Nutrient Agar Media Composition, Preparation, Uses

Nutrient Agar Media

Nutrient agar is an essential culture medium that is commonly used for the cultivation of non-fastidious microorganisms. It can also be used to ensure quality control and quality prior to serological or biochemical tests.

Nutrient media may be utilized for cultivation of fastidious microorganisms by adding blood or serum
Nutrient Agar is a perfect medium for demonstrations and teaching purpose since it permits the longer-lasting longevity of cultures in temperatures at ambient temperature, without the danger of growth overgrowth, which could occur in more nutritious media.

The media is based on a straightforward formula, which has been re-used and is utilized in the microbiological evaluation of many samples. It is also suggested by the traditional techniques.
Nutrient agar is a universal media, which is used primarily for routine cultivation or to guarantee the long-term survival of microorganisms.
It is among the most popular and widely employed non-selective mediums for the cultivation routine of microorganisms.

Nutrient agar is used to cultivate and count of numerous bacteria that aren’t particularly sensitive.
This medium can be modified to allow the development of various fastidious organisms through the addition of other biological fluids like blood of sheep or horse egg yolk, serum and so on.

Nutrient agar Principle

Nutrient Agar is made up of diverse nutrients, which permit to grow a vast range of microorganisms which don’t usually require particular supplements or nutrients.

The main components of the media include Peptone, Beef Extract and Agar. Alongside these nutrients there are vitamins and important trace elements that aid in the development of bacteria are included.
The peptone is the main source of protein or nitrogen that serves as an amino acid source for bacteria.
The extract of beef is the main carbon source that is vital for the creation of carbohydrates within the bacteria. It also has other elements such as vitamins, various organic compounds, trace minerals and salts, which help to boost the growth of different organisms.

In addition the addition of sodium chloride to the medium to keep the balance of the medium and to prevent the pH change of the medium throughout the growth.
The distillation process provides an environment to dissolve nutrients, making it more easy for bacteria to take them in.
Agar is the agent that solidifies that creates a solid substrate for the organism to grow on. This permits the study of colony morphology as well as the an examination for the species.

The medium is composed of a basic nutrient which is necessary for a broad range of organisms, which is why it is a universal medium that can be utilized for a variety of uses.

Nutrient agar Composition

Ingredients	Amount (gm/L)
Beef extract	3.0 gm
Peptone	5.0 gm
Sodium chloride	8.0 gm
Agar	15.0 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: Beef extract can be described as an aqueous extraction of beef tissues that are lean. It is a water-soluble substance from animal tissues, including carbohydrates organic nitrogen compounds, salts, and water-soluble vitamins.

Peptone: Peptone is made by digesting proteinaceous materials e.g., meat, casein, gelatin, using acids or enzymes. Peptone is the main source of organic nitrogen , and can also contain vitamins and carbohydrates. Based on the nature proteins and their digestion method the peptones vary in their components, and differ in their capacity to help support the development of bacteria.
Agar: Agar is a complex carbohydrate that is derived from marine algae. It serves as a solidifying agent in media, but does not possess any nutritional value. It gels once the temperatures of the media is at 45 degrees Celsius and then melts once the temperature is 95 degrees Celsius.

Preparation of Nutrient agar

Nutrient Agar

The nutrient-rich broth and agar are readily available from many companies that sell cultures in powdered (free-flowing homogeneous) form.

Dissolve the medium that has been dehydrated in the proper amount of distillation water i.e. 23 grams dehydrated nutritional in agar (see the instructions of the manufacturer) in 1000 mL of distilled water.
Stir frequently and then boil for 1 minute until the powder.
Sterilize the medium using the process of autoclaving (121degC for 15 minutes)

Dispense the medium in tubes (i.e. 3ml for nutritional agar slopes; 5ml for deeps of nutrient agar) or onto plates.
The agar medium was left to harden.
Give the medium a date and it a batch code.

Store in a cool dark place.

*Shelf life: Up to 2 years provided there isn’t any change in the appearance or color of the media that could indicate damage or contamination.

Nutrient Broth Preparation

Prepare using ready-to-use dehydrated nutritional broth powder. The ingredients of nutrient broth are identical to the nutrient agar, with the exception that the agar is not present. The preparation and storage of the broth are identical to those described for the nutrient Agar.

Semisolid Nutrient Agar Preparation

1 Mix 0.75 grams of nutrient-rich agar and 1.3 grams of nutrient broth in 100ml of distilled water. Heat until 100°C to dissolve elements (place the flask into the bath of boiling water).
2 Dispense the liquid in 5-7 milliliter quantities in screw-cap bottles.

Sterilize using autoclaving (with caps loosening) at 121°C over 15 minutes.
After cooling, screw on the cap of the bottle. Note the date and provide it a batch #.
Keep as previously described for nutrient-rich Agar.

