Introduction to Medicine Cultivation and Normal Flora PDF

Bacterial Genetics Bacterial genetic material Is single circular DNA Bacteria are haploid (have a single chromosome). Bacteria has extra chromosomal genetic material called plasmid. Plasmid has different function:  Contain genes that code for antibiotic resistance.  Contain genes that...

Bacterial Genetics Bacterial genetic material Is single circular DNA Bacteria are haploid (have a single chromosome). Bacteria has extra chromosomal genetic material called plasmid. Plasmid has different function:  Contain genes that code for antibiotic resistance.  Contain genes that code for extracellular toxin.  For recombination of genetic material. 11/04/2024 1 Exchange of genetic information involve a unilateral transfer of genetic information from a donor cell to a receptor cell The transfer of genetic information can occur by either of the three methods:  Transformation  Transduction  Conjugation Transformation A process by which a bacterium acquire DNA fragments or genes from surroundings. Usually this occurs in microbial culture. Fig. Transformation process in bacteria Transduction is a method of gene transfer in which a virus (phage) acts as a vehicle for carrying DNA from a donor bacterium to recipient bacterium. 11/04/2024 5 Conjugation A process where by DNA is transferred from one bacterium to another by cell to cell physical contact using sex pili Plasmids are the genetic elements most frequently transferred by conjugation. NB: Conjugation is seen frequently in Gram-. negative rods (Enterobacteriaceae)  Cultivation of microorganisms 7  Culture media are artificially prepared media containing the required nutrients used for propagation of micro organisms. Once the bacteria are grown we can:  Identify them either by presumptive lab diagnosis like Gram stain or by definitive lab diagnosis like biochemical test  Test the antimicrobial sensitivity of the bacteria (drug testing). This helps to know whether the bacteria are sensitive or resistant to known antimicrobial drugs. 8  Common ingredients of culture media 1. H2O: is essential for growth of micro-organism and 80% of bacterial cell is H2O.  It must be free of mineral salts which can inhibit bacterial growth.  Either distilled or deionised water should be used. 2. Peptone: contains water soluble products obtained from break down of animal or plant protein (contains free amino acids, peptides, and proteoses (large sized peptides). Eg. lean meat, heart meat, milk etc.  Its the main source of nitrogen, some vitamins, carbohydrates, nucleic acid fraction and minerals. 9 3. Meat extract : this provides amino acids, minerals, and essential growth factors. 4. Yeast extract : is a very good growth stimulant.  It provides amino acids, water soluble vitamins (vit.B), inorganic slats, and etc. 5. Mineral salts: are essential for growth & for metabolism of living organism. E.g;- SO4: as a source of sulphur  PO42- :as a source of phosphorus  Nacl: is the common ingredient and source of Na & Cl  Other trace elements include Mg, Mn, etc 6. Carbohydrates : simple and complex sugars are used: As energy and carbon source. For differentiation of bacteria. 10 8. Accessory growth factors  Include vit B (thiamine, niacin, and biotin).These provide the necessary condition for growth 9. Other ingredients include, blood, serum, egg yolk etc depending on the organisms need. 7. Solidifying agents: are agents which are used to solidify the culture media. A. Agar : is a commonly used solidifying agent of culture media.  Is a mixture of two poly saccharide - agarose (70 -75%) & agaropectin (20- 25%).  It is produced from sea weeds 11  Agar is a good solidifying agent due to: 1. The protein is inert & undegradable by microorganism 2. Solid to gel transformation temperature is very high i.e it solidifies at 45oC and changed to gel > 45oC 3. It can form gel (solidify) at 1.5% w/v concentration and 4. It forms semi solid gel at 0.4 – 0.5% w/v concentration Uses of agar 5. Solidify culture media 6. Provide calcium and organic ions for the bacteria 7. Used for adding heat sensitive ingredients such as blood and serum while the agar is in gel phase at 45–50oC 8. The agar is very transparent so that growth of colonies is easily visible. 