GENERAL TOOLS AND

TECHNIQUES USED IN
CELL CULTURE
TECHNOLOGY

CENTRIFUGE
 Separate a mixture of two different miscible liquids.
 Study and analyze macromolecules and their hydrodynamic properties.
 Mammalian cells can be purified with the help of a special type of centrifuge.
 Fractionation of many subcellular organelles.
 Stabilization and clarification of wine.
 This technique, in combination with other purification techniques, is
extremely helpful while separating proteins.
 Centrifuges are widely used in the field of forensic chemistry. In this field,
the technique is employed for the separation of blood components from
blood samples. Furthermore, the technique is also employed in certain
laboratories for the separation of urine components from urine samples.
LAF (LAMINAR AIR FLOW) HOOD
1.Hospitals: Laminar flow hoods are used in hospitals to protect patients from
infection. They are often used in operating rooms and Intensive Care Units (ICUs).

2. Pharmaceutical industry: Laminar flow hoods are used in the pharmaceutical

industry to protect drugs and chemicals from contamination. They are used in research
and development laboratories.

3. Food industry: Laminar flow hoods are used in the food industry to protect food from
contamination. They are regularly used in restaurants, grocery stores, and food
processing plants.

4. Electronics industry: Laminar flow hoods are used in the electronics industry to
protect electronic components from contamination. They are often used in
manufacturing and assembly plants.

5. Cleanrooms: Laminar flow hoods are used in cleanrooms to protect sensitive

equipment from contamination. They are often used in semiconductor fabrication
facilities.

BIOSAFETY CABINETS

 The primary purpose of a BSC is to serve as a means to

protect the laboratory worker and the surrounding
environment from pathogens.
 All exhaust air is HEPA-filtered as it exits the biosafety
cabinet, removing harmful bacteria and viruses.
 This is in contrast to a laminar flow clean bench, which
blows unfiltered exhaust air towards the user and is not
safe for work with pathogenic agents.
pH meter

 A pH meter is an essential tool in laboratory settings to

measure the pH of solutions in pharmaceutical,
chemical, and biotechnology industries.
 A laboratory pH meter has a high measurement range,
providing the user with a more accurate and precise
reading.
 A pH meter is also used to monitor the growth of
pathogenic microorganisms that may spoil food or
beverages, or negatively impact the taste and quality of
the product.

Autoclave

 Autoclaves operate at high temperature

and pressure in order to kill microorganisms
and spores. (15 psi (pound per square inch)
121°C for around 15-20 minutes)
 They are used to decontaminate certain
biological waste and sterilize media,
instruments and lab ware.
Vortex mixer
 Mixing chemicals: Vortexing helps in most laboratories’ homogenous
mixing of chemicals. The mixing time is quicker than that of other
methods of mixing.
 DNA extraction: The vortexer is used for homogenous sample mixing
with extraction buffer. This method also helps in cell disruption.
 For tissue analysis and cell culture: The vortexer has been applicable
in making suspension of cell or tissue samples during tissue analysis
and cell culture.
 In the analysis of proteins and enzymes: Proper mixing of samples with
reagents and buffer is a critical step in studying proteins and enzymes.
So, a vortex mixer helps in homogenized mixing during analysis.

Hot air oven

 1. It is mainly used for the sterilization of glass
wares like pipettes, bottles test tubes, Petri dishes
etc.
 2. It is used for sterilization of powders as well as
oils which is not possible by moist heat
sterilization.
 3. Injectable can be sterilized by hot air oven.
 4. Hot air oven is used for sterilization of scalpels,
scissors, spatula, blades and glass syringes.
 Standard: 1.5 to 2 hr at 160°C
 15 to 30 min at 300°C
Magnetic stirrer
 It is widely used in chemistry laboratories to perform chemical
experiments and synthesis by mixing two or more components.
 It is used to prepare a medium to culture microorganisms in
microbiology laboratories.
 It is used to prepare samples and perform analysis in chemistry
and biology experiments.
 It is employed to achieve the maximum possible extraction
efficiency from plant material by reducing organic solvent
consumption.
 Other applications include oil analysis, soil suspending, buffer
solutions preparation, pH measurement, etc., where it
necessitates the blending of several types of liquids to achieve
uniform mixing.

