• There are various equipments used in animal cell culture and the basic equipments required to carry out the animal cell culture are enlisted as follows:

List of Basic Equipments needed in animal cell culture lab:

1. Sterile Work Area/Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
2. Incubator (humid CO2 incubator recommended)
3. Water bath
4. Centrifuge
5. Refrigerator and freezer (–20°C)
6. Cell counter (e.g. Automated Cell Counter or hemocytometer)
7. Inverted microscope
8. Liquid nitrogen (N₂) freezer or cryostorage container
9. Sterilizer (i.e., autoclave)

1. Sterile work area required for cell culture:

• In order to maintain clean cell culture work, it is necessary to prepare a separate room or space if possible.
• This room should be devoid of traffic, and if possible it should be equipped with an air flow cabinet that provides filtered air surrounding to the work surface.
• A HEPA (High Efficiency Particle Air Filter) filtered air is appropriate but is not economical.
• The laboratory must be especially designated for clean culture work and it should be strictly restricted to culture the primary animal tissue and micro-organisms in or near the cell culture laboratory.
• The laboratory coats should be placed at the entry gate of the laboratory and should not be used outside the lab.
• A laminar flow hood (i.e. biosafety cabinet) is supposed to the simplest and the most cost effective way to supply aseptic conditions.
• While permitting the containment of infectious splashes or aerosols produced by many microbiological processes, the laminar flow hood provides an aseptic work area.
• In order to meet the diversified research and clinical needs, Three kinds of laminar flow hoods, have been designated as Class I, II and III.
• When used with proper microbiological techniques, Class I laminar flow hoods supplies essential levels of protection to laboratory workers and to the environment, but they do not protect cultures  from contamination. 
• They are identical to chemical fume hoods in design and air flow characteristics.
• For work that involves BSL-1, 2, and 3 materials, Class II laminar flow hoods are designed and they also allow an aseptic environment essential for cell culture experiments. 
•  In order to handle exclusively harmful materials (e.g., primate-derived cultures, virally infected cultures, radioisotopes, carcinogenic or toxic reagents) a Class II biosafety cabinet should be employed.
• Class III biosafety cabinets are gas-tight, and they supply the highest achievable level of protection to personnel and the environment. 
• A Class III biosafety cabinet is needed for work that involves known human pathogens and other BSL-4 materials.
• Air-Flow Characteristics of Cell Culture Hoods:
  • The working environment is protected by the laminar flow hoods from dust and other airborne contaminants by retaining a constant, unidirectional flow of HEPA-filtered air over the work area. 
  • The flow can be both horizontal, blowing parallel to the work surface, and it can be vertical, blowing onto the working surface from the top of the cabinet.
• Clean Benches:
  • Vertical laminar flow or horizontal laminar flow “clean benches” are not biosafety cabinets.
  • These pieces of equipment discharge HEPA-filtered air from the back of the cabinet across the work surface toward the personnel, and the user might be exposed to potentially hazardous materials.
  • These devices only aids product protection.

2. Incubator:

• An incubator will be needed in order to supply the suitable temperature environment for cell growth at 30-40⁰ C.
• Depending on the type of cells being cultured, the  incubation temperature will vary.
• An incubator that has been designated to permit CO2 to be supplied from a main supply or gas cylinder is needed in order to maintain an atmosphere of between 2-5% CO2 is maintained in the incubator.
• In the medium, the concentration of CO2 is kept in the equilibrium with sodium bicarbonate.
• In general, several cell lines can be retained in an atmosphere of 5% CO2: 95% air at 99% relative humidity.
• Dry incubators are relatively cost-effective, but the cell cultures are needed to be incubated in sealed flasks to avoid evaporation.
• In a dry incubator, if the water dish is placed, it can supply some humidity however, they do not provide appropriate control of atmospheric conditions in the incubator.
• Humid CO₂ incubators are relatively expensive, however it allows superior control of culture conditions.
• They can be used to incubate cells that are cultured in petri-dishes or multiwell plates that needs a regulated atmosphere of high humidity and increased CO2 tension.

