Yashwantrao Chavan
Maharashtra
Open University
Graduate Degree Programme: B.Sc. in
Bio-Technology, Bio-Informatics and Genetics
SBT/ SBI/ SGS035: Lab Course B. Sc. (BT, BI, GS)
Semester 3 Lab
Workbook
1
Yashwantrao Chavan Maharashtra Open University
Vice-Chancellor-Dr. Rajan Welukar
Expert Advisory Committee
Mr. Manoj Killedar
Director, School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Chetana Kamlaskar
School of Science & Technology, Y.C.M. Open University, Nashik
Dr. Sunil Ganatra
135, Krushnakunj, Toata Colony, Lakadganj, Nagpur
Prof. Indira Ghosh
Bio-Informatics Center, University of Pune, Pune
Prof. Urmila Kulkarni-Kale
Bio-Informatics Center, University of Pune, Pune
Prof. Dr. Piyali Kar
Maharashtra Education Foundation, CBD Belapur, Navi Mumbai
Course Writers
Mrs Sheetal Mhaske and Ms Manjiri Ukey
G.H.Raisoni Institute of Interdisciplinery Sciences, Pune - 01
Course Editor
Mrs Nandini Joshi-Kotharkar
G.H.Raisoni Institute of Interdisciplinery Sciences, Pune - 01
Course Coordinator and IT Editor
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
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E-Version available at
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Prospectus =>Syllabus & Learning Resource => SBT => Sem03
© Yashwantrao Chavan Maharashtra Open University, Nashik
Printed & Published by: Shri. S.P. Kowale, Registrar, Y.C.M. Open University,
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2
CERTIFICATE
This is to certify that Mr./Mrs./Smt.
…………………………………………………………………….
has successfully completed experiment as per the following
details:
(1) Total number of experiment completed: ……………….…………………
(2) Serial number of experiment completed: …………………………………
Laboratory Instructor Name : ……………………………………….…………………….
Signature & date : ………………………………………….………………………
Programme co-ordinator Name: ………………………………….…………….…………
Signature & date : ……………………………………….………………………
External Examiner Name : …………………………………………….….…………………
Signature & date : ………………………………..…………….………………………………
3
List of Experiments of Semester 03 Biotechnology Lab
Sr.No
Name of Experiment
1.
Preparation of solutions of given molarity, molality and
normality.
2.
Preparation of buffers of specified pH using pH meter.
3.
Quantitative analysis of carbohydrates and non
carbohydrate: glucose, fructose, maltose, lactose, galactose,
protein, lipids, creatinine and urea
4.
Spot test for amino acids.
5.
Detection of sugar, glucose and albumin from urine.
6.
Demonstration of different equipment and techniques used in
microbiology
7.
Preparation of nutrient slant, and broth
8.
Preparation of cotton plugs
9.
Isolation of single colony on solid media : Streak plate and
Spread plate method
10.
Enumeration of bacteria by i)Breed method (ii) Pour plate
method
11.
Staining techniques : Simple staining, Gram staining, Acid
fast staining, Endospore staining and Viable count
12.
Demonstration of presence of α-amylase in saliva
13.
Estimation of blood glucose by Nelson-Somogyi method
14.
RBC count
15.
WBC count
16.
Measurement of blood pressure by spygmomanometer
17.
Estimation of protein by Lowry’s method
18.
Determination of acid value of fats
19.
Determination of clotting time of blood by capillary tube
method
20.
Assay of hemoglobin by haemoglobinometer
21.
Differential leukocytes count of blood
22.
Colorimetric estimation of protein by biuret method
23.
Detection of bacterial motility by hanging drop method
24.
Oligodynamic activity test of metals
Page
No.
4
BASIC RULES OF A MICROBIOLOGY LABORATORY
A microbiology laboratory is a place for working with a
variety of microorganisms. Since several culture media are
prepared and organic materials are present, the chances exist for
the presence of high spectrum of microbial community. Secondly,
while working with pure culture one should always follow the
microbiological rules so that neither the experiment should be
unsuccessful nor any hazard may occur. If a large number of
students are working in a microbiology laboratory, they should be
aware what to do or what not? What are the
apparatus/instruments/equipments present in the laboratory and
what is their functioning? What are the chemical solutions and
stains and how to handle? How should the students enter in the
microbiology laboratory and how should they work? Therefore, the
fresher such as students, teachers, laboratory assistants and
helper must follow the following guidelines:
1) Always wear an apron (a white coat or gown) before entering
the microbiology laboratory to protect from microbial
contamination and laboratory hazards. At regular intervals get the
apron washed.
2) Cut nails regularly.
3) Tie long hairs back to avoid contamination and fire hazard.
4) Keep your working laboratory bench clean of everything.
Nothing should be laying on the bench.
5) Never keep books, purses, bags, etc. on the working bench.
6) Always wash your hands with soap in running tap water before
and after the work.
7) Clean your working bench with ethanol (70%) or phenol
(1:100).
8) Never spit and smoke in the laboratory.
9) Don’t put anything of the laboratory (e. g. pencil, thread,
labels, inoculation needle, pins, etc) in your mouth, ears, nose
and eyes.
10) Don’t put your fingers in your eyes, ears, mouth. It may
facilitate the chance of infection by pathogenic microorganisms.
11) Don’t eat or drink or talk while working with microorganisms.
12) Don’t mishandle the chemical solution, stains, spirit lamp, UV
light, instruments/apparatus or electricity.
5
13) Always keep the burner at distance from the organic solvents.
Your sincere care will avoid fire accident. The burner must be
turned off soon after the use.
14) Always maintain aseptic condition while working with
microorganisms.
15) Always use flame sterilized inoculation needle/loop.
16) Don’t open the culture tubes/plates directly and never inhale
them nor observe with naked eyes.
17) Open the culture tubes/plates near the vicinity of flame of the
burner.
18) While working with broth culture don’t suck the suspension
with mouth. Always use pipette sucker.
19) After completion of work always label the cultures with names,
code and date of work. It will help recording the data.
20) Always keep plates in tiers and culture tubes in upright in
basket or racks. Finally, transfer all the culture in the incubator at
desired temperature of where ever to keep.
