Title: Preparation of Phosphate Buffered Saline and Tissue Culture Media

INTRODUCTION: Phosphate Buffered Saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer's phosphate groups help to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

PBS has many uses because it is isotonic and non-toxic to cells. These uses include substance dilution and cell container rinsing. PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. For these types of applications, Good's buffers are recommended.

There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium. The simplest way to prepare a PBS solution is to use PBS buffer tablets. They are formulated to give a ready to use PBS solution upon dissolution in a specified quantity of distilled water. They are available in the standard volumes: 100, 200, 500 and 1000 mL.

If used in cell culturing, the solution can be dispensed into aliquots and sterilized by autoclaving (20 min, 121C, liquid cycle). Sterilization may not be necessary depending on its use. PBS can be stored at room temperature, but may warrant refrigeration to prevent bacterial growth if the solution is not sterile and is kept for long periods of time. However, concentrated stock solutions mayprecipitate when cooled and should be kept at room temperature until precipitate has completely dissolved before use.

OBJECTIVES:

To introduce students with materials and techniques involve in preparation of phosphate buffered saline and tissue culture media

To introduce students with sterilization techniques involve in preparation of phosphate buffered saline and tissue culture media

A) PREPARATION OF PHOSPHATE BUFFERED SALINE (PBS)

MATERIALS:

1) Sterilized 250 ml bottles 2) Magnetic bar and stirrer 3) Petri dish (small) 4) Chemicals: i) NaCl ii) KNO3 iii) KH2PO4 (8.00g) (0.20g) (1.44g)

iv) NaH2PO4 (0.24g) v) Phenol red (0.1g) 5) Distilled water

METHODOLOGY:

1) 200 ml of phosphate buffer saline (PBS) is prepared. Based on the given value of chemicals for preparation of PBS in 1000 ml, the quantity of each chemical in order to prepare 200 ml PBS is calculated. 2) All the chemicals in a 250 ml bottle is dissolved with distilled water and stirred on a magnetic stirrer. 3) pH of the solution is adjusted to 7.4. 4) 5 ml of PBS is transferred into a petri dish. The remaining solution is steriled by autoclaving at 121C for 20 mins. 5) After finish the autoclaving, the PBS is let cool in room temperature. 6) 5 ml of sterile PBS is transfered into a petri dish. The petri dish in 37C is is put incubator. 7) The remaining PBS in a chiller/refrigerator is kept at 4C.

B) PREPARATION OF TISSUE CULTURE MEDIA

MATERIALS:

1) Filtration unit 2) Serological pipettes 3) Tissue culture flask 4) Dulbeccos Modified Eagle Medium (DMEM) in powder form 5) NaHCO3 (3.7g) 6) Distilled water 7) Antibiotics (penicillin-streptomycin)

METHODOLOGY:

1) The DMEM powder + NaHCO3 is dissolved in distilled water. 2) Using a magnetic bar and magnetic stirrer, the mixture is stirred until fully dissolve. 3) pH of media is adjusted between to 7.2. 5 ml of media is transfered into a petri dish. 4) The media (200 ml) is filtered using a filtration unit. Another 5 ml of media is transfered into a petri dish. 5) After filter, 1 ml of antibiotics (penicillin-streptomycin) is added. 6) The sterility of media is checked. 5 ml of filtered media is transfered into a petri dish. 8) The filtered media is kept in a refrigerator at 4C. 7) All the media (in petri dish) is incubated at 37C incubator and observed for the presence of contamination after 3 days incubation.

RESULTS:

a) Observations of prepared PBS Sample PBS Non sterile PBS Magnification 400 x Observations

Sterile PBS

400 x

b) Observations of prepared tissue culture media

Type of media Unfiltered media

Antibiotics No

Observations (400 x)

Filtered media wihout antibiotics

No

Filtered media + antibiotics

Yes

DISCUSSION:

1) Describe the purpose of preparation of phosphate buffer saline (PBS) in animal tissue culture procedure. Phosphate-buffered saline provides buffer so that the pH stays approximately constant and just as many ions per unit volume as the inside of a cell, so that the cells don't swell or shrink.

2) State the purpose of adding phenol red in the PBS. Phenol red act as a pH indicator which allows constant monitoring of pH. During the cell growth, the medium changes color as pH is changed due to metabolites released by the cells. At low pH, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple. Medium should be bright red for pH 7.4 which is optimum for cell culture work.

3) Describe two types of sterilization techniques for preparation of buffer and media.

Heat sterilization

A widely used method for heat sterilization is the autoclave.Autoclaves commonly use steam heated to 121134 C (250273 F).Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature (unnecessary in machines that grind the contents prior to sterilization). Following sterilization, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released.Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions. Advantages of heat sterilization are it is good penetration and maintains integrity of liquids (e.g. Lubricants) due to the 100% humidity within the chamber.

