Laboratories Solution preparation

0.1499 g
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Unit One: Introduction
Objective

● Define terminologies.
● Measurement.
● Measurement units.
● Physical units.
● Chemical units.
● Estimate uncertainty of measurement.
● Accuracy and precision.
 Prepare a Working Solutions
Introduction

● A concentrated solution to be diluted is called a stock solution.

● A working solution is used routinely for experiments and is often
prepared by diluting a stock solution.

● Preparing working solutions from stocks saves work and reduces

error and since you don’t have to measure powdered chemicals
each time you want to make a working solution.
Example:-to dilute a stock solution with water to make a working
solution that is one tenth as concentrated.
● This could also be called a ten-fold dilution or a “10X” dilution,
because the working solution will be ten times as dilute as the stock.
Prepare a Working Solutions
Define Terminologies

● Solution: is a homogeneous mixture of two or more

substances.
Solution: composed of a solute and the solvent
● Solute: is the dissolved substance, or the substance found in
small amount
● Solvent: is a substance in which solutes dissolves to make
the mixture or the substance that is present in the greatest
amount.
■ Water is the Universal Solvent but there are many things it cannot
Prepare a Working Solutions
Define Terminologies
● Mixtures: combinations of different substances where each
substance retains its chemical properties.

● Concentration: amount of a substance dissolved in a given

amount of solvent
● Compound: composed of two or more substances
(elements) but in a ratio that cannot vary.
E.g. Carbon dioxide (CO2), there is 6grams of carbon for
each eight gram of oxygen. It won't be Carbon dioxide if that
ratio changes.
Measurement Units
Measurement
● A measurement tells us about a property of something.
● It might tell us how heavy an object is, or how hot, or how long it
is.
● Ascertain an amount, size, or extent obtained by using an
instrument or device marked in standard units.
● Measurements are always made using an instrument of some
kind.
● Rulers, stopwatches, weighing scales, and thermometers are all
measuring instruments.
● The result of a measurement is normally in two parts: a number
and a unit of measurement,
Measurement Units
Measure/measuring:
● As certain the size, amount, or degree of(something) in
comparison with a standard unit or with an object of known
size.
● There are many measurement units used to describe
measurements.
■ for example, Traditional units, cgs units and Metric units (SI units).
● Majority of medical laboratory test methods and reagent
preparations are required an appropriate measurement unit.
■ In 1996, the international Federation of clinical chemistry
(IFCC) recommended the use of SI units for the clinical laboratory.
● And Also World Health Organization recommendations SI
Measurement Units
A. International System of Units(SI)
● Is the standard modern form of the metric system
● It is the world's most widely used system of measurement
used in both everyday commerce and science.
■ It comprises a coherent system of units of measurement built
around seven base units, 22 named and an indeterminate number
of unnamed coherent derived units
● A set of prefixes that act as decimal-based multipliers.
■ The International System of Units has been developed and agreed
internationally to make uniform
● In reporting of test results language(over comes language barriers)
Measurement Units
A. International System of Units(SI)
● In enabling an exchange of health information within health
institute, country or nation It is therefore important for health
authorities and laboratories to adopt SI units

■ The International System of Units is based on the meter-kilogram-

second system and replaces both the foot-pound-second (Imperial)
system and the centimetre-gram-second (cgs) system.

■ The system used almost globally .only Myanamar, Liberia and

United States do not use SI as official system of measurement but
in these countries, though ,SI is commonly used in Science and
medicine.
Measurement Units
The seven SI base units from which all the other units are derived
areas follows.
SI base units Symbol Quantity measured
meter m Length
kilogram Kg Mass
second s Time
mole mol Amount of substance
ampere A Electric current
kelvin K Temperature
candela Cd Luminous intensity
Measurement Units
A. International System of Units(SI)
● SI derived units consist of combinations of base units. For
ease of understanding and convenience Special names and
symbols have been given to those derived units with complex
base combination.
Table 2: The SI derived units.

Derived quantity Name Symbol

2
Area square meter m
3
Volume cubic meter m
Speed or velocity meter per second m/s
Measurement Units
B. SI Unit Prefixes
● To enable the measurement of quantities larger or smaller
than the base unit s or derived units, the SI Unit System
also includes a set of prefixes.

● The use of a prefix makes a unit larger or smaller

● The range of SI unit prefixes commonly used in laboratory

work is listed as follow.
Measurement Units
Table 3: SI unit smaller prefixes
Symbol Function
Prefixes Divide by
-1
deci d 10 10
-2
centi c 10 100
-3
milli m 10 1000
-6
micro µ 10 1,000,000
-9
nano n 10 1,000,000,000
-12
pico pf 10 1,000,000,000,000
-15
femto 10 1,000,000,000,000,000
Measurement Units
Table 4: SI unit larger prefixes.
Symbol Function
Prefixes Multiply by
1
deka da 10 10
2
hecto h 10 100
3
killo k 10 1000
6
mega M 10 1,000,000
9
giga G 10 1,000,000,000
12
tera T 10 1,000,000,000,000
15
peta P 10 1,000,000,000,000,000
18
exa E 10 1,000,000,000,000,000,000
Measuring units
Physical Units
A. Percent(%W/V):Weight of solute per unit volume of
solution.
Or mass of a substance in a mixture as a percentage of the
volume of the entire mixture.
■ Example:40 %w/v glucose solutionmeans,40 gm of glucose is
dissolved in 100 ml of a given solvent

B. Percent(%W/W):Weight of solute per weight of solvent. Or

Denotes the mass of a substance in a mixture as a
percentage of the mass of the entire mixture.
■ Example,30%w/wHClmeans,each100gm of hydrochloric acid
solution contains 30 gm of HCl and the rest 70 gm is the solvent
Measuring units
Physical Units

C. Percent(%V/V):Volume of solute per volume of [Link]

■ Describes the volume of the solute in mL per100 mL of the
resulting solution.
■ This is most useful when a liquid - liquid solution is being
prepared.
√ E.g. Beer is about 5%ethanol by volume.
√ This means every100 mL beer contains 5 mL ethanol
Measuring units
Parts by Part

Some times when solutions are too dilute,

their percentage concentrations are too low.
So, instead of using really low percentage
concentrations such as 0.00001% or 0.000000001%
choose another way to express the concentrations.
Used to denote the relative abundance of trace
elements in
■ the Earth's crust,
■ trace elements inforensics or
 Measuring units
Parts by Part…
2
A. Parts per hundred (denoted by'%' and very rarely'pph'1partin 10 ):
Denotes one particle of a given substanceforevery99otherparticles.

B. Parts per thousand

3
(denoted by'‰'[the permil symbol],and occasionally
'ppt'1 part in 10 ):
Denotes one particle of a given substance for every 999 other
particles.

[Link] per million ('ppm’)

D. Parts per billion ('ppb’)

E. Parts per trillion ('ppt’)

 Measuring units
Chemical Units
● Most common acids and some basic solutions like
ammonium hydroxide are usually found with their
concentrations expressed in specific gravity and percentage
by weight of the specific solution.
● These two information‘s (specific gravity and percentage by
weight) should be changed to the commonly known
expressions of concentration, like morality and normality.

● Molarity(M)
Molarity: defined as the number of moles of solute in each liter of
solution
A molar solution is a solution that contains one mole of the solute
Measuring units
● Molarity(M)
For example, the molar weight of sulfuric acid ( H2SO4) is 98
 Measuring units
● Molarity(M)
Example:- A solution contains 5.7 grams of potassium nitrate
dissolved in enough water to make 233 mL of solution. What is
its molarity?

● Liters of liquid, containing 2.0 moles of dissolved particles,

constitute a solution of 0.5M Such a solution may be
 Measuring units
Normality(N)
Normality: is defined as the number of equivalent weight of a
solute in a liter of solution.
A normal solution is a solution that contains one-gram
equivalent weight of the solute in one liter of solution.
 Measuring units
Normality(N)
In practice, this simply means one multiplies the molarity
of a solution by the valence of the ionic solute. It has
advantages when carrying out titration calculations.
The equivalent weight of is 98 divided for 2 (valancy of ),
which is 49.
Therefore, one normal solution of contains 49gram of
per liter of solution.
 Measuring units
● Molality(m)
Molality(m): is defined as the number of moles of solute in
1kilogram of solvent.
A molal solution is a solution that contains one mole of the solute in
one kilogram of solution.

