Media preparation and its types

Basics of Culture Media preparation along with their types and uses. Useful for basic understanding for freshers in biotechnology.

1. Media preparation, media types and culture methods

2. Media and its Need Media: A  growth medium  or  culture medium  is a solid, liquid or semi-solid designed to support the growth of  microorganisms  or  cells .   It is either an organic or a synthetic substance that provides both the biophysical and the biochemical factors necessary for the growth of bacteria . WHY DO WE REQUIRE A MEDIA? Microorganisms need nutrients, a source of energy and certain environmental conditions in order to grow and reproduce. In the environment, microbes have adapted to the habitats most suitable for their needs, whereas in laboratory, these requirements must be met by a culture medium. This is basically an aqueous solution to which all the necessary nutrients have been added. 

3. Composition   The general composition of a medium is as follows : H-donors and acceptors (approximately 1-15 g/L ) C-source (approximately 1-20 g/L ) N-source (approximately 0.2-2 g/L ) Other inorganic nutrients e.g. S, P (50 mg/L ) Trace elements (0.1-1 µg/L ) Growth factors (amino acids, purines, pyrimidine's, occasionally 50 mg/L, vitamins occasionally 0.1-1 mg/L ) Solidifying agent (e.g. agar 10-20 g/L ) Solvent (usually distilled water ) Buffer chemicals

4. Preparation of Media Required amount of chemicals is dissolved in distilled water using Erlenmeyer flasks or rimless clean test tubes. pH is adjusted by adding 1N of NaOH or N/10 th of HCl 15ml capacity of test tube  5ml of liquid medium is taken 250ml flask  100ml of media These are plugged with non adsorbent cotton and are covered using brown paper. This is kept for autoclaving – sterilization done at 15lbs/inch^2 at a temperature of 121 degree Celsius for 15min Later cooled down and poured in petri plates or test tubes which solidifies to form a medium.

5. Eg : Preparation of nutrient agar media. Nutrient Agar is a general purpose, nutrient medium used for the cultivation of microbes Composition of Nutrient Agar   0.5% Peptone - principal source of organic nitrogen for the growing bacteria . 0.3% beef extract - water-soluble substances which aid in bacterial growth, such as vitamins, carbohydrates, organic nitrogen compounds and salts . 1.5% agar - solidifying agent . 0.5% NaCl - maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms . Distilled water - Water is essential for the growth of and reproduction of micro-organisms and also provides the medium through which various nutrients can be transported . pH is adjusted to neutral (7.4) at 25 °C.

6. Preparation of Nutrient Agar Suspend 28 g of nutrient agar powder in 1 liter of distilled water . 2 . Heat this mixture while stirring to fully dissolve all components . 3.  Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes . 4 . Once the nutrient agar has been autoclaved, allow it to cool but not solidify . 5.  Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified . 6 . Replace the lid of each Petri dish and store the plates in a refrigerator .

7. Uses of Nutrients Agar: 1. It is frequently used for isolation and purification of cultures. 2 . It can also be used as a means for producing the bacterial lawns needed for antibiotic sensitivity tests.  In actuality, antibiotic sensitivity testing is typically performed on media specially formulated for that purpose.

8. Types of Media These media are prepared by mixing all the pure chemicals of known composition e.g. Czapek Dox medium. Such are those media, where exact chemical composition is unknown e.g. potato dextrose agar or MacConkey agar medium.

9. When 5-7% agar agar or 10-20% gelatin is added the liquid broth becomes solidified. used for making agar slants or slopes and agar stab. no agar is added or used while preparing the medium . After inoculation and later incubation, the growth of cells becomes visible as small mass on the top of the broth. prepared by adding half quantity of agar i.e. about 0.5% in the medium . This type of medium may be selective or diffrential type of medium

10.   Culture media   Culture media contain all the elements that most bacteria need for growth and are not selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.   Minimal media Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow "wild-type" microorganisms . Major types

11. Selective media Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain  antibiotic , such as  ampicillin  or  tetracycline , then that antibiotic can be added to the medium to prevent other cells, which do not possess the resistance, from growing. YM  ( yeast extract ,  malt extract  agar) has a low  pH , deterring bacterial growth. MacConkey agar  is for  Gram-negative  bacteria.

12. Differential media Differential or indicator media distinguish one microorganism type from another growing on the same medium. [7]  This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as  neutral red ,  phenol red ,  eosin y , or  methylene blue ) added to the medium to visibly indicate the defining characteristics of a microorganism. Examples of differential media include: Blood agar  (used in  strep  tests) contains bovine heart blood that becomes transparent in the presence of β- hemolytic organisms such as  Streptococcus pyogenes  and  Staphylococcus aureus . MacConkey agar  is differential for lactose fermentation.

13. Transport media Transport media should fulfill these criteria: Temporary storage of specimens being transported to the laboratory for cultivation Maintain the viability of all organisms in the specimen without altering their concentration Contain only buffers and salt Lack of carbon, nitrogen, and organic growth factors so as to prevent microbial multiplication Transport media used in the isolation of anaerobes must be free of molecular oxygen Examples of transport media include: Thioglycolate broth  is for strict  anaerobes . Stuart transport medium  is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralize. Certain bacterial inhibitors are used for gonococci, and buffered glycerol saline for enteric bacilli.  

14. Enriched media   Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen.  Blood agar  is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients.  Chocolate agar  is enriched with heat-treated blood (40–45 °C), which turns brown and gives the medium the color for which it is named.

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