3. Refrigerators and freezer (-20 °C) for specimen storage:

• Both refrigerators and freezer are very essential for storage of liquid media at 2–8°C and for enzymes (e.g. trypsin) and some media components (e.g., glutamine and serum) at –5°C to –20°C.
•  To store medium and buffers, a refrigerator or cold room is needed. 
• A freezer is required for keeping pre-aliquoted stocks of serum, nutrients and antibiotics. 
• Cryogenic Storage
  • There is high possibility for genetic instability in cell lines of continuous culture as their passage number increases, hence, it is necessary to prepare working stocks of the cells and preserve in cryogenic storage.
  • It is to be noted that the cells should not be stored in 20^oC or -80^oC freezers as their viability reduces when they are not stored at these temperatures.
  • Liquid nitrogen freezers permit storage in the vapor phase just above the liquid at temperature between -140^oC and -180^oC, or submerged in the liquid at a temperature below -196^oC.
  • The possibility of leaky vials or ampules exploding during removal is highly reduced by use of vapor phase storage, however, the liquid phase systems generally have longer static holding times, and are thus, more cost-effective.

4. Microscopes:

• In order to examine the cultures in flasks and dishes, a simple inverted microscope is needed.
• The morphological changes in cultures should be recognized as they are the first indicators for the identification of deterioration of a culture.
• Although, a microscope of very high quality will be needed for chromosome analysis or autoradiography work, a very simple light microscope with X100 magnification will suffice for routine cell counts in a hemocytometer.

5. Tissue culture ware:

• A diverse tissue culture plasticware is found, the most general being specially treated polystyrene. 
• Even if all tissue culture plasticware should support cell growth maximally, it is necessary to make sure that the new supplier facilitates the growth of cultures.
• Cells can be kept in petri dishes or flasks (25 cm2 or 75 cm2) , that have added the benefit that the flasks can be gassed and then sealed so that a CO2 incubator should not be used.
• This is especially useful in case if incubators fail.

6. Washing up and sterilizing facilities:

• Glassware such as pipettes should be immersed in a suitable detergent, then passed through a strict washing procedure with thorough soaking in distilled water prior to drying and sterilizing.
• Pipettes are often stuffed with non-absorbent cotton wool before being placed in sterilization containers.
• Glassware such as pipettes, conical flasks, beakers (covered with foil of aluminum) is sterilized for one hour in a hot air oven at 160 ° C.
• All other equipment, like automatic pipette tips and bottles (lids loosely attached) are sterilized by autoclaving at 121 °C for 20 min.

7. Water still or reverse osmosis apparatus:

• For preparation of media, and rinsing glassware, a double distilled or reverse osmosis water supply is required.
• The pH of the double distilled water should be checked regularly, as this can vary in some instances.
• Variations in the quality of water used may account for variations in outcomes, so it is necessary to use water from one source.
• Water is sterilized for 20 minutes at 121 °C by autoclaving.

8. Filter sterilization

• Media which can not be autoclaved must be sterilized through a membrane filter of 0.22 μm pore size.
• These can be obtained in different designs to filter a wide range of volumes.
• They can be bought as sterile disposable filters, or they can be sterilized in appropriate filter holders by autoclaving.

9. Centrifuge

• Periodically, to increase the concentration of cells or to wash off a reagent, cell suspensions require centrifugation.
• For most purposes, a small bench-top centrifuge, preferably with proportionally controlled braking, is enough.
• Refrigeration is not necessary, although, set at room temperature, it can be used to prevent overheating of cell samples.
• At 80 to 100 g, cells sediment satisfactorily; higher g may cause damage and encourage pellet agglutination.

