Microbiology for the Health Sciences PDF

Microbiology for the Health Sciences  FIELDS OF Microbiology : 1- Medical microbiology: This field focuses on pathogens, diseases, and body defenses. Immunology, Virology, Bacteriology, Mycology, and Parasitology. 2.Industrial microbiology: This field focuses on the production of alcohol, enzym...

Microbiology for the Health Sciences  FIELDS OF Microbiology : 1- Medical microbiology: This field focuses on pathogens, diseases, and body defenses. Immunology, Virology, Bacteriology, Mycology, and Parasitology. 2.Industrial microbiology: This field focuses on the production of alcohol, enzymes, vitamins, and antibiotic. 3.Agricultural microbiology: This filed is concerned with Soil fertilization, nitrogen, carbon, sulfur, and phosphorous cycles, as well as plant disease. 4.Food microbiology: This field focuses on food poisoning, toxicity and spoilage. 5.Molecular microbiology: deals with molecular mechanisms and physiological processes of microbes and utilization in production of biotechnology products such as vaccines, and antibodies. 6- Sanitary microbiology: is a science based on the detection of risks associated with the production, manufacture and consumption of foods and water. It has been established that environment facts determine the survival, growing and inactivation of the microorganisms. 7- environmental microbiology: is the study of the composition and physiology of microbial communities in the environment.  Microbes are related to all life. – In all environments – Many beneficial aspects – Related to life processes (nutrient cycling) – Only a minority are pathogenic. – Most of our problems are caused by microbes Introduction to medical microbiology  MEDICAL MICROBIOLOGY Medical Microbiology: is a science of studying micro-organisms that are associated with human disease. Bacteria Fungus Parasites Viruses – Most can only be seen with the microscope! – Commonly called “germs, Smaller than 0.1mm ** There are four classes of organisms that can cause disease: 1- Viruses: Their size < 0.3 microns in diameter, they are totally dependent on infected cells for replication. They cause intracellular infection. 2- Bacteria: Usually measure about one micron or more, multiply by binary fission, and they can cause intercellular or extracellular infection. 3- Fungi, these can be of two varieties: a. Yeasts are unicellular organisms measuring (2-20) microns. b. Molds are large multicellular organisms. 4- Parasites: these can be of two classes: a- Protozoa, these are unicellular organisms that vary in size, some are very small (about 3 microns) and can cause intercellular infection. Others are large (80 microns) and cause extracellular infection. b- Helminthes, these are multicellular and can reach several meters in lengths.  Microbiology Micro means very small, viewed by microscope The biology comes from bios referring to living organisms and logy means the study of, thus biology is the study of living organisms. Microbiology is the study of very small living organisms called microorganisms or microbes, these include bacteria, algea, protozoa, fungi and viruses.  scientist do not consider viruses as living organisms the terms infectious agents or infectious particles are often used in reference to viruses A disease-causing microorganisms called pathogens (3% of all known microbes) The others are nonpathogenic (97%), the beneficial microbes are 87%  Microbes live on and in our bodies e.g. skin, in the mouth and intestine are known as indigenous microflora (or indigenous microbiota). Some of them cause disease accidentally and known as opportunistic pathogens (10%) Diseases caused by microbes are called infectious disease  Portal of entry Microorganisms that cause disease are said to be pathogenic. ⚫Respiratory: via inhalation. ⚫Alimentary (GIT): by ingestion. ⚫Genital tract: sexual contact. ⚫Skin: abrasions, bites… ⚫Others: Conjunctiva, blood transfusion, injections and organ transplants. ⚫Congenital infections ( vertical transmission). Infection with microorganisms can be  Many bacteria and fungi are Saprophytes, which aid in fertilization by returning inorganic nutrients to the soil. Saprophytes * break down dead and dying organic materials (plants and animals) into nitrates, phosphates, carbon dioxide, water and other chemicals necessary for plant growth * Saprophytes also destroy papers, feces and other biodegradable matters, although they cannot break down most plastics or glass  MICROBES AND HUMAN WELFARE: The majority of Microbes Benefit human, animals and plants. ⚫Normal Body Flora. it is used to describe the various bacteria and fungi that are permanent residents of certain body sites especially the skin, colon, oropharynx and vagina. The members of normal flora vary in both number and kind from one to another site  MICROBES AND HUMAN WELFARE: ⚫ Although the normal flora extensively populates many areas of the body, the internal