Lecture 6 Principles & Functions of Culture Media PDF

LECTURE 6 PRINCIPLES & FUNCTIONS OF CULTURE MEDIA SBGA_300424 Mar-Aug24  Importance of media for culturing microbe. Methods of obtaining pure cultures. Liquid, solid and semisolid media. Types of media.  Media Used for Bacterial Growth The pr...

LECTURE 6 PRINCIPLES & FUNCTIONS OF CULTURE MEDIA SBGA_300424 Mar-Aug24  Importance of media for culturing microbe. Methods of obtaining pure cultures. Liquid, solid and semisolid media. Types of media.  Media Used for Bacterial Growth The primary objective of cultivating microorganisms, specifically bacteria, is to maintain viable populations of these organisms under controlled laboratory environments. Challenging process due to highly specific nutritional and environmental requirements and the diversity of these requirements among different species.  Nutritional Requirements All-purpose medium - tryptic soy broth (TSB). Specialised media - are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. e.g. enriched media - contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious (fussy) organisms, organisms that cannot make certain nutrients and require them to be added to the medium. Chemically defined medium - complete chemical composition of a medium is known. e.g. EZ medium Complex media - contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. e.g. nutrient broth, TSB, brain heart infusion (BHI). BHI is made by combining an infusion from boiled bovine or porcine heart and brain with a variety of other nutrients. BHI broth is often used in food safety, water safety, and antibiotic sensitivity tests.  Nutritional Requirements Selective medium - inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition. e.g. MacConkey agar that contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favour the growth of gram-negative bacteria, particularly the Enterobacteriaceae. Entero – the intestine; Enterobacteriaceae species (enterics) reside in the intestine that adapted to bile salt. Quick think 1: Bile salt are stored in the _______. What is lobe? part of an organ that appears to be separate in some way from the rest.  Lactose-fermenting organisms, such as E. coli and Klebsiella spp, grow as pink to red colonies with or without a zone of precipitated bile. Do E. coli & Klebsiella ferment lactose? Lactose-non-fermenting organisms, such as Salmonella, Shigella and Proteus spp., form colourless or clear colonies. MUCOID is resembling mucus. microbenotes.com/macconkey-agar/ https://microbiologyinfo.com/macconkey-agar-composition- principle-uses-preparation-and-colony-morphology/  Nutritional Requirements Enrichment medium - general purpose enrichment agar which can nourish and support the growth of gram-positive as well as gram-negative bacteria. e.g. blood agar (5% defibrinated mammalian blood – horse, sheep, human) is added to the autoclaved basal media (TSA or Columbia Agar). Why enriched? Supports the growth of fastidious bacteria and inhibits the growth of some bacteria like Neisseria and Haemophilus. e.g. chocolate agar - nonselective, enriched growth medium used for isolation of pathogenic bacteria. It is a variant of the blood agar plate, containing red blood cells that have been lysed by slowly heating to 80°C. as a result, the cell lysis releases intracellular nutrients such as hemoglobin, hemin (“X” factor), and the coenzyme nicotinamide adenine dinucleotide (NAD or “V” factor) into the agar which is utilized by fastidious bacteria. Red blood cell lysis gives the medium a chocolate-brown coloration when prepared from which the agar gets its name. The most common bacterial pathogens that require this enriched medium for growth include Neisseria gonorrhoeae and Haemophilus species. https://microbenotes.com/chocolate-agar/ https://microbeonline.com/x-v-factor-test-haemophilus-principle-procedure-results/  Nutritional Requirements Differential medium e.g. Eosin Methylene Blue (EMB) - slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella). Certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen (most E. coli strains). Other lactose fermenters produce larger, mucoid colonies, often purple only in their centre. Non-lactose fermenter demonstrated colourless colonies. https://microbenotes.com/eosin-methylene-blue-emb-agar/ https://www.labtestsguide.com/eosin-methylene-blue-agar-emb-agar  Mueller Hinton Agar (MHA) commonly used for the routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique. “ZOI” What is your comment? Smaller zone vs. larger zone? Which concentrations indicate more sensitive/ resistant results? How about if no ZOI observed? Fungal Culture Media https://microbeonline.com/common-fungal-culture-media-uses/ Exercise https://microbiologyinfo.com/list-of-culture-media-used-in-microbiology-with-their-uses/ Autoclave  Obtaining Pure Culture is a laboratory culture containing a single species of organism. a pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many fold before inoculating the new medium. obtaining a pure culture of bacteria is usually accomplished by spreading bacteria on the surface of a solid medium so that a single cell occupies an isolated portion of the agar surface. This single cell will go through repeated multiplication to produce a visible colony of similar cells, or clones.  Solid vs. semi solid vs. liquid media other than nutrients, a solidification agent (agar, an inert substance extracted from sea algae. It does not show any nutritional value) is used during the preparation of solid and semi solid media. semi solid media (deep culture) are microbial culture media that are prepared to add less amount of agar (solidifying agent at 0.2 to 0.5 %) to observe motility of bacteria. Are the results macroscopic? Exercise: Compare between solid and semi solid media.  Solid vs. semi solid vs. liquid media broth culture is any type of liquid used to grow bacteria. Nutrient agar (NA) vs. nutrient broth (NB) culture: NA - contains solidifying agent NB – remains in liquid form  Spread Plate Technique is the method of isolation and enumeration of microorganisms in a mixed culture and distributing it evenly. The technique makes it easier to quantify bacteria in a solution. https://microbeonline.com/spread-plate-technique/ Spread Plate Technique  Pour Plate Technique The pour plate method is a microbiological laboratory technique for isolating and counting the viable microorganisms present in a liquid sample, which is added along with or before molten agar medium prior to its solidification. A spread-plate assay produces a plate with colonies distributed across the agar surface, while a pour plate assay produces a mixture of colonies embedded within the agar layer and colonies presenting at the agar surface End of Lecture 6