What is a growth medium?

It is a culture medium or nutrient broth for all kinds of microorganisms to grow properly and create their colony. This matrix provides support to increase the population of microbes through the method of cell proliferation. It is regarded as the food material for those organisms. It is a type of in vitro mechanism, in which the development of life is processed outside the host body. It can be solid, semi-solid, or liquid in nature. Different kinds of cells develop in different types of media.

Composition of culture media

Several components are required to prepare the cell culture medium. A typical medium is made up of certain components like-

• Amino acids or nitrogen source
• Vitamins
• Inorganic salts or mineral salts
• Glucose or carbon source
• Water
• Specified energy sources
• Exclusive growth factors

Types of culture media

The classification is mainly based on two distinct factors. These factors are physical state and constructive ingredients.

Physical state types

Solid medium

It is a medium which is solid at room temperature. A mixture of agar and other nutrients is used as a solid medium. Agar is yellowish golden granular powder, collected from the seaweeds. Solid Media are useful for enumerating and isolating bacteria and determining their colony characteristics. 

Semi-solid medium

It is obtained by reducing the amount of agar to 0.2-0.5%. It helps in separating the motile organism from the non-motile ones. For example, peptone water.

Liquid medium

It is a "broth" composition. Generally, the bacteria grow uniformly in this medium. For example blood culture.

Constructive ingredients

As per the types of ingredients utilized, the constructive media are divided to be in two types. These are- routine laboratory media and synthetic media.

Routine laboratory media

• Basal media: These media are utilized for the growth of bacteria that do not need any exclusive enrichment. Some examples are nutrient broth, nutrient agar, and peptone water. Staphylococcus and Enterobacteriaceae can develop in this way.
• Enriched media: These media are enriched by adding blood, serum, or egg. Examples are blood agar, Lowenstein-Jensen media. Streptococci species of bacteria grow better in blood agar media.
• Selective media: These media dominate the surge of a particular desired bacterium by stopping the growth of unwanted bacteria. Examples: MacConkey agar, tellurite media, and some more. Antibiotics may be put to a medium as an inhibitor.
• Indicator or differential media: This medium involves an indicator. A specific organism instigates a change. For example, blood, neutral red, tellurite, and a few others. Blood and MacConkey belong to this category.
• Transport media: These media are important when specimens are not allowed to be cultured soon after collection. Some examples are Cary-Blair, Amies, and Stuart media.
• Storage media: This is perfect for storing microbes for a long time. For example, egg saline, chalk cooked meat broth medium, and some more.

Synthetic media

It is recognized as a chemically defined medium. In this medium, all known chemicals are utilized. Yeast, animal, or plant tissue are not found here. This growth medium is widely accepted for the culture of microbes, animal cells, and human cells also. These cells can be utilized due to their known chemical constituents. It is completely devoid of any animal component and accepted as the most consistent and purest cultural environment for living bodies.

The components of synthetic media are- 1% peptone + 0.5% NaCl (Sodium Chloride) in water.

Preparation of culture medium

In microbiology, the method of the preparation of a basic growth medium is not much complicated. It can be prepared with equipment and proper nutrient sources available in major schools. Sterilization is an important process, which is carried out for about 15 minutes at 121 degree Celsius. Inoculum preparation is also a mandatory stage. It requires obtaining microorganisms in an appropriate state so that they may be easily compatible with inoculation into cell culture. The following mixtures are prepared through certain components:

• Nutrient agar: Approximately 28 grams of nutrient agar powder is suspended in 1 liter of distilled water. This mixture is brought to a boil to be dissolved. The required amount is dispensed and sterilized.
• Nutrient broth: 13 grams of nutrient broth powder is added to 1 liter of distilled water and well mixed. The required amount is taken for the further procedure.
• Malt extract agar: About 18 grams of agar powder is dissolved in 1 liter of distilled water. It is brought to a boil to dissolve completely. About 15 grams of malt extract is added per liter and well mixed. A necessary amount is taken for the next step.
• Mannitol yeast extract broth: It is prepared in the same way as malt extract, except the agar part.
• Glucose nutrient broth: Nutrient broth is made up as it is mentioned before and 10-gram glucose per liter is added.
• Sugar peptone water: About 10 grams of peptone, 5 grams of NaCl (Sodium Chloride), 5 grams of sugar, and 20 cube centimeters of 1 liter distilled water are mixed. The potential of Hydrogen or pH of the mixture should be maintained within 7.4. The required amount is taken out for future reference.
• Tributyrin agar: It is a supplied form of agar and is always ready to use. It is given heat to melt and is dispensed aseptically. 1% of tributyrin can be added to nutrient agar to prepare it.
• Glucose yeast extract broth: Approximately 10 grams of peptone, 5 grams of NaCl, 3 grams of yeast extract are added to 1 liter of distilled water to create this broth.
• Glucose yeast extract Lemco broth: About 10-gram Lemco or meat extract is mixed with the glucose yeast extract broth.
• Milk agar: Firstly, nutrient agar is prepared but only in 900 cube centimeters of distilled water. Then, 20 grams of dried skimmed milk is dissolved in 100 cube centimeters of distilled water. These are sterilized separately. Milk is transferred after cooling to 45 to 50 degree Celsius. It is dispensed as required.
• Starch agar: 15 grams of nutrient agar is suspended in 100 cube centimeters of distilled water. It is then brought to a boil to dissolve. About 40 grams of soluble starch is heated in 100 cube centimeters of distilled water to create a suspension. These are then allowed to cool and then mixed with the nutrient agar. Dispensation and sterilization are then carried out as per need.

