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.
 Normally 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 H2O 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 0

12. SERIES NO ABSOLUTE
ALCOHOL IN
ml
95%ETHYL
ALCOHOL
TERTIARY
BUTYL
ALCOHOL IN
ml
DISTILLED
WATER IN ml
1 0 50 10 40
2 0 50 20 30
3 0 50 35 15
4 0 50 50 0
5 25 0 75 0
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 0 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 0 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
ALCOHOLI
N ml
XYLENE
/TRICHLOR
OETHYLEN
E IN ml
DURATION
IN HOUR
1 75 25 3
2 50 50 3
3 25 75 3
4 0 100 3

18.  Xylene/ trichloroethylene for cytological work
SERIES NO ETHYL
ALCOHOL
IN ml
XYLENE/TR
ICHLOROE
THYLENE
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 0 100 30

19.  CHLOROFORM
 Does not make tissue brittle
 Chloroform/benzene series.
SERIAL NO ETHYL
ALCOHOL
CHLOROFO
RM IN ml
DURATION
IN Hr
1 65 35 ½-3
2 35 65 ½-3
3 0 100 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.