1. BASIC STAINING TECHNIQUES
LECTURE 1

2. OBJECTIVES: At the completion of this section the student will be able to:
Know the steps involved in tissue processing and discuss the different types of microscopy.
lnterprete the cytological significance of differential staining produced by hematoxylin and eosin (H&E).
Recognize and identify cells in microscopic tissue preparations.
Understand that morphology reflects the function of cells.

3. Methods Histochemistry Cytochemistry Immunocytochemistry
Organ & tissue culture
Differential centrifugation
Specialized microscopic techniques

4. Histology
is the study of tissue sectioned as a thin slice, using a microtome.
It can be described as microscopic anatomy.
Histology is an essential tool of biology.

5. Histopathology, the microscopic study of diseased tissue, is an important tool of anatomical pathology since accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples.

6. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue.
antibodies are used to specifically visualise proteins, carbohydrates and lipids: this is called immunohistochemistry.

7. Histochemistry and Cytochemistry
Tissue Preparation
Methods for the observation of
- living cells
- dead cells
Example- micro-organism like bacteria, virus, fungi etc

8. Living Cells
Cells from tissue cultures and unicellular organisms observed with a phase-contrast microscope

9. Dead cells
Sample sources
Autopsy
Biopsy
Blood samples

10. Stages of the Histology Technique
Light microscopy
Sampling
Fixation
Dehydration
Clearing
Infiltration (impregnation)
Sectioning
Staining
Mounting
Embedding

11. Aim – is to provide a representative specimen
Sampling
Aim – is to provide a representative specimen

12. Fixation
Aim – to preserve the cells with the “least alteration” possible from the living state
Use of substances that coagulate the protoplasm
Simple – formaldehyde, alcohol, acetic acid, osmic acid, picric acid
Complex – Bouin’s fluid, Zenker’s fluid, special mixtures
Some organs needs fixation even before the macroscopic preparation takes place
10 % formaldehyde is the cheapest and easiest to keep fixative solution

13. Dehydration Aim – to remove water from the sample
to allow paraffin impregnation
Done by passing tissue sample through
gradually increasing concentrations of alcohol

14. Clearing
Aim – to take the alcohol out of the sample to allow paraffin to impregnate the tissue
The tissue is immersed in xylene (xylol) that is miscible both in the dehydrating and the embedding agent
The name of the process come from the clear appearance the sample gets
Benzene, cedarwood oil, chloroform and others can also be used

15. Infiltration
Aim – to incrust the sample inside a material that allow easy cutting
The sample goes inside a bath of warm paraffin
The tissue embedded in paraffin is put in a plastic frame that can be used later in the microtome
The embedded specimen in the plastic frame can be (and should be) safely conserved for many years
It is very important to properly label each one of the plastic frames

16. During this 12 to 16 hour process, paraffin wax will replace the water: soft, moist tissues are turned into a hard paraffin block, which is then placed in a mould containing more molten wax (embedded) and allowed to cool and harden.
Embedding can also be accomplished using frozen, non-fixed tissue in a freezing medium.
This freezing medium is liquid at room temperature but when cooled will solidify.

17. Microtome
A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination.

19. Sectioning Aim – to cut the sample in slices thin enough to be useful
Sections of the tissue are cut with the aid of the microtome
The thickness, between 3 and 10 µm, is selected depending the stain that is going to be used
The section is transferred to a glass slide

20. Sectioning Tissue block is cut into 1~20μm thick sections by
microtome, and laid out on the surface of warm
water and are then adhered to gel-coated slides.

21. Staining
Aim – to add color to the structures of the sample in order to differentiate them
There are several phases of staining
Every stain has a different procedure (phases, time, extra procedures, etc.)
Special stains color different structures
Most common, easiest to use & cheapest stain is hematoxylin and eosin (H and E)

22. Dehydration and clearing
Before
After

23. Mounting
Aim – to put the final sample in a medium where it is protected and can also be observed under the microscope
The sample is dehydrated again
A drop of mounting agent with a similar refractive index to that of glass is placed on the glass slide, and covered with a glass coverslip is added
Mounting medium - DPX
- Canada Balsam diluted in xylene (xylol)

24. (Important during surgical biopsy diagnosis)
Freeze Drying Method
(Important during surgical biopsy diagnosis)
Aim – to quickly prepare a glass slide for rapid study of specimens during a surgical procedure
Steps involved in specimen preparation:
Freeze the tissue
Dehydrate the sample under a vacuum
Embed the dehydrated sample
The quality of the section by this method is poor, and a regular histological technique should followed at the end of surgery.