Chemical sterilization

Chemicals are also used for sterilization. Low temperature gas sterilizers function by exposing the articles to be sterilized to high concentrations (typically 5 - 10% v/v) of very reactive gases (alkylating agents such as ethylene oxide, and oxidizing agents such as hydrogen peroxide and ozone). Liquid sterilants and high disinfectants typically include oxidizing agents such as hydrogen peroxide and peracetic acid and aldehydes such as glutaraldehyde and more recently o-phthalaldehyde. While the use of gas and liquid chemical sterilants/high level disinfectants avoids the problem of heat damage, users must ensure that article to be sterilized is chemically compatible with the sterilant being used. The manufacturer of the article can provide specific information regarding compatible sterilants. In addition, the use of chemical sterilants poses new challenges for workplace safety. The chemicals used as sterilants are designed to destroy a wide range of pathogens.

4) Describe the function of tissue culture media and the main constituents of complete media The function of tissue culture media is to allow the growth of cell culture. These media are designed to give appropriate nurients to the organism for their development. the various parts as carbohydrates(usually a sugar glucose or fructose or complex media), amino acids, various salts etc are present.

The main constituent of complete media are amino acid,vitamins,salts,glucose,organic supplement,hormons and growth factor and antibiotics.Amino acids are essential amino acid (amino acid which are not synthesized in the body)together with tyrosine and cysteine.Other non-essential amino acid are added as well.Glutamine is required by most cells as source of energy.

Many vitamins are essential for growth and proliferation of cells. Vitamins cannot be synthesized in sufficient quantities by cells and are therefore important supplements required in tissue culture. Again serum is the major source of vitamins in cell culture, however, media are also enriched with different vitamins making them suitable for a particular cell line. The B group vitamins are most commonly added for growth stimulation.Hormones and growth factors frequently added in serum-free media .It

replace serum.Example heparin-binding growth factor.Inorganic salt in the media help to retain the osmotic balance of the cells and help in regulating membrane potential by providing sodium, potassium and calcium ion.Glucose included in most of media as source of energy.Antibiotics are often used to control the growth of bacterial and fungal contaminants.Example,penicillin G for activity agaist bacteria gram positive and streptomycin against bacteria gram positive and gram negative

Media usully supplemented with serum.The sera used most in tissue culture are calf(bovine),fetal bovine,horse and human serum.The major component of serum is protein.Serum also contains growth factor,inhibitors and hormones,lipid and minerals.Example of protein in serum are alumin which is a carrier of lipids or mineral and globulins promotes cel attachment.Before use,serum is heat inactivated by incubating it for 30 mn at 56 celcius.Serum is inactivated in order to inactivate the complements( a substance in blood). Serum is an important component because it is a universal growth supplement,complex mixture of components and absolutely essential for cell growth and proliferation.It increase viscosity of medium thereby protecting the cells from shear.Seru bind and neutrilises toxin.

5) Explain why a tissue culture media should be supplemented with NaHCO3. Sodium bicarbonate is act as buffer to maintain physiological pH 7.2 - 7.4 in a culture environment. This buffer improves the pH control of media incubated in a 5-10% CO2 atmosphere and provides cells with carbonate ions that are essential for the metabolic functions of most cells. For cells that grow rapidly, only media with a high bicarbonate content are apt or a corresponding addition of sodium bicarbonate to bicarbonate-free media is necessary, if the cell culture is performed in a CO2 incubator. The required CO2 concentration is related to the desired pH-value and the NaHCO3 concentration of the medium.

6) Describe the importance of adding antibiotics to the tissue culture media. Antibiotics are added to cell culture media as a prophylactic to prevent contamination, as a cure once contamination is found, to induce the expression of recombinant proteins, or to maintain selective pressure on trasfected DNA.Example of antibiotics that commonly used are penicillin-G for activity agaist bacteria gram positive.It distrup cell wall integrity.Streomycin against bacteria gram positive and gram negative.It distrup protein synthesis.kanamycin against bacteria gram positive and gram negative,myoplasma.

7) Describe any findings on contaminants present in both non-sterilized or sterilized PBS and tissue culture media. Discuss about your findings and your expected results.

CONCLUSION: Phosphate buffered saline (PBS) is a balanced salt solution commonly used in the biolaboratory .The essential function of balanced salt solution is to maintain the ph and osmotic balance as well as provide cells with water and essential inorganic ions. Culture media is the media in which necessary elements for the growth of cells or organism.

these media are designed to give appropriate nurients to the organism for their development. the various parts as carbohydrates(usually a sugar glucose or fructose or complex media), amino acids, various salts etc are present.A Culture medium can be prepared on solid or liquid media form with nutrient organisms. All media prepared should be sterilized according to instruction given for each type of media. PH adjustment should be correct for all media.

REFERENCES: Phosphate buffered saline in wikipedia,the free encyclopedia.Retrived April 21,2013,from http://en.wikipedia.org/wiki/phsphate_buffered_saline. Retrieved( April 21,2013) from http://www.atlantabio.com/catalog/antibiotics-supplements&-reagents/biological-buffers/sodium-bicarbonate-powder

Retrived April 22,2013 from http://www.atcc.org/support/faqs/216ac/Adding%2Bantibiotics%2Bor%2Bantimycotics%2Bt o%2Bcell%2Bculture%2Bmedium-79.aspx