Mikroteknik

Table of Contents
Preface ..................................................................................... i
Preface Expert ........................................................................ ii
Table of Contents .................................................................. iii
Chapter 1 Material Safety Data Sheet ................................... 1
1.1 Reading chemical label on MSDS and Pictogram ............2
1.2 International norm of MSDS ............................................8
Chapter 2 Clearing and Whole Mount in Plant ................... 13
2.1 Clearing definition ......................................................... 13
2.2 Clearing by dissolution of protoplasm ............................ 14
2.3 Clearing tissue without removing protoplasm ................ 19
Chapter 3 Smears and Squash.............................................. 22
3.1 General procedure of smear or squash ............................ 23
3.2 Smear ............................................................................ 23
3.3 Squash .......................................................................... 30
Chapter 4 Paraffin Embedding Method in Plant ................ 33
4.1 Collecting ...................................................................... 33
4.2 Fixing ............................................................................ 34
4.3 Dehydrating ................................................................... 35
4.4 Infiltration and Embedding ............................................ 36
Chapter 5 Paraffin Embedding Method in Animal ............. 42
5.1 Fixation ......................................................................... 42
5.1.1 Fixatives may be classified: ..................................... 43
5.1.2 Fixatives Components: ............................................ 43
5.2 Dehydration ................................................................... 44
5.3 Clearing ......................................................................... 44
5.4 Infiltration ..................................................................... 44
5.5 Embedding/Blocking ..................................................... 45
5.6 Cutting/Sectioning ......................................................... 45
5.7 Mounting the trimmed block on a paraffin table ............. 46
5.8 Floating out sections, Affixing and Deparaffinizing ....... 48
5.9 Staining (Hematoxylin and Eosin) ................................. 49

5.9.1 Two staining procedure that common use in animal
technique ......................................................................... 50
5.9.2 Varies Stain Compositions ...................................... 52
5.10 Mounting .................................................................... 53
Chapter 6 Animal Whole Mount ......................................... 57
6.1 What is whole mount? ................................................... 57
6.2 An important note on fixation ........................................ 58
6.3 Obtaining images .......................................................... 58
6.4 Choosing the age of the embryo .................................... 58
6.5 Whole mount fluorescent immunohistochemistry .......... 59
6.6 Antibody staining of whole mount Drosophila embryos 61
6.7 Zebrafish whole mount immunohistochemistry ............. 63
6.8 Troubleshooting tips whole mount ............................. 64
6.8.1 High background .................................................... 64
6.8.2 No signal ................................................................ 65
6.8.3 Patchy staining........................................................ 66
6.8.4 Morphology of the tissue is not good. ..................... 67
Chapter 7 Blood Smear ........................................................ 69
7.1 Making a smear ............................................................. 69
7.2 Preparing staining buffer ............................................... 71
7.3 Blood Smear Preparation with Romanowsky Staining ... 72
7.3.1 Slide Preparation Procedural notes .......................... 74
7.3.2 Biologic causes of a poor smear .............................. 75
7.3.3 Staining Procedure .................................................. 75
7.3.4 Low power (10x) scan ............................................ 76
7.3.5 High power (40x) scan ............................................ 76
References ............................................................................. 79
Appendix: Material Safety Data Sheet ................................. A
CHAPTER 1
MATERIAL SAFETY DATA SHEET
Objective
After read this chapter we expect you will be able to:
1. Explain why people have to read MSDS before work
with hazardous substance
2. Recognize and understand symbol and code number
of hazardous substance according NFPA or HIMIS
3. Understand pictogram of hazardous substance
4. Summarize some important information regarding
with personal and environment safety from MSDS
Person or people that working using hazardous chemical
or dangerous goods have to know the risk substance in order to
organize safety for her/his self and environment. To obtain
safety information, people have to read material safety data
sheet (MSDS) first. The warning of safety level can read from
container of substance.
Material safety data sheet (MSDS) which is also called
safety data sheet (SDS) is document that contain information
about the potential hazards (health, fire, reactivity and
environmental) of the chemical and how they affect health and
safety in the workplace. MSDS can be obtained from companies
that produce the substance or internet by typing name substance
as key word. For example: HCl + MSDS.
The aims providing MSDS are to give information
around proper storage of a substance, first aid, spill response,
safe disposal, toxicity, flammability, and additional useful
information. By reading and understanding some information
that stated in MSDS, at least three points can be deduced:
a. How deep hazardous associated with the substance?
b. What should I do to protect myself, area around me, and
environment?
c. What should I do if an accident occurs?
Previously, MSDS was common consumed by hygienist
and safety professional, but now the user of MSDS is also
researcher, employers, supervisors, nurses, doctors, and
emergency personnel. MSDS give those peoples procedures for
handling or working with that substance in a safe manner.
1.1 Reading chemical label on MSDS and Pictogram

In the scope of hazardous substance, MSDS also contain
symbol that represent type and level of particular hazard. The
symbol is a shortcut to raise awareness about hazardous a
substance (Figure 1). Generally two style-symbols are applied in
MSDS, National Fire Protection Association (NFPA) and
Hazard Materials Information System (HMIS) respectively.
NFPA has diamond format (Figure 2A) and HMIS has rectangle
format (Figure 2B). Each color in both format represent a
different type of hazard.
Blue = Health hazard
Red = Fire hazard
Yellow = Reactivity hazard
White = Special hazard or personal protective equipment
(PPE)
Figure 1. Label notifications give warning regarding hazardous

degree of a substance.
Blue, Red, and Yellow color also contain number that denote
degree of hazard. The number move from 0-4, each level has
meaning as below:
0
= minimal hazard
1
= slight hazard
2
= moderate hazard
3
= serious hazard
4
= severe hazard
2
Figure 2. Style hazard symbol based on National Fire Protection

Association (NFPA) (A), and Hazard Materials Information
System (HMIS) (B) on 2-Mercaptoethanol according
ScienceLab.com MSDS.
In above figure, we can see slight different between figure 2 A
and B. Reactivity of figure 2A at yellow place has number 1, in
the other hand figure 2 B has number 0. Apparently NFPA and
HMIS have a little bit different consideration of hazardous
degree related with number denotation on the same substance
(Table 1).
Table 1. Explanation of hazardous degree according National
Fire Protection Association (NFPA) and Hazard Materials
Information System (HMIS).
NFPA
The Blue Section - Health Risks
4 The substance is a severe
health risk if the substance
is not handled safely. The
substance could cause
death
or
irreversible
injury.
3 The substance could cause
serious temporary or
irreversible injury.
2 The substance could cause
temporary incapacitation.
3
HMIS
Life-threatening, major or
permanent damage may result
from single or repeated over
exposures
Major injury likely unless

prompt action is taken and
medical treatment is given
Temporary or minor injury
may occur
The substance could cause

irritation.
0 There is no health hazard.
The Red Section - Fire
4 A flammable vapor or gas
which burns readily.
Irritation or minor reversible

injury possible
No significant risk to health
Flammable gases, or very

volatile flammable liquids
with flash points below 73 F
(23 C), and boiling points
below 100 F (38 C).
Materials
may
ignite
spontaneously with air
3 A flammable liquid or Materials capable of ignition
solid which can be readily under almost all normal
ignited.
temperature
conditions.
Includes flammable liquids
with flash points below 73 F
(23 C) and boiling points
above 100 F (38 C), as well
as liquids with flash points
between 73 F and 100 F.
2 The substance must be Materials which must be
heated for ignition.
moderately heated or exposed
.
to high ambient temperatures
before ignition will occur.
Includes liquids having a flash
point at or above 100 F
(38 C) but below 200 F
(93 C)
1 The substance must be Materials that must be
preheated before ignition preheated before ignition will
can occur
occur. Includes liquids, solids
and semi solids having a flash
point above 200 F (93 C)
0 There is no fire hazard
Materials that will not burn
The Yellow Section - Reactivity Hazards
4 The substance is readily Materials that are readily
capable of detonation or capable of explosive water
explosive reaction.
reaction,
detonation
or
.
explosive
decomposition,
polymerization,
or
selfreaction at normal temperature
and pressure
3 The
substance
may Materials that may form
4
detonate when exposed to explosive mixtures with water

heat or an ignition source and are capable of detonation
or explosive reaction in the
presence of a strong initiating
source.
Materials
may
polymerize, decompose, selfreact, or undergo other
chemical change at normal
temperature and pressure with
moderate risk of explosion.
The substance is readily Materials that are unstable and
capable of non-explosive may undergo violent chemical
reaction.
changes at normal temperature
and pressure with low risk for
explosion. Materials may react
violently with water or form
peroxides upon exposure to
air.
The
substance
may Materials that are normally
become unstable at high stable but can become
temperatures.
unstable (self-react) at high
temperatures and pressures.
Materials may react nonviolently with water or
undergo
hazardous
polymerization in the absence
of inhibitors
The substance is stable.
Materials that are normally
stable, even under fire
conditions, and will not react
with
water,
polymerize,
decompose, condense, or selfreact. Non-explosives
The White Section - The White Section - personal
Special Hazards
protection*)
OX = Oxidizer
A : safety glasses
ACID = Acid
B:
Safety glasses
and
protective gloves
ALK = Alkali
C: Goggles, protective gloves,
and a laboratory apron should
use
COR = Corrosive
D: A face shield, goggles,
protective gloves, a laboratory
5
apron, and an exhaust fume

hood (for reactivity hazard
no.3 or 4
E: Safety glasses, protective
gloves, and an exhaust fume
hood
H:
Splash
goggles,
a
laboratory apron, protective
gloves, and an exhaust fume
hood should be used when
handling this material.
= Use no water
= Radioactive
NOTE *)
.
Pictogram is hazardous label that convey specific information
about hazard of chemical by using a symbol plus other graphic
elements, such as a border, background pattern, or color .
Pictogram has three destination labeling. First, it label on
container or work place. The second, it label during delivery or
transportation the dangerous goods. And the third. it appear on
MSDS sheet. The pictogram according to Euro style include as
below :
Corrosive
Explosive
Highly Flammable
F+
Extremely flammable
Dangerous for the

environment
Oxidizing
T+
Xi
Toxic
Very toxic
Irritant
Xn
Harmful
Figure 3. Pictogram according to European style.

The similar pictogram is developed by Work Health and Safety
(WHS) Australia at 2012. The new labeling system derived from
7
the United Nations Globally Harmonised System (GHS) of

Classification and Labelling of Chemicals.
The following table shows these new pictograms and the types
of hazards that they represent :
Severe
health
hazards
Health
hazards
Acute
toxicity
Explosi
ve
Flamma
ble
Oxidising
Corrosi
ve
Gases
under
pressure
Environmen
tal hazard
Figure 4. Pictogram according to Work Health and Safety

(WHS) Australia.
1.2 International norm of MSDS
Writing format for MSDS has two style formats,
Occupational Safety and Health Administration (OSHA) and
American National Standards Institute (ANSI) formats. Mostly
MSDS format comply ANZI style. The style as following table:
Section Item
Section 1: Identification
Section 2 : Hazard(s)
identification
Section 3 : Composition and
information on ingredients
Explanation
This section links the chemical
name on the label to the MSDS.
The MSDS also lists the name,
address and the phone number
of the company, manufacturer or
distributor who provides the
chemical.
This section must identify all the
hazardous ingredients of the
material.
This section discusses the health
effects one may encounter when
exposed to the material. The
8
section will describe the

appearance of the material, the
potential health effects and
symptoms associated with
exposure, routes of entry, target
organs that could be affected,
and so on.
Section 4: First-aid measures This section will describe
possible first aid procedures for
each route of entry. The
procedures will be written so
that untrained individuals can
understand the information.
Section 5: Fire-fighting
This section will describe
measures
information on the fire and
explosive properties of the
material, extinguishing items,
and general fire-fighting
instructions.
Section 6: Accidental release This section gives information
measures
on how to respond when a
material spills, leaks or is
released into the air. This
information may include how to
contain a spill or the types of
equipment that may be needed
for protection.
Section 7 : Handling and
This section discusses
storage
information on handling and
storage of the material. Topics
that could be described are:
general warnings to prevent
overexposure, handling
procedures, and hygiene
instructions to prevent continued
exposure
Section 8 : Exposure
controls/ personal protection engineering controls and
personal protective equipment
that would help reduce exposure
to the material. The necessary
personal protective equipment
should be considered for
9
Section 9 : Physical and

chemical properties
Section 10 : Stability and

reactivity
Section 11 : Toxicological
information
Section 12 : Ecological
information
Section 13 : Disposal
considerations
eye/face protection, skin

protection and respiratory
protection.
This section will include
information about the physical
and chemical properties of the
material. The following
characteristics should be
detailed: appearance, odor,
physical state, pH, vapor
pressure, vapor density, boiling
point, freezing/melting point,
solubility in water and specific
gravity or density. Indicate if
these characteristics do not
apply to your material.
This section requires that
potentially hazardous chemical
reactions be identified. It
addresses chemical stability,
conditions to avoid,
incompatibility with other
materials, hazardous
decomposition and hazardous
polymerization
This section discusses data used
to determine the hazards that are
given in Section 3, Hazard
Identification. The following
information can be addressed:
acute data, carcinogenicity,
reproductive effects, target
organ effects, etc
This section will help determine
the environmental impact should
the material ever be released
into the environment.
This section gives important
information that may be helpful
in the proper disposal of the
material. The information can
cover disposal, recycling and
reclamation
10
Section 14 : Transport
information
Section 15 : Regulatory
information
Section 16 : Other
information
This section is designed to give

