Histological techniques involve fixing, processing, sectioning, and staining tissue samples to examine their microscopic structure. Fixation preserves tissues from degradation. Processing dehydrates tissues and embeds them in paraffin wax or other materials to allow thin sectioning. A microtome is used to cut thin sections for staining with dyes like hematoxylin and eosin, which impart color to different tissue components to reveal their structure under a microscope. These techniques prepare tissues for microscopic analysis while minimizing artifacts from handling.

1. 1
Chapter 2 :Techniques in
Histology

2. BIO 300
BIOLOGICAL TECHNIQUES AND SKILLS
SARINI BINTI AHMAD WAKID
FACULTY OF APPLIED SCIENCE
Chapter 2 :Techniques in
Histology
2

3. CHAPTER 2
TECHNIQUES IN HISTOLOGY
Chapter 2 :Techniques in
Histology
3

4. HISTOLOGICAL TECHNIQUE
A. Histology involves the preparation of tissues for examination with a
microscope.
1. Basic methods of histological preparation of tissues.
a. Fix tissue (e.g. 4 % paraformaldehyde + buffer)
b. Dehydrate tissue (alcohol series followed by toluene)
c. Embed tissue in “hard” medium (e.g. wax)
d. Section embedded tissue on a microtome
e. Mount sections on a supportive structure (e.g. slide) that can be
placed on a microscope stage
f. Usually remove the embedding medium
g. Stain tissue (e.g. hematoxylin-eosin)
h. Examine tissue with microscope.
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5. Artifacts
1. Preparative techniques are often “harsh” and can traumatize and
change the natural structure of a tissue.
2. As a result, what you see is not always real !
3. Tissue/cellular structures that are “created” by preparative techniques
for histological specimens are called “artifacts.”
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6. Fixation


Need to prevent autolysis of tissue samples by
enzymes/bacteria present
Fixation may be chemical or less frequently,
physical methods


chemical fixatives stabilize or cross-linking agents
 need small to get complete penetration of tissue –
intravascular perfusion
 routine fixative is 4% formalin, gluteraldehyde – interact
with amine groups of proteins
 EM requires additional fixation – gluteraldehyde and
OsO4 (stains lipids and proteins)
physical fixation – freezing sample
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7. TISSUE FIXATION




Fixation is a complex series of chemical events that differ for the
different groups of substance found in tissues.
Main purpose: to preserve material in a life-like manner.
The aim of fixation:
1- To prevent autolysis and bacterial attack.
2- To fix the tissues so they will not change their volume and shape
during processing.
3- To prepare tissue and leave it in a condition which allow clear staining
of sections.
4- To leave tissue as close as their living state as possible, and no small
molecules should be lost.
Fixation is coming by reaction between the fixative and protein which
form a gel, so keeping every thing as their in vivo relation to each other.
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8. The fixation solutions used for microscopy
intended to:




Penetrate rapidly to prevent post mortem
changes in the cells
Coagulate the cell contents into insoluble
substances
Protect tissues against shrinkage and
distortion during subsequent processing
Allow cell parts to become selectively and
clearly visible when stained.
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9. Factors affect fixation:
- PH.
- Temperature.
- Penetration of fixative.
- Volume of tissue.
According to previous factors we can determine the concentration
of fixative and fixation time.
Types of fixative:
Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and
Picric acid.
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10. Why fix tissue?
Preserve structure. Essentially to make the structural components of
the tissue more durable so that the tissue can be manipulated in
various ways.
• Fixed material is dead. You want to preserve the structure
(chemical and morphological) of the living material so that it
appears the same as it was in life.
• It will never be exactly the same. Important to choose fixative that
does the best job. Fixative used will depend on type of tissue to be
fixed.
Why dehydrate the fixed tissue
Most fixatives are water soluble, most embedding media are nonpolar and are not miscible with water. So, you have to move the
tissue from a polar (water-based) medium to a non-polar medium
(e.g. toluene) that is miscible with the embedding medium.
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11. TISSUE PROCESSING
The aim of tissue processing is to embed the tissue in a solid
medium firm enough to support the tissue and give it sufficient
rigidity to enable thin sections to be cut , and yet soft enough not
to damage the knife or tissue.
Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.
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12. Dehydration
To remove fixative and water from the tissue and replace
them with dehydrating fluid.
There are a variety of compounds many of which are
alcohols. Several are hydrophilic so attract water from
tissue.
 To minimize tissue distortion from diffusion currents,
delicate specimens are dehydrated in a graded ethanol
series from water through 10%-20%-50%-95%-100%
ethanol.
 In the paraffin wax method, following any necessary
post fixation treatment, dehydration from aqueous
fixatives is usually initiated in 60%-70% ethanol,
progressing through 90%-95% ethanol, then two or
three changes of absolute ethanol before proceeding to
the clearing stage.
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13. Types of dehydrating agents:
Ethanol, Methanol, Acetone.
• Duration of dehydration should be kept to the
minimum consistent with the tissues being processed.
Tissue blocks 1 mm thick should receive up to 30
minutes in each alcohol, blocks 5 mm thick require up
to 90 minutes or longer in each change. Tissues may
be held and stored indefinitely in 70% ethanol
without harm
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14. Clearing
Replacing the dehydrating fluid with a fluid that is totally miscible with
both the dehydrating fluid and the embedding medium.