organs usually are sterile such as CNS, blood, lower bronchi, alveoli, liver, spleen, kidney and bladder are free of all but the occasional transient organisms. Summary of the members of normal flora and their anatomic location MICROBES AND HUMAN WELFARE: ⚫ Bacteria participate in recycling vital elements in the environment such as nitrogen, carbon, oxygen, sulfur, phosphorus, etc. ⚫ Bacteria is used in sewage treatment, recycling water. (Bioremediation) ⚫ Fermentation of some products, in food industry. ⚫ Antibiotics production  MICROBES AND HUMAN WELFARE: Microorganisms are used in insect pest control: (viruses, bacteria and fungi) or their bioactive agents can be used as active substances and therefore are referred as Microbial Pest Control Agents (MPCA) ⚫Bacteria is used now in modern biotechnology such as genetic engineering, insulin, enzymes, vitamins production. Micro-organisms are abundant in our environment, not all are harmful, and indeed some are useful.  Introduction to Microbiology All living cells can be classified into two groups: 1-Prokaryotes (prenucleus): such as Bacteria and archea 2-Eukaryotes (true nucleus): such as Animals and plants also Fungi and protozoa.  Humans use the differences between bacterial (prokaryotes) and human cells (eukaryotes) to protect themselves from disease e.g., certain drugs kill or inhibit bacteria while not harming human cells. Viruses are non cellular elements; do not fit into any organizational scheme of living cells. They are genetic particles that replicate but are unable to perform the usual chemical activities of living cells. Characteristics Prokaryotes Eukaryotes Size Smaller (typically, 0.2 - 2 um Larger (typically, 10 - 100 m in diameter in diameter Nucleus No nuclear membrane nor True nucleus with nuclear nucleolus membrane and nucleolus Reproduction Asexual Asexual Or Sexual Cytoplasm organelles Absent Many, including lysosomes, Golgi apparatus, ER, mitochondria & chloroplasts Cell wall Usually present, chemically When present, usually simple complex (peptidoglycan) includes cellulose and chitin, NO peptidoglycan Ribosomes Smaller size (70S) Larger size (80S) Chromosome (DNA) Single, circular, typically Multiple, linear chromosomes lacks histones. with histone proteins Cell division Binary fission Mitosis Sexual reproduction No meiosis; transfer Meiosis fragments of DNA only  Fungi ◦ Eukaryotic (have membrane-bound nucleus) ◦ Obtain food from other organisms ◦ Possess cell walls ◦ Composed of  Molds – multicellular; have hyphae; reproduce by sexual and asexual spores  Yeasts – unicellular; reproduce asexually by budding; some produce sexual spores  Protozoa ◦ Single-celled eukaryotes ◦ Similar to animals in nutrient needs and cellular structure ◦ Live freely in water; some live in animal hosts ◦ Asexual (most) and sexual reproduction ◦ Most are capable of locomotion by  Pseudopodia – cell extensions that flow in direction of travel  Cilia – numerous, short, hairlike protrusions that propel organisms through environment  Flagella – extensions of a cell that are fewer, longer, and more whiplike than cilia  Algae ◦ Unicellular or multicellular ◦ Photosynthetic ◦ Simple reproductive structures ◦ Categorized on the basis of pigmentation, storage products, and composition of cell wall ⚫ The term "prion" is derived from proteinacious infectious particle and refers to the pathogen that causes transmissible spongiform encephalopathies (TSEs). ⚫This small infectious particle is a disease-causing form of a protein called cellular prion protein (PrPc). ⚫ PrPc is mainly found on the surface of cells in the central nervous system, but it is also located in other bodily tissues. ⚫A prion is composed of abnormally folded protein that causes progressive neurodegenerative conditions, with two of the most notable being Bovine spongiform encephalopathy (BSE or mad cow disease) seen in cattle, and Creutzfeldt- Jakob disease (CJD) seen in humans. Transmitted by ingestion. blood transfusion, THE PROKARYOTIC CELL(Bacteria)  They have three basic shapes: 1. Coccus ( spherical). 2. Bacillus (rod-shaped). 3. Spiral (twisted).  Bacteria come in a great many sizes and several shapes  Most bacteria range from 0.2 to 2.0 μm in diameter and from 2 to 8μm in length.  Bacteria and Archaea ◦ Unicellular and lack nuclei ◦ Much smaller than eukaryotes ◦ Found everywhere there is sufficient moisture; some found in extreme environments ◦ Reproduce asexually ◦ Two kinds  Bacteria – cell walls contain peptidoglycan; some lack cell walls; most do not cause disease, and some are beneficial  Archaea – cell walls composed of polymers other than peptidoglycan  Classification of bacteria Division I: Gracilicutes. Bacteria having thin cell wall.  e.g. Gram-negative bacteria (E. coli, Pseudomonas). Division II: Fermicutes. Bacteria having thick cell wall.  eg. Gram-positive bacteria. a. Firmibacteria: include Gram positive cocci as Streptococci and Staphylococci. b. Thalobacteria: Filamentous as Corynebacteria and Nocardia Division III: Tenericutes (Mollicutes)  Bacteria lack cell wall.  e.g. Mycoplasma; contain only plasma membrane around cytoplasm. Therefore, antibiotics which act on cell wall can not affect on Mycoplasma Division IV: Mendosicutes.  