basic shipping information.
The basic shipping information
could include: the hazardous
materials description, hazard
class and the identification
number (UN or NA numbers).
information on the regulations
under which the material falls.
This section should include any
other important information
concerning the material. This
information can include: hazard
ratings, preparation and
revisions of the MSDS, and
label information.
By consideration that safety is a major concern, five main

information should be underlined in MSDS. The information
include:
the identity of the chemical.
health and physicochemical hazards.
safe handling and storage procedures.
emergency procedures.
disposal considerations.
To get a better understanding on the format of MSDS, refer to
the appendix at the end of this book. After the students read the
real example of MSDS, we hope they will more understand
about MSDS and can learn to withdraw five major information
from MSDS.
SELF-QUIZ
1. What is MSDS?
2. What alert hazard of following color of chemical hazard
label?
a. Red
b. yellow
c. blue
d. white
11
3. What interpretation highest number or lowest number of

chemical hazard label?
4. Why should an individual working with chemicals
understand the hazard coding system on a chemical
label?
5. Regarding with safety for your self, area around your
working place, and environment, what you should put
major attention when you read MSDS?
12
CHAPTER 2
CLEARING AND WHOLE MOUNT
IN PLANT
Objective
1. Define meaning terminology of whole mount and
clearing in plant tissue
2. Distinguish clearing process category one and two
3. Describe role substance with high index refractive in
clearing process
4. Understand clearing procedure using principle of
removal cytoplasmic component
5. Understand clearing procedure using principle of
without removal cytoplasmic component
6. Prepare whole mount specimen
Based on the word root, whole mount is composed two
word, whole and mount. Whole has intact connotation , while
mount has meaning attached to material surface. In practice
whole mount has a meaning placing a whole organism or
specimen on slide for microscopic observation or examination.
Terminology whole not absolutely describe intact body or
morphology such as individual bryophyte, however, part of
intact body for example small flower, small bud, and other
organ also are considered as whole. How is large leaf? In whole
mount, principally material or specimen can be observed under
microscope. And it is compulsory that the material should be
transparent in order allow light can pass to optic system of light
microscope. The clear tissue is beneficial for studying of
vascular anatomy, elucidating three-dimensional organization
plant organ, calcium oxalate type, haustorium or hyphe of fungi
in infected tissue, and screening embryo or ovule mutant .
2.1 Clearing definition
Literally, clearing has meaning easily to see because
transparency of medium. In nature plant tissue doesnt
transparent while some microscopy work require transparent
condition. Therefore some clearing method were introduced to
clear plant tissue. All we know that nature of plant cell and
13
tissue contain varies pigment, ergastic substance, organelles.

Those substances and organelles have different refractive index
and light scattering therefore the cell and tissue look opaque or
semiopaque. They need a clearing procedure to improve
visualization. There are many clearing method that already
develop to make tick tissue or small organ visible or transparent.
These methods are classified into two categories.
In the category one, a clearing is performed by remove
cytoplasmic content using harsh chemical (e.g NaOH). In the
category two by contrast, cytoplasmics component are not
removed also tissue structure is unaltered by treatment process.
However refractive index of the components are changed to
more uniform their refractive by chemical agent. Making
cytoplasmic component transparency degree is almost equal.
Finally the clearing agent work to make cell or tissue translucent
both through their refractive index and though their abiliy
dissolved cytoplasmic component. In a cleared cell or tissue can
stand out some character because of their special chemical or
physical properties. The characters include such feature vascular
anatomy, schlereid distribution, crystals, silica cells, tanniferous
idioblasts, trichomes, laticifers, stomata, and even nuclei (in
embryos).
2.2 Clearing by dissolution of protoplasm
In normal standard, application 5% NaOH or KOH for 3
days or more produce cleared tissue. However, a weak solution
NaOH or KOH (2 %) is used for soft tissue to avoid cellulose
degradation. During hydrolysis and oxidation by NaOH,
phenolic oxidation almost occurred with dark pigment result.
According to Singleton (1972), dark pigment formation due to
three mechanisms:
a. Oxidative phenol-phenol coupling to give quinones and
related compounds.
b. Phenol-aldehyde condensation to give very stable polymers
whose constitution depends on reaction conditions.
Naturally-occurring aldehydes or those produced in
protoplasm breakdown, e.g. furfural from carbohydrates,
may be involved.
c. Phenol-amine coupling, involving quinoid phenol
derivatives and amine-containing substances, e.g. peptides.
Affinities phenol polymeric to protein seem to protect tissue by
making slow down NaOH attack.
14
The phenolic pigment that produce during alkali

treatment ban specimen transparency even though protoplasm
already gone. The phenolic pigment can be removed by repeated
washing in succession with warm water, warm 70% ethanol,
warm 95% ethanol.
Dark pigment that may be formed in earlier clearing
stage is recommended to bleached using bleaching agent. And
some bleaching agent that were known to work well are :
hydrogen peroxide , chromium trioxide as in Stockwell's
solution , hypochlorite , chlorine , acidified sodium chlorite,
commercial household bleach. However, application bleaching
agent should be put in consideration for fragile tissue because
they can dissolve cellulose and lignin especially in acid
condition. Therefore acid environment also both concentration
and length incubation of bleaching agent are important to put in
attention.
Alkali treatment can be applied to three type tissue :
- Fresh
- Dry
- Fixed
Fixed tissue have varied responses to alkali treatment depend on
kind of fixative. Carnoy or 70% alcohol can function as
pretreatment event, because they slightly accelerate clearing by
extracting various constituent of protoplasm. However, fixative
containing formaldehyde produce contra clear effect because
such follow:
1.Formaldehyde make cross-linking protein in tissue which
enable tissue resistant to alkali (NaOH or KOH) attack and the
whole specimen may disintegrate because degrade the
cellulose before it clears.
2.Formaldehyde reacts with many plant phenols, especially the
condensed tannins (leucoanthocyanins and catechins), to form
dark polymers .
Dry tissue is quicker to clear compare fresh tissue because of
disruption protoplasm during drying. As consequence, drying
tissue usually produce dark pigment resulting from air and
enzymic oxidation of phenolic compounds. Even drying leaf has
weak point, Ruzin (1999) strong suggested to dry first before
treating with NaOH.
15
Example clearing
component
tissue
by
removing
cytoplasmic
A. Clearing leaf tissue in general

A leaf as object of research may be in dry or fresh
condition. The fresh leaf may treat first in boil alcohol or
Carnoys solution (Table 2) to remove chlorophyll (Khasim,
2002). The dry leaf, however, submerge in NaOH or KOH
directly. The order steps of clearing and staining leaf tissue is
described below (Khasim, 2002):
1. Submerge the specimen (treated fresh leaf or dried leaf)
in 5% NaOH or KOH at 37oC for 24 h or more until
transparent.
2. Wash briefly to omit any left of pigment with water, then
it dip in Stockwell solution for one to several hour.
Stockwell composition
Water
90 ml
Potassium bichromate
1g
Glacial acetic acid
10 ml
Chromic acid
1g
The above solution has function to bleach tissue. If the
leaf was not bleah/clear, dipping in chloral hydrate
solution (2.5 g/ml) is additional step to clear specimen.
3. Dehydrate tissue in 30% and 50% ethanol for 5 minute
each.
4. Stain tissue in 1% safranin (in 50% ethanol) for few
minutes.
5. Continue dehydrate in 70%-95% and absolute of ethanol
briefly to avoid de-staining.
6. Give additional clearing in xylene. Make sure the
specimen dehydrated, if still contain water the specimen
look milky white. Put back in ethanol absolute to solve
milky problem.
7. Mount leaf with solution that soluble in xylene such as
Canada balsam or Enthellan
B. Clearing leaf tissue to observe Calcium oxalate crystal
Clearing tissue of whole mount specimens has different
method depend on the aim of research target. The clearing
method for observation crystal Ca-Ox mostly is conducted by
dissolving protoplasm as basic principle. The following section
describe some example good clearing method for preparation
calcium oxalate slide.
16
1. Endriyani (2009) using A. Muelleri

The specimen (leaf, petiole and corm) fix in farmer solution (
70% ethanol : glacial acetic acid = 3:1) for while, remove
them to 95% ethanol for 24 h, wash briefly before transfer to
10% NaOH until the specimen transparent, usually take 24 h.
wash once more before dehydrate in ethanol series 30-50-8095-100% for 10-15 minute each. Repeating clearing using
mixture of ethanol : xylene in varies proportion , 3:1 1:1
1:3. Mounting using Hoyer solution.
2. Handayani (2009) using A.muelleri
The specimens (leaf and petiole) were submerged in in 5%
NaOH at 37oC for 24 hour , move to 50% house hold
bleaching for 1 h, dehydrate in alcohol series 25-50-70-80%
for 10 minute each, and 5 minute in 96% ethanol. Then
mounting using Hoyer solution.
3. Fatmawati (2012) using A.Campanulatus (wild)
Leaf and petiole clearing
Submerged specimen (leaf, petiole: 1x1 cm2) in 5% NaOH at
37oC for 24 hour. Replace NaOH with 50% house hold
bleaching , leave it there for 1 h. After bleach, wash three
times with aquades, dehydrate in alcohol series 25-50-7095% for 10 minute each, wash in alcohol abs, then mounting
using glycerine.
Slice corm clearing
Submerged specimen in 5% NaOH at 37oC for 24 hour, move
to 50% house hold bleaching for 1 h, wash three times with
aquades, transferred to 30% alcohol for 1 h, then directly
mounting in glycerin
4. Rohmiati (2012) using A.Campanulatus (domesticated)
Leaf clearing
Submerged specimen (leaf, 1x1 cm2) in 5% NaOH at 37oC
for 24 hour, move to 80% house hold bleaching for 30 minute
and wash in tap running water. Then, transfer the leaf to 30%
and 50% ethanol for 15 minute each. The process clearing is
finish. Mounting the leaf using glycerin. The specimen ready
to observe under microscope.
Petiole clearing
Submerged specimen (petiole 1x1 cm2) in 5% NaOH at 37oC
for 24 hour, move to 50% house hold bleaching for 1 h, wash
using aquades for 5 minute, dehydrate in alcohol series 3050-70-80-100% for 10 minute each. Finally mounting the
specimen in glycerin.
17
Slice corm clearing

Submerged specimen in 5% NaOH at 37oC for 24 hour, move
to 50% house hold bleaching for 20 minute, then directly
transfer to 30% ethanol for 15 minute and finally mounting
using glycerin.
5. Aulia (2012) using A.variabilis
Leaf clearing
Submerged specimen (leaf, 1x1 cm2) in 5% NaOH at 3739oC for 43 hour, move to 50% house hold bleaching for 1 h
and wash in running tap water. Then, transfer the leaf to 30%
and for 10 minute. Mounting the leaf using Hoyer solution.
The specimen ready to observe under microscope.
Petiole clearing
Submerged specimen (petiole 1x1 cm2) in 5% NaOH at 37oC
for 24 hour, move to 50% house hold bleaching for 1 h, wash
using aquades for 5 minute, transfer to 30 and 50% alcohol
for 10 minute each. Finally mounting the specimen using
glycerin solution.
Sliced-corm clearing
Fix the sliced corm in 70% ethanol for 30 minute, Submerged
specimen in 5% NaOH at RT for 20 minute, move to 50%
house hold bleaching for 20 minute (shaking some times to
omit bubble), then directly transfer to slide glass and
mounting using glycerin.
10 m
30 m
Figure.6 Druse crystal (A) and Raphide crystal (B) in corm of

A.variabilis (Aulia, 2011)
C. Clearing tissue for sieve tube (Lersten, 1986)
1. Treat soy bean leaf or other dicotyledenous species with
more concentrated NaOH (10% in water) for 2-4 weeks.
Continuing clearing in chloral hydrate (2.5 g/ml)
2. Stain cleared tissue with chlorazol E black (1% in
ethanol absolute) for 3-6 minutes
18
3. Mount sample with lower epidermis upward. The aim

position is phloem more close with objective lens so that
it will see first.
2.3 Clearing tissue without removing protoplasm
Acidified of chloral hydrate has been used successfully
to clear tissue without remove cytoplasmic component in many
experiment. There are many clearing solution formula already
developed with chloral hydrate as a important component. BB41/2 is the famous one of clearing solution . The Composition
BB- 41/2 is 9 part of 41/2 solution and one part of benzyl
benzoate (Ruzin, 1999). Detail of BB-41/2 clearing solution is
as below :
41/2 solution
by weigh
85% Lactic acid
2
Chloral hydrate
2
Phenol
2
Clove oil
2
Xylene
1
BB-41/2 solution
41/2 solution
Benzyl benzoate
by weigh
9
1
Chloral hydrate itself is potential to clear tissue (Harijati, 2007).