Other expanded equipments and additional supplies:

• Other additional equipments and supplies needed in animal tissue culture lab are:
  • Aspiration pump (peristaltic or vacuum)
  • pH meter
  • Roller racks (for scaling up monolayer cultures)
  • Confocal microscope
  • Flow cytometer
  • Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multiwell plates)
  • Pipettes and pipettors
  • Syringes and needles
  • Waste containers
  • Media, sera, and reagents
  • Cells
  • Cell cubes

1. Aspiration pump:

• An aspirator is a form of ejector-jet pump, that creates vacuum by means of the Venturi effect.
• Fluid (liquid or gaseous) passes through a tube in an aspirator that gets narrower and then expands in the cross-sectional area and thus volume.
• The pressure of the fluid diminishes as the tube narrows.
• The most popular type of aspirator is the cheap and simple water aspirator.
• It is used in laboratories for chemistry and biology and consists of a tee fitting connected to a tap and has a hose barb on one side.
• The water flow passes through the tee’s straight portion, which at the intersection where the hose barb is attached has a restriction.
• To this barb, the vacuum hose should be attached.
• Although historically popular for low-strength vacuums used in chemical bench work, they use a lot of water, and depending on what the vacuum is being used for, i.e. removal of solvents, by mixing these potentially dangerous solvents into the water stream, they can breach environmental protection laws such as RCRA, then rinsing them down a drain which often directly leads to the municipal sewer.
• The intensity of the vacuum generated is restricted by the vapor pressure of the liquid (for water, 3.2 kPa or 0.46 psi or 32 mbar at 25^o C or 77 F) if a liquid is being used as the working fluid.
• This restriction does not exist if a gas is used.
• The industrial steam ejector (also named the ‘steam jet ejector’, ‘steam aspirator’, or ‘steam jet aspirator’) uses steam as a working fluid.

2. pH meter:

• PH meter is an electrical instrument for calculating the activity of hydrogen ions (acidity or alkalinity) in the solution.
• A pH meter comprises necessarily of a voltmeter connected to a pH-responsive electrode and a reference (unvarying) electrode.
• The pH-responsive electrode is normally glass, and a mercury-mercurous chloride (calomel) electrode is usually the reference, although sometimes a silver-silver chloride electrode is used.
• The two electrodes act like a battery when they are submerged in a solution.
• The electrical potential (charge) of the glass electrode is directly related to the hydrogen ion activity in the solution (59.2 millivolts per pH unit at 25^o C [77 °F]) and the potential difference between the glass and reference electrodes is determined by the voltmeter.

3. Confocal microscope:

• Confocal microscopy is a specialized type of standard fluorescence microscopy (also termed widefield fluorescence microscopy) that produces high-resolution images of material stained with fluorescent probes using specific optical components.

4. Flow cytometer:

• Flow cytometry is a method for cell analysis that was first used in the 1950s to determine the volume of cells in a fluid stream that circulated quickly as they flowed in front of a viewing aperture.
• Fluidics, optics and electronics are the three principal components of a flow cytometer.
• The transport of the sample from the sample tube to the flow cell is the function of the fluidic system of the flow cytometer.
• The sample is either sorted (in the case of cell sorters) or transported to waste after it has been through the flow cell (and past the laser).
• The optical system components include excitation light source, lenses, and filters used to capture and move light around the instrument and the photocurrent-generating detection system.
• The brains of the flow cytometer are the electronics.
• Here the photocurrent from the detector is digitized and analyzed to be saved for subsequent analysis.

