Nutrient Agar

Intended Use

Nutrient Agar (BS004) is used for the cultivation of a wide variety of microorganisms, can be enriched with blood or other biological fluids.

Product Summary and Explanation

In the early 1900’s, the American Public Health Association (APHA) suggested the formula of Nutrient Agar as a standard culture medium used in water testing.1 Nutrient media are basic culture media used for maintaining microorganisms, cultivating fastidious organisms by enriching with serum or blood and are also used for purity checking prior to biochemical or serological testing (6,7). This relatively simple formula has been retained and is still widely used in the microbiological examination of variety of materials and is also recommended by standard methods. It is one of the several non-selective media useful in routine cultivation of microorganisms (8,9). It can be used for the cultivation and enumeration of bacteria which are not particularly fastidious. Addition of different biological fluids such as horse or sheep blood, serum, egg yolk etc. makes it suitable for the cultivation of related fastidious organisms. If required, enrichments can be added to this medium. Nutrient Agar, modified by incorporating 4-methylumbelliferyl-β-D-glucuronide (MUG), is used for fluorogenic detection of Escherichia coli.2
Nutrient Agar meets APHA and Association of Official Analytical Chemists (AOAC) standard methods.2,3
Nutrient Agar is specified in many standard methods procedures for the examination of food, dairy products, water, and other materials.2-5

Principles of the Procedure

Nutrient Agar is prepared using specially selected raw materials to support good growth of a wide variety of microorganisms. Peptic digest of animal tissue, beef extract and yeast extract provide the necessary nitrogen compounds, carbon, vitamins and also some trace ingredients necessary for the growth of bacteria. Sodium chloride maintains the osmotic equilibrium of the medium.

Formula / Liter

Ingredients	: Gms / Litre
Peptic digest of animal tissue	: 5.00
Beef extract	: 1.00
Yeast extract	: 2.00
Sodium chloride	: 5.00
Agar	: 15.00
Final pH: 7.4 ± 0.2 at 25°C
Formula may be adjusted and/or supplemented as required to meet performance specifications

Precautions

1. For Laboratory Use only.

Directions

1. Boil the bottle media at 100°C for 10 minutes in boiling Water bath.
2. Heat if necessary, to dissolve the medium completely.
3. Mix well and pour into sterile petri plates.
4. DO NOT OVERHEAT.
5. The surface of the medium should be dry when inoculated.

Quality Control Specifications

Prepared Medium	: Pale to Light Yellow – slightly opalescent.
pH	: pH 7.4 + 0.2 at 25°C
Gel Strength	: Firm, compared to 1.5% Agar Gel.
Reaction of 2.8% Solution	: pH 7.4 + 0.2 at 25°C
Quantity	: 100ml

Expected Cultural Response: Cultural response on Nutrient Agar at 32-35°C after 18 – 24 hours incubation.

Sr. No.	Organisms	Results to be achieved
Sr. No.	Organisms	Inoculum (CFU)	Growth	Recovery
1.	Bacillus subtilis ATCC 9372	50-100	Good-Luxuriant	>=70%
2.	Escherichia coli ATCC 25922	50-100	Good-Luxuriant	>=70%
3.	Salmonella typhimurium ATCC 14028	50-100	Good-Luxuriant	>=70%
4.	Staphylococcus aureus ATCC 25923	50-100	Good-Luxuriant	>=70%
5.	Streptococcus pneumoniae ATCC 6305	50-100	Good-Luxuriant	>=70%
6.	Streptococcus pyogenes ATCC 19615	50-100	Good-Luxuriant	>=70%

The organisms listed are the minimum that should be used for quality control testing.

Test Procedure

1. Inoculate directly onto the surface of the medium. Streak for isolation with an inoculating loop.
2. Use standard procedures to obtain isolated colonies from specimens. Incubate plates at 35 ± 2°C for 18-24 hours or longer, if necessary.
3. Tubed slants are used primarily for the cultivation and maintenance of pure cultures. They should be inoculated with an inoculating loop and incubated under the same conditions as the plated medium.