The Chloral hydrate solution was applied after clearing
staining infected leaf produced very nice whole mount leaf-hair
slide ( Figure 5).
Figure 5. Infected chickpea hair by Ascochyta rabiei

(Harijati, 2007)
19
Clearing-staining tissue also contain more chloral hydrate (Table

2). By watching staining- clearing solution we realize why the
tissue gave clear appearance. Some component of solution has
high refractive index (Table 3).
Table 2. Composition clearing-staining solution for infected
tissue (Harijati , 2007)
Component
Volume or weight
300 ml 99% ethanol
300 ml
50 ml chloroform
50 ml
125 ml lactic acid
125 ml
450 g chloral hydrate
450 g
2 g trypan blue or chlorazol Black E
2g
Table 3. Refractive index of some common microscope media
Media
Refractive index ( n D20 )
Water
1.33
Ethanol
1.36
Chloral hydrate
1.60
Glycerol
1.47
Xylene
1.50
Methyl salicylate
1.54
Acidifi ed chloral hydrate in 1.43
glycerol
Lactic acid
1.43
Visikol
1.45
The high refractive index of medium can allow the light
the medium without refraction between the boundary of the
glass and objective lens of microscope. Clearing agents with
high refraction index allow more light more light to continue
through the microscope to the observer. A high refractive index
also create improvement dept of field. It means that more
vertical planes can observed in the microscope in a particular
focal plane. The depth of field is proportionate to the refractive
index.
Even usually initial step for microtechnique is fixation,
Garner (1975), however, introduce one stop clearing which
involves infiltration of material with high refractive index.
Those material include a lactic acid, strong solution of phenol
and chloral hydrate or mixture of these substance. In this
procedure quite simple, just leave the specimens in one of those
20
solution or mixture of substance until they are transparent then

mounting them in medium which contain high refractive index
as well such Hoyer medium.
Example clearing tissue without removing cytoplasmic
componen
Combination of lactic acid and chloral hydrate is favorite
solution without removal cytoplasmic component. There are
many procedure available. In general Ruzin (1999) describe as
below :
1. Fix tissue in either FAA, FPA, or CRAF
2. Transfer the tissue to BB-41/2 clearing solution then
continued transfer to 95% ethanol
3. Stain in basic fuchsin (0.02 g in 2 ml 95% ethanol,
diluted to 100 ml in deionized water) at 60oC for 12-14
hour.
4. Wash 12 h in deionized water, change many times
5. Dehydrate to 95% ethanol for 12 hour
6. Transfer to mixture ethanol acid solution ( EtOH : HCl =
3:1) for 1-5 minute
7. Wash in 100% ethanol for 24 h
8. Clear in xylene and mount using Canada balsam
SELF-QUIZ
1. Please explain about whole mount in microscopy
discipline!
2. What should do if you want omit protoplasmic
component or not remove them?
3. What do you think about substance with high refractive
index?
4. Could you describe procedure to clear leaf tissue by aim
to see Ca-Ox crystal?
21
CHAPTER 3
SMEARS AND SQUASH
Objective
1. Distinguish between smear and squash
2. Prepare clean slide glass when reuse slide glass
3. Conduct preparation cytology specimen using smear or
squash technique
4. Prepare some solution for fixation and staining
In general, chromosome slide is prepared using smear or

squash technique. And actually both smear and squash technique
share in same process but there is a little deviation (Table 4).
Table 4. The equation and difference between smear and squash
smear
Pre-treatment
no
Dissolving middle lamella yes
The compactness of origin less
tissue
squash
yes
yes
more
Smear and squash definitely require clean microscopy slide

because during preparation process of those technique not need
adhesive. Therefore if someone feel worried about cleanness of
slide, the following solution formula can apply to clean slide :
Potassium dichromate
Conc. Sulphuric acid
Distillated water
20 g
100 ml
100 ml
Dissolved 20 g potassium dichromate in 100 ml dH2O, add 100

ml H2SO4 slowly then stir low beat. During that process will
produce a heat. After potassium dichromate dissolve well, stop
stirring and let the solution cool. Now the solution is ready to
use. Cleaning slide is performed by immerse the slide for while
(depend on dirty level of slid) and wash thoroughly under
running tap water. It recommended some time immerse in strong
22
ethanol containing few drops ammonia solution, then wash in

running tap water and drying in free dust area.
3.1 General procedure of smear or squash
The source material that suitable for smear or squash is
microsporocytes, root tip, young leaf and others meristematic
tissue. To conduct smear or squash action, flow chart below is
general pattern to obtain specimen
Killed and fixed tissue
Hydrolysis
Maceration
Stain
Cover
3.2 Smear
Such in blood smear, plant smear has principal spreading
evenly of material as thin as possible. The most popular
example smear in plant is anthers microsporocyte. Take anthers
some days before pollen grain appear or disperse. For example
anther of Tradescatia sp suppose harvest 15 days before pollen
grain shedding. Also important note, harvest material is
conducted in mid day in order to obtain good meiosis.
There is some method that applied for smear. The
famous one is iron-acetocarmine. Example of Iron acetocarmine
method as below:
Method I
1. Harvest and fix anther in FAA solution (Table 5).
2. Place fixed anther on a clean slide, give few drops
acetocarmine staining (Table 6) using Pasteur pipette over the
anther.
3. By press gently, squeeze out microsporocyte, remove anther
wall and others debris
23
4. Put cover slip and remove excess stain using tissue paper
5. Seal cover slip with transparent nail polish
6. Leave preparation for few days to facilitate the stain into
microsporocyte.
Method II
1. Place anther in center of clean slide
2. Put another slide over anther, press gently and spread evenly
using that slide
3. Pour Carnoys solution (Table 5) in petri dish that previously
equipped with two or three glasses (Figure 1). Put slide with
invert face touch fixative solution. Keep it at least 10 minute.
Figure 7. Rectangle petri dish equipped with rod glasses

4. Put few drop acetocarmine staining (Table 6) on smear and
cover with cover slide
5. Remove excess solution with tissue paper and seal cover slip
with transparent nail polish
Table 5. The fixatives that common use in cytology studies
Fixative name
Carnoy (type I) or
Farmer
Carnoy (type II)
CRAFT (chromic
composition
volume
Absolute ethyl alcohol
15 ml
Glacial acetic acid
5 ml
Absolute ethyl alcohol
15 ml
Glacial acetic acid
5 ml
Chloroform
15 ml
Chromic acid (1% chromium
20 ml
24
acid acetic acid

formalin) (type I)
trioxide)
1 % Acetic acid
37-40% formalin
CRAFT (type II)
trioxide)
10 % Acetic acid
37-40% formalin
Water
CRAFT (type III)
trioxide)
10 % Acetic acid
37-40% formalin
Water
CRAFT (type IV)
trioxide)
10 % Acetic acid
37-40% formalin
Water
CRAFT (type V)
trioxide)
10 % Acetic acid
37-40% formalin
Navashin
10% chromic acid
2 % osmic acid in 2 % chromic
acid
10 % acetic acid
Distillated
Note : add 1% saponin to
mixture
FAA (Formalin,
70% ethyl alcohol
acetic acid, alcohol) * Glacial acetic acid
Formalin
*). Wok well during experiment
75ml
5ml
20 ml
10 ml
5 ml
65 ml
30 ml
20 ml
10 ml
40 ml
40 ml
30 ml
10 ml
20 ml
50 ml
35 ml
15 ml
1 ml
7.5 ml
10 ml
41.5 ml
90 ml
5 ml
5 ml
Table 6. The staining that common use in cytology studies

Snows solution
Composition
Carmine
Conc. HCl
Distilled water
85% ethanol
4g
1 ml
15 ml
95 ml
Preparation
Add Carmine and conc. HCl to
dH2O.
Mix well using magnetic bar
gently while boil ( 10 minute).
After cooled, add 85% EtOH. Mix
25
well then filtered.

Store in brown bottle
Feulgen (30 mL)
Composition
Basic Fuchsin 150 mg
1 N HCl
4.5 ml
dH2O
30 mL
K2S2O5 (Pot.metabisulfit)
0.45 g
Aceto-orcein
Composition
Orcein
Glacial acetic acid
Preparation
Dissolved 150 mg basic fuchsin
using 30 ml boiling dH2O.
Cool the mixture to 50oC then
filter through Whatman No. 1
paper.
Add 4.5 mL 1N HCl to filtered
substance, mix well. Add and
dissolve 0.45 g K2S2O5. Let it for
24 h in dark to bleach.
Add 0.5 g activated charcoal (e.g.
Norit), shake 2 minute, and filter.
Storage in brown bottle
preparation
2.2 g
Dissolve 2.2 g Orcein dissolve in
100 ml 100 ml glacial acetic acid.
Gently boil the mixture while
stirring
Cool and filter
Storage in brown bottle
Note : when use , dilute up to 45
% using dH2O
Aceto-Carmine
Composition
Carmine
1g
Glacial acetic acid
90 ml
Conc Ferric acetate
few
drops
dH2O
110 ml
preparation
Add 110 m dH2O to 90 ml glacial
acetic acid.
Boiling the mixture up to boiling
point in fume hood.
Stop boiling, add 1 g carmine.
Cool and add few drop ferric
acetate until appear
wine-red
color. Let it stand for 12 hour
Filter and store as main stock
Method III
1. Fix anther in Famers solution for 12 hours or some week to
enhance the staining ability of chromosome
2. Place the anther in center of clean slide and put a drop of
acetocarmine (Table 5) over it.
26
3. Squeeze out microsporocyte by pushing gently , remove cell

wall and debris
4. The slide over fire gently for a second to spread cell flatten
(Figure 8). Avoid boil the cell
Figure 8. Passing the slide over fire

5. Transfer slide to jar that containing acetic acid : alcohol
absolute = 1:1 for 5 minute
6. Moving the slide in following jar for 1 minute each
a. Jar 1 containing 1:3 acetic acid and alcohol absolute
b. Jar 2 containing 1:9 acetic acid : alcohol absolute
c. Jar 3 containing 1:1 alcohol absolute and xylene
7. Mount with Canada Balsam or Enthelan (dilute with xylene).
Instead of anther, root tip also is suitable as smear material.
The popular root tip experiment is onions root. The root is
prepared by germinate onion on water (Figure 9). By this
technique , the root keep clean and easily to harvest. And in the
morning (around 7-9 am) is good time to harvest the root
because the root cells are active dividing mitoticly.
27
Figure 9. Onion germination method

Root- method I
1. Cut root tip 2 mm length , fix using CRAF fixative (Table
4)
2. Place the root in the center of clean slide glass and give 2
drops of acetocarmine staining (Table 5).
3. Passed the slide over fire 4-6 times with 1 second each to
allow cells to spread and flatten (Figure 8). Please take a note
: do not to allow the cell boiled.
4. Put cover slip, wrap around the slide with tissue/towel paper
(Figure 10) and press gently with finger or pencil bottom to
spread cell.
Figure 10. Sample spreading

5. Remove the paper and seal cover slip using transparent nail
polish
Root- method II
1. Fix roots in Famers solution for 12 hours or some week to
28
2. Remove the root to container that already filled with mixture

95 % ethanol : conc.HCl = 1 : 1 for 5-30 minute. This
solution has function to dissolve middle lamella.
3. Transfer the root to Carnoy solution type II (Table 4) for
hardening tissue.
4. Place the root on the center clean slide glass, drops
acetocarmine, chopped with razor blade or scalpel or cutter,
and pressed gently with the blade.
5. Put cover slip and passed the slide over fire (Figure 8) shortly
(1 second) for 3-4 times. Avoid boil the material.
6. Seal cover slip using transparent nail polish
Root- method III
1. Fix roots in Famers solution for 12 hours or some week to
2. Remove the root to container that already filled with mixture
95 % ethanol : conc.HCl = 1 : 1 for 5-30 minute. This
solution has function to dissolve middle lamella.
3. Transfer the root to Carnoy solution type II (Table 5) for
hardening tissue.
4. Place the root on the center clean slide glass, drops
acetocarmine, chopped with razor blade or scalpel or cutter,
and pressed gently with the blade.
5. Put cover slip and passed the slide over fire (Figure 8) shortly
(1 second) for 3-4 times. Avoid boil the material.
6. Put the slide in a dish that contains 10% acetic acid aq until
cover slip loose.
7. Take cover slip using forceps and put the slide in jar
containing mix solution acetic acid: ethanol abs. = 1 : 1 for 5
minute.
8. Moving the slide in following jar for 1 minute each
a. Jar 1 containing 1:3 acetic acid and alcohol absolute
b. Jar 2 containing 1:9 acetic acid : alcohol absolute
c. Jar 3 containing 1:1 alcohol absolute and xylene
9. Mount with Canada Balsam or Enthelan (dilute with xylene).
Root- method IV
1. Cut root tip 3 mm length and fix in Farmer solution for
at least 24 hour
2. Remove the root tip using Pasteur pipette or other tool to
test tube/watch glass containing 1 N HCl then digest at
60oC for 12 minute at constant temperature
3. Remove HCl, add 1 ml Feulgen staining (Table 6)
29
4. Incubate for 10 minute until the tip produce distinct dark

