Infection and Immunity 2024 PDF

Department: Anesthesia Module title: Basics of infection, immunity and neoplasia module Module ECTS: 3 ECTS Module code: BioMM – 2321  Introduction to Microbiology Microbiology is science which deals with living organisms that are individually too small to be...

Department: Anesthesia Module title: Basics of infection, immunity and neoplasia module Module ECTS: 3 ECTS Module code: BioMM – 2321  Introduction to Microbiology Microbiology is science which deals with living organisms that are individually too small to be seen with the naked eye. It considers the minute, microscopic forms of life and their relationships to humans and other organisms. Branches of microbiology ▪ Medical microbiology ▪ Industrial microbiology ▪ Food microbilogy ▪ Soil microbiology ▪ Agricultural ,water microbiology  Scopes of Microbiology Microbiology studies include subjects like ✓ cell structure, ✓ metabolism, ✓ genetics, ✓ growth and control, etc., which create Microbiology scope for jobs in various sectors like ✓ Healthcare, ✓ Biotechnology, ✓ Research, ✓ Environmental Science, and ✓ Agriculture ✓ Food science etc 3  Medical microbiology Deals with the microorganisms that have relation to human. It studies on etiologic agents, epidemiology, diagnosis, treatment and prevention of Pathogenic microorganisms to human and body defence to these pathogens. Some microorganisms are pathogenic others are non - pathogenic or normal flora. 4  Sub divisions of microbiology Bacteriology – which deals with bacteria Mycology- which deals with fungi Phycology - Which deals with Algae. Protozoology – which deals with Protozoa. Virology -studies about viruses  Historical Background of Microbiology Man kind has always been affected by diseases which were originally believed to be visitations by the Gods and meant to punish evil doers. The early Greeks believed that living things could originate from non-living matter (abiogenesis) and that the mother of life could create life from stones Aristotle discarded this notion, but he still held that animals could arise spontaneously from dissimilar organisms or from soil.  Historical Back… Hippocrates, ✓ Hippocrates, the Greek physician, and known as the father of medicine, did not provide much information about the origin of life. ✓ However, his ideas about the natural causes of illness were fundamental for his time ✓ Hippocrates was the first to suggest that illnesses were caused naturally, not by other supernatural means. ✓ He observed that ill health resulted due to changes in: ✓ Air, winds, water, climate, food, ✓nature of soil and habits of people. 7  Historical Back… Antony Van Leeuwenhoek (1632-1723 G.C.) ✓ Father of Microbiology, discovered the first microscope ✓Observed “animalcules” using simple microscope with one lens in samples of water. ✓He was the first who properly described the different shapes of bacteria. ✓Question raised - where did they originate?  Historical Back… Two major theories were formulated 1. Theory of Abiogenesis 2. Theory of Biogenesis  Historical Back… 1. Abiogenesis theory: deals with the theory of spontaneous generation; stating that living things originated “spontaneously” from non-living things. – Aristotle (384-322 BC): was an ancient Greek philosopher who was the founder of a theory spontaneous generation. – Aristotle believed that non-living matter contained "vital heat“ – Aristotle believed that spontaneous generation occurred when certain materials, such as mud, cheese, wood, or dung, began to spoil.  Historical Back… ▪ He observed spontaneous existence of fishes from dried ponds, when the pond was filled with rain. 11  Historical Back… John Needham (1713-1781) – Needham's Experiment ✓ Needham combined grain, broth and meat and then boiled for a few minutes. ✓ He assumed this mixture had thus been pasteurized, killing all microbial life ✓ Needham then poured the broth into flasks and sealed them with corks ✓ Then he waited several days, then examined the broth under a microscope and found living microorganisms. 