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Chapter 6: Microbial Growth
Microbial Growth:
4 Refers to an increase in cell number , not in
cell size.
4 Bacteria grow and divide by binary fission ,
a rapid and relatively simple process.
Requirements for Growth
Physical Requirements
1. Temperature : Microbes are loosely
classified into several groups based on their
preferred temperature ranges.
A. Psychrophiles : “Cold-loving”. Can grow at 0 oC. Two groups: u True Psychrophiles : Sensitive to temperatures over 20 oC. Optimum growth at 15oC or below. Found in very cold environments (North pole, ocean depths). Seldom cause disease or food spoilage. u Psychrotrophs : Optimum growth at 20 to 30oC. Responsible for most low temperature food spoilage.
Requirements for Growth
Physical Requirements
1. Temperature :
B. Mesophiles : “Middle loving”. Most bacteria. u Include most pathogens and common spoilage organisms. u Best growth between 25 to 40oC. u Optimum temperature commonly 37oC. u Many have adapted to live in the bodies of animals.
Requirements for Growth
Physical Requirements
1. Temperature :
C. Thermophiles : “Heat loving”. u Optimum growth between 50 to 60oC. u Many cannot grow below 45oC. u Adapted to live in sunlit soil, compost piles, and hot springs. u Some thermophiles form extremely heat resistant endospores. u Extreme Thermophiles (Hyperthermophiles): Optimum growth at 80oC or higher. Archaebacteria. Most live in volcanic and ocean vents.
Growth Rates of Bacterial Groups
at Different Temperatures
Food Spoilage Temperatures
Requirements for Growth
Physical Requirements
2. pH :
4 Most bacteria prefer neutral pH (6.5-7.5). 4 Molds and yeast grow in wider pH range, but prefer pH between 5 and 6. 4 Acidity inhibits most microbial growth and is used frequently for food preservation (e.g.: pickling). 4 Alkalinity inhibits microbial growth, but not commonly used for food preservation. 4 Acidic products of bacterial metabolism interfere with growth. Buffers can be used to stabilize pH.
Requirements for Growth
Physical Requirements
2. pH : Organisms can be classified as:
A. Acidophiles : “Acid loving”. u Grow at very low pH (0.1 to 5.4) u Lactobacillus produces lactic acid, tolerates mild acidity. B. Neutrophiles : u Grow at pH 5.4 to 8.5. u Includes most human pathogens. C. Alkaliphiles : “Alkali loving”. u Grow at alkaline or high pH (7 to 12 or higher) u Vibrio cholerae and Alkaligenes faecalis optimal pH 9. u Soil bacterium Agrobacterium grows at pH 12.
Requirements for Growth
Physical Requirements
3. Osmotic Pressure : Cells are 80 to 90% water.
A. Hypertonicsolutions : High osmotic pressure removes water from cell, causing shrinkage of cell membrane (plasmolysis). Used to control spoilage and microbial growth. u Sugar in jelly. u Salt on meat. B. Hypotonic solutions : Low osmotic pressure causes water to enter the cell. In most cases cell wall prevents excessive entry of water. Microbe may lyse or burst if cell wall is weak.
Isotonic Versus Hypertonic Solution
Plasmolysis
Effects of Osmosis on Bacterial Cells
Requirements for Growth
Chemical Requirements
5. Oxygen:
B. Facultative Anaerobes : Can use oxygen, but can grow in its absence. Have complex set of enzymes. Examples : E. coli, Staphylococcus , yeasts, and many intestinal bacteria.
C. Obligate Anaerobes: Cannot use oxygen and are harmed by the presence of toxic forms of oxygen. Examples : Clostridium bacteria that cause tetanus and botulism.
Requirements for Growth
Chemical Requirements
5. Oxygen:
D. 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. E. Microaerophiles: Require oxygen, but at low concentrations. Sensitive to toxic forms of oxygen. Example : Campylobacter.
Requirements for Growth
Chemical Requirements
Toxic Forms of Oxygen:
1. Singlet Oxygen : Extremely reactive form of oxygen, present in phagocytic cells. 2. Superoxide Free Radicals (O 2 -.): Extremely toxic and reactive form of oxygen. All organisms growing in atmospheric oxygen must produce an enzyme superoxide dismutase (SOD) , to get rid of them. SOD is made by aerobes, facultative anaerobes, and aerotolerant anaerobes, but not by anaerobes or microaerophiles. Reaction : SOD O 2 -.^ + O 2 -.^ + 2H+^ -----> H 2 O 2 + O 2 Superoxide Hydrogen free radicals peroxide
Requirements for Growth
Chemical Requirements
3. Hydrogen Peroxide (H 2 O 2 ): Peroxide ion is toxic and the active ingredient of several antimicrobials (e.g.: benzoyl peroxide). There are two different enzymes that break down hydrogen peroxide: A. Catalase: Breaks hydrogen peroxide into water and O 2. Common. Produced by humans, as well as many bacteria. Catalase 2 H 2 O 2 ----------> 2H 2 O + O (^2) Hydrogen Gas peroxide Bubbles B. Peroxidase: Converts hydrogen peroxide into water. Peroxidase H 2 O 2 + 2H+----------> H 2 O Hydrogen peroxide
Microbial Growth
Culture Media
Culture Medium: Nutrient material prepared for
microbial growth in the laboratory.
Requirements:
4 Must be sterile
4 Contain appropriate nutrients
4 Must be incubated at appropriate temperature
Culture: Microbes that grow and multiply in or on a
culture medium.
