Microbial Growth and Reproduction.pdf

Microbial Growth & Reproduction Dr. Kevin Harris NURS1111 Presentation topics 1. 2. 3. 4. 5. Antimicrobial Resistance Hand Hygiene Safe injection practices & sharps disposal Environmental cleaning Waste management and segregation Presentation topics 6. Chain of Infection 7. Standard precautions 8. T...

Microbial Growth & Reproduction Dr. Kevin Harris NURS1111 Presentation topics 1. 2. 3. 4. 5. Antimicrobial Resistance Hand Hygiene Safe injection practices & sharps disposal Environmental cleaning Waste management and segregation Presentation topics 6. Chain of Infection 7. Standard precautions 8. Transmission based precautions 9. PPE 10. Use of the microbiology lab Presentation topics 11. Types of isolation 12. Antibiotics 13. Sterilisation 14. Pathogenicity Presentation topics 15. Rashes, skin and soft tissue infections 16. Respiratory infections 17. Blood stream infections 18. Urinary tract infections 19. Brain and CSF infections 20. STIs What is growth? Definition of growth The increases in cell size and number that take place during the life history of an organism. Microbial growth and reproduction Refers to an increase in cell number, not in cell size. Microbial reproduction - How does growth occur? Bacteria grow and divide by binary fission. a rapid and relatively simple process. asexual reproduction by a separation of the body into two new bodies Microbial reproduction - Binary fission Microbial reproduction - Binary fission Microbial reproduction Microbial growth - bacterial culture Bacterial Division occurs mainly by binary fission. Generation Time: Time required for a population to double. Generation time varies considerably: E. coli divides every 20 minutes. Most bacteria divide every 1 to 3 hours. Some bacteria require over 24 hours to divide. Phases of growth - lag phase Period of adjustment to new conditions. Little or no cell division occurs, population size doesn’t increase. Phase of intense metabolic activity, in which individual organisms grow in size. May last from one hour to several days. Phases of growth - log phase Cells begin to divide and generation time reaches a constant minimum. Period of most rapid growth. Number of cells produced > Number of cells dying Cells are at highest metabolic activity. Cells are most susceptible to adverse environmental factors at this stage. Radiation Antibiotics Phases of growth - stationary phase Population size begins to stabilize. Number of cells produced = Number of cells dying Overall cell number does not increase. Cell division begins to slow down. Factors that slow down microbial growth: Accumulation of toxic waste materials Acidic pH of media Limited nutrients Insufficient oxygen supply Phases of growth - death phase Population size begins to decrease. Number of cells dying > Number of cells produced Cell number decreases at a logarithmic rate. Cells lose their ability to divide. A few cells may remain alive for a long period of time. Four Phases of Bacterial Growth Curve Basic requirements? Food Clothes Shelter Water Requirements for growth and reproduction Physical requirements Biochemical requirements Physical requirements Requirements for growth - Physical Temperature pH Osmotic pressure Physical Requirements - temperature Microbes are loosely classified into several groups based on their preferred temperature ranges. Psychrophiles Mesophiles Thermophiles Physical Requirements - temperature Psychrophiles – “Cold-loving” Can grow at 0oC. Two groups True Psychrophiles Sensitive to temperatures over 20oC. Optimum growth below 15oC Found in very cold environments (North pole, ocean depths). Seldom cause disease or food spoilage. Psychrotrophs Optimum growth at 20 – 30oC. Responsible for most low temperature food spoilage. Food Spoilage Temperatures Physical Requirements - temperature Mesophiles – “Middle loving” Includes most pathogens Best growth between 25 to 40oC. Optimum temperature commonly 37oC. Many have adapted to live in the bodies of animals. Physical Requirements - temperature Thermophiles – “Heat loving”. Optimum growth 50 – 60oC. Many cannot grow below 45oC. Adapted to live in sunlit soil, compost piles, and hot springs. Some thermophiles form extremely heat resistant endospores. Hyperthermophiles Optimum growth at 80oC