Chapter 6 Microbial Nutritions PDF

CHAPTER 6 MICROBIAL NUTRITION Nutrient Requirements Uptake of Nutrients Culture Media Isolation of Pure Cultures  Nutrients What are nutrients? Chemical substances acquired from the environment by cellular activities and used for growth Why are nutr...

CHAPTER 6 MICROBIAL NUTRITION Nutrient Requirements Uptake of Nutrients Culture Media Isolation of Pure Cultures  Nutrients What are nutrients? Chemical substances acquired from the environment by cellular activities and used for growth Why are nutrients important? Nutrients are used to form hundreds of chemical compounds inside a living cell  95% of cell dry weight is made up of major elements:  Carbon  Oxygen  Hydrogen  Nitrogen  Sulfur  Phosphorous  Potassium  Calcium Microbial cell composition  Magnesium  Ferum Nutrients Requirements Source of carbon for basic structures Source of cellular energy to drive metabolic reactions (ATP) Source of high energy electrons/H, reducing power (NADH/NADPH) In order for bacteria to grow, they need a source of raw materials and energy – Source can be the same (e.g. glucose) or different (e.g. carbon dioxide and sunlight).  Where do raw materials come from? Microbes acquire energy from oxidation of organic or inorganic molecules, or from sunlight Growth requires raw materials: some form of carbon Autotrophs vs. heterotrophs – Auto = self; hetero = other; troph = feeding – Autotrophs use carbon dioxide – Heterotrophs use pre-formed organic compounds (molecules made by other Nutrients Requirements Macronutrients (macroelements): – elements required in large amounts – C, O, H, N, S, P, K, Ca, Mg, and Fe Micronutrients (trace elements): – metals and organic compounds needed in very small amounts – Mn, Zn, Co, Mo, Ni, and Cu Macronutrients K – enzymatic activity in protein synthesis Ca – heat resistance of bacterial endospores Mg – co-factor for enzyme, stabilizer to ribosome and cell membrane Fe – ATP synthesis by electron transport Micronutrients (Mn, Zn, Co, Mo, Ni,  The acronym CHNOPS, which stands for carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur, represents the six most important chemical elements whose covalent combinations go to make up most biological molecules on Earth! Pyrimidi Purines nes  Requirement of Carbon, Hydrogen, Oxygen and Electron source Carbon is backbone of all organic components present in a cell Hydrogen and oxygen are also found in organic compound Electrons play a role in energy production and reduction of CO2 to form organic molecules  Nutritional Types of Organisms Microorganisms can be classified based on: Energy source - Phototrophs use light - Chemotrophs obtain energy from oxidation of organic or inorganic compounds  Nutritional Types of Organisms Electron source – Lithotrophs use reduced inorganic substances – Organotrophs obtain electrons from organic compounds Carbon source – Autotrophs use CO2 as their sole or principal carbon source – Heterotrophs use organic molecules as carbon sources which often obtained from  Energy - Electron - Carbon Light (photo) Organic (organo) CO2 (auto) Organic or inorganic (chemo) Inorganic (litho) Organic (hetero)  Requirements for Nitrogen, Phosphorus, and Sulfur N, P and S is needed for synthesis of important molecules (e.g. amino acids, nucleic acids and carbohydrates)  Growth Factors growth factors are organic compound that are essential cell components (or precursors) that the cell cannot synthesize must be supplied by environment if cell needs to survive and reproduce 3 major classes: Amino acid Purines & Vitamins pyrimidines  needed for protein synthesis Amino acid Purines & Vitamins pyrimidines needed for nucleic acid function as enzyme cofactors synthesis  How are these nutrients taken into the cell ?  Uptake of Nutrients Nutrients enter cells by: 1. Simple diffusion 2. Facilitated diffusion 3. Active transport 4. Group translocation 5. Endocytosis  Simple Diffusion also called passive diffusion results from molecular kinetic energy and random movement of particles movement from higher to lower concentrations transport small molecules across lipid bilayer not energy dependent (passive)  Simple Diffusion Extracellular Intracellular Example of molecules: H2O, O2 and CO2  Facilitated Diffusion Similar to simple Differ from simple diffusion: diffusion: – direction of – uses protein channels movement is from or carrier proteins in higher to lower plasma membrane concentration – carrier proteins may – movement of change its molecules is not conformational energy dependent structure to transport (passive) molecules – transport larger molecules Facilitated Diffusion Example of molecules: glucose, amino acids, fatty acids, glycerols Facilitated Diffusion Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. Facilitated Diffusion What does this plateau line indicate?  Active Transport movement of molecules from lower to higher concentrations energy-dependent process (ATP or proton motive force) involves carrier proteins in plasma membrane hydrolysis of ATP causes conformational changes in the carrier protein resulting in the forced movement of substances concentrates molecules inside cell Active Transport Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.  