Cultivation of Bacteria PDF

CULTIVATION OF BACTERIA Kasun Aththanayake BSc. (Biological Science), MPhil in Microbiology  CONTENT Media- types, cultivation of aerobic and anaerobic bacteria Pure cultures and cultural characteristics: Maintenance and preservation of pure cultures- subculturing overlaying wi...

CULTIVATION OF BACTERIA Kasun Aththanayake BSc. (Biological Science), MPhil in Microbiology  CONTENT Media- types, cultivation of aerobic and anaerobic bacteria Pure cultures and cultural characteristics: Maintenance and preservation of pure cultures- subculturing overlaying with mineral oil and lyophilization Physical and chemical control of microorganisms; antimicrobial agents- penicillin and tetracycline What is a Culture in Microbiology? Any growth or cultivation of an organism in a medium. Usually employed in reference to the deliberate growth of microorganisms.  Type of cultures Pure culture Mixed culture Contains only one single type Contains more than one type/ of microorganism, rarely species of microorganisms. found outside the laboratory.  Laboratory Cultivation of Microorganisms Should provide a favorable environment for their growth. Necessary nutrients must be provided to be used as building materials for new cells. Environmental conditions (pH, Oxygen tension, temperature, humidity, light) should be provided. Importance of culturing: Direct laboratory methods (ex: Microscopy)- provide only preliminary information. Culture method- required for definitive identification and characterization Components of a culture media Composition of growth media 7  Microbiological Media The food or nutrient material prepared for the growth of microorganisms in the laboratory. Can be classified into several categories depending on their; 1. Physical nature (consistency) 2. Chemical composition 3. Purpose/ functional type TYPES OF CULTURE MEDIA Solid Media Plate and Slope (Slant) Cultures Based on Liquid Media Broth Cultures Consistency Semi-Solid Media Stab Cultures Enrichment Media General Purpose Culture Media Media Based on Purpose Selective Media Special Purpose Media Natural/ Complex Differential Media Media Transport Media Based on Semi-Synthetic Composition Media Synthetic Media 1. Physical Nature Solid media Semi solid media Liquid media Solid media Used to separate microbes by allowing them to grow as isolated colonies. Also used to observe colony characteristics and maintain stock cultures. Solidifying agents are used. – Agar (1.5-2%) Silica gel Example: plates and slants Semi solid media Prepared with 0.5% or less agar Have a soft custard like consistency Used for the cultivation of microaerophilic bacteria Used for the determination of bacterial motility Liquid/ broth media Used to isolate microbes Do not contain agar Cultivate microbes in large quantities To obtain products industrially To carryout different biochemical tests/ fermentation studies To obtain microbial extracts Composition of nutrient broth, a medium for the growth of heterotrophic bacteria: Media for the growth of fungi Media compositions 16 17  2. Chemical Composition Three categories according to their components Chemically undefined media/ natural media Semi-synthetic media Chemically defined media/ complex media 1. Natural media Contain natural products such as dil. blood, urine, milk etc. The exact chemical composition is not known (chemically undefined). 2. Semi-synthetic media Contain nutrient sources in which the nature and quantity of constituents are partly known. E.g: Potato extracts, peptones, yeast extracts, beef extracts, soya bean extracts This type of media is very useful as a simple complex medium May be sufficiently rich to completely meet the nutritional requirements of many different microbes. These types of media are needed when the nutritional requirements of a particular microbe is unknown.  PDA Yeast Extract Beef extract Soya Bean Extract Synthetic media This is prepared by combining precisely determined compositions of components. The exact concentration of every chemical in the medium is known. 3. Purpose/ Functional Type Media can be categorized depending on the function or the purpose of the medium General purpose media Minimal media Selective media Differential media Assay media Enrichment media Reducing media Transport media  1. General Purpose Media Simple media that support the growth of a large variety of microorganisms, mostly non- fastidious microorganisms. Generally used for the primary isolation of microorganisms. Ex: Nutrient Agar, Nutrient Broth, Potato Dextrose Agar Nutrient Agar Nutrient Broth 21  Special Purpose Media Many special-purpose media are needed to facilitate the enumeration and isolation of certain types of bacteria. To meet these needs, numerous media are available. 1. Enrichment Media Used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective media. Typically these are liquid (broth) media. Ex: Selenite F Broth, Tetrathionate Broth, Alkaline Peptone Water 23  2. Selective Media A selective medium has components added to it, which will inhibit/ prevent the growth of certain types/ species of bacteria and promote the growth of the desired. Physical conditions of the medium (pH, temperature) can also be adjusted to render it selective. That is, the medium is being selective for one particular type of microorganisms. Ex: MacConkey Agar 24 MacConkey Agar Used for Enterobacteriaceae members contains bile salt that inhibits most Gram-positive bacteria. Consists of - Crystal violet Bile salts pH indicator – Neutral red Agar Lactose Water Selective components are crystal violet and bile salts. Both of them Inhibit the growth of Gram +ve bacteria. This too acts as a differential medium. Differentiation is given by lactose and pH indicator (neutral red).  3. Differential Media A differential medium allows the investigator to distinguish between different types of bacteria based on some observable trait in their pattern of growth on the medium. Ex: Eosin Methylene Blue (EMB) Agar E. coli on EMB Agar Enterobacter on EMB Agar 26 EMB agar In EMB agar, selective components are Eosin and Methylene blue dyes. Inhibit the growth of Gram +ve bacteria Dyes react with acidic products released by gram –ve bacteria when they use lactose as a carbon and energy source Eg: Escherichia coli produces colonies with a green metallic sheen Selective/ Differential media Some culture media are both selective and differential Eg: MacConkey agar contains bile salts and crystal violet dye to inhibit the growth of Gram +ve bacteria and allow Gram –ve bacteria to grow Blood agar - contains bovine heart blood that becomes transparent in the presence of β-hemolytic organisms such as Streptococcus pyogenes and Staphylococcus aureus. 