12  Historical Back… Based on this information, he concluded that microorganisms had spontaneously generated in the flask. Thus, he believed he had proved the spontaneous generation hypothesis to be correct This was not, however, the case, because there were two fundamental defects in Needham's Experiment. – First, the mixture he used was not completely pasteurized. – Second, he hadn't properly sterilized the flask containers before he poured the partially pasteurized broth into them. 13  Historical Back… 2. Biogenesis theory: - ▪ States that life comes from pre- existing life. ▪ The controversy on spontaneous generation took 200 years. Francesco Redi (1626-1697): ✓ He is the scientist who first tried to set an experiment to disprove spontaneous generation. ✓ In 1668, Italian physician Francesco Redi, disproved the theory of abiogenesis by demonstrating that maggots come from fly eggs, not from rotting meat  Historical Back… Sealed jars: No maggots appeared Open jars: maggots appeared on meat Covered jars: Maggots appeared on the cloth, but not on the meat 15  Lazzaro Spallanzani (1776): Repeated Needham’s experiments to disproof spontaneous generation, but with a few important changes. The Spallanzani experiment used aseptic techniques, ✓ By boiling the experimental mixture for a longer period of time than Needham ✓ By sanitizing the flasks the mixture was held in for the experimental period. ✓ Spallanzani's Experiment thereby proved spontaneous generation does not exist.  Historical Back… The foundation of microbiology was securely laid during the period from about 1880 to 1900. Students of Pasteur, Koch, and others discovered in rapid succession a host of bacteria capable of causing specific diseases (pathogens). They also elaborated many techniques and laboratory procedures for revealing the ubiquity, diversity, and abilities of microbes. 17  Historical Back… Louis pasture (1822- 1895) The scientist who disproved the theory of abiogenesis once and for all. Performed experiment to disprove theory of spontaneous generation. In his experiment, he filtered air through cotton plug. ✓ He placed plug in infusion broth, broth became cloudy ✓ Organisms present in the air 18  Historical Back… He designed a large curved flask/swan-necked (pasture goose neck flask) and placed a sterile infusion broth. ✓ Flasks remained sterile unless tilted or neck broken. 19  * In ‘a’ air freely moved through the tube, but dust particles were trapped in the curved portion of the flask, and no microbial growth was observed..  Historical Back… Therefore, Pasteur proved that microorganisms entered to the broth with the air and micro organisms did not evolve spontaneously.  Other major contribution of Louis Pasteur. Microbial theory of fermentation Principles and practices of sterilization and pasteurization Culture techniques Development of vaccines against anthrax and rabies Discovery of streptococci.  Historical Back… The Germ theory of diseases Pasture has also developed the germ theory of diseases, which states that a specific disease is caused by a specific type of microorganism. Robert Koch, a German physician, in 1876 established an experimental procedure to prove the germ theory of disease, which states that specific disease is caused by specific pathogen. The scientific procedure is known as Koch’s Postulate.  Koch’s Postulate: proof of germ theory of disease A Micro-organism can be accepted as a causative agent of an infectious disease only if the following conditions are satisfied. 1. The microorganism should be found in every case of the disease and under conditions, which explain the pathological changes and chemical features. 2. It should be possible to isolate the causative agent in pure culture from the lesion 24  Koch’s postulates… 3. When such pure culture is inoculated in to appropriate lab animal, the lesion of the disease should be reproduced. 4. It should be possible to re-isolate the bacterium in pure culture from the lesion produced in the experimental animal 25  Koch’s postulates… Fig. Koch’s postulate 26  Exceptions to Koch’s postulate Many healthy people carry pathogens but do not exhibit symptoms of the disease. Some microbes are very difficult or impossible to grow in vitro (in the laboratory) in artificial media. Eg. Treponema pallidum. Many pathogens are species specific. Eg. Brucella abortus cause abortion in animals but not in humans. Certain diseases develop only when an opportunistic pathogen invades imuno-compromised host. 27  Historical Back… Major achievements of Robert Koch 1. Discovery and use of solid medium in bacteriology 2. Discovery of causative agents of tuberculosis and cholera 3. Koch’s postulate  Historical Back… Progress in the 20th century All of these developments occurred in Europe. Not until the early 1900s did microbiology become established in America. Many microbiologists who worked in America at this time had studied either under Koch or at the Pasteur Institute in Paris. 29  Historical Back… Once established in America, microbiology flourished, In 1923 American bacteriologist David Bergey established that science’s primary reference, updated editions of which continue to be used today. 