Nutrient Agar: Introduction, Composition, Test Requirements for Preparation, Test Procedure, Colony Characteristics, Uses, and Nutrient Agar Footages

Introduction of Nutrient Agar

Nutrient agar is a simple medium which uses to grow bacteria. It is devoid of indicators, selective agents, differential ingredients, and enriching substances therefore uses for better expression of pigmentation, biochemical test, and even serotyping. The short form of nutrient agar is NA.

Composition of NA

Constituents	Gm/Liter
Lab-Lemco’ powder	1.0
Yeast extract	2.0
Peptone	5.0
Sodium chloride	5.0
Agar	15.0
Distilled water	1000 ml
pH	7.4 ± 0.2 @ 25°C

Principle of Nutrient Agar

Peptone is an enzymatic digest of animal protein and the principal source of organic nitrogen for growing bacteria. Lab-Lemco powder( beef extract) and yeast extract are water-soluble ingredients of nutrient agar that contribute to vitamins, carbohydrates, nitrogen, and salts. Sodium chloride maintains the osmotic equilibrium of the medium. The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms. Agar acts as the solidifying agent.  Water is an essential ingredient for the growth and reproduction of organisms and also serves as a transport medium for the agar’s various substances.

Preparation of NA

1. Suspend 28.0 grams in 1 liter of purified/distilled or deionized water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. After autoclaving,  leave for cooling to 45-50°C.
5. Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified.
6. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of NA

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature sensitive; it protects from light, excessive heat, moisture, and freezing.

Test Requirements

• Test specimens ( samples or growth of bacteria)
• Inoculating loop
• Bunsen burner
• Incubator
• Control strains (Escherichia coli ATCC 25922 and Staphylococcus aureus
   ATCC 25923)

Test procedure (specimen/organism inoculation)

1. Allow the plates to warm at 37°C or to room temperature, and the agar surface to dry before inoculating.
2. Inoculate and streak the specimen as soon as possible after collection.
3. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
4. Streak for isolation with a sterile loop.
5. Incubate plates aerobically at 35-37ºC. for 18-24 hours.
6. Examine colonial characteristics.

Result and Interpretation

Control strains i.e. Escherichia coli ATCC 25922 and Staphylococcus aureus
ATCC 25923): good-luxuriant

Presence of non-fastidious bacteria in specimen: Presence of  growth on nutrient agar

Uses of Nutrients Agar

1. For the cultivation and maintenance of non-fastidious bacteria.
2. Preparation of blood agar
3. It is also used in antibiotic sensitivity testing
4. Concentrated agar up to 3 more % prevents swarming of Proteus species as well as Clostridium tetani.
5. Preparation of chocolate agar ( heating blood agar changes to chocolate agar).
6. It uses for better expression of pigmentation.
7. It is also used for serotyping of organisms.
8. It also uses for the isolation of pure cultures from mixed growth.
9. Nutrient agar is also beneficial for the enumeration of organisms in water, sewage, dairy products, feces, and other materials.

Keynotes

• Nutrient broth contains the same ingredients except for agar.
• Earlier it was used as blood and chocolate agar base medium but nowadays replaced by various manufacturers.

Limitations

1. Individual organisms differ in their growth requirement and may show variable growth patterns in the medium.
2. Each lot of the medium has been tested for the organisms specified on the certificate of analysis. It is recommended to users validate the medium for any specific microorganism other than that mentioned in the certificate of analysis (COA) based on the user’s unique requirement.
3. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
4. It is a general-purpose medium and thus for recovering fastidious organisms like S. pneumoniae and H. influenzae nutrient agar should be modified.

Nutrient Agar Footages

Staphylococcus aureus colony morphology on nutrient agar

Micrococcus luteus colony morphology on nutrient agar

Vibrio cholerae colony characteristics on nutrient agar

Micrococcus roseus growth on NA

Pyocyanin and pyorubrin pigments of Pseudomonas aeruginosa on NA

Further Readings

1. https://labmal.com/product/nutrient-agar-500g/
2. http://himedialabs.com/TD/M001.pdf
3. https://catalog.hardydiagnostics.com/cp
4. https://en.wikipedia.org/wiki/Nutrient_agar
5. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
6. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
7. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
8. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
9. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
10. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
11.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.