Subjects included;• Cultivation• Medium• Inoculation of media• The Requirements for Growth• Preparation• Classification• Eplanation of types of media functionally.
Cultivation; The propagation of living organisms, appliedespecially to the growth of microorganisms or othercells in artificial media.Medium Any preparation that contains nutrients essentialfor microorganisms growth.Culture medium: A nutrient substance that is used to growmicro-organisms .
Inoculation of media Introduction of infected material to the medium ,forcultivation of organisms. Agar Is Complex polysaccharide, Used as solidifyingagent for culture media in Petri plates, slants, anddeeps. Generally not metabolized by microbes Liquefies at 100°C Solidifies at ~40°C
The Requirements for Growth1.CHEMICAL REQUIREMENTS(NUTRITIONAL FACTORS)• Carbon• Nitrogen, sulfur, and phosphorous• Trace elements• Organic growth factor• Vitamins (e.g. folic acid, vitamin B-12, vitamin K)• Oxygen
Preparation• Wash hands & wear gloves• Sterilize all equipments• Pour D/W in glassware (required amount)• Add powder ingredients (required amount)• Heat to dissolve completely• Autoclave• Dispense the medium into tubes ,bottles &plates• Store at required temperature• Sterilizing Culture Media
2.Nutritional a ) Simple e.g peptone water b ) Complex e.g blood agar c ) Synthetic e.g Davis /mingioli medium
3.Functionala) Basic mediab) Selective mediac ) Differential mediad ) Enriched mediae ) Enrichment media f ) Transport media
Basic media• This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown.• Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.
Basic media• This media contains; 1.A carbon source such as glucose for bacterial growth 2.Water 3.Various salts needed for bacterial growth
There are two types:1. nutrient broth which is liquid media
Selective media• culture medium that allows the growth of certain types of organisms, while inhibiting the growth of other organisms• Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen.• Various approaches to make a medium selective include addition ofantibiotics, dyes, chemicals, alteration of pH or a combination of these medium
Selective media• Thiosulphate citrate bile salt sucrose (TCBS) which is selective for Vibrio cholerae• MacConkey agar contain bile salt which is selective for Enterobacteriaceae
Differential media• distinguishs one microorganism type from another based on a difference in colony appearance includes (color, shape, or growth pattern) on the media.• uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red , or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism
Differential media• is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria• Examples; Mannitol salt agar Eosin methylene blue MacConkey agar
• Mannitol salt agar which is differential for mannitol fermentation Like Staphylococcus aureus change the color of agar from pink to yellow• Eosin methylene blue which is differential for lactose and sucrose fermentation
• MacConkey agar which is differential for lactose fermentation Ex: E.coli From non lactose fermentersEx: salmonella,shigella
Enriched media• Enriched with Blood,serum,extrapeptones,egg& vitamins• Used for: cultivation of all fastidious organisms, (haemophilus influenza&Streptococcus spp.)• Examples:• Blood agar &chocolate agar
7.Mixing4.Adding the the bloodcontentstobottle 1.agar powders with molten medium 2.Weighing out at 45 c the ingredients 5.Autoclaving 8.Pouring plates 3.Measuring the desired 6.Adding volume of blood to the water for Preparation of blood agar out 9.Laying medium bloodmedia base plates to set medium
Chocolate agar fastidious respiratory bacteria, such as Haemophilus• Originally, red growth factors, like NADto a meltedinfluenzae need blood cells were added and hemin,which are insideand heatedcells; thus, a prerequisite to nutrient base red blood to approximately 75°Cgrowth is lysis the red blood cells which lysed of the red blood cells. The agar isnamed for the color and contains no actual chocolate.
Enrichment media• A liquid culture medium promotes the growth of a particular organism and prevent the growth of normal competitors.• Prevent non pathogenic bacteria from overgrowing the pathogenic bacteria• Example: selenite-Fbroth alkaline-peptone water
SELENITE –F BROTH• Intended Use :is used for the selective enrichment of Salmonella spp.• Prepared Appearance: Prepared medium is clear, with no to light precipitate and very pale yellow.
ALKALINE PEPTONE WATER• Intended use: is used for the enrichment of Vibrio cholera and Vibrio species from food, water, feces and clinical studies.• Prepared appearance: The color is amber
Transport media• Preserve viability and ratio of microbes during its transportation over 24_48 hours.• Example: CARY-BLAIR MEDIUMSTUART TRANSPORT MEDIUM
CARY-BLAIR MEDIUM• Intended use;• for the collection and transport of fecal and rectal samples, maintaining viability of Salmonella and Shigella.• Survival depends upon: 1. bacterial type &concentration , transport medium formulation, transport temperature and duration.• the color is white opalescent
STUART TRANSPORT MEDIUM• Intended use: is a semisolid medium used in the transport and preservation of specimens for the cultivation of diverse organisms such as gonococci, streptococci, EnterobacteriaThe color : is blue in surface.