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Medical Laboratory Technology: Volume 1
Figure 4.45 (a-c) Single-pan physical balances of dierent types. The triple beam balance (c) is of
superior quality and most commonly used. Each beam bears dierent range of weights.
is balanced. The poising nut adjusts the balance before weighing the substance; at the end of
the adjustment, all the slide weights are at ‘0’ and the pointer is at ‘0’. A tare beam is supplied
with some of the improved balances; this is used to ‘zero’ substance during weighing. The
three beams are illustrated in Figure 4.46.
Introduction to Laboratory Equipment and Basic Laboratory Operations
Figure 4.46 Use of triple beam balance (single pan). The reading is 79.89.
119
Use of double-pan physical balance
The use of a double-pan physical balance with two beams of sliding weight is described here
(Figure 4.47). This type of physical balance is more commonly seen in the clinical laboratories
of developing countries.
1. Zero seing:
• Move the poising nut to the middle of its screw and push all the weights to their ‘0’
position, the extreme left notch.
• Check that the pointer is swinging freely. If it is touching the side of the pillar,
move the pointer a lile forward. The pointer should move equally to both sides
of the ‘0’ mark in the centre. If not, move the poising nut for ‘zeroing’ of the
balance.
2. Determining the container weight:
• Model with tare weight: Put the container on the pan and move the tare weight until
the pointer swings equally to both sides of ‘0’. The balance is again poised and the
weight of the container is nullied.
• Model without tare weight: Find out the weight of the container by moving the
weights on the arms at dierent ranges until the pointer shows equal swing on
both sides of ‘0’. For example, if after the weight-seing for the container, the
middle beam weight (range 0–500 g) is at ‘0’, tare beam weight (range 0-100 g)
at 10 and the front beam weight (range 0–10) at 5.1, the weight of the container is
15.1 g.
3. Calculation of nal weight:
Make a note of the weight of the container (15.1 g, in the above example) and add this
weight to the required weight of the substance (e.g., 384.2), which comes to 384.2 + 15.1 =
399.3 g.
4. Seing the weight:
• Set the middle beam weight (range 0–500 g) to the 300 g position.
• Then set the tare beam weight (range 0–100 g) to 90 g position.
• Finally, set the front beam weight (range 0–10 g) to 9.3 g position.
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Medical Laboratory Technology: Volume 1
Figure 4.47 Use of double-pan physical balance: (a, b, c) Examine the balance and its scale, (d)
Locate the zero adjustment knob, (e) Clean the pans and balance them with the zero
adjustment screw; check the sensitivity by putting equal weights on both pans and
gradually increase the small weight (mg) until the pointer begins to move, (f) Put the
weighing paper on the left pan and balance the pan by moving the weight on the scale,
(g-l) Record the weight; add the reagent on the weighing paper and weigh again. Add the
weights placed on the pan and weights indicated on the scales (1–10 and 1–200 gm).
Actual weight of the reagent is the dierence of the nal weight and the weight of the
weighing paper.
Note Always check the scale of the graduation on each beam. If there are ten divisions
between 0 and 1 g on the front beam (0–10 g range), each division is equivalent to 0.1 g or
100 mg. If there are ve divisions, it will be equivalent to 0.2 g or 200 mg.
Introduction to Laboratory Equipment and Basic Laboratory Operations
121
Analytical balance
Analytical balances are more accurate, with a sensitivity of 0.1 mg or lower. Most analytical
balances have a maximum weight limit which means that the balance should not be used for
weighing substances beyond the tolerance point. The balance is enclosed in a glass case to
avoid air draft. The balance should be placed on a rm table, preferably made of concrete, to
minimize disturbance during weighing.
There are two basic types of analytical balances, the double-pan type and the single-pan
type. In the former case, two pans are suspended from a cross-beam, material to be weighed
is put on the left pan and counterweights are put on the right pan (Figure 4.44). Counterweights of less than 100 mg are manipulated by the rider which is placed on the cross-beam.
This type of balance is more common, although the sophisticated single-pan balances are
seen in advanced private laboratories. Single-pan automatic balances have the weights built
inside and are added by manipulating dials which indicate the weights added (Figure 4.44).
Analytical balances are expensive and must be handled carefully.
• Select a balance that suits the requirement.
• Never put the substance directly on the pan. Use the watch glass or weighing paper;
beakers and other containers are also used provided they are not too heavy.
• The balance must be in an area which is least disturbed.
• All substances must be weighed at room temperature.
• Load and unload the balance only when the pan is arrested.
• If the standard weights are to be placed manually, always use forceps to pick up the
weights.