5. Cell culture vessels:

• In order to shield cultures from the external environment while retaining the correct internal environment, culture vessels provide a contamination barrier.
• The vessels have an effective and consistent cell attachment substrate for anchorage-dependent cells.
• Simple access to cultures and optically transparent viewing surfaces are more features of vessels.
• All cultural vessels were originally glass.
• In comparison to plastic, glass drawbacks include heavy weight, cost, labor-intensive cleaning, and poor microscopic viewing.
• Surface treatment techniques were developed for polystyrene in the 1960s, enabling plastic vessels to replace glass for most applications of cell culture.
• a. Glass flasks:
  • In the 1920s, the first glass flasks were developed by Alexis Carrel.
  • The more conventional straight neck rectangular (also hexagonal) glass T-flasks were created by Harry Earle in the 1940s.
  • Today, with a number of growing areas, a variety of shapes, with several different neck designs, plastic flasks are available.

b. Cell culture dishes:

• The best economy and access to the surface of growth are provided by cell culture dishes.
• Hence, they are regarded as the vessels of choice for cloning or other manipulations such as scraping that need the direct access to cell monolayer.
• They should be used with incubators that regulate CO2 and humidity.
• The majority of manufacturers provide dishes in four sizes: 35 mm, 60 mm, 100 mm and 150 mm.
• These are nominal diameters and may not be the growth surface’s true diameter.
• Cell culture dishes are accessible for growing anchorage-dependent cells with either specially treated surfaces, or untreated (native) surfaces for growing suspension cultures where attachment is not required.

c. Roller bottles:

• To cultivate large numbers of anchorage-dependent cells, the roller bottle was created.
• Today, they suppy a more cost-effective means of growing large volumes of cells using basically the same culture techniques as with flasks, but with substantially less labor.
• In addition to the conventional smooth wall design, roller bottles are available with small ridges that roughly double the available surface area for developing cells without raising the bottle dimensions.

d. Pipettes and pipettors:

• The serological pipette is a relatively common laboratory instrument used for transferring milliliter volumes of liquid.
• In order to calculate the volume of liquid being aspirated or dispensed, serological pipettes usually have gradations along their sides.
• These instruments are most commonly used with a pipette dispenser, which enhances the liquid transfer through the development of a partial vacuum.
• Depending on the amount of volume you want to pass, the same pipette dispenser can be used with a number of serological pipette sizes.
• Serological pipettes are usually sterilizable and reusable, either plastic, sterile, and disposable or glass.
• For the transfer of fluids, all serological pipettes involve the use of a pipette dispenser.
• This primitive liquid transfer method is not recommended, as it may lead to liquid entering the oral cavity and some severe adverse side effects may be caused.
• One type of dispenser, the pipette bulb supplies the least amount of accuracy and is usually used with glass serological pipettes in order to transfer non-specific volumes of liquid.
• The pipette pump is often used for glass pipettes and enables the liquid volume to be controlled more precisely.
• For dispensing repeated volumes of solution, pipette pumps are especially useful.
• The most popular pipette dispenser type is the pipet-aid.
• It consists of several primary components: the nose cone is where the pipette is attached and where the filter is also located, which protects the inside of the pipet-aid from fluid and retains sterility.
• Two triggers can be found on the pipet-aid handle; the top trigger for aspirating liquids is depressed, the bottom for dispensing.
• Pipet-aids are also fitted with settings that monitor the speed at which fluid is dispensed.
• For example, the instrument can be fixed to dispense liquid using pressurized air, in a blow-out setting, and with no force, in a gravity setting.
• Although there are cords in some pipet-aids, most are battery operated.
• Some pipet-aids have a stand attached to the handle that allows the pipet-aid to rest on its side without removing the pipette.

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• Cell culture is the technique where cells are allowed to grow under controlled conditions, usually outside of their natural environment.
• Likewise, animal cell culture is a technique in which the cells are removed and are allowed to grow in a favorable artificial environment.
• The removal of tissue can either take place from tissue directly or from disaggregation by enzymatic or mechanical means before culture, or they may be originated from a cell line or cell strain that has been established earlier.
• Even if the basic mammalian/animal cell culture techniques are similar to those that are applied to bacteria, fungi, and yeast, there are however some characteristic differences.
• Usually, mammalian cells are found to be more delicate and more susceptible to mechanical damage.
• They have lesser growth rates and need more complex culture media along with special substrates.

