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PREPARATION OF NUTRIENT AGAR
Bacteriological media come an a wide range of types. Nutrient Agar is a complex
medium because it contains ingredients with contain unknown amounts or types of nutrients.
Nutrient Agar contains Beef Extract (0.3%), Peptone (0.5%) and Agar (1.5%) in water. Beef
extract is the commercially prepared dehydrated form of autolysed beef and is supplied in the
form of a paste. Peptone is casein (milk protein) that has been digested with the enzyme pepsin.
Peptone is dehydrated and supplied as a powder. Peptone and Beef Extract contain a mixture of
amino acids and peptides. Beef Extract also contains water soluble digest products of all other
macromolecules (nucleic acids, fats, polysaccharides) as well as vitamins and trace minerals.
Although we know and can define Beef Extract in these terms, each bach can not be chemically
defined. There are many media ingredients which are complex: yeast extract, tryptone, and
others. The advantage of complex media is that they support the growth of a wide range of
microbes.
Agar is purified from red algae in which it is an accessory polysaccharide (polygalacturonic
acid) of their cell walls. Agar is added to microbiological media only as a
solidification agent. Agar for most purposes has no nutrient value. Agar is an excellent
solidification agent because it dissolves at near boiling but solidifies at 45oC. Thus, one can
prepare molten (liquid) agar at 45oC, mix cells with it, then allow it to solidify thereby trapping
living cells. Below 45oC agar is a solid and remains so as the temperature is raised melting only
when >95oC is obtained.
In this experiment each student will prepare 200 ml of Nutrient Agar to be used in
Experiment 4. In subsequent experiments, the media ingredients can be found in the Appendix.
It is important for you to know how each medium works: what is the energy source? what is the
carbon source? what is the nitrogen source? does the medium have selective or differential
ingredients? why can only some types of bacteria grow on the particular medium?
MATERIALS
1. Electronic or beam balances.
2. Weigh boats, tongue depressors.
3. Tripods, asbestos wire-gauze, asbestos gloves.
4. 10 ml nonsterile pipettes.
5. pH paper or pH meter with standard buffers.
6. 4 13x100 mm screw capped culture tubes.
7. Graduated Cylinder, 250 ml.
8. 2 500ml Erlenmeyer Flasks
9. Beef Extract, Peptone, Agar.
10. 3 N HCl, 3 N KOH.
11. 16 x 150 mm screw cap culture tubes.
12. Nonabsorbent cotton and gauze to make cotton stoppers.
Nutrient Agar
Beef Extract: 0.3%
Peptone: 0.5%
Agar: 1.5%
PROCEDURE
1. You will be making 200 ml of Nutrient Agar. To weigh out Beef Extract, first tare a tongue
depressor, then dip it into the Beef Extract and weigh. Adjust the amount of Beef Extract until
the correct amount is obtained. Be sure to be careful not to get Beef Extract on to the balance!
You need to weight out enough Beef Extract to get a 0.3% solution. Place the tongue depressor
into the flask, beef extract side down.
2. Tare a weigh boat and weigh out enough Peptone and add that to the flask.
3. Add 200 ml of distilled water and swirl to dissolve the peptone and beef extract. Check the
pH, it should be 7.0.
4. Tare a weigh boat and weigh out enough Agar and add that to the flask.
5. With a Bunsen burner, tripod, asbestos wire-gauze, heat the medium to boiling to dissolve the
agar. CAREFUL: 1) keep the rotating the flasks to prevent the agar from cooking onto the
bottom of the flask and 2) watch out: boiling agar can froth and boil out all over the lab bench.
As soon as it begins to boil take it off the heat and put it on to the bench. Allow it to cool a few
minutes.
6. While the agar is still warm, but not hot, pipette 3 ml each into 4 13x100 mm screw cap
culture tubes.
7. Label the flask and your tubes with your name.
8. After preparation of your medium, the instructor will take you to the autoclave.
9. Place your media in the autoclave with those of the rest of the class.
10. After discussion of the parts of the autoclave, autoclave the medium for 20 minutes.
11. The media will be saved and used in Experiment 4.
What else can I help you with?
What is the advantage of using a nutrient agar plate over a nutrient agar slant?
Slants are better suited than agar plates, because they can be capped, preventing the agar and the culture from drying out. The cap also prevents airborne contaminants from entering the slant. Also, slants take up less storage space than an agar plate.
Will s aureus grow on a crystal violet agar plate?
Staphylococcus aureus can grow on crystal violet agar plates as crystal violet agar is a selective medium that inhibits the growth of Gram-negative bacteria and allows the growth of Gram-positive bacteria like S. aureus.
If an organism can grow on both a nutrient agar and a MacConkey Agar on which would you expect it to grow better?
You would expect the organism to grow better on nutrient agar because it is a general-purpose medium that supports the growth of a wide range of organisms. MacConkey agar, on the other hand, contains inhibitors that selectively inhibit the growth of Gram-negative bacteria, so the organism may not grow as well on this medium.
What is the color of nutrient agar?
Nutrient agar is a clear pale buff colour.
If you strongly suspect a nutrient agar slant culture is contaminated what method would you use to ascertain whether you are right or not?
You have to make subculture from this slant and after incubation you can observe how many types of microorganisms are present in the nutrient agar slant. If you have one colony shape so you have a pure nutrient agar slant but if you have more than one type of colonies so the nutrient agar slant is contaminated.
What is the purpose of of agar in nutrient agar?
To make it semi-solid
What is the colony color of serratia on nutrient agar plate?
Yellowish
What is responsible for solidifying nutrient agar plates?
Agar, a type of polysaccharide derived from seaweed, solidifies nutrient agar plates when it cools below 45°C. This process forms a gel matrix that helps to support bacterial growth on the surface of the plate.
What color are the colonies of E Coli when growing on a nutrient agar plate?
whitw to whitish gray
What is the method called when the bacteria sample and nutrient agar are mixed before pouring?
Pour plate method
What is the advantage of using a nutrient agar plate over a nutrient agar slant?
Slants are better suited than agar plates, because they can be capped, preventing the agar and the culture from drying out. The cap also prevents airborne contaminants from entering the slant. Also, slants take up less storage space than an agar plate.
If an organism can grow on both a nutrient agar and a MacConkey Agar on which would you expect it to grow better?
You would expect the organism to grow better on nutrient agar because it is a general-purpose medium that supports the growth of a wide range of organisms. MacConkey agar, on the other hand, contains inhibitors that selectively inhibit the growth of Gram-negative bacteria, so the organism may not grow as well on this medium.
Will s aureus grow on a crystal violet agar plate?
Staphylococcus aureus can grow on crystal violet agar plates as crystal violet agar is a selective medium that inhibits the growth of Gram-negative bacteria and allows the growth of Gram-positive bacteria like S. aureus.
What is nutrient agar?
simply agar medium
What is the difference between nutrient agar culture media and MacConkey agar culture media?
nutrient agar is used generally for culturing any organism.But Muller hinton agar is specifically used for testing antibiotic sensitivity as it does not contain any inhibitory substances for the growh of the organism
What is the color of nutrient agar?
Nutrient agar is a clear pale buff colour.
Why was a blood agar plate used instead of a nutrien agar plate for the hand washing experiment?
because blood agar contain only blood, the culture from the mouth might not grow properly, whereas nutrient agar contain mixture of nutrient and blood, therefore culture from the mouth will properly.
