Topic 4: Culture Media and Reagents

Culture media and reagents are essential components in microbiology, cell biology, and molecular biology, providing the nutrients, conditions, and environment required for the growth, maintenance, and study of microorganisms, cells, or tissues.

1. Culture Media

Culture media are formulations designed to support the growth of specific types of organisms or cells. They can be broadly categorized based on their purpose and composition.

a. Types Based on Purpose

1. General-Purpose Media
   • Support the growth of a wide variety of organisms.
   • Examples: Nutrient agar, tryptic soy broth.
2. Selective Media
   • Contain substances that inhibit certain organisms while allowing others to grow.
   • Examples: MacConkey agar (selects for Gram-negative bacteria), Mannitol salt agar (selects for Staphylococcus species).
3. Differential Media
   • Contain indicators to distinguish between organisms based on metabolic activity.
   • Examples: Blood agar (differentiates hemolytic activity), Eosin methylene blue (EMB) agar.
4. Enriched Media
   • Supplemented with additional nutrients for fastidious organisms.
   • Examples: Chocolate agar, blood agar.
5. Transport Media
   • Maintain and preserve specimens during transport to the laboratory.
   • Examples: Stuart’s transport medium, Amies medium.

b. Types Based on Composition

• Defined (Synthetic) Media
  • Exact chemical composition is known.
  • Used for specific research or when studying nutrient requirements.
  • Example: Minimal salts medium.
• Complex Media
  • Contain extracts or digests (e.g., peptones, yeast extract), making the exact composition unknown.
  • Example: LB (Luria-Bertani) medium.
• Agar-Based Media
  • Solidified with agar, used for growing colonies or specific isolations.

Natural media

• Consist solely of naturally occurring biological fluids
• Natural media are very useful and convenient for a wide range of animal cell culture
• The major disadvantage of natural media is its poor reproducibility due to lack of knowledge of the exact composition of these natural media

Artificial or synthetic media
Are prepared by adding nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors
Artificial media: serves to:

• provide a balanced salt solution, with specific pH and osmotic pressure for immediate survival
• provide a balanced salt solution supplemented with various formulation of organic compounds and/or serum for prolonged survival;
• Facilitate indefinite growth

Artificial media are grouped into four categories:
1) Serum containing media

• Fetal bovine serum (FBS) is the most common supplement in animal cell culture media
• It is used as a low-cost supplement to provide an optimal culture medium
• Serum provides carriers or chelators for labile or water-insoluble nutrients, hormones and growth factors, protease inhibitors, and binds and neutralizes toxic moieties

2) Serum-free media

• Serum has many drawbacks and can lead to serious misinterpretations in immunological studiesThey are formulated to support the culture of a single cell type
• It incorporates defined quantities of purified growth factors, lipoproteins, and other proteins, which are otherwise usually provided by the serum
• These media are also referred to as ‘defined culture media’ since the components in these media are known

3) Chemically defined media

• These media contain contamination-free ultra pure inorganic and organic ingredients, and may also contain pure protein additives, like growth factors
• Their constituents are produced in bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, specific amino acids, and fatty acids

4) Protein-free media

• Protein-free media do not contain any protein and only contain non-protein constituents
• Compared to serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of any expressed product
• Formulations like MEM, RPMI-1640 are protein-free and protein supplement is provided when required
• Culture media contain a mixture of amino acids, glucose, inorganic salts, vitamins, and other nutrients

-Provide nutrients and energy source

-Maintain pH and osmolarity (260-320 mOsm/L)

• The requirements for these components vary among cell lines
• Each component performs a specific function

Inorganic salt: Inorganic salt in the media helps to

• maintain osmolarity
• regulate membrane potential by providing sodium, potassium, and calcium ions
• provide ions for cell attachment and enzyme cofactors

Amino acids: are the building blocks of proteins

• Essential amino acids must be included in the culture media as cells can not synthesize these by themselves
• Nonessential amino acids may also be added to the medium to replace those that have been depleted during growth

L-glutamine

– Essential amino acid (not synthesised by the cell)

– Energy source (citric acid cycle), used in protein synthesis

– Unstable in liquid media – added as a supplement

Non-essential amino acids (NEAA)

– Energy source, used in protein synthesis

– May reduce metabolic burden on cells

Growth Factors and Hormones (e.g.: insulin)

