Chapter 3 Material And Methods

3. MATERIAL AND METHODS

3.1.Materials:

3.1.1 Equipment’s

• Laminar airflow cabinet

• Fungal incubator
• Bacterial incubator
• Autoclave
• Hot air oven
• Sonicator

3.1.2 Glass wares

• Petri plates
• Conical flask
• Beaker
• Measuring cylinder
• Test tube

3.1.3 Miscellaneous:

• Micropipettes
• Bunsen burner
• Spatula
• Inoculating needle
• Cork borer

3.1.4 Reagents:

• Distilled water

• Bacterial antibiotic ( Gentamicin)

• 3.2. NA (Nutrient Agar)

Nutritional Agar is a multipurpose nutrient medium that may be used to cultivate

microorganisms that support the growth of a variety of non-fastidious organisms. The reason
why nutritional agar is so well-liked is because it supports the development of several bacterial

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species and includes a variety of nutrients Composition of Nutrient Agar 0.5% , Peptone:-It is
an enzymatic digest of animal protein. Peptone is the principal source• of organic nitrogen for
the growing bacteria. 0.3% beef extract/yeast extract:-It is the water-soluble substances which
aid in bacterial growth, such as vitamins, carbohydrates, organic nitrogen compounds and salts.
1.5% agar :-It is the solidifying agent

• 0.5% NaCl:-The presence of sodium chloride in nutrient agar maintains a salt

• concentration in the medium that is similar to the cytoplasm of the microorganisms.

3.3. Nutrient Broth?

Basically, the nutrient broth is the nutrient agar that lack of the solidifying agent, agar powder.
They remain in liquid form at room temperature and are usually used to maintain the stocks of
microorganisms. In general, they are used to grow fastidious organisms. Also, you can enrich
your nutrient broth with blood, serum, sugars etc. for special purposes.

3.4.How to prepare nutrient broth?

• Add 13g of nutrient broth powder (CM0001B) in 1L of distilled water.

• Mix and dissolve them completely.

• Pour them into the final containers (e.g. conical flask)

• Sterilize by autoclaving at 121°C for 15 minutes.

•

3.5. STRAINS
Bacterial and fungal strains were collected from the microbiology lab of ShooliniUniversity.
Staphylococcus aureus, E.coli, Candida albicans pathogenic bacteria used in antimicrobial
assay.
3.6.METHODS
• Collection of the sample
• Washing with distilled water
• Grinding
• Filtration
• Heating
• Centrifuge
• Drying
• Characterization

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• Dilutions
• Antimicrobial assay

3.7.Sampling

Fig 2: samples of beat root and mint

Beetroot and mint sample was collected from local market of the kotla nala solan Himachal
Pradesh, the beetroot sample was collected with leaves because it will not let it dry and we can
keep it for 3-5 days, For the mint sample fresh mint leaves are required
3.8.Washing with distilled water

The Beetroots and mints were washed and cleaned with distilled to remove any pollution. In
the next step, the Beetroot and Mint was cut into small pieces.

3.9.Grinding

The beetroot and mint was grinded for 10 minutes for 3 times separately.

3.10. Filtration

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Chapter 3 Material And Methods

The beetroot and mint was filtered with Whatman filter paper after filtration the aqueous extract
and boiled for 25 minutes at 60- 70 degree Celsius and 363 rpmAfter boiling add 3.4 gm silver
nitrate in 200 ml solution of both mint and beetroot

Fig 3:Aqueous extract of Beetroot

After adding silver nitrate the colour of beetroot changes from red to ruby red

Fig 4: Mint colour changes from green to brown – green

3.11. Centrifuge

Centrifugation is a very common technique to separate solid particles dispersed in liquid

mediumThe liquid sample is placed in a special vial or holder, which is rotated very fast.
Sample components are separated due to the centrifugal force, based on their density
difference. the sample should be centrifuged for 15 minutes at 4000 rpm .

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Chapter 3 Material And Methods

Fig 5: After centrifugation dry the samples in hot air oven for two days

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