Steps to culture animal cells:

• Harvest cells
• Isolation of the cells with the use of appropriate enzymes.
• In a culture dish with appropriate growth media, the cells are placed.
• The culture dish is now kept in incubator for the culture of cells.
• Cells can be sub-cultured in order to fix the problem or to get the pure culture.
• Now, cells are ready to be manipulated or modified for lab procedures.

Media Composition for animal cell culture:

• The basic components for the animal cell culture are enlisted as bellows:
• Sources of Energy: Glucose, Fructose, Amino acids
• Nitrogen sources: Amino acids
• Vitamins: Usually, water soluble vitamins B & C.
• Inorganic salts: Na+, K+, Ca²⁺, Mg²⁺
• Fat and Fat soluble components: Fatty acids, cholesterols
• Antibiotics
• Growth factors and hormones.
• Oxygen and CO₂ concentration.
• Physical environment: The physical environment consists of the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses.
• CO₂ incubators are employed and designed to resemble the environmental conditions of the living cells.
• For visualizing cell cultures in vitro, an inverted microscope is used.
• Low speed centrifuges are needed for most animal cell cultures.
• Cryopreservation is the storage of cells using liquid nitrogen at a very low temperature (-180 °C to -196 °C).
• DMSO is a cryo-preservative molecule that prevents cells from being harmed.
• For the culture of animal cells, serum is necessary and contains growth factors that promote cell proliferation.
• Even if animal cell culture media differ in their complexity, most include:
  • Amino acids:   0.1-0.2 mM
  • Vitamins:  1 microM
  •  Salts (NaCl) :  150 mM
  • KCl : 4-6 mM 
  • CaCl₂ :  1 mM
  • Glucose : 5-10 mM

Animal Culture media:

• In animal tissue culture, 2 types of culture media are used:
  • Natural media
  • Artificial media
• The type of medium relies basically on the type of cells to be cultured and its objectives.

1. Natural media:

• These media include the naturally occurring biological fluids and are of the following three types:
  • Clots
  • Biological fluids
  • Tissue extract

i) Clots:

• Plasma clots are the most commonly used clots and has been employed for a long time.
• In the present time, plasma is commercially found in liquid state that can be prepared in the laboratory.

ii) Biological Fluids:

• Several biological fluids can be employed as culture media such as amniotic fluid, pleural and ascetic fluids, hemolymph of insects, aqueous humoral from eye, serum etc.
• Among them, serum is the mostly preferred.

iii) Tissue Extracts:

• The most commonly used tissue extract is chick embryo extract, however, bovine embryo extract is also used.
• In the culture media, the extracts from spleen, liver, bone marrow and leucocytes were also used.
• The substitution for tissue extract can be a mixture of amino acids and certain other organic compounds.

2. Artificial media:

• For the following purposes, various artificial media have been employed:
• Immediate survival (a balanced salt solution with specified pH and adequate osmotic pressure)
• Prolonged survival (a balanced salt solution in addition with serum, or appropriate formulation of organic compounds.
• Indefinite growth
• Specialized functions
• Artificial media may be classified  into following types:
  • Serum containing media
  • Serum-free media
  • Protein free media
  • Chemically defined media