12 Types of culture media The main types of culture media are: 1. Basic media 2. Enriched 3. Selective media 4. Indicator (differential) media 5. Transport media 13 1. Basic Media These are simple media that will support the growth of micro organisms that do not have special nutritional requirements. Example - Nutrient Agar - Nutrient broth 14 2. Enriched/Enrichment media These media are required for growth of organism with extra nutritional requirements such as H. influenza, Neisseria spp, and some streptococcus species. The media can be enriched with whole blood, lyzed blood, serum, vitamins, and other growth factors. E.g:- Blood Agar (contain whole blood) - Chocolate agar (contain lyzed blood) Enrichment media: this term is usually applied to fluid selective media E.g. Tryptosoya broth, Selenite F broth 15 3. Selective media These are solid media which contain substances which inhibit the growth of one organism to allow the growth of the other to be more clearly demonstrated. The medium is made selective by incorporation of certain inhibitory substances like bile salt, crystal violet, antibiotics, etc. E.g. Addition of crystal violet favors the growth of gram negative bacteria and slows the growth of Gram positives. Selective medium is used when culturing a specimen form a site having normal microbial flora to prevent unwanted contaminants overgrowing a pathogen. 16 Examples of Selective agar 1. Thiosulphate citrate bile salt sucrose agar(TCBS)  is alkaline medium and selective for V. cholera. 2. Xylose Lysine Deoxy Cholate (XLD) agar  selective for Salmonella and Shigella. 3. Modified New York City (MNYC) medium  Selective for Neisseria gonorrhoeae. 4. Butzler medium  Selective for Campylobacter species. 5. Salmonella and Shigella agar (SSA)  Selective for Salmonella and Shigella species. 17 5. Differential (Indicator) media These are media to which dyes or other substances are added to differentiate micro-organisms. Many differential media distinguish between bacteria by incorporating an indicator which changes colour when acid is produced following fermentation of a specific carbohydrate.  E.g. Mac Ckonkey agar - contain neutral red as an indicator and lactose as carbohydrate. Lactose fermenting bacteria will become pink and other bacteria become colourless. 18  Figure: differential media (Triple sugar iron/TSI agar media) Figure: differential media (MacConkey agar media , pink colonies are 19  6. Transport media These are mostly semisolid media that contain ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic & anaerobic pathogens when specimens cannot be cultured immediately after collection. Their use is particularly important when transporting microbiological specimens form health centres to the district microbiological laboratory or regional public health laboratory Example  Cary-Blair medium :– is used for preserving and transporting enteric pathogens.  Amies transport medium:- is used for transportation of gonococci. 20 Culture media can be classified by their consistency (form) as: A. Solid media  Are solidified by agar  Are used mainly in Petri dishes as plate cultures, in bottles or tubes as stab (deeps) or slope cultures. B. Semi solid media  Are culture media prepared by adding small amount of agar  Semi solid media are used mainly as transport media, and for motility and biochemical tests. C. Fluid culture media  Fluid media are most commonly used as enrichment when organisms are likely to be few E.g. blood culture 21 22 CULTURING OF BACTERIA Materials for Culturing Bacteria Basic materials used for culturing bacteria. Culture media Petri dishes Test tubes Inoculating loops, straight wire loops Bunsen burner Incubator 23 Appearance of Bacterial colonies on solid Media Bacterial colonies should be examined in a good light & a low power magnifying lens can help to see morphological details. 1. When viewed form above: colonies may appear round, irregular crenated, or branching. They may be transparent or opaque and their surface may be smooth or rough, dull or shiny. Eg. The colonies of pneumococci have a ringed appearance. 24 2. When viewed form the side: Colonies may appear flat or raised in varying degrees some times with beveled edges or with a central elevation or depression. 25 3. When touched with wire loop: Some colonies are soft and easily emulsified such as S.aureus. Where as others are difficult to break up such as S. pyogens. 4. The colour of colonies: this also helps to identify bacteria, especially when using differential media containing indicators. E.g. - V. cholera in TCBS agar appear Yellow - Corny bacterium diphteriae in Tellurite agar appear black. Figure:TCBS Agar - Vibrio chol. 26 Changes which may occur in the medium when bacteria are cultured on solid agar. These include hemolytic reactions, pigment production, color changes surrounding carbohydrate fermenting colonies, & blackening due to hydrogen sulphide production  An example of pigment forming organism is pseudomonas aeroginosa which produces a yellow – green color in media such as blood agar and MacConkey agar.  