or higher. Archaebacteria Most live in volcanic and ocean vents. Growth Rates of Bacterial Groups at Different Temperatures Physical Requirements - pH Most bacteria prefer neutral pH (6.5-7.5). Molds and yeast grow in wider pH range Acidity inhibits most microbial growth used frequently for food preservation (e.g.: pickling). Alkalinity also inhibits microbial growth not commonly used for food preservation. Acidic products of bacterial metabolism interfere with growth. Buffers used to stabilize pH. Physical Requirements - pH Acidophiles: “Acid loving”. Grow at very low pH (0.1 to 5.4) Lactobacillus produces lactic acid, tolerates mild acidity. Neutrophiles: Grow at pH 5.4 to 8.5. Includes most human pathogens. Alkaliphiles: “Alkali loving”. Grow at alkaline or high pH (7 to 12 or higher) Vibrio cholerae optimal pH 9. Soil bacterium Agrobacterium grows at pH 12. Physical Requirements - osmotic pressure Cells are 80 to 90% water. Physical Requirements - osmotic pressure Osmolarity the proportion of dissolved particles in an amount of fluid and is generally the term used to describe body fluids. As the dissolved particles become more concentrated, the osmolarity increases. Osmolality The proportion of dissolved particles in a specific weight of fluid. The terms osmolarity and osmolality are often used interchangeably in clinical practice. Physical Requirements - osmotic pressure Hypotonic solutions Low osmotic pressure causes water to enter the cell. Cell wall prevents excessive entry of water. Physical Requirements - osmotic pressure Physical Requirements - osmotic pressure Isotonic solutions At same osmotic pressure of cells Physical Requirements - osmotic pressure Physical Requirements - osmotic pressure Hypertonic solutions High osmotic pressure removes water from cell, causing plasmolysis Used to control spoilage and microbial growth. Sugar in jelly. Salt on meat. Physical Requirements - osmotic pressure Isotonic vs Hypertonic Solution Plasmolysis Physical Requirements - osmotic pressure Halophiles Require moderate to large salt concentrations. Ocean water contains 3.5% salt. Extreme or Obligate Halophiles Require very high salt concentrations (20 to 30%). Bacteria in Dead Sea Facultative Halophiles Do not require high salt concentrations for growth How is this relevant to you? Type IV Solution Uses 0.9% Normal Saline (0.9% NaCl) Fluid resuscitation for Monitor closely for hypervolemia, hemorrhaging, severe vomiting, diarrhea, GI suctioning losses, especially with heart failure or renal wound drainage, mild hyponatremia, failure. or blood transfusions. Isotonic Lactated Ringer’s Solution (LR) Should not be used if serum pH is Fluid resuscitation, GI tract fluid greater than 7.5 because it will losses, burns, traumas, or metabolic worsen alkalosis. May elevate acidosis. Often used during surgery. potassium levels if used with renal failure. Isotonic 5% Dextrose in Water (D5W) Provides free water to help renal *starts as isotonic and excretion of solutes, hypernatremia, then changes to and some dextrose supplementation. hypotonic when dextrose is metabolized Isotonic Nursing Considerations Should not be used for fluid resuscitation because after dextrose is metabolized, it becomes hypotonic and leaves the intravascular space, causing brain swelling. Used to dilute plasma electrolyte concentrations. How is this relevant to you? Type Hypotonic Hypotonic IV Solution 0.45% Sodium Chloride (0.45% NaCl) 5% Dextrose in Water (D5W) Uses Nursing Considerations Used to treat intracellular dehydration and hypernatremia and to provide fluid for renal excretion of solutes. Monitor closely for hypovolemia, hypotension, or confusion due to fluid shifting into the intracellular space, which can be life-threatening. Avoid use in patients with liver disease, trauma, and burns to prevent hypovolemia from worsening. Monitor closely for cerebral edema. Provides free water to promote renal excretion of solutes and treat hypernatremia, as well as some dextrose supplementation. Monitor closely for hypovolemia, hypotension, or confusion due to fluid shifting out of the intravascular space, which can be life-threatening. Avoid use in patients with liver disease, trauma, and burns to prevent hypovolemia from worsening. Monitor closely for cerebral edema. How