Nutrient agar: Introduction, Composition, Preparation, Test Procedure, Colony Characteristics and Uses

Nutrient agar

Nutrient agar with Micrococcus luteus

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 for serotyping. The above image shows the growth of Micrococcus luteus which has a  characteristic feature of yellow pigment and therefore yellow colony.

Further supporting features are-

Composition of Nutrient Agar

(Oxoid)
Typical Formula        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 Nutrient agar

  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 Nutrient agar

  • 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; protects from light, excessive heat, moisture, and freezing.

Test Requirements

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 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

Colony Characteristics of various organisms in Nutrient Agar

Colony morphology of various bacteria as shown in the following video-Colony of Staphylococcus aureus on nutrient agar golden yellow large i.e. greater than 1 mm in size, smooth, convex, opaque, and easily emulsifiable

Pyocyanin and pyoveedin pigments of Pseudomonas on nutrient agar: Pigment production of Pseudomonas, some strains produce diffusible pigments like Pyocyanin (blue), pyoverdin or fluorescein ( greenish-yellow), pyorubin (reddish brown) and pyomelanin ( brown to black). There are 6 different types of Pseudomonas aeruginosa colonies that may be observed- Type 1. Large and leafy 2.  smooth, circular, and coliform type Type 3. Rough Type 4.  rogose Type 5. Mucoid due to exopolysaccharide as shown above image and Type 6.  Dwarf and smallest

Colony morphology of bacteria || Micrococcus roseus || Red pigment on nutrient agar as shown below-

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. Uses for better expression of pigmentation.
  7. It is also used for serotyping of organisms.
  8.  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 of Nutrient Agar

  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. The medium 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. This is a general-purpose medium and thus for recovering fastidious organisms like S. pneumoniae  and H. influenzae nutrient agar should be modified.

References

  1. https://labmal.com/product/nutrient-agar-500g/
  2. http://himedialabs.com/TD/M001.pdf
  3. https://en.wikipedia.org/wiki/Nutrient_agar
  4. https://catalog.hardydiagnostics.com/cp
  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. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  9. 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.
  10. 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.
  11.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.

 

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