Cell wall have faults.  eg. Halophilic bacteria have the ability to live in high sugar or salt concentrations. Ultra-Structure of bacterial cell 1-Essential parts 2-Accessories 1. Cell wall. 1. Volutin granules 2. Cytoplasmic membrane. 2. Bacterial capsule. 3. Cytoplasm. 3. Extracellular slime layer. 4. Nuclear bodies. 4. Flagella. 5. Fimbriae. 6. Endospores.  I. Essential parts  It is a complex, semi-rigid structure, present in most prokaryotes, that gives shape and protection to the cell. Chemically, it consists of murien network (peptidoglycan) 1.Cell wall A. Cell wall of Gram positive bacteria:  Very thick peptidoglycan layer representing 30-70%of dry weight of cell wall.  Teichoic acid presents only in Gram positive bacteria carrying somatic antigens. B. Cell wall of Gram negative bacteria:  Triple layer structure (murien like substance in addition to lipopolysaccharides; and lipoprotein).  Peptidoglycan layer represents less than 10% of dry weight of cell wall.  There is no teichoic acid. Structure mainly is lipoprotein, lipopolysaccharides (LPS; somatic antigens) and responsible for much of the toxicity of Gram-negative bacteria.  Non typical cell wall Mycobacterium and Nocardia contain peptidoglycan and stain gram positive, but their cell wall is composed of unique types of lipids. Mycoplasmas are bacteria that naturally lack a cell wall. Its cell membrane contains sterols (sterols and fatty acid) waxy lipoid material vary from 3-5% which forms the thick cell wall and leads to irritation of the tissue resulting in granulomatous tissue. that make it resistant to lysis. Functions of cell wall: 1. Support the skeleton of bacteria and give the main shape of bacterial cell. 2. Prevent bacterial cell from destruction or rupture due to metabolism or enzymatic reaction resulting in osmotic pressure imbalance. 3. Plays an important role in the cell reproduction. Characteristics Gram Positive Gram Negative Peptidoglycan Very thick layer (30-70 %of dry Thin layer (10 %of dry weight weight of cell wall). of cell wall). Teichoic acid Present Absent - Lipid& Lipoprotein Low High -Lipopolysaccharides Absent Present Gram's Stain Violet colour Red colour  2. Cytoplasmic membrane.  Triple laminar structures of phospholipids.  Acting as a permeability (osmotic) barrier for most molecules.  Also, site of enzymatic activities and function in energy conservation. 3. Cytoplasm  Bacterial cytoplasm consists of two parts (soluble and particular). a. Soluble fractions: mainly containing enzymes and RNA. b. Particulate fractions: consist mostly of ribosomes, the sites of protein formation; and membranous molecules. 4. Nuclear apparatus: The bacterial chromosome and plasmids Unlike eukaryotes, bacteria contain a single DNA chromosome. Bacterial chromosome is not enclosed inside of a membrane bound nucleus but instead resides inside the bacterial cytoplasm. The bacterial chromosome is mostly long, closed, single, circular strand 1mm in length. Along with chromosomal DNA, most bacteria also contain small independent pieces of DNA called plasmids. Plasmids can be easily gained or lost by a bacterium and can be transferred between bacteria.  II. Accessories 1. Volutin granules(Metachromatic granules) Intracellular granules present in cytoplasm of many species of bacteria. Varying in shape and size also in number. Also, volutin granules differ in their site inside bacterial cell. It may be either condensed at poles (Pasteurella species) or scattered allover cytoplasm (Corynebacterium). Volutin granules have strong affinity for nuclear stains as Leishman's stain. Functions: 1. It is thought that it is a site for energy metabolism. 2. Volutin granules help in the diagnosis especially in Pasteurella species (bipolarity). Volutin or metachromatic (Babes’ Ernst) granules which are polymetaphosphate energy storage depots (food reserves) They are visualized by staining with methylene blue show (beading), Neisser’s or Albert stain appear green and bluish black. Corynebacteria with Methylene blue stain show beading metachromatic granules stained with Albert stain (green and bluish black).  2. Capsule and Slime layer. Chemical impermeable structure surrounding the cell wall. Pathogenic capsulated bacteria show maximum capsule formationin the host tissue. S-layer (surface layer) is a cell surface protein layer found in many different bacteria. It has the same chemical nature of the capsule butit is less viscous. Chemical structure of the capsule: 1. Polysaccharides: as Streptococci. 2. Hyaluronic acid: as Pneumcocci. 3. Polypeptide: as Bacillus anthracis.  Importance of capsule: 1. Capsule of pathogenic bacteria has antiphagocytic activity so it increases the virulence. 