An examples of organism that produces a color change is Vibrio chorera which is sucrose fermenting, giving a yellow color in TCBS agar. 27  Blacking due to hydrogen sulphide production is seen with many Salmonella cultured in kligler iron agar(KIA). Hemolytic reaction in blood agar is seen with beta hemolytic Streptococci (complete hemolysis) and alpha hemolytic pneumococci(partial hemolysis) Figure: Beta hemolysis Figure: Alpha hemolysis NB. Morphological appearance of colonies on blood agar can vary depending on the species of blood used. E.g. Horse, sheep, or goat blood. 28 Reporting culture results The manner of reporting culture depends on whether the specimen is either from a sterile site or from site with a normal microbial flora. Sites normally Sterile: Identify and report all bacteria isolated up to their genera. And if helpful identify the actual species using bio chemical tests. Sterile sites include blood, bone marrow, CSF, pleural and peritoneal fluids. NB. For isolating organism from sterile sites use culture media which is non selective, and enriched. 29 Sites Having a Microbial Flora: Interpretation requires patients’ clinical data to judge whether an isolate is a pathogen which causes the patients illness. Sites with normal flora include faeces (stool), sputum, skin, throat and nose swabs, vagainal, cervical, and urethral swabs. NB: For isolating organism from sites with normal flora selective media are much better than general media. 30 NORMAL FLORA 31  The term "normal microbial flora" denotes the population of microorganisms that inhabit the skin and mucous membranes of healthy normal persons. Viruses and parasites are not considered members of the normal microbial flora by most investigators because they are not commensals and do not aid the host. 32  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 non-pathogenic or potentially pathogenic microorganisms that inhabit the skin or mucous membranes for hours, days, or weeks;  it is derived from the environment, and does not establish itself permanently on the surface. Members of the transient flora are generally of little significance as long as the normal resident flora remains intact. However, if the resident flora is disturbed, transient microorganisms may colonize, proliferate, and 33 FIGURE: Numbers of bacteria that colonize different parts of the body. Numbers represent the number of organisms per gram of homogenized tissue or fluid or per square centimeter of skin surface. NB: The human body, which contains about 1013 cells, routinely harbors about 1014 bacteria 34 Significance of the Normal Flora Can cause infection  When misplaced, e.g., fecal flora to urinary tract or abdominal cavity, or skin flora to catheter  if person becomes immunocompromised, normal flora may overgrow (e.g. oral thrush). Contributes to health  Protective host defense Produce antimicrobial substances against pathogens Compete for attachment and nutrient with pathogenic bacteria  Serve as nutritional resource by synthesizing: vitamin K & B. 35 Normal Flora of the Skin Because of its constant exposure to and contact with the environment, the skin is particularly apt to contain transient microorganisms. Nevertheless, there is a constant and well-defined resident flora, modified in different anatomic areas by secretions, habitual wearing of clothing, or proximity to mucous membranes (mouth, nose, and perineal areas). The predominant resident microorganisms of the skin are:  aerobic & anaerobic diphtheroid bacilli (eg, corynebacterium, propionibacterium);  Non-hemolytic aerobic & anaerobic staphylococci (Staphylococcus epidermidis & other coagulase-negative staphylococci, occasionally S aureus, & Peptostreptococcus spp); 36  Gram-positive, aerobic, spore-forming bacilli that are ubiquitous in air, water, and soil; alpha-hemolytic streptococci (viridans streptococci) and enterococci (Enterococcus species); and gram-negative coliform bacilli and acinetobacter. Fungi and yeasts are often present in skin folds; acid-fast, nonpathogenic mycobacteria occur in areas rich in sebaceous secretions (genitalia, external ear). 37  Among the factors that may be important in eliminating non-resident microorganisms from the skin are the  low pH, the fatty acids in sebaceous secretions, and  the presence of lysozyme. Neither profuse sweating nor washing and bathing can eliminate or significantly modify the normal resident flora. Placement of an occlusive dressing on skin tends to result in a large increase in the total microbial population and may also produce qualitative alterations in the flora. 38  Normal Flora of the Mouth & Upper Respiratory Tract The flora of the nose consists of prominent corynebacteria, staphylococci (S. epidermidis, S. aureus), and streptococci. The mucous membranes of the mouth and pharynx are often sterile at birth but may be contaminated by passage through the birth canal.  Within 4–12 hours after birth, viridans streptococci become established as the most prominent members of the resident flora and remain so for life. They probably originate in the respiratory tracts of the mother and attendants. 