is this relevant to you? Type IV Solution Uses Nursing Considerations Hypertonic Used to treat severe 3% Sodium hyponatremia and Chloride (3% NaCl) cerebral edema. Monitor closely for hypervolemia, hypernatremia, and associated respiratory distress. Do not use it with patients experiencing heart failure, renal failure, or conditions caused by cellular dehydration because it will worsen these conditions. Hypertonic 5% Dextrose and Used to treat severe 0.45% Sodium hyponatremia and Chloride (D50.45% cerebral edema. NaCl) Monitor closely for hypervolemia, hypernatremia, and associated respiratory distress. Do not use it with patients experiencing heart failure, renal failure, or conditions caused by cellular dehydration because it will worsen these conditions. Hypertonic 5% Dextrose and Lactated Ringer’s (D5LR) D10 Monitor closely for hypervolemia, hypernatremia, and associated respiratory distress. Do not use it with patients experiencing heart failure, renal failure, or conditions caused by cellular dehydration because it will worsen these conditions. Used to treat severe hyponatremia and cerebral edema. Chemical requirements Chemical requirements Carbon Other elements Oxygen Chemical Requirements - Carbon Carbon Makes up 50% of dry weight of cell. Structural backbone of all organic compounds. Chemoheterotrophs Obtain carbon from their energy source lipids, proteins, and carbohydrates. Chemoautotrophs and Photoautotrophs Obtain carbon from carbon dioxide. Chemical Requirements - nitrogen Nitrogen: Makes up 14% of dry cell weight. Used to form amino acids, DNA, and RNA. Sources Protein Ammonium: Found in organic matter Nitrogen gas (N2) Obtain N directly from atmosphere. Important nitrogen fixing bacteria, live free in soil or associated with legumes (peas, beans, alfalfa, clover, etc.). Nitrates: Salts that dissociate to give NO3-. Chemical Requirements - sulphur Sulfur Used to form proteins and some vitamins (thiamin and biotin). Sources: Protein Hydrogen sulfide Sulfates: Salts that dissociate to give SO42-. Chemical Requirements - phosphorus Used to form DNA, RNA, ATP, and phospholipids. Sources Mainly inorganic phosphate salts and buffers. Chemical Requirements - other elements Potassium, magnesium, and calcium are often required as enzyme cofactors. Calcium is required for cell wall synthesis in Gram positive bacteria. Trace Elements: Used as enzyme cofactors. Commonly found in tap water. Iron Copper Molybdenum Zinc The in-between Growth requirements - oxygen Both physical and chemical requirement Organisms that use molecular oxygen (O2), produce more energy from nutrients than anaerobes. Can classify microorganisms based on their oxygen requirements Growth requirements - oxygen Obligate Aerobes Require oxygen to live. Examples Pseudomonas, common nosocomial pathogen. Growth requirements - oxygen Facultative Anaerobes Can use oxygen Can grow in its absence Examples: E. coli, Staphylococcus species, yeasts, and many intestinal bacteria. Growth requirements - oxygen Aerotolerant Anaerobes: Can’t use oxygen, but tolerate its presence. Can break down toxic forms of oxygen. Example: Lactobacillus carries out fermentation regardless of oxygen presence. Growth requirements - oxygen Microaerophiles Require oxygen, but at low concentrations Sensitive to toxic forms of oxygen. Example: Campylobacter. Growth requirements - oxygen Obligate Anaerobes Killed by the presence of oxygen Examples: Clostridium Microbial growth Microbial growth - how do we grow them? Culture Media Microbial growth - what’s required in media? Nutrients material prepared for microbial growth in the laboratory. General Requirements Must be sterile Contain appropriate nutrients Must be incubated at appropriate temperature Culture Microbes that grow and multiply in or on a culture medium. Microbial growth - class of culture media Consistency Nutritional value Application Microbial growth - class of culture media Consistency Solid Semi-solid Liquid Microbial growth - class of culture media Nutrition Simple Complex Synthetic Microbial growth - class of culture media Application Basal Enriched Selective Differential Transport Anaerobic Assay Storage Media by consistency Microbial growth - Solid media Nutrient material with a solidifying agent Plates and slants. The most