• Always balance the empty pans before using the balance. In case of a suspended dou-
ble-pan balance, a screw is aached to each end of the cross-beam (Figure 4.44), which
is screwed out (increases weight on that side) or screwed in (decreases weight on that
side). Initial adjustment of the unloaded balance to a reading of zero is necessary.
Colorimeters and Spectrophotometers
Colorimeters and spectrophotometers are mostly used in the clinical biochemistry laboratory. The instrument measures absorbance (A) or optical density (OD) of coloured solutions in
the visible range. The use of spectrophotometers in the ultraviolet range is not yet popular
in the laboratories of developing countries. If the measurement is done visually with lters,
the instrument is called comparator (Figure 4.48). Modern spectrophotometers are capable
of providing specic quality of light (expressed in ‘nm’ wavelength) with the help of prisms
or diraction grating. Colorimeters use lters that provide ranges close to the selected wavelengths. There are several types of colorimeters and spectrophotometers in the market; a few
are illustrated in Figure 4.48.
Figure 4.48 Commonly used photometers in laboratories
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Medical Laboratory Technology: Volume 1
Refrigerators
A refrigerator is necessary in any clinical laboratory. It prolongs the life of perishable materials by cooling. Low temperature slows down biochemical activities and thereby arrests deterioration. In the clinical laboratory, the refrigerator stores media, reagents antisera, antibiotic
disks and other material. Special refrigerators are used in the blood bank, which maintain
temperatures close to 4°C (± 1°C). With the blood bank refrigerator, temperature must be
constantly recorded on a chart.
For smaller laboratories performing routine diagnostic tests, household refrigerators are
satisfactory, whereas in large laboratories commercial refrigerators or walk-in cold rooms are
provided. Choose two smaller refrigerators rather than one large one with double capacity.
This is because in case of mechanical breakdown of one of the refrigerators there will be less
interruption and stored materials can be saved.
The refrigerator is divided into two compartments—the freezing compartment and the
cooling compartment. The freezing compartment is used for keeping substances in frozen
state (sera, certain antibiotic discs and others). The temperature of the freezing compartment
is usually between –15 and –20°C. The cooling compartment outside the freezing compartment should be 4–6°C. Check the temperature of the cooling compartment daily—place a
thermometer, dipped in a bole of water at a corner of the cooling compartment.
LabOratOry water
Water supply to the laboratory is a basic requirement. The water supplied to the laboratory
can be classied as tap water, deionized water, distilled water and deionized distilled water.
The clinical laboratory needs an abundant supply of clean water, which may not always be
available. Because the composition of tap water varies widely it cannot be used for preparing
reagent solutions. The primary use of tap water is washing, but it is the source of other forms
of puried water.
Storage of water is important for areas where water can be scarce in certain parts of the year
or the water comes from a tank or well which may go dry. Hence, the laboratory must have
its own reserve supply probably in plastic containers. Decant the water that has been stored
before ltering.
Obtaining Clean Water
Tap water can be ltered to reduce crude contaminations and if minerals (electrolytes) are
removed by chemical treatment, tap water becomes deionized water. The deionized water,
however, is not free from organic maer. Distillation is another process to get puried water,
it may be free from organic and inorganic materials dissolved in water but it may contain
volatile gases. Deionized distilled water or double-distilled water is the purest form and may
be used in preparing standard solutions. If tap water shows a deposit after storing in a bole
for about 3 h, it requires ltration.
Filtration
A porcelain or sintered glass lter can be aached to the tap. Alternatively, water is rst kept
in a bucket, a lter with a siphon arrangement is immersed in the bucket and the clean water
is drawn out into a bole (Figure 4.49). It is important that the lter is cleaned at least once
a month by dismantling and washing in boiling ltered water. In dry seasons, water may be
scarce; it should then be stored in plastic containers or glass containers (Figure 4.49). Water
used for washing can be stored in metal drums. Stored water must be decanted for ltering
so that the sediments can be avoided.
Introduction to Laboratory Equipment and Basic Laboratory Operations
123
Figure 4.49 (a) Plastic water storage container, (b) Water ltration through sand lter and gravel, (c)
Solar still and its components to get distilled water, (d) Water ltration through porous
unglazed porcelain
Using a sand lter with gravel at the boom (Figure 4.49b) provides reasonable amounts of
clean water but the water is not free from minerals and volatile organic compounds.
Tap water and ltered water contain undesirable minerals and organic maer. Hence, they
are not always suitable for the preparation of many reagents and solutions in the laboratory.
Solar still
Distilled water is used for the preparation of reagents and as a nal rinse for some glassware before drying. For laboratories in remote areas and with limited resources, a simple
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Medical Laboratory Technology: Volume 1
solar-powered water still (Figure 4.49) can provide water for the preparation of reagents.
