Histopathology and Cytopathology(Practical) part 2nd

THIS TOPIC IS FOR MEDICAL LABORATORY 3RD YEAR DIPLOMA STUDENTS

1. Histopathology and Cytopathology (Practical) Prepare reagents & stains used for Hematoxylin & Eosin stain, PAS stain, Alcian Blue stain and Ziehl-Neelsen stain. Prepare reagents & stains used for Giemsa and Papanicolaou stains. Stain sections by H/E stain, PAS stain, Alcian Blue stain and Ziehl-Neelsen stain. Prepare cytological fixatives and fixation of cells . Prepare cytological smears and stain with pap method.

2. 6. Prepare reagents & stains used for Hematoxylin & Eosin stain, PAS stain, Alcian Blue stain and Ziehl-Neelsen stain Prepare reagents: 1. Hematoxylin & Eosin stain: Hematoxylin and eosin staining technique functions to recognize different types of tissues and their morphological changes, especially in cancer diagnosis. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue,  nuclei  are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining . Preparation of Harris’s hematoxylin : Ingredients : Hematoxylin - 5gm Absolute alcohol - 50ml

3. Hematoxylin solution Bluing buffer Eosin stain solution

4. Ammonium alum - 100gm Distilled water - 1000ml Mercuric oxide - 2.5gm Glacial acetic acid - 40ml Method :- Dissolve the hematoxylin in absolute alcohol and ammonium alum in hot water. Mix the two solutions and heat to boiling. Remove from flame, and add mercuric oxide and cool rapidly. Glacial acetic acid if added gives brisk nuclear staining, but life of the solution is reduced. Hence if acetic acid is to be added, it should be added in working solution. Preparation of Mayer’s hematoxylin : Ingredients : Hematoxylin - 1.0gm Distilled water - 1000ml

5. Ammonium alum- 50gm Sodium iodate - 0.2gm Citric acid (reduces pH)- 1.0gm Chloral hydrate (preservative)- 50gm Method :- Hematoxylin is dissolved in distilled water using gentle heat. Then alum is added and dissolved. Then sodium iodate , citric acid and chloral hydrate are added respectively Preparation of EOSIN: Ingredients : Eosin Y (water soluble)- 1.0gm Distilled water - 80ml 95% alcohol - 320ml Glacial acetic acid- 0.4ml

6. Preparation – Dissolve eosin in water and then add this to 95% alcohol (one part eosin solution with 4 parts alcohol). To the final mixture add a few drops of acetic acid (0.4ml). The acetic acid increases the staining intensity of eosin. When ready to use, the stain should be cloudy; if clear, add a few drops of the acetic acid. The solution should be standardized by staining the control slides. Stains used: It is widely used in Histopathology, Immunochemistry, and Immunopathology to analyze and demonstrate morphologies of tissues, and cells.

7. 2. PAS stain: PAS stain, which stands for Periodic Acid-Schiff, is a common histological staining technique used to detect carbohydrates like polysaccharides, glycogen, and glycoproteins in tissue samples . Solutions and Reagents: 0.5% Periodic Acid Solution: Periodic acid ---------------------- 0.5 g Distilled water -------------------- 100 ml Mix well and store in amber bottle Schiff Reagent : Basic fuchsin – 1gm Distilled water – 200ml HCL( conc )- 2ml Sodium metabisulfite -2gm Heat , cool, add metabisulfate , stand 24hrs , filter and refrigerate Test for Schiff reagent: Pour 10 ml of 37% formalin into a watch glass. To this add a few drops of the Schiff reagent to be tested. A good Schiff reagent will rapidly turn a red-purple color. A deteriorating schiff reagent will give a delayed reaction and the color produced will be a deep blue-purple.  Mayer’s Hematoxylin Solution:

8. Periodic acid Schiff reagent Hematoxylin

9. Lillie's Cold Schiff's Reagent: Basic fuchsin - 10.0 gm Sodium metabisulfite - 18.0 gm Distilled water - 1000 ml Hydrochloric acid- 10.0 ml Stir solution for 2 hours, set in a dark cool place overnight. The solution is now a clear light brown to yellow color. Add: Activated charcoal 500.0 gm (or two heaping spoons) Stir Filter through Whatman #2 filter paper into a 1000 ml graduated cylinder. Change the filter paper often. Restore volume to 1000 ml with distilled water. Store in Refrigerator, solution is stable for 6 months.

10. Stain Use: Cytology: This stain has also been used in undifferential identification of tumors hence used in the diagnosis of glandular carcinomas ( adenocarcinoma ). Pathology: It is also used in the diagnosis of liver and kidney pathologies. Fungal studies: It is used to demonstrate the fungal hyphae and yeast-forms of fungi in tissue samples to identify  Candida albicans ,  Aspergillus fumigatus , and  Cryptococcus neoformans  infections. Gastrointestinal pathology: To detect the presence of mucins in the gastrointestinal tract. Lung studies: the stain studies the amorphous or granular globules of the pulmonary alveolar proteinosis . Skin: to study the eosinophilic globoid bodies or Kamino bodies. Muscle biopsies: to demonstrate glycogen components. To detect prostate cancer, pancreatic pathologies, effects of the small intestines, testis. Application in enzymatic cytochemistry for the detection of granules.