Growth of microorganisms in a culture medium

Cell culture media generally exhibit necessary nutrients to give support to the microbes to live in a better way. Culture media vary in several ingredients that allow selecting for or against organisms. Glucose or glycerol are often utilized as the carbon source, and ammonium salts are needed to act as inorganic nitrogen reagents. MacConkey agar is appropriate for gram-negative bacteria. The selective agar is used in the form of Hektoen enteric agar for the gram-negative. The gram-positive bacteria grow best in the nutrient medium full of mannitol salt agar (MSA). Most anaerobes grow better in blood agar plates. Microorganisms that can synthesize their nutrient from inorganic material are called prototrophs. When a microbe loses its ability to absorb or synthesize any kind of organic compound, they are considered auxotrophic. Protein hydrolysates are used as the growth-promoting factors for the microorganisms, which are unable to synthesize their nutritional material or nutrient from the growth medium properly.

Significance of cell culture media

Culture media provide fundamental importance mainly to the microbiological tests. The importance is most commonly found-

• To acquire the purest culture.
• To generate and calculate the number of microbial cells.
• To choose the appropriate microbes to proceed further.

Quality control of the culture media

Quality control can be divided into two parts-

Physical characteristics

• The color of a sterilized medium should be compared with a non-sterilized medium to notice if any difference exists.
• Crystallization clarity is needed to be judged.
• The gel-based medium should be kept firm and usable.
• The potential of Hydrogen (pH) is a significant factor to notice very precisely.
• Plates and bottles should be examined for damage like cracks or defects.

Microbiological characteristics

• The media sterility test is designed for detecting microbial contamination. A small number, usually 2% of the batch of the uninoculated items are incubated. The temperature and time selected for the incubation will depend on the nature of the medium. For the general-purpose one, it is kept as 30 to 35 degree Celsius for three days.
• The most important test is the challenge of carrying out the culture method in the microbiology laboratory. This key test is undertaken by the media creator and that is undeniable.

Context and Applications

Students practically learn to prepare this growth medium in their educational institutions. Often they do not take further care after preparing them, which results in damage in the medium. Skin, eyes, or any kind of mucous membranes should not be touched with any laboratory reagent, stain, fixative, and any other chemicals.

Common Mistakes

The nutrient growth medium study is included mainly in streams like-

• Masters in Science in Microbiology
• Masters in Science in Biotechnology
• Masters in Science in Zoology
• Masters in Science in Botany
• Bachelors in Science in Zoology
• Bachelors in Science in Botany

Currently, the SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2), which is responsible for the pandemic disease COVID-19, also undergoes the cell culture process at CDC (Center for Disease Control and Prevention). The organization cultures this virus enough to ensure its availability to research and prepare vaccines for human health

Practice Problems

Q1. What type of media is Lowenstein-Jensen?

1. Basal media
2. Selective media
3. Enriched media
4. Complex media

Correct Answer: c.

Explanation- Enriched media. In this kind of medium, eggs, and blood are added to the nutrient solution to make it better. Lowenstein-Jensen media consists of coagulated eggs along with other ingredients.

Q2. From where one can collect the Agar?

1. Basil leaves
2. Seaweed
3. Algae
4. Dried blood

Correct Answer: c.

Explanation- Algae. Red algae secrete a jelly-like substance. This substance is agar.

Q3. How much nutrient agar is used to prepare the milk agar?

1. 300 cube centimeter of distilled water
2. 1 liter of tap water
3. 100 cube centimeter of river water
4. 900 cube centimeter of distilled water

Correct Answer: d.

Explanation- 900 cube centimeter (cm) of distilled water. The nutrient agar is prepared in 900 cube cm of distilled water. After then, 20 grams of dried skimmed milk and 100 cube cm of water are mixed.

Q4. What is the full form of CDC?

1. Central Defence Council
2. Center for disease control and prevention
3. Center of disease counseling
4. Center for diagnosing and control

Correct Answer: b.

Explanation- Center for disease control and prevention. It is the national public health agency of the United States.

Q5. What is Lemco?

1. Milk extract
2. A mixture of sugar and agar
3. Meat extract
4. Synthetic product without any natural ingredients

Correct Answer: c.

Explanation- Meat extract. Lemco is a meat extract. 10gm of Lemco can prepare the glucose yeast extract agar. It is mixed with glucose yeast extract broth in a particular amount to get a better result.