Nutrient agar- Principle, composition, preparation, results, uses

What is Nutrient agar?

Nutrient agar is a basic culture medium commonly used for the culture of non-fastidious microorganisms, and for quality control and checking purity prior to biochemical or serological testing.

  • Nutrient media can also be used for the cultivation of fastidious microorganism by enriching the medium with serum or blood
  • Nutrient Agar is an ideal medium for the demonstration and teaching purposes as it allows more prolonged survival of cultures at ambient temperature without the risk of overgrowth that might occur with more nutritious mediums.
  • This media has a relatively simple formula that has been retained and is still widely used in the microbiological examination of a variety of samples and is also recommended by standard methods.
  • Nutrient agar is a general-purpose media that is mostly used for routine culture or to ensure prolonged survival of microorganisms.
  • It is one of the most important and commonly used non-selective media for the routine cultivation of microorganisms.
  • Nutrient agar has been used for the cultivation and enumeration of many bacteria that are not particularly fastidious.
  • The media can be made suitable for the cultivation of other fastidious organisms by the addition of different biological fluids such as horse or sheep blood, serum, egg yolk, etc.

Principle of Nutrient agar

  • Nutrient agar is made with various nutrients which allow the growth of a wide variety of microorganisms that do not usually require specific nutrients or supplements.
  • The primary constituents of the media are peptone, beef extract, and agar. In addition to these nutrients, some vitamins and some trace ingredients necessary for the growth of bacteria are also added.
  • The peptone is the source of nitrogen or protein that acts as a source of amino acids for the bacteria.
  • The beef extract is the primary source of carbon which is essential for the formation of carbohydrates in the bacteria. It also contains other components like some vitamins, different trace minerals, organic compounds, and salts, which further enhance the growth of different organisms.
  • Besides, sodium chloride is added to the medium in order to maintain the osmotic equilibrium of the medium and prevent the change in pH of the medium during growth.
  • The distilled water provides a medium to dissolve the nutrients so that it is easier for the bacteria to absorb them.
  • Agar is the solidifying agent that provides a stable surface for the organism to grow on, which allows for the observation of colony morphology and enumeration of the organism.
  • The medium is made up of basic nutrient that is essential for a wide variety of organism, which makes it a general media that can be used for various purposes.

Composition of Nutrient agar

  • Most nutrient agar used these days in laboratories is prepared from the dehydrated powder supplied by different vendors. The composition of the media, however, remains the same.
  • It can also be prepared in the lab if the necessary constituents of the media are available.
  • The following is the composition of the nutrient agar:
S.N Ingredients Gram/liter
1. Peptone 5.0
2. Yeast extract 1.5
3. Beef extract 1.5
4. Sodium chloride 5.0
5. Agar 15.0
Final pH at 25°C: 7.4 ±0.2

Preparation of Nutrient agar

  1. In a beaker, 28 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water.
  2. The suspension is then heated to boiling to dissolve the medium completely.
  3. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes.
  4. Once the autoclaving process is complete, the beaker is taken out and cooled to a temperature of about 40-45°C.
  5. If enrichment is desired, the addition of blood or biological fluids can be done after the autoclaving process.
  6. The media is then poured into sterile Petri plates under sterile conditions.
  7. Once the media solidifies, the plates can be placed in the hot air oven at a lower heat setting for a few minutes to remove any moisture present on the plates before use.

Storage of Nutrient agar

  • The media in the powder form should be stored between 10 to 30°C in a tightly closed container, and the prepared medium should be stored at 20-30°C.
  • After opening, the product should be appropriately stored when dry, after tightly capping the bottle in order to prevent lump formation as the medium is hygroscopic in nature and thus, absorbs moisture relatively quickly.
  • The container should be stored in a dry ventilated area protected from extremes of temperature and sources of ignition.
  • The product should be used before the expiry date on the label.

Result Interpretation on Nutrient agar

Nutrient Agar

Image Source: Eukaryotica and TM Media.

The media forms light yellow colored clear to slightly opalescent gel on Petri plates after colling. The following table demonstrates the growth of important medical bacteria with their colony morphologies on Nutrient Media:

S.N Organism Growth Colony Morphology
1. Escherichia coli Good-luxuriant Greyish to white-colored large, circular and convex colonies; smooth and rough colonies.
2. Salmonella Typhi Good-luxuriant Smooth colorless colonies with a diameter range of 2-4 mm.
3. Staphylococcus aureus Good-luxuriant Golden yellow colored circular, convex and smooth colonies of the diameter range of 2-4 mm; opaque colonies.
4. Streptococcus pyogenes Good-luxuriant Circular, pinpoint colonies of the size 0-5 to 1 mm in diameter; light yellow colored with low convex elevation; matt surface in virulent strains but glossy surface are seen in non-virulent strains; mucoid colonies in the case of capsule production.
5. Pseudomonas aeruginosa Good-luxuriant Large, opaque, flat colonies with irregular margins and distinctly fruity odor; variable pigment production; virulent strains might produce mucoid colonies.
6. Klebsiella pneumoniae Good-luxuriant Circular, dome-shaped, mucoid, translucent or opaque greyish white colonies; 2-3 mm diameter
7. Yersinia pestis Good-luxuriant Tiny, almost invisible, shiny grey, translucent “spots’; 1 to 2 mm irregular, grey-white to slightly yellow in color with raised, irregular, “fried egg” appearance, which becomes prominent as the culture ages.

Uses of Nutrient agar

  • Nutrient agar is used for the culture of less fastidious organisms as a general medium.
  • It is also used for routine culture of microorganisms from typical environmental samples like water, food, and even air.
  • Nutrient agar is often used for the demonstration and teaching purposes as it doesn’t contain harmful substances and can be used for the isolation of multiple microorganisms.
  • The use of Nutrient agar is recommended by standard methods as it has a simple composition which can even be prepared within a laboratory.
  • It can also be used for the preservation of microorganisms for an extended period of time without contamination that might occur in a more nutritious medium.
  • It is used as a purity testing method prior to the different biochemical tests and serological tests.
  • It is one of the most common media used for the enumeration of bacteria from environmental samples.
  • The addition of biological fluids like horse or sheep blood, serum, egg yolk, etc. to the nutrient agar makes the medium more selective for certain fastidious organisms.

Limitations of Nutrient agar

  • Different organisms might differ in their growth requirement and may show variable growth patterns on the medium. This makes the medium quite unreliable during isolation.
  • Because nutrient media supports the growth of many microorganisms, chances of contamination are quite high during isolation.
  • It cannot be used as a selective medium for the cultivation of fastidious organisms that have particular nutrients requirements.
  • Nutrient agar mostly only allows the isolation of bacteria and not other microorganisms like fungi.
  • In some cases, some microorganisms might demonstrate similar colony morphologies on nutrient agar which makes it difficult to distinguish them without microscopic examination.

References and Sources

  • Nutrient Agar. TM Media.
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