Bio244 6
- 1. MICROBIAL GROWTH REQUIREMENTS: INCREASE IN NUMBER OF MICROBIAL CELLS Phosphorus Organic growth factors Trace elements Physical TEMPRATURE pH Osmotic pressure CHEMICAL Hydrogen Oxygen (O) Nitrogen (N) Sulfur (S) CHAPTER 6 MICROBIAL GROWTH-
- 2. PHYSICAL REQUIREMENTS for GROWTH : • MINIMUM GROWTH TEMPERATURE – Lowest temperature at which a species will grow • OPTIMUM GROWTH TEMPERATURE – Temperature at which species will grow best • MAXIMUM GROWTH TEMPERATURE – Highest temperature at which growth is possible TEMPERATURE
- 3. CLASSIFICATION OF MICROBES: • PSYCHROPHILES - Cold loving microbes • Optimum growth temperature: 15o C • Capable of growing at 0o C, but not at 250 C • Found in ocean depths, polar regions – Psychrotrophs-less temperature sensitive, some foods spoilage • MESOPHILES - moderate temperature loving microbes – Optimum growth temperature: 25c -40 c – Most common microbes – Food spoilage and disease • THERMOPHILES heat loving microbes • Optimum growth temperature: 50-60o C • Minimum growth temperature: 45o C • Found in hot springs, compost piles TEMPERATURE RANGE of GROWTH
- 4. ACID BASE CHEMISTRY – ACID – a substance that dissociates into hydrogen ions (H+) and negative ions in aqueous solution Ex. Hydrochloric acid, citric acid – BASE – a substance that dissociates into hydroxide ions (OH- ) and positive ions in aqueous solution Ex. Sodium hydroxide
- 5. • pH – potential hydrogen Logarithmic scale used to measure H+ concentration – Most bacteria grow between pH 6.5 to 7.5 – Molds and yeast grow between pH 5 to 6 – Acidophiles grow in acidic environments BUFFERS – compounds that keep pH from changing drastically; ex. peptones, amino acids, phosphate salts-very important in the preparation of microbial Media PHYSICAL REQUIREMENTS for GROWTH cntd.: pH
- 6. PHYSICAL REQUIREMENTS for GROWTH cntd. Osmotic Pressure the force used by a solvent in moving from an area with a lower solute concentration to an area of higher solute concentration – Hypertonic environments - concentration of solute (ex. salt or sugar) is higher outside cell, causes plasmolysis • Results in loss of water from a cell –Inhibits bacterial growth –Used to preserve food, ex.Salted fish Halophiles - require higher salt concentrations in their environment
- 7. CHEMICAL REQUIREMENTS for GROWTH Carbon – Structural, organic molecules, energy source Chemoheterotrophs use organic carbon sources Ex. Humans, fungi, protozoa, most bacteria, helminths Autotrophs-use CO2 as carbon source Ex. Plants Nitrogen In amino acids, proteins Sulfure In amino acids, thiamine, biotin Phosphore In DNA, RNA, ATP, membranes PO4 3− is a source of phosphorus Trace Elements Inorganic, required in small amounts; Ex.: Fe, Cu, Mb, Zn Organic Factors Organic, from environment Vitamins, amino acids, purines pyrimidines
- 8. CHEMICAL REQUIREMENTS for GROWTH cntd. - OXYGEN Obligate aerobes-O2 is required for growth Facultative anaerobes- growth can occur when O2 not present Obligate anaerobes -no growth when O2 present- harm by O2 Aerotolerant anaerobes- can tolerate O2 but can’t use it for growth Micro- aerophiles need very low O2 concentrations
- 9. TOXIC FORMS of OXYGEN Produced in small amounts during normal metabolic processes-harmful to cells Some Forms of Toxic Oxygen – Superoxide free radicals (O2 -1 ) – very unstable, steal electrons from cellular molecules – Peroxide anion (O2 -2 ) – contained in hydrogen peroxide – Hydroxyl radicals (OH- ) – most reactive MECHANISMS of ELIMINATION Superoxide free radicals (02 -1 ) 02 - + 02 - + 2H+ superoxide H202 + 02 dismutase Peroxide anions (02 -2 ) 2H2O2 catalase 2H2O + O2 peroxidase
