Culture Media Composition PDF

CULTURE MEDIA COMPOSITION  Culture media supply the nutritional needs of microorganisms ( C ,N, Phosphorus, trace elements, etc)  Defined medium : precise amounts of highly purified chemicals  Complex medium (or undefined) : highly nutritious substances.  In clinical micr...

CULTURE MEDIA COMPOSITION  Culture media supply the nutritional needs of microorganisms ( C ,N, Phosphorus, trace elements, etc)  Defined medium : precise amounts of highly purified chemicals  Complex medium (or undefined) : highly nutritious substances.  In clinical microbiology,  Selective : contains compounds that selectively inhibit  Differential: contains indicator  terms that describe media used for the isolation of particular species or for comparative studies of microorganisms. 2 TYPES OF MEDIA  Media can be classified on three primary levels 1. Physical State 2. Chemical Composition 3. Functional Type 3 PHYSICAL STATES OF MEDIA  Liquid Media  Semisolid  Solid (Can be converted into a liquid)  Solid (Cannot be converted into a liquid) 4 LIQUID MEDIA  Water-based solutions  Do not solidify at temperatures above freezing / tend to be free flowing  Includes broths, milks, and infusions  Measure turbidity  Example: Nutrient Broth, Methylene Blue Milk, Thioglycollate Broth 5 SEMI-SOLID MEDIA  Exhibits a clot-like consistency at ordinary room temperature  Determines motility  Used to localize a reaction at a specific site.  Example: Sulfide Indole Motility (SIM) for hydrogen sulfide production and indole reaction and motility test. 6  SOLID MEDIA  Firm surface for discrete colony growth  Advantageous for isolating and culturing  Two Types 1. Liquefiable (Reversible) 2. Non-liquefiable  Examples: Gelatin and Agar (Liquefiable) Cooked Meat Media, Potato Slices (Non-liquefiable) 7 CHEMICAL COMPOSITION OF CULTURE MEDIA 1. Synthetic Media  Chemically defined  Contain pure organic and inorganic compounds  Exact formula (little variation) 2. Complex or Non-synthetic Media  Contains at least one ingredient that is not chemically definable (extracts from plants and animals)  No exact formula / tend to be general and grow a wide variety of organisms 8  SELECTIVE MEDIA  Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe.  Example: Mannitol Salt Agar [MSA] encourages the growth of S. aureus. MSA contain 7.5% NaCl which inhibit the growth of other Gram + bacteria 9 Growth of Staphylococcus aureus on Mannitol Salt Agar results in a color change in the media from pink to yellow. 10  DIFFERENTIAL MEDIA  Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates.  Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that develops in the colonies. Blood agar for hemolysis (α,β,and γ hemolysis), EMB, MacConkey Agar, …etc. 11 12  ENRICHMENT MEDIA  Is used to encourage the growth of a particular microorganism in a mixed culture.  Ex. Manitol Salt Agar for S. aureus  Blood agar , chocolate agar, Slenite F broth 13 BACTERIAL COLONIES ON SOLID MEDIA P. aeruginosa (TSA) S. Marcescens (Mac) S. Flexneri (Mac) 14  LABORATORY CULTURE OF MICROORGANISMS  Microorganisms can be grown in the laboratory in culture media containing the nutrients they require.  Successful cultivation and maintenance of pure cultures of microorganisms can be done only if aseptic technique is practiced to prevent contamination by other microorganisms. 15  MICROBIAL GROWTH  Microbesgrow via binary fission, resulting in exponential increases in numbers  Thenumber of cell arising from a single cell is 2n after n generations  Generationtime is the time it takes for a single cell to grow and divide 16 Binary Fission 17 Rapid Growth of Bacterial Population 18  GROWTH CURVE  During lag phase, cells are recovering from a period of no growth and are making macromolecules in preparation for growth  During log phase cultures are growing maximally  Stationary phase occurs when nutrients are depleted and wastes accumulate (Growth rate = death rate)  During death phase death rate is greater than growth rate 19  METHODS USED TO MEASURE MICROBIAL GROWTH Plate Count Method: Turbidity Measurement (Spectrophotometry): This method involves spreading a known volume of a This method measures the cloudiness of a liquid microbial sample onto the surface of a solid agar culture, which is directly proportional to the microbia medium. cell density. After incubation, colonies formed by individual A spectrophotometer is used to measure the viable cells are counted. absorbance of light passing through the culture at a Results are expressed as colony-forming units (CFUs) specific wavelength. per unit volume. Turbidity measurements are expressed in optica density units (OD) or absorbance units. Impedance Microbial Growth Analyzer: Flow Cytometry: This method measures the electrical impedance of a This technique uses lasers and detectors to analyze culture medium, which changes as microorganisms individual cells in a liquid sample. grow and metabolize nutrients. Cells are tagged with fluorescent dyes, and the 20 resulting signals are used to determine cell size complexity, and viability. Biomass Determination: Viable Cell Count: This method involves direct measurement of the In addition to plate counting, other methods like microbial biomass. the Most Probable Number (MPN) method or the Dry weight, protein content, or other cellular use of fluorescence-based dyes (e.g., acridine components can be used to estimate the biomass orange) can be used to count viable cells. concentration. The MPN method involves statistical estimation It requires harvesting and drying the cells before based on the presence or absence of growth in measurement. multiple tubes or wells. Real-time PCR (Polymerase Chain Reaction): Metabolic Activity Measurement: Quantitative PCR can be used to measure the Monitoring the metabolic activity of amount of DNA in a sample, providing an indirect microorganisms, such as the production of carbon measure of microbial growth. dioxide, can provide an indirect measure of microbial growth. Resazurin reduction assays are an example of a metabolic activity measurement.  Flow Cytometry Impedance Microbial Growth Analyzer Metabolic Activity Measurement: Real-time PCR  VIABLE COUNTS  Each colony on plate or filter arises from single live cell  Only counting live cells 23 Direct Count Pour Plate 24 25  Direct Count Spread or Streak Plate 26 27  MICROSCOPIC COUNTS  Need a microscope, special slides, high power objective lens  Typically only counting total microbe numbers, but differential counts can also be done 28 TURBITITY  Cells act like large particles that scatter visible light A spectrophotometer sends a beam of visible light through a culture and measures how much light is scattered  Scales read in either absorbance or % transmission  Measures both live and dead cells 29  INOCULATION  Sample is placed on sterile medium providing microbes with the appropriate nutrients to sustain growth.  Selection of the proper medium and sterility of all tools and media is important.  Some microbes may require a live organism or living tissue as the inoculation medium. 30  INCUBATION  An incubator can be used to adjust the proper growth conditions of a sample.  Need to adjust for optimum temperature and gas content.  Incubation produces a culture – the visible growth of the microbe on or in the media 31  ISOLATION  The end result of inoculation and incubation is isolation.  On solid media we may see separate colonies, and in broth growth may be indicated by turbidity.  Sub-culturing for further isolation may be required. 32  INSPECTION  Macroscopically observe cultures to note color, texture, size of colonies, etc.  Microscopically observe stained slides of the culture to assess cell shape, size, and motility. 33  IDENTIFICATION  Utilize biochemical tests to differentiate the microbe from similar species and to determine metabolic activities specific to the microbe. 34