11. 3. Alcian Blue stain: Alcian blue stain is  a histological dye that selectively stains acidic polysaccharides like mucopolysaccharides and glycosaminoglycans , making it useful for identifying mucins , cartilage, and connective tissues in a lab setting . Alcian Blue pH 2.5 Solution: Prepare the buffer:  Mix 15 mL of 100% acetic acid with 485 mL of distilled water to create a 3% acetic acid solution.  Dissolve the dye:  Add 1 gram of alcian blue (8GX) to the 500 mL of 3% acetic acid buffer. Note: This recipe makes 500 mL of 1% solution, a common alternative is 1g to 100mL for 0.1% as seen in other sources. A common procedure suggests 1g in 100mL of 3% acetic acid for a pH 2.5 solution.  “8GX” refers to the specific grade or type of dye Mix:  Stir the solution until the alcian blue is completely dissolved.  Filter:  Filter the solution before use . Alcian Blue pH 1.0 Solution: Prepare the buffer:  Use 0.1 M hydrochloric acid.   

12. Alcian Blue stain reagent

13. Dissolve the dye:  Add 1 gram of alcian blue (8GX) to 100 mL of the 0.1 M hydrochloric acid buffer.  Adjust pH:  The solution should have a pH of 1.0.  Filter:  Filter the solution before use.  Important Safety Precautions: Always work in a fume hood and wear gloves, goggles, and a lab coat.  Acids can be corrosive and irritating; handle with care.  Avoid contact with skin and eyes.  Refer to the specific manufacturer's instructions and safety data sheets (SDS) for more detailed safety information.  Use of stain: Identifying acidic mucins :   The stain is a basic dye that binds to acidic components in tissues, making them visible under a microscope. 

14. Visualizing GAGs and proteoglycans :   It helps researchers study the distribution of these compounds, which are important in cartilage and other connective tissues.  Differentiating between types of mucins : At a pH of 1.0, the stain selectively binds to strongly sulfated GAGs.  At a pH of 2.5, it binds to both sulfated and carboxylated mucins Pathology: It can be used to help diagnose conditions like Barrett's esophagus, where mucins in the goblet cells stain blue.  It aids in the diagnosis of certain diseases involving the accumulation of glycosaminoglycans , such as some forms of mesothelioma or atherosclerosis. 

15. 4. Ziehl - Neelsen stain The Ziehl - Neelsen stain in histopathology is a differential staining technique used to identify acid-fast bacteria, particularly Mycobacterium species, in tissue samples. Prepare reagents: Carbol-fuchsin solution: Ingredients:  Basic fuchsin : 1gm 100% ethyl alcohol: 10ml Phenol crystals: 5ml Distilled water: 100ml Preparation:   Dissolve the basic fuchsin in the alcohol.  Separately, dissolve the phenol crystals. Combine the solutions and add the distilled water.  Note:   Some protocols suggest a higher concentration or melting the phenol crystals first. Always filter before use. 

16. Carbol-fuchsin solution Acid-alcohol decolorizer Methylene blue counterstain

17. 2. Acid-alcohol decolorizer : Ingredients:  95% ethyl alcohol : 95ml  Concentrated hydrochloric acid: 3ml  Preparation:  Slowly add the concentrated hydrochloric acid to the ethyl alcohol, swirling the flask to mix and dissipate heat.  Note: Always add acid to water or alcohol, never water to acid.  3. Methylene blue counterstain : Ingredients: 0.1% to 1% aqueous methylene blue solution.  Preparation:  Dilute methylene blue stock solution in distilled water to the desired concentration.  Note:  Some protocols specify preparing a higher concentration methylene blue solution (e.g., 1.4%) in alcohol and then diluting it with water for the working solution. 

18. Safety precautions Phenol is toxic and can cause burns, so handle with care and use gloves.  Carbol-fuchsin is a potential carcinogen; work in a fume hood when preparing and using it.  Wear gloves and eye protection when handling all reagents.  Always add acid slowly to water or alcohol, not the other way around.  Use in histopathology: Identifying bacteria:   The primary purpose is to identify the presence of acid-fast bacilli, such as Mycobacterium tuberculosis (causing  tuberculosis ) and  Mycobacterium leprae  (causing  leprosy ).  Early diagnosis:   It is crucial for the early detection of infections caused by these organisms.  Monitoring treatment:   The stain can be used to monitor a patient's response to therapy by assessing the bacterial load in a sample over time.  Tissue preservation:   Care must be taken during tissue processing, as strong acid decalcification can destroy the acid-fast property of the bacteria. Formic acid is often recommended for this purpose

