Culture Media Preparation PDF

Culture Media Preparation Mark Jerwin F. Fabrero Biological Sciences Department Centro Escolar University A microbial culture medium is a mixture of substances that promotes and supports the growth and differentiation of microorganisms. The microorganisms that grow in...

Culture Media Preparation Mark Jerwin F. Fabrero Biological Sciences Department Centro Escolar University A microbial culture medium is a mixture of substances that promotes and supports the growth and differentiation of microorganisms. The microorganisms that grow in a culture media are referred to as the culture. Components of culture media - Culture media are formulated such that it mimics the organism’s natural habitat or environment, one that meets their necessary requirements. protein nitrogen carbohydrates agar (solidifying agent) dyes & indicators enriching substances Agar - Developed by Fanny Hesse - Extracted from red seaweed - No nutritive value - Melts at 98˚C and sets at 42˚C - Used as solidifying agent of solid medium  TYPES OF CULTURE MEDIA BASED ON CONSISTENCY A. Solid Media - 1.5% to 2.% agar - used to grow microorganisms in their full physical form, prepare bacterial pure cultures, or isolate bacteria to study colony characteristics. - e.g. blood agar, nutrient agar, McConkey agar, and chocolate agar TYPES OF CULTURE MEDIA BASED ON CONSISTENCY B. Semi-solid Media - 0.2% to 0.5% agar - appears as a soft, jelly-like substance - used to study the motility of microorganisms and to cultivate microaerophilic bacteria - e.g. Hugh and Leifson’s oxidation fermentation medium, Stuart’s and Amies media, and Mannitol motility media.  TYPES OF CULTURE MEDIA BASED ON CONSISTENCY C. Liquid Media - no solidifying agent - also called broths - large growth of bacterial colonies can be observed. - used for the profuse growth of microorganisms and fermentation studies. - e.g. Tryptic soy broth, phenol red carbohydrate broth, MR-VP broth, and nutrient broth. TYPES OF CULTURE MEDIA BASED ON NUTRITIONAL COMPONENT A. Simple media - general-purpose media that supports the growth of non-fastidious microbes - primarily used for the isolation of microorganisms. - e.g. nutrient broth, peptone water, and nutrient agar. B. Complex media - contains nutrients with unknown concentration that are added to bring about a particular characteristic of a microbial strain. - e.g. tryptic soy broth, blood agar, and nutrient broth. TYPES OF CULTURE MEDIA BASED ON NUTRITIONAL COMPONENT C. Synthetic media: - chemically defined media produced from pure chemical substances. - with known concentration of ingredients. - It is generally used in scientific research - e.g. Czapek Dox Medium TYPES OF CULTURE MEDIA BASED ON APPLICATION OR CHEMICAL COMPOSITION A. Basal media - routinely used simple media having carbon and nitrogen sources that boost the growth of many microorganisms. - general-purpose media and are considered non-selective media. - do not require enrichment sources for the growth of non-fastidious bacteria. - used to isolate microorganisms in labs or in sub-culturing processes. - e.g nutrient broth, nutrient agar, and peptone water. TYPES OF CULTURE MEDIA BASED ON APPLICATION OR CHEMICAL COMPOSITION B. Enriched media - prepared by adding additional substances like blood, serum, or egg yolk in the basal medium. - used to grow fastidious microorganisms as they require additional nutrients and growth- promoting substances. - e.g. Chocolate media, Blood agar (prepared by adding 5-10% blood by volume to a blood agar base) used to identify hemolytic bacteria TYPES OF CULTURE MEDIA BASED ON APPLICATION OR CHEMICAL COMPOSITION C. Selective media - Allows the growth of certain microbes while inhibiting the growth of others. - Used to isolate microorganisms. - The selective growth of microbes is decided by adding substances like antibiotics, dyes, bile salts, or by pH adjustments. TYPES OF CULTURE MEDIA BASED ON APPLICATION OR CHEMICAL COMPOSITION D. Differential or Indicator media - contain certain indicators like dyes or metabolic substrates in the medium composition which gives different colors to colonies of different microbial species when they utilize or react with these components. - allows the growth of more than one microorganism, but bacterial colonies are differentiated based on their color.  DIFFERENTIAL MEDIA EXAMPLES Blood agar - three types of blood cell lysis or hemolysis are observed: alpha, beta, and gamma - used to differentiate microorganisms based on their ability to lyse blood cells.  DIFFERENTIAL MEDIA EXAMPLES Mannitol salt agar - The fermentation of mannitol by Staphylococcus aureus causes the media to change to yellow, however, coagulase-negative staphylococci that can’t cause fermentation appear in pink.  DIFFERENTIAL MEDIA EXAMPLES MacConkey agar - differentiates the gram-negative bacteria based on their lactose metabolism. - The lactose fermenting bacteria, such as Escherichia coli, Klebsiella spp, Citrobacter, and Enterobacter forms pink-red colonies - lactose non-fermenters, like Salmonella, Shigella, Proteus, Providencia, Pseudomonas, and Morganella form pale or colorless colonies.  DIFFERENTIAL MEDIA EXAMPLES Thiosulfate citrate bile salts sucrose (TCBS) agar - used to differentiate organisms that ferment sucrose. - V. cholerae that ferment sucrose form slightly flattened yellow colonies having opaque centers and translucent peripheries. - V. parahaemolyticus can’t ferment sucrose and forms green to blue-green colonies. TYPES OF CULTURE MEDIA BASED ON APPLICATION OR CHEMICAL COMPOSITION E. Transport media - used for clinical specimens which are required to be transferred immediately to maintain viability. F. Anaerobic media: - used for anaerobic bacteria G. Assay media - used for amino acids, vitamins, and antibiotics assays. - e.g. antibiotic assay media is used to determine the antibiotic potency of microorganisms.  HOW TO PREPARE CULTURE MEDIA A. Nutrient Broth 1. Mix ingredients in an Erlenmeyer flask by calculating the amount of needed powder for desired volume (usually 8g/L). 2. Dissolve by heating over flame. Dispense 10 ml into each test tubes, plug with cotton plugs. 3.Autoclave at 15 psi for 15-20 minutes. Store in refrigerator for future use.  HOW TO PREPARE CULTURE MEDIA B. Nutrient Agar in Test Tubes 1. Mix ingredients in an Erlenmeyer flask by calculating the amount of needed powder for desired volume (usually 28g/L). 2. Boil until completely dissolved. Dispense 10 ml into each test tubes, plug with cotton plugs. Then autoclave at 15 psi for 15-20 minutes. 3. Solidify by slanting the test tubes (Agar Slants) or solidify in upward position (Agar Butts/Stabs). Store in refrigerator for future use.  HOW TO PREPARE CULTURE MEDIA C. Nutrient Agar in Petri Dishes 1. Mix ingredients in an Erlenmeyer flask by calculating the amount of needed powder for desired volume (usually 28g/L). 2. Boil until completely dissolved. Autoclave at 15 psi for 15-20 minutes. 3. Dispense 20 ml of the autoclaved media in petri dishes. Then Store in refrigerator for future use. Thank you!