Nutrient agar media is used as a general-purpose medium to grow a wide variety of non-fastidious microorganisms. It consists of peptone, beef extract, and agar. This relatively simple formulation provides the nutrients necessary for the replication of a large number of non-fastidious microorganisms.
Nutrient agar/broth is used for the cultivation and maintenance of non-fastidious organisms and the enumeration of organisms in water, sewage, dairy products, feces, and other materials.
Table of Contents
Composition of Nutrient Agar
|Beef extract||3.0 gm|
|Sodium chloride||5.0 gm|
|Distilled water||1000 mL|
Final pH 6.8 ± 0.2.
Composition of nutrient broth: Nutrient broth contains all these ingredients except agar.
Characteristics of the components used in Nutrient Agar/broth
- Beef extract is an aqueous extract of lean beef tissues. It contains water-soluble substances of animal tissue, which include carbohydrates, organic nitrogen compounds, water-soluble vitamins, and salts.
- Peptone is made by digesting proteinaceous materials e.g., meat, casein, gelatin, using acids or enzymes. Peptone is the principal source of organic nitrogen and may contain carbohydrates or vitamins. Depending upon the nature of protein and method of digestion, peptones differ in their constituents, differing in their ability to support the growth of bacteria.
- Agar is a complex carbohydrate obtained from certain marine algae. It is used as a solidifying media agent and has no nutritive value. Agar gels when the temperature of media reaches 45°C and melts when the temperature reaches 95 °C.
Preparation of Nutrient Agar
Nutrient agar and broth are available from most suppliers of culture media in powdered (free-flowing, homogeneous) form.
- Dissolve the dehydrated medium in the appropriate volume of distilled water i.e., 23 gm dehydrated nutrient agar (see the manufacturer’s instruction) in 1000 mL distilled water.
- Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
- Sterilize the medium by autoclaving (121°C for 15 min)
- Dispense the medium into tubes (i.e. 3 ml to make nutrient agar slopes, 5 ml to make nutrient agar deeps) or plates.
- Leave the agar medium to solidify.
- Date the medium and give it a batch number.
- Store in a cool dark place.
Shelf life: Up to 2 years, providing there is no change in the appearance of the medium to suggest contamination or deterioration.
pH of medium: The pH of nutrient agar should be within the range of pH 6.6-7.0 at room temperature.
Prepare from ready to use dehydrated nutrient broth powder. The contents of nutrient broth are the same as those for nutrient agar except that the agar is omitted. Its preparation and storage are the same as described for nutrient agar.
Semisolid Nutrient Agar
- Mix 0.75 g nutrient agar and 1.3 g nutrient broth in 100 ml distilled water, and heat to 100°C to dissolve the ingredients (place the flask in a boiling water bath).
- Dispense the medium in 5–7 ml amounts in screw-cap bottles.
- Sterilize by autoclaving (with caps loosened) at 121°C for 15 minutes.
- When cool, tighten the bottle caps. Date the medium and give it a batch number.
- Store as described previously for nutrient agar.
The amount of medium mentioned in this procedure is adequate to make about 20 bottles. Please manage the amount accordingly, based on your requirements.
Uses of Nutrient Agar/broth
- For the enumeration of organisms in water, sewage, dairy products, feces, and other materials.
- For the cultivation and maintenance of non-fastidious species.
- The color of prepared nutrient agar will be light amber, very slightly to slightly opalescent
- pH of the prepared media should be 6.8 ± 0.2.
- Media should be checked for their performance using stable, typical control cultures. When the prepared media are inoculated with Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and incubate at 35 ± 2°C for 18-48 hours, media should show good growth of these organisms.