common solidifier is agar (1.5 – 2%) Unique Properties of Agar: Melts above 95oC. Does not solidify until it reaches 40oC. Cannot be degraded by most bacteria. Polysaccharide made by red algae. Originally used as food thickener (Angelina Hesse). Microbial growth - Semi-solid media 0.2-0.5% agar concentration Appears as a soft, jelly-like substance Mainly used to study the motility of microorganisms and cultivate microaerophilic bacteria bacteria on this media appear as a thick line. Examples: Hugh and Leifson’s oxidation fermentation medium Stuart’s media Amies media Mannitol motility media Microbial growth - liquid media (broth) No solidifying agent Used to enhance growth and fermentation studies Examples Tryptic soy broth Phenol red carbohydrate broth MR-VP broth Nutrient broth. Media by nutritional content Microbial growth - simple media a general-purpose media that supports the growth of non-fastidious microbes, and it is primarily used for the isolation of microorganisms. Examples nutrient broth peptone water nutrient agar. Microbial growth - complex media Nutrient material whose exact chemical composition is not known. Widely used for heterotrophic bacteria and fungi. Made of extracts from yeast, meat, plants, protein digests Composition may vary slightly from batch to batch. Energy, carbon, nitrogen, and sulfur requirements are primarily met by protein fragments (peptones). Vitamins and organic growth factors provided by meat and yeast extracts. Examples Nutrient broth: Liquid media Nutrient agar: Solid media Microbial growth - synthetic media Chemically defined media Produced from pure chemical substances. Known concentration of ingredients, like sugar (glucose or glycerol) and nitrogen source (such as ammonium salt or nitrate as inorganic nitrogen). Generally used in scientific research, and an example is Czapek Dox Medium Media by application Microbial growth - class of culture media Application Basal Enriched Selective Differential Transport Anaerobic Assay Storage Microbial growth - Basal media Routinely used simple media having carbon and nitrogen sources that boost the growth of many microorganisms. General-purpose media Considered non-selective media. Suitable for growing Staph and Enterobacteriaceae Examples: nutrient broth, nutrient agar, and peptone water. Microbial growth - Enriched media Adding additional substances e.g. blood, serum, or egg yolk in the basal medium. Used to grow fastidious microorganisms Examples Chocolate agar - used to grow N. gonorrhea & Haemophilus species blood agar - used to identify hemolytic bacteria Loeffler’s serum slope. Microbial growth - Selective media Allows the growth of certain microbes while inhibiting the growth of others. Selective growth is decided by adding substances antibiotics, dyes, bile salts, or by pH adjustments. No Culture media Inhibiting substances Bacteria 1 Thayer Martin Agar Contains antibiotics; vancomycin, colistin, and nystatin Used for Neisseria gonorrhoeae 2 MacConkey’s Agar Contains bile salts Used for Enterobacteriaceae members 3 Lowenstein Jensen Medium Addition of malachite green Used for M.tuberculosis 4 Mannitol Salt Agar Contains 10% NaCl Used to recover S.aureus 5 Crystal Violet Blood Agar Contains 0.0002% crystal violet Used for Streptococcus pyogenes 6 Thiosulfate citrate bile salts sucrose (TCBS) agar Have elevated pH of about 8.5-8.6 Used for isolating Vibrio cholerae 7 Wilson and Blair’s Agar Addition of dye brilliant green Used for recovering S. typhi 8 Potassium tellurite medium Contains 0.04% Potassium tellurite Used to recover C.diphtheriae 9 Pseudosel Agar (cetrimide agar) Contains cetrimide (antiseptic agent) Used to recover Pseudomonas aeruginosa 10 Salmonella-Shigella Agar Contains bile salts, brilliant green, and sodium citrate Used for the isolation of Salmonella, which causes typhoid Microbial growth - Selective media Microbial growth - enrichment media Liquid medium used to increase the relative concentration of certain microbes Inhibits the growth of commensal in the clinical specimen. It’s also used in isolating fecal and soil microorganisms. Examples Selenite F broth which is used to isolate Salmonella typhi Tetrathionate broth Alkaline peptone water. Microbial growth - differential media contains indicators e.g. dyes or metabolic