Weighing balance:

 Analytical balances are precision measuring instruments

used in quantitative chemical analysis, to determine the
mass of solid objects, liquids, powders and granular
substances.
Micro filtration unit:
MFS examples can be found in
pharmaceutical, chemical and semi-
conductor industries for:
 Separation of catalysts
 Separation of enzymes and yeasts
 Clarifying of extraction fluids
 Separation of glass cutting and metal
particles from oil / fluid

Incubator
 Incubators are used to grow microbial culture or cell cultures.
 Incubators can also be used to maintain the culture of organisms to be
used later.
 Some incubators are used to increase the growth rate of organisms,
having a prolonged growth rate in the natural environment.
 Specific incubators are used for the reproduction of microbial colonies
and subsequent determination of biochemical oxygen demand.
 These are also used for breeding of insects and hatching of eggs in
zoology.
 Incubators also provide a controlled condition for sample storage
before they can be processed in the laboratories.
CO2 INCUBATOR

 CO2 incubators are enclosures designed to

precisely control the environment needed to
grow biological or cell cultures.
 Adding additional CO2 at the right level you
balance the Oxygen and prevent the pH inside
the cells from becoming either alkaline or
acidic, which both inhibit cell growth.

Inverted microscope:

 Inverted microscopes are useful

for observing living cells or organisms
at the bottom of a large
container (e.g., a tissue culture flask)
under more natural conditions than on
a glass slide, as is the case with a
conventional microscope.
 T-FLASK

 The T-flask is the most widely

used vessel for routine
maintenance of cell cultures.
Its secure screw cap offers
best protection of valuable
cells.

Sterilization techniques for culture room

 Tissue culture lab is compartmentalized into work elements in
separately specified areas such as media preparation, glassware
washing, sterilization, microscopy and aseptic transfers in order to
facilitate all operations and enhances cleanliness.
 Large transfer rooms are best sterilized by exposure to ultraviolet
(UV) light. Sterilization time varies according to the size of the room
and should be done when no experiment is in progress.
 Transfer rooms can also be sterilized by washing them 1-2 times a
month with a commercial brand of antifungal liquids.
 Culture rooms should be initially cleaned with detergent –brand
soap and then carefully wiped down with a 2% sodium hypochlorite
solution or 95% ethyl alcohol.
Sterilization techniques for apparatus

 Glassware: such as petri plates, vials, culture tube,

pipettes etc. are sterilized in a hot air oven at 160-
180C for 2-4 hours.
 Instruments: The metallic instruments such as forceps,
scalpels, needles etc. are flame sterilized and then
cooled in 95% ethanol under the process called
incineration. It can also be achieved by autoclaving.
 Large equipment can be sterilized with UV light or by
treatment with bactericides/ fungicides.
Sterilization techniques for transfer area

 Transferarea should be cleaned by

detergent followed by 2% sodium
hypochlorite and 95% ethanol. It can
also be achieved by exposure to UV
light.
Sterilization techniques for media
 In general, vegetative cells are destroyed at a lower
temperature in a short time (around 60°C in 5-10 minutes).
 However, the destruction of spores requires higher
temperature and relatively longer time (around 80°C for 15-
20 minutes).
 Spores of Bacillus stearothermophilus are the most heat
resistant.
 Moist heat provided by an autoclave or pressure cooker is an
efficient way to sterilize most materials. At a pressure of 15
psi above atmospheric pressure, water reaches a
temperature of approximately 121° C before it boils. Most
materials are effectively sterilized by 15 minutes exposure
to this temperature.

Sterilization techniques for vitamins

 Vitamins can be sterilized by the

method of autoclaving. For this a
concentrated stock solution is
required to be prepared followed by
filtration and subsequent addition in
sterile medium at the temperature of
growth (or room temperature).
Sterilization techniques for living
material (explant)
 Rinse thoroughly under running tap water for 10-30
minutes.
 Wash explant in a mild detergent such as Tween-20 before
treatment with the disinfecting solution.
 Submerge explants into the disinfectant solution. Seal
bottle and gently agitate. The explants are sterilized by
disinfectants (e g., sodium hypochlorite- NaOCl, mercuric
chloride-HgCl2)
 Under sterile conditions, decant the solution and rinse
explants several times with sterile distilled water.

