Practical 1 : Study of laboratory reagents

Introduction:
Various chemicals are used for the preparation of solutions and reagents in a laboratory. A chemical
is a substance produced by a chemical process by various manufacturers. It is of known composition.
Example: Sodium carbonate, potassium hydroxide, hydrochloric acid, glacial acetic acid, etc.

A Solution:
It is a combination of two substances-a solute and a solvent. The dissolved substance is called solute
and most of the times the substance present in a relatively greater proportion in the solution is
called the solvent

A Reagent:
This term can be applied in particular to any chemical compound or mixture of compounds, usually
in solution, employed in chemical analysis or for the detection of biological constituents.

Stock and Working Solutions:

A stock solution is a concentrated solution from which different types of working solutions can be
prepared by simple dilutions.

Acids and Bases:

Acids are proton donors and bases are proton acceptors.

Alkali:
These compounds yield hydroxyl ions on dissociation.

Strong Acids and Alkalies:

These compounds are completely ionized in solution.

Examples Strong acids: HCI, H,SO,, HNO, etc. Strong alkalies: NaOH, KOH, etc.

Weak Acids and Alkalies:

A Solution These compounds are dissociated only to a limited extent.

Examples Weak acids: Acetic acid, oxalic acid, etc.

Weak alkalies: Ammonium hydroxide, calcium hydroxide, sodium carbonate, etc.

PREPARATION OF REAGENTS
General Consideration
The reagents are required in laboratories for qualitative and quantitative tests involving
photometric, electrochemical, immunochemical, electrophoretic, chromatographic and titrimetric
techniques. Although nowadays diagnostic kits (ready-made reagents) manufactured by various
companies are used in various pathology laboratories, a laboratory worker should know, how to
prepare various types of the reagents

BASIC REQUIREMENTTS
1. Solid and liquid chemicals
These are commercially available in the following forms-
• Anala 'AR: Analytical grade chemicals
• GR: Guaranteed Reagent
Anala 'R' and GR chemicals are highly purified and are mainly used for the
preparation of quantitative reagents and also for the preparation of other standard
reagents such as normal, molar and primary standards
For general purpose where Anala 'R' or GR chemicals are not necessary but purity
within certain limits is required, general purpose chemicals (GPR) can be used.
Laboratory reagents (LR) or LAB products are mainly used for the preparation of
qualitative reagents such as 'Benedict's reagent, Fouchet's reagent, etc. LR or LAB
chemicals are of a degree of purity making them suitable for general laboratory use.
2. Solvents
Reagent grade water is used for the preparation of most of the reagents and solutions in the
laboratory. For the preparation of certain specific reagents, required for electrolyte and
hormonal studies, glass distilled water (distiled water prepared by using glass distillation
apparatus) is used. The other solvents which may be required are glacial acetic acid,
isopropanol, chloroform, ethanol, etc.
• Reagent Grade Water
The preparation of most reagents and solutions used in the clinical laboratory
requires pure water. The National Committee for Clinical Laboratory Standard
(NCCLS, USA) has established a standard for reagent grade water According to these
guidelines, single distilled water fails to meet the specifications for type I clinical
laboratory reagent water.
The term reagent grade water followed by designation of the type I, defines the
specifications of the water and is independent of the method of preparation (i.e.,
distillation or deionization). Any combination of methods (distillation or
deionization) may be used in the preparation of reagent grade water as long as the
final product meets the specifications suggested by NCCLS
3. Glassware
• Conical flasks (100 ml)
• Beakers (1000 ml)
• Measuring cylinders (1000, 500, 100 ml)
 • Volumetric flasks (100 ml, 1000 ml)
• Funnels and glass rods
• Amber colored glass and polyethylene bottles (100, 250, 500, 1000 ml)
4. Instruments
5.
• Physical balance and analytical balance
• Hot plate (or a gas burner) and magnetic stirrer
6. Other requirements
• Weight box
• Rubber gloves
• Safety spectacles

THE VARIOUS TYPES OF SOLUTIONS AND REAGENTTS

• Normal solutions
• Molar solutions
• Percent solutions
• Buffer solutions
• Buffered substrates
• Indicators
• Primary standards and
• Other complex reagents
Practical 2: Preparation of Molar, Normal and Percent solutions.

Introduction:
Exercise 1: Preparation of10 ml _____ Molar sodium chloride
solution.
• Requirements:
o Sodium chloride, anhudrous(AR)
o 50 ml beaker
o 10 ml volumetric pipettes
o 5 ml volumetric pipettes
o Graduated cylinder
o Analytical balance
o 50 ml reagent bottle

• Procedure:
o Calculate the amount of NaCl required to prepare given molar concentration.
o Carefully weight ___ g of NaCl required.
o Dissolve it in about 5 to 7 ml of water after transferring it into the 50 ml
beaker.
o Make final volume 10 ml by using graduated measuring cylinder.
o Transfer it into 50 ml reagent bottle and label it accordingly.

