MICROBIOLOGY Introduction Microbiology should be an easy word

MICROBIOLOGY Introduction: ‘Microbiology’ should be an easy word to define: the science (logy) of small (micro) life (bios), or to put it another way, the study of living things so small that they cannot be seen with the naked eye. Microbiology is the science that deals with the study of micro-organisms, their activates and their influences on different aspects of life.

The following list of some of the general areas in which the expertise of a microbiologist might be used: 1 -Medical laboratory Sciences 2 - Medicine 3 - Environmental science 4 - Food and drink production 5 - Fundamental research 6 - Agriculture 7 - Pharmaceutical industry 8 - Genetic engineering.

Microbiology comprises many disciplines, that are categorized on either taxonomic or functional bases: (1) Taxonomic disciplines of microbiology are: ØBacteriology (Study of bacteria) ØMycology (Study of fungi) ØVirology (Study of viruses) ØParasitology ( Study of parasite) (now in separated section)

(2) Functional disciplines of microbiology are: ØImmunology (host reaction to micro-organisms) ØMedical Microbiology (role of organism in infectious disease) ØGenetic engineering

Historical background: • WE NEED TO KNOW OUR PAST SO WE KNOW THE DIRECTION THAT WE NEED TO FOLLOW IN THE FUTURE. • Microorganisms had been on the Earth for some 4000 million years, when Antoni van Leeuwenhoek started out on his pioneering microscope work in 1673. Leeuwenhoek was an amateur scientist who spent much of his spare time grinding glass lenses to produce simple microscopes. • the Italian Francesco Redi (1626– 1697) showed that the larvae found on putrefying meat arose from eggs deposited by flies, and not spontaneously as a result of the decay process. This can be seen as the beginning of the end for the spontaneous generation theory, • Pasteur noticed that when lactic acid was produced in wine instead of alcohol, rod-shaped bacteria were always present, as well as the expected yeast cells. This led him to believe that while the yeast produced the alcohol, the bacteria were responsible for the spoilage (swan-necked flasks).

• The first proof of the involvement of bacteria in disease and the definitive proof of the germ theory of disease came from the German Robert Koch. In 1876 Koch showed the relationship between the cattle disease anthrax and a bacillus which we now know as Bacillus anthracis. • These results led Koch to formalise the criteria necessary to prove a causal relationship between a specific disease condition and a particular microorganism. These criteria became known as Koch’s postulates, and are still in use today.

Koch’s postulates: (1) The microorganism must be present in every instance of the disease and absent from healthy individuals. (2) The microorganism must be capable of being isolated and grown in pure culture. (3) When the microorganism is inoculated into a healthy host, the same disease condition must result. (4) The same microorganism must be re-isolated from the experimentally infected host.

• Table (1) The discovery of some major human pathogens • Year Disease Causative agent Discoverer • 1876 Anthrax Bacillus anthracis Koch • 1879 Gonorrhoea Neisseria gonorrhoeae Neisser • 1880 Typhoid fever Salmonella typhi Gaffky • 1880 Malaria Plasmodium sp Laveran • 1882 Tuberculosis Mycobacterium tuberculosis Koch • 1883 Cholera Vibrio cholerae Koch • 1883/4 Diphtheria Corynebacterium diphtheriae Klebs & Loeffler • 1885 Tetanus Clostridium tetani Nicoaier & Kitasato • Year Disease Causative agent Discoverer • 1886 Pneumonia (bacterial) Streptococcus pneumoniae Fraenkel • 1892 Gas gangrene Clostridium perfringens Welch & Nuttall • 1894 Plague Yersinia pestis Kitasato & Yersin • 1896 Botulism Clostridium botulinum Van Ermengem • 1898 Dysentery Shigella dysenteriae Shiga • 1901 Yellow fever Flavivirus Reed • 1905 Syphilis Treponema pallidum Schaudinn & Hoffman • 1906 Whooping cough Bordetella pertussis Bordet & Gengou • 1909 Rocky Mountain spotted fever Rickettsia rickettsii Ricketts

Leeuwenhoek’s microscope

The lens (a) was held between two brass plates and used to view the specimen, which was placed on the mounting pin (b). Focusing was achieved by means of two screws (c) and (d).