Cryopreservation
• Cryopreservation is a process of preserving or storing cells, tissues, organs or
any other biological materials from any potential damage by maintaining the
materials at very low temperature (typically -80 °C using solid CO2 or
−196 °C using liquid Nitrogen.
• Cryopreservation is based on the conversion of water present in the cells
from a liquid to a solid state.
• When cooling below 0°C, the biological effects are dominated by the freezing
of water, which typically constitutes at least 80% of the tissue mass.
• The cell water requires much lower temperature to freeze (even up to -68°C)
due to the presence of salts and organic molecules in the cells, in comparison
to the freezing point of pure water (around 0°C).
• The metabolic processes and biological divisions in the cells/tissues are almost
stopped when stored at low temperature.
Need of cryopreservation

 This technique can be used to preserve sperm, eggs,

embryos, and tissues.
 It can maintain the viability of cells and tissues for long
periods of time.
 It can be used to preserve tissue from a wide variety of
species.
 It can be used to preserve cells and tissues for
research purposes.
 It can be used to preserve cells and tissues for medical
treatments.

The cryopreservation of plant cell culture

followed by the regeneration of plants
involves the following steps:
1. Development of sterile tissue cultures
2. Addition of cryoprotectants and pre-treatment
3. Freezing
4. Storage
5. Thawing
6. Re-culture
7. Measurement of viability
8. Plant regeneration
step I: Development of sterile tissue culture:

• Any tissue from a plant can be employed for

cryopreservation e.g. meristems,
endosperms, embryos, ovules, seeds,
cultured plant cells, calluses, protoplasts.
• Out of these, meristematic cells and
suspension cell cultures which are in the late
lag phase or log phase are most appropriate.

step II: Addition of cryoprotectants and pre-

treatment:
• The compounds that can prevent the damage caused to
cells by freezing or thawing are called as cryoprotectants.
• Cryoprotectants reduce the freezing point and super-
cooling point of water.
• As a result, the ice crystal formation is delayed during the
process of cryopreservation.
• Cryoprotectants used are dimethyl sulfoxide (DMSO),
glycerol, ethylene, propylene, sucrose, mannose, glucose,
proline and acetamide.
• Among them, DMSO, sucrose and glycerol are most
commonly used.
• Generally, a mixture of cryoprotectants instead of a
single one is preferred for more effective
step III: Freezing:
The different types of freezing methods used are as follows:
1. Slow-freezing method:
• The tissue or the essential plant material is allowed to
slowly freeze at a slow cooling rates of 0.5-5°C/min from 0°C
to -100°C.
• Then it is transferred to liquid nitrogen.
• Slow-freezing method facilitates the flow of water from the
cells to the outside.
• This avoids intracellular freezing and promotes extracellular
ice formation.
• Because of this, the plant cells are partially dehydrated and
can survive better.
• The slow-freezing technique is successfully employed for the
cryopreservation of suspension cultures.

2. Rapid freezing method:

• In this technique, the vial containing plant
material is plunged into liquid nitrogen.
• The freezing process occurs so quickly that small
ice crystals are formed within the cells.
• Rapid freezing technique is applied for the
cryopreservation of shoot tips and somatic
embryos.
3. Stepwise freezing method:
• This technique is a combination of slow and rapid freezing
procedures having the advantages of both, and occurs in a
stepwise manner.
• Firstly, the plant material is cooled to an intermediate
temperature.
• Then it is kept there for about 30 minutes.
• Finally, it is rapidly cooled by plunging it into liquid nitrogen.
• Stepwise freezing method has been successfully applied for
cryopreservation of suspension cultures, shoot apices and buds.
4. Dry freezing method:
• It has been reported that the non-germinated dry seeds can
survive freezing at very low temperature in comparison to water-
imbibing seeds which are sensitive to cryogenic injuries.
• Low temperature dehydration process that involves freezing the
product and lowering pressure, thereby removing the ice by
sublimation.
• In a similar way, dehydrated cells are observed to have a better
survival rate after cryopreservation.