Commercially available distilled water may have absorbed volatile gases. Freshly collected distilled water from solar still or distilled water prepared in the laboratory is best suited for preparing laboratory reagents. Always store the distilled water in glass or plastic
containers and use the distilled water, prepared the same week, for preparing laboratory
reagents.
Solar water stills can be easily constructed using a clean plastic container with two compartments (one large and one small) and a large surface area, over which is placed a glass
cover in a sloping position. Water is poured into the large compartment from which it is
evaporated by the sun. It condenses on the glass cover and drops into the small compartment.
The small compartment has an outlet at the boom through which the distilled water can
pass into a glass bole placed underneath the container. In tropical climates, 2–7 L of distilled
2
water can be produced daily from a solar still with a surface area of 1 m
.
Important Collect the distilled water in a glass or plastic container, not in a metal contain-
er. Replace or replenish the water when the level reaches a height corresponding to the last
quarter of the compartment. It will contain residue.
Distillation equipment
Distilled water prepared in most laboratories is by boiling ordinary water. The steam generated from the boiling water is then cooled down while passing through a condenser. The
condenser carries cold running tap water which forms a jacket around the central tube in
which steam condenses into water. This distilled water is then collected in a ask or bole
(Figure 4.50).
The distillation apparatus (Figure 4.50) can be made of glass or metal (alembics). Heating
of the distillation apparatus can either be accomplished with gas or kerosene or electricity. If
properly done, single-distilled water is suciently pure for most laboratory work. Doubledistilled water is used only for special purposes. Discard the rst 10% of the distillate which
contains the volatile gases. Similarly, distillation should be discontinued when the last 10%
of the water is remaining. Hence, if 2 L of water are distilled, the rst 200 mL of distillate
and the last 200 mL of residual water should be discarded. The water may be freed of
organic or nitrogenous compounds by adding a lile potassium permanganate to the water
(1 g/L) before distilling. Glass stills are expensive, require electric heating and are more
fragile. The glass-distilled water is more pure and the still yields higher quantities of the
distillate. While the glass still is in operation, it should be aended occasionally in order
to check the ow of water as well as the water level of the distillation ask. Use only the
round boom ask for boiling the water during distillation. If the glass distillation ask is
dry, it will crack.
If the heating is done with a gas burner, gas stills, made of copper or stainless steel, can
be used which provide single-distilled water. It should be of a capacity that will yield 1–2 L
of distillate per hour. The size of the distillation set must be adequate to meet the needs of
the laboratory. The choice of the distillation equipment depends on the facilities available.
Distilled water should not be made in large quantities and stored over a long period of time.
Prepare only enough distilled water to last a few days. Store the distilled water in large plastic or glass boles which should always be kept stoppered. Distilled water is only free from
electrolytes and other substances but it is not sterile.
Testing the distilled water
It is a good practice to test distilled water occasionally for the presence of chloride and
sulphate ions (Figure 4.50).
Introduction to Laboratory Equipment and Basic Laboratory Operations
125
Figure 4.50 Laboratory water–demineralized and distilled, (a) Ion exchange columns in the water
line of the laboratory, (b-d) Various types ofdeionizers are used in order to get mineralfree water; the purity of deionized water is tested by the resistance metre or by chemical
procedure, (e) When the deionized water produces turbidity with silver nitrate, presence
of chloride (and mineral) is suspected, (f) Distilled water is prepared on a small scale
with the help of a laboratory distillation set, (g) Metal stills and (h) Glass stills are used to
obtain distilled water in a large scale; the latter is more pure
126
Reagents
• Nitric acid, concentrated. Keep in a dropping bole.
• Glacial acetic acid. Keep in a dropping bole.
• Silver nitrate solution (2.5% aqueous). Dissolve 2.5 g of silver nitrate in distilled water
and dilute to 100 mL with distilled water. Keep the solution in a dark-coloured bole.
• Barium chloride solution (10% aqueous). Keep in a dropping bole.
Procedure
1. Place in a small size beaker, 10 mL of the distilled water to be tested.
2. Add to it two drops of nitric acid and 1 mL of silver nitrate solution. The presence of a
white cloud or white precipitate indicates the presence of chloride ions. Pure distilled
water remains clear.
3. In another beaker, place 10 mL of the distilled water to be tested.
4. Add ve drops of acetic acid and mix.
5. Then add ve drops of 10% barium chloride solution and mix.
6. The formation of a white granular cloud or precipitate indicates the presence of sulphate ions. Pure distilled water remains clear.