Choice of a clearing agent depends upon
the following:
- The type of tissues to be processed, and the type of processing to be
undertaken.
- The processor system to be used.
- Intended processing conditions such as temperature, vacuum and
pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .
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15. • Some clearing agents:
- Xylene.
- Toluene.
- Chloroform.
- Benzene.
- Petrol.
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16. Embedding

Is the process by which tissues are surrounded by a medium such as agar,
gelatin, or wax which when solidified will provide sufficient external support
during sectioning.

Paraffin wax


properties :
Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced
during the refining of coal and mineral oils. It is about two thirds the density
and slightly more elastic than dried protein. Paraffin wax is traditionally
marketed by its melting points which range from 39°C to 68°C.
The properties of paraffin wax are improved for histological purposes by the
inclusion of substances added alone or in combination to the wax:
- improve ribboning.
- increase hardness.
- decrease melting point
- improve adhesion between specimen and wax
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17. Precaution while embedding in wax
• The wax is clear of clearing agent.
• No dust particles must be present.
• Immediately after tissue embedding, the wax must be rapidly
cooled to reduce the wax crystal size.
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18. 
There are four main mould systems and associated
embedding protocols presently in use :
1- Traditional methods using paper boats
2- Leuckart or Dimmock embedding irons or metal
containers
3- the Peel-a-way system using disposable plastic
moulds and
4- systems using embedding rings or cassette-bases
which become an integral part of the block and serve
as the block holder in the microtome.
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19. Tissue processing
Embedding moulds:
(A) paper boat;
(B) metal bot mould;
(C) Dimmock embedding mould;
(D) Peel-a-way disposable mould;
(E) base mould used with embedding ring ( F) or cassette
bases (G)
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20. 
General Embedding Procedure
1- Open the tissue cassette, check against worksheet entry to ensure the correct
number of tissue pieces are present.
2- Select the mould, there should be sufficient room for the tissue with allowance
for at least a 2 mm surrounding margin of wax.
3- Fill the mould with paraffin wax.
4 Using warm forceps select the tissue, taking care that it does not cool in the
air; at the same time.
5- Chill the mould on the cold plate, orienting the tissue and firming it into the
wax with warmed forceps. This ensures that the correct orientation is maintained
and the tissue surface to be sectioned is kept flat.
6- Insert the identifying label or place the labeled embedding ring or cassette
base onto the mould.
7- Cool the block on the cold plate, or carefully submerge it under water when a
thin skin has formed over the wax surface.
8- Remove the block from the mould.
9- Cross check block, label and worksheet.
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22. Why embed?
a. Tissue will be sectioned. Needs to be durable enough to
withstand the sectioning process. Also, want components
of tissue to remain in their natural positions. Don't want
them to be moved to new positions.
b. Embedding in wax or plastic immobilizes structural
components of tissue. Holds them in place as sectioning
is done.
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23. 

ORIENTATION OF TISSUE IN THE BLOCK
Correct orientation of tissue in a mould is the most important step in
embedding. Incorrect placement of tissues may result in diagnostically
important tissue elements being missed or damaged during microtomy.
elongate tissues are placed diagonally across the block

tubular and walled specimens such as vas deferens, cysts and
gastrointestinal tissues are embedded so as to provide transverse
sections showing all tissue layers

tissues with an epithelial surface such as skin, are embedded to provide
sections in a plane at right angles to the surface (hairy or keratinised
epithelia are oriented to face the knife diagonally)

multiple tissue pieces are aligned across the long axis of the mould, and
not placed at random
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24. Processing methods and routine
schedules

Machine processing

manual processing
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25. Sectioning