coloring
5. Put 1 drop 45% acetic acid on the center of clean slide
glass
6. Place the soft root to 45% acetic acid on slide, remove
un-stain root.
7. Add cover slip and press gently on cover slip using
rubber part of pencil or other friendly tool to spread out
the cells.
3.3 Squash
Pretreatment is beneficial in squash method. The pretreatment
has some purpose :
Stops formation spindle
Increase number of metaphase stage by arresting the
chromosome at metaphase plate.
Contracts the chromosome length with distinct
constriction
Increase the viscosity of the cytoplasm
Numerous pretreatment has been applied and developed
described below :
1. Ice-cold water
Pretreatment Ice-cold water is applied to plant that has a lot
chromosome. The pretreatment is suitable for cereal
chromosome. The material (for example root) put in vial that
already filled cold water. Cover the vial with a thick layer of
ice. Keep in refrigerator for 12 to 24 hour. Pretreatment for
barleys root or wheats root are recommended 16 to 18 h and
24 h respectively. Longer pretreatment will shorten
chromosome.
2. 8-Hydroxyquinoline
Pretreatment with 8-hydroxyquinoline is effective for plant
with small- size chromosome. The root keep in 0.002 M 8hydroxquinoline solution for 3-5 h at 16-18oC. Warmer
temperature cause sticky of chromosome.
8-hydroxyquinoline is prepared by dissolved 0.3-0.5 g 8-hydroxyquinoline
in one liter of ddH2O .
3. Colchicine
Low concentration of colchicine (0.1-0.5 %) is recommended
for pretreatment (1-2 h, at RT). Application
high
concentration of colchicines cause polyploidy. Soybeans root
that incubated for 1.5 h produce the best result.
30
4. -Bromonaphthalene
The effect of -bromonaphthalene is similar with colchicines.
Pretreatment using -bromonaphthalene is in saturated for 2h at room temperature. The pretreatment is suitable for wheat
and barley chromosome.
5. Paradichlorobenzene (PDB)
Similar with 8-hydroxyquinoline, PDB is the best when
applied for plant with small-size of chromosome. The
treatment require 1.5 % PDB (in dH2O). Dissolving of 1.5%
is conducted at 60oC for overnight. Pretreatment root is
performed when the solution cool with length treatment 2-2.5
h at 15-20 oC or RT.
Some example squash proceed is described below :
Oxyquinoline aceto-orcein squash method
1. Prepared HCl-Orcein mixture:
Mix aceto-orcein stain (Table 6) : HCl = 9:1
2. Pretreatment the root tip in 0.03 % 8-hydroxyquinoline
aqueous in cool room for 4 h.
3. Remove the root to watch glass and give a few drops of
HCl-Orcein solution. Passed gently on fire 3-4 times with
interval 1 minute. Leave the specimen in this solution for 2030 minutes. Note : keep care heating, because a heat
accelerate maceration effect of HCl.
4. Incubate root tip in aceto-orcein solution for 20-39 minute or
longer for small chromosome (12-16 h). Do not heated.
5. Transferred the root to clean slide glass, cut and omit undark part of root. Chopped the dark part and cover with
cover slip immediately and press gently to spread cell.
6. Before cover slip sealed , let it un-cover in refrigerator
overnight.
Alcoholic Hydrochloric Acid-Carmine squash method

1. Fix root tip or shoot tip or other material in Farmers solution
(Table 5) or mixture of ethanol : glacial acetic acid:
chloroform = 3:1:1
2. Wash in 70% ethanol three times , one hour for each wash.
3. Incubate in capped vial containing Snows solution (Table 5)
at 50-60 oC for 24-48 h.
4. Rinse material with water or 70% ethanol, move material in
45% acetic acid before squash.
31
5. placed the material in the center of clean slide glass, put a few
drop mixture of Hoyers mounting medium : 40 % acetic acid
= 1: 1.
6. Put cover slip and squash the material.
SELF-QUIZ
1. Why squash and smear technique are suitable for cytological
studies?
2. What is the advantage pretreatment?
3. What condition /status plant tissue is suitable for cytological
studies?
4. What is function of Carnoy solution?
5. Why some time after fix in fixative solution, the material
have to dip in solution that contain mixture of concentrated
HCl and ethanol?
32
CHAPTER 4
PARAFFIN EMBEDDING METHOD
IN PLANT
Objective
1. Create a permanently microscopic slide of plants
part by paraffin embedding method.
2. Understand steps in preparation paraffin- embedding
method
3. Conduct trouble shooting when facing with unexpected result
Permanent slide is the top slide in plant microtechnique.
The permanent slide can be kept longer for years and offered
good quality slide specimen. In fresh sectioning, the slide only
produce satisfy slide in short time (hours until some days). Also
it need tissue which is rigid physically in nature to allow razor
blade cut the tissue as thin as possible. If the tissue soft and wet
such Aloe vera, fresh sectioning in thin size is hard and often
failure to obtain good anatomy observation even already cut in
more 100 m thick. The soft or fragile tissue needs supporting
material to restrain microtomes knife compression for
obtaining thin sectioning (5-15 m). There are some material
used to support tissue and the famous one is paraffin embedding.
The paraffin embedding has several steps which include
fixation, dehydration, clearing, infiltration /impregnation,
embedding/
blocking,
cutting/sectioning,
affixing,
deparaffinizing and staining, and mounting. Before run the first
step, collecting material is critical work that should put in
attention. Because if people careless or do it the wrong way,
result will deviate from expected result.
4.1 Collecting
Plant materials which will make a permanent
microscopic slide should be choose in good sense such as no
wound, healthy and representative plants. When we collect plant
materials from field, place it between sheets of wet toweling
paper and keep in closed container such as a tin can or a zip
plastic. It is important to recognize the size of plant material that
will be cut for sample. The subdividing of soft fresh material is
33
best done with a razor blade, with the material placed on a sheet
of wet blotting paper or held carefully against finger. Leaves are
almost invariably cut into small pieces for processing. Narrow
leaves that are not much over 5 mm wide, may be cut into
complete transverse pieces measuring 2 to 4 mm along the rib.
Broad leaves should be cut into small pieces, selected to include
midrib, lateral veins, fungus pustules, fern sori, or other desired
structures. Herbaceous stems, roots, petioles, and other more or
less cylindrical organs are usually cut into short sections or disks
(Figure 11).
Figure 11. Part of plant that will be used as sample.

A. Whole young plant, B. Section of plant
4.2 Fixing
Fixing is a process that fix the plant tissues in a fixative
or killing solution. Several kind of fixative solution are simple
fixative solution such as ethanol 70 % and compound fixative
solution such as FAA (Table 7), Nawaschin or Craf (Table 8),
etc. The aim of fixing is to preserve the plant tissues as well as
plant tissues alive. When we fix plant materials we also evacuate
the air of plant tissues with desiccator or aspirator that
connected to vacuum pump (Figure 12). The aim of aspiration is
to remove the air from air cavity of plant tissues.
Table 7. Reagent of FAA
No
Stock solution
1 Ethyl alcohol (50 or 70 %)
2 Glacial acetic acid
3 Formaldehyde (37-40 %)
Quantity (mL)
90
5
5
Table 8. Reagents of Nawaschin/Craf

No Stock solution
Nawaschin or Craf type (mL)
Nawacschin
I
II III IV
V
(soft)
(hard)
34
1
2
3
4
5
1 % chromic
acid
1 % acetic
acid
10 % acetic
acid
Glacial acetic
acid
Formaldehyde
(37 40 %)
aqueous
Water
75
20
75
20
65 40 20
20 30 40
-
10 20 30
-
10 10
50
35
15
Figure 12. Fixing and evacuating the air from air cavity of
plant tissues. A. Vacuum pump that connected to aspirator jar,
B. Section of plant that float to above fixative solution
4.3 Dehydrating
Dehydrating is a process to extract the water from plant
tissues and to facilitate the fixative solution fills the plant tissues
(Figure 13). The aim of dehydrating is to extract the water from
plant tissues. Several kind of dehydrating solution include
ethanol series (Table 19), Johansen solution (Table 10) and
combination solution ethanol and TBA (Tertiary Butyl Alcohol)
(Table 11).
Table 9. Reagents of ethanol series
No
Stock solution
1 50 % ethanol (if use 50 %
ethanol in FAA)
2 60 % ethanol
3 70 % ethanol (if use 70 %
35
Volume (mL)
50
50
50
ethanol in FAA)
80 % ethanol
90 % ethanol
100 % ethanol
100 % ethanol
4
5
6
7
50
50
50
50
Table 10. Reagents of Johansen

No
Stock solution
1
2
3
4
Water
95 % ethanol
TBA
100 % ethanol
Johansen type (mL)

I
II
III
IV
V
50
30 15
40
50 50
45
10
20 35
55
75
25
Table 11. Reagents of combination ethanol and TBA

No Stock solution Ethanol TBA (mL)
Distilled
equal with
(mL)
water
ethanol
(mL)
1 50 %
40
10
50
2 70 %
50
20
30
3 90 %
50
40
10
4 100 %
50
50
5 100 %
100
(three times
replacement)
Figure 13. Series of dehydration solution

4.4 Infiltration and Embedding
Infiltration is a process of filling plant tissues with
paraffin or other embedding agents gradually. The early solution
of infiltration depend on the kind of dehydrating solution. For
example if we use ethanol series for dehydrating, the infiltration
begin with combination of ethanol-xylene and then xyleneparaffin (Table 14), but if we use Johansen solution and
combination between ethanol and TBA for dehydration, the
infiltration begin with TBA-paraffin (Table 15). Paraffin that
36
use for embedding has melting point 58 C. All of infiltration

and embedding process conducted in incubator. In one block
may contain one sample or more than one sample (Figure 15).
The container for sample embedding or sample block is called
paraffin boat, it is formed from slightly thick paper like a cube,
or we can use plastic box (Figure 14)
Table 14. Reagents of infiltration type 1 and embedding
No
Stock solution
Ratio
1 100 % ethanol : xylene
3:1
1:1
1:3
4 Xylene : paraffin
3:1
5 Xylene : paraffin
1:1
6 Xylem : paraffin
1:3
7 Paraffin
Two times replacement
Table 15. Reagents of infiltration type 2 and embedding
No
Stock solution
Ratio
1 TBA : paraffin
3:1
2 TBA : paraffin
1:1
3 TBA : paraffin
1:3
4 Paraffin
Two times replacement
Figure 14. Embedding sample in plastic box. A. Pouring liquid

paraffin in plastic box, B. Sample is embedding in paraffin
block, C. Various type of plastic box , D. Embedding sample in
stainless steel box
37
A B
Figure 15. Sample of part of plant that embedded in paraffin
block . A. Single paraffin block consist of one sample, B. Big
paraffin block consist of more than one sample
4.5 Trimming, Sectioning, and Affixing
Trimming is a process to cut the part of sample block that no
filled plant sample. The piece of material to be sectioned is
fastened to a mounting block, which is clamped into the rotary
microtome. Inexpensive mounting blocks can be made of hard
and porous wood. The most useful sizes range from 1 x 1 x 2 cm
to 2 x 2 x 3 cm. Soak the wood blocks in hot canning wax. After
that the sample block is mounted on wood block as a holder
(Figure 16 and 17). The next step is sectioning the sample
block. Sectioning is cutting the sample block by rotary
microtome. The angle of the razor-blade holder and the trimmed
sample block holder in microtome is 45 . The knife should be
sharp and clean. The thickness of sample section approximately
5 m (Figure 18). Next stage is affixing, it is mounting the
paraffin ribbon that formed from sectioning on slide glass with
an adhesive prior to staining. New slides should be cleaned,
although they may seem to be clean. Adhesive for mounting
usually egg albumen. Drop one drop of egg albumen on slide
glass and place the paraffin ribbon on it. Then the slide glass is
placed on hot-plate (40 C) until dry (Figure 19).
38
Figure 16. Trimming of paraffin block . A. Single paraffin

block consist of one sample that begin to trimming although still
in the plastic box, B. Paraffin block slices layer by layer, C. The
result of paraffin block that has been trimmed, D. Wood holder
B
C
Figure 17. Mounting of paraffin block. A. Cutter razor is heated

above Bunsen, B. Paraffin block that is facilitated by hot cutter
razor mounted on wood holder, C. The result of paraffin block
that has been mounted on wood holder
39
Figure 18. Sectioning of paraffin block. A. Sectioning process,

left hand hold a rod to facilitate the paraffin ribbon, right hand to
rotating the wheel of microtome , B. The angle of razor blade
and the knife holder of microtome about 45 , C. The paraffin
ribbon that generated from sectioning process, D. Good paraffin
ribbon approximately 5 m thick and no rupture.
Figure 19. Affixing of paraffin ribbon on slide glass. A. Series

of slide glass that contain sample above the hot plate , B.
Paraffin ribbon that contain sample on slide glass begin
melting
4.6 Staining
Staining is to stain the slide glass that contain sample of
plant tissue. The aim of staining is to make a contrast among
plant tissues. Stain consist of basic base and acid base, natural
40
base and synthetic base. In plant tissue usually use safranin-fast

green stain. Safranin will stain unlignified cell wall, the color is
green, and in contrast fast green will stain lignified cell wall, the
color is red. Staining step begin with xylene for deparaffin
paraffin ribbon and then rehydration to stain that dissolve in
water, and the next dehydration to stain that dissolve in ethanol,
and finish in xylene again to clearing the slide glass.
4.7 Mounting and Labeling
Mounting is mount a slide glass with an adhesive and
cover by cover slip. Examples of mounting agent are Canada
balsam and entellan. Labeling is give an identity to the slide
glass, involve name of plant specimen, the kind of section, the
kind of stain, etc.
SELF-QUIZ
1. Explain the steps of paraffin embedding method in plant!
2. What is fixation?
3. What is dehydration?
4. What is infiltration?
5. Explain the steps of staining!
41
CHAPTER 5
PARAFFIN EMBEDDING METHOD
IN ANIMAL
Objective
1. understand the steps of paraffin method in animal tissue
processing
2. process animal tissue with paraffin method based on available
protocols.
Before animal tissues can be visualized using light

microscope, it needs a series processing. One of methods for
tissue processing is paraffin method. Paraffin plays an crucial
role, fill intercellular spaces and embed the tissue. Without
paraffin, tissues will be pressed or cracked and can not be
examined properly. Paraffin method include some processses:
- Fixation
- Dehydration
- Clearing
- Infiltration / impregnation
- Embedding / blocking
- Cutting / sectioning
- Affixing
- Deparaffinizing and staining
- Mounting
5.1 Fixation
Fixation is a process to prevent post-mortem changes of
the tissues. Fixation also necessary to separate the solid phase of
protoplasm from aqueous phase, to convert the cell parts into
materials that will remain insoluble during susequent treatment
and protect the cells from distortion and shrinkage when they
are subjected to fluids. Fixation increase permeability and are
more receptive to staining. Unfixed tissue elements have limited
binding sites for dyes. Fluids used for fixation are called fixing
solutions or fixatives. Fixatives should:
- penetrate the tissue rapidly
- poagulate cell contents into insoluble substances
- protect tissues against shrinkage and distortion
during dehydration, clearing, infiltration and
embedding
42
allow cell parts to become selectively and clearly

visible by means of dyes.
5.1.1 Fixatives may be classified:

