BIOL2010 Week 7 Culturing Bacteria PDF

www.udst.edu.qa BIOL2010 Microbiology Unit 4: Culturing Bacteria Media and Culturing Microorganisms A culture: a group of microorganisms that grow in a medium. A medium: A solid, liquid or semi-solid that is designed to support the growth of microorganisms: Can be broth, Agar, or some othe...

www.udst.edu.qa BIOL2010 Microbiology Unit 4: Culturing Bacteria Media and Culturing Microorganisms A culture: a group of microorganisms that grow in a medium. A medium: A solid, liquid or semi-solid that is designed to support the growth of microorganisms: Can be broth, Agar, or some other enriched medium Culturing Bacteria Bacteria can live in Soil, Water, Food, & on and in the Human body One purpose of a microbiologist is to play detective and identify microorganisms Need to know characteristics of the organisms Useful when presented with an unknown sample of material Culturing Bacteria Culturing bacteria requires a pure sample Pure cultures are defined as: A culture that contains cells from a single species of an organism The medium selected to grow that culture must fit the organism enzyme and nutrition requirements Streak Plating is one method of collecting pure samples Pour Plates are another method Streak Plate Method Isolate pure colonies. Effective for separating mixed cultures Pour Plate Method Used primarily as a method for counting bacteria A serial dilution is performed from a liquid specimen Each dilution in the series is used to pour a plate Inoculate with bacteria in agar Plates are incubated until growth appears and colonies counted At some point you will have a plate with single, pure colonies This can be used to select out a single colony for growth: Colony forms from one originating bacteria – pure culture Can culture microaerophiles as well as obligate anaerobes. Serial Dilution – Pour Plate Estimating bacterial Numbers Choose plate with between 30 and 300 colonies. Multiply by the reciprocal of the dilution factor. Express answer in scientific notation. Culturing bacteria Culture Media: Media containing all nutrients needed for growth To grow we need to know the nutritional needs Have to have the ability to provide those needs in an artificial environment Culturing Bacteria There is no single media that will permit the growth of all bacteria. Some bacteria require anaerobic conditions for incubation There are numerous types of media available for culturing Culturing Media Media can be classified in two ways: 1. Composition – the type of substances that make up the media 2. Function – how the media affects the bacterial growth Culturing Media Media composition can be classified in two ways: 1. Natural Media – naturally occurring substances: blood, soil, etc. 2. Synthetic media – Media Prepared in a laboratory Further divided into: 1. Defined synthetic 2. Complex media – chemically non-defined Culturing Media Defined synthetic media: Contains known specific kinds & amounts of chemicals Prepared from purified ingredients & exact composition is known Defined Synthetic Media To use defined synthetic media: You must know the exact nutritional requirements of the organism Complex Media  Contains familiar materials in media  Chemical composition varies  Often contain nutrients in the form of extracts or enzyme digested products of milk, meat, plants or yeast (therefore not chemically defined) Complex Media Nutrient Broth – contains peptone (partially digested protein sources) and beef extract. Tryptic Soy Broth – contains tryptone (digested casein), peptone (digested soy bean), glucose, sodium chloride, and dipotassium sulfate. Cooked Meat Medium – contains beef heart, peptone (casein and meat peptides), glucose, and sodium chloride. Isolating and Identifying Bacteria As a microbiologist, much of what you do will be dedicated to identifying organisms based on specific criteria. These can include: The source of the culture specimen (water source, gut, lungs, etc) The microscopic appearance of the organism (shape, chains, etc) Its pattern of growth on selective media The organism’s metabolic, hemolytic, and fermentative properties on media The results of specific biochemical tests Isolating and Identifying Bacteria Selective media – Promote the growth of some organisms and inhibit the growth of others They select out an organism based on their ability to survive in the media Differential media – They allow the growth of organisms but determine whether an organism has specific enzyme capabilities to utilize the media. These do not select. Can produce coloured colonies Byproducts of metabolism can alter the media of indicators found in the media Enrichment media – Promotes the growth of SPECIFIC organisms that may be present in small numbers and outcompeted by other organisms Media enables growth Bacterial Enzymes Catalase – can digest peroxide Sucrase – degrades sucrose Amylase – can digest long chain