GMP Sop 03b

VERSION 1 – EFFECTIVE DATE: 01/01/2016
PREPARATION OF WATER BUFFERED

TO pH 7.2 WITH BUFFER TABLETS
MALARIA MICROSCOPY STANDARD OPERATING PROCEDURE – MM-SOP-03B
1. PURPOSE AND SCOPE

To detail the procedure for preparing buffered water from buffer tablets to pH 7.2 for use in preparation
of Giemsa stain working solution for routine staining of malaria blood films
This procedure is to be modified only with the approval of the national coordinator for quality assurance of
malaria microscopy. All procedures specified herein are mandatory for all malaria microscopists working
in national reference laboratories, in hospital laboratories or in basic health laboratories in health facilities
performing malaria microscopy.
2. BACKGROUND
Malaria parasites can be seen clearly under the microscope on properly stained blood films. The quality of
staining depends strongly on the pH of the working stain solution. Use of buffered water at the correct pH
(7.2) to dilute the stock stain solution helps to ensure good staining and ensures recognition of the specific
features of malaria parasites. Buffered water must be prepared and quality-checked for the correct pH
before staining blood films.
pH is the measure of the acidity or alkalinity of a fluid. It is based on a scale of near 0 (very acid) to 14 (very
alkaline). Liquids that are neither acid nor alkaline are described as neutral, at pH 7.0. The pH of a liquid
can be measured with a pH meter or with a colour indicator, such as the Lovibond comparator. Paper
indicator strips can also be used, but they are rapidly affected by high humidity and become unreliable.
In this SOP, use of commercially available buffer tablets that indicate a specific pH when mixed in a fixed
amount of distilled water (usually 1 L) is described. Buffer tablets do not have to be weighed and are useful
in laboratories with limited facilities. They must, however, be kept in an airtight tube under dry conditions;
otherwise, they rapidly absorb moisture and must be discarded. Some workers consider that the results of
staining are inferior when buffer tablets are used, but there is no evidence to support this perception.
3. SUPPLIES, MATERIALS AND EQUIPMENT

• commercial phosphate buffer tablets for 1 L of water (pH 7.2);
• commercial distilled or deionized bottled water, 1 L;
• a graduated cylinder, 1-L capacity;
• a conical flask or beaker, 1-L capacity;
• a screw-capped glass bottle, clean and dry, of 1-L or 500-mL capacity and
• small tongs or tweezers.
MM–SOP–03B: PREPARATION OF WATER BUFFERED TO PH 7.2 WITH BUFFER TABLETS
4. PROCEDURE
FLOW CHART DESCRIPTION OF ACTIVITY
1. Measure 1 L of distilled or de-ionized water
into a flask or beaker.
1. Pour 1 L of distilled or de-ionized
water into a flask or beaker.
2. Using small tongs or a tweezer, place one

2. Place one buffer tablet into the flask phosphate buffer tablet into the flask or
or beaker. beaker. Be careful not to touch the tablet
with your hand.
3. Mix by gently swirling the flask or beaker

3. Mix by gentle swirling. until the tablet is completely dissolved.
4. Label the bottle of buffered water as below,

4. Label the bottle of buffered water, and
and document the procedure in the quality
document the procedure in the quality
control log-book.
control log-book.
Buffered water pH 7.2
Date prepared: 17 Aug 2015
Expiry date: 24 Aug 2015
Prepared by: First name last name
5. Use water buffered to pH 7.2 to 5. Use water buffered to pH 7.2 to prepare the
prepare the Giemsa working solution. working solution of Giemsa stain.
5. PROCEDURE NOTES
• The buffer tablets (pH 7.2) must be kept in an airtight container, away from sunlight and damp.
They cannot be used if water vapour or moisture has reached them. Packed desiccant or silica gel
(not containing cobalt chloride) should be placed in the buffer tablet bottles to prevent moisture.
• Always check the expiration date of the buffer tablets before use.
• Store the prepared buffered water in a cool place away from direct sunlight. Use of a dark bottle
or a clear glass bottle wrapped in brown paper is recommended to prevent bacterial, fungal and
algal growth.
• Check continuously for contamination.
• Do not keep buffer solution for more than 7 days to avoid a change in pH and to prevent
contamination.
• Follow the manufacturer’s guidance for use and storage of buffer tablets. Use only the amount of
water specified by the manufacturer. Failure to do so will result in poor staining results.
• If facilities are available, check the pH of buffered water routinely before use, and record the result
in the quality control log-book.
2
MM–SOP–03B: PREPARATION OF WATER BUFFERED TO PH 7.2 WITH BUFFER TABLETS
6. QUALITY CONTROL AND DOCUMENTATION

Perform a quality control check of every new batch of buffered water and before every use, and record
the result in the quality control log-book. See MM-SOP 3c: Quality control of Giemsa stock solution and
buffered water.
7. RELATED SOPs
MM-SOP 3c: Quality control of Giemsa stock solution and buffered water.
8. REFERENCE
WHO. Basic malaria microscopy. Part I. Learner’s guide. Second edition. Geneva; 2010.
9. DOCUMENT HISTORY
Date Version Comments Responsible person

(mmm/yyyy) (First name, last name)
Reviewed and finalized Glenda Gonzales,
Jan 2016 1 by experts, edited and Technical Officer,
formatted WPRO

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VERSION 1 – EFFECTIVE DATE: 01/01/2016

PREPARATION OF WATER BUFFERED

1. PURPOSE AND SCOPE

3. SUPPLIES, MATERIALS AND EQUIPMENT

2. Using small tongs or a tweezer, place one

3. Mix by gently swirling the flask or beaker

4. Label the bottle of buffered water as below,

6. QUALITY CONTROL AND DOCUMENTATION

Date Version Comments Responsible person

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