step IV: Storage:

• Generally, the frozen cells/tissues are maintained at
temperatures in the range of -70 to -196°C for storage.
• Although, with temperatures above -130°C, ice crystal growth
may take place inside the cells which decreases viability of
cells.
• The ideal storage is done in liquid N2 refrigerator at 150°C in the
vapour phase, or at -196°C in the liquid phase.
• The final aim of storage is to halt all the cellular metabolic
activities and preserve their viability.
• The temperature at -196°C in liquid nitrogen is regarded as
ideal for long term storage.
• A regular and constant supply of liquid nitrogen to the liquid
nitrogen refrigerator is necessary.
• It is essential to check the viability of the germplasm time and
again in some samples.
step V: Thawing:
• Thawing is usually performed by plunging the frozen
samples in ampoules into a warm water
(temperature 37-45°C) bath with robust swirling.
• By this process, rapid thawing (at the rate of 500-
750°C min-1) takes place, and this preserves the cells
from the damaging effects from ice crystal
formation.
• As soon as the thawing occurs (ice completely
melts), the ampoules are transferred to a water
bath at temperature 20-25°C at the same instant.
• The cells get damaged if left in warm (37-45°C) water
bath for long time.

step VI: Re-culture:

• To remove cryoprotectants, the thawed
germplasm is washed various times.
• Following standard procedures, this material
is then re-cultured in a fresh medium.
• In some cases, the direct culture of the
thawed material is preferred without
washing.
• It is so because certain vital substances,
released from the cells during freezing, are
assumed to enhance in vitro cultures.
step VII: Measurement of viability:
• The measurement of survival or viability of
the frozen materials can be performed at any
stage of cryopreservation or after thawing or
re-culture.
• The commonly used techniques are staining
techniques using triphenyl tetrazolium
chloride (TTC), Evan’s blue and fluorescein
diacetate (FDA).
• The entry of cryopreserved cells into cell
division and regrowth in culture is the best
indicator to measure the viability of them.

step VIII: Plant regeneration:

• The regeneration of the desired plant is the

ultimate purpose of cryopreservation of
germplasm.
• The cryopreserved cells/tissues have to be
carefully nursed, and grown for appropriate
plant growth and regeneration .
• Along with maintenance of proper
environmental conditions, addition of
certain growth promoting substances is
often essential for successful plant
regeneration.
Cryopreserving steps

Thawing the cells

Cell counting (direct counting by
haemocytometer)
 A hemocytometer (also known as a haemocytometer or a cell
counting chamber) is a tool used for manual cell counting.
 The Improved Neubauer has an H-shaped indent at the center
of the slide that separates the space into two counting
chambers.
 Grids are engraved onto the surface to make cell counting
easier and more precise.
 The 3×3 mm counting grid on the Improved Neubauer is
subdivided into nine 1×1mm squares.
 Each square is further divided into 16, 100, or 400 smaller
squares. The various grids allow you to count cells of different
sizes.
Steps:
 You can ensure your sample is representative by resuspending the solution
by pipetting up and down a few times before taking your sample.
 If you want to determine the viability of your cells as well as the total cell
count, you can use a dye exclusion test. This involves staining your cells
with a dye that can differentiate between viable and non-viable cells.
 Some commonly used stains are trypan blue, propidium iodide,
erythrosine B, acridine orange, and 4′,6-diamidino-2-phenylindole
dihydrochloride (DAPI).
 If there are too many or too few cells in your counting chamber, this will
impact your cell count.
 The recommended cell concentration for counting with a hemocytometer
is around 106 cells/ml.
 As a result, you may need to dilute or concentrate your sample before
counting.
 Clean the hemocytometer and the cover glass with ethanol. Ensure the
ethanol evaporates completely so it does not affect your cells.
 Place the cover glass on top of the hemocytometer’s chambers to stop
your sample from evaporating.
 Load 10 µl of your stained sample into one or both counting chambers
with a micropipette. Capillary action ensures even distribution of the
suspension within the chamber.
 Place the hemocytometer under the microscope.
 Adjust the microscope’s focus until you can clearly see the cells.
 Count the cells using a tally counter. Keep track of both the total
number of cells and the number of dead cells.
 When you are finished, clean the hemocytometer and the cover glass
with ethanol.
 A common and representative approach is to count the
cells in the four corner squares and the middle square of
the haemocytometer’s grid. This is called a ‘logical count.’