2. Diagnostic as in B. anthracis (MacFadyean's reaction). 3. New antigen; capsular antigen (K antigen) as in E.coli. 4. S-Layer protect bacteria from dryness. Blood smear, Polychrome MB MacFadyean's reaction of B. anthracis(blue bacilli with red capsule)  3. Flagella:(Organ of Locomotion) Flagella are composed protein called flagellin. It is whip-like structures originated from the cytoplasm of motile& swimming bacteria. Flagella are relatively thin and long. It is responsible for bacterial motility. Arrangement of Flagella: 1. Monotrichous: have a single flagellum at one pole. e.g. Vibrio cholera. 2. Amphitrichous: have a single flagellum at each pole. e.g. other Vibrio. 3. Lophotrichous: have a group or tuft of flagella found at one pole of the cell. e.g. C. botulinum. 4.Peritrichous: have multiple flagella around the whole body of the cell. E.g. Enterobacteriaceae and Bacillaceae. Arrangement of Flagella  4. Fimbriae and pili. (Organ of adhesion and conjugation) Fimbriae are thread like (protein in nature). Fimbriae are very shorter and thinner than flagella. Present in motile and non-motile bacteria. Fimbriae usually function to facilitate the attachment of a bacterium to a surface (e.g. to form a biofilm) or to other cells (e.g. animal cells during pathogenesis). A few organisms (e.g. Myxococcus) use fimbrae for motility. Pili are similar in structure to fimbrae but are much longer and present on the bacterial cell in low numbers. It is responsible for conjugation between bacteria. Non-sex pili also aid bacteria in gripping surfaces. Types of fimbriae: 1. Type I (Common fimbriae) From 10-1000 in number present in many species of bacteria such as Enterobacteriaceae has the ability to agglutinate RBCs. 2. Sex Fimbriae Two in number present only in donor cell (male cell). e.g. E. coli K12 i.e. in strains that carry the mating factor (F) only.  Bacterial spores (Endospores Mainly in Genus Bacillus and Genus Clostridium It is a resting phase against adverse environmental condition. Endospores are bacterial survival structures that are highly resistant to many chemical and environmental stresses that would be lethal for these cells in their normal vegetative form. It is not a reproductive stage and occurs at end of logarithmic phase. Bacterial spores (Endospores) Structure of bacterial spores: 1-Nuclear material + part of cytoplasm + inner plasma membrane 2-Germ cell wall (Mucopeptide) 3-Cortex (Mucopeptides) 4-Outer plasma membrane 5-Spore coat; double or triple layer structure composed of protein.  Types of spores I. According to location 1. Terminal e.g. Clostridium tetani 2. Subterminal e.g. Clostridium perfringens 3. Central e.g. B. anthracis. II. According to diameter of spores 1.Bulged: diameter of spores larger than the diameter of the bacteria. e.g. Anaerobic spore forming bacilli 2. Not bulged: diameter of spores is equal to diameter of bacteria. e.g. aerobic spore forming bacilli III. According to shape 1. Spherical 2. Oval 3. Ellipsoidal IV. According to number Mainly one spore except few species which contain more than one spore. Staining of spores Spore is unstained using Gram's staining technique; the vegetative body of the bacillus is deeply coloured, where as the spore appear as a clear unstained area in the organism. Spores need special staining techniques * Fleming& Nigrosine stain, * Schoeffer& Fulton's stain, and Hansen stain. Importance of spores: 1. Purification of sporulated bacteria mixed with non- sporulated (by heat). 2. Diagnostic importance (help in identification and differentiation between spore forming bacteria). 3. Formation of new antigen. Resistance of spores: Spores of B. subtilis can resist boiling for one hour and can live for 200-300 year in dry soil. Spore of C. tetani can live 15 years. Spores of B. anthracis can live 50 years. Bacterial Nutrition The growth of bacteria involves the synthesis of complex materials such as proteins, carbohydrates and lipids. Certain non pathogenic bacteria utilize inorganic nitrogen and CO2 of the atmosphere. The pathogenic bacteria require organic material e.g. amino acids and energy is obtained mainly by oxidative decomposition of carbohydrates. Certain accessory organic compounds are still required for growth with little amount. These are known as accessory growth factors or bacterial vitamins. These compounds have to be obtained from the body of the host or in culture media from ingredients of the medium e.g. meat extract,blood or serum.  Examples of such compounds as: 1. Nicotinic acid and Pimilic acid for diphtheria. 2. Biotin for Staphylococci. 3. Muracil and Riboflavin for Tetanus bacteria. 4. Minerals: K, Na, Ca, Mg, Fe, phosphate, carbonate and chloride.  Host-Parasite relationship According to the interactions between Bacteria and host bacteria are divided into: 1. Saprophytic bacteria: These are bacteria which normally live on dead organic mater. e.g. Clostridia. 