• Serum:
  • Serum is the yellowish liquid and is a transparent content that remains left over after the removal of fibrin and cells from the blood.
  • 2-10% of serum is often contained by normal growth media.
  • The most commonly employed supplement in animal cell culture is fetal bovine serum (FBS).
  • It supplies the basic nutrients for cells.
  • It also contains several hormones and various growth factor.
  • In addition to it, it also acts as a buffer.
• Serum containing media:
  • In animal cell culture media, fetal bovine serum is the most common supplement.
  • In order to supply an optimal culture medium, it is employed as an economical supplement.
  • Serum supplies carriers water-insoluble nutrients, protease inhibitors, hormones and growth factors and binds and neutralizes the toxic moieties.
• Serum free media:
  • In case of immunological studies, presence of serum in media can result to serious misinterpretations.
  • In general, these media are specifically designed to promote the culture of a single type of cell and incorporate specified amounts of purified growth factors, lipoproteins and other proteins normally supplied by the serum.
  • As the components of these media are known, thus it is referred to as ‘defined culture media’
• Chemically defined media:
  • These media contain ultra-pure inorganic and organic ingredients free of contaminants and may also contain pure protein additives, such as growth factors.
  • Their constituents are produced by genetic modification in bacteria or yeast with the addition of vitamins, cholesterol, particular amino acids, and fatty acids.
• Protein free media:
  • Protein-free media is devoid of any protein and only include non-protein constituents.
  • Usage of protein-free media enhances superior cell growth and protein expression in contrast to serum-supplemented media and enables downstream purification of any expressed product.

Development of animal culture media:

• Early efforts to culture cells, such as chick embryo extract, plasma, serum, and lymph, were conducted in natural media based on tissue extracts and body fluids.
• Demand for greater quantities of a medium of more reliable quality has led to the emergence of chemically defined media.
• However, Eagle’s Basal medium and subsequently Eagle’s Minimal Essential Medium (MEM) were broadly accepted, variously supplemented with human, horse or calf serum, protein hydrolysates, and embryo extract.
• In order to choose a medium, the minimum criteria required usually involve:
  • The medium should supply the cells with all of the nutrients.
  • Keep the physiological pH about 7.0 with ample buffering.
  •  The medium should be sterile, and isotonic to the cells.
  • The balanced salt solution that was initially used to establish a physiological pH and osmolarity needed to sustain cells in vitro was the basis for the cell culture media.
  • Various components (glucose, amino acids, vitamins, growth factors, antibiotics etc have been added to promote cell growth and proliferation, and several media have been created.

Physicochemical properties of culture media

• In order to promote good growth and proliferation of the cultured cells, the culture medium is required to possess certain physicochemical properties (pH, O2, CO2, buffering, osmolarity, viscosity, temperature etc.).
• pH:
  • Most cells can develop in the 7.0-7.4 pH range, but there are minor differences depending on the cell type.
  • Phenol red is usually used as an indicator.
  • It becomes orange at pH 7.0, yellow at pH 6.5, lemon yellow below pH 6.5, more pink at pH 7.6, and purple at pH 7.8. It is red at pH 7.4.
•  CO₂, bicarbonate and buffering:
  • The medium’s carbon dioxide is in a dissolved state, the concentration of which depends on the atmospheric CO₂ tension and temperature.
  • As seen below, CO2 in the medium occurs as carbonic acid (H2CO3), and as bicarbonate (HCO3-) and H+ ions.                                    

                             CO₂ + H₂O ↔ H₂CO₃ ↔ H⁺ + HCO₃^– 

• The CO2, HCO3-and pH concentrations are interrelated, as is obvious from the above equation.
• The pH would be decreased by increasing atmospheric CO2, rendering the medium acidic.
• Bicarbonate ions are neutralized by the addition of sodium bicarbonate (as a part of bicarbonate buffer).

