MACERATION AND SMEARS

1. MACERATION &
SMEARS

2. MACERATION
 A methods by which tissues are separated into
individual celled or their group by dissolving the
middle lamella with which the cells are held.
 The separation of fixed plant or animals material
through the hydrolysis of their interstitial tissue or
cement is known as maceration (Peter Gray)

3.  Maceration is resorted to when attempting to
visualise a three dimensional nature of
structural elements, to make a study of
individual cell and group of cells.
ADVANTAGES OF MACERATION
o Helps to view cells in their entirely, which is not
possible while sections are used
o Helps to study the nature of cell wall thickening
including pores

4.  The reagent and technique used in the process
depends upon the middle lamella. The middle
lamella in herbaceous plants consists of mainly
pectic substances, which can be hydrolysed by
mere boiling in water. In wood tissue the
middle lamella is more ligneous and can be
hydrolyzed by treatment with alkali, acid or
enzymes
 THREE COMMON METHODS ARE GIVEN
BELOW :
 SCHULTZE’S METHOD :
The material is treated with concentrated nitric acid
and few crystals of potassium chlorate(kclO3)
and warmed (avoid fumes). When the materials
turn completely bleached was thoroughly water

5.  HNO3+ KClO3 KClO4
potassium per chlorate-> highly reactive &
toxic ……. So, aviod fumes
 This method is used for very hard , woody
materials.
 JEFFREY’S METHOD
 Cut fry/fresh material into small slices (.5mm
thick)
 Boil & cool repeatedly
 Treat with mixture of equal volume of 10%
chromic acid , 1-2 day at 30-40°C
 Tap material gently using a glass rot with
rounded end to loose the cells

6.  Wash throughly in water to remove the
acids(usually the macerated tissue along with
the fluid mixture is poured into a large
volume of H2o & the macerated bits are
picked up with the brush)
 Stain with suitable stain
 Mount in 50% glycerine
Temporary /semi-permanent preparations
 Staining with 1% safranin for 6 hours
 Rinse thoroughly with water
 Dehydrate by rapid addition of hygrobutol
 Give 2 more changes of hygrobutol

7.  Add a little balsam highly diluted with
hygrobutol
 Evaporate it down to a mounting consistency
 Mount on a neat glass slide
 Used for woody & herbaceous materials
 Aviod inhaling the, vapour since HNO3
vapour since HNO3 vapour is highly toxic
HARLOW’S METHOD :
 The material is treated with chlorine water for
15-30 min. wash again and is macerated to
obtain cells.
 These cells are dehytrated by using
hygrobutol & is later infit treated by canada
balsam & then mount in the same balsam

8.  SMEARS:
 Cytological method used to study the internal details of
individual cells especially nuclear structure.
 Smears are prepared from cells which are not firmly
attached by middle lamellae
 Frequent materials for this study are sporocytes of
plants which give pollen grains after meiosis & cells of
testis in animals
 This can be easily prepared because it doesnot need
sectioning
 In plants smears are usually prepared from the
microsporocytes of the anther of rheo discolor or
chlorophylum
 Smears are prepared by smearing a material under
study on a clean glass slide to which is fixed and
mounted

9.  2 common used staining methods are the
acetocarmine method and Feulgen method
1. Acetocarmine method
 This method, originally developed by
BELLING involves killing, fixing and staining
all in one process and easy also
 The preparations reveals details regarding
structure and association of chromosome to
a great extent
 Carmine dye is obtained by adding alum to
cochineal(a yellowish red powder obtained
by grinding the fried bodies of female
Dactyl opius cacti insect)

10.  PREPARATION OF ACETOCARMINE
 Take 200cc, dil.acetic acid(90cc acetic in
110cc dis.H2o)
 Add 1g carmine dye to it
 Cool &decant
 Add a few drops of aqu.ferric acetate, till the
colour becomes dark wine red
 Keep it in well-stoppered dark bottle

11.  2. FEULGEN METHOD
 This method was originally developed in
1924 by feulgen and Rossenbeck. Later Lille
adopted a modified error proof technique
using Schiff’s reagent for feulgen stain
 PREPARATION OF BASIC FUCHSIN/FEULGEN
STAIN
 Dissolve 1g basic fushsin & 1.9g sodium
metasulphite in 100ml 0.15N HCl in conical
flask & shake well for 2hrs
 Add 500mg activate charcoal & shake
 Filter & store in dark bottles

12. MAKING SMEARS PERMANENT
1) Invert the smear slide in a petridish containing a
mixture of 10 parts acetic acid & 90 parts alcohol to
separate cover glass from the slide(1:9-acetic
acid:alcohol)
2) Take coverslip using a pointed forceps
3) Flood the coverslip with absolute alcohol & drain off the
alcohol. Repeat until the coverglass is clear
4) Flood with methyl benzoate & drain off this fluid &
invert the cover glass over a drop of DPX mountant or
Euparol on a clean warm slide
5) Follow the same procedure with the slide & finally
mount with a fresh clean & warm cover glass.