Preparation of reagents REQUIRED FOR ROUTINE DIAGNOSTIC PURPOSE

THIS TOPIC IS FOR PRARSITOLOGY

1. 4.2 Preparation of reagents required for routine diagnostic purposes ( iodine solution, brine solution, 33% zinc sulphate solution, normal saline solution, Stooll's reagent, Giemsa and Leishman's stain and benzidine solution).

2. Iodine solution: Potassium iodide (KI) 10 g Iodine 5 g Distilled water 100 ml Dissolve KI in about 20-30 ml of distilled water. Add iodine and heat gently with constant mixing until iodine is dissolved. Dilute to 100 ml with distilled water. Store in amber glass- stoppered bottle in the dark

3. Brine solution: A brine solution is typically a concentrated mixture of water and salt, most commonly sodium chloride, but can also include other salts like potassium chloride, calcium chloride, and magnesium chloride.  The concentration of salt in brine can range from about 3.5% (like seawater) to up to 26% (a saturated solution). 

4. Water: The primary component of brine is water, acting as the solvent for the dissolved salts.  Salt: The most common salt in brine is sodium chloride ( NaCl ), also known as table salt.  Other Salts: Depending on the application and source, brines may also contain potassium chloride ( KCl ), calcium chloride (CaCl2), magnesium chloride (MgCl2), and other salts.  Concentration: Brine concentration is typically expressed as a percentage of salt in water, ranging from low concentrations like 3.5% in seawater to higher concentrations like 26% for saturated solutions. 

5. 33% zinc sulphate solution: To prepare a 33% zinc sulfate solution, you typically start with zinc sulfate monohydrate (ZnSO4·H2O) and dissolve it in water .  The exact process depends on the desired concentration, but it generally involves weighing out the appropriate amount of zinc sulfate and adding it to a calculated amount of water.  Process: Calculate the required amount: For a 33% solution, you'd need 33 grams of zinc sulfate monohydrate for every 67 grams of water (since 33 + 67 = 100). This gives you a 33% solution by weight. 

6. Weigh the zinc sulfate: Accurately weigh out the calculated amount of zinc sulfate monohydrate.  Dissolve the zinc sulfate: Add the zinc sulfate to the appropriate amount of water in a container, like a beaker or flask.  Stir or shake: Stir or shake the solution until all the zinc sulfate has dissolved. The solution should appear clear and colorless.  Important Considerations: Solubility:  Zinc sulfate is soluble in water, but the solubility can vary depending on temperature.  Specific gravity:  The specific gravity of a 33% zinc sulfate solution is around 1.375, meaning it's denser than water.  pH:  The pH of a zinc sulfate solution is typically acidic. 

7. Normal saline solution: Normal saline, a common medical solution, is prepared by dissolving a specific amount of salt (sodium chloride) in water.  To make a 0.9% normal saline solution at home, you'll need approximately 2 teaspoons of non-iodized table salt for every 1 liter (4 cups) of water.  The water should be boiled and cooled to room temperature to ensure it's sterile .  This solution is used for various purposes, including wound cleaning, nasal irrigation, and even as a component in some medications. 

8. Boil the water:   Boil approximately 1 liter (4 cups) of tap water for 15 minutes to ensure it's sterile. Cool the water:  Let the boiled water cool to room temperature. Dissolve the salt:  Add 2 teaspoons of non-iodized table salt to the cooled water. Stir to dissolve:  Stir thoroughly until the salt is completely dissolved. Store the solution:  Transfer the solution to a clean, airtight container. Use within 24 hours:  Store the solution in the refrigerator and use it within 24 hours. 

9. Important Notes: Sterility: While this homemade method provides a basic saline solution, it's not as sterile as commercially prepared solutions. Always ensure the water is properly boiled and cooled to reduce the risk of contamination.  Non-iodized salt: Use non-iodized salt as iodized salt can cause issues, especially with specific medical conditions.  Freshness: Since this is a homemade solution, it's crucial to use it within 24 hours and discard any remaining solution. 