Example:-80 grams of a simple sugar is added to 750 g of water. The sugar

is glucose, with the composition . What is the molality of glucose in the
Measuring units
EstimateUncertaintyofMeasurement

What is uncertainty of measurement?

The uncertainty of a measurement tells us something about its
quality.

Uncertainty of measurement is the doubt that exists about the

result of any measurement.

You might think that well-made rulers, clocks and thermometers

should be trustworthy, and give the right answers

But for every measurement - even the most careful - there is always
a margin of doubt.
Measuring units
Error versus uncertainty

It is important not to confuse the terms ‘error’ and

‘uncertainty’.

■ Error is the difference between the measured value and the

‘true value’ of the thing being
measured.

■ Uncertainty is a quantification of the doubt about the

measurement result.
Whenever possible we try to correct for any known errors:
for example, by applying corrections from calibration
Measuring units
Error versus uncertainty
Many things can undermine a measurement.
Flaws in the measurement may be visible or invisible.
Because real measurements are never made under perfect conditions,
errors and uncertainties can come from:

The measuring instrument - instruments can suffer from errors including

bias, changes due to ageing, wear, or other kinds of drift, poor readability,
noise e.t.c

The item being measured - This may not be stable. (trying to measure the
size of an ice cube in a warm room.)

The measurement process - the measurement itself may be difficult to

make. E.g. measuring the weight of small but lively animals presents
 Measuring units
Where do errors and uncertainties come from?
Operator skill - some measurements depend on the skill and judgement of the
operator.
One person may be better than another at the delicate work of setting up a
measurement,
or at reading fine detail by eye. The use of an instrument such as a stopwatch
depends on
the reaction time of the operator.

Sampling issues - the measurements you make must be properly

representative of the
process you are trying to assess. If you want to know the temperature at the
work-bench,
don’t measure it with a thermometer placed on the wall near an air
conditioning outlet. If you are choosing samples from a production line for
measurement, don’t always take the first ten made on a Monday morning.
Measuring units
The general kinds of uncertainty in any measurement
The effects that give rise to uncertainty in measurement can be
either:

Random - where repeating the measurement gives a randomly

different result. If so, the more measurements you make, and
then average, the better estimate you generally can expect to
get.

Systematic - where the same influence affects the result for

each of the repeated measurements (but you may not be able to
tell). In this case, you learn nothing extra just by repeating
Accuracy and Precision of Measurement
Precision(Reliability)
● Is defined as the extent to which a questionnaire, test, observation
or any measurement procedure produces the same results on
repeated trials.
● In short, it is the stability or consistency of scores over time or
across raters. Keep in mind that reliability pertains to scores not
people.

Accuracy(Validity)
● Is defined as the extent to which the instrument measures what it
purports to measure. Or
● is the ability of a test to indicate which individuals have the disease
and which do not have the disease.
Accuracy and Precision of Measurement
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Accuracy and Precision of Measurement
A. measures of validity
1. Sensitivity.
2. Specificity.
3. Predictive value.

1. Sensitivity: is the ability of the test to identify

correctly those who have the disease from all
individuals with the disease.
Accuracy and Precision of Measurement
2. Specificity: is the ability of the test to identify correctly
those who do not have the disease from all individuals
free from the disease.

● To determine the sensitivity and specificity of a new

test Gold standard test is required.
● This helps to know the correct disease status of an
individual. Use a 2x2 table to compare the
performance of the new test to the gold standard test.

3. Predictive value: Is the probability that the test result

(positive or negative) will give the correct diagnosis
(has a disease or does not have).
Accuracy and Precision of Measurement
A. Positive predictive value
B. Negative predictive value

Table 5: Positive and negative predictive value.

Gold standard test (disease)
+ -
New test + a (True B
positive)
- C d (True negative)
Accuracy and Precision of Measurement
.
Accuracy and Precision of Measurement
● Positive Predictive Values:-The positive predictive value
(PPV) of a test is defined as the proportion (probability) of
people with a positive test result who actually have the
disease
Accuracy and Precision of Measurement
● Negative predictive value (NPV) is the probability that a
person with a negative(normal) test result is truly free of
disease.
 Unit Two: Chemicals and Solutions
Objective: ● Chemistry of acids, base,
buffer and chemical reaction
● Chemicals ● Standardize solution
● Making dilution
● Definition of chemicals
● Simple dilution
● Characteristics of
● Serial dilutions
chemicals
● Standardizing the solution to
● Grade of chemicals specified range and precision
● Solutions ● Determining concentration of
● Definition of solution standardize solutions
● Types of solutions ● Labeling and storing
standardized Solutions
● Expressing concentration
of solutions
Introduction
.
● Chemicals

● Chemicals-is a form of matter that has constant chemical

composition and characteristic properties can be elements,
compounds, ions and alloys
.
● Characteristics of Chemicals

It can not be separated into components.

Chemical can be pure or any mixture.
Has the same properties and ratio.
Exist as solids, liquids, gases.
Phases of matter may change with changes in
temperature or pressure.
Maybe combined or converted to others by means
of chemical reactions.
.
● Grade of Chemicals

● Lab Grade-Aline of solvents suitable for histology

methods and general laboratory applications.

● AR: The standard grade of analytical reagents; suitable

for laboratory and general use.

● Guaranteed Reagent(GR)-Suitable for use in analytical

chemistry, products meet or exceed
American Chemical Society (ACS) requirements where
applicable.
●
. Solution
● Solution is a homogenous mixture of two or more
components that can be varied in composition with in certain
limit. Every solution consists of two parts, the solvent and
solute

● Characteristics of Solutions
● Homogeneous
● The particles of solute in a solution cannot be seen by
naked eye.
● Does not allow beams of light to scatter.
● Stable.
. ● Functions of Solutions
● Involved in a chemical reaction, especially one used
to detect, measure, or produce another substance.
● Preserve/fix substances to protect from deterioration.
● Maintain Ph of a solution.
● Decrease concentration of a substance.
● Maintain osmotic pressure of a substance.
● Cleaning, disinfecting and sterilizing materials.
● Increase refractive index of light.
● Gives artificial color to be visualized
. ● Expressing Concentration of Solutions
● Concentration- amount of a substance dissolved in a
given amount of solvent.
● Concentration of solutions should be accurately
expressed for the appropriate use in the desired procedures.

● Concentration of a solution can be expressed in three ways

1. Qualitative Expressions of Concentration
2. Semi-Quantitative Expressions of Concentration
3. Quantitative Expressions of Concentration
. ● Qualitative Expressions of Concentration
● A solution can be qualitatively described as
Dilute: a solution that contains a small proportion of solute
relative to solvent.
Concentrated: a solution that contains a large proportion of
solute relative to solvent.
.
. ● Semi-Quantitative Expressions of Concentration
● A solution can be semi-quantitatively described as:

● Unsaturated: a solution in which more solute will

dissolve.

● Saturated: a solution in which no more solute will

dissolve.

● Supersaturated Solution-a solution that contains more

dissolved substance than does a saturated solution; the
solution is not in equilibrium with the pure substance.
. ● QuantitativeExpressionsofConcentration
● Quantitative notation of concentration is far more
informative and useful from a scientific point of view.
● Many units of concentration require measurement of a
substance’s volume, which is variable
depending on ambient temperature and pressure.
● Unless otherwise stated, all the following measurements
are assumed to be at standard
state temperature and pressure (that is,25degrees Celsius at1
atmosphere).
● There are a number of different ways to quantitatively
express concentration.
. ● Chemistry of Acids, Base, Buffer and Chemical
Reaction.

● Acid: Substance that produces ions (protons) in aqueous

solutions
+ -
● HCL(aq) H (aq) + Cl (aq)
3 + -
● HNO (aq) H (aq) + NO3 (aq)

● Base: Substance that produces (hydroxide) ions aqueous

solutions
+ -
● NaOH(aq) Na (aq) + HO (aq)
●Buffers
.
● Buffers: Buffers are defined as a compound or a mixture
of compounds that resists the pH upon the addition of
small quantities of acid or alkali.
● Buffer have definite pH value.
● The pH will not change after keeping it for a long period of
time. The pH value altered negligibly by the addition of small
quantities of acid or base.

● Buffer action: The resistance to a change in pH is known

as buffer action.
● So buffers can be added to show buffer action.

