Microbial Growth and Control PDF

**[Microbial grow and control]** **Bacterial Growth** - The rate at which bacteria grow and divide depends: - on the species (example: Escherichia coli 20 minutes, Mycobacterium tuberculosis 2 weeks) - on the nutritional status of the environment (example E. coli, o...

**[Microbial grow and control]** **Bacterial Growth** - The rate at which bacteria grow and divide depends: - on the species (example: Escherichia coli 20 minutes, Mycobacterium tuberculosis 2 weeks) - on the nutritional status of the environment (example E. coli, oscillates from a minimum of 20 min up to 2 h) it means that bacteria can adjust their growth speed depending on the conditions they find themselves in **Bacterial reproduction: asexual, by binary fission** - In most prokaryotes growth of a cell continues until the cell divides into two new cells **Unusual division patterns** - There are groups of bacteria that use unusual forms or patterns of cell division to reproduce.  - Some of these bacteria grow to more than twice their starting cell size and then use multiple divisions to produce multiple offspring cells.  - Some other bacterial lineages reproduce by budding.  small new cell** **forms and grows out of the surface of the parent cell, eventually pinching off to become a separate, independent cell - Still others form internal offspring that develop within the cytoplasm of a larger \"mother cell". Budding: **Genetic changes in bacterial populations** - Mutation - Spontaneous - Mutagens-induced - transposons - Horizontal or lateral genetic transference - Transformation - Conjugation - Transduction - Vesicles **Mutations -- changes in the DNA** - Point mutation -- addition, deletion or substitution of a few bases - Missense mutation -- causes change in a single amino acid - Nonsense mutation -- changes a normal codon into a stop codon - Silent mutation -- alters a base but does not change the amino acid **Bacterial Mutants** - Antimicrobial resistant mutants: can grow on medium that contains antimicrobial agent (For instance, AmpR mutation causes bacteria to be resistant to ampicillin, a common antibiotic related to penicillin). This means that even when ampicillin is present, these bacteria can survive and continue growing. - Antibiotic sensitive mutants, the opposite, they cannot grow on antimicrobial presence. - Nutritional mutants: - Prototrophic -- can synthesize own nutrients from minimal media. - Auxotrophic- need a supplement added to media. **Horizontal or lateral genetic transference** - Bacteria do not exchange genes by meiosis (sexual reproduction) - Important for bacteria- to adapt to new toxins, new phages, new environments. - bad for human host- new strains resistant to antimicrobials. - 1990 - reappearance of Tuberculosis in New York - strain resistant to 7 antimicrobials (related to strain in China) - Four main mechanisms: - Transformation - Conjugation - Transduction - Vesicles **Bacterial transformation** - A donor DNA molecule is taken up from the external environment and incorporated into the genome of the recipient cell. - It was the first mechanism of genetic transfer discovered in bacteria (Griffith, 1928). - Most bacteria do not have a natural ability for DNA uptake, however, chemical methods or electroporation can be use to introduce plasmids. **Conjugation** - Occurs usually by direct contact between bacterial cells of the same species. - It has been described also between bacterial cells and plants, animals and fungi. - Conjugation results in one-way transfer of DNA from a donor (or male) cell to a recipient (or female) cell through the sex pilus. = Bridge between two bacteries - The mating type (sex) of the cell depends on the presence (male) or absence (female) of a conjugative plasmid, such as the plasmid F in Escherichia coli. **Transduction** - Transduction is the process by which a virus transfers genetic material from one bacterium to another. Viruses called bacteriophages are able to infect bacterial cells and use them as hosts to make more viruses. After multiplying, these viruses assemble and occasionally remove a portion of the host cell\'s bacterial DNA. Later, when one of these bacteriophages infects a new host cell, this piece of bacterial DNA may be incorporated into the genome of the new host. - There are two types of transduction: - Generalized: the bacteriophages can pick up any portion of the host\'s genome - Specialized: the bacteriophages pick up only specific portions of the host\'s DNA.  **General transduction**  [Culture methods] **Techniques in Microbiology** - To gain greater insight into microorganisms, microbiologists commonly attempt to isolate and culture the organism of interest. - Identification takes five basic steps: - Inoculation (into suitable growth media) - Incubation (under suitable growth conditions) - Isolation (onto selective media) - Inspection (microscopic, biochemical, genetic\...) - Identification **First bacterial growth media** - The first media for bacterial growth was used by Louis Pasteur -- urine or meat broth - Problem: both liquid medium, which produce diffuse growth - Developing idea: to create a solid medium, which will allow the growth of individual, discrete colonies. - A colony is a macroscopically visible collection of millions of bacteria originating from a single bacterial cell - Cooked cut potato by Robert Koch -- earliest solid medium - Gelatin -- not satisfactory, it liquefies at 24ºC **Agar** - dried amorphous, gelatin-like, non-nitrogenous extract from Gelidium and other red algae, a linear galactan sulfate, insoluble in cold but soluble in hot water - Used for preparing solid medium - No nutritive value - Not affected by the growth of the bacteria - Melts at 98ºC and sets at 42ºC - 2% agar is employed in solid medium  **Types of growth media** 1.Based on their consistency: - solid medium - liquid medium - semi solid medium 2\. Based on the chemical composition: - simple medium - complex medium - synthetic or defined medium - special media (Enriched; Enrichment; Selective; Indicator; Differential; Sugar media) 3\. Based on oxygen requirement: - Aerobic media - Anaerobic media **Types of media based on their consistency:** generic, when we don't now witch one using. - Solid media -- contains 2% agar - Allows to see colony morphology, pigmentation, hemolysis. - Eg: Nutrient agar, Blood agar - Liquid media -- no agar - For inoculum preparation, blood culture, for the isolation of pathogens from a mixture. - Eg: Nutrient broth - Semi solid medium -- 0.5% agar - Eg: Motility medium **Types of media based on their composition** - Simple or basal media - Undefined, used to grow all kind of microorganisms from the environment - Nutrient broth: consists of peptone, meat extract, NaCl, - NB + 2% agar = Nutrient agar - Complex media - Media other than basal media - They have added ingredients - Provide special nutrients - Synthetic or defined media always the same composition - Media prepared from pure chemical substances and its exact composition is known - It does not contain any yeast, animal or plant tissue - Eg: Lee' s medium - (NH4)2SO4, MgSO4.7H2O, K2HPO4, NaCl, Glucose, Prolin, Biotin - Special Media: - Enriched - Enrichment - Indicator.... - Substances like blood, serum, egg are added to the basal medium. - Used to grow bacteria that are demanding (fastidious) in their nutritional needs. - Eg: Blood agar, Chocolate agar - Liquid media used to isolate pathogens from a mixed culture - Media is incorporated with inhibitory substances to suppress the unwanted organisms - Selenite F Broth -- for the isolation of Salmonella, Shigella - Alkaline Peptone Water -- for Vibrio cholerae - The inhibitory substance is added to a solid media - Mac Conkey's medium for gram negative bacteria - TCBS (thiosulfate citrate bile salts sucrose) -- for V. cholerae - Potassium tellurite medium -- Diphtheria bacilli - LJ (Lowenstein-Jensen) medium -- M. tuberculosis - Wilson and Blair medium -- S. typhi - - These media contain an indicator which changes its color when a bacterium grows in them. - Christensen's urease medium (contains Phenol Red): Test carried out to differentiate urease-positive Proteus species from other members of Enterobacteriaceae - Positive result: purple/ pink color - Negative result: unchanged - A media which has substances incorporated in it enabling it to distinguish between bacteria. - Eg: Mac Conkey's medium: distinguishes between lactose fermenters and non lactose fermenters - Lactose fermenters -- Pink colonies - Non lactose fermenters -- colourless colonies - Media containing any fermentable substance - Eg: glucose, arabinose, lactose, starch etc - Media consists of 1% of the sugar in peptone water - Contain a small tube (Durham's tube) for the detection of gas by the bacteria. - Anaerobic media - These media are used to grow anaerobic organisms. - Eg: Robertson's cooked meat medium - In this case normal media are used but they are incubated under anaerobic conditions - Methods to generate anaerobiosis: - Production of vacuum - Displacement of oxygen with other gases - Chemical method: Gaspak - Biological method - Commercially available disposable envelope. - Contains chemicals which generate H2 