- aldehydes:
formaldehyde, glutaraldehyde
- oxidizing agents:
osmium tetroxide, potassium permanganat,
potassium dichromat
- protein-denaturing agents or coagulant:
methyl alcohol, ethyl alcohol, acetic acid
miscellaneous:mercuric chloride, picric acid
5.1.2 Fixatives Components:
Aldehyde
10 % formalin ( 4 % formaldehyde)
40 % formaldehyde
distilled/tap water
Paraformaldehyde
2.26 % NaH PO
2
100 ml
900 ml
41.5 ml
2.52 % sodium hydroxide

8.4 ml
paraformaldehyde
2g
Alcoholic fixative
Carnoys fixatives
absolute ethanol
60 ml
glacial acetic acid
10 ml
chloroform
30 ml
Fixation : 1 5 hours
Picric acid fixative
Gendres fluid
100 % ethanol saturated with picric acid
80 ml
40 % formaldehyde
15 ml
glacial acetic acid
5 ml
Fixation: 4 hours
Bouins fluid
saturated aqueous picric acid solution
40 % formaldehyde
glacial acetic acid
Fixation: 24 hours
Mercuric chloride-containing fixative
Zenkers fluid
43
75 ml
20 ml
5 ml
distilled water
potassium dichromate
mercuric chloride
glacial acetic acid
Fixation : 4 24 hours
950 ml
25 g
50 g
50 g
5.2 Dehydration
Dehydration is a process to remove water and fixatives
from tissue and replace it with dehydrating fluid. Dehydration
achieved in a series of gradually increasing percentage of
alcohol in water. Gradual changing through 30%, 50%,
70%,80%, 90%, 95% and absolute alcohol reduce some tissue
shrinkage. Dehydration can be done by following procedure:
.................
30 minutes
- ethanol 30 %
.................
30 minutes
- ethanol 50 %
30 minutes
- ethanol 70 %
30 minutes
- ethanol 80 % ...
30 minutes
- ethanol 90 %
ethanol
95
%
30 minutes
1 hour
- ethanol(absolute) 1 ..
ethanol(absolute)
2..
1
hour
5.3 Clearing
Alcohol used for dehydration will not mix with paraffin,
some fluid that miscible with both alcohol and paraffin must be
used to remove alcohol. Paraffin can infiltrate the tissue if
alcohol completely removed. Xylene and creosote usually used
for clearing. One example of clearing procedure:
-
xyleneand absolute ethanol(1: 1) 30 minutes

xylene 1 .. 1 hour
xylene 2 .. 1 hour
5.4 Infiltration
Infiltration is a process by which the tissue is filled with
paraffin. Tissue directly tranferred from clearing agent to melted
paraffin. If thin sections (5-7 microns) are desired, infiltration
used paraffin with melting point 56-58 oC. For extremely thin
sections ( less than 5 microns), hard paraffin with melting point
60-68 oC can be used to obtained the good result.Tissues are
kept in melted paraffin 0.5- 1 hour in the oven. Two changes of
paraffin are sufficient. Horny skin, bone and brain need third
44
changes and infiltration time may be extended to six hours or

even overnight.
5.5 Embedding/Blocking
Infiltrated tissues are immediately transferred to
container or block and fill with melted paraffin to block the
tissues. The tissues are oriented based on the tissue section
desired. Placing and orienting the tissue is conducted in warm
temperature to prevent paraffin harden rapidly. Hard paraffin
block can be cooled by placing it in water. Cooled paraffin block
is ready for trimming and cutting by using rotary microtome.
Figure 20. Cooling paraffin block

5.6 Cutting/Sectioning
After fixing, dehydrating, infiltrating the specimen and
embedding it, a paraffin block containing the specimen
surrounded with paraffin wax should be obtained. The surplus
paraffin needs to be removed and the paraffin block trimmed in
its proper form prior to sectioning.The paraffin layer
surrounding the specimen is not too thick. The larger the surface
of the paraffin block to be sectioned, the higher the risk of
problems in section cutting and it serves no purpose to cut blank
paraffin. Cutting cause microtome knives/blades dull at the
end.Knife /blades is usedonly for sectioning the embedded
specimen so that there should be minimize paraffin surrounding
the specimen.
If mounting several serial sections under a common
cover slip, the surrounding paraffin should be kept to 1 or 2 mm.
The thinner the surrounding paraffin coat, the more sections can
be mounted under a single cover slip. For sections to be
45
mounted individually, specimen aresurrounded with a layer of 35mm paraffin. If only a single specimen is embedded in a single
block, trimming amounts of paraffin wax off the block every
slide with a scalpel or a razor blade gentlyuntil the specimen is
surrounded with a thin layer of wax. Trying to cut too thick a
layer of paraffin can cause the block and the specimen to crack.
The specimen should be in the exact middle of the paraffin
block.The upper and lower sides of the trimmed block should be
parallel. Improper trimming of upper and lower block sides will
result in curved ribbons of sections.
Figure 21. Trimming the paraffin block using a scalpel

5.7 Mounting the trimmed block on a paraffin table
Paraffin block has to be attached to paraffin table made
of pieces of hardwood, aluminum or hard plastic that fit with
specimen clamp (block holder).
Figure 22. Paraffin tables made of hardwood about 3cm x 2,5

cm x1 cm.
46
Mounting the trimmed paraffin block on the table can be done

by warming the knife in the flame of a Bunsen burner. The
trimmed blocks is put on warm knife, and place it to paraffin
table. Paraffin block can be stabilized by melting some small
paraffin shavings around the block. The paraffin block should
cool down completely.Final trim can be done after which the
paraffin block is ready to be mounted on the microtome.
Figure 23. Warming the knife in the flame of a Bunsen burner
Figure 24. The trimmed block is put on warm knife
47
Figure 25. The paraffin block is ready to be mounted on the

microtome
Figure 26. Sectioning: creating a ribbon of sections
5.8 Floating out sections, Affixing and Deparaffinizing

Before specimen ribbon is mounted to slide glass, it lay
on the water surface to remove folds. Single or multiple sections
can be separated from the ribbon drawn up onto the albuminized
(with Mayers albumen) slides (affixing process). Other folds
and air bubbles can be removed when section attached to slide
glass. Slides kept in warm condition (40-45 oC)until water
completely removed. Dried slides are ready for further
processing (deparaffinizing). Deparaffinizing can be done be by
place section in xylene , two times changes, 10 minutes each.
48
Figure 27. Floating out sections
Figure 28. Picking up the ribbon onto the slide glass

5.9 Staining (Hematoxylin and Eosin)
Hematoxylin is a basic dye, stains acidic structure of the cell
(nucleus, ribosome and endoplasmic reticulum). Hematoxylin is
extracted from the logwood tree (Haematoxylincampechianum).
. It is oxidized and combined with a mordant to allow it to bind
to the cell structures. Harris's hematoxylin and Mayer's
hematoxylin are often used in histology staining. Eosin is an
acidic dye , stains basic structures (cytoplasm, cell walls, and
extracellular fibres). Eosin is formed by a reaction between
bromine and fluorescein. There are two eosin: eosin Y which is
slightly yellowish and eosin B which is slightly bluish.
49
Figure 29. Hematoxylin chemical structure
Figure 30. Eosin Y chemical structure
5.9.1 Two staining procedure that common use in animal

technique
StainingProcedure 1 (Mayershematoxylin):
1. Deparaffinize sections, 2 changes of xylene, 10 minutes
each.
2. Re-hydrate in 2 changes of absolute alcohol, 5 minutes
each.
3. 95% alcohol for 2 minutes and 70% alcohol for 2 minutes.
4. Wash briefly in distilled water.
5. Stain in Mayershematoxylin solution for 8 minutes.
6. Wash in warm running tap water for 10 minutes.
7. Rinse in distilled water.
8. Rinse in 95% alcohol, 10 dips.
9. Counterstain in eosin-phloxine B solution (or eosin Y
solution) for 30 seconds to 1 minute.
50
10. Dehydrate through 95% alcohol, 2 changes of absolute

alcohol, 5 minutes each.
11. Clear in 2 changes of xylene, 5 minutes each.
12. Mount with xylene based mounting medium.
StainingProcedure 2 (Harrishematoxylin):
1. Deparaffinize sections, 2 changes of xylene, 10 minutes
each.
2. Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.
3. 95% alcohol for 2 minutes and 70% alcohol for 2 minutes.
4. Wash briefly in distilled water.
5. Stain in Harrishematoxylin solution for 8 minutes.
6. Wash in running tap water for 5 minutes.
7. Differentiate in 1% acid alcohol for 30 seconds.
8. Wash running tap water for 1 minute.
9. Bluing in 0.2% ammonia water or saturated lithium
carbonate solution for 30 seconds to 1 minute.
10. Wash in running tap water for 5 minutes.
11. Rinse in 95% alcohol, 10 dips.
12. Counterstain in eosin-phloxine B solution (or eosin Y
solution) for 30 seconds to 1 minute.
13. Dehydrate through 95% alcohol, 2 changes of absolute
alcohol, 5 minutes each.
14. Clear in 2 changes of xylene, 5 minutes each.
15. Mount with xylene based mounting medium.
Results:
Nuclei ---------------------------------------- blue
Cytoplasm ---------------------------------- pink to red
Figure 31. Histology of kidney (H & E Staining)

51
5.9.2 Varies Stain Compositions

Mayer's haematoxylin:
Haematoxylin - 1g
Distilled water - 1 litre
Potassium or ammonium alum - 50g
Citric acid - 1g
Chloral hydrate - 50g
Sodium iodate - 0.2g
Add the haematoxylin, potassium alum and sodium iodate to the
distilled water and dissolve by warming and stirring, or by
standing overnight at room temperature. Add the chloral hydrate
and citric acid and boil the mixture for 5 minutes. Coolandfilter.
The solution is then ready for use.
Harris' haematoxylin:
Haematoxylin - 5g.
100% alcohol 50 mls
Potassium alum - 100g.
Distilled water - 1 litre
Mercuric oxide - 2.5g.
Glacial acetic acid - 40mls
Dissolve the potassium alum in the water by warming and
stirring. Dissolve the haematoxylin in the alcohol and add.
Bring rapidly to the boil remove from the heat and add the
mercuric oxide. Cool, add the acetic acid and filter. The Staining
ready for use immediately.
Eosin Y Solution:
Eosin Y Stock Solution (1%):
Eosin Y --------------------------------------- 10 g
Distilled water ------------------------------- 200 ml
95% Ethanol ---------------------------------- 800 ml
Mix to dissolve and store at room temperature.
Eosin Y Working Solution (0.25%):
Eosin Y stock solution ------------------ 250 ml
80% Ethanol ------------------------------ 750 ml
Glacial acetic acid (concentrated) ----- 5 ml
Mix well and store at room temperature.
Eosin Stock Solution:
Eosin Y ------------------------------------ 1 g
Distilled water --------------------------- 100 ml
52
Mix to dissolve.
Phloxine Stock Solution:
Phloxine B ------------------------------1g
Distilled water -------------------------- 100 ml
Mix to dissolve.
Eosin-Phloxine B Working Solution:
Eosin stock solution ------------------ 100 ml
Phloxine stock solution -------------- 10 ml
Ethanol (95%) ------------------------- 780 ml
Glacial acetic acid -------------------- 4 ml
Mix well.
5.9.3 Clearing (Differentiation) Reagent for H&E Staining
1% Acid Alcohol Solution:
Hydrochloric acid ---------------- 10 ml
70% ethanol ---------------------- 1000 ml
Mix well and store at room temperature
Differentiate 30 second to 2 minutes after hematoxylin
staining
Bluing Reagent
Lithium Carbonate Solution (Saturated):
Lithium carbonate ---------------------------- 1.54 g
Distilled water -------------------------------- 100 ml
Mix to dissolve and store at room temperature.
Bluing for 30 seconds to 1 minute after hematoxylins taining
and clearing/differentiation
0.2% Ammonia Water Solution (Bluing):
Ammonium hydroxide (concentrated) ------ 2 ml
Distilled water -------------------------------- --1000 ml
Mix well. The pH will be around 10.0 Store this solution at
room temperature.
Bluing for 30 seconds to 1 minute after hematoxylin staining
and clearing/differentiation.
5.10 Mounting
Mounting is the final stage in the preparation of tissues
for microscopy. Mounting media is called mountant.
a mountant should possess certain characteristics:
53
- colourless and transparent