25. ***immersion of tissues in xylene dissolves the tissue lipids, which is undesirable effect when these compounds are to be studied.
To avoid loss of lipids, a freezing microtome has been devised in which the tissues are hardened at low temperatures to provide rigidity necessary to permit sectioning.
The freezing microtome is a more elaborate and efficient successor of the cryostat.***

26. Cell Smears
Cell smears are a form of histological preparation that does not require sectioning. Smears can be made for example of the blood or bone marrow. Smears are also common for swabs or scrapings of epithelial cells (e.g. from the oral cavity, cervix uteri).

27. MATERIALS
- sterilized lancet or needle - 20 clean microscope slides and coverslips - Canada balsam or other medium for permanent preparations - 95% ethyl or methyl alcohol - distilled water - Giemsa stain - low containers (you can make them with aluminum sheet also) - microscope which magnifies 200 times at least

28. TAKING THE BLOOD
MAKING THE SMEAR
FIXING
STAINING
CHECKING
COVER-SLIPPING
OBSERVATION

29. MAKING THE SMEAR

30. FIXING
fixing technique consists of dipping the smear in a vessel containing 95% ethyl or methyl alcohol for 3-5 minutes.

31. STAINING
To be able to observe and recognize the different kinds of leukocyte, you must stain them. For this purpose, normally Giemsa stain is used.
It is a mixture of stains, based on methylene blue and eosin

32. Electron Microscopy

33. Stages of the Histology Technique
Electron microscopy
Small samples (1 mm3 or less)
Tissue must be fresh
Double fixation (glutaraldehyde for proteins plus osmium tetroxide for lipids [osmium also stains])
Quick dehydration and clearing
Embedding in plastic resins (Epon or Araldite)
Cut with ultramicrotome (glass or diamond knifes) produces 30 – 50 nm thick specimens
Mounting in copper grids
Stain with heavy metals

34. Tissue Fixation in Electron Microscopy
Same principles as in Light Microscopy with
the following differences:
Double fixation in glutaraldehyde and osmium tetroxide
Ultra-thin sections (< 90nm thick) from plastic embedded blocks.
Thus, all the original EM pictures are black-white images.

35. TEST YOURSELF
The agents in which procedure are intended to stabilize tissue structure by coagulating proteins and promoting cross-linking in?
Clearing
Dehydration
Embedding
Fixation

36. Stains
Acid dyes stain basic structures: (proteins, membranes, cytoplasm). The commonest acid dye is eosin; it is pink
Basic dyes stain acid structures: Nucleic acids (RNA, DNA, rER, nuclei). The commonest basic dye is hematoxylin; it is blue
NOTE: tissues that stain with basic dyes are termed basophilic; tissues that stain with acid dyes are termed acidophilic
Nuclei are basophilic, since they are mainly made of DNA
Cytoplasm is acidophil, since most proteins are basic in nature

37. Useful stains in histology
Regular Stains:
Hematoxylin & Eosin (H&E)
Trichromes (differentiate intercellular structures)
Iron hematoxylin (for iron-containing cells, like muscle & red blood cells)
Specific stains:
Proteoglycans (PAS, cresyl violet, toluidine blue, methylene blue)
Neurons (Nissl, Ag, Osmium, cresyl violet)
Elastic fibers (orcein or resorcein)
Reticular fibers (silver stain)
Blood cells (Romanovsky stains)
Polysaccharides (PAS)

38. Haematoxylin-eosin staining
H&E is the most common dye used in the study of histology
Hematoxylin stains acidic structures (nucleic acids, nuclei, rER) blue
Eosin stains basic structures (proteins, membranes) pink

39. Structures that are not readily stained by hematoxylin or eosin, are called neutrophilic structures.

40. Which of the following statements is false about the rough endoplasmic reticulum(RER)?
(A) Prominent in cells specialized for protein secretion
(B) Presence of polyribosomes confers basophilia
(C) Presence of polyribosomes confers acidophilia in the cell
(D) This organelle can be viewed with a light microscope

41. Trichrome stain
Uses three dyes to differentiate intercellular structures
Particularly helpful in highlighting red blood cells within blood vessels

42. Trichrome stain Gomori trichrome Mallory’s trichrome
Masson’s trichrome
Red keratin and muscle fibers,
Blue or green collagen and bone,
Light red or pink cytoplasm, and
Dark brown to black cell nuclei

43. Trichrome stain
Note the blue nuclei, the pink/purple cytoplasm, the red blood cells and the tuquoise material that is non-cellular.