5% Sheep Blood α-hemolysis : Partial Haemolysis β-hemolysis : Complete Haemolysis γ-hemolysis : No Haemolysis **Produce clear zones around their colonies because of red blood cell destruction** 33 4. Transport Media Transport media are essentially solutions of buffers with carbohydrates, peptones, and other nutrients (excluding growth factors) designed to preserve the viability of bacteria during transport without allowing their multiplication. Ex: Viral Transport Medium (VTM) 5. Assay medium Used for the assay of vitamins, amino acids, and antibiotics 6. Minimal Medium Contains the minimum nutrients possible for colony growth Generally without the presence of amino acids Used to grow “wild–type” microorganisms. Contains only inorganic salts, a carbon source, and water. Labeling process The base of the plate should be labeled with, Date/Name of the sample or method/Dilution (if any)/Name of the person or strain Keep open the agar plate for a few minutes until the media solidifies. Agar plates should stored upside down (inverted) to prevent condensation. Laboratory cultivation of microorganisms Importance of cultivating microbes in a laboratory: To study their growth patterns To study their colony formation Isolation of pure cultures of microorganisms Pure culture Pure culture is one in which only one strain of microbes is growing. This is important because if there were two species or strains being cultivated together, it would be difficult to identify which effect is being brought about by which species. A pure culture is obtained by isolating a single cell of a microorganism and permitting that cell to grow and form a colony. For a fungus, this usually means obtaining a single fungal spore and permitting that spore to germinate and produce mycelium. For a bacterium, a single cell is separated and permitted to form a colony. Viruses must be isolated within a suitable host. Methods to obtain a pure culture 1. Streak plate 2. Pour plate 3. Spread plate 1. Streak plate method This establishes a concentration gradient. That is, decreasing the quantity of microorganisms across the surface of a solidified medium. Streaking a plate involves drawing a loopful of microorganisms forward and backward across the surface of a solid medium until a single microbe falls off the loop at a time There are several methods available: Zig-zag method Quadrant method Zig- zag method Quadrant method ‘T’ method 2. Pour plate method This involves diluting a microbial sample in several tubes of liquid agar media or a suitable diluent (serial dilution). A 10-fold dilution series is prepared. Each tube contains fewer and fewer bacteria. The final diluent contains the least number of bacterial cells. 1 mL or 0.5 mL of the diluent can be used to prepare pour plates. Here, first add the diluent into the empty Petri plate and then pour the liquid medium on top of the sample and mix it properly. The petri plates prepared from the highest dilution will have the lowest number of microorganisms and form the most isolated colonies. Each colony represents a pure culture.  Pipette tips Micropipette Serial dilution Test tubes Petri plates Distilled water Nutrient agar 3. Spread plate method A diluted sample can be used in this method. During the inoculation, the cells are spread over the surface of a solid medium using a bent glass rod. Colony characteristics of microorganisms Purpose To determine the cultural characteristics of microorganisms. When grown on a variety of media, microbes will exhibit differences in the macroscopic appearance of their growth. These differences are called cultural/ colony characteristics. Used as a basis for separating microbes into different taxonomic groups. The cultural characteristics for all known microbes are found in Bergey’s Manual of Systematic Bacteriology. They are determined by culturing the organisms on nutrient agar slants and plates, or in nutrient broth. Colony Morphology Colony morphology of bacteria Elevation of bacterial colonies Colony morphology can vary dramatically with the medium in which the bacteria are growing. These beautiful snowflake-like colonies were formed by Bacillus subtilis growing on nutrient- poor agar. Tabulation of colony morphology/ characteristics Code of the Form Elevation Margin Surface Opacity Colour Size isolate Light KCB01A05 Circular Flat Entire Smooth Opaque yellow Small Creamy KCB02A11 Circular Flat Entire Smooth Opaque yellow Small KCB03A12 Circular Raised Entire Smooth Opaque White Medium Creamy KCB04B3 Circular Raised Entire Smooth Opaque white Small KCB05BTZ I Circular Raised Entire Smooth Opaque White Medium Creamy KCB06C9 Circular Flat Entire Smooth Opaque yellow Small KCB07C10 Circular Raised Entire Smooth Opaque Yellow Small Exercise Report the colony morphology of the following bacterial colonies in a table. Code Size Form Margin Surface Opacity Color 1 2 6 1 3 5 2 4 5 4 3 6 Exercise Report the colony morphology of the following bacterial colonies in a table. Code Form Margin Surface Opacity Color Size 1 Filamentous Undulate Rough Opaque Pale Medium yellow and orange 6 2 Circular Entire Smooth Opaque Lite pink Small 1 3 Filamentous Undulate Rough Opaque Pale Large brown 5 4 Circular Entire Smooth Opaque Yellow Small 2 5 Circular Entire Smooth Opaque Orange Small 6 Circular Entire Smooth Opaque Lite pink Small 4 3 Growth Patterns in a Slant  Preservation of cultures Sub culture/Passaging Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultures conditions for extended periods of time. Refrigeration Pure cultures can be successfully stored at 0-4 0C either in refrigerators or in cold-rooms. Applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) as the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continue slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime. Paraffin Method/Preservation by overlaying cultures with mineral oil A simple and most economical method of maintaining pure cultures of bacteria and fungi. Sterile liquid paraffin is poured over the slant (slope) of culture and stored upright at RT. Ensure anaerobic conditions and prevent dehydration of the medium. It helps microorganisms or pure culture to remain in a dormant state and therefore culture can be preserved form months to years (varies with species). Cryo-preservation Cryo-preservation or cryo-conservation is a process where organelles, cells, tissues, extracellular matrix, organs, or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures. Lyophilization A process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase The principle involved in lyophilizer The basic principle in lyophilization is sublimation, in which the conversion from a solid directly into a gas occur. Just like evaporation, sublimation occurs when a molecule gains enough energy to break free from the molecules around it. This process is commonly used in the food industry, biological sample preservation, and pharmaceutical applications, being a dehydrating technique suitable for heat-sensitive samples. Advantages of Lyophilization Processing a liquid with ease (and thereby simplifying aseptic handling) enhancing the stability of a dry powder as well as the product stability in a dry state. Removing water without having to heat the product excessively.  Microbial Growth Kasun Aththanayake MPhil in Microbiology (University of Kelaniya), BSc.in Biological Science (University of Kelaniya)  Microbial Growth Increase in number of cells, not cell size Division of bacterial cells occurs mainly through binary fission (transverse) – Parent cell enlarges, duplicates its chromosome, and forms a central transverse septum dividing the cell into two daughter cells. Alternative means Budding Conidiospores Fragmentation  Rate of Population Growth Time required for a complete fission cycle is called the generation, or doubling time. Each new fission cycle increases the population by a factor of 2 – exponential growth Generation times vary from minutes to days from species to species. Population is in balanced growth, therefore, doubling of DNA, RNA and protein of the cell population also occur.  