21) Never leave your cultures on working table or seat.
22) Clean the working table/bench when the work is completed.
23) Clean lenses of objective with tissue paper.
24) Keep the stains, reagents, stock cultures to their respective
places when the work is completed.
25) After completion of work keep your slides/pipette/culture
tubes/plates in container and steam sterilize before washing.
26) Record your result at time.
27) For any difficulty, ask your laboratory assistant or concerned
teachers.
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Experiment No: 1
Aim: Preparation of solution of given Molarity, Molality and Normality.
Theory and Principle:
Normality: The number of gram equivalent of the solute present
per liter of its solution .
N = Weight in gram of solute/ litre of solution
Equivalent weight in gram.
Or = equivalent weight/ 1000.
Molarity: Gram molecular weight of solute per litre of solution .
M = Molecular weight of solute
1000ml of solvent .
Molality : It represent the number of moles of solute present in
1000g of solvent .
1m = Molecular weight of compound
1000g of solvent.
Procedure :
1. Weigh the required amount of solute.
2. Dissolve in required amount of solvent.
A. Prepare 0.1N, 1M, 0.2M Solution of NaOH.
Molecular weight of NaOH = 23+16+1 = 40
Equivalent weight of NaOH = molecular wt.
No.of replaceable H/OH = 40
a. To prepare 0.1 N solution of NaOH .
To prepare 1N solution: Dissolve 40g NaOH in 1000 ml of solvent.
0.1N NaOH=4g of NaOH in 1000 ml of solvent (Distilled Water)
Molarity = Molecular weight / 1000ml of solvent .
For 1M of NaoH : Dissolve 40g NaOH in 1000ml of solvent (D.W.)
Molality = Molecular weight of compound
100g of solvent
For water; volume = mass/ density
= 1000ml = mass of water 1g/ml
=1000g = mass of water
For 1M of NaoH = 40g of NaoH dissolved in 1000ml of water
Therefore, 0.2M Of NaoH= 8g of NaoH dissolved in 1000ml of water.
B. To prepare 0.5N, 2M, 0.4M of HCL in 1000ml of solvent
Molecular Weight of HCl = 1+35.5=36.5
7
For HCl specific gravity = 1.18
Volume = Mass/specific gravity
=36/1.18
= 30.51ml
Therefore for 1N HCl = Dissolve 30.51ml of HCl in 1000ml of solvent
(D.W.)
For 0.5N HCl = 15.25ml Of Hcl in 1000ml of solvent (D.W.)
For 1M HCl =Dissolve 30.51ml of Hcl in 1000ml of solvent(D.W.)
For 2M HCl =Dissolve 61.02ml of Hcl in 1000ml of Solvent(D.W.).
Result:
8
Experiment No: 2
Aim: - Preparation of buffer of specified pH using pH meter.
Theory:- Buffers are defined as substances that resist changes in the
pH of the system. Weak acids or bases,in the presence of their salts
with strong bases or strong acids respectively form buffer system.
Ex: Phosphate/monohydrogen phosphate.
Carbonic acid/bicarbonate
Proteins/proteinate.
pH:- The pH of the solution is the value with which defines its
hydrogen ion concentration in aqueous solution, it is relative strength
of hydrogen ion reach species called acid and the hydrogen ion
deficient species called base which determines the net pH of a
solution.
Measurement of pH :pH indicators:- These are usually organic compounds of natural or
synthetic origin whose colour is dependent on the pH of the solution.
Indicators are dependent on the pH of the solution.
Indicators are usually weak acids which dissociates in solution.
pH meter:- The most reliable and accurate method for the routine
measurement of pH is the pH meter in which a change in pH is
measured as change in electrical potential. If a metal rod is
placed in solution of its salt, it acquires potential. If two dissimilar
metals are dipped into the solution of their salts, the difference in
potential can be measured or calculated from the two separate
potentials. The standard electrode is thus required against which the
potential of other electrodes can be compared. This is the “standard
hydrogen electrode”, consisting of platinum rod dipped in the aqueous
solution with a given H+ activity in which hydrogen gas is bubbled
continuously at 1 atmospheric pressure. But this is too cumbersome to
be used, as reference electrode for routine use,
other secondary reference electrode of known potential in relation to
standard hydrogen electrode are used.
Example:- Calomel electrode, glass electrode.
Precautions:1. The glass electrode is fragile and must be handled with care.
2. Electrode must not be left to dry.
3. The temperature compensation dial must be set before it is
9
Calibrated as potential is produced dependent on temperature.
4. The meter must be calibrated first with a standard buffer of pH
7 and then with pH 4 or pH 9.
Procedure:1. Prepare the solution of given molarity as per the table.
2. Do the addition for specific buffer as per the table.
3. Make up the volume up to 1000ml.
4. Check the pH with the help of pH meter.
Observation Table:S.No
1
2
pH
2
4
3
6
4
5
9
10
Solution To Be Used
0.2M KCl + 0.2M HCl
0.2M succinic acid + 0.2M
NaOH
0.2M succinic acid + 0.2M
NaOH
0.1M KCl + 0.1M H3BO3
1M NaHCO3 + 1M Na2CO3
Volume In ml
65ml + 250ml
250ml + 100ml
250ml + 435ML
250ml+250ml+208ml
13.8ml + 12ml
Result:-
10
Experiment No.3
Aim: Quantative analysis of carbohydrates and non carbohydrate:
glucose ,fructose, maltose lactose, galactose protein, lipids,
creatinine and urea.
Principle: The Benedict’s quantitative reagent contains potassium
thiocynate and potassium Ferro cyanide in addition to sodium
citrate,sodium carbonate and CuSO4.Glucose reduces cupric ions in
solution to cuprous ions which react with potassium thiocynate to form
white colored cuprous thiocynate. The small amount of potassium
thiocynate acid is keeping cuprous oxide in solution. As precipitate
formed is white. The loss of all blue color is readily observed which
indicates complete reduction of cupric ions.
Requirements:
I Chemicals
1. Benedict’s reagent
2. 1% Glucose
3. 1% fructose
4. 1% Lactose
5. 1% Maltose
6. 1% Galactose
II Apparatus:
1. Test tubes
2. Pipettes
3. Water Bath
Procedure:
1. Take 5ml of Benedicts solution reagent and 2ml of
Carbohydrate sample.
