Nutrient Agar- Principle, Composition, Preparation, Results And Uses

Introduction

Nutrient agar is a general purpose medium for the cultivation of organisms that are not demanding in their nutritional requirements e.g. organisms that can be isolated from air, water, dust etc.It is the most popular media that are generally available in all laboratories for enumeration of different bacteria and their maintenance.Nutrient is made up of:

• 0.5% Peptone – this provides organic nitrogen
• 0.3% beef extract/yeast extract – the water-soluble content of these contribute vitamins, carbohydrates, nitrogen, and salts
• 1.5% agar – this gives the mixture solidity
• 0.5% Sodium Chloride – this gives the mixture proportions similar to those found in the cytoplasm of most organisms
• distilled water – water serves as a transport medium for the agar’s various substances
• pH adjusted to neutral (6.8) at 25 °C (77 °F).

Principle

Bacteria are routinely cultured in a solid medium i.e. Nutrient Agar Medium (NAM) to obtain the discrete colonies of the bacteria present in the specimen or to get the information about cultural characteristics of bacteria on a solid medium, colony morphology and patterns of growth etc. The Solid basal medium, used in bacteriology laboratory constitutes the 4 essential components –

• Beef Extract – It is the beef derivative which is a rich source of Organic Carbon, Nitrogen, Vitamins and Inorganic Salts that supports the rapid growth of bacterium in the laboratory at an optimum temperature, pH, and Osmotic Pressure.
• Peptone – It is a semi-digested protein which is soluble in water and easily metabolized by the bacterial cell, provides the rich source of protein to the bacterial cell of the rapid growth.
• Sodium Chloride – It maintains the osmotic pressure in the agar medium so that the movement of molecules takes place in and out of the bacterial cell. It must be present in right proportion otherwise it will lead to the lysis of the bacterial cell.
• Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-Anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling at 40-45 °C.

Preparation Of Nutrient Agar

Composition of Nutrient Agar

Materials

• Sterile Conical Flask / Erlenmeyer Flask
• Spatula
• Beef Extract
• Peptone
• Sodium Chloride
• Agar-Agar
• Measuring Cylinder
• 1N HCl
• 1N NaOH
• pH Strip
• Weighing Scale
• Distilled Water
• Butter Paper

Procedure

1. Take a clean and dry Conical Flask/ Erlenmeyer flask.
2. Pour 500 ml of distilled water to the flask and add the weighed quantity of Peptone, Beef Extract, and Sodium Chloride.
3. Now add the weighed quantity of Agar-Agar to the above solution.
4. Mix well the content and Heat it with continuous agitation to dissolve the constituents.
5. Now add more distilled water to the medium and make the volume 1000 ml.
6. Check the pH of the solution using pH strip, it should be 7.2 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
7. Mix well the content and apply the Non-absorbent cotton plug to the flask.
8. Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
9. Allow the content to cool down to 40-45 °C and pour in the empty media plates under the strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
10. Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.

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), contamination.
• Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

Precautions To Consider When Preparing Nutrient Agar

• Carefully measure the quantity of peptone, beef extract, Agar-Agar and sodium chloride as per the quantity of medium to be prepared.
• Maintain the pH of the medium carefully. A single extra drop can change the pH of the solution.
• Acid and Alkali are corrosive to the skin, handle with care.
• Store the Medium at low temperature in dust and contamination free environment for later use.

Result Interpretation on Nutrient agar

The media forms light yellow colored (golden yellow), clear to slightly opalescent gel on Petri plates. The table below explains the growth of some of the most common bacteria with their colony characteristics on Nutrient Media:

Uses of Nutrients Agar

1. For the cultivation and maintenance of non-fastidious bacteria.
2. Preparation of blood agar
3. It is also used antibiotic sensitivity testing
4. Concentrated agar upto 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 for better expression of pigmentation.
7. It is also used for serotyping of organisms.
8. It also uses for 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.

Limitations of Nutrient Agar

1. It is general purpose medium,thus it cannot be used as a selective medium for the cultivation of fastidious organisms that have particular nutrients requirements.
2. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
3. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
4. Nutrient media supports the growth of many microorganisms, chances of contamination are quite high during isolation.
5. Nutrient agar mostly only allows the isolation of bacteria and not other microorganisms like fungi.

Differences Between Nutrient Agar And Nutrient Broth

Further References

1. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Hand book Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4.  MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
6. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
7. 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.
8. 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.
9. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI – formerly NCCLS), Wayne, PA.
10.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.