30  Historical Back… Since the 1940s microbiology has experienced an extremely productive period during which ✓Many disease-causing microbes have been identified ✓Methods to control them developed ✓Microorganisms have also been effectively utilized in industry 31  Historical Back… ✓ The study of microorganisms has also advanced the knowledge of all living things. ✓ Microbes are easy to work with and thus provide a simple vehicle for studying the complex processes of life; as such ✓ They have become a powerful tool for studies in genetics and metabolism at the molecular level ✓ Knowledge of the basic metabolism and nutritional requirements of a pathogen often leads to a means of controlling disease or infection 32  Types of microorganisms ▪ The organisms that constitute the microbial world are characterized as either prokaryotes or eukaryotes 1. Eukaryotes a. Algae (except blue green algae) b. Protozoa c. Fungi d. Slim molds 2. Prokaryotes a. Bacteria b. Blue-green algae/cyanobacteria c. Archaebacteria 1. Prokaryotes:( pro= primitive, karyot= nucleus) ❖ are simpler than eukaryotes at every level except for their complex cell wall. ❖ eukaryotes are 5-20 times larger than bacteria Fig. Prokaryotic cell  Prokaryotes vs eukaryotes Property Eukaryotes prokaryotes Size 10-100 um in diam 0.2-2um Chromosom no multiple Single/one Cell division mitosis Binary fission nucleus true Nuclear material Nuclear membrane present Absent Sexual reproduction present Absent ribosoms 80s 70s Organells(mitochon present absent dria,golgi app,ER) Histons present absent  Bacterial classification and nomenclature Bacteriology: the discipline of biology related to the study of bacteria. Bacteria are prokaryotes There is no official classification of bacteria, but classification is based on several methods. 1. Phenotyping : includes microscopic, macroscopic morphology and biotyping of bacteria. 2. Analytical: mass spectrophotometer 3. Genotypic: nucleic acid hybridization, plasmid analysis, ribotyping  Bacteria are classified in to different categories in Bergey’s manual of determinative bacteriology, (1974), and the classification is based on: 1. Morphology 8. Amino acid sequencing of proteins 2. Staining 9. Genetic composition 3. Motility 4. Growth 5. Nutritional requirement 6. Bio chemical and metabolic activity 7. Pathogenicity Morphology: - ❖Bacteria vary widely in size, ranging from 0.2 um to 10um long -There are three basic morphological forms/shapes 1. Spherical or coccoid/cocci- (singular –coccus) 2. Rods or bacilli (singular - bacillus) 3. Spirals or spirilla Fig. Different bacterial morphologies A. Cocci, B. Bacilli, C. Spiral shape Fig. different bacterial arrangements  Thus the science of taxonomy (biological classification) was devised based on the binomial system developed in the 18th century. In binomial system, each organism is given two names. ( eg. homo sapiens for humans ) The first is the genus or genera (plural) and the second is the species.  To express genus capitalize the fist letter of the word for example Esherchia. To express genus and species together, capitalize the first letter of the genus name Example: Eschelchia coli. The genes can be abbreviated as E.coli  Basic features of Bacterial Cell General property – Typical prokaryotic cell – Contain both DNA and RNA – Most grow in artificial media – Replication is by binary fission – Contain rigid cell wall  Structure of Bacteria Bacteria have characteristic size, shape, consistency, texture and color. Bacterial cell structures have 3 architectural regions: 1. Cell envelope ✓ Capsule, cell wall and cell membrane 2. Cellular element enclosed with in the cell envelope: ✓ Mesosomes, ribosome, nuclear material, polyamines and cytoplasmic granules. 3. Cellular element external to the cell envelope (appendages) : ✓ Flagellum, Pilus  1. Cell Envelop 1. Cell envelope consists of a capsule, cell wall and plasma membrane ❖Cell wall: - A basic frame, maintain the shape(rigidity) of bacteria - protects the delicate cell protoplast from osmotic lysis. - Its strength is due to a complex amino sugar polymer (peptodoglycan or mucopeptide) – Multi layered structure and constitutes about 20% of the bacterial dry weight  Chemical Composition of cell wall The major component of cell wall is peptidoglycan (PG) It consists of a polymer of disaccharides cross- linked by short chains of amino acids (peptides). peptidoglycan is made up of N-acetyl muramic acid and N-acetyl glucoseamine attached by by glycosidic linkage. – Peptidoglycan found only in bacterial cell walls – Some times called back bone of bacteria.  