19. 7. Prepare reagents & stains used for Giemsa and Papanicolaou stains. Giemsa stain: Giemsa stain is a differential stain used in histopathology and other fields to differentiate cells and identify microorganisms Prepare reagent: To prepare Giemsa stain solution, first create a stock solution by dissolving Giemsa powder in a mixture of methanol and glycerin, then allow it to stand for at least one to two months.  Next, prepare the working solution by diluting the stock solution with buffered water or a buffer solution at room temperature to the correct ratio for staining, typically 1:10 (1 part stock to 9 parts buffer).  1. Prepare the stock solution (500 mL )  Dissolve 3.8 g of Giemsa powder into 250mL of methanol. Heat the solution to approximately 60∘C is equivalent to 140∘ (degrees Fahrenheit)

20. Slowly add 250mL of glycerin to the heated solution. Filter the solution to remove any debris. Allow the solution to stand for at least one to two months before use.  2. Prepare the working solution Combine 10 mL of the stock solution with 80 mL of distilled water and 10 mL of methanol.  Alternatively, mix 1 part of the Giemsa stock solution with 9 parts of buffered water (pH 7.2) for a 1:10 dilution.  Use the working solution immediately or within 24 hours. Safety precautions: Work in a well-ventilated area or fume hood, as methanol is highly toxic and flammable. Wear appropriate personal protective equipment, such as gloves, safety glasses, and a lab coat. Store methanol in a locked cupboard when not in use. 

21. Applications in histopathology: Blood and bone marrow:  Used to differentiate blood cells (erythrocytes, leukocytes, platelets) in hematopoietic tissues.  Microorganism detection:  Can be used on tissue sections to identify microorganisms that are difficult to see otherwise. Examples include:  Helicobacter pylori in gastric biopsies  Leishmania parasites in tissues  Fungi like Aspergillus niger   Malaria parasites in blood smears  Cytology:  Applicable to clinical cytology specimens and tumor samples. 

22. 2. Papanicolaou stain The Papanicolaou stain, or  Pap stain , is a multi-color cytological staining technique that helps pathologists diagnose disease by highlighting cell structures. It produces a polychromatic (multiple colors) and transparent stain, making it easy to see nuclear and cytoplasmic details.  While most famous for its use in  cervical cancer screening , it is used in cytology to detect a range of abnormal cell changes and infections.     To prepare Papanicolaou stain reagents: 1.Orange G (OG-6) stain preparation: Orange G powder: 5grams Absolute alcohol: 950 ml Phosphotungstic acid: 1.5grams Glacial acetic acid:10ml Distilled water: 50ml Dissolve the Orange G powder: Dissolve 5grams of Orange G powder in 950 ml of absolute alcohol. 

23. Add other reagents: Add 1.5grams of phosphotungstic acid and10ml of glacial acetic acid to the solution.  Add water: Incorporate 50ml of distilled water into the mixture.  Mix thoroughly: Stir the solution until everything is completely dissolved and mixed homogeneously.  Store: Store the completed solution in a dark bottle.  Filter: Filter the solution before each use.  2. EA-50 (or EA-36) stain preparation: Dissolve dyes: Separately dissolve Light Green SF, Eosin Yellow, and Bismarck Brown in distilled water.  Combine with other reagents: Combine the three solutions and add 95% ethanol and glacial acetic acid.  Add phosphotungstic acid and lithium carbonate: Add 2 grams of phosphotungstic acid and a few drops of saturated aqueous lithium carbonate to the mixture. 

24. Mix thoroughly: Mix the final solution thoroughly.  Store: Store the completed solution in a dark bottle.  Filter: Filter the solution before each use.  Important considerations: Filtering: Always filter the stains before use to ensure a clear stain.  Storage: Keep the prepared solutions in dark bottles away from light to prevent degradation.  Quality: Use high-quality reagents from reputable sources for consistent results.  Safety: Be mindful of the flammability of alcohol and the cost of waste disposal, especially for Xylene . 

25. Applications of Papanicolaou Staining (Pap stain) Used in the Pap smear (or Pap test). Screening for cervical cancer. Examination of myeloma cancer cells of the liver. Screening for thyroid cancer. Screening for cell carcinomas. Examination and characterization of benign tumors. Identification of  Candida  species. Identification of  Chlamydia trachomatis .

26. 8. Stain sections by H/E stain, PAS stain, Alcian Blue stain and Ziehl-Neelsen stain. 1. H/E stain: Hematoxylin and eosin stain (H and E stain or HE stain) Hematoxylin and eosin staining technique functions to recognize different types of tissues and their morphological changes, especially in cancer diagnosis. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue,  nuclei  are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Hematoxylin and Eosin stain is the primary diagnostic stain used in histology and Histopathology including application in the Fine-needle Aspiration Biopsies, Paraffin Fixed embedded tissue. Hematoxylin is used to demonstrate the nuclear of the cell and its content, by observing the depth of the color of dye and the time of sample suspension in the dye.