and CO2 on addition of water. - Cold catalyst -- in the envelope - Indicator is used -- reduced methylene blue. - Colourless -- anaerobically - Blue colour -- on exposure to oxygen - Incubation of inoculated culture under growth conditions which may vary in: - Temperature - Gas composition - Agitation - Light availability - Humidity - It is often up to the scientist to find the best conditions for culturing the organism of interest - A common strategy routinely employed during identification of microorganisms is to establish growth on general, non-specific media. - The cells can then be transferred to growth on selective or differential media to enhance the survival of particular species above others - Thus a microbiologist may arrive at a 'pure culture' of an organism, which contains only cells of one single species - The microbiologist may then visually inspect cells from the pure culture by microscopy to confirm / identify / record (in the case of new species) the physical features of the microorganism - This may be supported by genetic (sequencing), and / or biochemical (enzyme activity, substrate metabolism, staining) tests to characterise the microorganism - Most cells are colourless and thus difficult to view with microscope. - The samples require smearing and fixing. - Smear is a distribution of bacterial cells on a slide for the purpose of viewing them under the microscope - Method: - A staining can be: - Simple staining involves the use of only one dye and is used primarily as a means to study the morphology and structure of organisms. - Differential staining uses more than two dyes and is also used to differentiate the organisms into one of two groups. - positive staining - where the actual cells are themselves colored and appear in a clear background - negative staining -- where the cells remain clear (uncolored) and the background is colored to create a contrast to aid in the better visualization of the image. - Gram staining - Acid fast staining - Spore staining 1. Primary stain is crystal violet. All cells stain purple. 2. Treat with iodine, which helps the stain to penetrate deeper in the cell wall. All cells remain purple. 3. Decolorize with ethanol and acetone. Breaks down Gram negative cell wall outer layer so the purple stain can be washed away. Gram negative cells are now colorless, Gram positive cells remain purple. 4. Stain with safranin, which stains cells pink. Gram positive cells remain purple, gram negative cells are now pink. This second stain is referred to as a counterstain - Used to stains cells that have a high level of waxy material (such as mycolic acids) in their cell walls which will not stain with water-based stains (crystal violet, methylene blue, etc.) - Stain first with carbolfuchsin (pink). - Decolorize with hydrochloric acid. Only the non waxy cells will decolorize. - Counterstain with methylene blue (stains non-acid fast cells blue) - Some bacteria can turn into endospores to survive adverse conditions. - Endospores don't stain easily with normal water-based dyes. Steps: 1. Stain with malachite green that is driven into spore with heat. 2. Decolorize with water - Growth is defined as an increased in the number of microbial cells in a population (also measured as an increase in microbial mass) - Growth rate is the change in cell number or cell mass per unit time - The interval for the formation of two cells from one is called a generation. Therefore the generation time is the time required for the generation to double (doubling time) - Before it can divide bacterial cells must duplicate its DNA - When we inoculate a microbial population in fresh medium, growth does not begin immediately , but only after a period of time called the lag phase. This time is required to synthesize new enzymes. - In the next phase (exponential) the number of cells doubles during each unit time period. It is the consequence of each cell dividing to form two cells, each of which divides to form two more cells. - Stationary phase starts when an essential nutrient of the culture medium is used up or some waste product of the organism builds up in the medium to an inhibitory level and exponential growth ceases. - Death phase: if incubation continues after a population reaches the stationary phase, the cells may remain alive and continue to metabolize, but they may also die. In some cases death is also accompanied by cell lysis. [Determination of growth] **Measurement of growth** - Total cell count - Viable count - Dilutions - Cell mass - Turbidity **Total cell count (direct microscopic count)** - A special counting chamber is required, in