- able to completely permeate tissue
- resistant to contamination
- set without crystallising, cracking or shrinking
- not react with or induce fading in stains and reaction
products
- miscible with dehydrant or clearing agent
Mounting media are divided into categories: hydrophobic and
hydrophilic. Hydrophobic mountants are used for sections that
need to be dehydrated and cleared before they are applied.
Canada balsam, DPX (distrene, plasticiser, xylene), and
permount are hydropobic mountants. Sections are mounted in
hydrophilic media directly from water. Water, glycerol and
phosphate buffer glycerol are hydrophilic mountants.
After staining process, section is covered with mountants
coverslip. Coverslip was pressed gently to ensure that mountants
are not too much and all tissue parts are covered with mountant.
Too thick mountant can cause the image of tissue is not focusly
examined.
Figure 32. Coverslipping
SELF-QUIZ
1. Which one of the following subtances is not a fixative?
a. osmium tetroxide
b.potassium permanganat
c. methanol
d.formaldehyde
e. hidrochloric acid
54
2. Fixatives functions in animal tissue processing is :
a. coloring tissue
b. dehydrating tisssue
c. protecting tissue against distortion
d. clearing tissue
e. allowing cell parts to shrink
3. Which one of the following substances is a clearing
agent?
a. ethanol
b. xylene
c. canada balsam
d. aldehyde
e. lithium carbonate
4.Mounting media should posses the following
characteristics, except:
a. permeable to tissue
b. tranparent
c. induce stain fading
d. miscible with clearing agent
e. set without shrinking
5. Which one of the following fluid should be absent in the
end of infiltration process?
a. water
b. ethanol
c. paraffin
d. water and ethanol
e. ethanol and paraffin
6. Clearing agent used in clearing process must:
a. miscible to water
b. miscible to ethanol
c. miscible to ethanol and paraffin
d. miscible to water and ethanol
e. miscible to water and paraffin
7. Imersing haematoxylin- stained tissue in tap water is to:
a. blue the tissue
b. redden the tissue
c. fix the tissue
d. dehydrate the tissue
e. decolorize the tissue
8. Cutting too thick layer of paraffin block can cause:
a. tissue can not be stained
b. tissue cracked
c. tissue poorly absorb stain
55
d. tissue easily be deparafinized

e. tissue hardly be examined
9. Haematoxylin:
a. can bind directly to the tissue
b. is acidic dye
c. needs oxidation to bind the tissue
d. stain cytoplasm
e. stain acidic stucture of the cell
10. Which one of the following tissues needs a longest time
during infiltration?
a. liver
b. kidney
c. gaster
d. bone
e. spleen
56
CHAPTER 6
ANIMAL WHOLE MOUNT
Objective
1. Understand general whole mount preparation
2. Understand whole mount fluorescent immunohistochemistry
3. Understand antibody staining of whole mount Drosophila
embryos
4. Understand whole mount immunohistochemistry of
zebrafish
5. Know the real internal structure of embryo and to look for
abnormalities of embryo without sectioning first
6. Conduct trouble shooting when facing with un-expected
result
6.1 What is whole mount?

Whole mount is placing a whole organism or specimen
on a slide for microscopic examination. Whole mount staining is
the staining of small pieces of tissue, usually embryos, without
sectioning onto slides first. This is often used on embryos by
stem cell and embryonic development researchers and also
neuroscientists who are able to stain the whole embryos at
various stages to follow the expression of target proteins through
the development of the animal.
Whole mount staining is very similar to staining of
cryosections or immunocytochemistry (ICC). If an antibody has
been used successfully on cryosections (IHC-Fr - this does not
including paraffin embedded sections), then the antibody should
work for a whole mount embryo. The difference being that the
sample being stained is much larger, and thicker, than a normal
section on a slide. Therefore, incubations for fixative, blocking
buffer, antibody, wash buffer, permeabilization and substrate
color development will need to be much longer to allow for
permeabilization right into the centre of the sample.
Researchers use different times, but the details in these
procedures provide a guideline for optimizing the experiment at
these stages if necessary. As introduction before describe in
details some specific methods and example preparation whole
mount, we will elaborate in brief about fixation, image
obtaining, and choosing embryo age.
57
6.2 An important note on fixation

Whichever fixative has been successfully used in IHC-Fr
with the antibody you have chosen should be suitable for whole
mount. However, most researchers use 4% paraformaldehyde
(PFA). Although this concentration of PFA is very low, this has
to be left on for a long period of time on whole mount samples
to allow for permeabilization to the centre of the sample.
Therefore, this will not be suitable for all antibodies, as the
protein cross linking formed by the fixative may block access of
the antibody to the epitope. Normally, in IHC-P, we could
perform antigen retrieval. This is not possible on embryo
samples as the heating procedure would destroy the sample. If
PFA fixation does not work for the whole mount tissue, then
there is a possibility the antibody is sensitive to the protein
crosslinking, and you will require another fixative. Methanol is a
popular second choice of fixative when optimizing whole mount
procedures. In case zebrafish, zebrafish embryo fixation and
preparation requires extra steps to fix and permeabilize to ensure
the egg membrane is permeabilized.
6.3 Obtaining images
Some researchers view and obtain images of embryos as
they are. The whole embryo can be imaged while floating in
glycerol buffer in a petridish, before mounting. If small enough,
the whole embryo can be mounted in glycerol before setting in a
coverslip. In this case, grease should be used around the corner
of the coverslip to help keep it in place and prevent damage to
the coverslip when using the microscope. However, they can
also be set in gelatin and sectioned if it is difficult to obtain a
clear view of the staining through the whole embryo
(particularly at larger late embryo stages or larger tissue
samples). If immunofluoresent labeling is used, then confocal
microscopy can be a useful tool to scan through the embryo,
rather than sectioning the whole embryo onto separate slides
after staining.
6.4 Choosing the age of the embryo
This is important as the embryo grows, it will become too large
to stain. The various reagents, including fixative, antibody and
developing solution will not be able to permeate to the centre of
the sample, and the number of stained cells will make obtaining
a clear image very difficult. However, larger and older embryos
can be dissected into segments before staining if necessary.
58
Recommended ages:
Chicken embryos: up to 6 days
Mouse embryos: up to 12 days
6.5 Whole mount fluorescent immunohistochemistry
The advantage of using fluorescence to stain whole
mount sections is that confocal microscopy can be used to
section through the larger embryo or tissue sample without
having to manually section onto slides. This gives a clearer idea
of where the target protein of interest is expressed within the
tissues.
Procedure:
1.
Obtaining the embryo:
Chicken: Gently break the egg into a medium sized clean
glass petridish. The embryo will naturally float to the top
of the yolk. It will then be visible for careful removal
using clean scissors and a Pasteur pipette with the tip
removed (this prevents any damage to the embryo from
the narrow end of the pipette).
Mouse: Operate on adult female to remove embryos.
Dissect the embryo in ice cold PBS removing as much
unwanted tissue as possible. \
We recommend to remove as much embryonic membrane
and excess tissue as possible as this can prevent the
antibody perfusing into the embryo.
2.
Place embryo in a 5 ml bijous in 4% paraformaldehyde.
Leave to fix 4oC. The time
required will need
optimization. We suggest trying between 2 hours and
overnight. OR fix in m-DMSO (80% methanol, 20%
DMSO) or other fixative of choice.
Generally whichever fixative has been used successfully
with the antibody when used in cryosections, this fixative
should be suitable for whole mount. However, this may
require some optimization.
When the sample is fully equilibrated with the fixative (i.e
the fixative has permeabilized the whole sample) then it
should sink to the bottom of the solution. Ensure the
sample has sunk to the bottom of the fixative before
proceeding.
3.
Wash 3X in PBS 0.5 - 1% Triton thirty minutes each time.
4.
Incubate the embryos twice for 1 hr in block (PBS 1%
Triton + 10% FCS + 0.2% Sodium Azide), room
temperature.
59
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Incubate embryos in peroxidase block (0.1% H2O2 diluted

in blocking buffer) overnight 4oC.
Wash embryos 2X in blocking buffer.
Transfer embryos using Pasteur pipette with the end cut
off to a 2 ml tube. Add primary antibody at the required
dilution /concentration. It is recommended that as
incubations
can be very long in whole mount
staining, the antibody should be diluted in blocking
buffer containing 0.02% sodium azide to prevent
microbial growth.
Incubate for 1 to 4 days on a gentle rotation devise at 4 oC.
This incubation time will require some optimization
depending on the antibody and also the size of the embryo.
Wash embryos 3X 1 hr in PBS 1% Triton + 10%
FCS0.2% sodium azide
Wash 3X 10 minutes in PBS 1% Triton Wash well to
remove traces of sodium azide as this will inhibit
peroxidase activity when developing
Add secondary antibody in blocking buffer (no sodium
azide)
Incubate for 2 to 4 days with gentle rotation 4oC
Wash 3X 10 minutes in PBS 1% triton
Incubate embryos in DAB substrate for 2 to 3 hrs RT.
Transfer embryos to a dish and add fresh DAB plus 5 ul
H2O2 per 1 ml of DAB.
Rinse three times in PBS once reaction and staining have
reached desired intensity
Mount and view embryos. Store at 40C until analysis.
Mounting:
1.
Place sample in 100% glycerol for 48 hours. When sample
is fully equilibrated with the glycerol (i.e it is fully
perfused with the glycerol) it will sink to the bottom of the
vial. Ensure the sample is at this stage before proceeding.
2.
75% glycerol has approximately the same density as
gelatin which is used to mount and set the samples on a
slide. Therefore, samples should be equilibrated in 75%
glycerol after staining for approximately 1(5 minutes)
(again, when equilibrated, the sample should sink).
3.
Place in 50% glycerol until the sample sinks. The embryo
can be imaged at this stage, or mounted whole in the
glycerol on a slide. Use grease around the edges of the
cover slip for protection. If the sample is to be embedded
60
in gelatin and sectioned on a vibratome; place 20% gelatin

pre-warmed to 65oC. Leave for approximately 30 minutes
to equilibrate before taking out the sample to mount.
When equilibrated in the gelatin, the sample should sink to
the bottom.
6.6 Antibody staining of whole mount Drosophila embryos
Procedure :
1.
Prepare fixation mix in 1.5 ml cap: 400 l PBS, 100 l
40% formaldehyde and 500 l n-heptane. For the detection
of some extracellular antigens it helps to add 0.02% SDS
or to fix the embryos in 500 l picric acid / 500 l nheptane. Vortex the fixation mix at highest speed for 1
min.
2.
Remove yeast and dechorionate embryos by covering the
apple-juice plate with 100% bleach for 2min; light
rotations of the apple-juice plate will bring the
dechorionated embryos to the surface.
3.
During bleaching: close the narrow opening of a funnel
with a nylon-mesh (diameter of holes: 40 m), attach
mesh by wrapping a rubber band around the funnel.
4.
Wash embryos into the funnel by squirting deionized
water over the plate, especially along the edges of the
plate; wash embryos in funnel by squirting water along the
funnel three times.
5.
Remove rubber band and mesh; pick up the embryos with
a very fine paint brush; dechorionated embryos will adhere
to the brush; dip brush with embryos into the fixation mix
and whisk the brush with the cap lying on its side fix
embryos for 15 min on a shaker with gentle rotation.
6.
During fixation: heat the tip of a glass Pasteur pipette over
a Bunsen burner; aim the flame at the middle of the tip and
hold the end of the tip with two fingers (glass is a bad heat
conductor therefore your fingers are safe); when the
middle glows red remove the tip from the flame and
immediately pull; break away the long drawn out and very
thin part from the tip. As an alternative to the glass pipette
it is possible to use a 1 ml Gilson pipette.
7.
To stop fixation remove about 80% of the lower phase of
the fixation mix with the pipette; fill up the cap with 1 ml
of methanol and vortex on highest speed for about 1min.
This procedure will remove the extra embryonic
membrane.
61
8.
9.
10.
11.
12.
13.
14.
15.
16.
2 min after vortexing the majority of the embryos will be

at the bottom of the cap. If not all embryos sink to the
bottom of the cap, remove as much of the liquid as
possible and refill the cap with methanol. Do not vortex
again. After most of the embryos are at the bottom of the
cap, remove all liquid and wash embryos three times with
methanol.
Rehydrate the embryos with three washes in PBT (PBS
with 0.3% Triton added); incubate in 20% newborn calf
serum / PBT on shaker for at least 10 min. Note that
Triton only slowly dissolves in PBS i.e. set up the solution
about 20 min in advance. PBT can be kept at room
temperature indefinitely. Calf serum should be stored in
the fridge and 0.02% sodium azide should be added.
Add primary antibody diluted in 10% NCS / PBT and
0.02% sodium azide; most primary antibodies can be used
at least three times i.e. store the primary after the first
incubation at 4oC for further stainings; diluted 1:1000
(Abcam) ab290 can be re-used five times.
Depending on the quality of the primary antibody,
incubations can last from 2 hr at room temperature (high
affinity antibodies) to overnight at 4oC (low affinity
antibodies). Overnight incubation at 4oC aides the
perfusion of the antibody. For (Abcam) ab290 2h at room
temperature are sufficient.
Wash procedure: three rinses with PBT, 10min incubation
with 30% NCS / PBT, three rinses with PBT,10 min
incubation with 30% NCS / PBT, three rinses with PBT.
Add secondary antibody diluted in 10% NCS / PBT for 2
hr at room temperature, lay cap on its side on a gently
moving shaker.
Repeat step 13.
Non-fluorescent detection: use Diaminobenzidine assay
(normal signal) or Alkaline Phosphatase assay (weak
signals); develop Alkaline phosphatase signal in the dark
and take an aliquot of embryos out of the cap with a 1ml
Gilson pipette to follow the staining; postfix embryos with
4% formaldehyde / PBT for 10 min to stabilize alkaline
phosphatase signal.
Enhancement of very weak signals: use Vectastain ABC
Elite Kit, or TSA Kit Fluorescent detection: use secondary
antibodies coupled to Alexa488, Alexa568 or Cy5
62
17.
Incubate embryos in 50% glycerol / PBS until they sink to

the bottom of the cap (about 10 min); replace the 50%
glycerol l /PBS with 70% glycerol / PBS wait again until
embryos are at the bottom of the cap (about 1hr); replace
70% glycerol/ PBS with 90% glycerol / PBS and allow
embryos to sink to the bottom of the cap.
Embryos can be stored in 90% glycerol / PBS at 4oC for at
least three years. There is no need for any light protection
for fluorescent staining.
6.7 Zebrafish whole mount immunohistochemistry