● Buffer capacity: The amount of acid/base required to

produce a unit change in pH in a solution is called buffer
capacity.
●
. Types of buffers:
● Generally buffers are of two types:
1. Acidic buffers
2. Basic buffers

● There are some other buffer system:

1. Two salts acts as acid-base pair.
● E.g. Potassium hydrogen phosphate and potassium
dihydrogen phosphate.

2. Amphoteric electrolyte.
E.g. Solution of glycine

3. Solution of strong acid and solution of strong base.

. ● Acidic Buffers:
An acidic buffer is a combination of weak acid and its salt
with a strong base.i.e. Weak acid & salt with strong base
(conjugate base).
EXAMPLES:
CH3COOH / CH3COONa
H2CO3 / NaHCO3
H3PO4 / NaH2PO4
HCOOH / HCOONa

● Basic Buffers:
A basic buffer is a combination of weak base and its salt
with a strong acid. i.e. Weak base & salt with strong acid
(conjugate acid).
 Prepare a Working Solutions
Making dilution

● Types of dilution
1. Simple dilution
2. Serial dilution

● Simple Dilution:
A general process of preparing less concentrated solutions from a
solution of greater concentration, a unit volume of a liquid material
of interest is combined with an appropriate
volume of a solvent liquid to achieve the desired concentration. To
dilute a solution means to add more solvent without the addition of
more solute to bring a solution into the desired concentration. The
resulting solution is thoroughly mixed so as to ensure that all parts
of the solution are identical.
Theratioofconcentratedorstocksolutiontothetotalvolumeequalsthed
 Prepare a Working Solutions
Making dilution

● Simple Dilution:
The df is inversely related to the concentration
thus, the dilution factor increases as the
concentration decreases.
A 1:5 dilution (verbalize as"1 to 5" dilution)
entails combining 1 unit volume of solute(the
material to be diluted) + 4 unit volumes of the
solvent medium
hence, dilution factor couldbe:1+ 4 =5
Mathematically this relationship can be shown
in the equation:
Prepare a Working Solutions
Making dilution

● In the performance of dilution, the following equation is used

to determine the volume (V2 ) needed to dilute a given
volume(V1 )of solution of a known concentration(C1 ) to the
desired lesser concentration ( C2).

● Likewise,thisequationalsoisusedtocalculatetheconcentrationof
thedilutedsolution when a given solution is added to the
starting solution.
● Inmakingasimpledilution,thelaboratorytechnicianmustdecideon
 Prepare a Working Solutions
Using Proportion

● It is used when reagents are prepared by adding a specific

amount of one solution to a specific amount of another
solution.
.

● Where:
C–total volume of final reagent
A–total parts of solution A
B – total parts of solution B
 Prepare a Working Solutions
Example1: a buffer made by adding two parts of ‗solution A‘to five
parts of solution B would be required to make 70 mL of the buffer.

Example2:a100mg/mLN2 standard is diluted1:[Link] the

concentration of the resulting solution is100mg/mLx1/10=10mg/mL
Prepare a Working Solutions
● UsingC1V1=C2V2

● This formula is useful only if the units for concentration &

volume are the same & if three of the four variables are known.

● Example-1:- What volume is needed to make 500ml of 0.1M

solution of tris-buffer from a solution of 2 M tris-buffer?
:
.
.
Prepare a Working Solutions
● Example-2:-To make 45 ml of 30% Solution from
70% solution.

● Therefore,19.3ml of 70% solution must be diluted with 25.7ml

of distilled water to obtain 45ml of a 30% solution
Prepare a Working Solutions
● Diluting body fluids/standards
● Example: To make 8ml of a 1 in 20 dilution of blood.
C1xV1=C2V2
=>20xV1=1x8V1(blood volume)=0.4
Therefore, to prepare 8ml of a 1in 20 dilution, add 0.4ml of
blood to7.6ml of the diluting fluid.

● To make 4ml of a 1 in 2 dilution of serum in physiological

saline.
C1xV1=C2V2 =>2xV1=1x4=>V1(serum volume) = 0.4
To prepare 4ml of a 1in 2 dilution, add 2ml of serum to 2ml of
physiological saline.
Calculating the dilution of a body fluid
Prepare a Working Solutions
● Diluting body fluids/standards

● Calculate the dilution of urine using 0.5 ml of urine and 8.5 ml

of diluting fluid (physiological saline)
● Total volume of urine and diluting fluid,0.5+8.5= 9.0μl=>0.5:9=
1in18 dilutions.

● Example1: 10 dilution = ratio of 1 : 9 = 1 : 10 = one part of serum + 9

parts of diluent.
100 μL + 900 μL of saline.
1 mL serum + 9 mL of saline.
2 mL serum + 18 mL of saline.
Prepare a Working Solutions
● Serial Dilutions
● A serial dilution may be defined as multiple progressive dilutions
ranging from more concentrated solutions to less concentrated
solutions.

● Simply a series of simple dilutions which amplifies the dilution factor

quickly beginning with as mall initial quantity of material (bacterial
culture, a chemical, orange juice, etc.).
● Final dilution factor (DF)= DF1 *DF2 *DF3etc.

● It is the stepwise dilution of a substance in solution. Usually the

dilution factor at each step is constant, resulting in a geometric
progression of the concentration in a logarithmic fashion.
Used to accurately create highly diluted solutions as well as
solutions for experiments resulting in concentration curves with a
logarithmic scale.
Prepare a Working Solutions
●
Serial Dilutions
It is required for certain quantitative tests
Serial dilution is extremely useful when the volume of the
concentrate &/or diluents is in short suppl
Large dilutions may be difficult to make because of the
amount of diluent that needs to be added.
Forexample,a1/1000dilutionmaybedifficultto create accurately
even with 0.1mLof serum & 99.9 mL of diluent. A series of
dilutions, also called serial dilutions, may be a better way to
make the dilutions.
The dilution fold of a system can be determined by the
formula:
 Prepare a Working Solutions
● Serial Dilutions…
● Example 1.
What is the dilution fold of the following serial dilution system consisting
of five tubes? The
following amount of diluents have been added to the tubes; 0.5 mL to
tube 1& 0.5 mL to tube 2 to 5. Next, 0.5 mL of patient serum is added to
tube 1 and 0.5 mL is serialy transferred through tube 5.
finally, 0.5 mL is discarded from tube 5.
1/Y =0.5/1.0
Y x0.5=1
Y =1/0.5= 2

● Example2.
It is often desirable to determine the dilution of a given tube (Y) in a serial
dilution system. This dilution can be calculated by Solution of tube 1 = dilution
of Y x [ 1/dilution fold]
What is the dilution of tube3 in the preceding example? Y= ½ x (½) (Y-1)
= ½x (½)2
=½x½x½
 Prepare a Working Solutions
Example 3
In a typical microbiology exercise the students perform a three step 1:100 serial
dilution of a bacterial culture in the process of quantifying the number of viable
bacteria in a culture.
Each step uses a 1 ml total volume. The initial step combines 1 unit volume of
bacterial culture (10 ul) with 99 unit volumes of broth (990 ul) = 1:100 dilution.
In the second step, one unit volume of the 1:100 dilution is combined with 99 unit
volumes of broth now yielding a total dilution of 1:100x100 =1:10,000 dilution.
Repeated again (the third step) the total dilution would be 1:100x10,000
=1:1,000,000 total dilution. The concentration of bacteria is now one million times
less than in the original sample.
 Prepare a Working Solutions
● Example 4
If you want to make a serial dilution of 1: 2,
1: 4, 1: 8, 1: 16, and so on.
1. The total volume is fixed; suppose it is 1
mL.
2. (Initial dilution factor) (next dilution factor)
= final dilution factor
3. 1 : 2 x 1 : 2 = 1 : 4.

● Procedure:
1. First tube = (1 mL serum + 1 mL diluent) =
1:2
2. Second Tube = 1 mL from first tube + 1
mL diluent = 1 : 4
3. Third tube = 1 mL from tube 2 + 1 mL
diluent = 1 : 8
4. Fourth tube = 1 mL from tube 3 + 1 mL
diluent = 1 : 16
Standard Solutions
● These are solutions in which the concentration of a given
chemical is precisely known

● They are used to determine the value of an identical chemical

with unknown concentration of a given solution.