Use a microtome to section embedded
samples 1-10 µm thick
Float on surface of warm water to remove
wrinkles and load on to slide
Frozen samples are cut with a cryostat

use this method when wanting to study enzyme
activity which can be damaged by fixation
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26. CUTTING
• using the microtome
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27. • A microtome is a mechanical instrument
used to cut biological specimens into
very thin segments for microscopic
examination. Most microtomes use a
steel blade and are used to prepare
sections of animal or plant tissues for
histology. The most common applications
of microtomes are :
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28. 1- Traditional histological technique:
Tissues are hardened by replacing water with paraffin. The tissue is
then cut in the microtome at thicknesses varying from 2 to 25
micrometers thick. From there the tissue can be mounted on a
microscope slide, stained and examined using a light microscope
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29. 2- Cryosection:
Water-rich tissues are hardened by freezing and cut frozen;
sections are stained and examined with a light microscope. This
technique is much faster than traditional histology (5 minutes vs.
16 hours) and are used in operations to achieve a quick diagnosis.
Cryosections can also be used in immunohistochemistry as
freezing tissue does not alter or mask its chemical composition as
much as preserving it with a fixative.
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30. 3- Electron microscopy:
After embedding tissues in epoxy resin, a microtome equipped with a
glass or diamond knife is used to cut very thin sections (typically 60 to
100 nanometers). Sections are stained and examined with a
transmission electron microscope. This instrument is often called an
.
4- Botanical microtomy:
ultramicrotome


hard materials like wood, bone and leather require a sledge microtome.
These microtomes have heavier blades and cannot cut as thin a regular
microtomy.
Microtome blades are extremely sharp, and should be handled with great
care. Safety precautions should be taken in order to avoid any contact
with the cutting edge of the blade.
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31. Microtome knives
• STEEL KNIVES
• NON-CORROSIVE KNIVES FOR
CRYOSTATS
• DISPOSABLE BLADES
• GLASS KNIVES
• DIAMOND KNIVES
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32. Why section?
a. Allows you to see internal structure of tissue.
b. Allows stains, or specific markers such as
antibodies to more easily infiltrate the tissues.
c. Allows light to pass through tissue making
structure visible.
d. While sectioning is useful in many instances, in
some cases tissues are stained and examined
without sectioning.
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33. STAINING
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34. Purpose
1. Add contrast to the image –
separation of bacteria in terms of
morphological characteristics and
cellular structures.
2. Identify chemical components of
interest (visualize)
3. Locate particular tissues, cells or
organelles (differentiation)
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35. Staining


Need to stain samples to impart contrast to
various structures, without hard to identify
tissues
Dyes will stain more or less selectively – usually
acids or bases and form electrostatic linkages
with tissue components


basic dyes – attach to acid components, basophilic
 toluidine blue, methylene blue and hematoxylin
acid dyes – attach to basic components, acidophilic
 orange G, eosin, acid fuchsin
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36. Stains

Hemotoxylin and Eosin – (H&E) most common
stain for general morphology and structures



nucleus, RNA-rich areas in cytoplasm and matrix of
hyaline cartilage is blue while cytoplasm and collagen
are pink
Trichromes – additional dyes that will
differentiate other structures
Counterstains – use to give general structure
when doing immunohistochemistry – do not want
to overpower the complex attached to what we
are looking for
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37. Hematoxylin and Eosin (H & E)
H & E is a charge-based, general purpose stain. Hematoxylin stains
acidic molecules shades of blue. Eosin stains basic materials shades of
red, pink and orange. H & E stains are universally used for routine
histological examination of tissue sections.
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38. Staining machine
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39. Why stain the tissue?
a. Creates higher contrast that allows observation of
structure that is not visible in unstained tissue.
b. May reveal differences in chemical nature of
regions of the tissue.
http://www.ipass.net/grc/dimpg9.htm
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40. Staining
Dyes create contrast by imparting a color to
cells or cell parts




Basic dyes – cationic, positively charged
chromophore
Acidic dyes – anionic, negatively charged
chromophore
Positive staining – surfaces of microbes are
negatively charged and attract basic dyes
Negative staining – microbe repels dye, the dye
stains the background
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40

41. Staining reactions of dyes
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41

42. Staining



Simple stains – one dye is used; reveals
shape, size, and arrangement
Differential stains – use a primary stain
and a counterstain to distinguish cell types
or parts (examples: Gram stain, acid-fast
stain, and endospore stain)
Structural stains – reveal certain cell
parts not revealed by conventional
methods: capsule and flagellar stains
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42