Mother cell 0 2 1st generation 21 2nd generation 22 3rd generation 23 4th generation 24 Number of cells in ‘n’ number of generation = 2 n  Growth Curve Bacteria growing in batch culture produce a growth curve. Lag Phase  1. Lag Phase Increase in cell size but not multiplication. (There is no growth in terms of an increase in the number of cells.) Time is required for adaptation (synthesis of new enzymes) to a new environment. During this phase vigorous metabolic activity occurs but cells do not divide. (Rapid biosynthesis of cellular macromolecules, primarily enzymes and RNA.) This is a preparation for the next phase of the life cycle)  Enzymes and intermediates are formed and accumulate until they are present in a concentration that permits growth to start. Antibiotics have little effect at this stage. One hour to several days. The duration depends on the composition of the medium, age of inoculated cells, type of culture, etc. Log Phase  2. Log Phase The lag phase is followed by a period of rapid, balanced growth as a consequence of binary fission, characterized by cell doubling. There is a rapid increase in population which doubles regularly until a maximum number of cells is reached. There is an orderly increase in the chemical constituents of the cells.  Cells are mostly uniform in chemical composition, metabolic and physiological activities. It is the peak time of physiological activities and efficiency. The organism divides at a regular rate in a determined interval called as “Generation time” (time taken for the population to double). It can be calculated by the slope of the log phase.  The cells multiply at the maximum rate in this exponential phase, i.e. there is a linear relationship between time and logarithm of the number of cells. Mass increases in an exponential manner. This continues until one of two things happens: 1. one or more nutrients in the medium become exhausted 2. toxic metabolic products accumulate and inhibit growth Importance: Antibiotics act better at this phase. Stationary Phase  3. Stationary Phase Due to a growth-limiting factor such as the limited nutrients, insufficient oxygen supply, accumulation of toxic waste materials, and acidic pH of the medium slow down the microbial growth. This leads to the cessation of growth. The stationary phase results from a situation in which the growth rate and death rate are equal.  The number of new cells created is limited by the growth factor and as a result, the rate of cell growth matches the rate of cell death. The result is a “smooth,” horizontal linear part of the curve during the stationary phase. Death Phase  4. Death/ Decline Phase Continuous depletion of nutrients and building up of metabolic waste. Population size begins to decrease in a logarithmic rate. (Microbes die at a rapid and uniform rate.) Theoretically the entire population should die during a time interval equal to that of the log phase. However, practically this doesn’t occur. A small number of highly resistant organisms persist for an undetermined length of time.  Cells lose their ability to divide due to lack of nutrients, environmental temperature above or below the tolerance band for the species, or other injurious conditions.  Physical and Chemical Factors Affecting Growth Temperature Light pH Oxygen Water Saline requirements Pressure  1. Temperature Bacteria vary in requirement of temperature for growth. Temperature influences the rate of a chemical reaction through its action on cellular enzymes. Low temperatures slow down enzymatic actions, thereby slowing down the cellular metabolism and consequently cell growth. High temperatures cause denaturation and above a certain temperature organisms cannot live.  For each species there is a temperature range and growth does not occur above maximum or below minimum. Any organism will exhibit a range of temperatures over which it can grow, defined by 3 cardinal points. 1. Minimum temperature 2. Optimum temperature 3. Maximum temperature Optimum temperature: the temperature at which growth occurs best. There are several classes of microorganisms on the basis of the three cardinal points. 1. Psychrophiles – Cold-loving organisms - Minimum temp: below 0 0C Optimum temp: 10 - 15 0C Maximum temp: below 200C E.g: Photobacteria (marine), Gallionella (iron bacteria) 2. Psychrotrophs – Also found in cold environments – Minimum temp: 0 0C Optimum temp: 15 - 30 0C Maximum temp: 35 0C E.g: Aeromonas, Psychrobacter, Carnobacterium  3. Mesophiles – Most common type of microbes which can grow under room temperature. Minimum temp: 10 -15 0C Optimum temp: 20 – 40 0C Maximum temp: below 450C E.g: Common soil and aquatic bacteria 4. Thermophiles – Grow best at high temperatures. Minimum temp: 45 0C Optimum temp: 55 - 80 0C Maximum temp: above 100 0C E.g: Bacillus stearothermophilus 5. Extreme thermophiles – Grow at extremely high temperatures. Microbes are found near hydrothermal vents, and volcanoes. Minimum temp: 60 0C Optimum temp: 80 0C or higher Maximum: 115 0C or can even go up to 250 0C E.g: Sulfolobus acidocaldarius Growth Rates of Bacterial Groups at Different Temperatures  2. Light Bacteria (except phototropic) grow well in the dark. They are sensitive to UV light and other types of radiation. Cultures die if exposed to sunlight in a laboratory setting.  3. pH Bacteria are sensitive to variation in pH/ log [H +] ion concentration. Each type of bacteria has a pH range. The range of pH over which organisms grow is defined by 3 cardinal points. 1. The minimum pH below which organisms cannot grow. 2. The maximum pH above which organisms cannot grow. 3. Optimum pH at which bacteria grow best. Microbes can be categorized into several classes depending on their pH requirement. Neutrophiles: Organisms grow best at neutral or slightly alkaline pH (pH 7.2-7.6). Acidophiles: Organisms which grow at acidic pH (pH 2 or below or 5) Alkaliphiles: Organisms which grow at alkaline pH (pH roughly 8.5-11.5) Neutrophile e.g. Escherichia coli Acidophile e.g. Picrophilus oshimae Alkaliphile e.g. Bacilllus firmus  4. Oxygen (O2) Requirement Depending on the oxygen requirement, microbes can be categorized into 5 major classes. 1. Obligate aerobes: require oxygen for growth. 2. Obligate anaerobes: grow in the absence of oxygen. 3. Microaerophiles: grow best in low oxygen concentrations. 4. Facultative anaerobes: ordinarily aerobic, but can grow in the absence of oxygen as well. 5. Aerotolerant: considered anaerobic, but can tolerate the presence of O2, strictly fermentative. Obligate/ Strict Aerobes Require the presence of oxygen for their growth. Oxygen acts as the final electron acceptor in respiration (electron transport chain). The presence of atmospheric oxygen in the cell results in the formation of toxic products like free radical oxygen or superoxide radicals, hydrogen peroxides, and hydroxy radicals. These toxic products oxidize and rapidly destroy cellular constituents. Thus these organisms have enzymes catalase, peroxidase, and superoxide dismutase to degrade these toxic products. E.g: Fungi, Cyanobacteria, Pseudomonas Obligate anaerobes Cannot tolerate oxygen and die in the presence of oxygen. Similar to aerobes, toxic products are formed in the presence of atmospheric oxygen. But they lack the enzymes to degrade these toxic products. Therefore, in the absence of these enzymes, even small amounts of oxygen are very lethal. E.g: Clostridium, Methanobacterium Facultative Anaerobes Do not require oxygen for growth. But grow better