27. Hematoxylin and eosin are both dyes, and naturally, dyes have a high affinity for tissues, depending on the Acidity and/or alkalinity of the dyes. Objective: To demonstrate the morphologies of tissues and cells. Principle: Hematoxylin is extracted from  Hematoxylon campechianum  tree upon which on oxidation, hematein is produced from hematoxylin , the dye used in the hematoxylin and Eosin staining technique. Adding a mordant enables hematein to bind to the anionic elements of the tissues. Hematoxylin without eosin acts a counterstain in immunohistochemical and hybridization protocols that use colorimetric substrates (such as alkaline phosphatase or peroxidase ) Eosin dye is acidic dye hence it as a negative charge ( eosinophilic ). Therefore it stains the basic structures of a cell ( acidophils ), giving them a red or pink color, for example, the  cytoplasm  is positively charged, and therefore it will take up the eosin dye, and appear pink.

28. Hematoxylin dyes are basic dyes, hence they are positively charged.  Therefore it will stain the acidic structures of tissues and cell structures (basophilic), purplish-blue. Hematoxylin is not basic by itself. It has to be conjugated with a mordant (aluminum salt) before it is used so as to strengthen its positive charge for efficiency in binding to the tissue components. The mordant, which also defines the color of the stain, will bind to the tissue, then the hematoxylin will bind to the mordant to form a tissue-mordant- hematoxylin complex link. This will stain the nuclei and chromatin bodies purple. H &E stain can be classified into three types: progressive, modified progressive, and regressive. Progressive staining takes place without a differentiator for removing any excess dye after adding hematoxylin . This cause background staining to occur in charged slides. Its mainly used to stain Mucin . While the Modified progressive and regressive, use a differentiator in removing excessive dyes. It is used for the demonstration of nucleus content.

29. OR H&E staining is a two-step process that uses two dyes, haematoxylin and eosin, to color tissue samples.  Hematoxylin , a basic dye, stains the cell nuclei blue-purple by binding to the acidic DNA within them.  Eosin, an acidic dye, stains the cytoplasm and extracellular matrix pink or red by binding to basic cellular proteins.  This creates a contrast that allows for the visualization of cellular and tissue structures under a microscope.   Reagents: Distilled water Alum hematoxylin Acid alcohol Scott’s tap water Eosin dye

30. Procedure The procedure is simplified into various processes: Dewaxing , Dehydration, Hematoxylin , Differentiation, Bluing, Eosin, Dehydration, Clearing, Cover-slipping The steps include: Clean the sections to distilled water. Then stain nuclei with the alum hematoxylin (Mayer’s) to fix the tissue, for about 5 minutes. Rinse the stain with smoothly running tap water Using the differentiator, 0.3% acid alcohol, and note the endpoint i.e the correct endpoint is after bluing up, the background becomes colorless. Rinse the stain in smoothly running tap water. Rinse the satin in Scott’s tap water substitute which shortens the time for the correct end-point. Rinse with  running tap water Flood the smear with eosin for 2 mins , and since eosin is highly soluble in water, use enough quantify of it. The over stained eosin can be removed or washed off with running tap water. Dehydrate the smear, clear, and mount using a clean coverslip .

31. Results and Interpretation: Nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin ( hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast.

32. Nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining.

33. Applications: It is widely used in Histopathology, Immunochemistry, and Immunopathology to analyze and demonstrate morphologies of tissues, and cells. Advantage: The method is quick to execute, cheap and can be altered Limitation: Hematoxylin and Eosin are inefficient in that not all features of a substance can be received and special stains must be used.

34. 2. PAS (Periodic Acid-Schiff)stain: Periodic-Acid Schiff (PAS) staining technique is used in histochemistry and histological studies to demonstrate the presence of  carbohydrates  and carbohydrate compounds such as polysaccharides, mucin , glycogen, and fungal cell wall components. It has been used to detect glycogen in tissues such as the skeletal muscles, the liver, the cardiac muscles. It uses formalin-fixed, paraffin-embedded tissue sections, or/and frozen tissue sections. Principle of Periodic Acid-Schiff (PAS) Staining: The periodic acid of Schiff’s reaction with carbohydrates is an oxidative process where some polysaccharides react with the periodic acid producing an oxidized compound, an aldehyde .