Types of cell wall I. Gram positive cell wall of bacteria ❖ has two layers (Peptidoglycan cross linked with teichoic acid) – The peptidoglycan layers is much thicker than Gram negative bacteria ❖ The peptidoglycan layer comprises 50 – 90% of the cell wall and 20 – 40% of the cell wall weight  Gram positive cell wall …Cont’d ❖The large amount of peptideglycan make gram- positive bacteria susceptible to the enzyme lysozyme and penicillin. ❖Penicillin specifically inhibits peptidoglycan synthesis ❖Teichoic acids and cell well- associated protein are the major surface antigens of the gram- positive cell well.  II. Gram negative Cell wall of bacteria Is some what complex than Gram positive bacterial cell wall Has thin peptidoglycan layer Has high lipid content (lipopolysaccharied) in the outer membrane Has periplasmic space.  Gram negative cell wall …Cont’d Outer membrane Contains receptors (sites) for bacteriophage attachment or bacteriocine (bacteriocine – are antibacterial agents produced by bacteria) It participates in cell division Used in transport of materials (either out of or towards the cell) Lipopoly saccharides It is responsible for antigenicty of the outer membrane Periplasmic space Found between outer membrane and the cell membrane Mostly contain enzymes and endotoxins.  (a) (b) Fig. Gram-Positive (a) and Gram negative (b) cell wall of bacteria  Capsule Capsules are often regarded as portion of the cell envelope Capsular constituents vary among the different species of prokaryotes. serve to enable the bacteria to attach to tissues and to resist phagocytic digestion.  Cytoplasmic membrane (Plasma membrane) ▪ It is the actual barrier between the interior and exterior of the bacteria cell. ▪ The cytoplasmic membrane exhibits a well- defined selective permeability, excretion of enzyme, and biosynthesis of cell well and other proteins ▪ The bacterial transport system and the principal energy system (oxidative phosphophorylation) are located.  Cytoplasmic membrane … Cont’d It accounts for 30% of the dry weight of bacterial cell Chemically, the plasma membrane consists of proteins and phospholipids. It is 60% protein, 20 – 30% lipid and 10-20% carbohydrate.  2. Cellular Element Enclosed with in the Cell Envelope Mesosomes: are complex invaginations of cytoplasmic membrane in to the cytoplasm seen in many bacteria, but not in all. – Increase in the total surface area of the membrane. – Mesosomes are attached to chromosomes and are involved in DNA segregation during cell division. – Others are involved in to secretion of proteins and active transport. – It is involved in respiratory enzyme -activity. (Site of oxidative phosphorulation) Ribosomes sites of protein synthesis composed of ribosomal RNA (rRNA) (70%) and proteins (30%) constitutes 90% of the RNA and 40% of the total protein. The ribosomes of procaryotes are smaller than cytoplasmic ribosomes of eucaryotes. Procaryotic ribosomes are 70S in size, being composed of 30S and 50S subunits. – S or Svedberg unit designates the sedimentation coefficient of the rRNA  Cytoplasmic inclusions are distinct granules that may occupy a substantial part of the cytoplasm. are usually reserve materials of some sort. For example, – carbon and energy reserves may be stored as glycogen (a polymer of glucose) – polybetahydroxybutyric acid (a type of fat) granules. – elemental sulfur (sulfur globules) are stored by some phototrophic as reserves of energy. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm which contain photosynthetic pigments or specialized enzyme complexes.  Nuclear material is concentrated in the cytoplasm as a nucleoid. The nucleoid consists of one long double-stranded circular DNA molecule (chromosome). The chromosome serves as the control center of the bacterial cell, carries the genetic information needed for producing several thousand enzymes and other proteins. The prokaryotic nucleoid is considered primitive nucleus, – it is not surrounded by a nuclear membrane – It does not have a definite shape, and has little or no protein material. Apart from nucleus, the bacteria may have some extra chromosomal genetic material in the form of DNA, which is known as Plasmid.  3. Cellular Elements External to the Cell Envelope Flagellum It is the organ of locomotion in bacterial cell and consists of filament is free on the surface of bacterial cell It is composed of protein named as flagellin The flagellar antigen in motile bacterium is named as H (Hauch) antigen. Fig. ? Different flagellar arrangements  4. Pilli and Fimbriae interchangeable terms used to designate short, hair-like structures (finer filaments) on the surfaces of procaryotic cells. are extruding from the cytoplasmic membrane are shorter and stiffer than flagella, and slightly smaller in diameter. Like flagella, they are composed of protein called pilin arranged in helical strand Two functional types of pili may occur independently or together on some cell. I. Common pili – almost always called fimbriae – Help for attachment of bacteria to epithelial cell – They considered as virulence factor in some species of bacteria, because they allow pathogens to attach to (colonize) tissues and to resist attack by phagocytic white blood cells. II. Sex pili or F pili – occur less commonly – appear to be specifically involved in bacterial conjugation, i.e transfer of genetic material (DNA) from one bacterium to another.  