 Once you have counted your cells, you can use your
cell count and your dilution factor to determine the
total cell count or concentration in your original
sample using the following formula:
 Each of the nine squares in the Improved Neubauer grid has a volume of 0.1
mm3.
 The multiplication factor of 104 in the formula above converts the count from
cells per 0.1 mm3 to cells per ml.
 Most hemocytometer squares have a volume of 0.1 mm 3, so the
multiplication factor will be 10 4 in most cases.
 The table to the left shows the multiplication factors for other counting
chambers.
 When you know the total number of
cells and the number of dead cells in
your sample, you can calculate cell
viability using the following formula:
Cell viability by Evans blue stain
 More specifically, Evan’s blue dye can penetrate through
ruptured or destabilized membranes and stain cells.
 Thus, when plant cells are subjected to stress that
compromises membrane integrity, the number of cells that
are permeated by Evan’s blue dye will be increased
compared to control cells that are not stressed.
 In contrast, live, healthy cells that are capable of
maintaining membrane integrity do not take up Evan’s blue
dye.
 Cells that have taken up Evan’s blue dye will have an
accumulation of a blue protoplasmic stain and these
stained cells can be qualitatively documented under bright
field microscopy with or without the use of a camera.

Cell sorting (FACS- Fluorescence-

activated cell sorting)
 Fluorescence-activated cell sorting (FACS) is a specialized type of flow
cytometry.
 It provides a method for sorting a heterogeneous mixture of biological
cells into two or more containers, one cell at a time, based upon the
specific light scattering and fluorescent characteristics of each cell.
 Interests are first labelled with an antibody which is individual for a
particular cell surface molecule.
 Antibody is coupled to a fluorescent dye, like when in a narrow stream
the individual cells pass a laser beam in single file, the fluorescence of
each cell is measured.
 A vibrating nozzle then forms small droplets which each containing a
single cell which are given a negative or positive charge based on
whether the cell they contain is fluorescing.
 A strong electric field defects the various charged droplets into separate
containers so that each container has a homogeneous population of cells
eventually with respect to the cell surface molecule tagged along
fluorescent antibody.

Types of media:
 Culture media is a gel or liquid that contains nutrients and is used to grow
different cell types.
Types of Culture Media
The culture media are classified in many different ways:
❑ Based on the physical state
 Liquid media
 Solid media
 Semisolid media
❑ Based on the presence or absence of oxygen
 Anaerobic media
 Aerobic media
❑ Based on nutritional factors
 Synthetic/ defined
 Semi-synthetic/ differential
 Complex/ natural
Synthetic/defined media:
 Such media are composed of the substances that are chemically known.
 These media are very useful in studying the physiology, metabolic nature
and nutritional requirements of microbes.
 Both autotrophs and heterotrophs can be grown in these media.
 Examples- Mineral glucose medium, Richard's solution, Raulins medium
etc.

Semi- Synthetic/differential
 Semi-synthetic: Culture media, the chemical
components of which are partially known and partially
obscure are termed as semisynthetic culture media.
 Examples- Potato dextrose agar (PDA), Czapek-Dox agar,
oat meal agar (OMA), corn meal agar (CMA), beef
peptone agar and nutrient agar.
Potato Dextrose Agar
 Potato 200g
 Dextrose 20g
 Agar Agar 20g
 Distilled Water 1 lit
Complex/ natural medium
 Natural medium: Culture media of which,
the exact chemical composition is not
known is called natural or complex
culture media.
 Examples- Milk, urine, diluted blood,
vegetable juices, meat extracts, beef and
tomato juice, blood etc.