35. Aldehydes are revealed by the red or pink or magenta coloration due to the fixation of the colorless Schiffs fuchsin . PAS is linked to its association with diastase enzymes which are responsible for the convention of starch to maltose and sequentially to glucose. During glucose conversion, the stain appears pink which defines the intra or extracellular mucins persistence. Hematoxylin or methyl green is used to stain the nuclei. Preparation of Solutions and Reagents: Periodic Acid solution (0.5%): Periodic acid- 0.5g Distilled water- 100ml

36. Testing Schiff’s Reagent: 10 ml of 37% formalin Add Schiff’s reagent to be tested. A good Schiffs reagent turns red-purple in color while a poor Schiffs reagent will have a delayed reaction producing deep blue-purple coloration Mayer’s Hematoxylin Solution: Procedure for Periodic Acid-Schiff (PAS) Staining: Deparaffinize and hydrate water. Oxidation: Add 0.5% of the Periodic acid solution for oxidation for 5 minutes. Rinsing: In distilled water. Aldehydration : Place the stain in Schiff reagent for 15 minutes, which turns light pink.

37. Washing: Using lukewarm tap water, wash the stain for 5 minutes, turning it dark pink. Counterstaining: Add Mayer’s Hematoxylin for 1 minute. Wash with running tap water for 5 minutes. Dehydrate and mount with a synthetic mount medium. Results and Interpretation: Glycogen, mucin , elastic fibers, basophilic granules of the pituitary gland, reticular fibers, thyroid colloids, basement membranes, bone cartilage, and other carbohydrate components stain pink or purple. The nuclei stains green or blue depending on the dye used. The background stains blue.

38. Applications of Periodic Acid-Schiff (PAS) Staining Cytology: This stain has also been used in undifferential identification of tumors hence used in the diagnosis of glandular carcinomas ( adenocarcinoma ). Pathology: It is also used in the diagnosis of liver and kidney pathologies. Fungal studies: It is used to demonstrate the fungal hyphae and yeast-forms of fungi in tissue samples to identify  Candida albicans ,  Aspergillus fumigatus , and  Cryptococcus neoformans  infections. Gastrointestinal pathology: To detect the presence of mucins in the gastrointestinal tract. Lung studies: the stain studies the amorphous or granular globules of the pulmonary alveolar proteinosis . Skin: to study the eosinophilic globoid bodies or Kamino bodies. Muscle biopsies: to demonstrate glycogen components. To detect prostate cancer, pancreatic pathologies, effects of the small intestines, testis. Application in enzymatic cytochemistry for the detection of granules.

39. 3. Alcian Blue stain Principle: Alcian blue stain is the electrostatic interaction between the positively charged Alcian blue dye and negatively charged acidic mucopolysaccharides and glycoproteins in tissues. The dye's blue color comes from its copper phthalocyanine structure.  By adjusting the pH, it's possible to differentiate between different types of acidic mucins : at a pH of 2.5, it stains both carboxylated and sulfated mucins , while at a lower pH of 1.0, it selectively stains only the sulfated mucins .   To perform the Alcian Blue stain procedure, first deparaffinize and hydrate the tissue sections.  Then, place slides in 3% acetic acid for 3 minutes, followed by the Alcian Blue solution for 30 minutes at room temperature.  After a brief rinse in acetic acid, wash in running water and then stain with Nuclear Fast Red for 5 minutes.  Finally, dehydrate, clear with xylene , and coverslip the slides. 

40. Procedure: 1.  Deparaffinize and hydrate :  Remove paraffin from the tissue sections and rehydrate them through a series of alcohols down to distilled water.  2.  Rinse in acetic acid :  Place slides in 3% acetic acid for 3 minutes to prepare the tissue for staining.  3.  Stain with Alcian Blue :  Immerse the slides in Alcian Blue solution for 30 minutes at room temperature. For faster results, you can use the stain at 37°C for 15 minutes.   4.  Rinse excess stain :  Briefly rinse the slides in 3% acetic acid for 10–30 seconds to remove any excess Alcian Blue stain.  5.  Wash in water :  Rinse the slides in running tap water for about 2 minutes, followed by a 1–2 minute rinse in two changes of distilled water.  6.  Counterstain :  Apply the Nuclear Fast Red solution for 5 minutes to stain the nuclei. 

41. 7.  Wash again :  Rinse the slides in running tap water for 1–2 minutes, followed by two changes of distilled water for 1–2 minutes each.  8.  Dehydrate :  Dehydrate the slides by passing them through a graded series of alcohols (e.g., 95% and 100%).  9.  Clear and coverslip :  Clear the slides with xylene and mount them with an appropriate mounting medium and a coverslip .  Interpretation: Alcian blue stain is interpreted as a blue or bluish-green color in the presence of acidic polysaccharides like mucins and glycosaminoglycans in tissues. The specific interpretation depends on the pH used: at pH 2.5, it stains both sulfated and carboxylated mucins blue, while at a lower pH of 1.0, it selectively stains strongly sulfated mucins , appearing blue or greenish-blue.  The intensity and type of blue can help differentiate between various polysaccharides and is used in diagnostics for conditions like intestinal metaplasia and adenocarcinomas .