10. Uses of Normal Saline: Wound cleaning:  Normal saline can be used to clean wounds and prevent infection.  Nasal irrigation:  It can be used in neti pots or other nasal irrigation devices to relieve congestion and clear nasal passages.  Medications:  It's used as a diluent for some medications and IV solutions.

11. Stooll's reagent: Stoll's reagents are a group of chemical solutions used for various laboratory procedures, especially in chemistry and microbiology .  They are prepared by dissolving specific chemicals in a solvent, typically water, ethanol, or other organic liquids, and can include acids, bases, salts, or other chemical compounds.  1. Identifying the Reagent: Determine the specific reagent needed for the intended experiment or procedure.  Consult reagent preparation protocols or recipes for the specific reagent

12. 2. Preparing the Solution: Choose the Solvent: The solvent used depends on the solute and the specific requirements of the reaction or test. Common solvents include distilled water, ethanol, and organic solvents like dimethyl sulfoxide (DMSO) Calculate the Concentration: Determine the required concentration of the reagent solution Weigh or Measure Solute: Use a balance or graduated cylinder to accurately measure the required amount of the solute (the chemical being dissolved).  Dissolve the Solute: Add the solute to the solvent and stir or heat until it is completely dissolved

13. Filter (if needed): If any undissolved particles remain, filter the solution to remove them.  Label and Store: Clearly label the reagent bottle with the name of the reagent, its concentration, and the date of preparation.  3. Safety Precautions: Always wear appropriate personal protective equipment (PPE),  such as gloves, lab coat, and eye protection Work in a well-ventilated area : to avoid inhaling any hazardous fumes. Be aware of any potential hazards : associated with the specific reagents and solvents being used. 

14. Examples of Common Reagents: Acid solutions:  Hydrochloric acid ( HCl ), sulfuric acid (H2SO4). Basic solutions:  Sodium hydroxide ( NaOH ), ammonium hydroxide (NH4OH). Buffer solutions:  Solutions that maintain a specific pH. Salt solutions:  Sodium chloride ( NaCl ), potassium chloride ( KCl ). Indicator solutions:  Solutions that change color to indicate the presence or absence of a specific substance. 

15. Giemsa stain: To prepare Giemsa stain, first create a stock solution by dissolving 3.8g of Giemsa powder in 250ml of methanol, heating the solution to 60°C, and slowly adding 250ml of glycerin.  Filter the solution and store it for 1-2 months before use.  For a working solution, dilute the stock solution with a buffer solution (like pH 7.2 phosphate buffer) or distilled water, depending on the specific staining procedure. 

16. Stock Solution: 1. Dissolve Giemsa powder: Add 3.8g of Giemsa powder to 250ml of methanol in a clean container.  2. Heat and add glycerin: Heat the solution to approximately 60°C, and slowly add 250ml of glycerin while stirring.  3. Filter: Allow the solution to cool and then filter it through a filter paper (e.g., Whatman #1).  4. Mature and store: Store the filtered solution in a dark bottle for 1-2 months to allow it to mature and the stain quality to improve

17. Working Solution Preparation: 1. Dilute with buffer: For a 10% working solution, dilute 1ml of Giemsa stock solution with 9ml of buffered water (pH 7.2). 2. Use fresh solution: Prepare the working solution just before staining and discard any unused stain within 15 minutes. 3. Alternative working solution: For a 3% working solution, dilute 3ml of Giemsa stock solution with 97ml of buffered water. 

18. Important Considerations: Methanol: Methanol is a volatile and flammable liquid, so use it in a well-ventilated area and avoid direct contact with skin.  Glycerin: Glycerin is a viscous and hygroscopic liquid, so handle it with care and store it properly to prevent it from absorbing moisture.  Maturation: The maturation period for the stock solution is crucial for achieving optimal staining quality.  Fresh working solution: Always prepare fresh working solutions before use to ensure the best staining results.  Buffer: Using a proper buffer solution, like phosphate buffer with a pH of 7.2, is essential for achieving optimal staining. 