Pioneers in Microbiology: • Aristotle (384– 322 BC) believed that life arose from muck, decaying food, warm rain, or even dirty shirts. • Antony van Leeuwenhoek (1632– 1723) used a primitive microscope to observe stagnant water, hay infusions, and scrapings from the teeth. • John Tyndall (1820– 1893) discovered that some bacteria existed in two forms: a heat stable form and a heat-sensitive form. THE GOLDEN AGE OF MICROBIIOLOGY • Mid to late 1800’s HISTORY CONT. • Louis Pasteur – France – Fermentation—with the final result being pasteurization. • Heating wine and milk to 145. 5 degrees to kill bacteria. Without killing this bacteria, both the wine and the milk would sour. Louis Pasteur cont. – Developed immunizations for anthrax in cattle and rabies in humans.

• Robert Koch – Koch’s Postulates • An organism must be present in ALL cases of the disease. • The organism must be isolated in pure culture. • The organism can be removed from the pure culture and placed in another human or experimental animal and the same symptoms are developed. • The organism can be removed from this new person and grown again in pure culture. • His postulates prove that certain organism cause certain diseases. • Ignaz Semmelweis—Vienna and Oliver Wendell Holmes—America – Discover the importance of hand washing prior to surgery and then between patients.

Other Introduction: The word Microbiology is a Greek one: It is the study of very small organisms (Microorganisms) that can not be seen by naked eye but by Microscope. Micro = very small bio = life logy = Science These microorganisms include four major groups: • Bacteria: studded under the name of Bacteriology. • Fungi: studded under the name of Mycology. • Viruses: studded under the name of Virology. • Protozoa: studded under the term of Parasitology & Protozoology.

Why do we study Microbiology? Because almost all diseases are caused by microorganisms. There is a classification for these major groups of microorganisms which can be distinguished on the bases of their structure and the complex of their organization. A- Prokaryotes: Which included Bacteria and blue green algae. Characteristics of prokaryotes: 1 - Very small in size. 2 - Lack of nuclear membrane. 3 - Lack of mitochondria. 4 - Multiplication is by binary fission. 5 - Has a single piece of genetic material known as plasmid.

B- Eukaryotes: Which included Fungi and Protozoa. Characteristics of eukaryotes: 1 - Large size. 2 - Lack of plasmid. 3 - Posses mitochondria. 4 - Multiply by sexual multiplication & others (mitosis). 5 - Posses nuclear membrane.

Classification of Micro-organisms: ØIn any discussion on biological classification, it is impossible to avoid mentioning Linnaeus, the Swedish botanist who attempted to bring order to the naming of living things by giving each type a Latin name. (Carl von Linn´e! It was Linnaeus) who was responsible for introducing the binomial system of nomenclature, by which each organism was assigned a genus and a species. ØThe science of taxonomy involves not just naming organisms, but grouping them with other organisms that share common properties.

ØIn the 20 th century, an increased focus on the cellular and molecular similarities and dissimilarities between organisms led to proposals for further refinements to the three-kingdom system. One of the most widely accepted of these has been the fivekingdom system proposed by Robert Whittaker in 1969. This took into account the fundamental difference in cell structure between procaryotes and eucaryotes, and so placed procaryotes (bacteria) in their own kingdom, the Monera, separate from single-celled eucaryotes. Another feature of Whittaker’s scheme was to assign the Fungi to their own kingdom, largely on account of their distinctive mode of nutrition.