in the presence of excess oxygen. In oxygen-poor environments, cellular respiration may occur anaerobically. E.g: Escherichia coli, Enterococcus, Saccharomyces Aerotolerant Anaerobes Grows equally well in the presence or absence of oxygen. E.g: Streptococcus pyogenes Microaerophiles Obligate aerobes. Grow best when oxygen concentration is less than atmospheric oxygen level (2-10%). Growth is damaged by atmospheric oxygen level. Eg: Campylobacter, Spirillum volutans, Treponema pallidum, Lactobacillus spp.  5. Water Availability/ Moisture Water is essential for bacterial protoplasm and drying is lethal. Treponema is highly sensitive to drying while some genera can withstand/tolerate drying for months. Endospores can survive in a dry state for several decades. Xerophiles: can tolerate low water levels, found in very dry environments such as deserts and salt beds. e.g: Trichosporonoides nigrescens Different organisms have different ranges of water activity (aw) over which they can grow.  6. Osmotic Effect Water diffuses from regions of high water concentration (low solute concentration) to regions of lower water concentration (higher solute concentration). Usually water tends to diffuse into the cell. This is because the cytoplasm contains many ions and other solutes dissolved in it. In an environment where the solute concentration exceeds that of the cytoplasm, water will leave the cell. Dehydrated cells cannot grow. Osmophiles- Live in environments with high solute concentrations. E.g: Molds that can survive on ham (salty)/sugary food.  7. Saline Requirements Halophiles- Require at least some NaCl for growth. Found in salt lakes, marine environments, heavily salted food and seafood, etc. E.g. Vibrio fischeri Extreme halophiles- Require 15-30% NaCl. They are organisms that live in marine environments such as Halobacterium salinarum. Halotolerant- They can survive as the concentration of NaCl increases, however, the optimum is at low NaCl concentration. The classic example is Staphylococcus aureus, and actually, this property can be used as a key diagnostic trait.  8. Pressure Hydrostatic pressure is a well-known physical parameter that is now considered an important variable of life since organisms can adapt to pressure changes. Humans consider that moderate values for hydrostatic pressure are those around 1 atm (= 0.1 MPa = 14.7 psi). A piezophile (barophile) is an organism with optimal growth under high hydrostatic pressure (usually living in deep-sea sediments, hydrothermal vents,…) or, more operationally, an organism that has its maximum rate of growth at a hydrostatic pressure equal to or above 10 MPa (99 atm). Hyperpiezophile is defined as an organism that has a maximum rate of growth above 50 MPa (493 atm). E.g. Thermococcus piezophilus  Obligate piezophiles refer to organisms that are unable to grow under lower hydrostatic pressures, such as 0.1 MPa. In contrast, piezotolerant organisms are those that have their maximum rate of growth at a hydrostatic pressure under 10 MPa, but that nevertheless are able to grow at lower rates under higher hydrostatic pressures. Measurement of Cell Growth by Cell Number and Cell Mass  Measurement of cell number To determine the rates of microbial growth and death, it is important to enumerate microorganisms (to determine their numbers). Estimating the number of microbial cells in a sample, is known as a count. It is essential to determine the number of microbes in a given sample. (e.g. to determine the safety of food, water, and other commercial products by knowing the levels of microbes in them)  Two major approaches are used to measure cell numbers. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells.  Bacterial Growth Estimation by Cell Number Direct Methods Indirect Methods 1. Total Cell Count Most Probable 2. Viable Count Number (MPN) 3.Membrane Filter Technique Technique  1. Total Cell Count (Direct Microscopy) Direct cell count refers to counting the cells in liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample. Cells are counted directly under the microscope or by an electronic particle counter.  Direct counts provide an estimate of the total number of cells in a sample; both live (viable) and dead cells. Two methods are available. 1. Counting chamber method 2. Electronic counting method  I. Counting Chamber method Special slides marked with a known area and known depth with a grid on the bottom are used. Constructed to retain a known volume of suspension. Specially used to count microbial cells, fungal spores and blood cells. The number of particles in a known volume can be calculated with the aid of a microscope by using the dilution factor. Then the original number in the samples can be calculated. There are several types of counting chambers available. Haemocytometer Petroff-Hauser counting chamber Counting Chamber Method: The chamber grid  Method 1. Shake the suspension to spread the cells evenly in the suspension 2. Transfer a small volume of the suspension onto the Haemocytometer, using a pasture pipette, and put the coverslip on the chamber. 3. Observe the Haemocytometer under the microscope, and count the number of cells present in several large squares. 4. Calculate the average number of cells per large square 5. Using the average number of cells per large square and the volume of the large square, calculate the number of cells present in 1 mL of the sample. 6. Express the cell concentration of the suspension as the number of cells per mL (1 mL = 103 mm3)  How to count cells using a Haemocytometer? Haemocytometer has 2 counting chambers and a special glass cover slip. Each chamber contains a grid with exact dimensions. There are two supports on either side of a counting chamber The cover glass is placed on the supports to maintain the depth of the chamber as 0.1 mm. Haemocytometer grid 1 mm Depth of Haemocytometer = 0.1 mm (total cells counted)/(squares counted)*10-4*initial volume*dilution factor = total number of cells Note: 10-4 is the volume of squares on the hemocytometer (0.1 mm3). The dilution factor will be 1 unless you have diluted your cell suspension with trypan blue. II. Electronic counting method An electronic counting device (Coulter counter) is used. The microbial suspension is forced through a small hole in the coulter counter. An electric current flows through the hole and electrodes placed on both sides of the wall measure its electric resistance. Every time a microbial cell passes through the hole, conductivity drops, and the cell is counted. This is largely used to count large microbes like protists, yeasts, fungal, and microalgal cells. Also used to count red and white blood cells. Coulter counter  2. Viable Count (Standard Plate Count – SPC) Microorganisms in a sample are diluted or concentrated. Grown on a suitable medium- the development of growing microorganisms (colony formation on agar plates). Assume one cell gives rise to a single colony of bacteria. The colonies are counted and the results are expressed as Colony Forming Units (CFUs). This is used to estimate the number of microorganisms in the original sample. So the number of living microbial cells is measured. - Viable count  Method 1. Prepare a dilution series of the sample to be estimated. 2. Plate on nutrient agar using either the pour plate method or spread plate method 3. Incubate the plates at 370 C and count the number of colonies. Dilution series  Pour Plate Method Prepare a dilution series of the sample. Pipette out 1 mL from each sample into agar plates. Pour molten agar at about 450 C into each petri plate and mix in circular motions. Allow the agar to set. Incubate at desired temperature. Use at least 3 plates for one dilution for greater accuracy. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/ mL in the original sample.  Spread Plate Method Pre-prepared agar plates are required. Prepare a dilution series of the sample. Pipette out 0.1 mL from each sample into agar plates. Dip the L-shaped glass/ metal spreader into alcohol. Flame the spreader over a Bunsen burner. Spread the sample evenly over the surface of the agar using the sterile spreader, carefully rotating the Petri dish underneath at the same time. Incubate the plates at the desired temperature. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/ mL in the original sample. Pour Vs Spread Results Count the number of colonies of plates with 30 – 300 colonies. CFU calculation CFU/mL = (no. of colonies x dilution factor) / volume of culture plate Dilution Factor = 1/ Dilution Counting of colonies on a plate Each Petri dish is taken for counting of colonies. For this purpose, various instruments such as the Quebec colony counter and electronic colony counter are used. The Quebec colony counter is one of the simplest colony counters used in small laboratories. In this, the Petri dish containing bacterial colonies is mounted on a platform.  When the Petri dish is illuminated from beneath, the visible colonies can be counted with the help of its lens that provides X1.5 magnification. Electronic colony counter is a highly improved device. The Petri dish is placed on its illuminated stage, the count bar is depressed, and the precise number of colonies is instantly displayed on a digital readout. Electronic (automatic) colony counter  3. Membrane Filter Technique The sample is passed through a membrane using a filter funnel and vacuum system. The membrane has a known uniform porosity of predetermined size (generally 0.45 µm) sufficiently small to trap microorganisms. Bacteria in the sample are concentrated on the surface of the membrane. The membrane is placed on an appropriate medium. Method Filter the sample through a membrane filter. Place the filter on an agar medium. Incubate at 370 C and count the colonies. Yellow color colonies on membrane filters – Red, maroon, or pink colonies - Presumptive Total coliforms and presumptive coliforms Enterococci Uses of Membrane Filters Membrane filters are used extensively in the laboratory and in the industry to sterilize heat-labile fluid materials. Effective and acceptable technique to monitor drinking water. Useful for bacterial monitoring in the pharmaceutical, cosmetics, electronics, and food and beverage industries. Indirect Measurement of Microbial Cell Number – The Most Probable Number (MPN) Technique This is an estimation of the number of cells in a population determined as a statistical probability falling within a particular range. The MPN is calculated by observing the number of tubes that display bacterial growth in broth media and bring about a metabolic change, such as lactose fermentation. Method Calculation using MPN Table  Measurement of Cell mass Direct method – Measurement of dry weight Cell mass is determined directly by weighing whole cells. Measuring both live (viable) and dead cells, it is a total measurement. Takes dry weight of all cells. Must wash the cells well to eliminate media. In this, cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. Centrifugation (cells)  Indirect methods Estimated by measuring relatively constant biochemical components of microbial cells (proteins, ATP, lipopolysaccharides, peptidoglycan, and chlorophyll). Can also be estimated by measuring turbidity. Various procedures based on the detection of specific microbial macromolecules or metabolic products can be used to estimate number of microbes. E.g: Peptidoglycan can be quantified, and because this biochemical occurs exclusively in the cell wall of bacteria, the concentration of peptidoglycan can be used to estimate bacterial numbers.  Such biochemical approaches for determining microbial mass depend on the development of analytical chemical procedures for quantifying the particular biochemical and determining what proportion of bacterial cell is composed of the specific biochemical constituent.  1. Nitrogen content Use biochemical assays to measure total amounts of nitrogen in the cell culture. Knowing the percentage of the cell mass that is generally nitrogen, one can now calculate to the total cell mass. 2. Turbidimetric assays Bacterial cells will absorb and/ or scatter light. The amount of light that is absorbed or scattered is proportional to the mass of cells in the culture. A colorimeter (monochromatic or uses just one wavelength) or a more versatile instrument known as a spectrophotometer (wavelength can vary) can be used to measure turbidity (cloudiness) by measuring the Optical Density value.  Measurement of the turbidity of the suspension Microbial cells scatter light passing through the suspension. Therefore, the suspension will look turbid (cloudy). The amount of scattering is directly proportional to the concentration of cells. The extent of light scattering is measured by a spectrophotometer.  Method Prepare a series of dilution from the given bacterial suspension and count the number of cells in each dilution using the Haemocytometer. Measure the turbidity (absorbance) of each dilution. Plot a graph- turbidity (absorbance) against the number of cells. Measure the turbidity of the given samples. Get the bacterial count using the standard graph.  Before measuring the absorbance, select the specific wavelength that gives the highest sensitivity via a filtered scan. Use the medium used in suspension without the bacteria as the blank. Measure the turbidity as soon as possible to avoid the cell number increasing through budding.  Growth measurement of filamentous fungi On a solid medium, a fungus grows radially producing an almost circular outline of the colony. One of the earliest methods of measuring fungal growth in a petri dish was to measure the diameter of the radius of the mycelium at the marked places right- angled to each other. Then the average of these crossed diameters can be taken.  Fungal growth rate tube The total length of the hyphae can be measured by using a fungal growth rate tube. Inoculation is done at one end. Measurements are made along one radius of the growing mycelium on the traveling microscope. Cellular Medium cap Day 1 Day 2 Day 3 Nylon Fungal  PRINCIPLES AND METHODS OF STERILIZATION - PART B Kasun Aththanayake MPhil in Microbiology (University of Kelaniya), BSc.in Biological Science (University of Kelaniya) LEARNING OUTCOMES To understand the; Mode of action of – alcohols, aldehydes, halogens, phenols, heavy metals, Detergents: quaternary ammonium compounds. 2 3 METHODS OF MICROBIAL CONTROL 4 CHEMICAL AGENTS FOR DISINFECTION Treatment of inert surfaces and heat-labile materials can be accomplished through the use of disinfectants that are chemical agents. The following factors should be considered when choosing the correct chemical agent for disinfection or antisepsis; Type and level of microbial contamination Concentration of active ingredient Duration of contact between disinfectant and item to be disinfected pH Temperature Humidity Presence of organic matter or soil load 5  FEATURES OF CHEMICAL AGENTS USED IN DISINFECTION AND ANTISEPSIS Resistance to inactivation - Be fast acting even in the presence of organic substances, such as those in body fluid. Broad spectrum - Be effective against all types of infectious agents without destroying tissues or acting as a poison if ingested. Non–toxic to organisms. Non–corrosive for materials and low tension – Easily penetrate the material to be disinfected without damaging or discoloring the material. 6  Stability upon storage - Stable even when exposed to light, heat, or other environmental factors. Ease of preparation/availability - Be inexpensive and easy to obtain, prepare and use or not have unpleasant odors. MECHANISMS OF ACTION OF CHEMICAL AGENTS Different chemical agents possess different mechanisms of action upon the interaction of the antiseptic or disinfectant with the cell surface followed by penetration into the cell and action at the target site(s). Different mechanisms include: Membrane damage and disruption. Release of lipopolysaccharide layer of Gram-negative bacteria. Cross-linking of proteins causing protein inactivation. Amino acid leakage. DNA disruption and damage. Oxidation of compounds – forming reactive species. 8 TYPES OF CHEMICAL AGENTS GASEOUS AGENTS Ethylene oxide Betapropiolactone Vapour phase Hydrogen peroxide CHEMICAL AGENTS LIQUIDS AGENTS Alcohols Aldehydes Halogens Phenols Quaternary Ammonium Compounds (QACs) are a type of chemical that is used to kill Heavy metals bacteria, viruses, and mold. They are often found in disinfectants and in cleaning products Quaternary ammonium compounds that are used in places such as hospitals, day care centers, restaurants, and homes. 9 10  ETHYLENE OXIDE Ethylene oxide (EtO) is both microbicidal and sporicidal and kills by combining with cell proteins. Rapidly penetrates packing materials, even plastic wraps. As alkylating agents, they attack proteins, nucleic acids, and other organic compounds; both are particularly reactive with sulfhydryl and other enzyme- reactive groups Takes place in an ethylene oxide sterilizer General conditions enzyme-reactive 5 to 8 hours at 38°C or 3 to 4 hours at 54°C with a relative humidity maintained at 40 to 50%, EtO concentration at 700 mg/liter Extensive aeration of the sterilized materials is necessary to remove residual EtO because it is so toxic. 11  ALCOHOLS Most widely used disinfectants and antiseptics. They are bactericidal and fungicidal but not sporicidal some lipid-containing viruses are also destroyed. Most widely used alcohols are - ethyl alcohol (ethanol, alcohol), isopropyl alcohol (isopropanol, propan-2-ol) and n-propanol. Alcohols work through the disruption of cellular membranes, solubilization of lipids, and denaturation of proteins by acting directly on S-H functional groups. 12  Because of the lack of sporicidal activity, alcohols are not recommended for sterilization but are widely used for both hard-surface disinfection and skin antisepsis. The antimicrobial activity of alcohols is significantly lower at concentrations below 50% and is optimal in the 60 to 90% range. Alcohol-water mixtures are additionally more penetrating than pure alcohols. 14  PHENOLICS Phenolics are phenol (carbolic acid) derivatives. These biocides act through membrane damage and are effective against enveloped viruses, rickettsiae, fungi and vegetative bacteria. Phenol induces progressive leakage of intracellular constituents, including the release of Potassium, the first index of membrane damage. They also retain more activity in the presence of organic material than other disinfectants. Mainly used on inanimate objects. Available commercial products are Lysol, Pine-Sol, Amphyl, O-syl, Tergisyl, Vesphene, L- Phase and Expose. 15  ALDEHYDES – GLUTARALDEHYDE Glutaraldehyde is an important dialdehyde that has found usage as a disinfectant and sterilant, in particular for low-temperature disinfection and sterilization. Shows a broad spectrum of activity against bacteria and their spores, fungi, and viruses. Involves a strong association with the outer layers of bacterial cells, specifically with unprotonated amines on the cell surface, possibly representing the reactive sites. Alkylation of sulfhydryl, hydroxyl, carboxyl, and amino groups of microorganisms, which alters RNA, DNA, and protein synthesis. ≥2% aqueous solutions of glutaraldehyde, buffered to pH 7.5–8.5 with sodium bicarbonate is effective. However, the time of exposure varies. 16 https://cmr.asm.org/content/cmr/12/1/147.full.pdf 17 ALDEHYDES – FORMALDEHYDE Formaldehyde solution (formalin) is an aqueous solution containing 34 to 38% (wt/wt) CH2O with methanol to delay polymerization. Can be also used in its gaseous form. Its clinical use is generally as a disinfectant and sterilant in liquid or in combination with low-temperature steam. Formaldehyde is bactericidal, sporicidal, and virucidal, but it works more slowly than glutaraldehyde. Disinfects by the interaction with protein results from a combination with the primary amide as well as with the amino groups. Formaldehyde acts as a mutagenic agent and as an alkylating agent by reaction with carboxyl, sulfhydryl, and hydroxyl groups. 18 19 HALOGENS Chlorine and iodine based compounds are the most significant microbicidal halogens that have been used for both antiseptic and disinfectant purposes. 20 HALOGENS - CHLORINE AND CHLORINE COMPOUNDS Hypochlorites, the most widely used of the chlorine disinfectants. Broad spectrum - (5.25%–6.15% sodium hypochlorite – household bleach.) Can be bactericidal, fungicidal, sporicidal, tuberculocidal and virucidal. Biocidal effects on mycoplasma and vegetative bacteria, at higher concentrations are sporicidal. The microbicidal activity of chlorine - undissociated hypochlorous acid (HOCl). Alternative compounds - chlorine dioxide, sodium dichloroisocyanurate, chloramine-T. 22  Mechanism of action - Oxidation of sulfhydryl enzymes and amino acids, ring chlorination of amino acids, membrane disruption, loss of intracellular contents, decreased uptake of nutrients, inhibition of protein synthesis, decreased oxygen uptake, oxidation of respiratory components, decreased ATP production, DNA breakage, and depressed DNA synthesis. 23 HALOGENS - IODINE AND IODOPHORS Iodine is rapidly bactericidal, fungicidal, tuberculocidal, virucidal and sporicidal. Iodophors - complexes of iodine and a solubilizing agent or carrier, which acts as a reservoir of the active “free” iodine. Iodine rapidly penetrates into microorganisms and attacks key groups of proteins, nucleotides and fatty acids which culminates in cell death. Lipid-enveloped viruses are more sensitive to iodine or iodophor attack. 24  HEAVY METALS Metals such as silver, iron, and copper could be used for environmental control, disinfection of water, or reusable medical devices or incorporated into medical devices (e.g., intravascular catheters). Antimicrobial activity of metals include antisepsis, disinfection, and anti-infective chemotherapy. Silver and silver containing compounds plays a major role in disinfection. Efficient bactericidal agents 25 HEAVY METALS - SILVER AND SILVER CONTAINING COMPOUNDS The most important silver compound currently in use is silver sulfadiazine (AgSD), although silver metal, silver acetate, silver nitrate, and silver protein, all of which have antimicrobial properties. Mostly bactericidal, however sometimes show virucidal properties. Silver nitrate - The mechanism of the antimicrobial action is closely related to their interaction with thiol (sulfydryl, SH) groups leading to protein denaturation. AgSD - a broad spectrum of activity 26 Possible mechanisms of antibacterial activity of silver nanoparticles (research gate) 27 MICROBIAL RESISTANCE TO ANTISEPSIS AND DISINFECTION 29  Different microorganisms respond to disinfectants and antimicrobial agents using different mechanisms. They develop resistance mainly due to the differences in cellular structure, composition, physiology and formation. Increased microbial resistance to disinfectants, sterilants and antimicrobial agents!!! 