• Calculations:
o 1 Molar solution = molecular weight of solute dissolved in grams
per liter of solution
o Therefor 1 Molar NaCl= 58.44 gm dissolved in 1000 ml D/W
(M.W. of Nacl= 58.44)

o For 10 ml final concentration…

1000 ml solution ----- 58.44 gm NaCl
10 ml solution ----- x gm

X= 58.44 * 10 /1000 = 0.5844 gm

o For Given Molarity

1 Molar solution ---------0.5844 gm NaCl
______Molar solution ------- x gm

X = 0.5844 * ____ / 1 = ______________

Exercise 2: Preparation of 100 ml 0.1 N NaCl solution.
• Requirements:
o Sodium chloride, anhudrous(AR)
o 50 ml beaker
o 10 ml volumetric pipettes
o 5 ml volumetric pipettes
o Graduated cylinder
o Analytical balance
o 50 ml reagent bottle

• Procedure:
o Calculate the amount of NaCl required to prepare given molar concentration.
o Carefully weight ___ g of NaCl required.
o Dissolve it in about 5 to 7 ml of water after transferring it into the 50 ml
beaker.
o Make final volume 10 ml by using graduated measuring cylinder.
o Transfer it into 50 ml reagent bottle and label it accordingly.

• Calculations:
o To calculate the normality of the given solution its molarity or amount of
substance added needs to known
o If molarity is given, then
Normality = molarity * No. of H+ or OH- Ions
o If the amount of substance added is given, then

Normality = amount of substance added * 1000/ gram eq. weight * volume of solutiom

Gram eq. weight = gram molecular weight / no. of H+ or OH- Ions

o To calculate the amount required to make 100 ml of 1 solution use N 1V1=N2V2

For our exercise, NaCl does not have any H+ or OH- Ions therefore, here Molarity of NaCl =
normality of NaCl.

Thus, for 1 N NaCl, 58.44 gm will be required for 1000 ml and therefor 0.5844 gm will be
required for 10 ml.
Exercise 3: Preparation of 10 ml _____ % w/v sodium chloride
solution.
• Requirements:
o Sodium chloride, anhudrous(AR)
o 50 ml beaker
o 10 ml volumetric pipettes
o 5 ml volumetric pipettes
o Graduated cylinder
o Analytical balance
o 50 ml reagent bottle

• Procedure:
o Calculate the amount of NaCl required to prepare given % w/v
concentration.
o Carefully weight ___ g of NaCl required.
o Dissolve it in about 5 to 7 ml of water after transferring it into the 50 ml
beaker.
o Make final volume 10 ml by using graduated measuring cylinder.
o Transfer it into 50 ml reagent bottle and label it accordingly.

• Calculations:
o 1 % w/v solution = 1 gm dissolved per 100 ml solution

o For 10 ml final concentration…

100 ml solution ----- 1 gm NaCl
10 ml solution ----- x gm

X= 1 * 10 /100 = 0.1 gm

o For Given Molarity

1 % w/v solution --------- 0.1 gm NaCl
______% w/v solution ------- x gm

X = 0.1* ____ / 1 = ______________ gm.

Practical 3: Preparation various dilutions from stock solutions.
Introduction:
In microbiology laboratories as in other science laboratories, solutions must be diluted to
achieve a desired final concentration of the active material contained in that solution.
A solution may be defined as a mixture of two or more substances (solute) in which the
molecules of the solute are evenly distributed and will not separate on standing or precipitate
from the solution. Solutes are dissolved in a solvent or diluent, such as water, alcohol, etc.
Solutions are usually referred to as stock solutions and may be diluted by a variety of
methods, depending upon the experimental requirements.
Some of these methods are listed as follows:
1. A dilution factor must be determined first in order to dilute a solution. This dilution
factor tells us how many times a solution must be diluted and is calculated by
dividing the initial concentration (IC) of the solution by the final concentration (FC)
desired.
Example: You wish to dilute a 10% stock solution to a final concentration of 2%
10%/2%= 5 (dilution factor) Take 1.0ml of the 10% stock solution plus 4.0m! of
solvent which equals a total of 5.0ml. Thus each ml of the final solution wili contain
2% of the original 10% initial solution
2. Simple Dilution (Dilution Factor Method)..
A simple dilution is one in which a unit volume of a liquid material of interest
an appropriate volume of is combined with a solvent liquid to achieve the desired
concentration. The dilution factor is the total number of unit volumes in which
your material will be dissolved. The diluted material must then be thoroughly
mixed to achieve the true dilution. For example, a 1:5 dilution (verbalize as "1 to
5" dilution) entails combining 1 unit volume of diluent (the material to be diluted)
+ 4 unit volumes of the solvent medium (hence, 1 4 5 dilution factor)
• Example: To dilute a streptavidin solution 1:3000
Mix 1 unit volume of streptavidin solution with 299 volumes of solvent medium.

