Growth Requirements and Microbial Cultures PDF

Growth requirements and microbial cultures Karolina Witek Department of Pharmaceutical Microbiology  Microbiological media provide suitable nutrients that are necessary for microbial growth  Microbiological media The idea of culturi...

Growth requirements and microbial cultures Karolina Witek Department of Pharmaceutical Microbiology  Microbiological media provide suitable nutrients that are necessary for microbial growth  Microbiological media The idea of culturing bacteria was first introduced by Robert Koch  Microbiological media agar resists digestion by bacterial enzymes agar melts when heated to 85°C Fannie Hesse  Function ✓ enrich the numer of microorganisms ✓ obtain pure cultures ✓ select for certain microorganisms and suppress others ✓ differentiate among different microorganisms ✓ preserves microorganisms for longer period of time  Basic requirements Nutrients Mineral salts Carbon sourse Nitrogen source Sulphate, phosphates etc. Suitable pH Growth (7,2-7,4) factors Oxygen Appropriate Temperature Sterility osmotic pressure  Components carbon sources energy source, synthesis of cell components nitrogen sources enzymes, other proteins and nucleic acids S sulfur-containing amino acids and proteins P ATP, phospholipids, and nucleic acids trace elements: Co, Cu, Fe, Mn, Mo, Zn supplied with water or contamination of other medium components, additives include: Ca, Mg, P, K, S, Cl (in the form of salts) cofactors in enzymatic reactions, synthesis of cell components vitamins typically used as coenzymes differentiating and selective substances  Classification 1. Consistency Culture media Liquid/broth medium Solid Semi-solid - specific amounts of nutrients - agar 1.5-2.0% - agar ≤ 0.5% (no gelling agents such as - allows bacteria to grow gellatin or agar) -cultivation microaerophilic bacteria in physically informative of microaerophilic or useful ways - various purposes (e.g. microaerophilic bacteria bacteria or determination propagation of large number bacterial motility - isolation of bacteria or of bacterial motility of organisms, research) for determination of the colony characteristics of the isolate e.g. agar plate, agar slant, agar stab (agar deep tube) e.g. LB broth (Luria Bertani broth) e.g. sulfite indole medium Solid media  Classification 1. Nutritional components Culture media Simple media Complex media support the growth of non-fastidious ingredients whose exact components are bacteria (e.g. peptone water, nutrient difficult to estimate (e.g. blood agar) agar)  Classification 3. Functional use of application Basal media Enriched media Selective media Differential media Chromogenic media Transport media  Basal medium general purpose media contains minimum nutrients possible for colony growth supports most non-fastidious bacteria often used to grow microorganisms Nutrient broth e.g. TSB tryptose-soy broth - liquid medium Nutrient agar e.g. TSA tryptose-soy agar - solid medium Peptone water  Enriched medium contains special nutrients that allow growth of particular organisms - Blood agar (contains 5% sheep blood) – used for growing most of the clinically important bacteria and detecting hemolytic activity -Chocolate agar (contains factor X, hemin and V NAD) – used for isolation of demanding bacteria, e.g. Haemophilus, Neisseria -Schaedler Agar – used for growing anaerobic bacteria  Enriched medium Chocolate agar Blood agar  HAEMOLYSIS α - haemolysis α β - haemolysis β γ-haemolysis  α-HAEMOLYSIS partial lysis of RBCs to produce a greenish-grey or brownish discoloration due to the reduction of RBC hemoglobin to methemoglobin in the medium surrounding the colony example: Streptococcus pneumoniae  β-HAEMOLYSIS complete lysis of RBCs, resulting in a distinct, clear,colorless zone surrounding and under the colony RBC membrane is destroyed example: Streptococcus pyogenes, Streptococcus agalactiae and Staphylococcus aureus  γ-HAEMOLYSIS no hemolysis of RBCs there is no change in the medium under and surrounding the colonies example: Enterococcus faecalis  Selective medium encourages the growth of some organisms but supresses the growth of others SF medium (selenine-phosphate) – used to grow bacteria of the genus Salmonella-Shigella (phosphate inhibits the growth of the accompanying flora, acidic sodium selenine enhances the growth of Salmonella and Shigella) Löwenstein-Jensen medium - used for culture of Mycobacterium spp., notably Mycobacterium tuberculosis (malachite green inhibits the growth of other bacteria)  Enriched medium Löwenstein-Jensen medium M. tuberculosis appears as brown, granular colonies  Sabouraud Dextrose Agar Dextrose provides a rich source of carbohydrate for the rapid growth of fungal cells slightly acidic pH and addition of antibiotics inhibits the growth of bacteria Selective and differential medium 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 this change allows to distinguish a certain type of colony from others growing on the same plate MacConkey agar – used for isolation of Gram-negative enterics - selective constituent – crystal violet and bile salts inhibit Gram- positive bacteria - differential constituent – lactose and pH indicator (neutral red) identify lactose fermenters as red (red when acidic) colonies and non-fermenters as light pink. Most intestinal pathogens are nonfermenters and hence do not produce acidic pH Selective and differential medium Selective and differential medium Chapman agar selective medium for isolation and differentiation of pathogenic staphylococci selective agent - 7,5% NaCl, differential agent - mannitol pH indicator - phenol red Formation of acid from mannitol is indicated by a colour change to yellow Selective and differential medium SS agar for isolation of Salmonella and Shigella Selective agent – high concentration of bile salts and ferric citrate Differential agent- sodium thiosulfate which is a source of sulfur for Salmonella producing hydrogen sulfide Salmonella - colorless colonies with a black center Shigella - colorless colonies Chromogenic medium  innovative  simultaneous isolation and identification of microorganisms  technology based on soluble colourless molecules (called chromogens), composed of a substrate (targeting a specific enzymatic activity) and a chromophore.  the target organisms are characterized by enzyme systems that metabolize the substrates to release the chromogen. The chromogen can then be visually detected by direct observation of a distinct colour change in the medium. CPS agar Media used in susceptibility testing Mueller-Hinton agar For non-fastidious bacteria Mueller-Hinton agar with 5% hourse blood for fastidious bacteria  Transport medium Transport media are special media formulated to preserve a specimen and minimize bacterial overgrowth from the time of collection to the time it is received at the laboratory Sterile swab with Stuart’s medium  Transport-growth medium Uromedium for determining urine bacterial count in the bacteriological diagnosis of urinary tract infections Aeromedium and anaeromedium liquid media that allows growth of bacteria isolated from blood Inoculation of culture plate Streaking method Spreading method Puring method  Inoculation of a culture plate plate provide a large surface for isolation and observation of colonies streaking is done using a loop or a swab as follow: 1. Streaking 2. Zigzag Streak plate technique – necessary equipment Streak plate technique Third streak area: individual isolated colonies First streak area: heavy, confluent growth Second streak area: growth starts to thin out, some individual colonies appear  Streak plate technique - purpose: to produce isolated colonies of an organism (mostly bacteria) on an agar plate to separate organisms in a mixed culture (to purify/isolate particular strain from contaminants) to identify the organism: - to study the colony morphology - to perform biochemical tests (valid when performed on pure cultures)  Streak plate technique - principle inoculum is diluted by streaking it across the surface of the agar plate the inoculum is diluted to the point where there is only one bacterial cell when these bacterial cells divide and give rise to thousands and thousands of new bacterial cells, an isolated colony is formed (colony forming units; CFUs) pure cultures can be obtained by picking well isolated colonies and re-streaking these on fresh agar plates  Streak plate technique - tips for the best results: use only a small amount of inoculum streak lightly so that you do not gouge the agar. flame the loop after you streak each quadrant. make sure surface of the plate is free of droplets of condensed moisture Streak plate technique Spread plate technique Pour plate technique  Inoculation of slant slant tubes - tubes containing nutrient medium plus agar the medium has been allowed to solidify at an angle in order to get a flat inoculating surface a loop is using to streak the surface