42. Uses: Identifying acidic mucins :   The stain specifically colors acidic mucins , which is useful for diagnosing various cancers and conditions.  Diagnosing specific diseases:   It aids in the diagnosis of conditions like: Adenocarcinomas :  Identifying mucin -producing tumors.  Barrett's esophagus:  Detecting changes in the esophageal lining.  Atherosclerosis :  Observing the increase of non-sulfated acidic mucus in blood vessel walls.  Mesothelioma :  Identifying excessive amounts of non-sulfated acidic mucus.  Macular corneal dystrophy :  Helping diagnose diseases characterized by glycosaminoglycan deposition.  Staining connective tissues:   It highlights glycosaminoglycans in connective tissues like cartilage.  Research applications:   It is used in developmental biology and biochemistry for visualizing acidic glycans on blots and in gels. 

43. 4. Ziehl - Neelsen stain Principle: The Ziehl - Neelsen stain uses basic fuchsin and phenol compounds to stain the cell wall of Mycobacterium species. Mycobacterium does not bind readily to simple stains and therefore the use of heat along with carbol-fuschin and phenol allows penetration through the bacterial cell wall for visualization. Mycobacterium cell wall contains high lipid content made up of mycolic acid on its cell wall making it waxy, hydrophobic, and impermeable. These are ß- hydroxycarboxylic acids made up of 90 carbon atoms that define the acid-fastness of the bacteria. Use of Carbol-fuschin which is basic strongly binds to the negative components of the bacteria which include the mycolic acid and the lipid cell wall. addition of acid alcohol along with the application of heat forms a strong complex that can not be easily washed off with solvents.

44. The acid-fast bacilli take up the red color of the primary dye, carbol-fuschin . While non-acid-fast bacteria easily decolorize on the addition of the acid-alcohol and take up the counterstain dye of methylene blue and appear blue This technique has been used in the identification of  Mycobacterium tuberculosis  and  Mycobacterium leprae . Procedure for tissue sections: 1. Prepare and fix the tissue smear Smear the tissue onto a slide. Air dry and then heat-fix the smear.  2. Stain with carbolfuchsin Cover the slide with carbolfuchsin dye. Apply gentle, dry heat for two minutes. Let the slide cool and then rinse with water. 

45. 3. Decolorize with acid alcohol Flood the slide with acid alcohol for about 30 seconds to remove the stain from non-acid-fast cells.  Rinse with water.  4. Counterstain with methylene blue Flood the slide with methylene blue for 30 seconds to 1 minute. Rinse with water and dry the slide.  5. Interpret the results Acid-fast bacteria will appear red. The background and other non-acid-fast cells will appear blue. 

46. OR PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. * Carbol-fuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. Filter solution once a week. 3. Wash in running tap water. 4. 1% Acid alcohol until light pink and color stops running. 5. Wash in running tap water for 5 minutes.. 6. Rinse in distilled water. 7. Working methylene blue for 30 seconds. 8. Rinse in water. 9. Dehydrate, clear, and coverslip .

47. Application : The Ziehl - Neelsen stain is applied to tissue sections to identify and diagnose acid-fast bacteria, such as Mycobacterium tuberculosis, which cause diseases like tuberculosis. 

48. 9. Prepare cytological fixatives and fixation of cells. Cytological fixatives are substances used in cytology to preserve cell structures for analysis by preventing decay. Common cytological fixatives 1. 95% Ethanol : The most commonly recommended and "gold standard" fixative for cytological preparations, particularly PAP smears.  It works by dehydrating the cell and causes a mild shrinkage, which helps preserve nuclear detail.  Its disadvantages include being flammable, expensive, and subject to misuse. 

49. Preparation steps: Gather materials:  You will need a container and 95 ml of ethanol (either absolute or 95% denatured alcohol) and 5 ml of distilled water. Measure components:  Carefully measure 95 ml of ethanol and 5 ml of distilled water. Combine and mix:  Pour the measured amounts into the container and gently mix them together.  2. 10% Neutral Buffered Formalin (NBF): A widely used fixative for both cytology and general histopathology.  It is effective for long-term preservation without excessive hardening.  A potential drawback is the formation of a formalin pigment, which can be prevented by buffering the solution.  The fixative is a good routine fixative. This solution is hypotonic in buffer ions and has a pH of 6.8

50. REAGENTS: Sodium phosphate, monobasic- 4.0 gm Sodium phosphate, dibasic - 6.5 gm Formaldehyde, 37% - 100.0 ml Distilled water - 900.0 ml Notes: Monobasic = (of an acid) Having one  replaceable  hydrogen atom. Dibasic = (of an acid) having two  replaceable  hydrogen  atoms Preparation steps for 1 liter of 10% Neutral Buffered Formalin: Gather materials:  Obtain commercial formalin (37-40% formaldehyde solution), distilled water, sodium phosphate monobasic, sodium phosphate dibasic (anhydrous), and a 1-liter container. Safety precautions must be followed when handling the chemicals.  Add water:  Pour approximately 900 ml of distilled water into the 1-liter container. 