39  Early in life, aerobic and anaerobic staphylococci, gram- negative diplococci (neisseriae, Moraxella catarrhalis), diphtheroids, and occasional lactobacilli are added. When teeth begin to erupt, the anaerobic spirochetes, Prevotella species, Fusobacterium species, Rothia species, Capnocytophaga species establish themselves, along with some anaerobic vibrios and lactobacilli. Actinomyces species are normally present in tonsillar tissue and on the gingivae in adults, and various protozoa may also be present. Yeasts (Candida species) occur in the adult mouth. 40  Small bronchi and alveoli are normally sterile. The predominant organisms in the upper respiratory tract, particularly the pharynx, are  non-hemolytic and alpha-hemolytic streptococci and neisseriae.  Staphylococci, diphtheroids, haemophili, pneumococci, mycoplasmas, and prevotellae are also encountered. 41  Infections of the mouth and respiratory tract are usually caused by mixed oronasal flora, including anaerobes. Periodontal infections, perioral abscesses, sinusitis, and mastoiditis may involve predominantly  Prevotella melaninogenica, fusobacteria, & peptostreptococci. Aspiration of saliva (containing up to 102 of these organisms and aerobes) may result in necrotizing pneumonia, lung abscess, and empyema. 42 Normal Flora of the Intestinal Tract At birth the intestine is sterile, but organisms are soon introduced with food. In breast-fed children, the intestine contains large numbers of lactic acid streptococci and lactobacilli. These aerobic and anaerobic, gram-positive, non-motile organisms (eg, Bifidobacterium species) produce acid from carbohydrates and tolerate pH 5.0. 43  In bottle-fed children, a more mixed flora exists in the bowel, and lactobacilli are less prominent. As food habits develop toward the adult pattern, the bowel flora changes. Diet has a marked influence on the relative composition of the intestinal and fecal flora. Bowels of newborns in intensive care nurseries tend to be colonized by Enterobacteriaceae, eg, klebsiella, citrobacter, and enterobacter. 44  In the normal adult, the esophagus contains microorganisms arriving with saliva and food. The stomach's acidity keeps the number of microorganisms at a minimum (103–105/g of contents). The normal acid pH of the stomach markedly protects against infection with some enteric pathogens, eg, cholera. As the pH of intestinal contents becomes alkaline, the resident flora gradually increases. 45  In the normal adult colon, 96–99% of the resident bacterial flora consists of anaerobes:  Bacteroides species, especially B fragilis; Fusobacterium species; anaerobic lactobacilli, eg, bifidobacteria; clostridia (C perfringens, 103–105/g); and anaerobic gram- positive cocci (Peptostreptococcus species). Only 1–4% are facultative aerobes  gram-negative coliform bacteria, enterococci, and small numbers of proteus, pseudomonads, lactobacilli, candida, and other organisms. 46  Intestinal bacteria are important in  synthesis of vitamin K,  conversion of bile pigments and bile acids,  absorption of nutrients and breakdown products, and  antagonism to microbial pathogens. Among aerobic coliform bacteria, only a few serotypes persist in the colon for prolonged periods, and most serotypes of Escherichia coli are present only over a period of a few days. 47 Normal Flora of the Urethra The anterior urethra of both sexes contains small numbers of the same types of organisms found on the skin and perineum. These organisms regularly appear in normal voided urine in numbers of 102–104/mL. 48 Normal Flora of the Vagina Soon after birth, aerobic lactobacilli appear in the vagina and persist as long as the pH remains acid (several weeks).  Mixed flora of cocci and bacilli At puberty, aerobic and anaerobic lactobacilli reappear in large numbers and  contribute to the maintenance of acid pH through the production of acid from carbohydrates, particularly glycogen.  This appears to be an important mechanism in preventing the establishment of other, possibly harmful microorganisms in the vagina. 49  If lactobacilli are suppressed by the administration of antimicrobial drugs,  yeasts or various bacteria increase in numbers and cause irritation and inflammation After menopause, lactobacilli again diminish in number and a mixed flora returns. The normal vaginal flora includes group B streptococci in as many as 25% of women of childbearing age.  During the birth process, a baby can acquire group B streptococci, which subsequently may cause neonatal sepsis and meningitis. 50  The normal vaginal flora often includes also  alpha hemolytic streptococci, anaerobic streptococci (peptostreptococci), Prevotella species, clostridia, Gardnerella vaginalis, Ureaplasma urealyticum, and sometimes listeria or Mobiluncus species. The cervical mucus has antibacterial activity and contains lysozyme. 