2. Parasitic bacteria: Bacteria which live and propagate on the host body causing tissue damage (disease). e.g. - Bacillus anthracis cause of anthrax. - Salmonella typhi cause of typhoid fever. - Brucella abortus cause of Malta fever in man & Brucellosis 3. Commensal and Opportunistic bacteria: Bacteria live on the host causing no harmful effect (commensal)except when the resistance of the host is lowered these bacteria will then take the upper hand and cause damage to the tissues (opportunistic). e.g. - Pasteurella multocida (in the upper respiratory tract of 10% when the host was under stress) e.g. bad ventilation, over crowding,... produce severe bronchopneumonia. - Also E. coli in the intestine of man under stress produce enteritis. 4. Symbiotic bacteria: Bacteria live on the host with sharing benefit. They are provided with a constant temperature and nutrients and they provide substances useful or necessary for its host. e.g. - Intestinal bacteria take food and produce vitamins (B12). - Lacobacilli on the vaginal epithelium produce lactic acid protecting from colonization and disease caused by yeast and other potentially harmful microbes. 5. Synergistic bacteria: When two or more bacteria produce certain effect which none of them could do alone. e.g. foot rot disease, produced by a group of organisms. 6. Antibiotic bacteria: Certain bacteria produce antibiotics that kill other bacteria. e.g. E. coli produces colicin  Bacterial Growth and Reproduction Means increase in the size and number of microbes. In the lab, bacterial growth and replication can be seen either by: - Development of colonies on solid media. - Transformation of a clear fluid medium to a turbid suspension. Bacterial Growth requirements (Factors affecting bacterial growth) 1. Bacterial Nutrition. 2. Oxygen requirements. 3. Temperature. 4. pH 5. Moisture  Bacterial Reproduction Almost all bacterial cells reproduce asexually by binary fission. Binary fission is a process in which bacterial cell divides into two identical daughter cells. This simple type of cell division is called a mitosis The first step in this process is replication in which increasing of bacterial cell size and duplication of bacterial chromosome.  Then the bacterial cell elongates and the cytoplasm begins to be constricted in the center. The two chromosomes move to opposing sides of the cell, then a double cell wall grows to fill in the furrow in the center as the cell divides into two separate identical daughter cells as the original parent cell. Under favorable conditions, a bacterial cell divides by fission once every 20 minutes.  Generation time: or Doubling time The time required for bacterium to give rise to two daughter cells under optimum condition (generally, not more than 20-30 min).  Bacterial Growth Cycle In the laboratory, when bacteria are introduced in a new suitable culture medium under appropriate condition of temperature, pH,nutrition…etc, their growth will follow a definite course. The growth curve may be divided into four phases. 1.Lag Phase: Lasting for 2-4 hours. It is a period of adaptation occurs immediately after inoculation of the cells into fresh medium during which there is no multiplication. Factors affecting Lag phase: 1. Species of bacteria. 2. Size of the inoculum. 3. Media used. 4. Age of the inoculum. 5. Temperature of incubation E. coli has much shorter lag phase (2-4h) than thoseof tubercle bacilli (2-3 weeks) and Listeria (4-6h). -The bigger size the shorter lag phase. Suitable media shorten the lag phase. Fastidious bacteria either not grow at all or even grow after prolonged lag phase on ordinary media. -The younger the inoculum the shorter lag phase. Inoculum form culture at log phase may be no lag phase on the new medium. Inoculum form culture at decline phase the lag phase may be prolonged. If temperature is lowered the growth cycle is prolonged 2. Exponential Phase: Logarithmic (Log) Phase Lasting for 6-8 hours under optimum temperature. Bacteria are dividing by simple binary fission, doubles by geometric progression at regular intervals. In this phase, bacteria show the typical morphology. 3. Stationary Phase: Lasting form few hours to several days. The number of bacteria reproduce is equal to those dying. Population growth is limited by: 1. Exhaustion of available nutrients. 2. Accumulation of inhibitory metabolites or end products. 3. Change of pH toward acidity. 4. Exhaustion of space, i.e lack of "biological space". Spore-forming bacteria have to induce the activity involved in sporulation process. 3-Decline Phase: Death Phase Lasting for few dayes to several weeks. The number of bacteria reproduce is very low while the death rate is very high (the viable cell population declines) until certain period of time there are no viable organisms in the medium. It occurs due to: nutrient exhaustion, highly acidic pH and accumulation of toxic metabolites.  