Preparation of Basic Laboratory Reagents and Solutions
Some of laboratory tests need the reagents and solutions for the purpose of preserving morphology, speed up of reactions and give color the features of cells, and parasites. Example Gram stains provide gram positive Bacteria blue or purple color and gram negative pinkish red color. So this start on preparation of reagents and solutions.
In this article you will learn:
- Apparatus and Material needed to prepare Laboratory reagents and solutions
- Special apparatus for dissolving reagents
- Weighing of Ingredients or solute in analytical balance
- Preparation of Common reagents and solutions (Drabkin’s, Field stain, Giemsa, Acridine orange, saline, iodine, ZN stain and distilled water)
- Quality of Equipment and Reagents
- Quality of ingredients
Apparatus and Material needed to prepare respective reagent
- measuring cylinder
- beakers or flasks
- weighing balance and spatula
- storage containers
- Powder or solid solute
- Distilled water or other solvent
For example: When preparing Field stain one needs Field Stain A powder and Field Stain B and distilled water.
Special apparatus for dissolving reagents
- Some reagents do not go into solution when simply mixed together.
The following special equipment may need to be employed:
o glass rod
o magnetic stirring bar
o magnetic stirrer
o hot plate
o water bath
A water bath
Example of preparation of reagents that requires heated diluents:
- Heat 600 mL distilled water to boiling.
- Weigh out 6 g of stain powder and add to 500 mL volumetric flask containing some hot water.
- Mix by swirling to dissolve.
- Add the remaining volume of distilled water to make 500 ml.
- Parafilm and mix thoroughly.
- Filter and place in brown bottle.
- Label with correct using tape or label and marker pen (name of reagent, amount prepared, date of preparation. Technician who prepared)
- Add the solute to the diluent. (A portion of the diluent is measured into the measuring cylinder. Some procedures require that the diluent is heated.)
- The solution is homogenously mixed until the solute goes into solution (this may require the use of stirring rod, or magnetic stirrer and magnetic bar.)
- The remaining diluent is added to make the final volume.
- The solution is mixed again.
Weighing of Ingredients or solute in analytical balance
- The Analytical balance is used to weigh out the specific amount of powder using a spatula to dispense the powder onto a filter paper.
- The measuring cylinder can be used to measure specific amount of distilled water or other diluents added to the flask.
- The powder is then added to the diluents to make the solution.
- The solution is mixed by swirling the flask.
Steps for weighing the ingredients
- Determine the balance is centered, clean and turn on power, if applicable
- Adjust the balance to zero with no sample.
- Place a weighing boat or filter paper on scale and zero or tare.
- Place the estimated amount of dry powder on the scale with a clean spatula.
- Read the mass after the reading has become stable.
- Adjust by adding more powder or subtracting away powder.
- Read the mass.
- Continue adding or subtracting powder with a spatula until the mass reads the desired amount.
- Carefully remove the filter paper or weighing boat containing the powder and pour into the measuring cylinder or flask containing the estimated amount of diluent.
- Turn off power or arrest the pan to neutral.
- Wipe any spilled powder off the balance with a soft brush.
The equipment and supplies needed to weigh the ingredient include the balance, spatula, weighing boat or filter paper.
Preparation of Common reagents and solutions (Drabkin’s, Field stain, Giemsa, Acridine orange, saline, iodine, ZN stain and distilled water)
- Drabkin’s solution: potassium cyanide, potassium ferricyanide and sodium bicarbonate, distilled water. This reagent is used to measure haemoglobin and the potassium ferricyanide is the main active ingredient.
- Field stain: Field A powder, distilled water, sodium azide used along with Field B powder, distilled water, sodium azide. Field stain is used to color cells in whole blood smears for viewing blood cells or parasites.
- Giemsa: Giemsa powder, absolute methanol glycerol, buffered water or distilled water (pH 7.2) Giemsa stain is used to color cells in whole blood smears for viewing blood cells or parasites.
- Acridine orange: Acridine orange powder, glacial acetic acid, distilled water. Acridine orange is a special stain for viewing blood parasites using a fluorescent microscope.
- Physiological Saline: sodium chloride and distilled water. Saline is used as a diluent for whole blood since it is the correct salt solution to prevent rupture of cells. It is also used as a diluent to blood plasma and in making up solutions and reagents used in the laboratory.
- Iodine Stain: potassium iodide, iodine, distilled water. This stain is used to color parasite egg components so that they can be better visualized with a microscope.
- Ziehl Neelsen stain: strong Carbol Fuchsin, absolute (70%) methanol, phenol crystals, 3% HCl in methanol, 25% sulphuric acid, (3% HCl in alcohol may be substituted), 0.3% Methylene blue. This stain is used to colourise the acid fast bacilli (AFB) that cause tuberculosis, Mycobacterium spp.
Quality of Equipment, Reagents and solutions
Verification of equipment function:
- Check that the balance maintenance is current and documented in the Maintenance log book.
- With each use: With the weighing pan secured (arrested) use a clean camel- hairbrush to sweep away remnants of dust or chemicals into a dustpan. Verify that weighing pan is free from dust and fingerprints prior to use.
- With each use: Verify placement on a vibration-free bench, in an area free from air drafts, and direct sun. Adjust the lengths of the centering screw legs using the level bubble, as available. Zero the balance with empty pan. Place a weighing vessel or paper on the pan and adjust the tare button to subtract out the mass of the vessel. Always secure the pan in standby mode before adding weight to the balance.
- Periodically: Check the accuracy and precision of the balance by weighing known weights (50, 100, 200, 500, 2000 and 4000 mg) in triplicate taking care to handle with clean cotton gloves or forceps in order to protect the standardized weights. Calculate the accuracy and precision based on the standard procedure or manual.
Quality of Reagents and solutions
- Reagents and solutions is properly labeled.
- Reagents and solutions are not expired.
- Reagents and solutions generally appear adequate:
-The quantity must enough amount to be dispensed within reagents and solutions according to manufacture manual.
- Reagents and solutions quality appears correct.
- Reagents and solutions must preserve morphology, chemical reactions and color to the cells, parasite and Bacteria.
- Reagents and solutions are prepared by Standard operating procedure.
- F.J. Baker, R.E. Silverton (2001): Introduction to Medical Laboratory Technology, 7thEdition, Oxford University Press; Oxford, England.
- Monica Cheesbrough (1981): Medical Laboratory Manual for Tropical Countries Volume I, 2nd Edition Butterworth & Co. (Publishers) Ltd;
- Monica Cheesbrough (2002): District Laboratory Practice in Tropical Countries Part 1 & 2, Cambridge University Press; Cambridge, England.