Bacterial spores Under conditions of limited supply of nutrition, vegetative forms of certain bacteria especially gram- positive bacilli and actinomycets form highly resistant and dehydrated forms, which are called endospores. These endospors are capable of survival under adverse conditions such as heat, drying, freezing, radiation, and actions of toxic chemicals. BACTERIAL METABOLISM AND GROWTH  Learning objective: At the end of this chapter the students will be able to: Explain bacterial nutritional and environmental requirement Discuss Bacterial metabolism Describe bacterial growth and growth curve  Bacterial metabolism and growth Nutrition ✓For optimal growth and multiplication, bacteria requires nutrients, such as water, energy, carbon, nitrogen and some inorganic salts. ✓Bacteria also require various environmental factors for growth in optimum concentration. - -These include Oxygen/Carbon dioxide, pH, temperature and light ✓All bacteria need some form of the element Carbon, H, O2, S, P, and N for growth. ✓Special elements such as K, Ca, Fe, Mn, Mg, Co, Cu, Z, Ur are needed by certain bacteria. ✓Some have specific vitamin, and growth factor requirements and others need organic substances secreted by other microorganisms during their growth.  1. Nutrient requirement ✓Autotrophs: - are free-living, non-pathogenic bacteria, most of which can use carbon dioxide as their carbon source 1.Photoauototrophs 2. Chemoautotroph ✓ Heterotrophs.are generally parasitic bacteria which require more complex organic compounds than carbon dioxide as their source of carbon and energy  2. Temperature requirement Most pathogenic bacteria grow best at an optimum temperature of 37 0C. Optimum temperature is the temperature at which growth occur best. ❖Based on temperature requirement, microorganisms can be broadly classified into Psycrophylic- are those bacteria, which grow in the range of -5 to 200C – These bacteria include those which cause spoilages of food at refrigeration temperature (2- 8oc).  Mesophilic- are those bacteria, which grow at 20-450C and show optimum growth at 37oC. – all medically important bacteria (pathogenic bacteria) belong to this group Thermophilic – are those organisms which prefer high temperature (50-800C) Hyperthermophilic – Those which grow at a temperature of above 800C – Some of them grow even at 2500C – are found in hot springs, geysers and industrial heated wastes  3. Oxygen requirement ✓ The need of oxygen for particular bacterium reflects its mechanism to meet the requirement of energy. On the basis of this requirement, bacteria have been divided in to: Obligate Anaerobes-these grow only in the environment devoid of oxygen – e.g. clostridium Facultative aerobes- these can grow under both aerobic and anaerobic conditions, e.g. enterobacteriaceae  Obligate aerobes- these cannot grow unless oxygen is present in the medium Microaerophilic- these organisms can grow under conditions with low oxygen tension Aerotolerant anaerobes – These bacteria oxidize nutrient substrates without using oxygen. Unlike obligate anaerobes, they can tolerate the presence of oxygen.  4. pH requirement Most pathogenic bacteria require a pH of 7.2-7.6 for their optimal growth. Based on pH requirement bacteria can be classified as Neutrophilic:- bacteria grow best at neutral pH (pH=7) – Most pathogenic micro-organism best grow at neutral pH (pH=7) Acidophilic – Bacterial grow best at acidic pH (pH7)  Bacterial Metabolism bacterial metabolism involves all the cellular processes required for the survival and replication of the organism. Most biochemical reactions fall into two categories: Catabolism and Anabolism.  Bacterial growth Bacteria divide by binary fission(asexualy). When a bacterial cell reaches a certain size, it divides to form two daughter cells. Generation time or population doubling time The time required for a bacterium to give rise to two daughter cells under optimum conditions  The generation time of bacteria ranges from as little as 20 minutes for E-coli to more than 20 hrs for Mycobacterium tuberculosis. The generation time varies not only with the species but also with the amount of nutrients, the temperature, the pH, and other environmental factors. The growth cycle of bacteria has four major phases. 