Preparation of media: microbial media-

LB [Luria-Bertani Medium] agar
 LB Broth , also known as LB Medium, Lysogenia Broth, Luria
Broth or Luria-Bertani medium, is a nutrient-rich medium commonly
used for the cultivation of bacteria.
 Making 1 L of LB Agar:
 5 g Yeast extract
 10 g Peptone
 10 g NaCl
 12 g Agar
 Mix this with 1 L of Distilled H2O
 Autoclave at 121 °C for 15 minutes.
 Pouring into plates to solidify.
Preparation of media: microbial media-
LB broth
 Making 1 L of LB Broth:
 25 g pre-mixed broth powder
 *This powder contains 10 g peptone, 5 g yeast
extract and 10 g NaCl*
 Mix and dissolve them completely.
 Pour them into the final containers (eg. conical
flask)
 Sterilize by autoclaving at 121°C for 15
minutes.

Plant media-MS [Murashige and Skoog

Media]
 MS or Murashige and Skoog Media is a nutrient
media used for the in vitro culture of plant
cells, tissues, and organs.
 It was first developed in 1962 by Toshio
Murashige and Folke Skoog and has since
become one of the most widely used media for
plant tissue culture.
 It contains a balanced mixture of
macronutrients, micronutrients, vitamins, and
plant growth regulators to provide optimal
conditions for the proper growth and
development of plants.
 MS media components:
 Macronutrient: The macronutrients in MS medium
are the primary elements that plants need in large
amounts for their growth and development.
 They include nitrogen (N), phosphorus (P),
potassium (K), calcium (Ca), magnesium (Mg), and
sulfur (S).
 These macronutrients are provided in the form of
inorganic salts, such as ammonium nitrate
(NH4NO3), potassium phosphate (KH2PO4), calcium
chloride (CaCl2), magnesium sulfate (MgSO4), and
potassium sulfate (K2SO4).

MS media components:
 Micronutrient: The micronutrients in MS medium are
trace elements that are required in smaller amounts for
plant growth and development.
 They include iron (Fe), manganese (Mn), zinc (Zn), copper
(Cu), boron (B), and molybdenum (Mo).
 These micronutrients are provided in the form of various
salts, such as iron sulfate (FeSO4), manganese sulfate
(MnSO4), zinc sulfate (ZnSO4), copper sulfate (CuSO4),
boric acid (H3BO3), and sodium molybdate (Na2MoO4).
 Vitamins: The vitamins added to the MS medium include
thiamine (vitamin B1), pyridoxine (vitamin B6), nicotinic
acid (vitamin B3), and riboflavin (vitamin B2).
 Supplements Of MS Medium
 Other than macronutrients, micronutrients, and vitamins, plants need other
supplements as well to grow properly in the lab environment. It includes:
 Sugar: Sucrose acts as a carbon source for plants. It avails the energy plants
need to grow and develop.
 Solidifying agent: Two most commonly used solidifying agents used in tissue
culture medium are: agar and gellan gum. They provide solid support and
supply nutrients for the growth of in vitro plants. They are also important for
maintaining the pH and osmotic balance of the medium.
 Plant Growth Regulators: These are also known as plant growth hormones or
hormones. The five classes of hormones naturally found in plants include:
auxin, cytokinin, gibberellic acid, abscisic acid, and ethylene. However,
normally only auxin, cytokinin, and gibberellic acids are only widely used in
tissue culture applications. Cytokinins are added to promote cell division and
shoot formation, while auxins can be added to promote root formation.
Gibberellins are plant growth regulators that facilitate cell elongation, help the
plants to grow taller. They also play major roles in germination, elongation of
the stem, fruit ripening and flowering.