43. Microbiological stains
Crystal Violet
Methylene Blue
Gram Stain
Capsule Stain
Acid Fast – Red
Fast, blue non
Flagella Stain
Spore Stain green
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44. Simple Stains
Bacteria have nearly the same refractive index as water, therefore, when
they are observed under a microscope they are opaque or nearly invisible
to the naked eye.
Different types of staining methods are used to make the cells and their
internal structures more visible under the light microscope.
Simple stains use one dye that stains the cell wall.
The cells are then visible against a light background.
Steps:
1.
Place the slide on the staining rack.
2.
Flood the slide with a basic stain: either crystal violet (1 min.), Safranin
(2 min.), or Methylene blue (2 min.).
3.
Wash the stain off the slide with deionized water.
4.
Blot the slide with bibulous paper.
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45. Differential Staining
 Differential Stains use two or more stains and allow the cells to be
categorized into various groups or types.
 Both techniques allow the observation of cell morphology, or shape,
but differential staining usually provides more information about the
characteristics of the cell wall (Thickness).
 The most common differential stain used in microbiology is the Gram
Stain.
 Basic stains, due to their positive (+) charge will bind electrostatically
to negatively charged molecules such as many polysaccharides,
proteins and nucleic acids.
 Acid stains ( - ) bind to positively charged molecules which are much
less common, meaning acidic stains are used only for special
purposes.
 Some commonly encountered basic stains are crystal violet, safranin
(a red dye) and methylene blue.
 Basic stains may be used alone (a simple stain) or in combination
(differential stain) depending on the experiment involved.
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46. Mounting sections
• Wet mounts – to observe fresh specimens
1. Isolate the specimen
2. Place the specimen in the small droplet of the relevant
fluid (fresh water, seawater) on a microscope slide.
3. Gently lower a coverslip onto the droplet, using forceps
of two needles with absorbent paper.
4. Remove any excess water on or around the coverslip
with absorbent paper.
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Histology
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47. Mounting sections
• Temporary mounts
-involve wet mounting in a mountant with a
short useful life – for identification
purposes.
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Histology
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48. Mounting sections
• Permanent mounts – protect sections during
examination and allow storage without deterioration.
Procedure
1. Apply little mountant to a coverslip of appropriate size.
2. Turn the coverslip over and place on its edge to one side of the
sections.
3. Lower the coverslip slowly down onto the sections so as to displace
all the air and sandwich the sections between the slide and the
coverslip.
4. Press firmly form the centre outwards to distribute the mounting
medium evenly.
5. Allow the solvent to evaporate – best results come from slow drying
when time allows, but many synthetic mountant will tolerate brief
heating when speed is essential.
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Histology
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49. The Gram Stain


Developed by Christian Gram in 1884
A staining procedure that differentiates between
bacteria based on the structure of their cell walls.


Thick Cell Wall
 Penicillin and derivatives of penicillin are used on
these bacteria because it attacks peptidoglycan (cell
wall) synthesis.
Thin Cell Wall with Outer Membrane
 Penicillin is ineffective on these bacteria because
they have a lipid membrane outside of their cell wall
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50. 




Gram Staining
The Gram Stain is a differential stain.
Four different reagents are used and the results are based on differences in
the bacterial cell wall.
Gram Positive bacteria have a relatively thick cell wall composed of a special
carbohydrate called Peptidoglycan.
Gram Negative bacteria have a much thinner cell wall composed of the same
carbohydrate, Peptidoglycan, but with certain chemical differences, such as
the presence of Lipopolysaccharides (LPS).
Notice that both Gram Positive and Gram Negative bacteria have a cell wall
composed primarily of Peptidoglycan.
Gram Staining Steps
1.
2.
3.
4.
Crystal violet acts as the primary stain. Crystal violet may also be used
as a simple stain because it dyes the cell wall of any bacteria.
Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell
wall).
Decolorizer is used next to remove the primary stain (crystal violet) from
Gram Negative bacteria (those with LPS imbedded in their cell walls).
Decolorizer is composed of an organic solvent, such as, acetone or
ethanol or a combination of both.)
Finally, a counter stain (Safranin), is applied to stain those cells (Gram
Negative) that have lost the primary stain as a result of decolorization.
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Histology

51. Gram Staining Procedure
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Histology
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52. 