2. Boil in water bath for 5 min.
Observation:
Observe the color change with respect to carbohydrate use,
Glucose, fructose gives dark brown color
Maltose galactose gives orange color
Sucrose turns colorless.
Result:
11
Experiment No.4
Aim: Spot test for amino acids.
Principle: Chromatography is a method by which members of groups
are separated using continuous distribution and redistribution between
two phases:
I. Stationary phase
II. Mobile phase
A liquid – Liquid chromatographic system is used to separate amino
acid. What man No.1 filter paper is used as a supporting medium.
Cellulose in What man No.1 filter paper makes an ideal support
medium where water is observed between the cellulose fibers and
forms a stationary hydrophilic phase. A small amount (2-3 µl) of amino
acid solution is applied on the What man no.1 sheet and it is
introduced to liquid- liquid system(Reservoir Buffer),various amino
acids get separated according to their respective Rf values. The
separated amino acids are stained by ninhydrin reagent which reacts
with the amino acid to from violet complex.
Requirements:
1.Alanine
2.Methionine
3.Valine
4.Lysine
5.Test amino Acid sample 1
6.Test amino Acid sample 2
7. Whatman No.1filter paper.
8.Capillary tube
9.Reservior Buffer
Procedure :
1. Add sufficient quantity of the reservoir buffer system in the
chromatography container. Cover it with an airtight lid.
2. Perform the experiment next day (i.e. after 18-24 hrs) so that
the container becomes saturated with the vapors of the
reservoir buffer system
3. Mark six points with the help of pencil leaving 2-3 cm
distance from the base on one of the side of the Whatman
No.1 paper for application of the standard and test amino
acid samples.
4. By using separate glass capillary for separate standards and
samples apply each solution on the marked point of application.
12
5. Tide the other end of the Whatman No.1 paper with the
thread or some other support to keep it exactly straight when
dipped in reservoir buffer.
6. Allow the reservoir buffer to run until it reaches near the
periphery of paper.
7. Remove the paper from the reservoir tank. Mark the solvent
front.
8. Dry this paper in hot air over(at 100 0C) for 5 to 10 minutes.
9. Spray dried paper with ninhydrin solution.
10. Make the spots of separated amino acid and determine their
Rf values.
Result:
13
Experiment No.5-a
Aim: Detection of glucose from Urine
Principle: When Benedict’s quantitative reagent (5ml) is heated with
eight drops of urine (about 0.5 ml) glucose present in urine reduces
cupric ions present in the regent to cuprous ions. Alkaline medium is
provide to the cuprous ions. Alkaline medium is provided to the
reaction by sodium carbonate present in the reagent. The original
colour of Benedict’s reagent is blue. It changes to green, yellow,
orange and red according to the concentration of glucose present in
urine.
Requirements:
Chemicals
1. Benedict’s reagent
2. Urine sample
Apparatus:
1. Test tubes
2. Burner
3. Water bath
Procedure:
1. Take 5ml benedict’s solution ,Add 0.5 ml or 8 drops of urine
sample.
2. Boil in water bath for 5 min
3. Observe in color change.
Result:
14
Experiment No.5-b
Aim:
Detection of albumin in urine.
Principle:All the methods are based on the principle of precipitation
of protein by chemical agents (acids) or co-agulation by
heat. If the turbidity is due to phosphate precipitation, it
will clear.
RequirementsChemicals:Glacial acetic acid
Urine Sample
Appratus:Test Tubes
Burner
Procedure :
1.Take 5-10 ml of clear urine in test tube.
2.Boil the upper portion over flame.
3.If the turbidity develops , add 1-2 drops of glacial acedic acid.
4. The turbidity is due to phosphate precipitation, it will clear.
5.Reboil the specimen.
Observation :No formation turbidity at upper portion of urine indicates absence of
protein in urine.
Result:-
15
Experiment No:- 6
Aim: - Demonstration of different equipments and techniques used
in microbiology.
Common Glassware: The most glassware used in a microbiological
laboratory are: test tubes, culture tubes, Petri dishes, Erlenmeyer
flasks, measuring cylinder, pipettes, glass spreader, volumetric flasks,
screw-capped glass bottles, haemocytometer, etc.
I Test tube, culture tube and screw-capped tubes:
1.These are made up of glass, one-end of which is closed and the
other end open.
2. If the side wall of the open end is slightly curved out side, it is
called test tube; if the side wall is smooth, it is called culture
tube. When the side wall of the tube has screws so that a
plastic cap may be fitted, it is called screw-capped tube.
3. These are used in microbiological laboratory.
4. The test tubes are used for testing the chemicals such as pH,
etc.Culture tubes are used for preparation of agar slants and
purification of microorganisms. The open end is plugged with
non-absorbent cotton plug.
5. Some times the microorganisms are purified and preserved in
screw-capped tubes.
II Petri dish:1. It consists of two shallow glass dishes, the upper half or lid
and the lower half or bottom half.
2. For isolation and cultivation of different types of
microorganisms these dishes are used.
3. According to the requirement, its diameter varies.
4. Molten agar medium is aseptically poured on the bottom half
of the sterilized Petri dish and then covered with the upper
half.
5. The Petri dishes are sterilized by putting them in a
Petri dish container and in turn in an oven/autoclave.
6. Now, disposable sterile plastic Petri dishes are also available
for the same purpose.
III. Pipette
1. It is a cylindrical and graduated glass apparatus.
2. Its one end (lower side) tapers, while the other end (mouth
piece) is normal. The middle portion is wider or of the same size
as mouth end.
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3. It is graduated with numbers 1, 2…..10.
4. It has different measuring capacity such as 0.1, 0.5, 1, 5, 10ml,
etc. hence measures different quantities.
5. It is used for transferring appropriate amount of liquid in other
containers.
6. It should be sterilized in an oven/autoclave before use by
keeping in pipette container after being plugged with cotton.
7. For safety point, liquid should be sucked by attaching pipettesucker at the normal end of pipette.
8. Pipettes should be sterilized by keeping them first in a steal can
(steal container) the steal can then is sterilized at 121o C for 30
minutes.