which a grid is marked on the surface of the glass slide, with squares of known small area **Total cell count: limitations** - Dead cells are not distinguished from living cells - Small cells are difficult to see under the microscope - Precision is difficult to achieve - The method is not suitable for cell suspensions of low or high density **Viable count: Spread-plate method** **Dilutions** **Cell mass** - Net cell mass can be measured by concentrating a known volume of culture and weighing the pellet obtained. - Usually dry weight is determined following drying of the pelleted cells at 90-100ºC overnight. - A faster method is using turbidity measurements: a cell suspension looks cloudy because cells scatter light passing through the suspension. **Turbidity** - A spectrophotometer is used, which passes light through the suspension and detect the amount of unscattered light that emerges. - The measurement comes in optical density units (OD) - A standard curve needs to be prepared for every species. [Effect on environmental factors on growth] **Factors influencing growth** - Temperature - Acidity and Alkalinity (pH) - Water availability - Oxygen **Temperature** - When temperature raises, chemical and enzymatic reactions in the cell proceed at more rapid rates and growth becomes faster - Above a certain temperature biomolecules can be damaged - For every organism there is: - a minimum T (below which growth does not longer occur - optimum T (at which growth is most rapid) - maximum T (above which growth is not possible) **classification according to the optimum temperature** - Psychrophiles: low optimum T (15 ºC) - Mesophiles: midrange optimum T (\*) - Thermophiles: high optimum T (45 ºC) - Hyperthermophiles: very high optimum T (80 ºC) - Psychrotolerant: can grow at 0 ºC but the optimum T is between 20º and 40 ºC - (\*) Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes **Classification on the basis of pH requirements** - Acidophiles: can grow at low pHs - Alkaliphiles have high pH optimum, between 10-11 - Neutrophiles: pH optimum between 6 and 8 **Water availability (water activity)** - Water diffuses from a region of high water concentration (low solute concentration to a region of lower water concentration: osmosis) - Water can become unavailable to an organism when the dissolved solute concentration in its environment increases - Microorganisms can be classified accordingly to the environmental solute concentration that they can tolerate **Classification on the basis of water requirements** - Halophiles: grow optimally in sea water, and they require some NaCl to grow - Halotolerant: can tolerate some solutes but grow better in the absence of the added solute - Extreme halophile: require 15-30 % NaCl - Osmophile: able to live in high sugar \[ \] - Xerophile: able to grow in very dry environments **Classification on the basis of oxygen requirements** - Aerobes: capable of grow at full oxygen tension (air is 21% oxygen) - Facultatives: they can grow both in the presence or absence of oxygen - Microaerophiles: they can only use oxygen when it is present at reduced levels - Obligate Anaerobes: are killed by oxygen **Chapter 2 Part II\ Microbial growth control** **Chapter overview** - Basic concepts - Physical methods of microbial growth control - Chemical methods of microbial growth control **Introduction** - Early civilizations practiced salting, smoking, pickling, drying, and exposure of food and clothing to sunlight to control microbial growth - Use of spices in cooking was to mask taste of spoiled food. Some spices prevented spoilage - In mid 1800s Semmelweiss and Lister helped developed aseptic techniques to prevent contamination of surgical wounds. Before then: - Nosocomial infections caused death in 10% of surgeries - Up to 25% mothers delivering in hospitals died due to infection **Definitions** - Sterilization: killing or removing all forms of microbial life (including endospores) in a material or an object - Heating is the most commonly used method of sterilization - Commercial Sterilization: Heat treatment that kills endospores of Clostridium botulinum, the causative agent of botulism, in canned food. Does not kill endospores of thermophiles, which are not pathogens - Disinfection: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. Usually involves the removal of vegetative or non-endospore forming pathogens. It may use physical or chemical methods - Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination - Asepsis: Absence of significant contamination - Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery. Aseptic techniques are also