Dehydrtating agents and clearing agents

LABORATORY TECHNIQUES

1. LABORATORY TECHNIQUES

2. DEHYDRATING AGENTS CLEARING AGENTS

3. Water will not mix with usual media in which stained and sectioned materials may be finally preserved. The purpose dehydration is therefore, to remove all traces of water from the cells and tissues before finally mounting. Dehydration : treating the material with the series of solutions containing progressively decreasing concentration of water and progressively increasing concentration of the dehydrant.

4. The material should not be allowed to become dry at any stage . Long interval in long concentrations make soft tissues Long interval in high concentrations make materials brittle. In the last but one grade of dehydrant,a very small quantity of eosin powder may be added to colour the material dehydrated for embedding in paraffin, this helps in locating materials.

5. REAGENTS IN DEHYDRATION Some are water removing Others are both water removers and solvents for paraffin.

6. DEHYDRATING AGENTS Ethyl alcohol Most commonly used. Isopropyl alcohol is used instead of this ,it is cheap and easily produced Methyl alcohol rarely used with plant materials due to high toxicity. Drastic dehydrating action that damage structures.

7. Strength of alcohol 60,70,80 percent alcohol by volume ,to be stored in glass stopper reagent-bottles ,next in this series will be 95 % and absolute alcohol. N ormally the time for which materials are left in each grade shall be 30 minutes for root tips and small pieces of leaf ,12 hours for large blocks of wood Up to 70% and 1 hr and 24hr respectively for 80,95,& 100% .

8. Acetone The series to be prepared with distilled H 2 O are 75 ,10,15,20,25,30,35,40,50,60,70,80,90,100 % Intervals can be less than one hour in each grade The procedure is exactly the same as with ethyl or isopropyl alcohol.

9. GLYCERINE It is a thick, colorless, sweet tasting liquid and is an excellent reagent for dehydrating delicate materials like filamentous algae and fungi High boiling point Water can be removed by evaporation. Material is washed well and transferred to a large volume of 5% glycerin take in a wide mouthed jar Leave this jar open but well protected from dust in a room until the water evaporates leaving the material covered by glycerin

10. BUTYL ALCOHOL AND TERTIARY BUTYL ALCOHOL Have additional advantage that they can be directly followed by paraffin impregnation since they are also solvents of paraffin.

11. Different grades are prepared in combination with ethyl alcohol and are tabulated below. SERIES NO ETHYL ALCOHOL 95% IN ml NORMAL BUTYL ALCOHOL IN ml DISTILLED WATER IN ml 1 20 10 70 2 25 15 60 3 30 25 45 4 30 40 30 5 25 55 20 6 20 70 10 7 15 85

12. SERIES NO ABSOLUTE ALCOHOL IN ml 95%ETHYL ALCOHOL TERTIARY BUTYL ALCOHOL IN ml DISTILLED WATER IN ml 1 50 10 40 2 50 20 30 3 50 35 15 4 50 50 5 25 75 The series with tertiary butyl alcohol

13. DIOXANE DIOXAN (diethylene dioxide) Does not require any other reagents to be mixed for dehydration. No drastic plasmolysing effect Rapid penetration capacity Does not make tissue brittle Reduces the number of separate steps in the process of dehydration

14. The following series are recommented for materials washed in water fixing. Dioxan series 1 SERIES NO DIOXAN IN ml DISTILLED WATER IN ml DURATION IN EACH IN hr 1 35 65 4-12 2 60 35 4-12 3 100 4-12 SERIES NO DIOXANE IN ml DISTILLED WATER IN ml DURATION IN EACH IN hr 1 50 50 4-12 2 65 35 4-12 3 100 4-12

15. Clearing or dealcoholization Transfer of materials after dehydration in the reagents that are not solvents of wax The clearing action is merely incidental to the function of the reagent to serve as solvent wax.

16. XYLENE (xylol) a conventional reagent Used for dealcoholization before materials transferred to solvents of paraffin Trichloroethylene is a good substitute for xylene ,it is non toxic and non-inflammable.

17. Xylene/ trichloroethylene series for large pieces of tissue SERIES NO ETHYL ALCOHOLIN ml XYLENE /TRICHLOROETHYLENE IN ml DURATION IN HOUR 1 75 25 3 2 50 50 3 3 25 75 3 4 100 3

18. Xylene/ trichloroethylene for cytological work SERIES NO ETHYL ALCOHOL IN ml XYLENE/TRICHLOROETHYLENE IN ML DURATION IN min 1 90 10 30 2 80 20 30 3 70 30 30 4 60 40 30 5 50 50 30 6 40 60 30 7 30 70 30 8 20 80 30 9 10 90 30 10 100 30

19. CHLOROFORM Does not make tissue brittle Chloroform/benzene series. SERIAL NO ETHYL ALCOHOL CHLOROFORM IN ml DURATION IN Hr 1 65 35 ½-3 2 35 65 ½-3 3 100 ½-3 Benzene is a good substitute in this series Chloroform and benzene vapors are inflammable and harmful in inhale too much.

20. CEDAR OIL Used like xylol. Easy to obtain but clearing action is low. CLOVE OIL Obtain from syzygium aromaticum. Eugenol is the major constituent. Used to clear sections in the slides before mounting. Give better transparency to tissues & hence visible clearly .

21. BEECH WOOD CREOSOTE fragile materials are dehydrated using creosote from 80% alcohol 2 changes are enough to have complete dehydration Later mounting is done using balsam . It has method of clearing. Counter staining can also be done Fern prothallus ,sections of bryophyte thallus etc…are double stained properly using creosote method.