Relationship between bacteria and the environmental conditions I. Influence of atmosphere (O2 and CO2): 1-Facultative anaerobes 2-Obligatory (Strict) 3-aerobes Obligatory (Strict) 4-anaerobes Microaerophilic bacteria 5-CO2 bacteria 1.Facultative anaerobes Bacteria can grow in the presence or absence of O2 (but more growth occurs in presence of O2). They include most of bacteria. e.g. Staphylococci, Streptococci, Salmonella, E. coli and Proteus. 2. Obligatory aerobes Bacteria can grow only in the presence of air or O2. e.g. Mycobacterium tuberculosis and Pseudomonas aeruginosa. 3-Obligatory anaerobes Bacteria can not grow in the presence of free O2 i.e. killed in the presence of O2 due to the absence of catalase enzyme (present normally in aerobic and facultative anaerobes). e.g. Clostridium species. N.B. Catalase enzyme in the presence of O2 form H2O2, also H2O2 by catalase turned to H2O and O2 but in the absence of catalase, accumulation of H2O2 which is lethal to the bacteria (effect of nascent O2). 4. Microaerophilic bacteria Some bacteria can grow only in a traces of O2. e.g. Actinomyces bovis and Helicobacter. 5. CO2 bacteria Some bacteria require high concentration of CO2 (5-10%) for their growth especially for primary isolation and after subculture may not needed. e.g. Brucella abortus and Campylobacter species. 4. Microaerophilic bacteria Some bacteria can grow only in a traces of O2. e.g. Actinomyces bovis and Helicobacter. 5. CO2 bacteria Some bacteria require high concentration of CO2 (5-10%) for their growth especially for primary isolation and after subculture may not needed. e.g. Brucella abortus and Campylobacter species.  II. Influence of Temperature: 1-On Bacterial growth 2- On viability Temperature acts as necessary growth factor as well as a mean of sterilization depending on the degree and time of exposure. 1-Influence of temperature on Bacterial growth For each bacterial species there is a definite range of temperature within which the growth takes place: A-Minimum temperature B-Optimum temperature C-Maximum temperature A-Minimum temperature It is the lowest temperature at which the growth takes place. B-Optimum temperature It is the best temperature at which the growth takes place. The optimum temperature is approximately that of the habitat (37C for human& animal pathogens and 40C for fowl pathogens). C-Maximum temperature I t is the highest temperature at which the growth takes place. Bacteria Minimum Optimum Maximum temperature temperature temperature B. anthracis 12c 37c 43c P. aeruginosa 5c 37c 43c Salmonella 14c 37c 44c Neisseria 30c 37c 39c  According to temperature required for growth, bacteria are classified into four groups: 1. Psychrophiles (0-20c) Non pathogenic for mammals. Grow best at low temperatures even below 0c with an opt. temp. of 20c. May cause spoilage of food in the refrigerator. Include fish pathogens. e.g. Vibrio anguillarum, Edwardsiella tarda and Listeria (the only G+ve can live in refrigerators). 2. Mesophiles (20-44c) Includes both saprophytic and pathogenic bacteria. Having an opt. temp. of 37 C. Some mesophiles have a wide range of temp. for its growth. e.g. Salmonella and E. coli. Others have a narrow range. e.g. Neisseria. 3. Thermophiles (55-80c) Bacteria can grow and multiply at high temp. Include non parasitic bacteria. May cause spoilage of food specially milk and milk products during pasteurization and canned meat e.g. Lactobacillus acidophilus. 4. Thermoduric bacteria Bacteria can live only in high temp. but without multiplication.  III. Influence of pH:  Most of bacteria of medical importance grow best at neutral or slight alkaline pH (between 7.2-7.6).  Some bacteria (acidophilic) and also yeast and fungi grow best in the presence of highly acidic pH (4-4.5). e.g. L. acidophilus & fungi (Candida, Cryptococcus and Aspergillus).  Other bacteria need slightly acidic pH (6.8- 6.9). e.g. Brucella species  Some bacteria (basophilic) need highly alkaline (pH 8- 8.9). e.g. Campylobacter and Vibrio which need higher pH (pH 9.6).  Generally strong acidic or alkaline solution is highly harmful to most of bacteria with exception of G. Mycobacterium which is highly resistant mainly to 8-10% HCl or 4% NaOH.  IV. Influence of moisture and desiccation  As 80% of bacterial cell consisted of water so moisture is absolutely necessary for bacterial growth.  Air drying is an injurious to many microbes.  According to effect of dryness bacteria may be: A-Sensetive die at once on drying 1.Spirochaetes: as Treponema pallidum (cause of Syphilis in man). 2.Neisseria gonorrhoea. N.B. Most of M.O causing Venereal diseases are sensitive to dryness B-Resistant Resist dryness 1.Sporulated bacteria: F. Bacillaceae (G. Bacillaus& G. Clostridium). 2.Non sporulated bacteria: T.B bacteria can resist dryness for month. S. aureus can survive for long time. Lyophilization Or (freeze-drying)  It is an excellent method for preservation of bacteria.  