used to prevent bacterial contamination in food industry. - Degerming: mechanical removal of most microbes in a limited area. Example: Alcohol swab on skin - Sanitization: Use of chemical agent on food-handling equipment to meet public health standards and minimize chances of disease transmission. Example: hot soap and water. - Disinfectant: applied to inanimate objects - Antiseptic: applied to living tissue - Bacteriostatic agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them - Germicide: An agent that kills certain microorganisms - Bactericide: An agent that kills bacteria. Most do not kill endospores. - Viricide: An agent that inactivates viruses. - Fungicide: An agent that kills fungi. - Sporicide: An agent that kills bacterial endospores of fungal spores. **Rate of microbial death** Several factors influence the effectiveness of antimicrobial treatment: - Number of microbes: The more microbes present, the more time it takes to eliminate population. - Type of microbes: Endospores are very difficult to destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control. - Environmental factors: Presence of organic material (blood, feces, saliva) tends to inhibit antimicrobials etc. - Time of exposure: Chemical antimicrobials and radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures. [Physical methods of microbial control] **Physical methods of microbial control** - Heat - Low temperature - Filtration - Desiccation - Osmotic pressure - Radiation **Heat** - Kills microorganisms by denaturing their enzymes and other proteins. Heat resistance varies widely among microbes. - Moist heat: kills microorganisms by coagulating their proteins. In general, moist heat is much more effective than dry heat. - Dry heat kills by oxidation. **Moist heat** - Boiling: Heat to 100ºC or more at sea level. Kills vegetative forms of bacterial pathogens, almost all viruses, and fungi and their spores within 10 minutes or less. Exceptions: - Hepatitis virus: Can survive up to 30 minutes of boiling. - Endospores: Can survive up to 20 hours or more of boiling. - Reliable sterilization with moist heat requires temperatures above that of boiling water. This is achieved with an autoclave, a chamber which is filled with hot steam under pressure. Preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure. - Temperature of steam reaches 121ºC at twice atmospheric pressure. - Most effective when organisms contact steam directly or are contained in a small volume of liquid. - All organisms and endospores are killed within 15 minutes. - Requires more time to reach center of solid or large volumes. - Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc. - Classic Method of Pasteurization: Milk was exposed to 65ºC for 30 minutes. - High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72ºC for 15 seconds. - Ultra High Temperature Pasteurization (UHT): Milk is treated at 140ºC for 3 seconds and then cooled very quickly in a vacuum chamber. - Advantage: Milk can be stored at room temperature for several months. **Dry heat** - - - **Low temperature** - - - - - - **Filtration** - High Efficiency Particulate Air Filters (HEPA): Used in operating rooms and burn units to remove bacteria from air. - Membrane Filters: Uniform pore size. Used in industry and research. Different sizes: - 0.22 and 0.45um pores: Used to filter most bacteria. Don't retain spirochetes, mycoplasmas and viruses. - 0.01 um Pores: Retain all viruses and some large proteins. **Desiccation** - - - - **Osmotic pressure** - Plasmolysis: As water leaves the cell, plasma membrane shrinks away from cell wall. Cell may not die, but usually stops growing. - Yeasts and molds: More resistant to high osmotic pressures. - Staphylococci that live on skin are fairly resistant to high osmotic pressure. **Radiation** - Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer). Dislodge electrons from atoms and form ions, causes mutations in DNA and produce peroxides. - Used to sterilize pharmaceutical and disposable medical supplies. Food industry is interested in using ionizing radiation. - Disadvantages: Penetrates human tissues. May cause genetic mutations in humans. - Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. - Used to disinfect operating rooms, nurseries, cafeterias. - Disadvantages: Damages skin, eyes. Doesn't penetrate paper, glass, and cloth - Microwave Radiation: Wavelength ranges from 1 millimeter to 1 meter. Heat is absorbed by water molecules. - May kill vegetative cells in moist foods. Bacterial endospores, which do not contain water, are not damaged by microwave radiation. - Solid foods are unevenly penetrated by microwaves. - Trichinosis outbreaks have been associated with pork cooked in microwaves. **Forms of Radiation** **Alcohols** - Kill bacteria, fungi, but not endospores or naked viruses. - Act by denaturing proteins and disrupting cell membranes. - Evaporate easily, so they lack residual action. - Used to mechanically wipe microbes off skin before injections or blood drawing. - Not good for open wounds, because cause proteins to coagulate. - Ethanol: Drinking alcohol. Optimum concentration is 70%. - Isopropanol: Rubbing alcohol. Better disinfectant than ethanol, cheaper and less volatile. **Aldehydes** - Include some of the **most effective antimicrobials.