CULTURE MEDIA AND TYPES

CULTURE MEDIA AND ITS TYPES What is Culture Media? The media is a source of nutrients to support the growth of the micro-organisms in-vitro. The media helps in the growth and counting of microbial cells, selection of microorganisms, and survival of microorganisms. The culture medium can be liquid or gel. Common ingredients of culture media • Peptone- source of carbon and nitrogen. • Beef extract- source of amino acid, vitamins, minerals. • Yeast extract- source of vitamin, carbon, nitrogen. • Distilled water • Agar- solidifying agent How to prepare culture media? 1. Weigh the amount of ingredients powder on weighing machine. 2. Dissolve the ingredients in distilled water. 3. Adjust PH of the medium if needed. 4. Add agar and boiled it to dissolve. 5. Pour the media into flask. 6. Autoclave the media when ingredients fully dissolve. 7. Sterilization is done in autoclave to prevent from contamination, at 121ºC for 15 min at 15lbs. 8. After the autoclave place the media flask in laminar air flow. 9. Sterilize the laminar air flow with 70% alcohol. 10. A bit cools down the media and pours into sterile Petri-plates for solidification. 11. Then sample is ready to spread(spreader) / streak 12. (Inoculation loop) on the medium for identification or isolation of microbes. 13. Sealed the Petri plates with paraffin, label them. 14. Keep them inverted in incubator at 37ºC for 24hrs. 15. Observe the result next day colonies formation is visible on the media.

What is a Defined medium? A defined medium has a known quantity of all ingredients, like carbon source (Glucose or Glycerol) and nitrogen source (Ammonium salt or Nitrate as inorganic nitrogen). The medium needs in metabolic, nutritional, and physiological growth experiments. (Czapek Dox Medium) What is an Undefined medium? This medium has different complex ingredients in unknown quantities, for example- yeast extract, beef, various salts, and enzymatic protein. (Potato dextrose agar, MacConkey agar) What is Complex media? This media is other than basal media; it has added ingredients to bring the characteristics of microorganisms with unique nutrients. Types of culture media based on consistency/ physical state 1. Solid medium 2. Semi-solid medium 3. Liquid medium 1. Solid media Principle of Solid Media It is for the isolation of bacteria as a pure culture on a solid medium. Robert Koch realized the use of solid media. Agar is used to hardening the media at 1.5- 2.0% concentration. Solid media allows the growth of bacteria as colonies by streaking on the medium. It solidified at 37 degrees Celsius. Agar is an un-branched polysaccharide extracted from red algae species like Gelidium. Colonies identification is done on this medium. Examples of Solid Media Nutrient agar, MacConkey agar, Blood agar, Chocolate agar. Growth of bacteria on solid medium appear as smooth, rough, mucoid, round, irregular, filamentous, punctiform. 2. Semi-solid media Principle of Semi-solid media This media shows the motility of bacteria and the cultivation of microaerophilic bacteria. This media has agar at a concentration of 0.5% or less. It has a jelly consistency. Examples of Semi-solid media Stuart’s and Amies media, Hugh and Leifson’s oxidation fermentation medium, and Mannitol motility media. The growth of bacteria in semi-solid appears as a thick line in the medium. 3. Liquid media Principle of Liquid media This media shows the growth of a large number of bacteria. It is called Broth that allows bacteria to grow uniformly with turbidity. The growth occurs at 37ºC in an incubator for 24hrs. Liquid media don’t have the addition of agar; it is for fermentation studies. Examples of Liquid media Nutrient broth, Tryptic soy broth, MR-VP broth, phenol red carbohydrate broth. Growth of bacteria in liquid media- Turbidity is seen at the end of the broth. Types of culture media based on chemical composition/application There are seven routine laboratory media. 1. Basal media 2. Enriched media 3. Selective media 4. Enrichment media 5. Indicator media or differential media 6. Transport media 7. Storage media 1. Basal media This media is simple as it enhances the growth of many microorganisms. It’s a routinely used medium in the lab, having Carbon and Nitrogen. This media allows the growth; of non- fastidious bacteria without any enrichment source; used for sub-culturing. It’s a non-selective medium. Staphylococcus and Enterobacteriaceae grow in this media. Examples of Basal media Nutrient Agar, Peptone water. 2. Enriched media This media requires the addition of other substances like blood, egg, or serum. An enriched media allows the growth of devised microorganisms but inhibits other and fastidious microbes grow as they require nutrients like vitamins and growth-promoting substances. Example of Enriched media Blood agar, Chocolate agar, LSS, Monsor’s taurocholate, Lowenstein Jensen media. Blood agar identifies hemolytic bacteria, chocolate media for N. gonorrhea. 3. Selective media As by name, we can tell, this media shows the growth of selective; microbes or desired microorganisms and inhibits the growth of unwanted microbes. The inhibition occurs by adding bile salts, antibiotics, dyes, PH adjustments. Media is agar-based; any media is possible to transform into selective by adding inhibitory agar. Examples of Selective media S.N. Media Bacteria 1. Mannitol Agar– It has 7% of sodium chloride that inhibits the growth of other microbes and promotes the growth of Staphylococci. It has phenol red dye that produces acid Staphylococcus used the mannitol for the acid production and the color of phenol red changes from red to yellow. Selective for Staphylococcus aureus 2. Salmonella-Shigella Agar Deoxycholate agar It is used for the isolation of Salmonella bacteria that causes typhoid. Selective for Shigella. 3. MacConkey Agar- It has bile salts that inhibit the growth of gram-positive bacteria Selective isolation for Enterobacteriaceae 4. TCBS Agar– Light green translucent media Bile salt inhibits the growth of unwanted bacteria Selective for Vibrio cholera. V. cholera produces acid by fermentation of sucrose that acts as indicator called bromothymol blue and yellow colonies appears. 