Bacteria is dried rapidly and completely while frozen in high vacuum sealed ampoules and stored at room temperature in dark.  Lyophilized bacteria for a period of a year.  How dryness affect bacteria? 1.Denaturation of bacterial protein. 2.Destruction of bacterial enzymes. 3.Increase of the salt concentration resulting in plasmolysis. 4.Increase of the germicidal effect of oxygen on dry matter i.e. oxidation.  Factors affecting dryness: 1. Sporulation of bacteria: Sporulated bacteria are more resistant to dryness. 2. Capsulation of bacteria : Capsulated bacteria are more resistant to dryness. 3. Species of bacteria: Some species resist dryness as T.B bacteria, while others are not as gonococci. 4. Nature of gas surrounding and temperature at time of dryness: In lyophilization, bacteria resist dryness when it occurs rapidly while frozen under vacuum.  Bacterial Products 1-Pigments a. Endopigments b. Exopigments 2-Toxins 3-Heat 4- Light Fluorescent bacteria more resistant to the action of ultraviolet rays 5-Miscellaneous products 1-Chromogenesis means the production of pigments by chromogenic bacteria. Endopigments Exopigments Bacteria produce pigment which remain Bacteria produce pigment that diffuse inside the cell and do not diffuse to the outside the cell. media. the colonies are pigmented The media is coloured while the colonies are not. e.g. S. aureus produce golden yellow e.g. Pseudomonas aeruginosa produces pigment. greenish blue pigments. 2-Bacterial Toxins: bacterial products which have a direct harmful action on tissue cells. Exotoxins Endotoxins Harmful bacterial products produced by Harmful bacterial products present within living bacteria and diffuse freely into the bacterial cell and not diffuse till the surrounding medium (extracellular toxins ) bacteria die and disintegrate (intracellular toxin). Produced mainly by Gram positive bacteria Produced mainly by Gram negative e.g. S. aureus and C. tetani. bacteria as S. typhi and S. paratyphi , E. coli. Highly toxic. Less toxic. Highly powerful (1g of tetanospsamin could Less powerful. destroy 30 million mice). Highly antigenic (host produce antitoxin) Weakly antigenic. Highly specific in their action: has specific Non specific in their action. target organ act on it e.g. Haemolysins (Haemolysis), Enterotoxins (Enteritis), Neurotoxin as tetanospasmin (Tetanus). Unstable i.e. easily inactivated by heat or Stable on treating with heat or chemicals chemicals forming toxoid (as 0.3% formalin i.e. for 2weeks) not converted into toxoid.  Exotoxins Endotoxins Produce specific disease as tetanus; Not produce specific diseases but caused produce by C. tetani. gastroenteritis symptoms i.e. vomiting and diarrhea. Preparation: Preparation: By growing the organism in a fluid Bacteria must be first disintegrated medium and then remove the bacteria by by: centrifugation or filtration. -Mechanically: by repeated freezing The fluid containing toxin is purified by and precipitation by one of the following: thawing or grinding with sterile sand. - Chemical: such as: ethanol, acetone or - Chemically: by chemical substances aluminum hydroxide. (extraction by diethylene glycol). -Salting out: by saturated solution of - By heat. ammonium sulphate. -Autolysis.  Dialysis. Then prepared as in exotoxins  Electrophoresis.  Separation by centrifugation or by bacterial filters. Importance of Toxins: 1. Plays an important role in bacterial virulence. 2. Diagnostic importance (help in identification of toxigenic bacteria. 3. Preparation of hyper immune sera or antitoxins.  Measurement of the potency of the toxin This is done by determination of the minimum lethal dose (MLD). MLD: is the smallest dose of toxin that kills the susceptible laboratory animal. LD50 (lethal dose 50): is the smallest dose of the toxin which kill 50% of susceptible lab animal.  Bacterial Virulence It is the degree of pathogenicity. Definition: It is a temporary natural property of a given strain in certain growth phase when introduced in a particular host under certain conditions to produce a disease or pathological state. Factors affecting bacterial Virulence (Components of bacterial virulence): 1. Toxigenicity It is the ability of the bacteria to produce exo- or endotoxins. It is the most important component due to the harmful effect of the bacteria is mainly due to the toxins. The more powerful is the toxin the higher is the virulence of bacteria.  e.g. C. botulinum and C. tetani.  2. Invasiveness It is the ability of the bacteria to invade the tissue of the host. The invasive bacteria are usually capsulated.  e.g. Pneumococci and Klebsiella pneumoniae. N.B. B. anthracis is 50% toxigenic and 50% invasive. 3. Communicability It is the ability of the M.O to establish itself in the host under natural condition. e.g. S. pyogenes produce streptokinase (fibrinolysin) which lyses fibrin formedby the tissue. Also C. perfringens produce hyalurinidase enzyme which breaks down the hyaluronic acid; so help in the spreading of the organism in the tissue. 