Ritu's Biology Hub

Continued from Part 1

Media – A culture medium is a solid or liquid preparation used to grow, transport and store microorganisms. It must contain all the nutrients required for microbes to grow. There are 3 types – liquid medium, solid medium, semi-solid medium. Knowledge of a microbe’s habitat is often useful in selecting the appropriate culture medium as its nutritional requirements is reflected by the kind of environment it is found in.

Pure Culture – A culture medium containing only one species of microorganism. Also known as axenic culture

Mixed Culture – A culture medium containing a mixture of microbes

Classification Of Media Based On Physical States

1. Liquid media
   • Water based solution that do not solidify at temperature above freezing and tend to flow freely
   • Also known as broths, milks or infusions
   • Cloudy or flaky appearances
   • Nutrient broth – peptones, meat extract, NaCl, distilled water
   • Methylene blue milk & litmus milk – contain dyes and whole milk respectively. Opaque liquids
   • Thioglycollate medium – Viscous broth for determining growth patterns in oxygen

2. Semi Solid media
   • Clot-like consistency as they contain an amount of solidifying agent like agar that thickens them but does not produce a firm substrate
   • Can determine motility of bacteria for example
   • SIM (sulfur indole motility test medium) – 0.3-0.5% agar

3. Solid media
   • Firm surface to allow the formation of discrete colonies
   • Used to isolate different microbes from each other to establish pure cultures
   • Isolating and culturing of bacteria and fungi
   • Hardened by adding 1-2% agar
   • Silica gel is used as a solidifying agent to grow autotrophs on solid media in absence of organic compounds and to determine carbon sources for heterotrophic bacteria by supplementing the medium with varieties of organic substances
   • 2 types:
     • Liquefiable solid medium – Reversible to liquid phase, contains a solidifying agent that changes its physical properties in response to change in temperature. Agar is obtained from a red alga – Gelidium. It is a sulphated polymer composed of D-galactose, 3, 6-anhydro-L-galactose and D-glucoronate. Advantages of agar:
       1. Solid at room temperature and liquefies only at 100°C and does not resolidify until it cools back to 42°C. Hence, microbes can be incubated at a wide range of temperatures that will not harm the microbes or the handler.
       2. Flexible and moldable and provides a basic framework to hold moisture and nutrients
       3. Not readily digestible and thus does not serve as a nutrient for most microbes
     • Non-liquefiable solid medium – Non reversible, less versatile applications than agar as they do not melt. Not flexible and compact. Example, rice grains used for cultivating fungi, cooked meat media for culturing anaerobes or potato slices.

Classification Of Media Based On Chemical Types

1. Synthetic / Defined media
   • A medium in which all the chemical components are known
   • Used to cultivate photolithotrophs like cyanobacteria and photosynthetic protists and chemoorganoheterotrophs
   • CO₂ in the form of sodium carbonate or bicarbonate serve as carbon source for photolithotrophs
   • Ammonium ion and nitrate serve as nitrogen source
   • Glucose serve as carbon source for chemoorganoheterotrophs
   • Used in research to know what the microbes are metabolizing

2. Non-synthetic / Complex media
   • Media that have some ingredients with unknown chemical composition
   • Needed when the nutritional requirement of a particular organism is unknown as a single a complex medium maybe sufficiently rich to completely meet the nutritional requirements
   • Used to culture fastidious bacteria
   • Blood, serum, meat extracts, milk, soybean digests, peptone etc. are added to the medium
   • Peptones are protein hydrolysates prepared by partial proteolytic digestion of meat, casein, soy meal, gelatin and other protein sources that serve as excellent sources of C, N and energy.
   • Example of complex medium – Nutrient broth, Tryptic soy broth, MacConkey agar

Functional Type Media

1. General Purpose media
   • Designed to grow a broad spectrum of microbes that do not have special growth requirements
   • Usually they are complex media
   • Example, Nutrient agar, Nutrient broth, Brain heart infusion, Trypticase soy agar (TSA)/broth
   • TSA contains partially digested milk (casein), soybean digest, NaCl and agar

2. Enriched media
   • Complex organic substances like blood, serum, haemoglobin or other growth factors added to a general purpose media
   • Complex media
   • Supports growth of fastidious bacteria
   • Example:
     1. Blood agar –Sterile animal blood with an agar base. Used to grow Streptococci and other pathogens
     2. Thayer-Martin medium / Chocolate agar – Used particularly for Neisseria

3. Selective media
   • Contains one or more agents that inhibit growth of one type of microbe not the other
   • Important for primary isolation of specific type of microorganisms from sample containing mixture of different species
   • Examples:
     • Mannitol salt agar (MSA) – 7.5% NaCl concentration allows growth of S.aureus while inhibiting all the other microorganisms
     • MacConkey agar, Hektoen enteric agar – Bile salts in them is inhibitory to gram positives and non-enteric gram negative microbes
     • Methylene blue and Crystal violet – Selects for the growth of only enteric gram negative organisms
     • Selenite, Brilliant green dye – Selects Salmonella from faeces
     • Sodium azide – Allows growth of only Enterococci (facultative anaerobe) from water and food samples kiing all other aerobic organisms
     • Sabouraud’s agar – Used for the isolation of yeasts

4. Differential media
   • Grows several types of bacteria but are designed to bring out visible differences among them
   • Distinguishes different types of microbes by showing variations in colony size or colour, change in media colour, formation of gas bubbles or precipitate etc.
   • Dyes are used as they are effective pH indicators
   • Example:
     1. Blood agar
     2. MacConkey agar
     3. Simmon’s citrate agar

Miscellaneous Media

1. Reduction Media – Growing anaerobic bacteria or for determining oxygen requirements of isolates
2. Carbohydrate Fermentaion Media– Contain sugars that can be fermented and a pH change is indicative of a positive reaction
3. Transport Media – Maintain and preserve specimens that need to be sustained that may die under unstable conditions eg., Stuart’s and Amie’s transport media
4. Assay Media – To test the effectiveness of antimicrobial drugs
5. Enumeration Media – Count the number of organisms in milk, water, food, soil etc.

Continued to Part 3