Preparation and Reagent

Preparation and Storage of Basic   Laboratory Reagents and Solutions 

    Definition of Terms

• Antiseptic: Substance used on humans and other animals that destroy harmful microorganisms or inhibit their activity. Anti: against. Septic: The presence of disease-causing microorganisms or their toxins in tissues or in the blood. So antiseptic also means against the presence of micro-organisms.
• Disinfectant: Substance used on inanimate objects that destroy harmful microorganisms or inhibit their activity.

·         Antiseptics are used to eliminate most bacteria and other micro-organisms from human skin such as the median cubital vein of arm prior to venipuncture.

·         Disinfectants are also used to eliminate most bacteria and micro-organisms from inanimate objects such as laboratory surfaces. Both are used to eliminate micro-organisms for clinical laboratory use.

·         Solution: mixture made up of a solute, the main component or active ingredient, and the solvent, inert diluting component.

• Solute: the main component of a solution
• Solvent: the inert diluting component

·         Dilution: A solution made less concentrated by the addition of more diluents or solvent. Diluent: solvent used to lower concentration of a solute in a solution

Step 3: Types antiseptics and disinfectants (5 minutes)

Common Antiseptics: 70% methanol and isopropyl alcohol

• 70% methylated spirit (methanol) and isopropyl alcohol. These two antiseptics are used for cleansing the skin of micro-organisms prior to venous or capillary puncture in blood collection.

Antiseptics:

Methanol

                                     

Common Disinfectants: 10% bleach and 5% Lysol ®

Bleach solution is a 30-35% solution of sodium hypochlorite.

·         10% bleach solution (Common brand name of Jik®) is a common laboratory disinfectant.

·         Lysol ® is a commercially prepared disinfectant containing phenols.

5% Lysol ® solution is used to cleanse laboratory surfaces such as benches, instrument covers to eliminate most harmful bacteria, viruses and other micro-organisms. 5% Lysol is commonly used for disinfection in microbiology laboratory areas.

Step 4: Types of concentration (30 minutes)

·         Volume per volume (V/V) in units such as millilitres or liter. For example V/V may be measured as mL/ mL or mL/ 100 mL. The numerator of the proportion equals volume of solute and the denominator equals the total volume made up of volume of solute + volume of diluent. Total volume = volume solute + volume diluents

·         A pipet or measuring cylinder is used to measure the volume of solute.

Measuring cylinder

·         Usually a measuring cylinder is used to measure the volume of diluents.  Conical flasks are used to mix the solution.

·         Weight/Volume stands for a proportion of weight or mass per volume in g/ mL or g/ 100 mL and is abbreviated W/V. The proportion equals g of solute/ total volume of solute + diluents. Gram/ millilitre or g/ mL also is the unit for specific gravity.

Step 5: Method for preparing V/V, W/V and W/W dilutions (60 minutes) Preparing V/V dilutions: 

·        Start with measuring out mL of stock antiseptic or disinfectant with a pipet for small volume or graduated measuring cylinder for larger volume. 

·        Next dilute the measured volume of stock to a final volume in a graduated cylinder. For example, if you are to dilute 5 mL of Lysol ® to the total volume of 100 mL, use a graduated cylinder that can hold 100 mL. You will be adding approximately 95 mL of diluents to the 5 mL of Lysol ®.

·        The tutor may bring a measuring cylinder and pipet to illustrate the point or refer to the pictures above of pipets and cylinder.

Preparing W/V dilutions:

·        Start with weighing out g of solute antiseptic or disinfectant powder with a balance.

·        Next, put the weighed powder into a graduated measuring cylinder that holds the final volume. Add enough diluent to dilute to the final volume.

Preparing W/W dilutions:

·        Start with weighing out grams of solute antiseptic or disinfectant powder.

·        Next, dilute to a final weight with diluent.

·        The density of pure water is 1 g/ mL. Since 1 gram of pure water has a volume of 1 mL, based on its density, gram/gram can use the solvent if it is pure water by measuring in either millilitres or grams.

Step 6: Key Points (5 minutes)

·         Definition of disinfectant is a substance that destroys harmful bacteria on inanimate objects while an antiseptic is a substance that destroys harmful bacteria on human skin or mucous membranes.

·         The methods for preparation of disinfectants and antiseptics is generally by pouring out a specific volume of concentrated stock antiseptic or disinfectant and diluting up to a final volume with distilled water.

·         This is called a volume/ volume or V/V dilution.

·         Another kind of dilution you may encounter is measuring the required weight of concentrated stock powder and diluting up to a final volume with distilled water to make a weight/ volume (W/ V) dilution.

Step 7: Evaluation (10 minutes)

·         Ask the question to students:

• How do you prepare 50 mL of 10 w/v% bleach solution from stock Jik®?

·         Answer for tutor to know: 10 w/v% x 50 mL = 5 mL. 5 mL of bleach is put in a 50 mL flask and diluted to final volume with approximately 45 mL distilled water.

References

·         F.J. Baker, R.E. Silverton (2001): Introduction to Medical Laboratory Technology, 7th Edition, 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.