Preparation and Sterilization of Culture Media

Microorganisms, like all other living organisms, require basic nutrients, for the sustenance of life.

The food material on which microorganisms are grown in the laboratory is known as a culture medium (pl. media) and the growth itself is called the culture.
In other words, the nutrient preparation on or in which culture (i.e. a population of microorganisms) is grown in the laboratory is called a culture medium.

Although all the microorganisms have the same basic requirements there is diversity as to the use of organic and inorganic compounds.
Thus cultural media vary in form and composition, depending on the species to be cultivated.
Some media contain solutions of inorganic salts and may be supplemented with one or more organic compounds while other media contain complex ingredients such as extracts or digest of plant and animal tissues.

These ingredients except for the agar, are used to prepare broth or liquid media.
Agar, which liquefies on heating to 960c and hardens into jelly on cooling to 40 450c, is used to solidify liquid media.
For the propagation of obligate intracellular parasites, rickettsias, and chlamydias, living hosts are required while bacteriophages propagate within certain bacterial cells, where the bacterial cells serve as the “medium” host for the bacteriophage.
Media have been classified variously using different criteria such as chemical composition, physical state, and utility purpose.

Three main types of culture media

On the basis of their composition, there are three main types of culture media:

Natural or empirical culture media
Semi-synthetic media

Synthetic or chemically defined culture media

What is natural or empirical culture media?

The exact chemical composition of the natural medium is not known.

The natural culture media include milk, urine, diluted blood, vegetable juices, meat extracts and infusions, peptone (digested protein rich in amino acids that provide nitrogen and carbon).

Nowadays, groups of living cells as tissue or callus or an organ are being used for viruses and rickettsias.

What is Semi-synthetic media?

Those media whose chemical composition is partially known are called semi-synthetic media. Any medium which contains agar becomes a semisynthetic medium.

Potato dextrose agar, Czapek-Dox agar, oatmeal agar, beef peptone, and nutrient agar are some of the semi-synthetic media.

What is Synthetic or chemically defined culture media?

Synthetic or a chemically defined culture medium is composed of special substances of known composition.

The synthetic medium may be a general purpose medium used for a wide variety of microorganisms or selective medium used for a selected microbe, or differential medium used for differential isolation of a microbe, or an assay medium used for the assay of vitamins, amino acids, and antibiotics.

Some of the commonly used synthetic media are the Czapek solution, Richard’s solution, Raulin’s solution, and Martin’s rose Bengal medium.

Cultivation methods of Bacteria

Nutrient broth and Nutrient agar have been considered as basic media for the cultivation and maintenance of most heterotrophic bacteria.

A liquid growth medium is generally used for the cultivation of microorganisms.

A solid medium is used for the isolation and cultivation of microorganisms.

What are the ingredients of Nutrient broth/agar?

Peptone serves as a source of nitrogen. Carbon is also provided by peptones and being proteinaceous and amphoteric in nature, peptone can also act as a buffer.
Meat / Beef extract serves as a source of organic carbon, nitrogen, vitamins, and inorganic salts.
Sodium chloride (NaCl) is required in the medium to maintain the isotonicity of the medium, which is necessary for the transport of nutrients into the cell.

Water is absolutely necessary for life processes and hence is very important in culture media. Hard and tap water containing calcium and magnesium can react with phosphates, contained in ingredients such as peptone and beef extract, and precipitates are formed. For such reasons, distilled water is preferred.
Agar is a mucilaginous substance extracted from aquatic algae belonging to the genus
Gelidium. Agar is a sulfuric acid ester of a linear galactan. It is insoluble in water but forms a viscous gel when heated in water at around 80 – 1000C. On cooling, the agar solidifies into a gel. In culture media, agar powder is used at 2-3 %( w/v).

What is Nutrient broth for Liquid medium?

Peptone – 1 g

NaCl – 0.5 g
Beef extract – 0.33 g
Distilled water – 100 mL

0.1N NaOH / 0.1 N HCl

Preparation of Nutrient broth for Liquid medium

Put the weighed amount of peptone, beef extract, and NaCl in 50 mL of distilled water. Heat with agitation to dissolve the constituents.
Add more distilled water to make 100 mL. Adjust pH of the medium 7.0, using pH meter or by pH indicator paper, by adding either acid or alkali, as the case may be.

Pour 10 Ml per tube. Apply cotton plugs. Autoclave at 1210C, 15 lbs pressure for 15 minutes.
Allow the autoclave to cool. Store the medium at low temperature in a dust-free environment.

What is Nutrient agar for Solid Medium?

Peptone – 1 g

NaCl – 0.5 g
Beef extract – 0.33 g
Distilled water – 100 mL

0.1N NaOH / 0.1 N HCl
Agar – 2- 3%

Preparation of Nutrient agar for Solid Medium

Dissolve all ingredients except agar.

