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Introduction:

Animal cell culture can be described as in vitro maintenance and propagation of animal cells using a suitable nutrient media. Culturing is a process of growing animal cells artificially. The most important and essential step in animal cell culture is selecting appropriate growth medium for invitro cultivation. The selection of the medium depends on the type of cells to be cultured and also the purpose of the culture. Purpose of animal cell culture can be growth, differentiation, or even production of desired products like pharmaceutical compounds. Animal cells are cultured using a completely natural media, or an artificial media along with some of the natural products.

Natural Media:
In the early years of this in vitro cultivation of animal cell culture technique natural media are obtained from biological sources were used. For example 1. Body fluid such as plasma, serum, lymph, amniotic fluid and much more are used. These fluids used as animal cell culture media after testing for toxicity and sterility. 2. Tissue extract such as extract of liver, spleen, bone marrow and leucocytes also used as animal cell culture media. But most commonly used tissue extract is from chick embryo. 3. Plasma clots are also used as media for animal cell culture and now they are commercially produced as culture media. 4. Bovine embryo extract are also prepared using bovine embryos of up to 10days age, and are used as animal cell culture media

Artificial Media:
1. The artificial media contains partly or fully defined components. 2. The basic criteria for choosing a artificial media for animal cell culture are that culture media should provide all the required nutrients to the cell. 3. Media should maintain the physiological pH at around 7 with the help of buffering system. 4. The animal cell culture media should be sterile, and isotonic to the culturing cells. The basis for the animal cell culture media is the balanced salt solution, which are used to create a physiological pH and osmolarity required to maintain the animal cells in vitro or in laboratory conditions. 5. For promoting cell growth and proliferation, many types of animal cell culture media are designed by adding or varying different constituents. For example serum containing media and serum-free media.

SERUM MEDIA

. Serum media is an example for natural media. Natural media are very useful and convinient for a wide range of animal cell culture. But they also have got some disadvantages such as poor reproductability due to lack of knowledge of exact composition of these natural media.
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2. Major reasons for using synthetic media are for immediate survival of cells, for prolonged survival, for indefinite growth and also for specialized functions. a. Balanced diet solution with specific osmotic pressure and pH are used for the immediate survival b. Serum or balanced salt solution along with amino acids, oxygen, vitamins and serum proteins are used for long survival. c. Minimum Essential Medium also known as Eagles media are used for mammalian cell culture d. Role of serum in animal cell culture is very complex and also it contains mixture of many bimolecule such as growth factors and also growth inhibitory factors.

Function of serum:
1. Serum is bound to proteins and basic nutrient in solution. 2. Serum also contains hormones and growth factors, which play a major role in stimulating cell growth and function 3. Serum also helps in attachment of the cells 4. Serum also acts as spreading factor. 5. Serum also function as binding protein such as albumin carrying factor 6. Serum also helps in carrying hormones, vitamins, minerals, lipids and much more biological substances. 7. Serum also minimizes mechanical damage and also damage caused by viscosity 8. Serum also acts as natural buffering agent and helps in maintaining the pH of the culture media.

Disadvantage of Serum in Media:


1. Serum may contain inadequate amount of cell specific growth factors and may need to suppliment the media or it may also contain in abundance of cytotoxic compounds. 2. Serum media has got high risk of contamination with virus, fumgi and mycoplasma. 3. There is no uniformity in the composition of serum, as still we do not know the exact composition of the serum 4. Special tests are done to maintain the quality of the each batch of serum before it is used in cell culture media. 5. Serum may also contain some of the growth inhibiting factors, these in turn will inhibit the cultured cell growth and proliferation. 6. Serum availability is restricted as they are extracted from cattle 7. Presence of serum in the culture media may interfere with the purification and isolation of cell culture products, such as pharmaceutical compounds. Due to this additional steps are added for the isolation of cell culture products.