44. Trichrome stain Note: Blue nuclei Pink color in cytoplasm
Deep red of blood cells

45. Diff btw H&E and Trichrome stain

46. Iron-haematoxylin
Highlights tissue and cells that contain iron, such as muscle & red blood cells

47. Specific stains for proteoglycans
Periodic acid-Schiff (PAS) reaction ALSO USEFUL FOR THE IDENTIFICATION OF POLYSACCHARIDES (Glycogen)
Cresyl violet
Toluidine blue
Methylene blue

48. Periodic Acid Schiff (PAS) staining
PAS reaction is a method to demonstrate glycogen-like substances.
Periodic acid oxidizes the glycol groups in the glucose residues into
aldehyde groups, which then react with Schiff’s reagent, producing
an insoluble compound with a reddish purple color.
PAS reaction in a hepatocyte
PAS reaction in mucosa of intestine

49. Specific stains used for NEURONS
Nissl (a specialized stain for rER of neurons)
Silver & Gold (for fibers & cytoskeletal elements)
Osmic acid (for myelin, a lipid)
Cresyl violet (proteoglycans)

50. A specialized stain for rough endoplasmic reticulum in neurons
Nissl staining
A specialized stain for rough endoplasmic reticulum in neurons

51. Neurofilaments stained
Silver Staining
Reticular fibres
Neurofilaments stained
with silver salts
Silver ions are deposited on the
structures and reduced to silver
particles, showing brown to black
color, (argyrophilic).
Reticular fibers cannot be
well seen with H&E staining

52. Reticular stain

53. Cresyl violet Highlights proteoglycans
Most commonly used for staining nervous tissue
This is a violet/purple stain

54. Elastin stains elastic fibers black

55. Verhoff stain for elastin
Elastic stains such as Verhoff's stain is more specialized, but can be useful to determine changes in vascular or respiratory function due to compromised elastin content. In these images, the elastic fiber content of small arteries is demonstrated

56. Romanovsky Stain for Blood Cells
Note the following:
Reddish-brown cytoplasm of red blood cells
Large (white) cell with deep blue granules
White blood cells are most readily identified with the use of Romanovsky – type stains
E g GIEMSA
Romanovsky stains (Giemsa, Wrights, etc.) are useful for highlighting various types of granules present in developing and mature white blood cells.

57. Metachromasia Phenomenon where a certain dye shows
structures in a different color from that of the dye.
The granules in cytoplasm of mast cells are shown in purple color when stained by toluidine blue, a blue dye.

58. Toludine blue

59. Osmic acid staining

60. ****The effect of osmium tetraoxide is to preserve and stain lipids and proteins.*****

61. Oil Red O
Oil Red O is used to stain lipids a red-orange color in unfixed frozen sections.

62. Methylene blue staining Periodic acid-schiff(PAS) reaction
. Q1. which technique is most commonly used to locate glycogen in cells?
Methylene blue staining
Periodic acid-schiff(PAS) reaction
Enzyme histochemistry
Q 2. frozen sectioning may be required to avoid the removal of which target substance when preparing tissues for paraffin sectioning?
Basic proteins
Lipids
Enzymes
carbohydrates
Q 1

63. ARTIFACTS

64. Pre-histology
These are features and structures that have being introduced prior to the collection of the tissues.
A common example of these include: ink from tattoos and freckles (melanin) in skin samples.
Post-histology
Artifacts can result from tissue processing. Processing commonly lead to changes like shrinkage, color changes in different tissues types and alterations of the structures in the tissue.
Because these are caused in a laboratory the majority of post histology artifacts can be avoided or removed after being discovered.

65. Artifacts (not natural occurences)
Caused by a bad histological technique
Autolysis
Poor sampling
Shrinkage
Folds
Stain precipitation and dust
Defects in the knife
The main cause of artifacts is poor fixation
Some tissues usually present artifacts, & wrong knowledge has been derived from them

66. Suprarenal gland showing with autolysis

67. Shrinkage and rupture

68. Shrinkage

69. Folds

70. Breaking and notches

71. Knife marks

72. Edge damage

73. Poor fixation

74. Dust