Lactase – breaks down lactose sugars carbohydrates Nitroreductase – reduces nitro groups Peptidase – can digest peptides Autolysin – degrading protease that breaks Lipase/esterases – can digest lipids/fats down cell walls of biological tissue Hyaluronidase – can digest collagen and Coagulase – clots blood. Used to evade hyaluronic acid phagocytes Haemolysins – can digest RBCs Streptokinase-collagenase – dissolves cell Keratinase – breaks down keratin – tissue fingernails, hoofs, skin Maltase – breaks down maltose  Enterobacteriaceae The family Enterobacteriaceae is part of the domain: Bacteria, phylum: Proteobacteria; class: Gammaproteobacteria; and order: Enterobacteriales. There are over 30 genera and 120 species of Enterbacteriaceae but more than 95% of clinically significant strains fall into 10 genera and less than 25 species. Nearly all are facultative anaerobes. They ferment glucose, reduce nitrates to nitrites, and are oxidase negative. With the exceptions of Shigella and Klebsiella which are nonmotile, these bacteria have peritrichous flagella. The Enterobacteriaceae include some of the normal inhabitants of the small and large gastrointestinal tracts and, therefore, are sometimes referred to as enterics. However, these terms are not synonymous as some of the species do not live in the gastrointestinal tract and many species in the gastrointestinal tract do not belong to the Enterobacteriaceae. Table 1 lists the genus and species of the most common human pathogenic Enterobacteriaceae. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/ enterobacteriaceae Common Human Pathogenic Enterobacteriaceae Genus Species Citrobacter freundii koseri Enterobacter cloacae aerogenes sakasakii Escherichia coli albertii Klebsiella pneumoniae oxytoca Proteus mirabilis vulgaris Salmonella enterica Serratia marcescens Shigella dysenterii flexneri sonnei boydii Yersinia pestis enterocolitica pseudotuberculosis Differential Media Eosin Methylene Blue Agar MacConkey Agar Hektoen Enteric Agar Mannitol Salt Agar Differential Media Eosin Methylene Blue Agar Used to detect fecal coliform bacteria. Has Eosin Y, and Methylene blue as pH indicators, turns dark in low pH. Also inhibits Gram positive bacteria. Has Lactose and Sucrose as fermentable carbohydrates. Rapid fermenters Green metallic colonies Medium fermenters Pink colonies Non coliforms Colorless colonies Differential Media McConkey Agar – Selective and Differential. Contains lactose, crystal violet, bile salts and neutral red indicator Selects out gram negative bacteria Gram positive cannot grow in crystal violet or bile salts Differentiates between those that can and cannot ferment lactose Lactose fermenters will produce red colonies Non lactose fermenters produce colourless colonies Differential Media Mannitol Salt Agar – Selective and Differential Isolates Staphylococci (gm +ve) Contains a high (7.5%) salt concentration Inhibits halophobic growth Pathogenic (medically significant) Staphylococci ferment mannitol Non pathogenic Staphylococci do not Differential Media Hektoen Enteric Agar – Selective and Differential Selects and differentiates Salmonella and Shigella species from other Enterobacteriaceae in fecal samples. Has bile salts and bromothymol blue which inhibits Gram positive bacteria Has ferrous compounds to indicate the production of Hydrogen sulfide gas. Salmonella colonies  Transparent green colonies with black centers Shigella  Transparent green colonies Lactose fermenters  Salmon colored colonies. Selective Media Bismuth Sulfite Agar Isolates a specific species of Salmonella (S. typhi - Typhoid Fever) Has bismuth sulfite and Brilliant green indicators prevent Gram positive and coliform bacteria from growing. Samples from food or stool are tested. Presence of S. typhi will reduce the sulfide resulting in black or green metallic colonies Enrichment Media Tryptic Soy Agar and Broth Blood Agar Chocolate Agar Enrichment Media Tryptic Soy Agar & Broth A general purpose non-selective media, with plenty of nutrients to support a wide range of bacterial species. Most commonly use media in general teaching microbiology labs. Enrichment Media Blood Agar Contains general nutrients and 5% Bovine (cow or sheep’s) blood Can differentiate between species of bacteria that are haemolytic (have hemolysins) and those that are not. Alpha haemolytic bacteria partially break down RBCs and produce a green colour Streptococcus pneumoniae Beta haemolytic bacteria completely break down RBCs and cause clearing Staphylococcus sp. and Streptococcus sp. Gamma haemolytic bacteria can grow but do not break the RBCs Staphylococcus sp. Enrichment Media Chocolate Agar Blood is added to tryptic soy agar and heated. The heated blood provides the necessary growth factors to support specific bacteria Used to grow fastidious respiratory pathogens Haemophilus influenzae (can cause meningitis) Neisseria meningitidis (meningitis