● Chemicals that are used to prepare these solutions should be

of analytical grade.
● Since poor standard solutions cause errors in the estimation
of the intended substances, their accurate preparation is of
utmost importance in order to obtain accurate and precise
laboratory findings in medical laboratories
Standard Solutions
● Primary Standard Solution

■ Primary standard solution is a chemical solution that has

the highest purity and
■ can be used directly for the exact measurement of
substances of unknown concentration in a given solution.
■ These solutions include sodium chloride, sodium
bicarbonate, potassium iodide, etc.
■ Primary standard solution should be made of substances
that are:
Free of impurities,
Stable on keeping in solid state and in solution,
Able to be accurately weighed or measured to give a
solution of exactly known concentration,
Standard Solutions
● Secondary Standard Solutions

■ Secondary standard solutions are solutions of lower purity and their

concentrations are determined by comparison to primary standard
solutions.
■ used for analytical procedures after their concentration is already
determined.
■ Some examples of these solutions are nitric acid, hydrochloric acid,
sulfuric acid, etc.

■ In the preparation of secondary standard solutions, the following

points should be taken into consideration:
Using analytical balance for weighing.
Dissolving the weighted substance in the prescribed volume of
solvent.

■ Determining the exact concentration by comparison against a primary

standard solution
Solutions…
● Buffer Solutions

■ A buffer is a solution of a weak acid or base and one of its respective

salts. Buffers are able to resist changes in the PH.

■ Buffers are used when the pH needs to be carefully controlled for the
diagnostic procedures, such
as in measuring enzyme activities.

● Classification of Solutions
■ Based on the States (Phase) of the Solution

■ Solutions can be gaseous, liquid and solid, solute can be gas, liquid or solid,
Solvents can also
be gas, liquid or solid.
.
 Classification of Solutions…

. Gas Liquid Solid

Gas Oxygen and other gases Water vapor in air Odor of solid
in nitrogen (air), Br2 gas ion air
dissolved in ar gas
Liquid CO2 in water Ethanol in water, various Sugar H2O,
(carbonated water) hydrocarbon in each NaCl in H2O
other (petroleum), Br2
liquid dissolved in H2O
Solid H+2 dissolved Water in activated Steel, Brass,
charcoal other metal
alloys
Classification of Solutions…
● Based on the Strength of the Solution:

■ Strength of a solution is measured by its solubility

■ Solubility is the ability of one compound to dissolve in
another compound at any one temperature, when a liquid
can completely dissolve in another liquid the two liquids
are miscible. But two substances that can never mix to
form a solution are called immiscible.
■ Here law of dissolution is applied―like dissolve like:-
Dilute or Weak Solution
Concentrated Solution
Unsaturated
Saturated Solution
Classification of Solutions…
● Based on the Types of Solvent of the Solution :

■ Aqueoussolutions:[Link],sugar
inwater,carbondioxide in water, etc.

■ Nonaqueoussolution:[Link],
benzene,petrol,carbontetrachloride etc., are some common
solvents. example, sulfur in carbon disulphide, naphthalene
in benzene, etc.

●Based on the Concentration of H3O+ of the Solution,

Solutions can be
Classification of Solutions…
● Based on the Osmotic Pressure (Solute Concentration) of the
Solution

■ Hypertonic
■ Isotonic
■ Hypotonic
 Classification of Solutions…
● Based on the Dye Content :

■ Based on the content of dye it can be classified as stains and non-stains.

■ Stains-reagent or dye used in treating a specimen for microscopic
examination.
■ Stains are used to bind and make visible specific structures within the cell
so that they are more easily visible in the microscope.
■ Stains(Dyes) are colored chemical compounds that are used to selectively
give (impart) color to the colourless structures of bacteria or other cells
■ Staining reactions are made possible because of the Physical phenomena
of capillary osmosis, solubility, adsorption, and absorption of stains or
dyes by cells of microorganisms.
■ Basic principle: The cellular components of mammalian as well as
microbial cell are different. For example the nuclei of cell are negatively
charged because of the presence of acidic component (DNA) hence it
combines with positively charged compounds,
(basic dyes). And the cytoplasm parts of a cell are generally positively
charged
Unit Three: Materials used to preparing solution
● OBJECTVE

■ Selecting standard procedure for stock and working

solution preparation.
■ Selecting Materials and solvent
■ Calculating and recording data
■ Measuring reagents for solution preparation
■ Selecting and assembling laboratory Equipment’s
■ Mixing or diluting working solution
■ Preparing Solutions
■ Labeling and storing Solutions
■ Recording Working solution
 Laboratory Wares
● Laboratory Glass Wares and Plastic Wares

■ Laboratory glass ware and plastic wares are materials used in clinical
laboratory for: measuring pipetting transferring Preparation of reagents
Storage etc.

■ Most of the routine laboratory wares used to be of glass, but recent

advantage made in the use of plastic resin to manufacture a wide range
of plastic ware having led to a gradual replacement of glass wares with
durable plastic ware.
■ The plastic ware used in the laboratory should be of high quality. Also
cheaper and safer to use than glassware.

■ The glass wares have the minor advantage of being re-usable and
autoclavable. But heavier, more costly and easily broken. In fact, in this
age of good awareness of the dangers posed by hepatitis and human
immuno deficiency viruses(HIV), most of the plastic wares are
Laboratory Wares…
● Classification of Laboratory Glass Wares

■ A. Can be divided into five main types according to their

composition.
1. Glass with high thermal resistance – borosilicate glass can
resist about 500oc and low alkaline contact.

2. High silica glass-contains 96% silicon, It is thermal endurable,

chemically stable and electric resistant.

3. Glass with high resistance to alkali-Boron free, used in strong

alkali low thermal resistance.

4. Low actinic glass– amber color to protect light.

Laboratory Wares…
● Classification of Laboratory Glass Wares

■ B. Based on their use

a) volumetric wares
b) Semi-volumetric Glass wares
c) Non-volumetric glass wares.

■ a) Volumetric wares: Apparatus used for measurement of

liquids Can be made either from glass or plastic. It includes:
Volumetric flasks
Graduated centrifuge tubes
Graduated serological pipette
Medicine dropper
Burettes
Micropipettes
Diluting or thoma pipettes etc
 Laboratory Wares…
■ b). Non-volumetric glass wares: are not calibrated to hold a particular or
exact volume, but rather are available for various volumes, depending on the
use desired.
Erlenmeyer flask
Round bottom flask
Flat bottom flask
Beaker
Centrifuge tube
Test tube
Pasture pipette

■ C).Semi-volumetric Glass wares: are used for approximate measurement.

It includes;
Graduated cylinder
Graduated specimen glass
Beakers
Conical flask
Medicine droppers with or without calibration mark
Graduated beaker with double beaks
Graduated glass
Laboratory Wares…
■ Pipettes

■ There are several types each having their own advantages and
limitations. They are designated as class
―A‖ or ―B‖ according to their accuracy.

1. Class “A” pipettes are the most accurate and the tolerance limits
are well defined that is, ± 0.01,
±0.02 and ±0.04 ml for 2, 25, and 50 ml pipettes respectively.

2. Class “B” pipettes: are less accurate but quite satisfactory for
most general laboratory.
Read the volume at lower meniscus
Significant errors will result if the temperature of the liquid
pipette is widely different from the temperature
o
of calibration.
The usual temperature of calibration is 20 and this is marked on
Laboratory Wares…

● Micropipettes

■ Micropipettes are frequently used in

Medical chemistry
Virology
Immunology and serology laboratories.

■ This is because in these laboratories often only small

quantities of materials are available for measurement. They
are found in different capacities such as 5, 10, 25, 50, 100 and
1000 micro liter.
■ There are also other kinds of pipettes that are used in medical
laboratories.
 Laboratory Wares…
● Volumetric Pipettes

■ Volumetricpipettesarecalibratedtodeliveraconstantvolumeofliquid.
■ The most commonly used sizes are 1,5, and 10ml capacities.
■ Less frequently used sizes are those which deliver 6, 8, 12,and soon ml.
■ They have a bulb mid–way between the mouth piece and the tip
■ The main purpose of the bulb is to decrease the surface area per unit volume and to
diminish the possible error resulting from water film.
■ The Volume(capacity) and calibration temperature of the pipettes are clearly written on
the bulb.
■ They should be used when a high degree of accuracy is desired.
■ The pipette is first rinsed several times with a little of the solution to be used, and then
filled to just above the mark.
■ Then the liquid is allowed to fall to the mark and the tip is carefully wiped with filter
paper.
■ [Link] liquid will
remain at the tip and this must not be blown out
N.B: The reliability of the calibration of the volumetric pipette decreases with an increase in size and
Laboratory Wares…
● Graduated or Measuring Pipettes

■ Graduated pipettes consist of a glass tube of uniform bore with

marks evenly spaced along the length. The interval between the
calibration marks depends up on the size of the pipette.
■ Two types calibration for delivery are available:
A. One is calibrated between two marks on the stem(Mohr).
B. The other has graduation marks down to the tip(serological
pipette)
■ These pipettes are intended for the delivery of predetermined
volumes. The serological pipette must be blown out to deliver the
entire Volume of the liquid and it has an etched ring (pair of rings)
near the mouth end of the pipette signifying that it is a blow out
pipette.
■ Measuring pipettes are common only in 0.1,0.2, 0.5,1.0 5.0,
and10.0ml sizes.
■ The liquid is delivered by allowing it to fall from one calibration mark
Laboratory Wares…
Laboratory Wares…
● Burettes

■ Burettes are used for measuring

variable quantities of liquid that are
used in volumetric titrations.
■ capacities from 1 to100 milliliters.
They are long graduated tubes of
uniform bore and
■ are closed at the lower end by means
of a
glass stopper, which should be lightly
greased for smooth rotation.
.
Laboratory Wares…
● Flasks

■ There are four types of flakshaving25 to6,000milliliter(ml)capacities.