– Stimulate glucose transport and utilization

– Uptake of amino acids

– Maintenance of differentiation

Antibiotics and Antimycotics

– Penicillin, streptomycin, gentamicin, amphotericin B

– Reduce the risk of bacterial and fungal contamination

– Cells can become antibiotic resistant – changing phenotype

– Preferably avoided in long term culture

Keto acids (oxalacetate and pyruvate)

– Intermediate in Glycolysis/Krebs cycle

– Keto acids added to the media as additional energy source

– Maintain maximum cell metabolism

Carbohydrates

– Energy source

– Glucose and galactose

– Low (1 g/L) and high (4.5 g/L) concentrations of sugars in

basal media

Vitamins

– Precursors for numerous co-factors

– B group vitamins  necessary for cell growth and proliferation

– Common vitamins found in basal media are riboflavin,

thiamine and biotin

Trace Elements

– Zinc, copper, selenium and tricarboxylic acid intermediates

Foetal Calf/Bovine Serum (FCS & FBS)

– Growth factors and hormones

– Aids cell attachment

– Binds and neutralize toxins

– Long history of use

Heat Inactivation (560C for 30 mins) – why?

– Destruction of immunoglobulins

– Destruction of some viruses (also gamma

irradiated  serum)

Care! Overdoing heat inactivation can damage growth factors, hormones & vitamins and affect cell growth.

2. Reagents

Reagents are chemicals or biological components used in media preparation, staining, and analysis.

a. Growth-Promoting Reagents

• Peptones: Protein hydrolysates used as a nitrogen source.
• Yeast Extract: Rich in vitamins and trace elements.
• Carbon Sources: Glucose, sucrose, or other sugars for energy.

b. Staining Reagents

• Used for microscopy and differentiation.
• Examples: Crystal violet, safranin, methylene blue, Gram’s iodine.

c. Selective Agents

• Substances added to inhibit unwanted organisms.
• Examples: Antibiotics (e.g., ampicillin), bile salts.

d. Indicators

• Show changes in pH or other metabolic byproducts.
• Examples: Phenol red, bromothymol blue.

Phenol red: Most of the commercially available culture media include phenol red as a pH indicator, which allows constant monitoring of pH

• During the cell growth, the medium changes color as pH is changed due to the metabolites released by the cells
• Phenol red changes color based on the pH of the medium:
  • At optimum pH value for cell culture (physiological pH), phenol red turns the medium Red-orange/ bright red  pH (7.2–7.4).
  • At low pH levels (acidic pH), phenol red turns the medium yellow  (<6.8).
  • At higher pH levels (basic pH) it turns the medium Pink-purple (>7.8).
• It allows researchers to visually assess the pH of the culture medium and detect shifts due to cellular metabolism or contamination.

Bromothymol blue (BTB) is a pH-sensitive dye commonly used in cell culture to monitor pH changes in the medium. It serves as an indicator of metabolic activity and environmental conditions.

• • Green: Neutral conditions (pH ~7.0)
  • Blue: Alkaline conditions (pH > 7.6)
• Purpose: Tracks pH changes due to cell metabolism. Cells produce acidic byproducts like lactic acid during growth, which lowers the pH, causing the color to shift towards yellow. As cells consume nutrients and excrete metabolic products, the pH of the medium changes. Bromothymol blue visually reflects these changes.

3. Buffering systems

Buffering systems regulating pH is critical for optimum culture conditions where gaseous CO2 balances with the CO3/HCO3 content of the culture medium

a) Natural: maintained by a CO2 incubator in an air atmosphere with 5-10% CO2

b) Chemical: Maintained by a zwitterion, HEPES (4,2-Hydroxy Ethyl-Piperazine Ethane Sulphonic acid)

-buffering capacity in the pH range 7.2-7.4 – No CO2 incubator

-Greatly increase the sensitivity of media to phototoxic effects induced by exposure to fluorescent light

Types of cell culture media

Culture medium is available in three forms from commercial suppliers:

1. Powdered form: it needs to be prepared and sterilized by the investigator
2. Concentrated form: to be diluted by the investigator
3. Working solution: to be used directly without further manipulation

Powdered medium is the least expensive but needs to be sterilized

Fetal bovine or horse sera can be added after filtration

• as the foaming that occurs in the presence of serum denatures the protein

Media should always be tested for sterility by placing it in a 37℃ CO2 incubator for 72 hours prior to utilization

• to ensure that the lot is contamination-free

Medium should be stored at 4℃

• several components of the medium are light-sensitive, it should be stored in the dark

Since several components of the medium are light-sensitive, it should be stored in the dark.