3 1 32 33 34 ACTIVITY Tutorial questions – Unit V 1. Advantages, disadvantages and limitations of hot air oven, autoclave 2. Compare and contrast dry heat sterilization and moist heat sterilization 3. Compare and contrast autoclave and pressure cooker 4. Highlight the working principle of pasteurization 5. Compare pasteurization and tyndallization 6. Highlight the low temperature methods of sterilization 7. Compare depth filtration and surface filtration 8. Explain the principle of laminar flow 9. Highlight the types of HEPA filters in microbiology 10. Explain the role of Ozone and Hydrogen Peroxide as a sterilant /disinfectant / antiseptic. 11. List the resistant mechanisms that microorganisms possess to develop resistance against sterilants and disinfectants. 35  Sterilization method Mechanism of action Antimicrobial Advantage Disadvantage Limitation Applications / Uses activity s s s PHYSICAL AGENTS Heat sterilization 1. Moist heat 2. Dry heat Radiation 1. Ionizing radiation a. Gamma rays 2. Non – ionizing radiation (UV rays) MECHANICAL AGENTS 1. Filtration b. Membrane filters c. Air filters CHEMICAL AGENTS 1. Ehylene oxide 2. Alcohols 3. Phenols 4. Aldehydes 5. Chlorine compounds 6. Iodine and iodophores 7. Silver containing 36 compounds THANK YOU! 37  PRINCIPLES AND METHODS OF STERILIZATION - PART A Kasun Aththanayake MPhil in Microbiology (University of Kelaniya), BSc.in Biological Science (University of Kelaniya) LEARNING OUTCOMES To understand the; Physical Methods and their mode of action Heat: Dry heat – Hot Air Oven, Incineration, Moist heat- Autoclave, Tyndallization(fractional sterilization) Filtration - Types of filters , Laminar air flow Radiation methods: UV radiation, γ - rays and cathode rays Chemical methods: Disinfectants, Antiseptics, Sanitizers, Microbicides – Bactericide, Virucide, Fungicide and Sporicide, Microbistatic-bacteriostatic and fungistatic agents. Use and mode of action of – alcohols, aldehydes, halogens, phenols, heavy metals, Detergents: quaternary ammonium compounds. 2 Importance of microbial control. WAY OF MICROBIAL CONTROL 4 TERMINOLOGY Sterilization Disinfection Antisepsis Sanitization Microbicide – Bactericide, Virucide, Fungicide and Sporicide, Microbistatic -bacteriostatic and fungistatic agents 5 Disinfection: The process of Sterilization: Any process that elimination of pathogenic eliminates, removes, kills, or microorganisms. So number of harmful deactivates all forms of life and microorganisms will be reduced other biological agents present in a specified region However, the process is NOT effective in case of spores. Made region completely free from microbes. Kill both vegetative cells Disinfectants are agents, usually and spores chemical, used to carry out disinfection and are normally used Sterilants are chemical agents used for only on inanimate objects. sterilization. Antisepsis: The process of reducing the viable number of microorganisms from any living surfaces to an extent that infection can be hardly produced This is usually done using optimum concentration of chemical substances (Antiseptics) Sanitization is closely related to disinfection. In sanitization, the microbial population is reduced to levels that are considered safe by public health standards. Sanitizers are used in inanimate objects and applied on living tissue. ANTIMICROBIAL AGENTS; MICROBICIDE OR MICROBISTATIC? Germicide / Microbicides are agents that kill pathogens (and many nonpathogens) but not necessarily endospores. It’s a form of disinfectant and can be a bactericide, fungicide, algicide, or viricide. 8 Microbistatics [Greek statikos, causing to stand or stopping] are agents that do not kill, but only prevent growth. If these agents are removed, growth will resume. They can be bacteriostatic and fungistatic 10 METHODS OF MICROBIAL CONTROL 11 PHYSICAL AGENTS: HEAT The most commonly used physical method of sterilization. Principle - At high heat / a large temperature rise, the microbial proteins undergo denaturation, the microbial lipids undergo melting and the DNA denatures lead to microbial death resulting in sterilization. Flaming Dry Hot air oven Heat Incineration Boiling Moist Autoclave 1. DRY HEAT Expose to direct dry heat. The simplest method is by direct heating through flaming. Less penetrating ability, therefore requires high heat and long hours to obtain accurate sterilization. Mainly used to sterilize laboratory glassware and equipment. Eg: flaming, hot air oven, incineration Sterilizing equipment by flaming (until it turns red hot) 1 1 Incineration: a waste treatment process that involves the combustion of organic substances contained in waste materials. This method also burns any organism to ash. It is used to sterilize medical and other biohazardous waste before it is discarded with non-hazardous waste HOT AIR OVEN Mechanical model: Made up of stainless steel or aluminum chamber. The chamber is enclosed within a thick layer of glass- insulation that makes up the outer wall. The door consists of a similar type of insulation and is generally of flanged type. Thermostatically controlled heaters are fixed for temperature control. A fan is fitted at the back of the chamber to circulate the air inside the chamber. 15 Equipment/materials sterilized by Hot air oven Glassware (Petri dishes, flasks, pipettes, and test tubes) Powders (starch, zinc oxide, and sulfadiazine) Materials that contain oils Metal equipment (scalpels, scissors, and blades) 16 Working Principle: Accomplished by conduction. (Conduction is the process by which heat energy is transmitted through collisions between neighboring atoms or molecules.) The heat is absorbed by the outside surface of the item, and then passes toward the center of the item, layer by layer. The entire item will eventually reach the temperature required for sterilization to take place. Dry heat does most of the damage by oxidizing molecules. The essential cell constituents are destroyed and the organism dies. 18  The temperature is maintained for almost an hour to kill the most difficult of the resistant spores. The most common time-temperature relationships for sterilization with hot air sterilizers are 170°C (340°F) for 30 minutes, 160°C (320°F) for 60 minutes, and 150°C (300°F) for 150 minutes or longer depending up the volume.  INCINERATION Incineration, or thermal oxidation is the process of oxidizing combustible materials by raising the temperature of the material above its auto-ignition point in the presence of oxygen and maintaining it at high temperature for sufficient time to complete combustion to carbon dioxide and water. Auto-ignition point: the lowest temperature at which the fuel will spontaneously ignite in a normal atmosphere without an external source of ignition such as a flame or spark. 