IV Pasteur pipette
1. The Pasteur pipette can be made by selecting a hollow glass tube
of similar diameter as of standard pipette or graduated pipette of
which one end is heated so as to blow the glass to form a narrow
end similar to 10 ml pipette. On another end, a rubber bulb is
fitted.
2. Before being sterilized all the pipettes are usually plugged with
cotton in order to avoid contamination.
Pasteur pipettes are generally used once and then transferred
into disinfectant.
V. Erlenmeyer flasks
1.It has narrow beak at top with a opening and a broad
bottom.
2.The flasks of different sizes, hence measure different
volume such as 100, 250, 500, 1000, 2000ml liquid.
3.The flasks are of round bottom or flat bottom.
4.Some times the flasks are also graduated to represent
the volume of liquid.
5.Certain modifications are made in Erlenmeyer flask
according to requirement, for e.g. a beak is fabricated near the
neck of the flask to connect to other equipment with rubber
tubing. Such flasks are called side-arm flasks.
6.During sterilization cotton plug is inserted in the mouth of the
flask.
7.It must be sterilized before microbiological usage.
VI. Volumetric flasks
1. It is used to prepare solution of accurate strength.
17
2. Its upper part is cylindrical and narrow, and marked at a point.
This mark denotes the water level to be maintained at this
point.
3. The lower half is rounded and voluminous.
4. Its base is flat so that it may be properly placed on the
surface.
VII. Glass spreader
1. Glass spreader is made by bending a glass rod and
making a L-shaped structure.
2. It is used to spread evenly the microorganisms on agar
surface present in liquid medium.
3. The long arm is hold in hand and the small arm is flamesterilized and put on agar surface.
4. It is brought forth and back so that microorganisms present in
liquid may be dissociated and evenly spread on entire surface
of agar.
VIII. Haemocytometer
1. It is used to measure the blood cells.
2. It is also used for counting the other cells viz, spores,
bacteria, etc.
3. It
consists
of
big
squares.
Each
square
has
1*1*0.1mm=0.1mm
4. There are 25 medium-sized squares in each large square
where each medium squares is 0.2mm length, 0.2mmwidth
and 0.1mm depth with a volume of 0.04mm3.
18
INSTRUMENTS
I. Inoculation needle and inoculation loop.
1. These are the most commonly used tools.
2. Inoculation needle/loop is made up of a long platinum
wire fixed into a metallic rod.
3. A wire loop has a handle with steel screw shaft in which
nichrome or platinum wire is to be fitted.
4. The loop should be such so as to retain a small circular
film in it by dipping in solution (5-7cm).
5. The needle straight wire is used for transferring culture
from solid medium.
II. Waterbath.
1. It is an instrument that is used to provide constant
temperature.
2. Temperature is controlled through a thermostat.
3. It is more useful to microbiologist because it provides a
uniform heat to the sample material mean for incubation.
III. Autoclave.
1. The killing action of heat on the organisms can be done
by using increase in the steam in a closed system.
2. Autoclave usually of pressure-cooker type made up of
gun metal sheets which is supported in a iron case.
3. The steam passes from below at the base. The side
walls are heated by steam jacket.
4. It is based on moist heat that used in sterilization.
5.Autoclave is usually operated at 15lb. /inch2 steam
pressure for 30 minutes. This temperature for 30 minutes is
19
enough to kill all spores and cells of microorganism.
IV. Laminar air flow.
1.It is an apparatus consist of an air blower in the rare side of
the chamber. There is a special filter system of highefficiency
particulate air filter (HEPA) which can remove as
0.3mn.Inside the chamber one florescent tube and the other
UV tube are fitted. Switch on the UV light for 30 minutes
before using it.
2.Due to uniform velocity and parallel flow of air current,
pouring of media, plating, slant preparation, etc, without any
kind of contamination are performed.
V Incubators.
1. It is an instrument that consists of copper/ steel chamber,
around which warm water or air is circulated by electric current
or by means of small gas flame.
2. It is generally design that can allow the desired micro organism
to grow at particular temperature.
I.
Hot air oven.
3. It is generally used for sterilization of glass ware, metal
20
devices and other articles which are spoiled by autoclaving.
For such purposes dry heat sterilization is used.It kills the
microbes by oxidizing there chemical constituent.
4.There is an in-built thermostat when required, it help in
regulating the temperature.
5.For sterilization, if the temperature of oven is 160o, the
holding time should be one hour but at 180oit should be 30
minutes.
6.The glass material should be wipe and dried before keeping
inside the chamber in the oven.
VI. pH meter
1. pH can be defined as a negative log of hydrogen ions
concentration pH=-log10(H+)=7.
2. pH is the degree of acidity and alkalinity of a solution on a scale
1 to 14.
3. pH values 1 to 7 show the acid values, pH 7 neutrality and pH 7
to14 alkalinity. pH of water is 7at 25o C.
4. The acid is proton donor and base is proton accepter i.e. acid
dissociates and produces hydrogen ion concentration(H+)
5. Maintenance of pH values is an important parameter for the
growth and process of any organism.
6. A standard ph meter has two electrodes, one glass electrodes
and second mercury-mercurous chloride (calomel) or silver-silver
chloride reference electrode. The reference electrode is emerge
in saturated KCl solution.
21
VII Water Distillation Unit - Metal (Wall Type)
Water distillers produce highly treated and disinfected water for
laboratory usage. The distillation process removes minerals and
microbiological contaminants and can reduce levels of chemical
contaminants.
Water distillers are neither intended to treat water that is visually
contaminated nor intended to convert waste water to safe,
microbiologically
Working Principle
A water distiller works by boiling water into water vapour, condensing
it and then returning it to its liquid state. It is collected in a storage
container.
The process occurs in several steps:
1. Municipal or well water is manually or automatically fed into
the distiller unit’s boiling chamber.
2. A heating element in the boiling chamber heats the water until it
boils.
3. The steam rises from the boiling chamber. Volatile contaminants
(gases) are discharged through a built-in vent. Minerals and salts are
retained in the boiling chamber as hard deposits or scale.