Serum Free Media:


As using serum in animal cell culture media has got some disadvantages, to overcome this serum free media are designed and developed. Advantages of Serum Free Media: 1. Main and important advantage of serum free media is scientists or researchers can control the growth of cultured cells as required by changing the composition of the media. 2. Serum free media can be designed using specific factors, which will help in differentiation of cultured cells with specific desired functions. Disadvantages of Serum Free Media: 1. cell proliferation is very slow in serum free media. 2. Cultured cells may need more than one type of media to obtain desired cell culture products. 3. Purity of reagents used in the serum free media 4. Availability

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y culture medium is a liquid or gel designed to support the growth of microorganisms , cells or small plants. y culture media consist of nutrients used to grow micro organism or cells outside of their natural habitats.

Basic requirements of culture media


Energy source , (carbon source,nitrogen source) Mineral salts ( sulphates phosphates and chlorides of K, Mg nd Ca ) pH - 7.2 to 7.4 Accessory growth factors ( e.g , tryptophan for salmonella typhi )

Agar agar : most important constituents of culture media


Agar- agar: It is used as solidifing agent , contains long chain poly saccharides , inorganic salt and proteins , solidify at 42c Melts at 98c Generally not metabolized by microbes, Obtained from sea weed (new zealand)

Types of culture media


 Nutrient media  Minimal media  Selective media  Differential media  Enriched media  Transport media

Nutrient media
source of amino acids and nitrogen is , beef yeast or extract.  Undefined medium ( amino acid source contains a variety of compounds with the exact composition being unknown)  Nutrient media contain all the essential elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections.

Nutrient media

Minimal media
 Contains the minimum nutrients possible for colony growth, without the presence of amino acids.  It is often used by microbiologists and geneticists to grow wild type microorganisms.  Minimal medium typically contains:  a carbon source (may be glucose or a less energy-rich source like succinate)  various salts, which may vary among bacteria species and growing conditions; these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid  water

Selective media
used for the growth of only select microorganisms. (e.g, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing).  Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite.

Selective media

some examples of selective media include:


y Eosin-methylene blue agar (EMB) contains methylene blue toxic to Gram-positive bacteria, allowing only growth of Gram negative bacteria y Yeast and mold has a low pH, deterring bacterial growth y blood agar contains bovine heart blood that becomes transparent in the presence of hemolytic Streptococcus y MacConkey agar for Gram-negative bacteria y Hektoen enteric agar (HE) which is selective for Gram-negative bacteria y mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol y xylose lysine desoxyscholate , is selective for Gram-negative bacteria

Differential media
Differential media or indicator media distinguish one microorganism type from another growing on the same media.]  This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue).  Examples of differential media include: y eosin methylene blue, which is differential for lactose and sucrose fermentation y MacConkey, which is differential for lactose fermentation y X-gal plates, which are differential for lac operon mutants

Enriched media
Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients.  Chocolate agar is enriched with heat-treated blood (40-45C), which turns brown and gives the medium the color for which it is named.

Transport media
y Transport media is used fortemporary storage of specimens being transported to the laboratory for cultivation. y maintains the viability of all organisms in the specimen without altering their concentration. y contain only buffers and salt. y lack of carbon, nitrogen, and organic growth factors prevents microbial multiplication. y transport media used in the isolation of anaerobes must be free of molecular oxygen. y Example: Thioglycollate broth for strict anaerobes, Stuart transport medium-a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, Venkat Ramakrishnan medium for v. cholerae

Sterilization of culture media


Culture media are sterlized at : Temperature 121c, Pressure 15 pascals, Time 15 t0 20 minutes, media containing heat sensitive substances like serum , fatty acid ,sugars are sterlized by free steam or filtration.

Storage of culture media


Individual media is stored in screw capped bottles . Culture media is preserved at low temperature (in refrigeratirs or cold rooms) Deep freeze refrigerators are used for preservation of media containing antibiotics and amino acids ( -10 to 400c).

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