substrates which give different colours to colonies of different microbial species when they utilize or react with these components. It allows the growth of more than one microorganism, Bacterial colonies differentiated based on their colour Microbial growth - differential media Blood Cystine Lactose Electrolyte Deficient (CLED) Mannitol Salt Agar MacConkey Thiosulfate citrate bile salts sucrose (TCBS) Chrome Agar Microbial growth - transport media useful for clinical specimens which are required to be transferred immediately to labs to maintain the viability of potential pathogens to prevent overgrowth of commensals or contaminating microorganisms. Examples Sach’s buffered glycerol saline – faeces from patients suspected to be suffering from bacillary dysentery. Cary Blair transport and Venkatraman Ramakrishnan media: Fecal samples collected from suspected cholera patients are transported using these media. Pike’s medium: A throat specimen containing Streptococci is transported using this medium Microbial growth - anaerobic media anaerobic bacteria require low oxygen levels, extra nutrients, and reduced oxidation-reduction potential. Usually supplemented with hemin and vitamin K nutrients Examples: Thioglycollate broth Robertson Cooked Meat (RCM) – Clostridium spp Microbial growth - the rest Assay media: It’s used for amino acids, vitamins, and antibiotics assays. For example, antibiotic assay media is used to determine the antibiotic potency of microorganisms. Storage media: It’s used to store microorganisms for a longer period, examples are BHI broth with 10% glycerol. Microbial growth - CO2 organisms Special Culture Techniques: Used to grow bacteria with unusual growth requirements. Bacteria that require high or low CO2 levels: Grow better at high CO2 levels and low O2 levels. Similar to environment of intestinal tract, respiratory tract, and other tissues. Equipment for Producing CO2 Rich Environments Microbial growth - pure culture Pure Culture: Contains a single microbial species. Most clinical and environmental specimens contain several different microorganisms. To obtain a pure culture, individual organisms must be isolated. The most common method of isolation is the streak plate to form colonies Measuring Microbial Growth - Direct Method Plate count: Most frequently used method of measuring bacterial populations. Inoculate plate with a sample and count number of colonies. Assumptions: Each colony originates from a single bacterial cell. Advantages: Measures viable cells Disadvantages: Takes 24 hours or more for visible colonies to appear. Only counts between 25 and 250 colonies are accurate. Must perform serial dilutions to get appropriate numbers/plate. Serial Dilutions are Used with the Plate Count Method to Measure Numbers of Bacteria Measuring Microbial Growth - Direct Method Pour Plate: Introduce a 1.0 or 0.1 ml inoculum into an empty Petri dish. Add liquid nutrient medium kept at 50oC. Gently mix, allow to solidify, and incubate. Disadvantages: Not useful for heat sensitive organisms. Colonies appear under agar surface. Spread Plate: Introduce a 0.1 ml inoculum onto the surface of Petri dish. Spread with a sterile glass rod. Advantages: Colonies will be on surface and not exposed to melted agar. Measuring Microbial Growth - Direct Method Filtration Used to measure small quantities of bacteria. Example: Fecal bacteria in a lake or in ocean water. A large sample (100 ml or more) is filtered to retain bacteria. Filter is transferred onto a Petri dish. Incubate and count colonies. Measuring Microbial Growth - Indirect Method Turbidity: As bacteria multiply in media, it becomes turbid. Use a spectrophotometer to determine % transmission or absorbance. Advantages: No incubation time required. Disadvantages: Cannot distinguish between live and dead bacteria. Requires a high concentration of bacteria (10 to 100 million cells/ml). Spectrophotometryoverview Measuring Microbial Growth - indirect Method Metabolic Activity: As bacteria multiply in media, they produce certain products: Carbon dioxide Acids Measure metabolic products. Expensive Measuring Microbial Growth - indirect Method Dry Weight: Bacteria or fungi in liquid media are centrifuged. Resulting cell pellet is weighed. Doesn’t distinguish live and dead cells. Goodbye!