Whole mount staining of Zebrafish embryos, now
commonly used, requires extra steps to fix and permeabilize to
ensure the egg membrane is permeabilized. Incubations for
fixative,
blocking
buffer,
antibody,
wash
buffer,
permeabilization and substrate color development will need to
be much longer than normal immunocytochemistry /
immunohistochemistry to allow for permeabilization right into
the centre of the sample.
Procedure:
1.
Place embryo in a 5 ml bijous in fixative for 1 hour. The
fixative should be chosen carefully, depending on what
has been previously used successfully with the antibody in
cryosections, and also on the target protein. Use 4%
paraformaldehyde (PFA), or perfix (this is very suitable
for neuro markers). Use a Pasteur pipette with the tip
removed to move the embryo from one vial to another or
to add or remove reagents. (this prevents any damage to
the embryo from the narrow end of the pipette).
2.
Wash at least 4X for 5 min in PBS 1% triton.
3.
Permeabilize the embryos in ice cold acetone / PBS for 8
minutes only. The acetone will help to permeabilize the
tougher egg membrane, which is not necessary for other
species such as chick or mouse
4.
Wash at least 4X for 5 min in PBS 1% triton.
5.
Incubate the embryos twice for 1 hr in block (PBS 1%
Triton + 10% FCS), room temperature.
6.
Incubate embryos in peroxidase block (0.1% H2O2 diluted
in blocking buffer) overnight 4oC.
7.
Wash embryos 2X in blocking buffer
8.
Transfer embryos using Pasteur pipette with the end cut
off to a 2 ml tube. Add primary antibody atthe required
63
9.
10.
11.
12.
13.
14.
15.
dilution /concentration. It is recommended that as

incubations can be very long in whole mount staining, the
antibody should be diluted in blocking buffer containing
0.02% sodium azide to prevent microbial growth.
Incubate for 1 to 4 days on a gentle rotation devise at 4oC.
This incubation time will require some optimization
depending on the antibody and also
the size of the
embryo.
Wash embryos 3X 1 hr in PBS 1% Triton + 10% FCS
Wash 3X 10 minutes in PBS 1% Triton
Incubate embryos in DAB substrate for 2 to 3 hrs RT.
.Transfer embryos to a dish and add fresh DAB plus 5 l
H2O2 per 1 ml of DAB.
Rinse three times in PBS once reaction and staining have
reached desired intensity
Mount and view embryos. Store at 40C until analysis.
Mount in melted 1% agarose in PBS. Once the agarose is
set, add 70% glycerol / PBS to cover the agarose and
place a coverslip on the sample.
6.8 Troubleshooting tips whole mount

Very similar difficulties to immunocytochemistry (ICC)
and immunhistochemistry (IHC) can occur when staining whole
mount tissue. The following tips are more specific to whole
mount staining. Most of these relate to the fact that incubation
times for all reagents, and wash steps, need to be much longer
that in ICC or IHC to allow penetration through the sample,
which will be much larger than a tissue section.
6.8.1 High background
Fixative used is not suitable for the antibody.
Most researchers use PFA for fixation. As antigen
retrieval methods are not recommended for whole mount (it can
destroy the tissue), ensure the concentration of this is no more
than 4%. This should cause fewer difficulties with protein cross
linking. Some antibodies will still be sensitive to the small
amount of protein cross linking at this lower percentage PFA,
and PFA fixation will not be suitable for some antibodies. The
usual alternative to PFA is methanol fixation. However, we
would recommend checking the antibody datasheet to obtain
information on fixation agents used successfully in whole mount
sections with the antibody you are using. If this information is
not available, fixatives used successfully in cryosections are
64
usually successful in whole mount. Fixation time may also

require optimization.
Antibody left on for too long.
The recommendation when optimizing antibody
concentration in whole mount is to start with 3 or 4 day
incubation. If this is not successful, work backwards to 1 day
(antibody will need to be on the sample for at least 24 hours to
ensure full penetration through the sample).
Microbial contamination.
As the incubations in whole mount staining are very
long, microbial contamination can become a problem. This can
lead to non specific background staining. Use clean glassware
(preferably sterile) and fresh reagents. 0.2% sodium azide can be
added to antibody buffers and blocking buffers wherever
possible. Please note this should not be added to peroxidase
conjugated secondary antibodies as it can inhibit the enzyme
activity. Follow washing guidelines carefully before adding
peroxidase conjugated secondary antibody to ensure any sodium
azide is washed away.
Wash steps not sufficient.
Follow the wash step guidelines provided in the
protocols. Wash steps will need to be long enough to permeate
and wash through the whole sample. Triton rather than Tween is
used as this is a stronger detergent which will permeate more
easily.
6.8.2 No signal
Antibody not left on for long enough www.abcam.com/technical
Antibody incubations should be much longer than when staining
sections on slides to ensure adequate permeation to the centre of
the sample. The recommendation when optimizing antibody
concentration in whole mount is to start with 3 or 4 day
incubation. If this is not successful, work backwards to 1 day
(antibody will need to be on the sample for at least 24 hours to
ensure full penetration through the sample).
Incorrect incubation times.
Incubations for fixative, blocking buffer, antibody, wash
buffer, permeabilization and substrate color development will
need to be much longer to allow for permeabilization right into
the centre of the sample.
Fixative used is not suitable for the antibody.
Most researchers use PFA for fixation. As antigen
retrieval methods are not recommended for whole mount (it can
65
destroy the tissue), ensure the concentration of this is no more

than 4%. This should cause fewer difficulties with protein cross
linking. Some antibodies will still be sensitive to the small
amount of protein cross linking at this lower percentage PFA,
and PFA fixation will not be suitable for some antibodies.
The usual alternative to PFA is methanol fixation. However, we
would recommend checking the antibody datasheet to obtain
usually successful in whole mount. Fixation time may also
require optimization.
Incorrect detergent used in buffers.
In order to allow full permeabilization of reagents and
antibody through the whole sample, Triton rather than Tween is
normally used. This is a stronger detergent which will permeate
more easily. It is also used at a relatively high concentration of
0.5 to 1%.
Zebrafish egg membrane not permeabilized.
Whole mount staining of Zebrafish embryos requires
extra steps to fix and permeabilize to ensure the egg membrane
is permeabilised. Fix for 1 hour, wash in PBS 1% triton then
permeabilize the egg membrane in in ice cold acetone / PBS for
8 minutes only. We recommend following the zebra fish whole
mount staining procedure provided.
Mouse and chick extracellular membrane not removed.
Remove as much embryonic membrane and excess tissue
as possible as this can prevent the antibody perfusing into the
embryo.
6.8.3 Patchy staining
Incorrect incubation times.
Incubations for fixative, blocking buffer, antibody, wash
buffer, permeabilization and substrate color development will
need to be much longer to allow for permeabilization right into
the centre of the sample. If any of these reagents have not
penetrated the whole sample, there may be areas of the tissue
not fully washed, fully fixed, or with full access to the antibody.
This will lead to patchy areas where staining is not sufficient.
Air bubbles in the tissue / Inadequate mixing of reagent and
sample.
Ensure the sample is placed on a gentle rotating or
rocking devise whilst incubating to prevent formation of air
66
bubbles and to ensure access of reagent to all the tissue. If any

of the reagents have not penetrated the whole, there may be
areas of the tissue not fully washed, fully fixed, or with full
access to the antibody. This will lead to patchy areas where
staining is not sufficient, or trapped air bubbles in which tissue
will not be stained.
Incorrect detergent used in buffers.
In order to allow full permeabilization of reagents and
antibody through the whole sample, Triton rather than Tween is
normally used. This is a stronger detergent which will permeate
more easily. It is also used at a relatively high concentration of
0.5 to 1%.
Sample is too large.
An embryo will grow to a stage where they are too large
for whole mount staining with good results, and other types of
sample will need to be kept within an optimized size range.
Reagents will not be able to fully penetrate the tissue if it is too
large, and the staining through the sample will be patchy. It is
sometimes possible to dissect the sample into sections which are
more manageable.
6.8.4 Morphology of the tissue is not good.
Inadequate fixation or over fixation.
Ensure the sample has been fixed for long enough to
allow penetration of fixative through the sample. The timing of
fixation may require some optimization. Fix at 4oC. We suggest
optimizing between 2 hours and overnight.
Sample has been heated or treated for antigen retrieval.
Heat treating the whole mount samples for antigen
retrieval is not possible, as it destroys the structure of the tissue.
Try using another fixative, rather than PFA. The usual
alternative to PFA is methanol fixation. However, we would
recommend checking the antibody datasheet to obtain
usually successful in whole mount.
Sample has been crushed whilst handling.
To move the tissue from one vial to another, or to add or
remove reagents, use a plastic Pasteur pipette with the tip
removed (this prevents any damage to the embryo from the
narrow end of the pipette). Avoid use of forceps where possible.
67
SELF-QUIZ
Why whole mount staining is very similar
immunocytoshemistry? Please explain your answer !
68
to
CHAPTER 7
BLOOD SMEAR
Objective
1. Make and stain a blood smear with Giemsa staining
2. Make and stain a blood smear with Romanowsky

staining .
A well-made blood smear is a beauty to be hold, and
likely to yield interesting and significant information for a
research project. A poor slide is a torment. The extra time and
care taken during the field season will be rewarded later when
the smears must be scanned, and parasites identified and
counted. Here, the methods for making and staining smears are
given, as well as a list of sources for high quality slides, stain,
and chemicals.
Dried blood samples for genetic studies should always be made
at the same time as the smears. The method is very easy and
modern research must combine studies of morphology under the
microscope with molecular methods. The technique for making
and storing dried blood samples is given in the section Dried
Blood Samples.
7.1 Making a smear
1. A single smear can be made per slide (smear running the
length of the slide) or two (or even three) smears can
share a slide, with the smears running the width of the
slide. Putting two smears per slide saves on weight (glass
is heavy) for field trips, and storage space.
2. It is easiest to use microscope slides with a frosted end,
so that identifying information can be written there with
pencil. Warning: Compare different pencils to find one
that does not yield labels that rub off or wash off in the
methanol dip.
3. Place a drop of blood approximately 4 mm in diameter
on the slide (near the end if one smear is to be made, or
at the proper location if two smears are to share a slide).
See the drawing below.
4. Spread the drop by using another slide (called here the
spreader), placing the spreader at a 45 angle and
BACKING into the drop of blood. The spreader catches
69
the drop and it spreads by capillary action along its edge.