5. Lowenstein Jensen Media- It is made selective by adding malachite green and stops the unwanted growth of pathogens. Selective for M. tuberculosis 4. Enrichment media It is a liquid medium, which also permits the growth of desired bacteria at a low density. The media provides an environment and conditions as selective media and inhibits unwanted bacteria from growing. It is for the isolation of the soil and fecal microorganisms. Examples of Enrichment media Selenite F-broth does the isolation of Salmonella Typhi from a fecal sample. Selenium allows the growth of desired organisms and, detection levels increase for intestinal flora. 5. Indicator or differential media This media shows visible changes due to the presence of an indicator. It differentiates bacteria based on colony color growing on the same plate; biochemical characteristics show organism’s growth with chemical indicators like neutral red, phenol red, methylene blue. Examples of Indicator or differential media Mannitol salt agar (mannitol fermentation shows yellow color colonies); blood agar is used to differentiate between hemolytic and non-hemolytic. MacConkey agar produces pink colonies due to lactose utilization and, non-lactose shows pale color colonies. 6. Transport media The media transport specimens after collection to control the overgrowth of organisms. For the cultivation, this media act as temporary storage. It also maintains the viability of pathogens in the specimen and prevents them from drying. Examples of Transport media Stuart’s transport medium (lacks carbon, nitrogen, growth factors). Cary Blair’s transport media and VR are used to transport feces samples from cholera patients. Pikes medium helps to transport streptococci from throat patients. 7. Storage media It maintains the longevity of bacterial culture. Examples are- cooked meat broth, NA egg saline. Types of culture media based on oxygen requirement Microorganisms have different requirements for growth depending on oxygen requirements. 1. Aerobic media In this media, it is easy to cultivate microbes, on solid media, the growth occurs by keeping the culture in the incubator. It shows the growth; of non-fastidious microorganisms. Examples of aerobic media are- liquid media, solid media Peptone water- 1%peptone + 0.5% Nacl +100ml water. Nutrient agar- nutrient broth +2% agar. 2. Anaerobic media The media cultivates anaerobic bacteria at low oxygen, reducing oxidation-reduction potential. Anaerobic media contains extra nutrients like vitamin K, hemin, and oxygen that get reduced by a physical or chemical process. The addition of glucose (1%), thioglycollate(0.1%), ascorbic acid (0.1%), cysteine (0.05%), or iron fillings added to cause the medium to reduce. The medium is boiled in a water bath to force out dissolved oxygen and packed with sterile paraffin. Examples of Anaerobic media RCM (Robertson cooked meat) isolation for Clostridium sp. Thioglycolate broth– It has sodium glycolate that maintains low oxygen. Types of special purpose culture media 1. Assay media The media assay vitamins, amino acids, and antibiotics. Example- Antibiotic sensitivity test the media used is Muller-Hinton agar has 1.7% agar for better diffusion of antibiotics. It also contains starch, which absorbs toxins released by bacteria. In this media plate Zone of inhibition is seen around antibiotics. 2. Minimal media Principal of minimal media Minimal media is a defined medium with different compositions depending on microorganisms cultured. It contains a carbon source like sugar/succinate and inorganic salts like magnesium, nitrogen, sulfur, phosphorus. Carbon is a source of energy; magnesium and ammonium salts are the sources of ions for metabolism stimulation. Phosphate is a buffering agent. The growth comparison of microbe culture and mutant forms- Minimal media and supplementary-minimal media- allow the differentiation of wild-type and mutant cells. Use- The selection of recombinants, for the growth of wild-type microorganisms. 3. Fermentation media The media is for optimum microorganisms. Fermentation media produce high yields of the product; media provide energy and nutrients for growth, and medium gives the substrate for the synthesis of products in the fermentation. Fermentation media contains major and minor components- Major components – Carbon and nitrogen for energy. Minor components- This contains inorganic salts, growth factors, vitamins, buffer, anti-foaming agents, dissolved oxygen, gases, growth inhibitors, enzymes. The nutrients in fermentation media depend on the organism and type of fermentation process. Two uses- Growth media It has low nutrients and creates raw material for further fermentation. Fermentation media It has high nutrients and creates end products. Example- The yeast requires 1% carbon, but the fermentation of alcohol, demands 12-13% carbon in the medium. Role of fermentation media The media has a high level of nutrients, microorganisms, and optimum conditions. During the incubation period under optimum conditions, microorganisms undergo metabolism. Fermentation organisms become hyperactive due to nutrients being in high quantities and, the result is nutrients getting consumed, media partially degraded. The waste effluent is the output product. The death of cells occurs if substrate-level reaches the inhibitory concentration and excess substrate causes them to inhibit vital enzymes. Excess substrate increases osmotic pressure and disturbs enzymatic activity in cells. Microbes release excess substrate as partially digested fermentation media and convert it into the insoluble inert compound as reserve food, which is harmless to cells. Example- yeast extract, beef extract, YPD, BMGY. Resuscitation culture media The resuscitation method is for the stressed bacterial recovery; this is a specialized medium that allows the growth of microbes that have lost the ability to produce because of the environmental harness. The culture provides nutrients and recovers their metabolism. For example- Bacteria require histamine for growth, and the medium lacks this component. Then it inhibits growth. The same bacterium is put in a medium having histamine, then it starts to grow again, and this medium acts as resuscitation media. For example- Tryptic Soy Agar.