General Microbiology Lecture Notes - BSFT

MODULE 5: MICROBIAL GROWTH Overview: Microbes that are “growing” are increasing in number, accumulating into colonies. Many bacteria survive and grow slowly in nutrient-poor environments by forming biofilms. Learning Outcomes: After successful completion of this module, the learner...

MODULE 5: MICROBIAL GROWTH Overview: Microbes that are “growing” are increasing in number, accumulating into colonies. Many bacteria survive and grow slowly in nutrient-poor environments by forming biofilms. Learning Outcomes: After successful completion of this module, the learner should be able to: 1. identify the microbial growth requirements. 2. determine how bacteria grow exponentially using generation time; and 3. describe different microbial growth phases. Course Material: Growth Factors may require small amounts of certain organic compounds for growth because they are essential substances that the organism is unable to synthesize from available nutrients. The requirements for microbial growth can be divided into two main categories: physical and chemical. I. Physical Requirements: 1. temperature - Most microorganisms grow well at the temperatures that humans favor. Classified into three primary groups: a. psychrophiles (cold-loving microbes) at 0⁰C. b. mesophiles (moderate-temperature– loving microbes) - 25–40°C, are the most common type of microbe. c. thermophiles (heat-loving microbes) – 50-60⁰C. d. hyperthermophiles - have an optimum growth temperature of 80°C or higher. e. extreme thermophiles – 121⁰C and above. 2. pH – refer to acidity or alkalinity of a solution. - Most bacteria grow best in a narrow pH range near neutrality, between \ pH 6.5 and 7.5. - Very few bacteria grow at an acidic pH below about pH 4. This is why a number of foods, such as sauerkraut, pickles, and many cheeses, are preserved from spoilage by acids produced by bacterial fermentation. - Acidophiles are bacteria that loves acids environment. 3. Osmotic pressure - require water for growth, and their composition is 80–90% water. - High osmotic pressures have the effect of removing necessary water from a cell. - Hypertonic - whose concentration of solutes is higher than in the cell. - Plasmolysis – shrinkage of cell cytoplasm. 39  Types of Halophiles: a. Extreme Halophiles – require high salt concentration b. Obligate Halophiles – require 30% of salt for growth. c. Facultative Halophiles – requires 15% of salt for growth II. Chemical Requirements: 1. Carbon 2. Sulfur, Nitrogen and Phosphorus 3. Trace elements such as iron, copper, molybdenum, and zinc 4. Organic Growth factors 5. Oxygen a. aerobes – microbes that use molecular oxygen b. anaerobes - microbes that do not use oxygen. Types of Oxygen Requirement: 1. Obligate aerobe - organisms that require oxygen to live e.g. Pseudomonas aeruginosa (Gram negative) Mycobacterium tuberculosis (acid-fast) Bacillus (Gram-positive). 2. Facultative anaerobe - ability to continue growing in the absence of oxygen e.g. E. coli and yeast 3. Obligate anaerobe - bacteria that are unable to use molecular oxygen for energy-yielding reactions e.g. Clostridium 4. Aerotolerant anaerobes - cannot use oxygen for growth, but they tolerate it fairly well. e.g. Lactobacilli 5. Microaerophile - they are aerobic; they do require oxygen. They grow only in oxygen concentrations lower than those in air. e.g. Borrelia burgdorferi, a species of spirochaete bacteria that causes Lyme disease in humans, Helicobacter pylori, a species of proteobacteria that has been linked to peptic ulcers and some types of gastritis Cell Division Two Types of Asexual Reproduction in Microbes 1. Binary fission - forms a totally new daughter cell, with the mother cell retaining its original identity. 2. Budding division - forms a totally new daughter cell, with the mother cell retaining its original identity. Generation time - When one cell eventually separates to form two cells, we say that one generation has occurred. - During one generation, all cellular constituents increase proportionally. 40  - Each daughter cell receives a copy of the chromosome(s) and sufficient copies of ribosomes and all other macromolecular complexes, monomers, and inorganic ions to begin life as an independent entity. *Biofilms- an attached polysaccharide matrix containing embedded bacterial cells. Microbial Growth and Quantification Growth - an increase in the number of cells. Culture medium or growth medium is a liquid or gel designed to support the growth of microorganisms. Laboratory cultures of microorganisms are grown in culture media Two broad classes of culture media: 1. Defined media are prepared by adding precise amounts of pure inorganic or organic chemicals to distilled water. Exact composition is known. 