20 It is used to sterilize medical and other biohazardous waste before it is discarded with non-hazardous waste. Bacteria incinerators / Micro incinerators are mini furnaces that incinerate and kill off any microorganisms that may be on an inoculating loop or wire. Sterilization usually occurs via infrared heat at a temperature of 815°C (1500°F). 2. MOIST HEAT The simplest method is by boiling. High penetrating ability, therefore requires less heat and short hours to obtain accurate sterilization. Moist heat destroys microorganisms by the irreversible denaturation of enzymes and structural proteins. Sterilization in saturated steam thus requires precise control of time, temperature, and pressure. Eg: boiling, autoclaving, pressure cooker, Tyndallization Sterilization by boiling 22 23  AUTOCLAVE Mechanical model: A double-jacketed steam chamber with devices that permit the chamber to be filled with saturated steam and maintained at a designated temperature and pressure for a period of time. It has a body, an internal heating system, a container to hold material, its cover fixed with a pressure gauge, a safety valve, pressure release valve. Lid is tightened with the help of screws and a gasket seals the body and lid. 24  A jacket, paddle lifter, timer, and indicator are also provided with large sized autoclaves. Autoclaves may be constructed of aluminum, mild steel, stainless steel or gun metal. Industrial autoclave can accommodate large trolley containing huge number of glassware’s or large bioreactors. Equipment/materials sterilized by autoclave Glassware (Petri dishes, flasks and test tubes) Growth media Pipette tips Micro-centrifuge tubes 26 Medical waste autoclave 28 Working Principle – Moist heat under pressure concept Heat moisture with saturated steam under pressure destroys bacterial endospores. Steam is described as saturated when it is at a temperature corresponding, to the boiling point (100°C), and when it is subjected to pressure the temperature goes above 100°C. 29 Bacterial endospores will be killed only if they are kept at 15pSI, 121°C for 15 minutes 30 How to check the accuracy of the autoclaving process?  PASTEURIZATION The process of heating liquids to destroy microorganisms that can cause spoilage or disease. Pasteurization does not kill all organisms and is therefore not a method of sterilization. Pasteurization does, however, reduce the microbial load, and the number of viable microorganisms in a sample. 32 34 TYNDALLIZATION Also called fractional sterilization. The process involves repeated cycles of boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. 35  The procedure only works for media that can support bacterial growth - it will not sterilize plain water. Drawback - thermophilic and anaerobic bacteria, whose endospores do not germinate in the broth during incubation period, generally escape killing during this process. PHYSICAL AGENTS - FILTRATION Filtration is an excellent way to reduce the microbial population in solutions of heat-sensitive material, and sometimes it can be used to sterilize solutions. Two main types – Depth filters and Membrane filters Depth filters are typically made up of layered fibrous material such that the outermost layer is designed to catch bigger particles, and the inner, more tightly packed layer is positioned to catch finer particles. 37 MEMBRANE FILTERS Circular filters that have porous membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, or other synthetic materials. Membranes with pores about 0.2 μm in diameter are used to remove most vegetative cells, from solutions ranging in volume from 1 ml to many liters. The membranes are held in special holders made of glass fibers to remove larger particles that might clog the membrane filter. 38  The solution is pulled or forced through the filter with a vacuum. Membrane filters remove microorganisms by screening them out much as a sieve separates large sand particles from small ones. These filters are used to sterilize pharmaceuticals, ophthalmic solutions (eye drops), culture media, oils, antibiotics, and other heat-sensitive solutions. 40 AIR FILTERS – HEPA FILTER Laminar flow biological safety cabinets employs high-efficiency particulate air (HEPA) filters, which remove 99.97% of 0.3 μm particles, are one of the most important air filtration systems. HEPA filters project a vertical curtain of sterile air across the cabinet opening. The laminar air flow apparatus sucks the air in the room continuously and blows out-the air through a pack of filters. The air is blown out uniform velocity and in parallel flow line. 41 https://www.youtube.com/watch?v=Q6qfZ2RR54Q 43 PHYSICAL AGENTS - RADIATION Various mechanisms are employed by different radiation types for sterilization. They primarily cause DNA damage leading to the destruction of the microorganisms. Types of radiation - Ionizing radiation and Non – ionizing radiations 44 45  IONIZING RADIATION Ionizing radiation is an excellent sterilizing agent and penetrates deep into objects. It will destroy bacterial endospores and vegetative cells, both prokaryotic and eukaryotic; however, ionizing radiation is not always as effective against viruses. Radiation of very short wavelength or high energy which can cause atoms to loss electrons or ionize. It breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, and polymerizes some molecules. Eg – Gamma rays, X rays, electron beams 46  - GAMMA RAYS The gamma rays cause both excitation and ionization by targeting vital molecules present in the cell. The death of a cell takes place because of ionization of target molecules (like water) present either inside or outside the cell. Gamma radiation from a cobalt 60 source is used in the cold sterilization of antibiotics, hormones, surgical threads, and plastic disposable supplies such as syringes. 47 NON-IONIZING RADIATION – UV RADIATION Naturally present in sunlight, however, most is filtered when it reaches the atmosphere. UV portion of the electromagnetic spectrum includes all radiations from 15-390 nm. UV-radiation around 260 nm is the most lethal to microorganisms. Short wavelength and high energy UV radiation have very little penetrating ability. Only the microorganisms present on the surface of an object where they are exposed directly to ultraviolet radiation are susceptible to destruction. 48  UV radiation from UV lamps is used extensively in hospital operating rooms, in aseptic filling rooms, in the pharmaceutical industry where sterile products are dispensed into vials or ampules, and in the food and dairy industries for treatment of contaminated surfaces. Commercial units containing UV lamps are available for water treatment. 49 51 ACTIVITY Tutorial questions 1. Advantages, disadvantages and limitations of hot air oven, autoclave 2. Compare and contrast dry heat sterilization and moist heat sterilization 3. Compare and contrast autoclave and pressure cooker 4. Highlight the working principle of pasteurization 5. Compare pasteurization and tyndallization 6. Highlight the low temperature methods of sterilization 7. Compare depth filtration and surface filtration 8. Explain the principle of laminar flow 9. Highlight the types of HEPA filters in microbiology 10. Advantages, disadvantages and limitations of Gamma rays, Cathode rays and UV rays in sterilization http://www.biologydiscussion.com/microorganisms/sterilizatiion/top-3-physical-methods-used-to-kill- microorganisms/55243 52 THANK YOU! 53