ØThe agents of human infectious belong to five major groups of microorganisms ; bacteria, fungi, viruses protozoa and helminthes. ØBased upon cell characteristic, living organism are recognized as having Euocaryotic or procaryotic cell type. ØBacteria belong to the procaryotic, fungi and protozoa are Euocaryotic cell type. Viruses are classified by some scientist as kingdom viruses they are acellular (non- cellular)

Differences between Euocaryotes and procaryotes Euocaryotes procaryotes Size Larger Relatively small Structure of cell Complex Primitive DNA content Nucleus Nucleoid Nuclear membrane Present Absent Mitochondria Present Absent Multiplication Mitotic Binary fission Enzyme system Complex Simple Golgi bodies Present Absent Respiration Via Mitochondria Cytoplasmic membrane

The kingdom procaryote is further divided into division orders families genera species classes strain (subspecies) Practically, only the name of genus and species are used the generic (genus) name is always given a capital letter the specific (species) name is given a small letter the generic and specific name are italicised, or, if this isn’t possible, underlined.

Introduction to Bacteria form a large group of unicellular parasitic, saprophytic and free-living microorganism, varying in size from. 01 – 10 µm long. They have simple cell structure (procaryotic), contain both DNA &RNA and multiply by binary fission They are classified by their morphology, staining reaction, culture characteristic, biochemical reaction, antigenic structure and genetic composition using specialized molecular biology techniques.

Morphology of Microorganisms: Morphology of Bacteria: 1 - Cocci: round or spherical in shape; singular known as Coccus. It's diameter ranges between 0. 5 – 1 µm. When microorganism occurs as single it is known as Micrococcus monococcus = single cell. When it occurs in pairs, it is known as Diplococci e. g. Meningococci which cause meningitis. When it occurs in chain which can be short or long, it is known as Streptococci e. g. Streptococcus pyogenes which cause throat inflammation.

When it occurs in clusters it is known as Staphylococcus e. g. Staphylococcus aureus (golden colour). These arrangements are determined by the orientation and degree of attachment of the bacteria at the time of cell division.

2 - Rods: in Latin are known as Bacilli. Rod = Bacillus ; it has a stick like shape and the end may be round cut or square cut (Value of identification). When rod is single, it is known as bacillus or may be chain known as streptobacillus

Sometimes the rods may occur in chains but branching, some have the shape of Chinese letters and is known as club shaped e. g. Corynebacterium diphtheriae which causes diphtheria.

3 -Vibrios: It has a comma or curved shape ،، It is known as Vibrio and e. g. Vibrio cholerae which causes cholera. 4 - Spirillum and Spirochetes: have the shape of spiral e. g. Treponema palidum which causes syphilis.

Molecular Structure of Bacteria 1 - Cell Wall: is a component of bacterial cell which is outside the cell, it is a rigid organelle and also it gives the bacteria it's shape and also prevents expansion of cell membrane (protects the cell against osmotic pressure). The other name of the cell wall is known as peptidoglycan which is composed of: polysaccharides:

the main polysaccharides which are found in the composition are: N – acetyl glucose amine. N – acetyl muramic acid. and this peptidoglycan is only found in the cell wall of bacteria. It is a target for antibiotics to act on. Some antibiotics act on the cell wall e. g. penicillins and cephalosporins. This is known as selective toxicity.

1. Protoplast: is a G+ve bacteria with defective cell wall which is killed by lysozymes or antibiotic (penicillin). 2. Spheroplast: Is a G-ve bacteria which lost its cell wall completely or partially due to the action of lysozymes or antibiotic (penicillin). 3. L-forms: It may be a G+ve or G-ve bacteria loss their cell wall due to mutation. They are able to grow and divide or multiply and this is possible in a hypertonic or concentrated media or isotonic in order to retain its contents and do not release them. In this medium shrinking may happened but it does not affect. But in hypotonic media it will expand then explode.

2. Cell Membrane: It is very important structure in the bacterial cell, also called cytoplasmic membrane or sometimes protoplasmic membrane. Chemically cytoplasmic membrane is composed of bilayers of phospholipids. Basic functions of cell membrane are : 1 to control the movement of substances. 2 to secrete hydrolytic enzymes. 3 responsible for secretion of transport protein and proteins involved in cell wall synthesis.

3 - Cytoplasm: which contains: a. Genetic Material: a single piece of DNA without nuclear membrane. b. Ribosomes: which are known as protein making organelle. c. Mesosomes: Which are attached to the cell membrane and is thought that it is associated in cell division (binary fission).