1. Conical (Erlenmeyer) flasks: Conical (Erlenmeyer) flasks are useful

for titrations and
also for boiling solutions when it is necessary to keep evaporation
to a minimum. Some have a side
arm suitable for attachment to a vacuum pump.

2. Flat bottomed round flasks: Flat-bottomed round flasks are

convenient containers to heat liquids.
These flasks are widely used in the preparation of bacteriological
culture media.

3. Roundbottomedflasks:Roundbottomedflaskscanwithstandhigherte
mperatures than the flat bottomed type. They may be heated in a
Laboratory Wares…
● Flasks
4. Volumetric flasks: Volumetric flasks are flat – bottomed, pear-shaped
vessels with long narrow necks fitted with ground glass stoppers.
Most flasks are graduated to contain a certain volume, and these are
marked with the liters.
A horizontal line etched round the neck denotes the stated volume of
water at given temperature. They are used to prepare various kinds of
solutions. The neck is narrow so that slight errors in reading the
meniscus results in relatively small volumetric differences(minimizes
volumetric differences or errors)
Laboratory Wares…
● Beakers

■ Beakers have capacities from 5 to 5,[Link] usually

made up of heat resistant glass and are available in different
shapes. The most commonly used is the squat form, which is
cylindrical and has a spout. There is also a tall form, usually
without a spout
Laboratory Wares…
● Cylinders

■ Cylinders are supplied in 10 to

2,000ml capacities.
■ Some are of heat resistant glass or
plastic.
■ Measurement of liquids can be
made quickly with these vessels, but
a high degree of
accuracy is impossible because of
the wide bore of the cylinders
Laboratory Wares…
● Test Tube

■ Test tubes are made of hardened glass or plastic materials that can
withstand actions of chemicals, thermal shock and centrifugal
strains. They are used to hold samples and solutions during medical
laboratory procedures.
■ These include simple round hollow tubes
conical centrifuge tubes, vaccutainer tubes.
■ Test tubes can be with or without rims (lips)
.
Laboratory Wares…
● Reagent Bottles

■ Reagent bottles are used to store different types of laboratory

reagents.
■ They are made from glass or plastics.
■ Depending on their use, they are available in various sizes and
type
Laboratory Wares…
● Funnels

■ There are two types of funnels that are widely used in a medical laboratory. These
are filter funnel and separating funnel.

■ Filter Funnels:
Filter funnels are used for pouring liquids into narrow mouthed containers, and for
supporting filter papers during filtration. They can be made from glass or plastic
materials

■ Separating Funnels:
They are used for separating immiscible liquids of different densities. Separating
funnels are used for separating immiscible liquids of different densities. Example,
ether and water
.
Laboratory Wares…
● Pestle and Mortar

■ Pestle and mortar are used for grinding solids,

■ for example, calculi and large crystals of chemicals.
■ After each use always clean the pestle and mortar thoroughly.
■ This is because chemicals may be driven into the unglazed
surfaces during grinding, resulting in contamination when the
apparatus is next used.
Laboratory Wares…
● Pasture pipette

■ They are non-volumetric glassware used in transferring liquid.

It has a long –drown-out tip
with a rubber bulb or teat to suction. Eye droppers or medicine
droppers can use instead of pasture pipettes
EquipmentforPurifyingWater
● Distiller

■ A process by which impure water is

boiled and the steam condensed on
cold surface
(condenser) to give pure distilled
water is called distillation. Distilled
water is free from
dissolvedsaltsandclearcolorless,odo
[Link]
pparatusis called distiller.

.
EquipmentforPurifyingWater …
● Deionizer

■ A deionizer is an apparatus used to produce ion free water.

A deionizer is an apparatus for demineralizing water by
means of cartridges filled with ion exchange resin.

Deionizationisaprocessinwhichchemicallyimpurewaterispasse
dthroughanionand cation
exchange resins to produce ion free water.
Deionized water has low electrical conductivity, near neutral
pH and is free from water- soluble salts but is not sterile.
Equipment for Weighing/Balances …
● Balances

■ Essential laboratory instruments that are widely used for weighing

of various substances
(powders, crystals and others) in the laboratory. For instance, to
prepare reagents, stains and
culture media, balances are required to weigh accurately and
precisely within the needed
range. They should be kept carefully clean and located in an area
away from heavy
traffic,largepiecesofelectricalequipment,[Link]
zeanyvibration,as
interference that may happen, a slab of marble is placed under the
balance
Balances in medical laboratory may be:
Rough balances(mechanical balances)
Analytical balances/electrical/
.
Equipment for Weighing/Balances …
● RoughBalances

■ Rough balances are several types. Some of them use sliding scale,
some have a single or
double pan (s) and others utilize dial - operated fractions. They are
used for weighing
substances, which do not call for extreme accuracy. While
operating, they do not require
mainselectricityorbatterypowerandarecurrentlylessexpensivethanan
alyticalbalances of the
similar sensitivity. Some rough balances weigh accurately to 0.1 gm
of a substance. Two -
pan balance is a rough balance, which has two copper pans
supported by shafts.
It is used:
To weigh large amounts(up to several kilo grams).
When a high degree of accuracy is not required.
The sensitivityofatwopanbalanceis0.5 gm.
Equipment for Weighing/Balances …
● RoughBalances

■ The sensitivity of a balance is the smallest weigh that moves the

pointer over one division of the scale. For routine laboratory
purposes the sensitivity of a balance can be considered to be the
smallest weigh that it will measure accurately. Usually the larger the
amount of substance to go into a reagent, the least accuracy is
required. For instance, if the sensitivity of balanceis1mg, this means
that a weight of at least1.0mgisneededtomove the pointer over one
scale.
Equipment forWe ighing/Balances …
● Analytical Balances

■ Nowadays analytical and electronic balances (single pan balances

that use an electron
magnetic force instead of weights) are the most popularly used
balances in medical
laboratories to provide a precision and accuracy for reagent and
standard preparation.
Analytical balance is a highly sensitive instrument. It may have two
pans suspended from a cross beam, inside a glass case. It requires
mains electricity or battery(D.C) supplied power.
These balances are used:
To weigh small quantities usually in milligram (mg) range.
When great accuracy is
required.E.g.,2.750mg,0.330mg,5.860mg,etc.
Its sensitivity is 0.5mg to1mg depending on the model.
Equipment for Weighing/Balances …
● Analytical Balances

■ It requires mains electricity or battery(D.C) supplied power.

These balances are used:
To weigh small quantities usually in miligram (mg) range.
When great accuracy is
required.E.g.,2.750mg,0.330mg,5.860mg,etc.
Its sensitivity is 0.5mg to 1mg depending on the model.
N.B: The accuracy of a balance should be checked regularly as
recommended by the manufacturer.
 Laboratory Wares…
● Use and Care of Balances

■ A balance is a delicate instrument that requires practical instruction in its correct use.
The following should be applied when using a balance:
Read carefully the manufacturer‘s instructions.
Always handle a balance with care.
Position the balance on a firm bench away from vibration, draughts and direct sunlight.
Before starting to weigh, zero the balance as directed by the manufacturer. If using a
beam
balance, check the position of the beam.
Weigh the chemicals at room temperature in a weighing scoop or small beaker. And
Never
put the chemicals directly on the balance pan.
When adding or removing a chemical, remove the container to avoid spilling any
chemical
on the balance.
When using an analytical double pan balance, bring the pans to rest before adding or
removing a chemical.
Always use forceps to add or remove weighs. Protect the weights from dust, moisture
and fungal growth.
Use small brush to remove any chemical, which may have been spilt on the balance.
A container of self -indicating silica gel should be kept inside the analytical balance case
to remove any moisture present in the atmosphere.
Laboratory Wares…
● Incubator

■ Incubationatcontrolledtemperatureisrequiredforbacteriological
cultures,blood transfusion, Serology, Hematology and clinical
Chemistry tests.
■ For bacteriological cultures, an incubator is required where as
for other tests a dry heat block or a water bath may be used.
■ For the incubator, the air inside is kept at a specific temperature
(usually at 37 c).
0

■ When tubes are kept inside the incubator, they take the temperature
of the incubator.
■ The appropriate temperature is obtained by means of
temperature regulator and is maintained by a thermostat. This
Laboratory Wares…
● Use and Care of Incubator

■ Read carefully the manufacturer‘s instruction.

Make sure the incubator is positioned on a level surface and that
none of the ventilation openings are blocked.
If the incubator does not have a temperature display, insert a
thermometer in the vent hole through the roof of the incubator.
Adjust the thermostat dial until the thermometer shows the correct
reading, i.e., 35 - 37Oc for the routine incubation of bacteriological
cultures.
Before incubating cultures and tests, check the temperature of the
incubator.
Clean the incubator regularly; making sure itis disconnected from
its power supply.
Every three to six months check the condition of the incubator
Laboratory Wares…
● Water bath

■ The water bath, like the incubator, is required for controlled

temperature incubation of culture and liquids, and many other
laboratory tests.
■ The temperature of the water bath is thermostatically
controlled and can be set at any desired level ranging usually
from 20 Cto100 C.
0 0

■ The heating coil may be of immersion type or enclosed in a

case, some models have propellers to help to circulated
water so that identical temperature is maintained throughout
the water bath.
Laboratory Wares…
● Use and Care of Water Bath

√ Maintain the minimum level in the water bath with chemically pure
water. Avoid use tap water.
√ Avoid use of water as salts from tap water may get deposited on
coil and so affect its function
√ Always use a thermometer to check that the temperature is stable
at the desired level.
√ Make sure that the substance being incubated is below the surface
of water in the bath.
√ It is advisable to cover the tubes, flasks or plates during incubation
to avoid
contamination and dilution as a result of condensation of water
from the lid of the water bath.
Laboratory Wares…
● Mixers

■ Are instruments used for preparation of reagents for mixing or

dissolving purpose.
■ Also used for homogenization.

.
 Laboratory Wares…
● Desiccators

■ Desiccators are instruments, which are used for drying of chemicals or

to keep other chemicals from being hydrated. As chemicals stay for
long period of timeout of dessicators, they
oc
sometimes absorb water.
The chemical is dried in an oven at 110 for 1 hour, and then it is placed
in a desecrator over night before weighing on the analytical balance.
The purpose of the oven is to remove the water and that of the
desicator is to store the
chemical at an ambient temperature where it cannot reabsorb water.
Desiccator contains substances called drying agents. These absorb
the water in the air of the desiccators. The most commonly used drying
agents (desiccants) are calcium chloride and concentrated sulfuric
acid. The chemical that is to be dried is placed in another bottle or test
 Materials used to preparing solution
● PH Meter

■ Definition: is an instrument which is used to measure Potentialof

ionhydrogen([Link] or
alkalinity of a substance) or is an instrument used to measure the PH or H+ ion
concentration.
PotentialofhydrogenpHscaleis0–14
AcidpH:0-6.9
NeutralpH:7.0
AlkalinepH:7.1-14.0
The pH meter is composed of
1. Glass bulb electrode(PH-electrode)
2. Reference(Calomel)electrode
3. Potentiometer(Sensitive meter) which measures the electric volt.
The glass bulb electrode contains a solution of a certain fixed PH or H+ conc.
When the
electrodes are placed in a solution of unknown PH, an electrical potential is produced
between them (i.e the solution and the H+ ions in the PH-electrode) This potential
which is
Materials used to preparing solution
● Glass Rod

■ Also called stirring rod, stir rod or solid glass rod, commonly
uses borosilicate glass and quartz as material.
■ Its diameter and length can be customized according to your
requirements.
■ Glass rod are corrosion resistant.
■ It can resist most acid and alkali.
■ It has strong hardness and can work in 1200 °C high
temperature for long time.
■ stirring rod is widely used in laboratory and industry. In
 Materials used to preparing solution
●Burette Clamp

● Spatula
■ Is a scientific ■ Can also refer to a tongue depressor are
small stainless steel utensils or wooden
equipment which
utensils,
used specifically to
■ used for scraping, transferring, or applying
hold and secure a powders and paste like chemicals or
burette on a stand, treatments.
so that a burette is ■ Many spatula brands are also resistant to
fixed and more acids, bases, heat, and solvents, which
convenient for the make them ideal for use with a wide range
Unit Four : Preparing Laboratory Solutions
Objective

● Selecting standard procedure for stock and working solution

preparation.
● Selecting Materials and solvent
● Calculating and recording data
● Measuring reagents for solution preparation
● Selecting and assembling laboratory Equipment’s
● Mixing or diluting working solution
● Preparing Solutions
● Labeling and storing Solutions
● Recording Working solution
 Prepare a Working Solutions
● Types of Clinical Laboratory Solution

● There are different types of solutions used in medical laboratory

procedures. These include reagent solution, staining solution,
standard solution and buffer solution.

● Reagents Solutions
● Any solution that is used in conjunction with a given sample and
expected to produce a measurable or noticeable change is called a
reagent solution.
● Necessary care, including the followings should be taken in
preparing a given reagent solution:
Chemical selection;
Following instruction of preparation;
 Prepare a Working Solutions
● There are two types of reagent solutions

● Stock Reagent Solution

●
Isaconcentratedreagentsolutionwhichisdilutedtoprepareaworkingsol
ution.
● Has a longer shelf life and occupies less space in storage than the
working solution.

● Working Reagent Solution

Prepare a Working Solutions
● Staining Solution

● Staining solutions are solutions that contain colored dyes.

● These solutions can contain basic, acidic or neutral dyes.
● give an artificial color for the substances to be identified from a
given biological specimen (whole blood, body fluids, urine, etc.).
● The substances may be identified by their characteristic reaction
with the staining solutions.
● Different types of blood cells, bacteria, parasites, and tissues
together with their cellular elements can be stained by using
appropriate types of stains (differential stains)
● such as Giemsa stain, Wright stain, Gram stain, Leishman stain, Acid
Fast Stain, etc. Simple stains are used to reveal the morphology
(shape, size and content) of an organism(s) and single dye is utilized
 Prepare a Working Solutions
● Basedontheirreaction,therearethreekindsofstains:
1. Basic stains
2. Acidic stains
3. Neutral stains

[Link] Stains
Are stains substance/cells the base part.
Example: Methylene blue stain, Safranin, Genetian violet, Carbolfuchsin etc

[Link] Stains
Are stains substance/cells the acidic part. E.g. eosin.

3. Neutral Stains
Are stains substance/cells both the acidic and basic components.
Neutral dyes stain both nucleic acid and cytoplasm. E.g. Giemsa‘stain,
Prepare a Working Solutions
● Basic Steps in Solution Preparation

Assemblingnecessarymaterialsandequipmentforpreparationofsolution
s.
Calculating and recording data.
Measuring chemicals and solvents.
Dissolving/diluting the solution in accordance SOPs.
Labeling and storage of reagents

● When preparing a solution decide whether the solution

requires.
An accurate volume preparation, E.g. a calibrate(standard).
Less accurate volume preparation, [Link]
Prepare a Working Solutions
● Analytical reagents (AR)
■ chemicals that are labeled Analar, Univar, AR (Analytical Reagent),
also known as GR(Guaranteed Reagent) which is prepared purely
and used for the preparation of calibrates or any reagent which
requires a pure chemical.

● Laboratory Grade(LG)
■ has lower purity but maybe Industrial grade chemicals. It‘s less
expensive, useful for some less pure solution preparation.

Deliquescent:-chemical dissolved in the moisture and go on taking in

moisture until the vapor pressure of the solution equals the pressure
of the water in the atmosphere.

Hygroscopic: a chemical absorbs water from the air but does not
Prepare a Working Solutions
●Preparing Accurate Solutions:
■ Use a balance of sufficient sensitivity
■ Weigh the chemical as accurately as possible.
■ The chemical should be of an analytical reagent grade.
■ Hygroscopic and deliquescent chemicals need to be weighed
rapidly
■ Use calibrated, chemically clean glassware
■ Read carefully the graduation marks and other information on
flasks and pipettes
√ Example: check whether a pipette is of the containing(rinsing-out)
type or of the delivery (nonrinsing-out) type.
√ It is best to use a delivery type
Prepare a Working Solutions
●Preparing Accurate Solutions:
■ Use a funnel to transfer the chemical into a volumetric flask.
■ Wash any chemical remaining in the container into the flask
with a little of the solvent.
■ Make the solution up to its final volume.
√ If warm, make up to volume only when the solution has cooled to
the temperature used to graduate written on the flask.
■ To avoid over-shooting the graduation mark,
√ use a Pasteur pipette or wash-bottle to add the final volume.
■ Make sure the bottom of the meniscus of the fluid is on the
graduation mark when viewed at eye level
■ Mix the solution well by inverting the flask at least twenty
times
 Prepare a Working Solutions
● Preparation of Calibrating Solutions(Standards):

■ When preparing calibrates the following are important:

√ Always use pure chemicals. use of impure can lead to serious errors in
test results.
√ Avoid weighing a very small quantity of a calibrate substance.
√ Instead prepare a concentrated stock solution which can be diluted to
make working solutions.
√ Use good quality distilled water. Electrolyte calibrates require deionized
water.
√ Use calibrated glassware and a volumetric technique.

■ N.B. calibrates should be prepared and standardized in a regional or

Prepare a Working Solutions
● Preparing Stains:

■ no need to use expensive volumetric glassware when preparing

stains.
■ Weigh the dye in a small container and transfer the weighed dye
direct to a leak-proof storage container, preferably a brown
bottle.
■ Add any other ingredients and the volume of solvent as stated
in the method, and mix well.
■ Adding a few glass beads will help the dye to dissolve more
quickly.
Prepare a Working Solutions
● Preparing Stains:

■ Note: Instead of measuring the volume of solvent each time the stain
is prepared, it is more practical to mark the side of the container with
the volume which needs to be added.
■ Transfer part of the stain to a stain dispensing container, filtering it if
required.
■ Always use dispensing containers with tops that can be closed when
not in use.
■ Label the container in a similar way to that described previously.
■ Store as instructed in the method of preparation.
■ Always protect stock containers of stain from direct sunlight.
 Prepare a Working Solutions
● Giemsa Stain Preparation:

■ Giemsa stock solution of about 500mlisprepared in the bottle of

stain by mixing Giemsa powder (3.8g), Glycerol (250 ml)and
Methanol (250ml) in a water bath at 50–60 °C.

■ Necessary Materials:
To make about 500 ml:
Giemsa powder............................................... 3.8g
Glycerol(glycerin).........................................250ml
Methanol (methyl alcohol).............................. 250ml
■ Materials & Equipment
Balance Funnel
Weighing paper Labeling Marker
 Prepare a Working Solutions
● Preparation of Giemsa working Solution(10%Giemsasolution):

■ Giemsa working Solution(10%) is prepared by diluting stock Giemsa

in a buffered Water (PH7.2)

● Calculate the total parts of Giemsa stock solution &water to be

added

● So 1part of Giemsa stock is mixed with 9parts of buffer water /

distal water.

■ Necessary Materials
Giemsa stock solution
Prepare a Working Solutions
● Preparation of EDTA Anticoagulant:

■ EDTA Solution(10%) is prepared by mixing EDTA in a Water

● So 2.5 part of EDTA is mixed with 25parts of distal water.

■ Necessary Materials/Reagent
To Make about 250ml
Di-potassium ethylene-diamine-tetra-acetic acid(K2–EDTA) ................2.5 g
Distilled water. .....................................................................................25ml

■ Necessary Tools & Equipment:

Balance
Funnel
Weighing paper
Micropipette
Measuring Cylinder
 Prepare a Working Solutions
● Preparation of Diluted Sodium Hypochlorite (Bleach)Disinfectants:

■ To prepare 10ml 0.5%bleach

■ Necessary materials:
Stock Sodium Hypochlorite Distilled water
Measuring Cylinder, Empty container
Gloves, Labeling Marker, Plastic Adhesive Tape

● Calculate the total parts of water to be added using the formula

● Total parts of water added=(%concentrate/%dilute)-1
=(5%/0.5%)-1
=9 parts

● So 1part of 5%bleach is mixed with 9parts of water

Prepare a Working Solutions
● Preparation of Diluted Sodium Hypochlorite (Bleach)Disinfectants:

● So 1part of 5%bleach is mixed with 9parts of water

Volume of water to be added = total volume×(parts of water/total
parts)
=10ml×(9/10)
=9ml
Volume of 5%bleach=total volume–volume of water added
=10ml-9ml=1ml
So 1ml of 5%bleach is mixed with 9ml of water
●procedures
2. Measure 1ml of 5%bleach into an empty container
3. Measure 9ml of water and mix well
 Prepare a Working Solutions
● Preparation of 70%Alcohol Disinfectant:

■ In order to create 70percent alcohol, a solution of ethanol alcohol

with a concentration greater than 70 percent must be diluted by a
calculated amount of water.

● The formula for this calculation is C1V1=C2V2,

● where
● C1 and V1 is the starting concentration and volume of the
solution and
● C2 and V2 is the final concentration and volume of the dilution

● So, V1= C2V2/ C1

 Prepare a Working Solutions
● Preparation of 70%Alcohol Disinfectant:

● For the purpose of this example, the initial solution is100percent ethanol
alcohol, creating a final volume of 500 mL of 70 percent alcohol
● Given
● C1=100%
● V2=500ml
● C2=70%
● V1=?
● So V1= C2V2/ C1 = 70%*500ml/100% = 350ml
● Volume of water to be added = total volume – ethanol (500ml –350)
=150ml
● So 350part of (95-100% ethanol) is mixed with 150parts of water
 Gram’s Stain Reagents Preparation Crystal Violet Stain:
● Crystal violet ................................................ 20g

● Ammonium oxalate.......................................... 9g
● Ethanol or methanol absolute......................... 95ml
● Distilled water .............................................. to1liter

● Acetone-alcohol

● To make1litre
● Acetone 500ml
● Ethanol or methanol absolute...................... 475ml
● Distilled water ............................................... 25ml
● Mix the acetone, ethanol and distilled water and transfer to a clean glass-
stoppered bottle.
 Gram’s Stain Reagents Preparation Crystal Violet Stain:
● Gram’s iodine ● Neutralred;1g/l(w/v)
• Potassium iodide.......................20g • To make 1 litter
• Iodine...................................... 10g • Neutral red........................1g
• Distilled water ........................ to1litre
• Should be stored in a brown bottle • Distilled water .............. 1litre

● Safranin
1. Prepare a stock solution
SafranineO............ 2.5g
Ethanol (95%) ............ 100ml
Mix until all the safranine is dissolved. Transfer the solution to a glass –
stoppered bottle.
Label the bottle (Safranine stock solution) and write the date.
2. Prepare a working solution in a glass stoppered bottle
Stock solution............ 10ml
Distilled water............ 90ml
Ziehl Neelson Stain Reagents Preparation
● A. Carbol fuschin
■ Solution A (saturated solution of basic fuchsin)
■ Basic fuchsin ......................3gm
■ Ethanol.............................. 100ml

■ Solution B (phenol aqueous solution, 50g/L (5%)

■ Phenol .......................................10gm
■ Distilled water ...........................200ml
■ Mix 10 ml of solution A with 90ml of solution B.
■ Transfer mixture to a glass stoppered amber bottle and
■ label the bottle― write the name& date.

Warning: Phenol is highly corrosive and poisonous

Ziehl Neelson Stain Reagents Preparation
● Preparation of Acid alcohol,3% v/v:

■ To make1liter:
■ Ethanol or methanol, absolute*...................................970ml
■ Hydrochloric acid, concentrated...................................30ml
■ procedures
1. Measure the ethanol or methanol and transfer to a1 liter capacity
leak-proof container.
■ Caution:
Ethanol and methanol are highly flammable, therefore use well
away from an open flame.
2. Measure 30ml of concentrated hydrochloric acid, add to the
solution, and mix well.
Ziehl Neelson Stain Reagents Preparation
● Preparation of Acid alcohol,3% v/v:

■ Caution:
Concentrated hydrochloric acid is a corrosive chemical with an
injurious vapor; therefore handle it with great care in a well-
ventilated room.

