Preparation and sterilization of Bacterial Nutrient media

The composition and preparation of bacterial nutrient media is the basic experiment in Microbiology. The Bacterial Nutrient media is composed of all essential nutrients that are required for the growth and multiplication of bacteria. In the given article, Composition & Preparation of Bacterial Nutrient Media is explained in simple and easy language.

Introduction Bacterial Nutrient Media:

Microorganisms are found everywhere; they are present in soil, water, air, and sewage and on the surface of animal and plant. And that’s why they are called as omnivorous. To isolate the microbes from natural environment and to grow them in laboratory, we need to provide them essential nutrients and physical environment. The Scientist have studied bacterial nutritional requirements and designed the synthetic media. The synthetic media are commercially available. The nutritional requirement varies from microbe to microbe and hence various types of media have been designed.

The growth of microbes in laboratory depends upon the supplied nutrients and physical optimum conditions. The growth of microbes is defined as increase in cell size and number of microbes. The designed media used for growing microbes are called as culture media.  

The culture media can be solid, semi solid or liquid state. The solid media can be prepared by using solidifying agent or gelling agent called as agar. The agar is extracted from seaweed (marine plants or algae). The agar is carbohydrate made of hetero-polysaccharides of agarose and agaropectin. The agar is also used in making food jelly. The Agar is used as solidifying agent because of its liquefying and solidifying ability. The agar powder is when added in water at room temperature, it gets partially dissolve but dissolves completely in boiling water i.e. at 100° C. and solidifies when it is cooled down. The agar serves as the best solidifying agent because the microbes cannot digest it. Semi-solid media contains less percentage of agar than solid media. The liquid media is prepared without agar and as called as Broth.  The prepared media can be poured into test tubes or petri plates and used for inoculation of desired microbes.

Principle of Bacterial Nutrient media Preparation:

The solid Nutrient media contains Beef Extract (0.3%), Peptone (0.5%), NaCl and Agar (1.5%) in water. These ingredients are commercially available. Beef extract provides Macro and Micro biomolecules, minerals and vital nutrients that support the growth of microbes. Peptones acts as source for nitrogen, amino acids and peptides. Sodium Chloride (NaCl) maintains the osmotic equilibrium of media.

Requirements for Bacterial Media Preparation:

1. Conical Flask
2. Pipette
3. Burner
4. Alcohol
5. Composition of Bacterial Nutrient Media
   • Agar – 15gm/L
   • Peptone – 5 gm/L
   • Beef extract – 3 gm/L
   • Sodium Chloride – 5 gm/L 
   • pH – 7.4 ± 0.2
   • Distilled water – 1L

Procedure of Bacterial Nutrient Media Preparation:

1. Weigh the above ingredients as per mentioned in the composition of Nutrient media.
2. The enlisted ingredients are dissolved in 800 ml and then boiled it using Bunsen burner and then volume is made up 1 Liter.
3. The ingredients are mixed properly by using magnetic stirrer.
4. The pH is checked and adjusted to 7.4 ± 0.2 by using acid or alkali.
5. The media is then autoclaved at 121° C for 20 min.

Assessment for sterility:

The sterility of nutrient media can be assessed by autoclave indicator tape. The tape are adhesive kind of tape. It is sensitive change in temperature. It changes the color when temperature reach 121°C. The change of tape assures the sterility of autoclaved media.

The other non expensive method is to keep the autoclave media for two days at room temperature. If media is sterile then there won’t be any microbial growth. But if you observe any microbial growth then it indicates that the autoclaving was properly done.

Result of Bacterial Nutrient Media Preparation:

The Nutrient media is prepared by using agar, peptone, beef extract and sodium chloride and its pH is adjusted to 7.4 approx. The prepared media is tested for sterility.

Conclusion of Bacterial Nutrient Media Preparation:

The sterile prepared media is now ready for microbial inoculation.

Meme –

Acknowledgement –

We Biokimicroki team appreciates Amrita Dinesh for contributing the meme for our article.