51 Normal Flora of the Conjunctiva The predominant organisms of the conjunctiva are diphtheroids, S epidermidis, and non-hemolytic streptococci. Neisseriae and gram-negative bacilli resembling haemophili (Moraxella species) are also frequently present. The conjunctival flora is normally held in check by the flow of tears, which contain antibacterial lysozyme. 52 Normal flora of the external auditory meatus It is an extension of skin normal flora and often profusely colonized.  Staphylococcus epidermidis  Diphtheroids  Alpha-hemolytic and non-hemolyic streptococci N.B there is no normal flora in blood and CSF 53 DISINFECTION , DECONTAMINATION AND STERILIZATION 54  Disinfection: Destruction of microbes that cause disease; may not be effective in killing spores. Antisepsis: destruction or inhibition of micro-organisms in living tissue there by limiting or preventing harmful effect of infection Sterilization: is the destruction or complete removal of all forms of micro-organisms including their spores. 55  The agents, which are used for sterilization and disinfection, can be divided into two broad groups: I. Chemical means’s of sterilization & disinfection II. Physical means’s of sterilization & disinfection 56  Classification of Chemical Mean’s of Sterilization and Disinfection 1. Chemical agents that damage the cell membrane  Surface Active Agents  Phenols  Organic solvents. 2. Chemical agents that denature proteins  Acids and Alkalis 3. Chemical agents that modify functional groups of proteins and nucleic acids  Heavy metals  Oxidizing agents  Dyes  Alkylating agents 57 1. Chemical Agents that Damage the cell Membrane 1.1 Surface Active Agents A. Cationic agents Quaternary ammonium compounds (Quates)  It causes loss of cell membrane semi permeability leading to loss of nutrients and essential metabolites B. Anionic agents Soaps and fatty acids  It causes gross disruption of cell membrane lipoprotein framework.  More active in Gram-positive bacteria than in Gram- negative bacteria. 58 Chemical Agents that Damage the cell Membrane cont…. 1.2. Phenolic Compounds  Phenol is highly effective in Gram positive bacteria.. 1.3. Organic Solvents Alcohol e.g. Ethyl alcohol, Isopropyl alcohol  Disorganize cell membrane lipid structure.  Denatures protein.  Active against Gram-positive bacteria, Gram-negative bacteria and Acid fast bacilli. Uses:  Potent skin disinfectants  Disinfects equipment's like clinical thermometer N.B: Ethanol is potent at concentration of 70% 59 2. Chemical Agents that Denature Proteins E.g. Acids and alkalies, Quates, Alcohol Causes conformational alteration of proteins. Acids like benzoic acid, citric acid and acetic acid are helpful as food preservatives: extending storage life of food products. 60 3. Chemical agents that modify functional groups of proteins and nucleic acids 3.1 Heavy metals Various metals and metal salts are commonly employed to prevent microbial growth or kill microbes Mercury compounds e.g. mercuric chloride- limited use because of its toxicity. - It can be used as antiseptics. 61  Silver compounds e.g. Silver nitrate, Used as ophthalmic and wound (e.g. in burn patients) antiseptic. 3.2 Oxidizing Agents Converts functional-S-H group into non-functional-S-S group. e.g. -Halogens like Chlorine and iodine - Hydrogen peroxide 62 Uses: Hypochlorite: sanitizing agent in dairy and food processing industries, households and hospitals. Organic or inorganic chloramines: Is effective water disinfectant acting by liberating chlorine. Iodine: effective skin disinfectant. Hydrogen peroxide (3-6%): Used for cleansing of wound, disinfecting medical-surgical devices and plastic contact lenses. 63 3.3 Dyes Example: Malachite green Brilliant green Crystal violet/gentian violet Uses: Highly selective for Gram-positive bacteria. For treatment of dermatological lesions. For formulation of selective culture media. 64 3.4 Alkylating Agents A. Formaldehyde - Formaldehyde 37% aqueous solution is named as Formalin. Uses:  Preservation of fresh tissues.  Preparation of vaccines from bacterial surfaces, viruses & toxins B. Glutaraldehyde  Is ten times more effective than formaldehyde  Used for sterilizing medical -surgical instruments C. Ethylene oxide is gaseous sterilizing chemical. Use:  Used to sterilize medical-surgical devices that would be damaged by heat. 65 II. Physical means of sterilization & disinfection Physical means of sterilization and disinfection include Heat, Filtration and Radiation 1. Heat: is the most reliable and universally applicable method of sterilization. Mechanism of Action: Dry heat-denatures protein. Moist heat-denatures and coagulates protein. 66 1.1. Dry heat It is less efficient and requires high temperature and long period heating than moist heat. A. Incineration It is anefficient method of sterilization and disposal of contaminated needles, syringes and cover slips at high temperature. B. Flaming Inoculating wires, loops and points of forceps are sterilized by holding them in the flame of a Bunsen burner until they are red hot. Scalpels , neck of flasks, bottles and tubes are exposed for a few seconds, but it is of uncertain efficacy. 67 C. Hot air sterilizer (hot air oven/ dry oven) It is essential that hot air should circulate between the objects being sterilized. These must be loosely packed and adequate air space to ensure optimum heat transfer. It is done by applying 140-160 0C for 45 to 60 min or 180 0 C for 30 minutes. Use: Usually used to sterilize glass wares and metallic objects. 68 1.2. Moist heat It is preferred to dry heat due to more rapid killing action. Moist heat can be used by the following methods. Temperature below 100 0c This includes the method of pasteurization where objects are subjected to a temperature of 72.oC for 15 seconds. This method does not destroy spores. - Pasturization: it is the process of application of heat at the temperature of 620c for 30 minutes or 720c for 15 seconds followed by rapid cooling to discourage bacterial growth. Uses: Pasteurization of milk ( disinfecting milk) Preparation of bacterial vaccines. 69  Temperatures around 100oC A. Boiling- (Hot water boilers) -are still a common methods in many hospitals. the maximum temperature is 100oC and will there for, not kill all the spores, but for 20 minute exposure all vegetative forms of bacteria and viruses can be destroyed provided instruments are cleaned before putting them in boilers. B. Tyndallization: Intermittent steaming Steaming of the material is done at 1000c for 30 minutes for three consecutive days. The principle is that spores which survived the heating process would germinate before the next thermal exposure and then would be killed. 70 Temperature above 100oC Autoclave: Steam under pressure It is based on the principle that when microorganism is boiled at increased pressure (in closed container), hot saturated steam will be formed which penetrates and gives up its latent heat when it condenses on objects. - Hot saturated stem in autoclaving acts as an excellent agent for sterilization because of: - High temperature - Wealth of latent heat (stem under pressure) - It destroys bacterial endospores and vegetative cells Uses: Sterilize solid and fluid culture media, gowns, medical and surgical equipment. 71 Temperature gauge 72 Table: Time-Temperature-Pressure Level Relationship in moist heat sterilization (Autoclaving) Temperature Time Pressure level 1210c 15 minutes 15 lb/inch2 1260c 10 minutes 20 lb/inch2 1340c 3 minutes 30 lb/inch2 N.B: Most autoclave work by 1210c ,15 minutes and 15 lb/inch2 (temperature, time and pressure respectively). 73 Other methods using low temperature A. Freezing (At 0oC or less temp)- is inactivation of living bacteria by cold. - It prevents active multiplication of bacteria by decreasing the metabolic activity of bacteria. B. Lyophilization: (Freeze-drying) is a process which involves rapid freezing with subsequent drying. Use: Preservation of microbial cultures. Preservation of vaccines. 74 2. Filtration Mechanical sieving through membrane filters. Used in Sterilization of thermolabile parental and ophthalmic solutions, sera and plasma. Have no microbicidal nature. Figure: Micropore filter apparatus 75 3. Radiation Ionizing and ultra violet radiation.  Ionizing radiation includes χ ray, γ ray and β ray. These induce break down of single stranded or sometimes double stranded DNA. ultra violet radiation is less active than ionizing radiation Uses of radiation: Sterilize surgical sutures, catheters, Petri dishes, and pharmaceutical products like hormones, enzymes and antibiotics. 76 LABORATORY DIAGNOSIS OF BACTERIAL INFECTION 77 Laboratory Diagnosis of Bacterial Infection Diagnosis requires a composite of information, including history, physical examination, radiographic findings, and laboratory examination. Infections may be caused by bacteria, viruses, fungi, and parasites. The pathogen may be exogenous (acquired from environmental or animal sources or from other persons) or endogenous (from the normal flora). 78 Laboratory Diagnosis of Bacterial Infection is used for Identification of causative agent Treatment: accurate antimicrobial treatment Surveillance purpose: to asses burden of disease in the community Outbreak investigation 79 Specimen Selection, Collection, and Processing Specimens are selected based on the type of underlying infection. Contamination of the specimen must be avoided by using only sterile equipment and aseptic precautions. The specimen must be taken to the laboratory and examined promptly. Special transport media may be helpful. Specimens must be collected before antimicrobial drugs are administered. 