1.The Lag Phase this phase is of short duration in which bacteria adapt themselves to new environment This is a period of active macro molecular synthesis like DNA, RNA, various enzymes and other structural components It is the preparation time for reproduction  No increase in cell number occurs, however, vigorous metabolic activity occurs and increase in size of bacteria. This can last for a few minutes up to many hours. The duration of lag phases varies with the species, nature of culture medium, temperature 2.The log or exponential phase During this phase, the population can double approximately every 30 minutes with fast growing bacteria It has limited duration because of:- – Exhaustion of nutrients – Accumulation of toxic metabolic end products – Rise in cell density – Change in pH and – Decrease in oxygen tension (in case of aerobic organisms) 3. Stationary Phase Occur when nutrients depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die resulting in a steady state There is almost a balance between the bacterial reproduction and bacterial death 4. The death/decline phase Due to depletion of nutrients and accumulation of toxic end products the number of bacteria dying is much more than those dividing and hence there is gradual decline in the total number of organism. There is drastic decline in viable cells. Fig. Bacterial growth curve  Continuous-culture to maintain a culture in exponential, steady- state (balanced) growth for long periods is to use a device in which fresh medium is continuously added but the total volume of culture is held constant by an overflow tube.  HOST PARASITE RELATIONSHIP General Concepts Host Susceptibility Resistance to bacterial infections is enhanced by phagocytic cells and an intact immune system. Initial resistance is due to nonspecific mechanisms. Specific immunity develops over time. Susceptibility to some infections is higher in the very young and the very old and in immunosuppressed patients  Bacterial Infectivity Bacterial infectivity results from a disturbance in the balance between bacterial virulence and host resistance Host Resistance Numerous physical and chemical attributes of the host protect against bacterial infection. These defenses include the antibacterial factors in secretions covering mucosal surfaces and rapid rate of replacement of skin and mucosal epithelial cells. Once the surface of the body is penetrated, bacteria encounter an environment virtually devoid of free iron needed for growth, which requires many of them to scavenge for this essential element.  Bacterial virulence factors may be encoded on chromosomal, plasmid, transposon, or temperate bacteriophage DNA; virulence factor genes on transposons or temperate bacteriophage DNA may integrate into the bacterial chromosome. Virulence Factors Virulence factors help bacteria to:- (1) invade the host, (2) cause disease, and (3) evade host defenses. The following are types of virulence factors: Adherence Factors: Many pathogenic bacteria colonize mucosal sites by using pili (fimbriae) to adhere to cells. Invasion Factors: Surface components that allow the bacterium to invade host cells can be encoded on plasmids, but more often are on the chromosome. – Capsules: Many bacteria are surrounded by capsules that protect them from opsonization and phagocytosis. – Endotoxins: The lipopolysaccharide endotoxins on Gram-negative bacteria cause fever, changes in blood pressure, inflammation, lethal shock, and many other toxic events. – Exotoxins: Exotoxins include several types of protein toxins and enzymes produced and/or secreted from pathogenic bacteria. – Major categories include cytotoxins, neurotoxins, and enterotoxins. – Siderophores: Siderophores are iron-binding factors that allow some bacteria to compete with the host for iron, which is bound to hemoglobin, transferrin, and lactoferrin.  NORMAL FLORA The term "normal microbial flora" denotes the population of microorganisms that inhabit the skin and mucous membranes of healthy normal persons The skin and mucous membranes always harbor a variety of microorganisms that can be arranged into two groups: (1)The resident flora consists of relatively fixed types of microorganisms regularly found in a given area at a given age; if disturbed, it promptly reestablishes itself. (2)The transient flora consists of nonpathogenic or potentially pathogenic microorganisms that inhabit the skin or mucous membranes for hours, days, or weeks; it is derived from the environment, does not produce disease, and does not establish itself permanently on the surface.  