To make a short smear, hold the spreader at a steeper
angle, and to make a longer smear, hold it closer to the
drop. Now, push the spreader across the slide; this
PULLS the blood across to make the smear. Do not push
the blood by having it ahead of the smearing slide! It
should take about one second to smear the drop. A
smooth action is required, with the edge of the spreader
held against the slide. This will yield a nice, even smear.
5. If doing one smear per slide, the spreader then becomes
the next slide to receive a smear. Thus, each slide serves
two duties, as a spreader, then as a slide to receive a
smear. If two smears are made per slide, be sure to flip
over the spreader to use the other edge for the second
smear produced. The spreader then is used to receive the
next two smears. Warning: If there is surplus blood on
the spreader, wipe it off carefully before flipping it over
to make the second smear on the slide
6. For blood taken from mammals, a THICK blood film
can also be made, but this is not possible with blood
from birds or reptiles. Only mammals have erythrocytes
that lack a nucleus. Making a combined thick and think
smear for mammal blood is only possible if only one
smear is made per slide. Make the thin smear starting
about 1/3 from the non frosted end of the slide. Then,
place another drop of blood at the clear end and use the
edge of the smearing slide to spread the drop out to about
a 1 cm circle. The thick smear will take longer to dry.
Because the erythrocytes of mammals lack a nucleus,
thousands of cells can be stacked, and parasites still seen
(not for identification, but simply to detect an infected
animal).
7. Smears should be air-dried, and then dipped into 100%
methanol. A coplin jar with a screw top is best for this.
We use a plastic version, which wont break in the field,
but has a poorly sealing top. Slides can be stored while
drying in a small plastic slide box (holds 25 slides).
Then, they are placed, two at a time, back-to-back, into
the slots in the coplin jar. Thus, ten slides can be dipped
at once. Be sure the alcohol does not reach the frosted
end of the slide. After one minute, the slides are removed
and placed on end to drain the alcohol. They can then be
placed into a plastic slide box for complete drying.
70
8. In the field, we place the plastic slide box or boxes into a

zip-lock bag with silica gel, and they are allowed to dry
overnight.
9. To store slides during long field trips, and where many
slides are to be made, they can be placed back into their
original cardboard boxes, with a piece of index card or
other clean paper between each slide.
Figure 33. Making the blood smear

7.2 Preparing staining buffer
Stock buffers (two)
The alkaline stock is Sodium phosphate, dibasic
anhydrous, H2HPO4. Mix 9.5 gm with distilled water to make
1000 mL. The acid stock is Potassium phosphate monobasic
71
anhydrous, KH2PO4, mix 9.07 gm with distilled water to make

1000 mL Working buffer: Mix 39 mL of acid stock with 61 mL
of the alkaline stock, and 900 mL of distilled water. Check pH,
and adjust to ph 7 or 7.2 by adding the acid buffer stock to lower
pH or alkaline to raise pH. Just a very few mL should be
necessary to reach the required pH.
Other supplies
Microscope slides. Good-quality slides seldom will retain any
oil from machines used in their manufacture, so cleaning should
not be required.
Giemsa. Not all Giemsa stains are equal in quality.
This plastic bottle has a pour spout that ALWAYS leaks. So,
store the bottle in a plastic bag and always handle the bottle
through the bag. Giemsa stain will color skin for several days!
Slide boxes. Use blue plastic slide boxes that hold 25 slides.
Coplin jars. The plastic jar used in the field for dipping into
methanol.
7.3 Blood Smear Preparation with Romanowsky Staining
Objectives:
1. To prepare at least five slide smears which are even, smooth
and have an acceptable feathered edge.
2. To stain and determine the acceptability of the Wright stained
blood smear by evaluating that all formed elements are
readily identifiable according to criteria outlined in the
textbook.
Principles:
Smear Preparation
A small drop of blood is placed near the frosted end of a
clean glass slide. A second slide is used as a spreader. The
blood is streaked in a thin film over the slide. The slide is
allowed to air-dry and is then stained.
Wrights Stain
The Wrights stain is a Romanowsky stain. A Romanowsky
stain is any stain combination consisting of eosin Y or eosin
B with methylene blue and/or any of its oxidations products.
Such stains produce the typical purple coloration of leukocyte
nuclei and neutrophilic granules as well as the numerous blues
and pinks found in other cell types. Methyl alcohol is used as
both a solvent and fixative in this procedure.
72
Specimen:
EDTA anti coagulated blood is preferred. Blood smears can
also be made from finger stick blood directly onto a slide.
Reagents, supplies, and equipment for Slide Preparation
1. Glass slides, 3 x 1 inch with frosted edge
2. Capillary tubes, plain or DIFF-Safe dispensers or wood
applicator sticks
Slide Preparation Procedure:
Three methods may be used to make blood smears:
1) the cover glass smear
2) the wedge smear
3) the spun smear. The spun smear requires an
automatic slide spinner.
For the purpose of this lab exercise, we will use the wedge
smear.
The wedge smear
1. Fill a capillary tube full with the anti coagulated
specimen.
2. Place a drop of blood, about 2 mm in diameter,
approximately inch from the frosted area of the slide.
Note: DIFF-SAFE dispensers or wood applicator
sticks can also be used to place the droplet of blood on
the slide.
3. Place the slide on a flat surface, and hold the narrow side
of the non frosted edge between your left thumb and
forefinger.
4. With your right hand, place the smooth clean edge of a
second (spreader) slide on the specimen slide, just in
front of the blood drop.
5. Hold the spreader slide at a 30 angle, and draw it back
against the drop of blood.
6. Allow the blood to spread almost to the edges of the
slide.
7. Push the spread forward with one light, smooth, and
fluid motion. A thin film of blood with a feathered edge
will remain on the slide.
8. Label the frosted edge with the date and two patient
identifiers (i.e.: patient name, patient ID, or date of
birth). If given, the sample accession number should also
be on the slide.
73
9. Allow the blood film to air-dry completely before

staining. (Do not blow to dry. The moisture from your
breath will cause RBC artifacts.)
10.Notify instructor once five acceptable slides are made.
Once the instructor has checked you off, the student can
proceed with staining two of the five acceptable slides.
7.3.1 Slide Preparation Procedural notes
1. A good blood film preparation will be thick at the drop
end and thin at the opposite end.
2. As soon as the drop of blood is placed on the glass slide,
the smear should be made without delay. Any delay
results in an abnormal distribution of the white blood
cells, with many of the large white cells accumulating at
the thin feathered edge of the smear.
3. The blood smear should occupy the central portion of the
slide and should not touch the edges.
4. The thickness of the spread when pulling the smear is
determined by the 1) angle of the spreader slide (the
greater the angle, the thicker and shorter the smear), 2)
size of the blood drop and 3) speed of spreading.
5. If the hematocrit is increased, the angle of the spreader
slide should be decreased.
6. If the hematocrit is decreased, the angle of the spreader
slide should be increased.
7. Common causes of a poor blood smear :
a. Drop of blood too large or too small.
b. Spreader slide pushed across the slide in a jerky
manner.
c. Failure to keep the entire edge of the spreader slide
against the slide while making the smear.
d. Failure to keep the spreader slide at a 30 angle with
the slide.
e. Failure to push the spreader slide completely across
the slide.
8. Although this is the easiest and most popular methods for
producing a blood smear, the wedge method does not
always produce a quality smear. The WBCs are
unevenly distributed and RBC distortion is seen at the
edges. Smaller WBCs such as lymphocytes tend to
reside in the middle of the feathered edge. Large cells
such as monocytes, immature cells and abnormal cells
74
can be found in the outer limits of this area. Spun smears

produce the most uniform distribution of blood cells.
7.3.2 Biologic causes of a poor smear
a. Cold agglutinin - RBCs will clump together. Warm the
blood at 37C for 5 minutes, then remake the smear.
b. Lipemia - holes will appear in the smear. There is
nothing you can do to correct this.
c. Rouleaux - RBCs will form into stacks resembling
coins. Document the abnormality. There is nothing that
can be done to correct this on whole blood.
Staining Reagents and Supplies
Slides can be stained manually or on automatic slide
stainers. For this lab, we will use the manual method.
a. Commercial Wrights stain
b. Commercial buffer
c. Deionized water
d. Coplin jars
e. Clothes pin or forceps for holding the slide
f. Drying racks for slides
7.3.3 Staining Procedure
1. Attach a clothes pin (or use forceps) to the thick edge of
the blood smear.
2. Place the slide in the Coplin jar with Wrights stain.
Allow to stand 5-10 seconds.
3. Raise the slide out of the stain and allow the majority of
the stain to run off the slide.
4. Place the slide in the first jar containing deionized water.
Allow to stand 10-20 seconds.
5. Remove the slide carefully and dip several times in the
second jar containing deionized water to rinse off the
excess stain.
NOTE: It may be necessary to change the DI water
frequently if many slides are being stained.
6. Wipe off excess fluid from the back of the slide. Place
the slide upright on a slide drying rack with the feathered
edge up and allow to air dry.
7. When completely dry, examine the smear with the
microscope as follows:
75
7.3.4 Low power (10x) scan

1. Determine the overall staining quality of the blood smear
by evaluating cells.
Cell Type
RBCs
Lymphocytes
Neutrophils
Monocyte
Eosinophils
Basophils
Appropriate Appearance on
Well-Stained Slide
reddish pink.
dark purple nuclei with varying
shades of blue cytoplasm
dark purple nuclei with reddish,
granular cytoplasm.
lighter purple nucleus with a grayblue cytoplasm.
bright red/orange granules
dark purple nuclei and granules.
2. Stain should not be too dark or too pale.

3. There should be no stain precipitate present on smear.
4. Determine if there is proper distribution of the cells on
the smear.
a. Scan the edges and center of the slide to be sure
there are no clumps of RBCs, WBCs or platelets.
Note clumps of similar cells in the feather because
they may be representative of an abnormal
population.
This is sometimes seen in
lymphoproliferative
and
myeloproliferative
disorders.
b. WBCs pulled to the feather end of the blood slide
should NOT exceed 2-3X the number of WBCs
present in the examination. Reject slides that do not
fit this requirement because large cells are
disproportionately dragged to the feather end which
may affect differential accuracy.
7.3.5 High power (40x) scan
1. Find an optimal area for the detailed examination and
enumerations of cells.
a. The area where approximately 50% of the RBCs
show minimal overlapping and 50% are individually
spaced.
b. There should be no area containing large amounts of
broken cells or precipitated stain.
c. The RBCs should have central pallor.
76
d. Nuclei and cytoplasm of WBCs should be the proper

color.
e. Platelets should be clearly visible.
2. Once the student has evaluated the quality of their smear,
the student should ask the instructor to examine the
smear based on stated criteria.
SELF-QUIZ
1. Why in each process on the blood smear preparation we
should keep the slide always in wet condition ? Please
explain your reason !
2. If blood smear is used to look for abnormalities within
the blood. What blood type should be focuses under
microscope observation ?
77
NOTES
78
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Special reference source of MSDS
http://www.safeworkaustralia.gov.au/sites/swa/whsinformation/hazardous-chemicals/sds/pages/sds
80
http://www.ccohs.ca/oshanswers/legisl/msdss.html
http://www.drs.illinois.edu/css/factsheets/msdss.aspx
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81
82
APPENDIX:
MATERIAL SAFETY DATA SHEET

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Table of Contents

5.9 Staining (Hematoxylin and Eosin) ................................. 49

1.1 Reading chemical label on MSDS and Pictogram

Figure 1. Label notifications give warning regarding hazardous

Figure 2. Style hazard symbol based on National Fire Protection

Major injury likely unless

The substance could cause

Irritation or minor reversible

Flammable gases, or very

detonate when exposed to explosive mixtures with water

apron, and an exhaust fume

Dangerous for the

Figure 3. Pictogram according to European style.

the United Nations Globally Harmonised System (GHS) of

Figure 4. Pictogram according to Work Health and Safety

section will describe the

Section 9 : Physical and

Section 10 : Stability and

eye/face protection, skin

This section is designed to give

By consideration that safety is a major concern, five main

3. What interpretation highest number or lowest number of

tissue contain varies pigment, ergastic substance, organelles.

The phenolic pigment that produce during alkali

A. Clearing leaf tissue in general

1. Endriyani (2009) using A. Muelleri

Slice corm clearing

Figure.6 Druse crystal (A) and Raphide crystal (B) in corm of

3. Mount sample with lower epidermis upward. The aim

Chloral hydrate itself is potential to clear tissue (Harijati, 2007).

Figure 5. Infected chickpea hair by Ascochyta rabiei

Clearing-staining tissue also contain more chloral hydrate (Table

solution or mixture of substance until they are transparent then

In general, chromosome slide is prepared using smear or

Smear and squash definitely require clean microscopy slide

Dissolved 20 g potassium dichromate in 100 ml dH2O, add 100

ethanol containing few drops ammonia solution, then wash in

Figure 7. Rectangle petri dish equipped with rod glasses

acid acetic acid

Table 6. The staining that common use in cytology studies

well then filtered.

3. Squeeze out microsporocyte by pushing gently , remove cell

Figure 8. Passing the slide over fire

Figure 9. Onion germination method

Figure 10. Sample spreading

2. Remove the root to container that already filled with mixture

4. Incubate for 10 minute until the tip produce distinct dark

Alcoholic Hydrochloric Acid-Carmine squash method

Figure 11. Part of plant that will be used as sample.

Table 8. Reagents of Nawaschin/Craf

Table 10. Reagents of Johansen

Johansen type (mL)

Table 11. Reagents of combination ethanol and TBA

Figure 13. Series of dehydration solution

use for embedding has melting point 58 C. All of infiltration

Figure 14. Embedding sample in plastic box. A. Pouring liquid

Figure 16. Trimming of paraffin block . A. Single paraffin

Figure 17. Mounting of paraffin block. A. Cutter razor is heated

Figure 18. Sectioning of paraffin block. A. Sectioning process,

Figure 19. Affixing of paraffin ribbon on slide glass. A. Series

base and synthetic base. In plant tissue usually use safranin-fast

Before animal tissues can be visualized using light

allow cell parts to become selectively and clearly

5.1.1 Fixatives may be classified: