Microbial Metabolism (Part 2) PDF

MICROBIAL GROWTH PHYSICAL AND CHEMICAL REQUIREMENTS & CULTURE MEDIA LEARNING OUTCOMES At the end of this topic, students should be able to: Explain the physical and chemical requirements of growth Distinguish between the various types of culture media and explain how they affect bacteria...

MICROBIAL GROWTH PHYSICAL AND CHEMICAL REQUIREMENTS & CULTURE MEDIA LEARNING OUTCOMES At the end of this topic, students should be able to: Explain the physical and chemical requirements of growth Distinguish between the various types of culture media and explain how they affect bacterial growth Review the standard growth pattern in a batch culture. Understand the various methods of growth measurement. Understand the mathematics and kinetics of growth.  PHYSICAL REQUIREMENTS The requirements for microbial growth can be categorized into physical requirements or chemical requirements. Physical requirements that are crucial in the growth of microbes: Temperature pH Osmotic pressure Biochemical requirements or also known as nutritional factors include the availability of: Carbon. Nitrogen, Sulfur, Phosphorus. Trace elements. Organic growth factor. Oxygen.  PHYSICAL REQUIREMENTS The temperature range over which an organism grows is determined largely by the temperatures at which its enzymes function. Within this temperature range, three critical temperatures can be identified: 1. The minimum growth temperature, the lowest temperature at which cells can divide. 2. The maximum growth temperature, the highest temperature at which cells can divide. 3. The optimum growth temperature, the temperature at which cells divide most rapidly—that is, have the shortest generation time.  PHYSICAL REQUIREMENTS Temperature: Microorganisms can be classified into FIVE groups 1. Psychrophiles Cold-loving microbes (0°C). Optimum at 15 °C, cannot grow at 25 °C and above. Mostly found in deep ocean or polar regions. Seldom cause problems in food preservation. Ex: Flavobacterium, Arthrobacter. 2. Psychrotrophs (facultative psychrophiles) Moderate or facultative psychrophiles. Can grow between 0 °C - 30 °C. Optimum between 20 °C - 30 °C. Cannot grow above 40 °C. Much more common than psychrophiles. Great interest to food microbiologist. Causes food spoilage in the refrigerator  PHYSICAL REQUIREMENTS 1. Mesophiles Most common type of microbe. Can grow between 10 °C - 48 °C Optimum between 25 °C - 40 °C Most pathogens fall into this category where their optimum temperature is at 37 °C. Ex: Escherichia. 2. Thermophiles Heat-loving microbes (40 °C - 70 °C ). Optimum temperature between 50 °C - 60 °C. Cannot grow below 45 °C. Some thermophiles form extremely heat resistant endospores. Found in sunlit soil, compost piles and natural hot springs. Ex: Thermus.  PHYSICAL REQUIREMENTS 1. Hyperthermophiles Also known as extreme thermophiles. Mostly are Archaea. Can grow between 65 °C - 110 °C. Optimum temperature at 80 °C. Found in hydrothermal vents and hot springs associated with volcanic activity. Sulphur is usually important in their metabolic activity. Ex: Thermococcus.  PHYSICAL REQUIREMENTS Typical growth rates at different types of microorganisms in response to temperature. Food preservation temperature  PHYSICAL REQUIREMENTS pH (The proper pH is needed to provide a proper environment for the ions to interact during synthesis and metabolic processes.) Most bacteria grow best at pH near neutral (6.5-7.5), however there are exceptions Acidophiles – highly tolerant of low pH, grow at a very low pH (0.1-5.4) Lactobacillus produces lactic acid, tolerates mild acidity. Neutrophiles – near neutral, grow at pH 5.4-8.5, includes most human pathogens. Alkaliphiles – highly tolerant of high pH, grow at alkaline or high pH (7-12 or higher), optimal pH for Vibrio cholerae, Soil bacterium Agrobacterium grows at pH 12. preserve food  PHYSICAL REQUIREMENTS Osmotic pressure When a bacterial call is placed in a hypertonic solution, the cellular water content will pass out through the plasma membrane and cause plasmolysis (shrinkage of cell’s cytoplasm) salting can help preserve food – salted fish Sweetened condense milk Extreme halophiles – adapted to salty environment that they require it for growth  aka obligate halophiles Facultative halophiles  do not require high salt conc, but can tolerate up to 2% salt (which most bacteria cannot) https://goo.gl/images/tNEgwp https://goo.gl/images/51cDpG https://goo.gl/images/fY7BJa NUTRITIONAL CLASSIFICATION The requirements and the forms taken in by bacteria leads to the classification of bacteria into: Autotroph: Organisms that derived their carbon requirement from CO₂. These are photosynthetic or Photosynthesis-like microorganism. Heterotroph: Derive their carbon from organic sources such as sugars, proteins and amino acids. Based on source of energy, Microorganism are subdivided into: Phototroph: Captures energy from light sources. These microorganism have pigments resembling chloroplast that can trap energy. Eg. Chromatin. Chemotroph/Organotroph: Energy is obtained by the breaking down of organic compound enzymatically (needs to be consumed or to be absorbed in). Energy is then trapped in the form of ATP and used as energy source. NUTRITIONAL CLASSIFICATION Photoautotroph: Autotroph that uses light as energy source. Photolithotroph: Phototroph that uses molecular hydrogen (H₂O) or sulphur compound as electron donor. Photoorganotroph: derives its energy from photosynthesis (uses light as energy source) and organic compounds. Eg. protozoan. **Chemoorganotroph: Uses energy derived from the chemical breakdown of organic nutrients.  CHEMICAL REQUIREMENTS Carbon – energy source, building blocks for cell components Carbon is needed because it is the backbone structure of any organic molecules It is the second most important requirement for growth after water Chemoautotrophs and photoautotrophs derive their carbon from carbon dioxide Chemoheterotroph get their carbon from their energy source – protein, carbs, lipid Nitrogen, sulfur and phosphorus Nitrogen and sulfur is needed for amino acids  backbone of protein Nitrogen and phosphorus is needed to make nucleotides  DNA molecule Phosphorus - required by the cells primarily for synthesis of nucleic acids, ATP and phospholipid. ATP  energy storage  CHEMICAL REQUIREMENTS Cont. - Nitrogen, sulfur and phosphorus Most organisms are not able to use molecular nitrogen (N₂) directly Some microbe have the ability to fix the molecular nitrogen into ammonium  nitrites which is readily used by most organism Trace elements Element that are needed in small amount Minerals  iron, copper, zinc (usually co-factors for enzymatic reaction) Oxygen Different microbe have different requirements for oxygen Microbe can be classified based on their oxygen requirements Water. All biological cells need water. Water provides the solutes for biochemical reactions to take place. All growth processes are biochemical reactions catalysed by enzymes. W.A (Aw) = water activity. It is an indicator of the content of water https://goo.gl/images/eRKSqs  CULTURE MEDIA Culture  microbes growing in or on culture media Culture media is the food used to grow and control microbes. Culture medium or growth medium is a liquid (broth) or gel (agar) designed to support the growth of microorganisms. It has to be sterile  only the intended microbe will grow There are different types of media suitable for growing different types of cells. Chemically defined  know the exact composition of the media Complex  contain extract and/or digest of yeast, meat or plant  CULTURE MEDIA Ideal characteristics of culture media Contain right nutrients for specific microorganisms. Contain sufficient moisture. Properly adjusted pH. Suitable level of O₂/ none at all. Must be sterile prior to inoculation. Incubated at proper temperature. Chemically defined media vs Complex media Chemically defined media p Complex media p g g Composed of pure biochemicals off the shelf. blood, milk, yeast extract, beef extract etc. contain organic source of carbon and energy Widely used for heterotrophic bacteria and fungi. (glucose/starch) p Nutrient broth What is fastidious organisms? A fastidious organism is any organism that has complex or nutritional requirements. In other words, a fastidious organism will only grow when specific nutrients are included in its diet. Heterotrophs? An organism that cannot manufacture its own food by carbon fixation and therefore derives its intake of nutrition from other source of organic carbon, mainly plant or animal matter. COMMONLY USED CULTURE MEDIA Nutrient agar/broth General purpose complex media use for cultivating bacteria Supports wide range of non-fastidious bacteria Contain 1. 0.5% peptone (nitrogen source), 2. 0.3% beef/yeast extract (vitamin, carbohydrates, nitrogen and salt), 3. 0.5% NaCl https://microbiologyinfo.com/nutrient-agar-composition- preparation-and-uses/  CULTURE Types of culture media: MEDIA Enrichment media Defined media which has been added with specifically required substances such as blood, serum or egg (eg. Blood agar) Helps in promoting the growth of targeted microbe which in normal circumstances may be difficult to grow (fastidious organism) Selective media Selects the growth of a specific microbe while inhibiting growth of others by adding certain salt or dyes or antibiotics Differential media Differentiate a specific bacteria from others Has a constituent that causes an observable change (a color change or a change in pH) in the medium when a particular biochemical reaction occurs. eg: Blood agar on hemolysis activity  MacConkey Agar MacConkey agar (MAC) was the first solid differential media to be formulated which was developed at 20th century by Alfred Theodore MacConkey. MacConkey agar is a selective and differential media used for the isolation and differentiation of non-fastidious gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas.  