VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

PREPARATION OF BUFFER

Materials required

• Triple ditillation water/Millipore water
• Chemicals (Tris, Citric acid monohydrate, Fructose)
• Glycerol
• Weigh the chemicals as per the requirement given above for preparation of 1 litre diluent

Buffer preparation (for 1 litre diluent)

(Click here for video demonstration)

• Take the triple distilled water in a sterile beaker according to the requirement
• Weigh the chemicals accurately
• Add the chemicals one by one and dissolve it by magnetic stirrer by required speed
• Finally add the glycerol at 7% level ie. 70 ml
• Make the volume upto 800 ml by adding distilled water
• Completely mix the glycerol by magnetic stirrer for at least 20 minutes
• Care should be taken while stirring that air bubbles should not be formed by over speed of the stirrer

The pH of buffer should be 6.8-6.9 Glycerol 7%

Sterilization of buffer

• The prepared buffer is sterilized by autoclaving at 100 ⁰ C for 15 min at 5 psi.
• Cool the buffer to room temperature
• Generally the buffer is prepared on previous day evening, sterilized and kept under laminar air flow over night.
• The diluent is prepared by adding egg yolk in the morning immediately before semen collection.

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