| Solutions Enzyme Solution 1. The enzyme solution is made up by adding 500mg of pectinase and 500mg of cellulase to 10cc of distilled water to which six drops of 1N hydrochloric acid are added. This solution should not be used for 24 hours after being made, but it will keep for one to two months in the refrigerator. Tubes containing the enzyme may be reused but should be kept corked and at about 2C when not in use. Barium Hydroxide : 1. 100cc distilled water+about 5 gm. barium hydroxide till solution is saturated. Shake vigorously while preparing. 2. Use fresh solution every time. 3. Keep bottle well stoppered. 2x SSC (saline sodium citrate) : 1. 8.716gm sodium chloride + 4.410gm sodium citrate + 6 drops 1N hydrochloric acid+500cc distilled water. 2. Heat to 60C before using. 3. Stock solution keeps about two weeks at 2C . Giemsa Stock Solution : 1. 1 gm Giemsa powder + 66cc glycerin + 66cc methanol. 2. Dissolve Giemsa powder in the glycerin at 60C for one hour with constant stirring. 3. Add methanol and continue stirring at 60C for one day (24 hours). 4. Keep refrigerated when hot in use. 5. Stock solution will keep one or two months. Citraie Buffer : 1. 2.1gm citric acid + 100cc distilled water=A. 14.2gm sodium phosphate + 500cc distilled water=B. 2. Mix 4.55cc of A with 15.45cc of B to prepare stock citrate buffer. Giemsa Staining Solution : 1. 5cc filtered Giemsa stock solution + 1.5cc methanol + 1.5cc stock citrate buffer + 60cc distilled water. 2. Before using skim the top of the staining solution. Sources of Chemicals : 1. Cellulase (2000 units/gn.) - E. Merck, catalog number 2329. 2. Pectinase (1.1 units/gm., polygalacturonase) - Sigma Chemical Company, catalog number P-4625. 3. Giemsa powder - Fisher Chemical Company, catalog number G-146. 4. The soruce of all other chemicals did not prove to be critical. |
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