BLOOD AGAR Blood agar – for microbes that require blood (trypticase soya agar enriched with 5% sheep blood) Chocolate agar – contains lysed red blood cells Both are considered enrichment media – contain blood or lysed red blood cells Helps in promoting the growth of targeted microbe which in normal circumstances may be difficult to grow - fastidious organism “Chocolate agar” sounds yummy but contains no actual chocolate. The colour comes from cooked blood! It is used to culture fastidious organisms. https://goo.gl/images/m6hD2a  BLOOD AGAR Blood agar – especially useful in identifying specific bacteria based on their ability to break down RED BLOOD CELLS – HEMOLYSIS Some species of bacteria produce extracellular enzymes which have the ability to lyse red blood cells in the blood agar. The extracellular enzymes is called hemolysins. HEMOLYSINS - It has the ability to destroy cells and release the red component of the blood (hemoglobin) into the medium. The red color of hemoglobin is altered as it is exposed to the chemicals in the blood agar Differentiation properties Alpha () hemolysis – partial hemolysis - a dark or discolored medium Beta () hemolysis – full hemolysis - medium is cleared under growth. Gamma () hemolysis – no hemolysis - no changes in the medium  HOW TO DETECT HEMOLYSIS? To find out if hemolysis has taken place, bacteria are streak on the blood agar plate. The medium is incubated overnight and will be inspected for any signs of hemolysis. Differentiation properties Alpha () hemolysis – partial hemolysis -color of the medium is altered as characterized by a dark or discolored medium Beta () hemolysis – full hemolysis - medium is cleared under growth. Gamma () hemolysis – no hemolysis - no changes in the medium https://goo.gl/images/PWqQvf Are blood agar plates selective or differential? Blood agar is both a selective and a differential medium. Selective? It is used for the isolation of Streptococcus and Moraxella species based on their ability to break down rbc. Differential? helps differentiate the type of hemolysis produced such as alpha-hemolysis, beta-hemolysis, and gamma-hemolysis. Selective and Differential medium MANNITOL SALT AGAR Mannitol salt agar (MSA)  contains high conc. of NaCl (salt) that selects for microbe that can withstand osmotic pressure The high concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels. Also contain the sugar mannitol and the pH indicator phenol red  microbe that can ferment mannitol and produce acid  will cause phenol red in the agar to turn yellow (differential properties) non-pathogenic staphylococci will not ferment mannitol. Selective and Differential medium MANNITOL SALT AGAR The Staphylococcus aureus ferments mannitol and turns the medium yellow. The Serratia marcescens does not grow because of the high salt content. Selective and Differential medium SALMONELLA-SHIGELLA AGAR Salmonella-Shigella (SS) agar  selects for growth of Salmonella spp. and Shigella spp. Selective properties  Bile Salts, Sodium Citrate and Brilliant Green (dye) serve to inhibit Gram-positive Differential properties Sodium Thiosulfate and Ferric Citrate permit detection of hydrogen sulfide production by the production of colonies with black centers. Contain lactose  permits the identification of lactose fermenters  pink colonies Non feremnters – form colorless colonies https://goo.gl/images/Q64rvv The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production as evidenced by colonies with black centers. Selective and Differential medium TCBS AGAR Thiosulfate Citrate Bile Salts-Sucrose (TCBS) agar Contain selective agents (sodium thiosulphate, sodium citrate) providing an alkaline pH to inhibit Gram-positive organisms and suppress coliforms Ox Bile: The bile salts inhibit growth of Gram-positive microorganisms Ferric citrate: Sodium Thiosulfate is also a sulfur source, and acts with Ferric Citrate as an indicator to detect hydrogen sulfide production. Differentiation: use to differentiates different species of Vibrio V. cholerae vs V. parahaemolyticus Selective and Differential medium Differentiation: V. cholerae large and slightly flattened, yellow colonies with opaque centers and translucent peripheries. V. parahaemolyticus  green to blue-green colonies as it does not ferment sucrose. https://goo.gl/images/QbkC9E