Group Translocation energy-dependent process chemically modifies molecule as it is brought into cell best known system: transports a variety of phosphoenolpyruvate: sugar phosphotransferase system (PTS), which transport various sugars while phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor Group Translocation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. Group Translocation - PTS  Endocytosis movement of very large substances into cell without travelling across plasma membrane membrane invaginates and forms vacuoles occur only in eukaryotic cells e.g: phagocytosis of  Endocytosis Two main types: phagocytosis – cell surface protrudes to surround and engulf particles – produces phagosome phagosome (phagocytic vacuole) which will fuse with lysosome for digestion pinocytosis – uptake of small amounts of pinosome liquid – produces pinosome  CHAPTER 6 MICROBIAL NUTRITION Nutrient Requirements Uptake of Nutrients Culture Media Isolation of Pure Cultures  Culture Media contain mostly all the nutrients required by the organism for growth can be classified based on: – physical nature – chemical composition – functional type Physical Nature Liquid, semi-solid, solid media Chemical Composition – Defined Media chemical compositions are precisely known can be used to culture fastidious organisms with specific requirements specifically prepared for research purposes e.g. minimal media used in bacterial genetics experiments Chemical Composition – Complex Media contain some ingredients of unknown chemical composition widely used to culture various microorganisms e.g. nutrient broth, Luria-Bertani agar, potato dextrose agar Source Ingredients Plant or animal Beef or yeast extracts extracts Soybean meal, casein or milk protein Digested proteins Peptone, tryptone Sulfated Agar used to solidify liquid Medium used to grow Leuconostoc mesenteroids What type of medium is this?  What type of media are these? Medium used to grow Medium used to grow Chemoheterotrophic Heterotrophic Bacteria Bacteria What type of medium is this? Blood agar Functional Types of Media 1. Supportive (general purpose media) – support the growth of many microorganisms – both Gram-positive and Gram-negative bacteria can grow on this media – e.g. tryptic soy agar, nutrient agar Supportive Media Klebsiella pneumoniae Bacillus cereus Tryptic soy agar Functional Types of Media 2. Enriched media – general purpose media supplemented with special nutrients or growth factors – addition of blood, serum, vitamins or eggs – support the growth of fastidious organisms – e.g. blood agar, chocolate agar, Lowenstein-Jensen (LJ) medium Enriched Media Neisseria Corynebacterium gonorrhoeae on sp. on blood agar chocolate agar Enriched Media Mycobacterium tuberculosis on LJ medium (enriched with coagulated eggs) Functional Types of Media 3. Selective media – allow the growth of some microorganisms and inhibit the growth of others – e.g. MacConkey agar and Eosin methylene blue (EMB) agar both media selects for Gram- negative bacteria and inhibits the growth of Gram-positive bacteria Selective Media – MacConkey contains bile salts and crystal violet dye that inhibit Gram- positive bacteria – EMB contains eosin and methylene dye which are toxic to Gram-positive bacteria E. coli on MacConkey agar Functional Types of Media 4. Differential media – distinguish between different groups of microorganisms based on their biological characteristics – e.g. MacConkey agar, EMB agar lactose fermenters vs non- fermenters – e.g. blood agar hemolytic vs non-hemolytic bacteria Differential Media – MacConkey contains lactose, peptone and neutral red dye – E. coli (Lac+) uses lactose in the media and produce acid that lowers pH of agar below 6.8 (pink colonies) – Pseudomonas sp. (Lac-) uses peptone and produce ammonia that raises pH of agar (colourless colonies) – Neutral red is a pH indicator, that changes from red toLow Indicator yellow pH Lowbetween pH range HighpH 6.8-8.0 pH range High pH Neutral Red Red 6.8 8.0 Yellow  MacConkey agar Pseudomonas E. coli aeruginosa Lactose-fermenter Non-lactose-fermenter PINK COLOURLESS  EMB agar E. coli E. coli and Pseudomonas sp. produces metallic green sheen colonies  Blood agar Beta hemolysis Alpha hemolysis Gamma hemolysis Beta hemolysis: complete lysis of red blood cells (RBC) and hemoglobin, results in complete clearing of blood around the colonies Alpha hemolysis: partial lysis of RBC and hemoglobin, results in a greenish-grey discoloration of blood around the colonies Gamma hemolysis: no hemolysis, results in no change in the medium  Blood agar Can you examine the type of hemolysis? Streptococcus Streptococcus viridans pyogenes  Mannitol salt agar Selective because of high salt – only members of Staphylococcus sp. can grow Differential because S. aureus ferments mannitol (turns yellow) but S. epidermidis does not. No growth of Micrococcus luteus  CHROMagar Chromogenic detection media Colonies with distinguishabl e colours Candida species differentiated when grown CHROMagar Identification of urinary tract pathogens with differential media Mechanisms of Action? https://www.foodqualitynews.com/Article/2014/07/01/Salmonella-s-need-for-nutrient-d uring-growth-in-inflamed-intestine How to purify a mixed culture?  Isolation of Pure Cultures Pure culture – population of cells arising from a single cell – consists of only a single species of microorganism Techniques used to isolate for pure cultures – streak plate – spread plate – pour plate 1. Streak Plate Technique Streak plate of Serratia marcescens 2. Spread Plate Technique involve spreading mixture of cells on agar surface using spreader to separate individual cells each cell can reproduce to form separate colony Spread Plate Bacterial Colonies 3. Pour Plate Technique sample is diluted and mixed with liquid agar mixture of cells and agar are poured into sterile culture dishes Pour Plate Bacterial Colonies  Morphological Observations very important criteria to identify a species must be supported by various Bacterial Colony Morphology  use of transillumination to determine whether colonies are hemolytic also to see slight color differences Optical property / density Margin Bacterial Colony Morphology E. coli lactose fermenter growing on MacConkey agar Shape: Circular Margin: Entire Elevation: Flat Dry appearance Klebsiella sp. colonies on MacConkey agar Shape: Circular Margin: Entire Elevation: Pulvinate, Mucoid cream-colored center after 48 hours’ growth Bacterial Colony Morphology Bacterial Colony Morphology  Storage of Cultures kept in 4C agar slant (last for few months) freeze in - 20C in glycerol stock (for years) End of Chapter 6