Culture media or growth media is a liquid or gel-like substance that provides essential nutrients and minerals required for organisms to grow under laboratory conditions.
All microorganisms have different requirements based on their habit and habitat. Thus, culture media needs to be formulated in such a way that they support the organism’s life in the lab.
Thus, a variety of culture media have been developed by scientists according to the type of organisms they want to study and the purpose of their experimentations. These include selective media, differential media, enriched media, complex media, defined media, and many more.
The mixture of nutrients is used in three consistencies: liquid without adding agar; semi-solid with lower agar percentage; and solid with a comparatively higher percentage of agar. Again, the preparation depends on the end goal of the experiment.
When cultures are being used for experiments, the culture medium works well, but when cells or microbial strains need to be preserved for future studies, advanced techniques are necessary.
This article discusses how to prepare culture media, how to maintain them, and how cultures are preserved (without culture medium) in labs so that they can be used in the long run.
Culture Media Preparation
Media preparation is the process of mixing nutrients, buffering agents, and other components that support the growth and development of an organism. It’s a routine task for labs working with plants, mammalian cells, and microorganisms.
The media should be formulated in a way that provides an optimal condition for the growth of microorganisms. Specific components of the media and their composition are decided based on the type of organism being studied.
What Are the Basic Components of the Culture Media?
A culture medium is a mixture of vitamins, amino acids, salts, glucose, and other nutrients. All these components are commercially available in either powder or liquid form. They all have essential functions to perform in the media such as providing nutrient support.
Here is a list of basic nutrient components and their functions in supporting the growth of organisms in laboratory conditions:
It provides sodium, potassium, and calcium ions that help the media to maintain osmotic balance and regulate the membrane potential of cultured cells.
It’s required to maintain optimum pH in cultures, which is 7.0 for most microorganisms. Some commonly used buffers in culture media are ammonia, calcium carbonate, and sodium hydroxide.
The appropriate pH is mainly achieved by using the following buffering systems:
- Natural buffering system: The system ensures a balance between gaseous CO2 and the CO3/HCO3 content of the culture medium. It’s a low-cost, non-toxic system, however, it requires a controlled gaseous environment. So, these cultures are incubated in CO2 incubators maintaining 5-10% CO2 in the air atmosphere around the cultures.
- Phenol red: It’s commonly present in commercially available culture media. It acts as a pH indicator and helps to monitor the media’s pH. As microbial species grow in the media, they release certain metabolites which change the media’s pH. Thus, if the media contains phenol red, it will change to yellow at low pH, purple at high pH, and at optimum pH of 7.4, it’s bright red. There are also some disadvantages of using phenol red in culture media:
- Phenol red mimics the action of steroids (especially estrogen), so estrogen-sensitive cells (like mammary tissue) should not be exposed to it.
- Phenol red interferes with the sodium-potassium balance of the serum-free culture media.
- Phenol red interferes with flow cytometric detection.
- HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid): It’s a zwitterionic sulfonic acid buffering agent. It maintains the media’s pH between 7.2 to 7.4 and doesn’t require a controlled gaseous environment. However, the chemical is expensive and toxic to cultures at a high concentration.
These are building blocks of proteins — an essential biomolecule required for life, and which cultured cells can’t synthesize on their own.
They promote cell proliferation and cell density in cultures, especially L-glutamine, a nitrogen source for cells, which helps in NADP and NAD synthesis that serve as a secondary source of energy for growing cells.
The amino acid should be added just before using the media, as it converts to ammonia over time, which kills cells.
Carbohydrates act as a carbon source for cultured cells or organisms and provide energy for their metabolic processes. In labs, glucose and galactose are the most common sugar sources, however, maltose and fructose are also often used.
Peptides and Proteins
They are essential in serum-free media because they are met in serum-containing media by serum, which contains fetuin, transferrin, albumin, aprotinin, and fibronectin.
Among the most commonly used proteins and peptides are fibronectin, albumin, and transferrin. These molecules play a significant role in culture media, such as removing toxic materials, transporting metabolites between cells and tissues, and inhibiting serine proteases.
They are essential for the proliferation and growth of cultured cells and organisms. The serum is a major source of vitamins in culture; however, for some cell lines, different vitamins are added in the place of serum. The most common vitamin supplement is vitamin B.
Fatty acids and lipids
They act as carbon sources and provide energy to cultured cells or organisms for growth and development. These biomolecules are provided by serum in serum-containing media, thus are usually added to serum-free media.
They are essential for many processes, including proper cell growth and enzyme activity. The trace elements commonly added to the media are selenium, zinc, copper, and tricarboxylic acid intermediates.
Certain cell lines require more than basal medium components and serum, they need supplements like growth factors, hormones, and signaling substances. They help cells and cultured organisms to grow, proliferate, and maintain normal cell metabolism.
After adding supplements, it’s vital to check the osmolality of the media since they can adversely affect osmolality and hinder cell growth. The optimum osmolality for most cell lines is between 260 mOSM/kg and 320 mOSM/kg.