In some instances, solutions are expressed in terms of "X" an indicator of

relative solute concentration. For example, the buffer saline sodium citrate
(commonly referred to as SSC) is usually made up as a 20X stock solution. However,
it is typically diluted before use.
The same rules discussed above apply for making dilutions from "X" stock
solutions.
• Example: solution of SSC can be prepared from a 20X SSC stock solution by
mixing one unit volume of 20X SSC with 19 volumes of water.
3. Mixing parts or volumes
 Mixing of parts or volumes is often confused with simple dilution. For example if
you are instructed to, "...add 1 part glacial acetic acid with 3 parts water" you are
literally mixing 1 unit volume of glacial acetic acid and 3 unit volumes of water You
would do the same if asked to mix 1 volume of glacial acetic acid with 3 volume of
water. Confusion can arise due to the use of a colon in some mixing recipes (see
example below).
Example: If you are instructed to make up a 1:3 acetic ethanol solution, you
are probably supposed to mix one unit volume of acetic acid and three unit
volumes of ethanol. However, if you are asked to make a 1:3 dilution of acetic acid
in ethanol, you would mix one unit volume of acetic acid with two unit volumes of
ethanol

4. Serial Dilution

A serial dilution is simply a series of simple dilutions which amplifies the dilution
factor quickly beginning with a small initial quantity of material (e.g, DNA, restriction
enzyme, etc.). The source of dilution material for each step comes from the diluted
material of the previous. In a serial dilution the total dilution factor at any point is the
product of the individual dilution factors in each step up to it.

Final dilution factor (DF) = DF1 * DF2 * DF3, etc.

Example: In the microbiology lab BIOLOGY 201 the students perform a three step
1:100 serial dilution of a bacterial culture. The initial step combines 1 unit volume
culture (10 u) with 99-unit volumes of broth (990 pl) =1:100 dilution. In the next step,
one unit volume of the 1:100 dilution is combined with 99-unit volumes of broth now
yielding a total dilution of 1:100100 1:10,000. Repeated again (the third step) the total
dilution would be 1:100 10,000 1:1,000,000 total dilution. The concentration of
bacteria is now one million times less than in the original sample.

5. Making fixed volumes of specific concentrations from liquid reagents: V1C1-V2c2

Very often you'll need to make a specific volume of known concentration due to
limited availability of liquid materials (some chemicals are very expensive and are only
sold and used in small quantities, e.g., micrograms) or to limit the amount of waste.
The formula below is a quick approach to calculating such dilutions where

V = Volume, N = concentration; in whatever units you are working

(source solution attributes) N1V1=N2V2 (new solution attributes)

• Example: Suppose you have 3 ml of a stock solution of 100 mg/ml

ampicillin (=C1) and you want to make 200 μL (= C2) of solution having 25
 mg/ml (= V2). You need to know what volume (V1) of the stock to use as
part of the 200 l total volume needed.

V1 = the volume of stock you'll start with. This is your unknown.

N1 = 100 mg/ ml in the stock solution

V2 = total volume needed at the new concentration 200 pl = 0.2 ml

N2 the new concentration = 25 mg/ml

By algebraic rearrangement:

V1 = (V2x C2) / C1

V1={0.2 ml x 25 mg/ml) / 100 mg/ml

After cancelling the units = 0.05 ml =50 μL

So, you would take 50 l of stock solution and dilute it with 150 l of solvent to get the 200 u of
25 mg/ml solution needed (remember that the amount of solvent used is based upon the final
volume needed, so you have to subtract the starting volume from the final to calculate it.

Exercise 1: Preparation of ____ % (w/v) dilution from 10 %(w/v)

stock solution.
• Requirements:
o Sodium chloride, anhudrous(AR)
o 50 ml beaker
o 10 ml volumetric pipettes
o 5 ml volumetric pipettes
o Graduated cylinder
o Analytical balance
o 50 ml reagent bottle

• Procedure:
o Prepare 10 % (w/v) of stock solution of NaCl by dissolving 1 gm of NaCl into 5
to 7 ml of water and then by making final volume 10 ml.
o Calculate the amount of stock solution required to prepare ____ % (w/v)
dilution.
o Make the final volume 10 ml by adding appropriate amount of water.
o Transfer the solution into appropriate bottle and label it.
• Calculations:
o Calculation for preparation of stock solution: 10 % (w/v)
o 1 % w/v solution = 1 gm dissolved per 100 ml solution

o For 10 ml final concentration…

100 ml solution ----- 1 gm NaCl
10 ml solution ----- x gm

X= 1 * 10 /100 = 0.1 gm

o For Given Molarity

1 % w/v solution --------- 0.1 gm NaCl
10 % w/v solution ------- x gm

X = 0.1*10 / 1 = 1 gm.

o Calculation for volume of 10 % (w/v) stock solution required to

prepare ___% (w/v) dilution.

V1 = the volume of stock you'll start with. This is your unknown.

N1 = 10 %(w/v) in the stock solution
V2 = total volume needed at the new concentration 10 ml
N2 the new concentration = _____% (w/v)

N1V1=N2V2

V1=N2V2/ N1
= ____ * 10 /10
Practical 3: Standardisation of 1 ml volumetric pipette.