and sepsis) Characteristic Media Triple Sugar Iron Media Used to differentiate Enterics (Enterobacteriaceae) based on carbohydrate fermentation, and reduction of Sulphur. Differential media, contains: Lactose, sucrose, glucose (small amount), ferrous sulfate and phenol red. Characteristic Media Triple Iron Sugar Media An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose fermentation only. An acid/acid (yellow slant/yellow butt) reaction: It indicates the fermentation of dextrose, lactose and/or sucrose. An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate fermentation results. Blackening of the medium: Occurs in the presence of H2 Gas production: Bubbles or cracks in the agar indicate the production of gas (formation of CO2 and H2) Other Media Cooked Meat Medium Thioglycollate Medium Sabouraud Medium Other Media Cooked Meat Medium Non selective broth used to grow a wide range of aerobic and anaerobic bacteria. Contains heart, peptone and a small amount of glucose. Other Media Thyioglycollate Medium An enriched non-selective broth used to grow a variety of microaerophiles and anaerobic bacteria. Can help determine the oxygen demand of bacterial species. Other Media Sabouraud Dextrose Medium (SDM) Used for the growth of pathogenic and non-pathogenic fungi. Contains peptones. Can also grow some species of bacteria.  Media Type Examples: Bacterial Media Differential Eosin Mehtylene Blue Agar Summary MacConkey Agar Hektoen Enteric Agar Mannitol Salt Agar Selective Media Bismuth Sulfite Agar Enrichment Media Tryptic Soy Agar/ Broth (TSA/B) Chocolate Agar Blood Agar Characteristic Media Triple Sugar Iron Media Other Media Cooked Meat Medium Thioglycollate Medium Sabouraud Medium Recall…Pour Plate Method Used primarily as a method for counting bacteria A serial dilution is performed from a liquid specimen Each dilution in the series is used to pour a plate Inoculate with bacteria in agar Plates are incubated until growth appears and colonies counted At some point you will have a plate with single, pure colonies This can be used to select out a single colony for growth: Colony forms from one originating bacteria – pure culture Can culture microaerophiles as well as obligate anaerobes. Biochemical Tests Media Tests for… Carbohydrate Phenol red broth with Durham tube Fermentation of particular carbohydrates Fermentation Litmus Test Litmus milk medium with litmus indicator. Ability of bacteria to use different milk products, could change color, release a gas or curdle the media. Indole Production Tryptophan broth and Kovacs reagent. If a bacteria can break down the amino acid tryptophan. Methyl Red test Methyl red – Voges-Proskauer (MRVP) If a bacteria can produce and maintain acid media and Methyl Red Solution. products from glucose fermentation. Urease test Media containing urea, if ammonia is Enzyme urease produced (turns litmus indicator basic). Enzyme is present. Nitrate reduction Media containing Nitrate is used. If nitrites Enzyme Nitrate reductase are produced bacteria has enzyme Oxidase reaction TSA/B + Oxidase reagent Presence of cytochrome-oxidase, an important part of the ETC, lets us know how effective a bacteria can produce energy aerobically. Egg-yolk reaction Egg-yolk agar Tests for lecithinase, an enzyme that can destroy animal tissue. Biochemical tests Carbohydrate Fermentation Tests Contains Phenol red pH indicator and a carbohydrate like glucose, dextrose, lactose, etc. Also contains a Durham tube. If the bacteria can ferment the carbohydrate they produce an acid which lowers the pH. Positive test: Broth turns yellow, acid (A) Negative test: Broth stays red/pink (-ve) If gas is present the record results as Acid-Gas (AG) Biochemical Tests Indole production Test to determine if a bacteria can break down the amino acid tryptophan to indole. Use to discriminate between different species of Enterobacteriaceae Bacteria are cultivated in tryptophan broth. Kovacs reagent (acid + alcohol) is added. If the bacteria can digest tryptophan, indole is produced. Indole will react with Kovacs reagent to turn pink, since the alcohol in not soluble the pink will float on the surface. Biochemical Tests Oxidase reaction Test used to determine if a bacteria has the cytochrome-oxidase enzyme. This is an enzyme in the ETC, tells us that the bacteria can produce energy aerobically by using the ETC. Effective way to identify some pathogenic species. Inoculate a broth tube or filter paper with bacteria, add the oxidase reagent and look for a blue color (+ve) or no change (-ve). Biochemical Tests Egg yolk reaction To test for lecithinase, an enzyme that can destroy animal tissue. Used to determine virulence of a species of bacteria. Uses Egg-yolk agar, has the molecule lecithin and a lipoprotein. If the bacteria has the enzyme, it breaks down the lecithin and causes a white precipitate to grow around the colonies (+ve). No white precipitate (-ve).