2. Complex media are made from digests of microbial, animal, or plant products. Microbial Growth Cycle Four Phases of Growth: a. lag phase - growth begins only after a period of time b. exponential or log phase - cell population doubles at regular intervals c. stationary phase – cells in the population grow while others die d. death phase - growth ceases Exponential growth is a repetitive pattern where the number of cells doubles in a constant time interval. - As one cell divides to become two cells, we express this as 20 -> 21. As two cells become four, we express this as 21 -> 22 and so on. - Bacteria grow quickly in batch culture (enclosed vessel), and cell numbers increase dramatically in a short period of time. - By measuring the rate of cell population increase over time, the growth depicts a certain “growth curve”. - In order to control both specific growth rate and cell density independently, continuous culture, a type of an open system must be done. - The most common type of continuous culture is the chemostat. - In the continuous culture growth vessel, a known volume of sterile medium is added at a constant rate while an equal volume of spent culture medium (which also contains cells) is removed at the same rate. Measuring Number of Microbes Microscopic counting is a quick and easy way of estimating microbial cell numbers. Microscopic counts can be performed either on samples dried on slides or on liquid samples. a. Stained samples to increase contrast between cells and their background 41  b. Liquid samples, counting chambers consisting of a grid with squares of known area etched on the surface of a glass slide are used. A. Direct Measurement of Microbial Growth 1. Plate count - most frequently used method of measuring bacterial populations. Often reported as colony-forming units (CFU). A viable cell is one that is able to divide and form offspring, and in most cell-counting situations. Methods of Viable Plate Count: a. spread plate method - a volume (usually 0.1 ml or less) of an appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader. - positions all the colonies on the surface and avoids contact between the cells and melted agar. b. pour plate method – a known volume (usually 0.1-1.0 ml) of culture is pipetted into a sterile Petri plate. (Refer to Figure 5.14 of the book). - colonies will grow within the nutrient agar (from cells suspended in the nutrient medium as the agar solidifies) as well as on the surface of the agar plate. 2. Serial Dilution - to ensure that some colony counts will be within this range, the original inoculum is diluted several times in a process. Refer to Figure 6.16. 3. Filtration - applied frequently to detection and enumeration of coliform bacteria, which are indicators of fecal contamination of food or water. 4. Most Probable Number (MPN) method - the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series. Uses of MPN: a. microbes being counted will not grow on solid media (such as the chemoautotrophic nitrifying bacteria). b. useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (such as coliform bacteria, which selectively ferment lactose to acid, in water testing). 5. Direct Microscopic Count - a measured volume of a bacterial suspension is placed within a defined area on a microscope slide. Petroff-Hausser cell counter – used for direct microscopic counting. Coulter counter – used for electronic cell counters. B. Indirect Methods by Estimating Bacterial Numbers 1. Turbidity - As bacteria multiply in a liquid medium, the medium becomes turbid, or cloudy with cells. 2. Spectrophotometer (or colorimeter) - instrument used to measure turbidity. 42  3. Metabolic Activity - assumes that the amount of a certain metabolic product, such as acid or CO2, is in direct proportion to the number of bacteria present. 4. Dry Weight - the fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator. It is then weighed. Activities / Assessments: In a separate sheet of paper, write the name, section, module number, title and questions. Then briefly discuss the following questions: 1. Compare and contrast the physical requirement in microbial growth. 2. Discuss the method of viable plate count. Grading System: In question No. 1, there will be a 10 points per physical requirement. In question No. 2, there will be a 10 points per viable plate count. RUBRICS FOR SCORING ESSAY QUESTION 5 4 3 2 1 The student has full The student has a good The student has a basic The student has some The student has no Level of Understanding understanding of the understanding of the understanding of the understanding of the understanding of question or problem question or problem question or problem question or problem the question or The response reflects a The response reflects The response provides problem. The The response addresses Synthesis of Information complete synthesis of some synthesis of little or no synthesis of response is the question information information information completely Total Score = _________ / 10 x 100 = _________% References: Madigan, M.T. et.al. Brock Biology of Microorganisms 15th ed. (2019). Pearson Education, Inc. Tortora, Funk and Case. Microbiology: An Introduction. (2013). 11th ed. Pearson Education, Inc. 43