Microbial Growth
Culture Media
Solid Media: Nutrient material that contains a solidifying agent (plates, slants, deeps). The most common solidifier is agar, first used by Robert Koch. Unique Properties of Agar: 4 Melts above 95oC. 4 Once melted, does not solidify until it reaches 40oC. 4 Cannot be degraded by most bacteria. 4 Polysaccharide made by red algae. 4 Originally used as food thickener (Angelina Hesse).
Microbial Growth
Culture Media
Chemically Defined Media: Nutrient material whose exact chemical composition is known. 4 For chemoheterotrophs, must contain organic source of carbon and energy (e.g.: glucose, starch, etc.). 4 May also contain amino acids, vitamins, and other important building blocks required by microbe. 4 Not widely used. 4 Expensive.
Microbial Growth
Culture Media
Complex Media: Nutrient material whose exact chemical composition is not known. 4 Widely used for heterotrophic bacteria and fungi. 4 Made of extracts from yeast, meat, plants, protein digests, etc. 4 Composition may vary slightly from batch to batch. 4 Energy, carbon, nitrogen, and sulfur requirements are primarily met by protein fragments ( peptones ). 4 Vitamins and organic growth factors provided by meat and yeast extracts. 4 Two forms of complex media:
- Nutrient broth : Liquid media
- Nutrient agar : Solid media
Microbial Growth
Culture Media
Anaerobic Growth Media: Used to grow anaerobes
that might be killed by oxygen. 4 Reducing media 4 Contain ingredients that chemically combine with oxygen and remove it from the medium. Example: Sodium thioglycolate
4 Tubes are heated shortly before use to drive off oxygen.
4 Plates must be grown in oxygen free containers (anaerobic chambers).
Microbial Growth
Culture Media
Special Culture Techniques: Used to grow bacteria with unusual growth requirements. 4 Bacteria that do not grow on artificial media :
- Mycobacterium leprae (leprosy): Grown in armadillos.
- Treponema pallidum (syphilis): Grown in rabbit testicles.
- Obligate intracellular bacteria (rickettsias and chlamydias): Only grow in host cells. 4 Bacteria that require high or low CO 2 levels:
- Capnophiles : Grow better at high CO 2 levels and low O (^2) levels. Similar to environment of intestinal tract, respiratory tract, and other tissues.
Equipment for Producing CO 2 Rich
Environments
Microbial Growth
Culture Media
Selective Media: Used to suppress the growth of unwanted bacteria and encourage the growth of desired microbes. 4 Saboraud’s Dextrose Agar: pH of 5.6 discourages bacterial growth. Used to isolate fungi. 4 Brilliant Green Agar : Green dye selectively inhibits gram-positive bacteria. Used to isolate gram-negative Salmonella. 4 Bismuth Sulfite Agar : Used to isolate Salmonella typhi. Inhibits growth of most other bacteria.
Bacterial Growth: Binary Fission Microbial Growth
Growth of Bacterial Cultures
Logarithmic Representation of Bacterial Growth : We can express the number of cells in a bacterial generation as 2 n , where n is the number of doublings that have occurred.
Microbial Growth
Phases of Growth
Bacterial Growth Curve : When bacteria are inoculated into a liquid growth medium, we can plot of the number of cells in the population over time. Four phases of Bacterial Growth:
1. Lag Phase:
4 Period of adjustment to new conditions. 4 Little or no cell division occurs, population size doesn’t increase. 4 Phase of intense metabolic activity, in which individual organisms grow in size. 4 May last from one hour to several days.
Microbial Growth
Phases of Growth
Four phases of Bacterial Growth:
2. Log Phase: 4 Cells begin to divide and generation time reaches a constant minimum. 4 Period of most rapid growth. Number of cells produced > Number of cells dying 4 Cells are at highest metabolic activity. 4 Cells are most susceptible to adverse environmental factors at this stage. - Radiation - Antibiotics
Microbial Growth
Phases of Growth
Four phases of Bacterial Growth:
3. Stationary Phase: 4 Population size begins to stabilize. Number of cells produced = Number of cells dying 4 Overall cell number does not increase.
4 Cell division begins to slow down. 4 Factors that slow down microbial growth:
- Accumulation of toxic waste materials
- Acidic pH of media
- Limited nutrients
- Insufficient oxygen supply
Microbial Growth
Phases of Growth
Four phases of Bacterial Growth:
4. Death or Decline Phase: 4 Population size begins to decrease. Number of cells dying > Number of cells produced 4 Cell number decreases at a logarithmic rate. 4 Cells lose their ability to divide. 4 A few cells may remain alive for a long period of time.
Four Phases of Bacterial Growth Curve Measuring Microbial Growth
Direct Methods of Measurement
1. Plate count: 4 Most frequently used method of measuring bacterial populations. 4 Inoculate plate with a sample and count number of colonies. Assumptions : - Each colony originates from a single bacterial cell. - Original inoculum is homogeneous. - No cell aggregates are present. 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 Methods of Measurement
1. Plate count (continued): A. Pour Plate : 4 Introduce a 1.0 or 0.1 ml inoculuminto an empty Petri dish. 4 Add liquid nutrient medium kept at 50oC. 4 Gently mix, allow to solidify, and incubate. Disadvantages:
- Not useful for heat sensitive organisms.
- Colonies appear under agar surface. B. Spread Plate : 4 Introduce a 0.1 ml inoculum onto the surface of Petri dish. 4 Spread with a sterile glass rod. 4 Advantages : Colonies will be on surface and not exposed to melted agar.
Pour Plates versus Spread Plates Measuring Microbial Growth
Direct Methods of Measurement
2. Filtration: 4 Used to measure small quantities of bacteria. - Example : Fecal bacteria in a lake or in ocean water. 4 A large sample (100 ml or more) is filtered to retain bacteria. 4 Filter is transferred onto a Petri dish. 4 Incubate and count colonies.
