PRACTICAL 1

TITLE
Preparation of Phosphate Buffered Saline and Tissue Culture Media.

INTRODUCTION
Phosphate buffered saline (PBS) is a buffer solution commonly used in biological research. It is a salt solution containing sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The osmolarity and ion concentrations of the solutions match those of the human body (isotonic). PBS has many uses because it is isotonic and non-toxic to cells. The uses are diluting substance, rinsing container containing cells, and separate attached and clumped cells by using EDTA with PBS.

OBJECTIVES
1) Students will be able to know the materials and techniques involve in preparation of phosphate buffered saline and tissue culture media. 2) Students will learn the sterilization techniques involve in preparation of phosphate buffered saline and tissue culture media.

A) Preparation of Phosphate Buffered Saline (PBS) MATERIALS

Sterilized 250 ml bottles Magnetic bar and stirrer Petri dish (small) Chemicals: i) NaCl (1.6g) ii) KNO3 (0.04g) iii) KH2PO4 (0.288g) iv) NaH2PO4 (0.048g) v) Phenol red (0.02g) Distilled water

METHODOLOGY
1) 200 ml of phosphate buffer saline (PBS) is prepared by calculating the quantity of each chemicals based on the given value of chemicals for preparation of PBS in 1000 ml.

2) All the chemicals is dissolved in a 250 ml bottle 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 the autoclaving is completed, the PBS is cooled in room temperature. 6) 5 ml of sterile PBS is transferred into a petri dish. The petri dish is placed in 37C incubator. 7) The remaining PBS is kept in a chiller/refrigerator at 4C.

B) Preparation of tissue culture media MATERIALS

Filtration unit Serological pipettes Tissue culture flask Dulbeccos Modified Eagle Medium (DMEM) in powder form NaHCO3 (3.7g) Distilled water Antibiotics (penicillin-streptomycin)

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

DISCUSSION 1. To remove unwanted cell debris and wash the cells during subculture. 2. Act as an indicator to indicate the presence of contamination or unwanted growth of microorganisms. 3. i) heating in an autoclave at 121C for 15 minutes, which kills all living organisms, including spores. ii) filtration method that filters media solution before used. 4. To provide area for cell growth and significant component for cell proliferation and also helps to maintain pH and osmolality. The main constituents of media are vitamin, hormone, amino acids, glucose, salts, antibiotics and growth factors. 5. NaHCO3 need to be supplemented because it balances the CO3 and HCO3 in the medium and let the medium become basic very rapidly due to metabolic activity. 6. To minimize or prevent the risk of contamination by pathogenic microbes. 7. In non-sterile, there are few lines that were overgrown by microbe, whereas for sterile PBS, there are no microbe found.

CONCLUSION The preparation of phosphate buffered saline media has thought the students on the crucial components that made up a media and the lab experiment thought students to carry out experiments in sterile environment to prevent contamination.

REFERENCES 1. http://protocolonline.com/recipes/phosphate-buffered-saline-pbs/