1. Lab 4 & 5 (Staining Technique)
بسم الله الرحمن الرحيم
Lab 4 & 5 (Staining Technique)

2. Objectives:
1.Understand the classification of Stains. 2.Understand basic tissue staining methods used in the clinical histology laboratory. 3. Identification the procedure of staining. 4. Understand special stains.

3. Overview
Most dyes used to visualize the membranes and organelles of the cell are water soluble.
The embedded wax must therefore be removed prior to staining.
Routine H&E staining and special stains play a critical role in tissue-based diagnosis or research.

4. By coloring otherwise transparent tissue sections, these stains allow highly trained pathologists and researchers to view, under a microscope, tissue morphology (structure) or to look for the presence or prevalence of particular cell types, structures or even microorganisms such as bacteria.

5. In the histopathology laboratory, the term “routine staining” refers to the hematoxylin and eosin stain (H&E) that is used “routinely” with all tissue specimens to reveal the underlying tissue structures and conditions.
The term “special stains” has long been used to refer to a large number of alternative staining techniques that are used when the H&E does not provide all the information the pathologist or researcher needs.

6. Classification of Stains
Acid Dyes:
In an acid dye the basic component is colored and the acid component is colorless. Acid dyes stain basic components e.g. eosin stains cytoplasm. The color imparted is shade of red.
Basic Dyes:
In a basic dye the acid component is colored and the basic component is colorless. Basic dyes stain acidic components e.g. basic fuchsin stains nucleus. The color imparted is shade of blue.

7. Neutral Dyes:
When an acid dye is combined with a basic dye a neutral dye is formed. As it contains both colored radicals, it gives different colors to cytoplasm and nucleus simultaneously. This is the basis of Leishman stain.

8. Special stains
When a specific components of tissue e.g. fibrous tissue, elastic tissue, nuclear material is to be stained, certain special stains are used which specifically stain that component tissue.

9. Types of special stains
1.PAS (Periodic Acid Schiff) stain: This stain demonstrates glycogen and neutral mucous substances, outlines basement membranes and reticulin and makes evident most types of fungi and parasites.
2.Alazarin Red: It is used to stain bone.
3.Sudan-Black: It is used for fat staining.
4.Masson’s Trichrome: It is used for differentiation of connective tissue elements.

10. 5. Papanicolaou’s stain: It is used to stain cells in cervical and sputum smear for cytology and to stain bone marrow and blood film.
6. Gymsa stain: it is used to stain blood film.
7. Phangeson stain: It is used to stain muscles.
8. Silver nitrate: It is used to stain skin human.
9. Mercury Brom Phenol Blue: It is used to stain total proteins.

11. 10. Stains for micro-organism: a
10. Stains for micro-organism: a. Gram-stain: Gram stain allows the separation of bacteria those that retain the crystal-violet-iodine complex (gram- positive) and those that are decolorized by alcohol treatment and counter stained by eosin, safranin or fuchsin. b. Ziehl_Neelsen stain: This stain detect acid fast bacilli. c. PAS stain: It is used for fungi, amoeba and Trichomonas. d. Modified Giemsa (2% Giemsa in water): Detects Helicobacter pylori.

12. Hematoxylin and Eosin Stain
Hematoxylin staining requires the use of a mordant (most commonly aluminum salts) and stains the nuclear components of cells a dark blue.
Hematoxylin is used in combination with eosin because eosin stains the cytoplasmic organelles varying shades of pink, red or orange.
The combination of the two stains provides a broad range of morphological information about the section.

13. Frozen Section
In this way tissue can be examined microscopically within minutes of its removal from the body. It reduces the time of processing from 18 hours to 5 minutes.
It has the disadvantage that only 8-16 micron thick section can be cut and finer details of tissue cannot be examined.
Frozen section is performed on a machine called cryostat.

14. what is microscope?????
Microscopy is the technology of making very small things visible to the human eye.
Used in the diagnosis of many diseases, pathological laboratories and medical, Educational microscope ( Used for research).

16. Microscope consists of 4 main parts:
frame work.
Adjustment system.
Magnification.
Lighting System.

17. Frame work Its includes three parts: Base Arm 3) Mechanical stage:
* It is the location of the specimen to be viewed
* Clips- utilized in holding the specimen in place

18. Stage Knobs Control * Top knob: is to move the forward and backward.
* Bottom knob: is to move the stage right and left.

19. Adjustment system It consists of the following parts: Optical tube.
Coarse adjustment.
Fine adjustment.

20. Focus and Resolution Parts
Coarse-adjustment knob
is the larger of the two knobs. It is used in bringing the object into quick focus.
Fine-adjustment knob
is used for improving the clarity of the image, especially when viewing under high power.

21. Interpupillary adjustment
To control the distance between the ocular lenses to adapt the distance between viewers eyes so, the eyepiece lenses will spread apart or get closer together to fit each individual.

22. Magnification
Microscope has two sets of lenses are :
Ocular lens (eyepiece).
Objective lens.
Microscope lenses
Ocular lens or eyepiece is used for viewing.
Revolving nosepiece contains objective lenses that are used to magnify the image in combination with the ocular lens.
The objective of microscopy is not just to increase magnification, but to do so while retaining sufficient resolution.

23. Oil immersion lens

24. How to use Microscope
Plug in microscope and turn on illuminator. Rotate nosepiece to lock 4X objective in place
Place smear on stage and center it under the 4X objective.
Using the course adjustment knob, move the objective lens to its lowest point. Look through the ocular and focus upward with the coarse adjustment until an image comes into view.
Rotate nosepiece to obtain the next objective lens 10x and repeat step 3.

25. Rotate nosepiece to obtain the next objective lens 40x .
Look through the ocular and focus upward with the fine adjustment until an image comes into view.
When using oil immersion lens put a drop of oil on the slide and Rotate nosepiece and repeat step 6.

26. How to properly carry the microscope
Always carry a microscope with one hand holding the arm and one hand under the base.

27. Microscope Maintenance

28. Microscope Storage
Proper storage of the microscope will prevent or reduce problems!
Optics and mechanisms of the microscope must be protected from:
Dust and dirt,
Fungus.
Store the microscope:
Under a protective cover,
In a low humidity environment.

29. Cleaning Solutions and Solvents
Cleaning Materials
Soap solution for cleaning of body and stage.
Ether-Alcohol, Alcohol, or Lens Cleaner Solution for cleaning of lenses.
Cleaning Solutions and Solvents
Lens paper
Commercial lens tissue for optics
Caution: Do not use paper towels or other rough paper products.
Alternatives include: Fine quality tissue paper , Muslin cloth, Silk

30. Step 1: Cleaning the Eyepieces
Microscope Cleaning Process
Step 1: Cleaning the Eyepieces
Blow to remove dust before wiping lens.
Clean in a circular motion inside out
Step 2: Cleaning the Eyepiece
Wipe the eyepieces dry with lens paper.
Repeat cleaning and drying if required.

31. Step 3:Cleaning the Objectives
Objectives should never be removed from the nosepiece.
Step 4: Cleaning the Microscope Stage
Step 5: Cleaning the Microscope Body
Unplug the microscope from power source.
Wipe the microscope body to remove dust, dirt, and oil.

32. Step 6: Cleaning the Condenser
Unplug the microscope from power source.
Clean the condenser lens and auxiliary lens using lint-free cotton swabs moistened with lens cleaning solution.
Wipe with dry swabs.

33. End of lecture