The amount of medium specified in this method is enough to make around 20 bottles. You should manage the amount in accordance with your needs.

Storage of Nutrient agar

The powdered media in its form must be stored between 10 and 30 degrees Celsius in a tightly sealed container. The prepared media should be kept between 20 and 30 degC.
Once the bottle has been opened, product must be properly stored once dry and sealed tightly around the bottle to prevent the formation of lumps as the product is hygroscopic by nature , and therefore retains moisture very quickly.

The container must be kept in a dry, ventilated location shielded from extreme temperatures and ignition sources.
The product must be used prior to the expiry date printed on the label.

Result Interpretation on Nutrient agar

The media is a transparent, light yellow-colored clear to slightly opaque liquid on Petri plates after colliding. The table below illustrates the development of key medical bacteria and their colony morphologies Nutrient Media:

Organism	Growth	Colony Morphology
Escherichia coli	Good-luxuriant	Greyish to white-colored large, circular and convex colonies; smooth and rough colonies.
Salmonella Typhi	Good-luxuriant	Smooth colorless colonies with a diameter range of 2-4 mm.
Staphylococcus aureus	Good-luxuriant	Golden yellow colored circular, convex and smooth colonies of the diameter range of 2-4 mm; opaque colonies.
Streptococcus pyogenes	Good-luxuriant	Circular, pinpoint colonies of the size 0-5 to 1 mm in diameter; light yellow colored with low convex elevation; matt surface in virulent strains but glossy surface are seen in non-virulent strains; mucoid colonies in the case of capsule production.
Pseudomonas aeruginosa	Good-luxuriant	Large, opaque, flat colonies with irregular margins and distinctly fruity odor; variable pigment production; virulent strains might produce mucoid colonies.
Klebsiella pneumoniae	Good-luxuriant	Circular, dome-shaped, mucoid, translucent or opaque greyish white colonies; 2-3 mm diameter
Yersinia pestis	Good-luxuriant	Tiny, almost invisible, shiny grey, translucent “spots’; 1 to 2 mm irregular, grey-white to slightly yellow in color with raised, irregular, “fried egg” appearance, which becomes prominent as the culture ages.

Applications of Nutrient Agar

Nutrient Agar is utilized to cultivate less fastidious organisms in general medium.
It can also be utilized for the regular cultivation of microorganisms from common environment samples, such as food, water, or even air.
Nutrient agar can be utilized for purpose of demonstration and for teaching as it does not contain harmful chemicals and is suitable to isolate a variety of microorganisms.

It is recommended to use Nutrient Agar is suggested by the standard method since it has a straightforward composition that is able to be made in an laboratory.
It is also employed to keep microorganisms over a long period of time, without contamination which could be present in a more nutritious environment.
It is utilized to determine the purity of the sample prior to performing the various serological tests and biochemical tests.

It is among the most widely used media to count organisms from samples of the environment.
The addition of biological fluids such as blood of sheep or horse egg yolks, serum etc. to the nutrient agar make the medium more suitable for specific fastidious organisms.

Limitations of Nutrient Gar

Different organisms may differ in their requirements for growth and can exhibit different growth patterns in the medium. This can make the medium insecure when isolated.

Because nutrient-rich media facilitates the development of many microorganisms, the risk of contamination is high in isolation.
It’s not recommended to use it as a filtering medium to cultivate species that are extremely meticulous and have specific nutritional requirements.
Agar for nutrients is mostly used to facilitate to isolate bacteria but it does not allow the isolation of other microorganisms, such as fungi.

In certain instances, microorganisms may exhibit similar colony morphologies and colony structures on nutrient the agar that makes it difficult to differentiate them without microscopic analysis.

Quality Control of Nutrient agar

The color of the prepared the nutrient agar should be mild amber, with a slight to lightly transparent.
The pH of the media is 6.8 + 0.2.

The media should be inspected to determine their performance by using stable, normal control strains. If the media you have prepared are inoculated with Escherichia Coli (ATCC 25922)), Pseudomonas aeruginosa (ATCC 27853) and incubated at 35 + – 2degC for 18-48 hours, the media will show a good growth rate of these microorganisms.

References

https://www.himedialabs.com/TD/M001.pdf
https://microbiologyinfo.com/nutrient-agar-composition-preparation-and-uses/

http://himedialabs.com/TD/EC002M.pdf
https://www.sigmaaldrich.com/analytical-chromatography/microbiology/microbiology-products.html?TablePage=17997595
https://us.vwr.com/assetsvc/asset/en_US/id/8040947/contents

https://microbeonline.com/nutrient-agar-composition-preparation-uses/