Supplements Of MS Medium
 Antibiotics or PPM (Plant Preservative Mixture): Contamination is the major
challenge in the tissue culture lab. Thus, you need to have something that can
fight those tiny organisms that we can’t see invading our plants through our
naked eyes. PPM in the media provide plants an all-round protection from all
types of contamination, such as airborne, waterborne, or those coming from
other sources.
 Plant Extracts: Some researchers use plant extracts such as coconut milk,
tomato juice, and banana pulp can be added to the MS medium to provide
additional nutrients and growth factors for plant tissue culture. They boost the
growth of plants or help in reducing the time plant cells take to start dividing
in vitro environments.
 pH indicators: Phenol red (Change colour from yellow below pH 6.8 to bright
pink above pH 8.2) or Bromothymol blue (yellow in acidic solution, blue in
basic solution and green in neutral solution) can be added to the MS medium to
monitor changes in pH during plant tissue culture. However, they are rarely
used.
Plants media-Whites media

 The medium was developed by P. R. White In 1963 for the

establishment of the root culture of tomato.
 This was the earliest plant tissue culture media developed
for root culture.
 It has a lower salt concentration and a higher concentration
of MgSO4.(Magnesium sulphate)
 The concentration of nitrate is 19% lesser than the MS
media.

Animal media- RPMI [Roswell Park

Memorial Institute]
 RPMI simply known as RPMI medium, is a cell culture medium commonly
used to culture mammalian cells.
 RPMI 1640 was developed by George E. Moore, Robert E. Gerner, and H.
Addison Franklin in 1966 at Roswell Park Comprehensive Cancer
Center (formerly known as Roswell Park Memorial Institute), from where
it derives its name.
 This medium contains a great deal of phosphate, amino
acids and vitamins. Normally, the medium contains no proteins or
growth factors, so it is commonly supplemented with 10% fetal bovine
serum.
 RPMI allows the cultivation of many cell types, especially human
lymphocytes, Jurkat cells, HeLa cells, bone marrow
cells, hybridomas and carcinomas.
Typically, one liter of RPMI 1640 contains:
 Glucose (2 g)
 A pH indicator (phenol red, 5 mg)
 Salts (6 g sodium chloride, 2 g sodium bicarbonate, 1.512 g disodium phosphate,
400 mg potassium chloride, 100 mg magnesium sulfate, and 100 mg calcium
nitrate)
 Amino acids (300 mg glutamine; 200 mg arginine; 50 mg each asparagine, cystine,
leucine, and isoleucine; 40 mg lysine hydrochloride; 30 mg serine; 20 mg each
aspartic acid, glutamic acid, hydroxyproline, proline, threonine, tyrosine, and
valine; 15 mg each histidine, methionine, and phenylalanine; 10 mg glycine; 5 mg
tryptophan; and 1 mg reduced glutathione)
 Vitamins (35 mg i-inositol; 3 mg choline chloride; 1 mg each para-aminobenzoic
acid, folic acid, nicotinamide, pyridoxine hydrochloride, and thiamine
hydrochloride; 0.25 mg calcium pantothenate; 0.2 mg each biotin and riboflavin;
and 0.005 mg cyanocobalamin)

DMEM (Dulbecco’s Modified Eagle’s Medium

 DMEM (Dulbecco's Modified Eagle Medium) is a widely used basal
medium for supporting the growth of many different mammalian
cells.
 Cells successfully cultured in DMEM include primary fibroblasts,
neurons, glial cells, HUVECs, and smooth muscle cells, as well as
cell lines such as HeLa, 293, Cos-7, and PC-12.
 DMEM has almost twice the concentration of amino acids and four
times the amount of vitamins as EMEM (Eagles Minimal essential
Medium).
 Dulbecco's Modified Eagle's Medium (DMEM) is modified to
contain 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium
pyruvate, and 1500 mg/L sodium bicarbonate. It has applications
for cell culture, cell growth, and viability.
FBS [Fetal Bovine Serum]
 Fetal bovine serum (FBS) is a byproduct of harvesting
cattle for the meatpacking industry.
 It is the liquid fraction of clotted blood from fetal calf.
It has no cells, fibrin, and clotting factors but contains
large number of nutritional and macromolecular
factors essential for cell growth.
 Bovine serum albumin is the major component of the
FBS.
 It also contains small molecules like amino acid,
sugars, lipids and hormones.
 FBS is used in cell culture of eukaryotic cells, and has
a wide range of applications.