4. Physiological state of the bacteria  Smooth phase is more virulent than the rough except B. anthracis.  Also bacteria in lag and log phase are highly virulent than stationary and decline phase. 5. Resistance to host defense mechanism Capsulation makes the organism more resistant to phagocytosis so increasing the virulence e.g. B. anthracis. S. aureus produce coagulase causing coagulation of plasma surrounding the organism forming a protective coat. Measurement of the virulence:  The virulence is measured by determination of the LD50.  LD50: it is the dose which kills 50% of injected animals.  This measure is more accurate than LD100. Generalized Infections Bacteremia : is circulation of bacteria in the blood. Septicemia : is circulation and multiplication of bacteria in blood, forming toxins and causing high fever. Pyemia: pyogenic bacteria produce septicemia with multiple abscesses in internal organs as liver and kidneys Toxemia: is circulation of bacterial toxins in blood e.g. C. diphtheriae toxemia  Morphology of viruses Do not possess cellular organization Contain one type of nucleic acid either RNA or DNA Lack enzymes necessary for protein and nucleic acid synthesis machinery of host cells They multiply by complex process and not by binary fission. They are unaffected by antibiotics. They are sensitive to interferon. Viruses are small (20-300nm), obligate intracellular infectious agents which can replicate only in living susceptible cells. Viruses infect all types of organisms either animals or plants and even bacteria and Archaea (bacterial viruses are called bacteriophages). The mature virus particle (virion) consists of central core of nucleic acid (DNA or RNA; not both) surrounded by protective coat of protein (naked virus). Some viruses contains envelope surrounded the protein coat (enveloped viruses). Virus structure Nucleic acid core Protein coat Envelope (Viral genome) DNA RNA Single strand Double strand Single strand Double strand The genes are few in number (3-100). They encode proteins needed for viral reproduction that the host cell will not supply. Some viruses are enveloped others are not. Envelope is a lipid layer surrounding the protein coat. Most enveloped viruses depend on the envelope for their infectivity. Function of envelope: 1. Protection from dryness. 2. It has sites of attachment (spikes). 3. It is used as a guide for virus classification. 4. Makes virus more susceptible for fat solvent chemicals Morphology of viruses Viral Capsid The capsid is an outer shell protein composed of smaller identical protein units called capsomers. (protein coat) Some capsids contain other ingredients (e.g. lipids, carbohydrates), but these are derived from their host cells. The arrangement of capsomers gives the virus structure its symmetry. Viral capsomers can acquire any of the following shapes: - Icosahedral (cubic) symmetry. - Helical symmetry. - Complex structure. - Head and tail viruses (bacteriophage Function of capsid: 1. Protect the viral genome. 2. It has sites of attachment to host cells (spikes). 3. It is responsible for the virus shape. Laboratory Diagnosis of Viral Infections 1. Identification of the virus in infected cell. 2. Microscopic identification directly in the sample using electron microscope. 3. Serologic procedures to detect a rise in antibody titer or the presence of IgM antibody. 4. Detection of viral antigens in blood or body fluids by ELISA test. 5. Detection of viral nucleic acid in blood or the patient's cells using PCR.  FUNGI A fungus (plural fungi) is a eukaryotic, spore forming organism. Only few of fungi are pathogenic. They include microorganisms such as yeasts and moulds as well as mushrooms. Morphologically 1. Yeasts: 2. Moulds (Filamentous fungi) 3. Diphasic fungi 1. Yeasts Grow as single cells and reproduce by budding e.g. Candida albicans. 2. Moulds These include fungi that grow as tubular filaments called hyphae and reproduce by spore production. 3. Diphasic Fungi Some fungi are dimorphic. They grow as yeasts at the body temperature (37C) and as filaments at 22C  Pathogenic fungi cause infections that range from superficial mycoses; affecting the horny layer of skin, subcutaneous mycoses, to deep systemic mycoses which may be severe and fatal. Usefulness of Fungi: Fungi are valuable economically as a source of antibiotics, vitamins, and various industrially important chemicals, such as alcohols, acetone, and enzymes. As well as for their role in fermentation processes, as in the production of vinegar, cheese, and bread dough. They are extremely important in soil renewal, through the decomposition of organic matter. Treatment of Fungi: Fungi are not affected by antibiotics. On the contrary, the prolonged course of broad spectrum antibiotics may cause some fungal infections, a condition known as super infection e.g. oral thrush caused by Candida albicans.