of the slant Do not gouge the agar with the loop, instead gently gaze the surface  Inoculation of stab tubes stab tubes (deeps) – tubes of solid or semi-solid medium which are inoculated by „stabbing” the inoculum into the agar using sterile needle for organisms that prefer reduced O2 evaluation of motility Inoculating needle is moved into the tube without touching the walls of the tube and the needle penetrates medium to its depth  Motility test Negative: organism does not Positive: move away from organism moves the stab away from the stab Inoculation of liquid media Liquid media are inoculated using sterile wire loop or pipette depending on whether the inoculum is colonial growth or a fluid culture or specimen Patterns of growth in liquid media 1. Sediment: a mass of organisms 1 2 3 appear as a deposit at the bottom of the tube (obligate anaerobes) 2. Turbidity: organisms appear as a general cloudiness throughout the broth 3. Pellicile: a mass of organisms is floating on the top of the broth (obligate aerobes) Phases of bacterial growth  Phases of bacterial growth – Lag phase no increase in living bacterial cells takes place when bacteria are inoculated into new fresh media bacteria try to adopt in new environment phase of adjustment necessary for the synthesis of enzymes and co-enzymes for physiological activities  Phases of bacterial growth – Log phase bacteria divides continuously at constant rate and the number of bacteria increase exponentially all bacteria are in their rapid stage of cell division and show balanced growth biochemical and physiological characteristics are commonly used for identification of bacteria  Phases of bacterial growth – stationary phase a constant bacterial population is maintained by balance between cell division and cell death induced by increased bacterial cell density, depletion of nutrition in media and accumulation of toxic secondary metabolic wastes production of antibiotics such as penicillin, streptomycin etc and enzymes by certain bacteria occur during stationary  Phases of bacterial growth – death/decline phase number of bacteria decrease continuously and exponentially death phase is brought about by depletion of nutrition and accumulation of toxic wastes. Factors affecting bacterial growth 1. Physical factors pH temperature oxygen concentration moisture hydrostatic/osmotic pressure radiation 2. Nutritional factors (Ca, S, P, trace elements, vitamins) Factors affecting bacterial growth – pH optimum pH is usually near neutrality most microbes do not grow at a pH more than 1 pH unit above or below their optimum according to their tolerance for acidity or alkalinity, bacteria are classified as: - acidophiles – grow best at a pH of 0.1 to 5.4 (e.g Lactobacillus) - neutrophiles – exist from pH 7.0 to 8.0 (pathogenic bacteria) - alkaliphiles – exist from pH 7.0 to 11.5 (e.g Vibrio cholerae) Factors affecting bacterial growth - temperature most species of bacteria grow over a 30 °C temperature range min and max temperatures vary according to their growth temperature range, bacteria can be classified as: psychrophiles – grow best at temperatures of 15 - 20° C mesophiles – grow best at temperatures 25 - 40° C thermophiles – grow best at temperatures 50 - 60 ° C Factors affecting bacterial growth – oxygen Obligate aerobes – must have oxygen for aerobic respiration Facultative anaerobes – ordinarily carry on aerobic metabolism when oxygen is present, but they shift to anareobic metabolism when oxygen is absent Microaerophiles – appear to grow best in the presence of a small amount of oxygen Aerotolerant anareobes – can survive in the presence of oxygen but do not use it in their metabolism Obligate anaerobes – killed by free oxygen Thank you for your attention  Colony morphology When describing a bacterial colony, we take into account: - size of the colony - colony shape - round, oval, lenticular -colony surface - shiny, matte, rough, smooth, wrinkled - shore of the colony - even, jagged - color of the colony - white, yellow, etc. -consistency of the colony - brittle, leathery, pulling etc. -transparency of colonies; transparent, opaque, opalescent etc. - growth of colonies: flat, convex - odor - e.g Pseudomonas aeruginosa – grape-like odor, acidic – staphylococci - acidic odor -surroundings of the colony: colored, unstained, hemolyzed, unhemolyzed -types of hemolysis: type α - partial haemolysis, greening around the colonies type β - total haemolysis type γ - no haemolysis