                              NaHCO₃ ↔ Na+ + HCO₃^–

•  The recommended bicarbonate concentration and CO2 tension for the appropriate pH are found in commercially available media.
• In recent years HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer being used in the culture media as it is considered more efficient than the bicarbonate buffer.
• However, due to the low cost, less toxicity and nutritional value to the medium, bicarbonate buffer is favored.
• In contrast to it, HEPES is expensive and along with it, it is toxic to the cells.
• The existence of pyruvate in the medium results in the excessive endogenous production of CO2 by the cells.
• This is good since the reliance on the exogenous supply of CO₂ and HCO^–₃ would be lower.
• In such a scenario, high amino acid concentrations can be used for buffering.
• In summary, cultures in open vessels need to be incubated in the CO₂ atmosphere, the concentration of which is in equilibrium with that of sodium bicarbonate in the medium.
• Oxygen:
  • A large majority of in vivo cells rely on the availability of O₂ for aerobic respiration, which is made possible by hemoglobin’s continuous supply of O₂ to the tissues.
  • The cultured cells mainly depend on the dissolved O₂ in the medium that may be toxic at high concentration due to the production of free radicals.
  • It is also completely necessary to supply appropriate amounts of O₂ in order to comply with the cellular requirements, preventing toxic effects.
  • The introduction into the medium of free radical scavengers such as glutathione,2-mercaptoethanol (β-mercaptoethanol) or dithiothreitol erases the toxicity of O₂.
  • To decrease O₂ toxicity, the addition of selenium to the medium is often recommended.
  • It is because selenium is a cofactor for the synthesis of glutathione.
  • In general, as compared with in vivo cells, glycolysis occurring in cultured cells is more anaerobic.
  • As the rate of diffusion of O₂ is affected by the depth of the culture medium, it is recommended to keep the depth of the medium in the range of 2-5mm.
• Temperature:
  • In particular, the optimum temperature for a provided cell culture is reliant on the body temperature of the organism, acting as the source of the cells.
  • Therefore the optimum temperature for cells obtained from humans and warm blooded animals is 37 ° C.
  • Higher temperatures cannot be handled by in vitro cells and most of them die if the temperature goes beyond 40 ° C.
  • For reproducible outcomes, it is therefore completely important to maintain a steady temperature (± 0.5°C).
  • If the cells are collected from birds, the optimum temperature for culture is slightly higher (38.5°C).
  • The culture temperature can be about 15-25 ° C for cold blooded animals (poikilotherms) that do not control their body heat.
  • In addition to directly affecting cell growth, temperature also influences the solubility of CO2, i.e. higher temperatures increase solubility.
• Osmolality:
  • Most cultured cells have a reasonably wide osmotic pressure tolerance.
  • This is comparable to human plasma osmolality (290mOsm/kg).
  • Once an osmolality is appointed for a culture medium, it should be sustained at that level (with an allowance of ± 10 mOsm/kg).
  • Osmolality is typically determined by the depression of the medium’s freezing point or vapor pressure elevation.
  • The instrument osmometer is used in the laboratory for osmolality measurement.
  • Osmolality is influenced if acids, bases, medications, etc are applied to the medium.
  • If we make up the medium ourselves, the measurement of osmolality is a useful quality control step, as it helps to protect against errors in weighing, dilution, and the like.
  • Osmolality is usually measured by depression of the freezing point or elevation of the vapor pressure, of the medium.
• Viscosity:
  • A culture medium’s viscosity is largely affected by the serum content and will have little impact on cell growth in most cases.
  • When a cell suspension is agitated (e.g. when a suspension culture is stirred), or when cells are dissociated after trypsinization, or in low serum concentrations/absence, viscosity becomes especially important.
  • Increasing the viscosity of the medium with carboxymethylcellulose (CMC) or polyvinylpyrrolidone (PVP) will minimize any cell damage that occurs under these conditions.
•  Surface Tension and Foaming:
  • The consequences of foaming have not been clearly identified, but the rate of denaturation of protein can increase, as might the risk of contamination if the foam enters the culture vessel’s neck.
  • If a film from a foam or spill gets into the capillary space between the lid and the base of a Petri dish or between a slack cap and the neck of a flask, foaming can also restrict gaseous diffusion.
  • In suspension cultures in stirrer vessels or bioreactors, foaming can occur when 5 percent of CO2 in air is bubbled via serum containing medium.
  • The addition of 0.01 percent to 0.1 percent silicone antifoam (Dow Chemical) or Pluronic F68 (Sigma) helps avoid foaming in this situation by minimizing surface tension and can also protect cells from bubble shear stress.

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