Agar should be adjusted only after adjusting the pH.
Prepare and dispense the required quantity (100 / 200mL) in conical flasks. Dispense 8 -10 mL in test tubes when slants or tubes are to be prepared.
Plug the flasks and tubes containing medium.

Autoclave at 1210C,15 lbs pressure for 15 minutes.
Allow the tubes to cool in slanting position(for agar slants) and upright position (for agar deep tubes).
The sterilized medium can be stored at room temperature in a dust-free environment (if to be used within a week) or in a refrigerator(if to be stored longer).

For the preparation of plates, allow the flasks to cool at around 50 – 600C. Pour around 15 -20 ml of the medium into Petri dishes quickly under aseptic conditions.
Allow the medium to gel to produce agar plates.
Do not pour media into Petri plates when too hot since it produces much condensed water on the Petri plate lid and thus can fall on to agar surface and may lead to culture contamination.

What is selective media?

A single medium or set of conditions cannot be constructed to support the growth of all types of microorganisms.

To isolate and grow particular organisms, simultaneously inhibiting /suppressing the growth of other types, it is necessary to devise media and cultural conditions which will selectively promote the growth of the desired organism.

Such Media are termed selective media. The liquid medium is used for enrichment purposes and the solid medium is used for isolation.

Examples of selective media are Brilliant green agar selective for Salmonella by inhibition of Gram-positive bacteria by brilliant green.

Sabouraud’s agar is selective for fungi due to its low pH (5.6) and high glucose concentration (3-4%).

What is Differential media?

These media are used for growing and differentiating between different types of bacteria on solid media.

Differential media, such as blood agar can be used to differentiate between hemolytic and non-hemolytic bacteria.

A classical example of such a medium is MacConkey’s broth and MacConkey’s agar.

MacConkey’s broth?

Peptone – 20.0 g

Sodium taurocholate – 5.0 g
Lactose – 10 g
Distilled water – 1 liter

pH – 7.2
Neutral red (1% sol.) – 4 mL

MacConkey’s agar

Peptone – 20.0 g

Sodium taurocholate – 5.0 g
Lactose – 10 g
Distilled water – 1 liter

Neutral red (1% sol.) – 4 Ml
Agar – 2-3%

What is yeast extract malt agar?

Yeast extract – 3g

Malt extract – 3g
Peptone -5g
Glucose – 10g

Distilled water – 1 liter
pH – 5.0-6.0

Sterilize by autoclaving at 1210C for 15 minutes. To eliminate bacteria present in the sample, the medium can be made selective by making the pH 3.5-4.0 and the temperature of incubation 20 -28C.

Such a medium can be used for direct isolation or a liquid medium for enrichment.

For the growth of osmotrophic/osmophilic yeasts, YM agar containing 30 -50% glucose is employed.

Cultivation methods for fungi

All fungi are generally heterotrophs.

For their cultivation and growth, fairly simple media containing sugar, inorganic nitrogen, and other inorganic salts supplying phosphates and other minerals can be used.
Complex media containing complex organic ingredients like peptone are also used. In general, media for the growth of molds have higher sugar concentration (3-4%) and a relatively low pH (3.5-6.0), as compared with media used for bacteria ( sugar not exceeding 1% generally and pH of 6.5-7.5).
Yeasts are in general mesophilic and can grow over a wide range of pH.

They can be isolated by direct plating on a rich, organic medium with moderately acid conditions.

Example – Yeast extract Malt extract agar.

What is sabouraud’s agar?

Mycological peptone – 10 g

Maltose/ Dextrose – 40 g
Distilled water – 1 liter
pH – 5.0

Preparation of sabouraud’s agar

General purpose of Sabouraud’s agar medium is the isolation and growth of fungi.
The medium is sterilized at 10 psi for 10 minutes (sterilization at lower pressure and temperature, as a medium, has relatively high sugar content).
Lower pH is essential, as it inhibits most of the bacterial population. High sugar content favors fungal growth.

What is Potato dextrose agar?

Potato Dextrose Agar contains following elements,

Potato (peeled) – 200.0g
Dextrose – 20.0g

Agar – 2-3 %
Distilled water – 1000 mL

Preparation of Potato dextrose agar

Potato dextrose agar is selective for the isolation of fungi.

Peel off the skin of potatoes cut them into small pieces, and boil them in 500 mL of water till they are easily penetrated by a glass rod.
Filter through cheesecloth. Add dextrose to the filtrate.
Dissolve agar in water and bring to the required volume by the addition of water.

pH is self-adjusted. Sterilize at 1210C for 20 minutes.