- 10. ANAEOBIC CULTURE METHODS 3. ANAEROBIC JAR 4. ANAEROBIC _________ 1.Reducing media contains chemicals (sodium thioglycolate) that combine with O 2 Media is heated to drive off O 2 2. OxyPlates enzyme (oxyrase) that reduces oxygen to water is added to growth media, transforms petri plate into an anaerobic chamber
- 12. Figure 6.5 BIOFILMS • Microbial communities • Form slime or hydrogels – Bacteria attracted by chemicals via quorum sensing • Share _________ • __________ from harmful factors
- 13. • Culture Medium: Nutrient (or any material) prepared for the growth of microbes in a laboratory • Sterile: No living microbes • Inoculate: Introduction of microbes into media • Culture: Microbes growing in/on culture medium • Pure culture - contains only one species or strain of bacteria • Agar: Complex polysaccharide (from marine algae) – Used as solidifying agent for culture media in Petri plates, slants, and deeps – Generally not metabolized by microbes – Liquefies at 100°C – Solidifies ~40°C CULTURE MEDIA important terms
- 14. CULTURE MEDIA Chemically Defined Media: Exact chemical composition is known Complex Media: Extracts and digests of yeast, meats (organs), or plants Ex. Nutrient broth, nutrient agar
- 15. CULTURE MEDIUM for Fastidious MICROORGANISMS
- 16. BOTH SELECTIVE and DIFFERENTIAL MEDIA 1.McConkey Agar 2.Eosin Methylen blue Agar DIFFERENTIAL SELECTIVE for gram negative
- 17. • Supress unwanted microbes and encourage desired microbes. Ex. Eosin Methylene Blue Agar (EMB) and MacConkey Agar-allow Gram (-)bacteria to grow but not Gram (+) bacteria. SELECTIVE MEDIA Figure 6.9b, c EMB Agar
- 18. • Used to distinguish colonies of different microbes: Ex. Lactose fermenting bacteria from lactose non-fermenting bacteria; Ex.: MacConkey agar and EMB agar DIFFERENTIAL MEDIA Figure 6.9a Lactose nonfermenter on MacConkey Agar Lactose Fermenter on Mac Conkey Agar
- 19. ENRICHED/DIFFERENTIAL MEDIA nutritionally fortified media which encourages the growth of a wide range of microorganisms Blood Agar Plate
- 20. STREAK PLATE METHOD PROCEDURE for STREAKING for isolation Isolated COLONIES on AGAR PLATE Colony- a population of cells arising from a single cell or spore or from a group of attached cells A colony is often called a colony-forming unit (CFU)
- 21. • Deep- Freezing: -50°to -95°C • Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum PRESERVING BACTERIAL CULTURES
- 22. Reproduction in PROKARYOTES • Binary Fission • Budding • Conidiospores (actinomycetes) • Fragmentation of filaments Binary fission
- 23. Figure 6.13 Bacterial Exponential Growth Curve Generation TIME:20mn Time required for a cell to double in number
- 24. Figure 6.14 FOUR TYPICAL PHASES of BACTERIAL GROWTH CURVE
- 25. METHODS to DETECT and MEASURE BACTERIAL GROWTH (numbers) • DIRECT – Plate Counts* – Filtration – Most Probable Number (MPN) –DIRECT Microscopic Count* • INDIRECT – Turbidity Measurements* – Dry Weight Determination – Metabolic Activity Measurements*
- 26. • Inoculate Petri plates from serial dilutions • Used to count living bacterial cells only. DIRECT PLATE COUNT METHOD Figure 6.16 Plate Count Method
- 27. DIRECT MICROSCOPIC COUNT The number of microbes in a specific volume of bacterial suspension are counted using a special slide Ex.: PETROFF HAUSSER Does not distinguish between living and dead
- 28. Turbidity Estimating Bacterial Numbers by Indirect Methods Figure 620 :Indirectly Measures number of cells present, dead or live
- 29. Estimating Bacterial Numbers by INDIRECT Methods • Metabolic activity – Amount of certain metabolic products is in direct proportion to number of bacteria present – Examples: » Oxygen Consumption » Acid Production • Dry weight – Weight of packed cell mass is proportional to the number of cells in culture – Used for filamentous Fungi