3. Label the bottle, and mark it Flammable.

4. Store at room temperature in a safe place. The reagent is stable

indefinitely.
For use: Transfer a small amount of the reagent to a dispensing
container that can be closed when not in use.
Ziehl Neelson Stain Reagents Preparation
● MethyleneBlueStain
■ Methylene blue chloride........................ 0.5g
■ Distilled water. ..................................... 100ml

●Malachite green
■ Malachite green .......................................... 0.5gm
■ Distilled water .............................................100ml
■ Using a pestle and mortar, grind the malachite green crystal
to a powder.
■ Dissolve the grind powder in 100ml distilled water and store
in a dark brown bottle.
Fluorescent Stain Reagents Preparation
●Auramine O

■ Auramine 0.1gm
■ 95% Ethanol 10 ml
■ Dissolve Auramine with Ethanol -------------------Solution1

●Phenol
■ Phenol crystal 3.0gm
■ Distilled water 87ml
■ Dissolve phenol with Distilled water --------------- Solution2
■ Mix solution 1 and2and store in amber bottle away from light
Fluorescent Stain Reagents Preparation
●Potassium permanganate

■ Potassium permanganate(KMnO4) -------0.5g

[Link]-------------------- 100ml
Dissolve and store in amber bottle

■Acridine orange

■ Acridine orange-------------------- 0.01g

■ Anhydrous dibasic sodium phosphate (NA2HPO4)
--------0.01g
■ Distilled water-------------------- 100ml
 Prepare a Working Solutions
●Lens Cleaning Solution (Ethyl Ether– Alcohol)
• Absolute ethanol..............................................20ml
• Ethyl ether ...................................................... 80ml
• Mix and stopper

●Boric Acid, Saturated Solution

• Boric acid ....................................................... 4.8g
• Distilled water ............................................1000ml
• Store in a glass-stoppered bottle.
• Label the bottle ―
 Prepare a Working Solutions
● Buffered Water, PH7.2
• Disodium hydrogen phosphate(Na2HPO4·2H2O).......................3.8g
• Potassium dihydrogen phosphate, anhydrous .............................
2.1g
• Distilled water .....................................................................1000ml
• Dissolve the salts in the distilled water, stirring well. Check the pH
using narrow range pH papers; it should be 7.0–7.2.
• Transfer the solution to a glass-stoppered bottle and Label the
bottle

● Eosin,10g/l(1%) Solution
• Eosin 1g
• Distilled water ............................................ q.s.100ml
 Prepare a Working Solutions
● Formaldehyde,10%Solution
• formaldehyde(CH2O)solution, 40%… .............................100ml
• Distilled water .................................................................... 300ml
• Transfer the solution to a glass-stoppered bottle.
• Labelthebottle―FORMALDEHYDE10%SOLUTION‖andwritethedate.
• Warning: Formaldehyde is corrosive and poisonous.

● PotassiumHydroxide,200g/l(20%)Solution
• Potassium hydroxide (KOH)pellets............................. 20g
• Distilled water ........................................................ 100ml
•Labelthevolumetricflask―POTASSIUMHYDROXIDE20%SOLUTION‖an
dwritethe date.
• Warning: Potassium hydroxide is corrosive.
 Prepare a Working Solutions
● PhysiologicalSaline,8.5 g/l(0.85%w/v)Solution(Isotonic Saline)
• Sodium chloride(NaCl) ............................................8.5g
• Distilled water .................................................... q.s.1000ml
•Label―SODIUMCHLORIDE 0.85% and write date.

● Trisodium Citrate, Anticoagulant

■ To make About 100 ml
•Trisodium citrate, anhydrous(Na3C6H5O7)......................... 3.8g
• Distilled water ..................................................................100ml
• Dissolve and Keep in the refrigerator.
● Use 1ml of the solution per 4ml of blood.
● Label ―TRISODIUMCITRATE 3.8%and write the date.
 Labeling and Storage of Reagents
● Labeling of Reagents
Label is An item used to identify something or someone with a
piece of paper, card, or give instructions or details concerning its
ownership, use, nature, destination, etc.; tag
Proper labeling is fundamental to a safe and effective laboratory
operation. Reagents created in the laboratory also require labeling.
All purchased reagent chemicals should be labeled with
√ Reagent
Chemical name √ Lot #
date received √ Concentration
date of initial opening √ Storage Temp
shelf-life √ Open/Prep Date
hazard warnings √ Expiration Date
Storage classification location
Name and address of manufacturer√ Prepared by
√ Location
√ Precautions
 Labeling and Storage of Reagents
● Storage of Reagents
Storing reagents in correctly are important causes of unreliable test results.
Reagents should be stored in a clean, cool, dry location to maximize shelf
life.
Refrigerate reagents only if indicated on the label.
Reagents that are stored at elevated temperatures or in humid locations
may experience accelerated
degradation and reduced shelf life.
Storage requirements are to be indicated on container labels:

A. Temperatures
Store reagents at required temperatures indicated by manufacturer.
Reagents with temperature storage requirements shall have these listed
on the labels.
Store reagents that must be refrigerated or frozen in freezers or
Refrigerators with the required
Labeling and Storage of Reagents
●Storage of Reagents
B. Light sensitivity
If indicated store in cabinets or in a dark container.
Secure storage are as against unauthorized removal of
chemicals.
Where possible, storage are as should have two
separate exits.
Maintain clear access to and from the storage areas.
Do not store chemicals in a is les or stair wells, on desks
or laboratory benches, on floors or in hallways, or in fume
hoods.
Prepare a Working Solutions
Use an appropriate "Acid Cabinet" for any acid solutions of 6 M
concentration or higher. Nitric acid needs to be isolated.
Label storage are as with a general hazard symbol to identify hazardous
chemicals and indicate correct fire fighting procedures.
File a Material Safety Data Sheet (MSDS) for every chemical stored in the
laboratory.
Store all reagent chemicals in compatible family groups. Do not
alphabetize.
Store all chemicals at eye level and below. The preferred shelving material
is wood treated with polyurethane or a similar impervious material.
All shelving should have a two-inch lip .If you use shelving with metal
brackets, inspect the clips and brackets annually for corrosion and replace
as needed.
Store chemical reagents prepared in plastic bottles(if possible and
appropriate to the chemical) to minimize the risk of breakage.
 Prepare a Working Solutions
Date containers upon receipt and again when opened.
Attach chemical labels with all necessary information to all containers.
When opening newly received reagent chemicals, immediately read the
warning labels to be aware of any special storage precautions such as
refrigeration or inert atmosphere storage.
Testperoxide-formingsubstancesperiodicallyforperoxidelevels;dispose of after
three months unless the MSDS for the substance indicates a longer shelf life.
Checkchemicalcontainersperiodicallyforrust,corrosion,andleakage.
Store bottles of especially hazardous and moisture-absorbing chemicals in
chemical-safe bags.
Maintain a complete inventory in the room where the chemicals are stored,
and make a copy available to fire fighters.
Keep storage are as clean and orderly at all times.
Have spill clean up supplies
Limit the amount of flammable and combustible materials stored to that
required for one year of laboratory work.
Record Working Solution Details in Laboratory Register
● Laboratory Solutions Register

■ All solutions prepared at Lab are recorded, by solution type, into the
Laboratory Solutions Register.
■ contains all identification details that are recorded on the label/
preparation.
■ Each solution is identified as either a primary or a secondary standard
■ If the solution is a primary standard, then all the raw data used to
calculate the concentration of the solution is recorded in the
Laboratory Solutions Register. This information includes:
details, including batch numbers, of the reagents used to make up
the solution
the amount of reagent used to make up the solution
the final volume of the solution.
Record Working Solution Details in Laboratory Register
●Laboratory Solutions Register

■ In the case of a primary standard, the label and the Laboratory

Solutions Register provide a comprehensive record of the
solution.
■ However, when a secondary solution is prepared, a
Standardized Solution Sheet is used to record the titration
results and the calculations of the concentration of the solution.
■ The solution identification details are then recorded on the label
and in the Laboratory Solutions Register.
■ The solution identification number is included in the
Standardised Solution Sheet for tracking purposes