4. The steam enters a coiled tube (condenser), which is cooled by cool
water.
5. Water droplets form as condensation occurs.
22
6. The distilled water is collected in a storage tank. If the unit is an
automatic model, it is set to operate to fill the storage tank.
Construction:
Made of heavy gauge stainless sheet inside and outside, with
immersion type heaters. The still provides continuous supply of
pyroxene free distilled water and is equipped with brackets for wall
mounting plug and cord and connector.
Output 2 ltr/hour
Output 4 ltr/Hour
Output 6 ltr/Hour
Power Requirements: To work on 220/230Volts, 50 Hz single phase AC
supply.
VIII Balance.
1. There are various types of balance used for weighing such as
single pan, chemical or analytical and electric balances.
2. These balances are used according to requirement and the
amount of the materials to be weighed.
23
IX Colorimeter
1. It is an instrument that utilizes light as a source of radiation and
measures changes in optical density or absorbance.
2. It has three basic principals (i) The source of radiation (ii) a unit
for dispersing the radiation at different wavelengths, (iii) a
device that detect the amount of radiation at different
wavelengths.
3. Spectrophotometer uses monochromatic (narrow wavelengths)
radiation, whereas colorimeter uses broad wavelength bands.
4. For quantitative estimation the absorbance of different
concentration of a substance can be measured following Beer’s
law. The mathematical expression of Beer’s law is Log Io/I=Kc.
Where,
Io= Original intensity of light beam
I = Intensity of beam passing through a solution
C = Concentration of the solution in moles/I
K = Constant A plot of absorbance (Y-axis) versus
concentration (X-axis) yields a calibration curve. This shows the
range of Beer’s law. It may be used for quantitative estimation.
X Centrifuges
1. Centrifuges are the instruments which operate through the
centrifugation technique. Centrifugation technique is based on
molecular mass, shape and density of the particles. The rate or
velocity at which particles sediment is proportional to force rate
and velocity of sedimentation applied forces. The apply of larger
force tan does the earth’s gravitational field, thus increase the
rate of sedimentation of particles. The different particles
sediment separately due to variation in their density, shape or
size.
2. Principle of sedimentation: The centrifugal force is applied which
induces the gravitational force of the materials present in a
solution. The centrifugal force acts in a direction away from the
centre of the axis. If the speed of rotation is faster, the force will
be higher accordingly. However, the rate of sedimentation
depends upon the applied centrifugal field (G). The G is the
function of square of the angular velocity (radians/sec.) of the
rotor and the radial distance (r, in cm) of the particles present
from the axis of rotation.
G=ω2 r.
3. Types of centrifuge :
i.
Small bench centrifuge.
ii.
Refrigerated centrifuge of large capacity.
24
iii.
iv.
High- speed refrigerated centrifuge.
Ultracentrifuge.
XI Microscope
1. For studying basic and applied microbiology, microscope holds
an important place among all the tools required in a laboratory.
The tiny microorganisms are visible only under microscope that
are invisible to the naked eyes. Some are even colourless but
some microscope with phase contrast attachment tends to
visualize them in high resolution. The basic microscope consists
of eyepiece (10Xmagnification) that is fitted inot the microscope
tube. The magnification varies in ocular eyepiece. For
identification, another low power lenses called objectives are
used which have varying power (10X, 40X).For high
magnification an oil immersion (100X) is also used. The oil
immersion objective may bring the objectives close to bring in
focus when rotated by the nosepiece carrier. The fine details are
easily visible by oil immersion (100X) objective. The other part
of microscope is stage with a mechanical holding device or
simple clips. It has an iris diaphragm and a filter that is moved
below the objective lens. The condenser consists of aplanatic
lenses, has a numerical aperture of 1.3 to1.4 under high
resolution. The microscope has light source (lamp) either built in
or externally separately movable device located under the
condenser, if fixed in position, directs the light up by tilting. The
use of lamp with a lens projects sharp image resulting in
improvement in observing the details of microorganism.
25
26
EXPERIMENT NO.7
Aim – I. Preparation of nutrient slant and broth.
Principle:-When the autoclaved agar medium is poured in to the
culture tubes and the later is placed in slanting positions, it gives agar
slants after solidification.
Requirements
1.Agar Medium (e.g. PDA(Potato dextrose agar), Nutrient agar
Medium.)
2.Culture tubes
3.Test tube stand.
4. Aluminum foil or paper
Procedure
1. Prepare nutrient medium as follows,
Peptone
5.0 grm
Beef extract
3.0 grm
NaCl
5.0 grm
Agar
15.0grm
Distilled water
1000ml
pH
7 - 7.4
2.Dispense 8- 10 ml of medium into each tube and put cotton
plug and tie it with a rubber band. The amount of medium may
be increased or decreased according to the volume of culture
tubes.
3.Transfer all the tubes in to the a test tube stand or iron basket
and autoclave at 121º C for 20 minutes.
4.Take out the culture tubes when temperature cools down and
place them in slanting position by giving a support .
Wait for about 30 minutes.
5.Thereafter, medium is solidified and agar slants are prepared.
6.Use the slants for culture transfer if required or store them for
further use.
II. Preparation of nutrient broth
Principle : Bacteria are generally cultivated in broth ,i.e the medium
devoid of agar. In fact requirement of nutrients is met by
27
supplementing beef extract(which is a source of mineral salts, organic
carbon and nitrogen,vitamins,etc) and peptone (which is semi-digested
protein)..
Requirements
HCL
1N
NaOH
1N
pH meter
Distilled water
Autoclave
Culture tubes
Glass rod
Beaker
Measuring cylinder
Method of preparation of nutrient broth is as follows.
Peptone
5.0 gm
Beef extract
3.0 gm
NaCl
5.0 gm
Distilled water
1000ml
pH
7.0
Procedure
1.Accurately weigh the chemical ingredients of the nutrient broth
and transfer them into a beaker containing 500ml distilled
water.
2.Dissolve the ingredients properly.
3.Add distilled water to make the volume to 1000ml.
4.Measure pH of the broth by using a pH meter and adjust the pH
to 7.0 by adding drops of either HCL or NaOH solution.
5.Dispense 10ml to each culture tubes.