51. Dissolve salts:  Add 4.0 g of sodium phosphate monobasic and 6.5 g of sodium phosphate dibasic (anhydrous) to the water and stir until completely dissolved.  Add formalin:  Measure 100 ml of commercial formalin and add it to the solution.  Finalize volume:  Add more distilled water to bring the total volume up to 1 liter.  Mix well:  Stir the solution thoroughly to ensure all ingredients are evenly distributed.  Check pH: Verify that the pH of the final solution is within the acceptable range of 7.2±0.5 Store properly:  Transfer the solution to a suitable container with a tight-fitting cap and store it in a well-ventilated area.  Label clearly:  Label the container with the solution's contents and date of preparation. 

52. 3. Methanol : Used as an alternative to ethanol, though it may have toxic potential.  Absolute methanol (100% ) uses: Blood smears / peripheral smear fixation (for Leishman / Giemsa prep), Gram stain smears, many cytology smears. Quick cell-fix for some immunostains Preparation Use absolute/anhydrous methanol (analytical grade or reagent grade). No dilution required—use as supplied. How to use Cover the smear or cells with methanol (or immerse slide) for 1–5 min (commonly 2–3 min for blood smears). Remove, let air-dry completely before staining. 4. Glutaraldehyde : An aldehyde fixative that is sometimes used, often in combination with other fixatives.  2.5% Glutaraldehyde in 0.1 M Phosphate Buffer (pH 7.4): Widely used for electron microscopy and histopathology .

53. Preparation (for 100 mL total): 25% Glutaraldehyde stock- 10 mL 0.2 M Phosphate buffer (pH 7.4)- 40 mL Distilled water - 50 mL Final concentration: 2.5% glutaraldehyde in 0.1 M phosphate buffer. Procedure: Measure 10 mL of 25% glutaraldehyde stock (handle in fume hood). Add 40 mL of 0.2 M phosphate buffer (pH 7.4) . Add 50 mL of distilled water to make final volume 100 mL. Mix gently — do not shake vigorously (to avoid foaming). Store at 4 °C in a tightly closed amber bottle . 5. Spray Fixatives : These are often used in a laboratory setting to quickly fix smears.

54. Spray fixatives are alcohol-based solutions used to instantly fix smears by spraying a fine mist over them. They prevent air-drying artifacts , preserve cell morphology , and are convenient for fieldwork or quick cytology . Composition of a Typical Spray Fixative: A spray fixative usually contains: Alcohol (main fixative) – denatures and coagulates proteins, preserving cell detail. Polyethylene glycol (PEG) or Carbowax – forms a thin protective film over the smear to prevent drying. Propellant – creates the spray pressure (in commercial aerosols; not used in lab-made versions). Laboratory Preparation of Spray Fixative: Formula (for 100 mL solution):

55. Component Quantity Purpose 95% Ethanol or Absolute Methanol 95 mL Main fixative agent Polyethylene Glycol (PEG-400 or PEG-600) 5 mL Coating/protective film (Optional) Glycerin (1–2 mL ) — Prevents over-drying and cracking Distilled water Add to 100 mL total volume — Preparation Steps: Measure 95 mL of 95% ethanol (or methanol). Add 5 mL of PEG-400 or PEG-600 (can slightly warm to mix well). Add a few drops (optional) of glycerin for smear protection. Mix thoroughly until solution is uniform. Pour into a fine-mist spray bottle (preferably glass or HDPE plastic). Label with: “Spray Fixative – 95% Ethanol + 5% PEG” Date of preparation Flammable – for cytology use only

56. Other types: Carnoy's solution :  A compound fixative that includes absolute ethanol, chloroform, and glacial acetic acid.  Zenker's fluid :  A compound fixative containing mercuric chloride, potassium dichromate, and acetic acid. Zinc formalin :  A less common fixative that contains zinc and formalin. Formal saline :  A less common fixative, though it can be used for long-term storage.  Acetic acid :  While not a primary fixative on its own, it is often combined with ethanol to prevent nuclear swelling and the loss of nucleic acids.  Ethanol/Ether :  A mixture of ethanol and ether that is used for some cytological preparations. 

57. Fixation of cells Methods for direct smears Direct smears are prepared from cell samples spread onto a glass slide, such as Pap smears and fine needle aspirations (FNA).  1. Wet fixation: This is the standard and preferred method for preserving nuclear detail.  Smear preparation:  After collecting the cell sample (e.g., with a spatula or brush), spread it thinly and evenly onto a clean, labeled glass slide. Avoid applying too much pressure, which can damage the cells. Immediate immersion:  Before the smear can air-dry, immediately place the slide into a container ( Coplin jar) filled with a liquid fixative. Fixative:  The most common fixative is 95% ethyl alcohol. Duration:  Leave the slide immersed for at least 15 to 30 minutes. The slide can be stored in the fixative for a longer duration if needed. 