19. Staining procedure (Thin Film staining): On a clean dry microscopic glass slide, make a thin film of the specimen (blood) and leave to air dry. dip the smear (2-3 dips) into pure methanol for fixation of the smear, leave to air dry for 30seconds Flood the slide with 5% Giemsa stain solution for 20-30 minutes. Flush with tap water and leave to dry

20. Staining Procedure ( Thick Film Staining): Add a thick smear of blood and air dry for 1 hour on a staining rack. Dip the thick blood smear into diluted Giemsa stain (prepared by taking 1ml of the stock solution and adding to 49ml of phosphate buffer or distilled water, but the results may vary differently). Wash the smear by dipping in in buffered water of distilled water for 3-5 minutes Leave it to dry Results: The Cytoplasm and cytoplasmic granules of blood cells appear red in color while the nucleus appears blue-purple in color

21. Leishman's stain: Leishman stain is typically prepared by dissolving Leishman stain powder in methanol, then diluting with a buffer solution like phosphate buffer.  The powder can be purchased commercially or prepared from a mixture of dyes like Azure- Eosinate salts and methylene violet.  The stain is used to differentiate cell types in blood smears, especially for identifying parasites like those causing malaria or Leishmaniasis

22. 1. Preparing the Stock Solution: Methanol: Dissolve Leishman stain powder in methanol. For example, 1.5g of Leishman stain powder can be dissolved in 500 ml of methanol,  Heating (Optional): Some protocols suggest gently heating the mixture in a water bath (40°C) to aid in dissolution.  Storage: Store the stock solution in a dark, tightly closed bottle at room temperature (15-25°C) to prevent oxidation and precipitation of dyes. 

23. 2. Preparing the Working Solution: Buffer: Dilute the stock solution with a buffer solution like phosphate buffer (pH 6.8, 6.4, or 7.0) and distilled water, Mixing: Mix the solutions thoroughly and allow them to stand for a few minutes before use. 

24. Procedure: 1. Place slides in methanol (fixative) for 30 seconds. 2. Place slides in Leishman Stain Solution for 3 minutes 3. Place slides in Stain-Buffer mixture for 6 minutes. See Reagent preparation section – “Stain Buffer Mixture”. 4. Remove slide from Stain-Buffer mixture and rinse with 10-15 ml of deionized water. 5. Allow slides to dry. 6. Examine microscopically

25. Result: Red blood cells (erythrocytes):  Stain yellowish-red. Lymphocytes:  Nuclei stain deep purple, while cytoplasm stains light blue. Neutrophils :  Nuclei stain dark purple, and granules stain reddish-violet. Eosinophils :  Nuclei stain blue, and granules stain red to orange. Basophils :  Nuclei stain purple to dark blue, and granules stain dark purple to black. Malaria parasites:  Stain red, with the cytoplasm appearing blue. 

26. Benzidine solution: A benzidine solution typically consists of  benzidine dihydrochloride dissolved in a solvent, often acetic acid, and sometimes with a small amount of hydrogen peroxide added just before use.  The concentration of benzidine can vary depending on the specific application, but a common concentration is 0.2% benzidine dihydrochloride in 0.5 M acetic acid.  Benzidine Dihydrochloride :  This is the active ingredient in the solution. It's a salt of benzidine , which is an organic compound with the formula (C6H4NH2)2. Acetic Acid:  This is used as a solvent to dissolve the benzidine dihydrochloride . Hydrogen Peroxide (Optional):  May be added just before use to enhance the reaction and color change in the benzidine test.

27. The benzidine solution is used in various applications, including: Detection of Blood:  It's a common reagent in forensic science and clinical laboratories for detecting the presence of blood.  Colony Staining:  It can be used to stain colonies of bacteria, particularly those that produce heme , which can react with benzidine .  Other Chemical Tests:  It can be used in other chemical reactions and assays. 

28. Principle of Benzidine test: Hemoglobin has peroxidase activity which releases nascent oxygen from H2O2 . Nascent oxygen oxidizes Benzidine in acid solution to form blue dye Procedure of Benzidine test: Take 2ml of Glacial acetic acid saturated with Benzidine and add 2ml of H2O2 To this add 4ml of urine in test tube Development of blue color indicates the presence of occult blood or  hemoglobin