Criteria for Selecting Culture Media

• The choice of cell culture media is extremely important, and significantly affects the success of cell culture experiments
• The selection of the media depends on
• —–the type of cells to be cultured,
• —–the purpose of the culture and
• —–cell density requirements
• —–resources available in the laboratory
• Different cell types have highly specific growth requirements, therefore, the most suitable media for each cell type must be determined experimentally

Preparation of culture media

1. To a mixing container that is as close to the final volume as possible, add 10% less distilled water than the desired total volume of medium
2. Add powdered medium to room temperature water with gentle stirring. Do not heat water
3. Rinse inside of package to remove all trace of powder
4. Add Sodium Bicarbonate as required
5. Dilute the medium to the desired volume with distilled water and stir until dissolved. Do not over mix
6. Adjust the pH to between 0.2 – 0.3 below the desired final working pH by slowly adding, with stirring, 1NaOH/ 1N HCI

–The pH usually will rise 0.2 – 0.3 units upon filtration

–Keep the container closed until the medium is filtered

7. Process the medium immediately into sterile containers by membrane filtration using 0.2 micron filter

Early developed media

• CMRL developed another media named it as CMRL1066 (Parker et al. 1957)
• Created by amending Medium 199  in order to culture mouse L cells under protein-free conditions
• The modifications included increased levels of reducing substances (cysteine, glutathione, and ascorbic acid), the elimination of fat- soluble vitamins, changes in the types of nucleic acid precursors, and the addition of coenzymes

Common media and their applications

1. Basal medium Eagle (BME) (Eagle 1955)

• Supplemented with the minimal components that are necessary for mouse L cells and human HeLa cells to reach the index of proliferative capacity
• They include 13 amino acids and eight vitamins
• It is unsuitable for cells whose cultures require many components because of its simple composition

2. Minimum essential medium (MEM) (Eagle 1959)

• BME modified according to the cellular need for  amino  acids, so the concentrations  of the  majority of amino acids are twofold, as compared to BME
• Non-essential amino acids, which cells can biosynthesize, were not  included in the original MEM formulation
• However, researchers can add non- essential amino acids to reduce the biosynthetic load

3. Dulbecco’s modified MEM (DMEM) (Dulbecco and Freeman 1959)

• Modified to have fourfold the  concentrations of  amino  acids and vitamins that are present in BME
• Developed to study the plaque-forming ability of the polyoma virus in mouse embryonic cells
• Various modifications have been made since, with supplementation, for example, of the non- essential amino acids, glycine and serine, iron, and pyruvate

What is usually in the DMEM?

DMEM,

• Contains glucose, some proteins, and essential salts
• Contains a pH indicator (phenol red) Media looks pink/red at pH 7.2
• Acidic -yellow or orange (cell growth, bacterial growth)
• Basic -purple (no cell growth, not enough CO2)

More media components

• Antibiotics (penicillin and streptomycin): Prevent bacterial contamination
• Salts and buffers: To simulate in vivo environment
• Serum: Portion of blood after the cells and fibers have clotted
• ———From cow, horse, sheep
• ———added to media as a nutrient source for growing cells
• ———Lipids, proteins

Preparation of DMEM

1. Prepare 900ml of distilled water in clean graduated cylinder.  Water temperature should be 15-30ºC
2. Put the beaker on magnetic stirrer
3. Add 15.4 g/L DMEM media powder to the water and stir gently Stir till completely dissolved.
4. Add 1.2 gram Sodium Bicarbonate (or 49.4 ml of 7.5% Sodium Bicarbonate Solution)
5. Add 10% (100ml) of Fetal Bovine Serum
6. Place the FBS in water bath to don’t prevent cell growth
7. Add 10ml from Antibiotics (penicillin, streptomycin)
8. Adjust pH to 0.2-0.3 units below the required pH using 1N HCl or 1N NaOH. The pH will rise by 0.1-0.3 units after filtration. The required media pH is 7.4
9. Add distilled water up to 1liter
10. Filter for sterility with 0.2µ filter into sterile bottles

4. Leibovitz’s L-15 Medium

• L-15 is buffered by phosphates and free base amino acids instead of sodium bicarbonate.
• This medium is designed for supporting cell growth in environments without CO2 equilibration
• It is modified as follows: with galactose, phenol red, L-glutamine, sodium pyruvate; without glucose, HEPES, sodium bicarbonate
• Contains no proteins, lipids, or growth factors
• Requires supplementation, commonly with 10% Fetal Bovine Serum (FBS)