They are required to control the growth of bacterial or fungal contaminants. However, antibiotics are not suggested for routine lab use as they can mask the growth of resistant bacteria and mycoplasma. Moreover, they also interfere with the growth of cultured organisms or cells.
It’s a complex mixture of growth factors, albumin, and growth inhibitors. It serves as a source of proteins, amino acids, carbohydrates, vitamins, lipids, trace elements, growth factors, and minerals.
In addition to protecting cells from mechanical damage, these molecules also act as a buffer, promote cell attachment to the substrate, and protect them from proteolysis. The normal concentration of serum used in media is between 2-10%.
How to Prepare Culture Media
Choosing the right media for a particular cell line or microbial culture is the first step in preparing culture media. It’s important as it impacts experimental successes. Media selection mostly depends on three factors:
- cell type;
- purpose of cell culture, and
- available resources in the laboratory.
The determination of the appropriate media and their suitability must be based on experiments.
After choosing the right media, media preparation follows. The procedures on how to prepare culture media vary depending on the type of media. However, basic steps for preparing culture media are:
- Select culture media recipes according to the medium that needs to be prepared and your available resources.
- Calculate all ingredients of the media according to your required volume.
- Weigh all main ingredients and trace elements using high accuracy balances.
- Take 80% volume of deionized or double-distilled water in a beaker.
- Dissolve the weighed ingredients in water by keeping the beaker on a magnetic stirrer.
- For some culture media, gentle heat is required, for which a hot plate with a magnetic stirrer can be used.
- Check the media’s pH and adjust it as required.
- Make up the total volume of the prepared culture media using double distilled water.
- Label the container containing media.
- Sterilize the media in an autoclave.
Heat-sensitive substances are added after sterilizing the media using membrane filters. Then, culture media are stored at a specific temperature to prevent any modification of their composition. Often, prepared culture media are stored for a few weeks by keeping in a fridge at 4ºC.
Maintenance and Preservation of Cultures
Cells are grown in a culture medium until they are used for experiments, but when they need to be maintained and preserved for future studies, other chemicals need to be added. The four techniques that are so far developed and available for storing and preserving cultures for future studies are:
In this technique, the bacterial cultures are stored at 4ºC in refrigerators or cold storage. In such conditions, the metabolic process of the cells is slowed down, which allows slow utilization of the provided media. Moreover, the process also protects cultures from getting damaged due to medium evaporation and helps in culture preservation for a longer time.
Nevertheless, the method has its limitation—as cells consume the media, they also produce waste products that build up in the vessel and can cause the cells to die. The solution for this problem is sub-culturing cells or microorganisms over a fixed interval. For example, bacterial species should be subcultured between 2-3 weeks, while fungal species need to be subcultured between 3-4 months.
2. Deep Freezing
In this method, cultures are kept in glycerol and stored for several years in a deep freezer at -40ºC. A 2ml glycerol is added onto the agar slope and shaken to make a suspension of cultures.
Then, the suspension is transferred into an ampoule (a storage vial) kept in a mixture of industrial methylated spirit and CO2, and frozen rapidly in a freezer at -70ºC. After this, they are removed from the -70ºC freezer and the mixture of methylated spirit and CO2, and stored in a freezer at -40ºC.
Before using the suspension cultures for experiments, they are removed from the freezer and placed in a water bath for a few minutes until the suspension melts and is ready to be used to prepare a streak plate.
3. Freezing Under Liquid Nitrogen
In this method, cells or microorganisms are stored in liquid nitrogen at -196ºC. It suspends the metabolism processes of cells and enables them to survive unchanged for a longer period.
Here, cells are suspended into glycerol or DMSO (dimethyl sulfoxide) — to prevent the formation of ice crystals that can kill them — kept in an ampoule and stored in liquid nitrogen. The method is preferable for cells that can not be preserved using the lyophilization technique.
4. Lyophilization or Freeze Drying
In this method, the cultures are rapidly frozen at -70ºC and then dehydrated by vacuum. Afterward, the frozen dried cultures are sealed and stored in the dark at a temperature of 4ºC.
It’s the most suitable method in labs for long-term culture preservation, including bacteria, fungi, and viruses. During use, cultures are revived by adding liquid media to the vial and then transferring it to a suitable culture/growth medium.
Culture media support the growth of cells and microorganisms in labs by fulfilling their need for nutrients and minerals. However, different organisms have different nutrient requirements and living conditions. Thus, several culture media are formulated to enable the growth of a selected cell or microbe in labs.
All the basic components of the media are formulated according to the cell or organisms that need to be grown. After preparing a mixture of nutrients, the media is sterilized using an autoclave and kept in suitable condition to prevent contamination.
If cultures are not in use, other preservation techniques are followed to preserve and maintain cultures, despite using culture media. However, there are some limitations to them that create scope for young scientists to work in the advancement of the area.
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