6.Put cotton plugs.
7. Transfer all the tubes in to the a test tube stand or iron basket
and autoclave at 121º C for 20 minutes.
8. Take out the culture tubes when temperature cools down.
9. Use the broth tube when required or store them for further use.
Result:
28
Experiment no.8
Aim : Preparation of cotton plugs.
Microorganism are ubiquitous in distribution. It is difficult to study a
particular type of microbe unless we know their ecological nature. The
anaerobic bacteria do not require oxygen, while aerobic require ,and
micro-aerophiles require but in a very low concentration .Therefore we
need cotton plugs to create aerobic environment and making the
growing culture free from undesired microbial contaminant. They are
adhered on the surface of cotton fibres and therefore, prevented to
enter inside the flasks, tubes, etc. Air facilitates the growth of
microorganisms in glass apparatus.
Requirements
Non – absorbent cotton rolls
Culture tubes
Flasks
Scissors
Procedure
1.Procure a non absorbent cotton roll and remove its paking.
2.Open the roll as per requirement
3.Cut a small piece of cotton sheet with the help of scissors.
Based on the requirement the following two types of cotton plugs
are prepared.
I. Cut, fold and rolled plug
1.Cut a small piece of cotton sheet with the help of scissors as
per requirement. i.e flask/culture tube, etc
2.Fold the cut piece from the centre.
3.Roll the sheet from any of ends. Opposite to the folded side
has cotton fibres that emerge out freely.
4.Insert the folded and rolled side in the neck/mouth of a
flask/culture tube, and the other end of plug bears cotton
fibres.
5.If it is tight, open the plug and remove some cotton from one
side and readjust as earlier.
II.Cut, rolled and fold plug
1. Cut a small piece of cotton sheet with the help of scissors.
29
2. Roll the piece from one end.
3. Fold the rolled piece from the centre.
4. Insert the folded end in the neck/mouth of a flask/culture tube,
and the other end bearing cotton fibres out in air.
Result:-
30
Experiment no.9
Aim:-Isolation of single colony on solid media: Streak plate and
Spread plate method.
Principle:-Generally, bacteria exist in mixed population. It is very rare
to get a single and pure form. For studying the cultural, morphological,
and physiological characters of an individual species, it is essential to
separate them from the others to get in the form called pure
culture.There are many important methods for isolating pure culture
from mixed culture.
I.
Streak Plate Method from Mixed Culture to Pure
Culture.
The colonies on a mixed plate are separated by spreading
on the plate with good spacing among each other using
streak plate method.
Requirements
Tripod and wire gauze
Bunsen burner
Beaker of water
Wire loop
Nutrient agar pour
Sterile Petri dish
Mixed culture.
Procedure
1. Liquefy a tube of nutrient agar and pour into the Petri dish,
rotate the plate gently for uniform distribution of the medium.
2. Streak the plate following quadrant or radiant or T-streak or
continuous streak.
3. Keep the streaked plates in inverted position at 250 c for
24-48 hrs.
4. Place the Petri dishes upside down to solve the problem of
water condensation because if it drops down on the
colonies, the organisms of one colony can spread on the
other colony.
Result
31
II.
Spread Plate Method
When inoculum is transferred into Petri dishes containing
nutrient medium cells are not separated from each other.
Therefore,there develop mixed colonies. Hence, isolation of pure
culture from mixed colonies is rather difficult. Therefore, spread
technique is employed. In this technique the propagules of
microorganism are spread over solidified agar medium with the help of
L-shaped glass tube called spreader when the Petri plate is spinning on
a turn table.
Requirements
Nutrient broth culture of streptococcus pneumonae and
Staphylococcus aureus
Nurient agar plates
Turn table
L-shaped bent glass
Ethanol(95%)
Bunsen burner
Beaker
Incubator
Inoculation loop.
Procedure
1.Take three nutrient agar plates and label them 1,2 and 3
respectively.
2.Aseptically, innoculate plate 1 with a loopful culture of
S.pneumonae, plate 2 with S.aureus and plate 3 with both
S.pneumonae and S.aureus.
3. Place plate 1 on turn table (revolution platform).
4. Sterilize the bent L-form glass rod by putting it first in ethanol
(95%)in a beaker, then on the flame of Bunsen burner and cool
the rod for 30 second.
5. Remove the lid of petri plate and spin the turn table.
6. Very gently touch the bent L-form of the glass tube on the
surface of agar and move it forth and back to spread bacterial
cells on agar surface when the turn table is spinning.
7. When turn table stops running, put the lid over the lower
half of petri dish.
8. Sterilise the L-shaped glass rod as in step 4.
9. Repeat steps 3 to 7 for plate 2 and 3 also.
10. Incubate all the plates at 250C for 24 hrs.
32
Result:-
33
Experiment No 10.
Aim:- Enumeration of bacteria by Breed Method and Pour plate
method.
Breed Method
Principle :-Term growth as commonly employed in bacteriology refers
to the magnitude of total population .growth can be determine by
various techniques one such technique is by determining the number
of cells .
Breed’s method is a direct method where the microscopic cells are
counted. In Breed’s method both viable as well as dead cells are
counted .
Requirements:
Bacterial culture
Glass slides
Methylene blue
Pour Plate Method
Principle:-The pour plate method is based on diluting the mixed
culture with liquefied nutrient agar in such a manner that the colonies
formed on the plate are countable. Generally,a loopful of bacteria is
inoculated into tube I and dilute into tube II and tube III. This method
has an advantage over the streak plate method as it does not require
much skill, but it has disadvantage due to the reasons that amount of
media and glassware are more.
Requirements:Mixed bacterial cultures
Nutrient agar
Petri plates
Hot plate
Beaker of water
Inoculation loop
Glass marker.
Procedure:1.Inoculate tube I with a loopful of bacterial and after thorough
mixing and transfer a loopful of bacterial in tube II. Shake the
tube vigorously between palms and transfer the loopful of
culture from tube II to tube III.
2.Note that all the three tubes contain liquefied nutrient agar and
are placed on a water beaker which are further kept on hot
34
plate.
3.Pour its content separately on the three plates. After
solidifying the medium, incubate at 250C for 24 hrs in inverted
position in an incubator.