Gram Staining Results
When reporting the results of the Gram stain you indicate the type of
stain used, the reaction, and the morphology of the cells observed.
Round (spherical), purple (or dark blue) cells are reported as Gram
positive cocci (GPC).
Rod-shaped, purple (or dark blue) cells are reported as Gram
positive bacilli (GPB).
The standard abbreviations for the four types of Gram stain and
morphology are:
1.
2.
3.
4.
Gram Positive Cocci (GPC)
Gram Positive Bacilli (GPB)
Gram Negative Cocci (GNC)
Gram Negative Bacilli (GNB)
Notice that both Gram Positive and Gram Negative bacteria have a
cell wall composed primarily of Peptidoglycan.
Spiral-shaped bacterial cells do not Gram stain well and are usually
observed using dark-field microscopy. There are no standard
abbreviations for Gram stain reactions of the spirilla of medical
importance.
Certain other types of bacteria may not Gram stain well, such as,
Acid-fast Mycobacteria (Mycobacterium tuberculosis).
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Histology

53. Bacterial Cell Shapes

Bacteria can have several different shapes, but the primary shapes we will
be observing are:
1.
2.
3.

Spherical or round cells – cocci (plural) or coccus (singular)
Rod shaped – bacilli (plural) or bacillus (singular)
Spiral shaped – spirilla
Some bacteria have characteristic clustering or arrangements, usually due
to how the cells divide and whether they remain attached together when
they divide.



Diplococci – divide in one plane and remain attached together after cell division.
Streptococci – divide in one plane and form long chains of attached cells.
Staphylococci – divide in many planes and remain attached together forming a
“grape-like cluster”
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54. The Microscope
Key characteristics of a reliable microscope
are:
 Magnification – ability to enlarge objects


Anybody have cheap telescope or binoculars?
Resolving power – ability to show detail
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54

55. Magnification in most microscopes results from interaction between
visible light waves and curvature of the lens



Angle of light passing through convex surface of glass
changes – refraction
Depending on the size and curvature of the lens, the image
appears enlarged
Extent of enlargement – magnification
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55

56. Light Microscopy


Mechanical and optical components make up
microscope
3 systems of lenses



condenser – collects and focuses light onto object
objective – enlarge and project the illuminated
image in direction of eyepiece
eyepiece – further magnification and project to the
retina or detector
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58. Principles of Light Microscopy

Magnification occurs in two phases –



The objective lens forms the magnified real image
The real image is projected to the ocular where it is magnified
again to form the virtual image
Total magnification of the final image is a product of the separate
magnifying powers of the two lenses
power of objective x power of ocular = total magnification
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59. Chapter 2 :Techniques in Histology
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59

60. Resolution
Resolution defines the capacity to distinguish or separate two adjacent
objects – resolving power

Function of wavelength of light that forms the image along with
characteristics of objectives
 Visible light wavelength is 400 nm–750 nm
 Numerical aperture of lens ranges from 0.1 to 1.25
 Oil immersion lens requires the use of oil to prevent
refractive loss of light
 Shorter wavelength and larger numerical aperture will
provide better resolution
 Oil immersion objectives resolution is 0.2 μm
 Magnification between 40X and 2000X
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60

61. Variations on the Optical Microscope



Bright-field – most widely used; specimen is darker than
surrounding field; live and preserved stained specimens
Dark-field – brightly illuminated specimens surrounded by dark
field; live and unstained specimens
Phase-contrast – transforms subtle changes in light waves
passing through the specimen into differences in light intensity,
best for observing intracellular structures
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61

62. Three views of a basic cell
Bright Field
Dark Field
Phase Contrast
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62

63. Phase Contrast and DIC Microscope

The differential
interference
microscope is similar
to the phase contrast
but has more
refinements
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63

64. Phase-Contrast and
Differential Interference Microscopy

Use phase-contrast for viewing images of
transparent samples


light changes speed when passing thru cellular
and extracellular structures with different
refractive indices
use to view living cells
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65. 2 Types of Electron Microscopes


Transmission electron microscopes (TEM) –
transmit electrons through the specimen. Darker
areas represent thicker, denser parts and lighter
areas indicate more transparent, less dense parts.
Scanning electron microscopes (SEM) – provide
detailed three-dimensional view. SEM bombards
surface of a whole, metal-coated specimen with
electrons while scanning back and forth over it.
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65

66. Transmission E.M.
Electrons pass through the sample
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66

67. Scanning EM
Scanning looks at
surface only
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67

68. NEXT CLASS:
Chapter 3
TECHNIQUES OF MICROBIOLOGY
THANK YOU
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Histology
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