms, sometimes referred to as a starter culture, which can be relied on to produce the desired fermentation, thus assuring a uniformly good product.  

·         Butter is made by churning cream until the fatty globules of butter separate from the liquid buttermilk. The typical flavour and aroma of butter and buttermilk are from diacetyls (a combination of two acetic molecules) that is a metabolic end-product of fermentation by some lactic acid bacteria (Streptococcus lactis and Leuconostoc spp.).

-          Buttermilk is made by inoculating skim milk with bacteria that form lactic acid and diecetyls. The inoculation is allowed to grow for 12 hrs or more before the buttermilk is cooled and packaged.

-          Sour cream is made from cream inoculated with organisms similar to those used to make buttermilk.

·         Yogurt is made from low-fat milk, from which much of the water has been evaporated in a vacuum pan. The resulting thickened milk is inoculated with a mixed culture of Streptococcus thermophilus (for acid production) and Lactobacillus bulgaricus to contribute the flavour and aroma.

-          The temperature of the fermentation is about 45^oC for several hours, during which time S. thermophilus outgrows L. bulgaricus. Maintaining the proper balance between the flavour-producing and the acid-producing organisms is the secret of making yogurt.

·          Several varieties of cheese are manufactured by the formation of a curd, which can then be separated from the main liquid fraction, or whey. The curd is made up of a protein, casein, and is usually formed by the action of an enzyme, rennin (or chymosin), which is aided by acidic conditions provided by certain lactic acid producing bacteria. These inoculated lactic acid bacteria also provide the characteristic flavours and aromas of fermented dairy products during the ripening process. The curd may undergo a microbial ripening process, except for a few unripened cheeses.

            Cheeses are generally classified as hard or soft cheeses;

-          The hard cheddar and Swiss cheeses are ripened by lactic acid bacteria growing anaerobically in the interior. Such hard, interior ripened cheeses can be quite large. The longer the incubation time, the higher the acidity and the sharper the taste of the cheese. A propionibacterium species produces CO₂ which forms the holes in Swiss cheese.

Food spoilage – Microbial growth can render a product unacceptable in three ways;

1.      The food may become spoiled as a result of enzymatic decomposition of the product, an increase in microbial numbers, or the release of compounds that result in unappetizing flavours, colours, and aromas.

2.      They can release toxins into the food during their growth. Ingestion of those toxins results in a brief but violent illness only a few hours after the chemical agents contact susceptible tissues. Such a food-borne illness is known as food poisoning or food intoxication.

3.      Foods may become unsuitable for consumption because of the presence of viable pathogens that can establish an infection after they have been ingested. That is, the food carries the pathogens into the host where they reproduce and cause disease. Outbreaks of that type of food-borne disease are known as food infections. These include;

a.       Staphylococcal food poisoning – Certain strains of Staphylococus aureus are able to release an exotoxin known as enterotoxin while growing in certain foods. This toxin shows symptoms in 2 – 4 hours but may be as long as 6 hours. Ingestion of the toxin results in an abrupt onset of an illness characterized by servere nausea, cramps, vomiting, diarrhea, etc. This can be controlled by practicing good personal hygiene.

b.       Bacillus cereus food poisoning – B. cereus has been identified as a cause of a staphylococcal –like food poisoning. These spore-forming bacteria may be transmitted in cereals, cereal products, re-heated fried rice, potatoes, vegetables, and meats. The clinical symptoms include vomiting, diarrhea, and abdominal cramping, but no fever.

c.        Botulism food poisoning – Clostridium botulinum is an anaerobic bacterium responsible for the food poisoning called botulism. This organism produces an exotoxin which has the most potent neurotoxins. Poisoning by this microorganism may be prevented by establishing such conditions or by boiling suspect foods for 15 – 30 minutes, since the toxin is heat liable. Symptoms of poisoning appear in 12 – 36 hours to several days. The victim progressively experience a swollen tongue, difficulty in swallowing and speaking, double vision, dizziness, acute nausea, vomiting, diarrhea, fatigue, and respiratory failure.

d.       Mycotoxins – Poisoning can result from the ingestion of fungi or their toxic products called mycotoxins. Several species of mushrooms are known to be highly toxic. Molds have the ability to produce mycotoxins. One of the most important mycotoxins, called aflatoxin is produced by members of the genus Aspergillus. This toxin is extremely heat stable and can withstand autoclaving. Aspergillus spp. commonly grows on seed crops such as peanuts, cottonseeds, cornmeal, oats and soybean. Aflatoxin is a source of fatal poisoning of turkeys, fish, and sheep fed on moldy grain; aflatoxins may also be responsible for cases of human intoxication. Aflatoxins have been found in milk from cows that were fed aflatoxin-containing grain and in human breast milk.