Specialized products outside the cell wall 1 - Capsule: this is a layer of loose slime material which surrounds some bacterial cells. The capsules are composed of mainly of polysaccharides or peptides. They resist phagocytosis and so their presence on a bacterium is associated with virulence. They are identified by negative staining due to their low affinity for simple staining.

* e. g. of capsulated bacteria: Klebsiella pneumoniae (polysaccharide) Bacillus anthraces (poly – D – glutamic acid) * The capsule is used for rapid and accurate diagnosis where agglutination reaction happens between the antibodies and the antigen (capsular antigen).

2 - Flagella: These are filaments that originate from the cytoplasm. They function as organs of motility. They are therefore seen only in organisms that are motile. They are made of protein (Flagellin). They have characteristic patterns of arrangement on the bacterial cell: A) Monotrichate: one flagellum at one pole. b)Amphitrichate: one flagellum at each pole c) Lophotrichate: tuff flagella at one or both poles. d) Peritrichate: flagella completely surrounding the bacterial cell.

preparations, but become visible only in silver impregnated preparation or in electron microscopy. The functions of flagella are: 1. motility (mainly) 2. attachment to site of infection e. g. stomach. 3. Diversity of antigens, this is used for diagnosis and this is mainly in Salmonella typhi (the causative agent of typhoid fever). 4. Invasion. 5. Colonization (form colony).

3 - Pili: Extended from cytoplasmic membrane, they are shorter and finer filaments than the flagella. These are the pili. They are made of protein (pilin). Pili are divided into two groups: a. Sex pili, referred to as sex pili because of their role during conjugation when genes are transferred from one cell(donor) to another cell (recipient). b. Common pili, or Fimbriae: these are thought to be the organs of adhesion that help bacteria to attach to the host cells (Virulent Factor).

4 - Spores: These are dense structure produced by some bacteria, e. g. the Bacilli and Clostridia groups, that enable them to survive adverse environmental conditions. They develop within and at the expense of the vegetative cell. The spore comprises the chromosomal material surrounded by several walls layers. Chemically, endospore has a large amount of Ca++ and less number of enzymes. Dpiclonic acid is also presents in spore.

Spores are resistant to heat, stains, desiccation, chemicals and disinfectants. Each spore germinates to produce a vegetative cell during favorable conditions. The location and shape of the spore in the cell may be of diagnostic assistance, e. g. the spores of Clostridium tetani are terminal, and the diameter is greater than that of the parent cell, so that they are characteristically of drum stick appearance. The positions of the spores are described as: Terminal , Sub terminal , or Central The shapes of spores are described as: Ovoid (Oval) , Spherical , Cylindrical .

Classification of bacteria: 1 - phenotypic classification: The microscopic and macroscopic morphologies of bacteria were the first characteristic used to identify bacteria. The phenotypic classification include: * Microscopic morphology (cocci, bacilli, curved, spiral, spirochetes and Vibrio), also the ability to retain the gram stain into gram positive and gram negative. * Macroscopic morphology (appearance of colonies of bacteria on culture media (colonies size, shape, pigment production and reaction on media. * Biotyping (using biochemical test or reaction) *Serotyping (by detection bacterial antigens using serological test. * phage typing (susceptibility to viruses that infect bacteria)

2 - Analytic classification: using chromatographic pattern include: * Cell wall fatty acid analysis * Whole cell lipid analysis * Whole cell protein analysis * Multilocus enzyme electrophoresis 3 - Genotypic classification: the most precise method for classifying bacteria is by analysis by their genetic material that include: * Guanine plus cytosine ratio * DNA hybridization * Nucleic acid sequence analysis * plasmid analysis * chromosomal DNA fragments * Ribotyping

• Others useful classification: * According to their oxygen requirement into: 1 - Aerobic 2 - Anaerobic 3 - Facultative anaerobic 4 - Microaerophilic. * According to temperature into: 1 - Thermophilic 2 - Mesophilic 3 - Psychrophilic * According to their growth factors into: 1 - Fastidious organism (required special growth factor) 2 - non fastidious (simple)