4.Evaluate the plates for obtaining pure culture.
Result:-
35
Experiment No 11.
Aim:- Staining Techniques :Simple Staining, Gram Staining, Acid fast
Staining, Endospore Staining and Viable Count.
I.Simple Staining:Principle: This technique is recommended to study morphology and
arrangement of bacterial cells. When a single dye is used, the process
is referred to as ‘simple staining’ or ‘monochrome staining’ since only
one staining solution is employed for colorization of bacterial smear. In
this case, basic stains with positively charged chroma gen (dye) binds
with negatively charged cell wall components (slightly acidic in
nature). Carbol fuchsin, crystal violet and methylene blue are
commonly employed for simple staining.
Requirements:
Inoculating loop
Bunsen burner
Staining tray
Microscope
Glass slide
Methylene blue (0.5%)
Crystal violet (1%)
Carbol fuchsin (diluted 10 times before use)
Procedure:1.Fix the smear on a slide by gentle warming over the flame of
spirit lamp.
2.Flood the smear with a solution of a basic dye such as carbol
fuchsin, crystal violet 2-60 seconds and methylene blue stains
within 1-2 minutes.
3.Then gently rinse tap water and dry with blotting paper.
4.Examine the smear under oil immersion objective.
Result:-
36
II.Gram Staining:Principle:-This is the most important differential technique used in
bacteriology. There are two groups, Gram-positive and Gram-negative
bacterial. The smear on the slide is prepared, stained with crystal
violet and then treated with iodine solution as a mordant The crystal
violet-iodine complex impart purple-black colour to the cells. In Grampositive cells this complex binds to the magnesium-ribonucleic acid
component of the cell wall, forming complex which is difficult to
remove. This serves as a lipid solvent and a dehydrating agent for
protein. The Gram-positive bacteria contains low lipid content, hence
the low amount of lipid is easily dissolved by alcohol. This makes
minute pores in the cell wall that are closed by dehydration effect of
alcohol.
In Gram-negative cells, large pores are formed that do not close
by dehydration of cell wall protein does not occur completely.This
facilitates the release of the unbound crystal violet complex leaving the
cell colourless or unstained. If the smear is counter stained with
safranine, the Gram-negative cells are easily seen due to absorption of
safranine and imparting the cells red colour; while Gram-positive cells
retain the blue colour of the primary stain.
Requirements:Bacterial slant culture
Crystal violet
Gram’s iodine
Ethyl alcohol
37
Safranine
Bunsen burner
Inoculating needle
Staining tray
Glass slide
Microscope
Procedure:1.Prepare a bacterial smear and heat fixed on the slide using
standard procedure. Pour a few drops of crystal violet on the
smear.
2.Wait for 1 minute and wash with tap water.Now, flood the
smear with Gram’s iodine for 1 minute and again wash with
tap water.
3.Decolourise the stain with ethyl alcohol (95%) by dropping
the reagent drop-wise until crystal violet fails to wash from
the smear.
4.Wash it with tap water and counter stain with safranine for 45
seconds and wash again with water.
5.After drying, examine under oil immersion.
Result:-
III Acid Fast Staining:Principle:-Certain bacterial and actinomycetes have waxy
components of the cell wall, hence their cell wall has limited
permeability. Majority of bacterial are stained with simple satin and
Gram stain but the members of the genus Mycobacterium can only be
stained by acid-fast stain. Once the stain enters inside the cell, it
cannot be readily removed even by acid alcohol decolorising agent.
Acid-fast staining is widely used as differential staining procedure in
bacteriology. However, some bacteria resist colourisation by both acid
and alcohol, and hence are referred to as acid-fast organisms.
Ziehl-Neelsen Staining method:- In Mycobacterium and some
38
species of Nocardia, the acid-fastness property is correlated with their
high lipid contents.Hence for staining of these bacteria heating with
strong dye is required. Once the bacteria are stained, it is difficult to
decolourise them even with acid and alcohol.Non-acid-fast bacteria
lose primary dye and take counter stain.
Requirements:Tryticase soy broth for Mycobacterium culture
Inoculating loop
Bunsen burner
Staining tray
Microscope
Glass slide
Decolourising solvent
Methylene blue
Carbol fuchsin
The reagents are prepared as given below:
Carbol fuchsin stain:
Basic fuchsin
0.3 gm
Ethanol(95%)
10 ml
Phenol (heat melted crystals)
5 ml
The basic fuchsin dissolved in ethanol, and then in phenol in water.
Mix them and keep for several days. Filter it before use.
Decolourising Solvent:
Ethanol (95%)
Hydrochloric acid (conc.)
Counter Stain:
Methylene blue chloride
Distilled water
97 ml
3 ml
0.3 gm
100 ml
Procedure:1.Place the slide with an air-dried and heat-fixed smear on a hot
plate until the steam rises with carbol fuchsin stained smear.
Keep the preparation moist with stain and steaming for 5 min.
Don’t boil it.
2.Wash the film with gentle stream of water until no colour
appears from effluent.
3.Decolourise with HCL (3%) and immediately wash with
tap water. Stain should appears faintly pink.
39
4.Now, counter stain with malachite green (1%) for 20-30
seconds and wash with water as before.
Result:-
IV Endospore Staining:Principle:-Endospores are the dormant structures formed in various
bacteria under unfavourable conditions. Their size is irregular and
highly refractile and heat-resistant. The differentiation between spore
and vegetative cell is made possible. Most of the anaerobes and
members of bacilli group of bacteria form spores under unfavourable
conditions.When the favourable conditions begin, spore starts
germination and gives rise a new vegetative cell.
Procedure
1. Using aseptic technique, prepare a bacterial smear on a clean
slide,
air dry and gently heat fix.
2. Prepare a boiling water bath.
3. Cover the slide with a piece of paper towel, and place on a
staining
40
rack over the water bath.
4. Flood the paper towel on the slide with Malachite green (primary
stain).
5. Steam the slide for five minutes.
6. Remove the slide from the water bath, and remove the paper
towel
from the slide.
7. Allow the slide to cool, and then rinse with deionized water until
the
water runs clear.