Members of the transient flora are generally of little significance so long as the normal resident flora remains intact. However, if the resident flora is disturbed, transient microorganisms may colonize, proliferate, and produce disease.  Significance of the Normal Flora Can cause infection - misplaced, e.g., fecal flora to urinary tract or abdominal cavity, or skin flora to catheter - if person becomes compromised, normal flora may overgrow  Contributes to health - protective host defense by maintaining conditions such as pH so other organisms may not grow - Produce antimicrobial substances against pathogens - Compete for attachment and nutrient with pathogenic bacteria - serve nutritional function by synthesizing: vitamin K and B vitamins  Bacterial staining ▪ Stains (Dyes) are coloured chemical compounds that are used to selectively give colour to the colourless structures of bacteria or other cells. ▪ Bacterial staining is the process of imparting colour to the colourless structures (cell wall, spore, etc) of the bacteria in order to make it visible under the microscope. Uses 1. To observe the morphology, size and arrangement of bacteria 2. To differentiate one group of bacteria from the other group. Type of staining methods Gram staining technique Ziehl-Neelsen technique to detect AFB Auramine-phenol technique to detect AFB bipolar staining of bacteria Albert staining of volutin granules Giemsa technique Gram’s stain This method was developed by the Danish bacteriologist Christian Gram in 1984. Basic concepts: Most bacteria are differentiated by their gram reaction due to differences in their cell wall structure. Procedure 1. Prepare smear from specimen or culture. 2. Allow the smear to air-dry. 3. Rapidly pass slide three times through flame. 4. Cover fixed smear with crystal violet for one minute and wash with tap water. 5. Tip off the water and cover the smear with Gram’s iodine for one minute. 6.Wash off iodine solution with tap water. 7. Decolorize with acetone-alcohol for 30 seconds. 8. Wash off the acetone-alcohol with clean water. 9. Cover the smear with safranin for one minute. 10. Wash off the stain and wipe the back of the slide. Let the smear air-dry. 11. Examine the stained smear with oil immersion objective to look for bacteria. Results Gram positive bacteria …………………….. purple Yeast cells …………………………………. Dark purple Gram negative bacteria …………………….. Pale to red Nuclei of pus cell …………………………… Red Epithelia cells ………………………………. Pale red B. Ziehl-Neelson (Acid fast) staining method Developed by Paul Ehrlich in1882, and modified by Ziehl and Neelson Ziehl-Neelson stain is used for staining mycobacteria which are hardly stained by Gram‘s staining method. Once the Mycobacteria is stained with primary stain it can not be decolorized with acid, so named as acid-fast bacteria. Procedure 1. Prepare the smear from the primary specimen and fix it by passing through the flame and label clearly 2. Place fixed slide on a staining rack and cover each slide with concentrated carbol fuchsin solution. 3. Heat the slide from underneath with sprit lamp until vapor rises (do not boil it) and wait for 3-5 minutes. 4. Wash off the stain with clean water. 5. Cover the smear with 3% acid-alcohol solution until all color is removed (two minutes). 6. Wash off the stain and cover the slide with 1% methylene Blue for one minute. 7. Wash off the stain with clean water and let it air-dry. 8. Examine the smear under the oil immersion objective to look for acid fast bacilli. Results AFB............. Red, straight or slightly curved rods, occurring single or in a small groups Cells......................................... Blue Background Material ……….. Blue  Culture media ❖Culture media are artificially prepared media containing the required nutrients used for propagation of micro- organisms or living tissue cells. Uses Isolation and identification of micro-organisms Performing anti-microbial sensitivity tests  Common ingredients of culture media 1. H2O 2. Peptone: 3. Meat extract : 4. Yeast extract : 5. Mineral salts : 6. Carbohydrates : 7. Solidifying agents: 8. Accessory growth factors Types of culture media The main types of culture media are: 1. Basic media 2. Enriched or enrichment media 3. Selective media 4. Indicator (differential) media 5. Transport media  BIOCHEMICAL TESTS Biochemical tests are used to differentiate different organisms based on their genus and species characteristics. Biochemical tests are performed on pure culture. The following are some of the common biochemical tests used for differentiation of different bacteria. 1. Catalase test 2. Coagulase Test 3. Oxidase test/Cytochrome oxidase test 4. Urease test 5. Indole test 6. Citrate utilization test 7. Triple sugar Iron (TSI) & Hydrogen sulfide production (H2S) etc