Medical Laboratory Technology: Volume 1
Demineralizer
Mineral salts are electrolytes and are in an ionized state in solution. When they are removed
from water, the laer is called demineralized water or deionized water. The electrolytes are
chemically removed from the tap water by passing it through a column of ion-exchange resins which retains all the mineral ions or dissolved mineral salts. The deionized water is not
free from organic maer and other non-ionizable substances. Preparation of deionized water
is cheaper than preparing distilled water and the former can be substituted for distilled water
in some laboratory operations. It is most useful in rinsing glassware and is pure enough for
preparing most laboratory reagents, including stains.
The commercially available deionizers are made of three components—the receiving end of
the deionizer, the resin column and the eluting end of the deionizer (Figure 4.50). The receiving
end is connected to the tap or water reservoir. When the demineralizer has to be run, open the
tap and allow the water to ran slowly (read the recommendation of the manufacturer for the
rate of running the water). The resin column receives the water from the receiving end and
puries it by removing the electrolytes as the water slowly passes through the column. The
eluting end of the deionizer is the outlet for the deionized water. It is connected to a closed
container to collect the puried water. Many deionizers are provided with a meter to measure
the resistivity or conductivity as an indication of the ionic purity of the demineralized water.
Resistivity and conductivity are reciprocally related. The demineralized water should be
of poor conductivity and high resistivity. The conductivity-testing meter is aached to the
eluting end. If the testing meter is not available, test the purity of water for chloride and
sulphate in the same way as described earlier. The resin column should be changed when the
column is discoloured or when the demineralized water shows the presence of chloride and
sulphate ions.
After using for a period of time, the resin column should be revived according to the manufacturer’s directions. Reviving is done by treating the resin with dilute acid or dilute alkali.
Always check the conductivity before re-using the resin column.
Quality control of demineralized water
One should keep in mind that demineralized water is free from ions but not necessarily free
from organic compounds. Use of a conductivity meter in the deionizer line is recommended
in order to obtain a dependable supply of deionized water (Figure 4.50). The conductivity
Introduction to Laboratory Equipment and Basic Laboratory Operations
meter registers the resistivity of the water resulting from the presence of ions. The more complete the demineralization, the higher the electrical resistivity of the water. If the needle stops
at a point below 2 mega-ohm/cm (a measure of resistivity), the cartridge of ion-exchange
resin granules has been used for too long and must be replaced or reactivated.
If the ion-exchange column does not have a conductivity meter, determine the pH of the
water supply owing into the apparatus and the pH of the water coming out from the other
end. If the pH remains the same, the resin column is no longer active. Demineralized water
should have a pH between 6.6 and 7.0.
Another way to test the demineralized water is to pass a weak solution of sodium chloride
(cooking salt) through the resin, then carry out the test of chloride with silver nitrate (1.7%).
The method is described with the testing of distilled water. Silver nitrate solution gives white
precipitate in the presence of chloride.
Some manufacturers advise to keep an eye on the colour of the resin column. It rums black
when inactive. It then needs to be reactivated.
Use of Demineralized Water
Demineralized water is lot cheaper to make than distilled water. It also replaces the use of
distilled water in many cases:
• Demineralized water can be used for rinsing glassware before drying.
• All the reagents used in medical laboratories, including stains, can be prepared from
demineralized water.
127
water fOr hUman cOnsUmptiOn
Occasionally the laboratory may be involved in testing the water which is meant for human
consumption. If the water is contaminated, government ocials must be immediately
informed so that appropriate measures can be taken, such as cleaning and disinfection
(‘shock chlorination’) with high doses of chlorine or bleaching powder. In some parts of
developing countries, wells may be the only source of water for consumption, which should
be well-protected.
Water should be considered as unt for human consumption if it contains:
• an undue amount of solid particles causing turbidity
• nitrites (indicating decomposition of organic maer)
• toxic substances (lead, arsenic, mercury, uoride)
• pathogenic organisms (e.g., Escherichia coli, indicating faecal pollution)
Sampling for Laboratory Testing
Water sample for laboratory testing should be collected carefully so that it is representative
of the water mass. For example, in case of well, tie the bole with a stone and lower the bole
(narrow neck) below the surface to collect the water sample. Do not touch the wall of the well.
For river stream or tank, collect from the middle. In case of tap water, cleanse the outlet of the
tap thoroughly after removing all aachments (particularly rubber hoses) and allow the tap
to run for a few minutes. If the specimen is meant for laboratory culture in search of pathogenic organisms, always use sterilized boles for sample collection. Also, ame the tip of the
tap with burning alcohol swab before the collection of the sample. Take a large amount of the
sample so that results are reliable. All water samples must be quickly processed in order to
avoid contamination. Chlorinated water, meant for laboratory culture, must be treated with
sodium thiosulphate solution. Sodium thiosulphate dechlorinates the water.