5. McCoy’s 5A

• Modification of Basal Medium 5A with high glucose, L-glutamine, bacto-peptone, phenol red; without sodium pyruvate, HEPES
• It supports the growth of primary mammalian cells
• Includes Dr. Hsu’s addition of Hank’s salts to enable use outside CO2 incubator
• Requires serum supplementation, commonly with 10% Fetal Bovine Serum (FBS)
• Uses sodium bicarbonate buffer system (2.2g/L), and therefore requires 5-10% CO2 environment to maintain physiological pH

6. RPMI-1640 culture media

• RPMI-1640 was developed at Roswell Park Memorial Institute (RPMI) in Buffalo, New York
• RPMI-1640 is a modification of McCoy’s 5A and was developed for the long-term culture of peripheral blood lymphocytes
• RPMI-1640 uses a bicarbonate buffering system and differs from the most mammalian cell culture media in its typical pH 8 formulation
• RPMI-1640 supports the growth of a wide variety of cells in suspension and grown as monolayers

Preparation of RPMI 1640 culture media

1. Prepare 900ml of distilled water in clean graduated cylinder. Water temperature should be 15-30ºC. Put the beaker on magnetic stirrer
2. Add equivalent powder to the water and stir gently. Stir till completely dissolved
3. Add 2.0 gram Sodium Bicarbonate (or 26.67 ml of 7.5% Sodium Bicarbonate Solution)
4. Adjust pH to 0.2-0.3 units below the required pH using 1N HCl or 1N NaOH. The pH will rise by 0.1-0.3 units after filtration
5. Add distilled water up to 1 liter and filter for sterility with 0.2µ filter into sterile bottles

7. Iscove’s Modified Dulbecco’s Medium (IMDM)

• IMDM is a highly enriched synthetic media well suited for rapidly proliferating, high-density cell cultures
• IMDM is a modification of DMEM containing selenium, and has additional amino acids, vitamins and inorganic salts as compared to DMEM
• It has potassium nitrate instead of ferric nitrate and also contains HEPES and sodium pyruvate
• It was formulated for the growth of lymphocytes, for example the differentiation of monocytes into macrophages, and hybridoma

Optimization of Cell Culture Media

• The complexity of the composition of cell culture media provides many challenges to optimize individual components of media
• Most of the classical culture media were devised for small-scale low-density cultures and often require serum as a key nutrient
• However, in the biotechnology industry where there is a need to sustain high cell densities and increase cellular productivity, development and optimization of culture media is very critical
• Typically, media for the biotechnology industry are serum-free and have a much higher concentration of nutrients than classical media
• Optimization of media requires the following parameters to be considered:

a) Product to be made

• Cell culture media should support maximal cell growth and sustain cell viability at increasing cell densities
• For the production of virus, not just high cell densities are required but there must be abundant nutrients in the media to sustain virus replication after infection
• For the production of recombinant protein, high cell density is required
• However, nutrients required for the cell growth can compete with those required for the production of proteins
• It is, therefore, very important to carefully determine the maximum cell densities a given medium can sustain for a required level of productivity

b) Cell lines to be used

• Different cell lines have different nutritional requirements because of the difference in metabolism which dictates media optimization methods
• Normal diploid fibroblasts require attachment factors to adhere and spread out on a surface for growth. They grow too much lower densities and therefore do not need nutrients in high quantities
• Hybridoma cells lines are generally highly dependent on glutamine. They typically lack a stationary phase after reaching a peak cell density and then decline rapidly in viability
• Optimization of medium, thus, would reduce the decline in viability and improve monoclonal antibody production

c) Manufacturing process involved

• Manufacturing process mode would not only affect the choice of cell culture medium but also approaches to optimization
• Different manufacturing processes used are:
• —-Batch Process: A single medium rich in nutrients is used to sustain cell growth and productivity
• —-Fed-batch: Several kinds of media are used over the course of the cell culture, depending on the stage of the process
• ———has lower nutrient concentrations when cell densities are low during inoculation but maintain high rates of cell growth during culture scale-up and early production

Applications

1. Microbiology: Isolating and identifying bacteria, fungi, and viruses.
2. Cell Culture: Supporting the growth of animal or plant cells (e.g., DMEM, RPMI-1640).
3. Molecular Biology: Recombinant DNA technology and protein expression (e.g., LB medium for E. coli).
4. Clinical: Diagnosing infections or monitoring contamination.