Nutrient agar and Nutrient broth

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    Introduction

    Nutrient agar (and broth version of it) is a general-purpose/basal medium that supports the growth of a wide range of non-fastidious organisms (microbes that can grow and thrive without specific nutritional or environmental conditions). It is commonly used for cultivation and maintenance of microbial cultures for scientific study or identification.

    However, the nutrient agar/broth media can be enriched with the addition of blood or other biological fluids (usually 10%) such as serum, to make the medium suitable for the cultivation of associated fastidious organisms.

    Uses

    • Nutrient Agar/broth is the simplest and most common medium in routine diagnostic laboratories.
    • It is ideally used for the isolation, cultivation and maintenance of non-fastidious organisms.
    • It is used for checking the purity of the culture prior to biochemical or serological testing.
    • It aids in the enumeration of organisms in water, sewage, dairy products, faeces and other materials.
    • It is used for producing bacterial lawns required for antibiotic sensitivity tests.
    • It provides a high-grade base for preparing special media.

    Principle

    The relatively simple formulation of nutrient broth/agar is ideal for the cultivation of microorganisms that are not specific in their nutritional requirements. The medium consists of beef extract (or yeast extract), peptone, sodium chloride and agar (in case of nutrient agar).

    The beef extract/yeast extract contains water-soluble substances, including carbohydrates, water-soluble vitamins, organic nitrogen compounds, and salts. Peptone (enzymatic digest of animal proteins) is the primary source of organic nitrogen, particularly amino acids and long-chained peptides for the growing bacteria. While sodium chloride maintains the osmotic equilibrium between the microorganisms and the medium, agar acts as a solidifying agent and has no nutritive value.

    pH

    The pH of nutrient broth/agar should be within the range of pH 6.8 +/- 0.2 at room temperature. Use 1N NaOH and 1N HCl for adjusting pH.

    There are other versions of nutrient agar (and broth) differ in pH between 7.2 - 7.4.

    Composition

    The composition of nutrient agar for 100 mL, 500 mL and 1L are provided in tabular form.
    ReagentFor 100 mLFor 500 mLFor 1 L
    Beef extract0.3 g1.5 g3 g
    Peptone0.5 g2.5 g5 g
    NaCl0.5 g2.5 g5 g
    Agar1.5 g7.5 g15 g

    There are other variations for the nutrient media which differ in the quantity of components. as an example, some venders use beef extract (1.5 g/L) and yeast extract (1.5 g/L) instead of beef extract (3.0 g/L) alone. 

    Nutrient broth

    The nutrient broth has the same formulation as above, without agar added. 

    Reagents

    Along with the reagents mentioned in the table

    • 1N NaOH
    • 1N HCl

    Materials and instruments

    • Glass beaker
    • Conical Flask / Erlenmeyer Flask
    • Spatula
    • Measuring Cylinder
    • pH meter
    • Weighing balance
    • Distilled Water
    • Butter Paper
    • Magnetic stirrer and pellet
    • Pipettes and tips
    • Petri plates and/or test tubes

    Preparation of media 

    1. Weigh the ingredients separately with respect to the volume of the media. (Here, we are considering 1L of the media).
    2. Suspend the ingredients (except agar) in a glass beaker containing about 900mL of dd H2O.
    3. Dissolve the components in the beaker using a magnetic stirrer. (Heat may be applied to dissolve the medium completely). 
    4. Adjust the pH of the medium to 5.6 or the desired value.
    5. Adjust the broth to a final volume of 1L using ddH2O.
    6. Transfer the broth to conical flask or aliquot into smaller volumes.
    7. Now add agar accordingly with respect to the volume of the media (i.e., 15 gms agar for 1L of the media). 
    8. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
    9. Transfer the broth to conical flask or aliquot into smaller volumes.
    10. If nutrient agar is required, add agar accordingly with respect to the volume of the media (i.e., 15 gms agar for 1L of the media). The concentration of the agar can be varied depending upon the purpose of the media (E.g. when the concentration of agar is reduced to 0.2-0.5gms/100mL, semi-solid agar is obtained which is ideal for motile organisms to spread).
    11. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
    12. Autoclave for 20 min at 15 psi (1.05kg/cm2) on liquid cycle.
    13. Mix well and pour into sterile Petri plates or tubes for slants.

    Alternatively, the commercially available nutrient broth media powders can be used (they are available as a homogenous mixture). Weigh the mixture of contents as prescribed by the manufacturer. If nutrient agar is required, add agar into the nutrient broth after the pH is adjusted and then autoclave.

    Cautions

    • Peptone, yeast extract and beef extract are hygroscopic. Store them in an air-tight container.
    • Seal the plates and tubes to avoid dehydration (drying) of the media.

    Storage

    Store the broth and plates at 4ºC until they are utilized, regardless of whether they contain antibiotics. They can be used up to 1-2 months.  

    References

    Wikipedia

    Science direct

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