80 Clinical samples 81 Microbial Examination A) Direct Examination :  Direct examination of specimens reveals gross pathology.  Direct examination can be. I) Macroscopic examination  Visual examination of specimen for color, consistency, presence or absence of blood/mucus. 82 Microbial Examination… II) Microscopic examination Is examination using magnifying lenses. This might include microscopic examination of wet mount preparation or stained preparation. Wet mount preparation  Is direct smear examination by adding normal saline under 10x, & 40x objectives of microscope. Staining  Is application of different colored dye to increase visualization organism.  Before staining the smears are fixed  Protects the internal structure of cells in fixed position  Done by two method  Heat  Methanol fixation 83 Type of staining methods 1. Simple staining method 2. Differential staining method 3. Special staining method 1. Simple staining method It is a type of staining method in which only a single dye is used. There are two kinds of simple staining methods A. Positive staining B. Negative staining A. Positive staining: The bacteria or its parts are stained by the dye.  e.g. Methylene blue stain, Crystal violent stain B. Negative staining- The dye stains the background and the bacteria component remain unstained. e.g. Indian ink stain. 2. Differential staining method A method in which multiple stains (dye) are used to distinguish different group of bacteria. e.g. Gram’s stain, Ziehl-Neelson stain. Gram’s stain Principle The principle of Gram’s stain is that cells are first fixed to slide by heat or alcohol and stained with a basic dye  (e.g. crystal violate), which is taken up in similar amounts by all bacteria. The slides are then treated with an Gram’s iodine (iodine KI mixture) to fix (mordant) the crystal violet stain on Gram positive bacteria, decolorized with acetone or alcohol, and finally counter stained with Safranin.  Gram positive bacteria: - stain dark purple with crystal violet and are not decolorized by acetone or ethanol.  N.B. The reason for the retention crystal violet by the gram positive bacteria after decolorization is due to the presence of more acidic protoplasm (PG layer) of these organisms which bind to the crystal violate  Gram negative bacteria: - stain red because after being stained with crystal violet they are decolorized by acetone or ethanol and take up red counter stain. (Neutral red, Safranin or dilute carbol fuchsin). Procedure of Gram staining 1. Add Gentian violet or crystal violet then wait for 1 min, wash with water. 2. Add mordant (iodine), then wait for 1 min, wash with water. 3. Add decolorizer (acetone/ethyl alcohol), then wait for 30s wash with water. 4. Add counter stain safranin/neutral red, then wait for 1 min, wash with water. 5. Air dry and examine under oil immersion microscope.  Notes: In Gram staining gram positive bacteria appear purple while gram negative bacteria appear red/pink. 89  Ziehl-Neelson (Acid fast Bacilli-AFB) staining method 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. The most striking chemical feature of mycobacteria is their extra ordinary high lipid content in the cell wall  Making it relative impermeability to stains, acid fastness, unusual resistance to killing by acid and alkali. The cell wall of Mycobacteria also contains a peptido- glycan layer, glycolipids, protein and Mycolic acid  Principle of Ziehl-Neelson (Acid fast) staining method Smear is heat–fixed, flooded with a solution of carbilfusin (a mixture of basic fuschin and phenol) and heated until steam rises.  The heating which facilitate penetration (entrance) of the primary stain into the bacterium. After washing with water, the slide is covered with 3% HCl (decolourizer). Then washed with water and flooded with methylene blue ( Mycobacterium tuberculosis) Procedure of Ziehl–Neelsen staining 1. Add carbolfuchsin and heat until vapor is observed then wait for 5 min, then wash with water. 2. Add decolorizer acid alcohol (3% HCl) wait for 2-3 min then wash with water. 3. Add counter stain methylene blue/malachite green wait for 1 min then wash with water. 4. Air dry and examine under oil immersion microscope. Notes: Mycobacterium tuberculosis appear pink in blue back ground. 93 94 Microbial Examination… B) Culture:  identify bacteria at genus and species level C) Antimicrobial Susceptibility:  Microorganisms, particularly bacteria, are tested in vitro to determine whether they are susceptible to antimicrobial agents D) Serodiagnosis:  A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis.  Bacterial antigen is detected using specific antibodies E) Molecular techniques (PCR)  Bacterial nucleic acid is detected 95