** Inactivate proteins by forming covalent crosslinks with several functional groups. - Formaldehyde gas: excellent disinfectant. Commonly used as formalin, a 37% aqueous solution. Formalin was used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines. Irritates mucous membranes, strong odor. Also used in mortuaries for embalming. - Glutaraldehyde: less irritating and more effective than formaldehyde. One of the few chemical disinfectants that is a sterilizing agent. Commonly used to disinfect hospital instruments. Also used in mortuaries for embalming. A 2% solution of glutaraldehyde is: - Bactericidal, tuberculocidal, and viricidal in 10 minutes. - Sporicidal in 3 to 10 hours. **Bisphenols, Biguanides, Diamidines** **Halogens** \- Iodine: - Tincture of iodine (alcohol solution) was one of first antiseptics used. - Combines with amino acid tyrosine in proteins and denatures proteins. - Stains skin and clothes, irritating. - Iodophors: Compounds with iodine that are slow releasing, take several minutes to act. Used as skin antiseptic in surgery. Not effective against bacterial endospores (Betadine) - Chlorine: - Used to disinfect drinking water, pools, and sewage. - Chlorine is easily inactivated by organic materials. - Sodium hypochlorite (NaOCl): it is the active ingredient of bleach. - Include copper, selenium, mercury, silver, and zinc. - Oligodynamic action: Very tiny amounts are effective. - Silver nitrate used to protect infants against gonorrheal eye infections until recently. - Mercury: organic mercury compounds like merthiolate are used to disinfect skin wounds, and thiomersal for vaccine preservation. - Copper: copper sulfate is used to kill algae in pools and fish tanks. - Selenium: kills fungi and their spores. Used for fungal infections, and also used in dandruff shampoos. (antiforfora) - Zinc: zinc chloride is used in mouthwashes and zinc oxide is used as antifungal agent in paints. **Peroxygens:** - Oxidize cellular components of treated microbes. Disrupt membranes and proteins. - Ozone: used along with chlorine to disinfect water. Helps neutralize unpleasant tastes and odors. More effective killing agent than chlorine, but less stable and more expensive. - Hydrogen Peroxide: used as an antiseptic. Not good for open wounds because quickly broken down by catalase present in human cells. Used by food industry and to disinfect contact lenses - Peracetic Acid: one of the most effective liquid sporicides available, kills bacteria and fungi in less than 5 minutes; kills endospores and viruses within 30 minutes. Used widely in disinfection of food and medical instruments because it does not leave toxic residues. - Phenol was first used by Lister as a disinfectant. - Rarely used today because it is a skin irritant and has strong odor. - Used in some throat sprays and tablets. Acts as local anesthetic - Biphenols: Effective against gram-positive Staphylococci and Streptococci. Used in nurseries in the past, however excessive use in infants may cause neurological damage. - Widely used surface active agents, cationic detergents. -  Effective against gram positive bacteria, less effective against gram-negative bacteria. Also destroy fungi, amoebas, and enveloped viruses. - Pseudomonas strains that are resistant and can grow in presence of Quats are a big concern in hospitals. - Advantages: Strong antimicrobial action, colorless, odorless, tasteless, stable, and nontoxic. - Diasadvantages: Form foam. Organic matter interferes with effectiveness. Neutralized by soaps and anionic detergents. - Chemicals that sterilize in a chamber similar to an autoclave. They denature proteins, by replacing functional groups with alkyl groups. - Ethylene Oxide: kills all microbes and endospores, but requires exposure of 4 to 18 hours. Toxic and explosive in pure form. Highly penetrating. Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment. **Efficiency of Different Chemical Antimicrobial Agents**