58. 2. Spray fixation This method is an alternative to wet fixation, particularly for Pap smears.  Smear preparation:  Prepare the smear on a labeled slide. Immediate spraying:  Hold the spray fixative bottle about 3 to 4 inches from the slide and apply an even, fine mist over the entire cell area. Drying:  Allow the fixative to dry completely, which leaves a protective wax-like coating over the cells.  3. Air-drying This technique is used when specific stains, such as Romanowsky -type stains (e.g., Diff- Quik ), are planned for better cytoplasmic and background visibility.  Smear preparation:  Create a thin, evenly spread smear. Allow to dry:  Wave the slide in the air for about 10 seconds to aid rapid and complete drying. Limitations:  Air-drying causes cells to enlarge and compromises nuclear detail, which can affect the accuracy of some diagnoses. 

59. 10.Prepare cytological smears and stain with pap method. Prepare cytological smears: Label the slide:  Use a pencil to label the frosted end of the slide with the patient's name, date of birth, or accession number. Apply the specimen:  Place a small drop of the specimen (e.g., from a fine needle aspiration, scrape, or fluid) onto the center of the clean glass slide. Spread the specimen: Place a second slide on top of the first, or use a spreader slide. Use the weight of the top slide to gently spread the material thinly and evenly across the surface of the bottom slide. Avoid using too much pressure, as this can crush the cells. Fix the smear: Immediately after spreading, fix the slide to prevent cell degradation. Spray-fix:  Apply a spray fixative and allow the slide to dry completely before staining. Alcohol fixation:  Place the slide in a Coplin jar containing 95% or 80% isopropanol for at least 10 minutes. Finalize and transport:  Ensure the slides are completely dry if air-dried, and then seal the container tightly to prevent leakage before sending to the lab. 

60. Frosted end of the slide

61. Stain with pap method The Papanicolaou (Pap) stain is a polychromatic stain that differentiates cells based on cytoplasmic transparency and nuclear features, making it a valuable tool in cytopathology . The standard method for Papanicolaou staining involves three primary staining solutions and several steps of fixation, hydration, and dehydration.  Part 1: Initial preparation Collect and prepare the smear:  Obtain a specimen using a brush or spatula and spread the cells evenly onto a clean glass slide. Fix the smear:  Immediately immerse the slide into a 95% ethanol fixative solution for a minimum of 15 minutes. This must be done before the specimen dries out to prevent cellular distortion. If using a spray fixative, apply it evenly to the wet smear. Ensure lab safety:  Wear appropriate personal protective equipment (PPE), such as gloves, throughout the process. 

62. Part 2: Nuclear staining ( Hematoxylin ) Hydrate:  Take the slide from the 95% ethanol fixative and rinse it gently in tap water to hydrate the cells. Stain with hematoxylin :  Place the slide in a hematoxylin solution (e.g., Gill's or Harris's) for 1–3 minutes to stain the cell nuclei blue or black. Rinse:  Wash the slide thoroughly in running tap water to remove excess stain. "Blue" the nuclei:  Place the slide in Scott's tap water substitute or a weak ammoniated water for about 20 seconds. This alkaline solution changes the hematoxylin color from purplish-red to a dark blue. Rinse:  Wash the slide again in running water.  Part 3: Cytoplasmic staining and differentiation Dehydrate:  Run the slide through a graded series of alcohol to dehydrate it. For example, use 95% ethanol with several dips to start the process. Stain with Orange G (OG-6):  Immerse the slide in the Orange G-6 (OG-6) stain for about 1–1.5 minutes. This stain colors keratinized superficial cells a brilliant orange

63. Rinse:  Rinse the slide in two changes of 95% ethanol to wash away the excess Orange G. Stain with Eosin Azure (EA):  Place the slide in the Eosin Azure solution (such as EA-50 or EA-65) for 2–3 minutes. This polychromatic stain differentiates other cell components: Eosin Y:  Stains superficial squamous cells, cilia, and red blood cells pink. Light Green SF:  Stains the cytoplasm of active cells, such as parabasal cells and intermediate cells, blue or green. Rinse and dehydrate:  Rinse the slide in several changes of 95% ethanol to differentiate the stains and remove excess EA. Follow with 100% (absolute) ethanol for more thorough dehydration.  Part 4: Clearing and mounting: Clear with xylene :  Immerse the slide in two changes of xylene for 2 minutes each. This step makes the specimen transparent in preparation for mounting. Mount the coverslip :  Apply a few drops of a permanent mounting medium (such as DPX) to the cleared slide and carefully place a coverslip over the specimen, avoiding air bubbles. Examine under a microscope:  Once the slide is properly mounted, it is ready for microscopic examination. 

64. Interpretation Blue/Purple:  The nucleus of a cell stains blue to black. A normal nucleus will have well-defined chromatin patterns, while abnormalities may show irregularities. Green:  The cytoplasm of metabolically active cells stains green. Orange:  Keratin, a protein found in squamous cells, stains bright orange. Pink:  Superficial squamous cells can stain pink or orange