8. Pour off any excess water and apply Safranin (counterstain) for
two
minutes.
9. Rinse excess Safranin off with deionized water, and blot the
slide dry
with paper.
10. Examine the slide with a light microscope under oil immersion.
The vegetative cells
are pink/red and the endospores are green.
41
Experiment No.12
Aim : Demonstration of presence of α - amylase in saliva.
Principle :Starch is made up of maltose unit which is hydrolysed by
enzyme amylase. Amylase is an extracellular hydrolytic enzyme it
breaks the 1,4 glycosidic linkages in starch and convert it in to dextrin
and finally in to maltose. α – amylase secreated by Aspergillus oryzae
and amylase is secreted by Bacillus subtilis.
The reduced products are determined by 3,5 dinitro salicylic acid
(DNS). The concentration of reduced DNS form by the amount of
starch hydrolyzed is directly proportional to enzyme activity .It turns
solution from yellow to brick red color which can be determined by
using colorimeter.
Protocol:Reagent
Phosphate Buffer (pH
6.7,0.1N)
Starch solution
NaCl, 1%
1
2.5
2
2.5
3
2.5
4
2.5
5
2.5
2.5
1.0
--1.0
---1.0
2.5
1.0
2.5
1.0
Mix well and keep tubes for 10 min at 370c
Water
1.0
1.0
0.5
0.5
Diluted saliva
-----0.5
0.5
0.5
0.5
Prepare tubes in duplicate
Immediately after the addition of saliva and diluting. Add 0.5ml
2N NaOH to tube 5 to stop the reaction. This is called the ‘Zero’
time control. The rest of the tubes are incubated at 370c for 15
minutes, at the end of which the reaction is stopped by the
addition of 0.5 ml 2N NaOH.
Now add 0.5ml of dinitrosalicylic acid reagent mix,well and heat
the tubes in a boiling water bath for 5 minutes .cool the tubes to
room temperature and measure their O.D at 520nm using tube 1
as blank. This does not contain any enzyme. Tube 2 is also a
control since this does not contain the substrate or the enzyme
source. Tube 3 contains the enzyme but not the substrate and any
reading in tube is due to contamination from the enzyme source. This
reading has to be subtracted from the value of readings for tubes 4
and 5 comparing tubes 4 and 5 ,it is seen that they contain complete
mixture. Expect that the reaction in 5 has been stopped immediately
on addition of enzyme .In other word it is the zero time control. So,
that amount of maltose formed in 15 min by the amount of saliva
added is :
42
(O.D. of tube 4 – O.D. of tube 3) – (O.D. of tube 5-O.D. of tube 3)
From this std .graph of maltose, one can calculate the corresponding
amount of maltose formed per ml of saliva.
Result:-
43
Experiment No.13
Aim:-The Determination of Blood Glucose by Nelson
Somogyi Method
Reagents-Analytical reagent grade or the equivalent.
1. Copper Reagent A.
Dissolve 25 gm. of Na2C03 (anhydrous),
25 gm. of Rochelle salt,
20 gm. of NaHCO3, and
200 gm. of Na2SO4 (anhydrous) in about 800 ml. of water and
dilute to 1 liter. Filter if necessary.
This solution should be stored where the temperature will not fall
below 200. Sediment may form after a few days. This may be filtered
off without detriment to the reagent.
2. Copper Reagent B.
15% CuSO4.5H2O containing one or two drops of concentrated sulfuric
acid per 100 ml.
3. Arsenomolybdate color reagent.
Dissolve 25 gm. of ammonium molybdate in 450 ml. of distilled water,
add 21 ml. of concentrated H2SO4 mix, add 3 gm. of Na2HAsO4.7H20
dissolved in 25 ml. of H2O, mix, and place in an incubator at 37” for
24 to 48 hours. If a reagent is needed quickly, an alternative
procedure is to heat to 55” for about 25 minutes. However, stirring
must be adequate to prevent local overheating; otherwise
decomposition of the chromogen may occur. This is accompanied by
the precipitation of a bright yellow compound. The first procedure has
been uniformly successful and is the recommended one; the second is
inconvenient, and with certain preparations of sodium arsenate
yields reagents which, though useful, are inferior in potential color
development to those prepared by the first procedure. This reagent
should be
stored in a glass-stoppered brown bottle.2
4. 5 per cent ZnS04.6H2O.
5. Approximately 0.3 N Ba(OH)2. The zinc and barium solutions should
be adjusted so that 5 ml. of zinc require between 4.7 and 4.8 ml. of
barium to produce a definite pink to phenolphthalein. The zinc should
be diluted to 20 or 25 ml. with H20 and the Ba(OH)2 added dropwise
with constant mixing during the titration. Store in a bottle protected by
soda lime from the carbon dioxide of the air. It is convenient to have
44
an arrangement for direct delivery of the Ba(OH) 2 into a 5 ml. burette
graduated to 0.01 or 0.02 ml.
Procedure
The blood filtrates are prepared as follows:1.Add 1 volume of blood to 15 volumes of water, mix, add 2
volumes of Ba(OH)2, mix, and after the mixture has turned
brown add 2 volumes of ZnSO4 and mix. After a few minutes
the mixture may be filtered; somewhat more filtrate may be
secured by a preliminary centrifugation.
2.For finger-tip blood, one may wash 0.1 ml. Of blood from a
pipette calibrated “to contain” into 1.5 ml. of water contained
in a small vial or test-tube. After 0.2 ml. each of Ba(OH)2 and
ZnS04 is added. the mixture is centrifuged. The filtrate is then
drawn into a 1 ml. pipette tipped with washed cotton.
3.1ml. of filtrate is pipetted into a narrow test-tube graduated at
25 ml.
4.1ml. of a mixture (prepared the day of use) of 25 parts of
Reagent A to 1 part of Reagent B is added. Since this latter
volume is not critical, a burette or measuring pipette may be
used. 1 ml. portions of appropriate standards and 1 ml. of
distilled water, to serve as a blank, are set up in the same way.
The solutions are mixed and heated for 20 minutes in a boiling
water bath. At the end of 20 minutes the tubes are cooled in a
pan of cold water. 1 ml. of the arsenomolybdate reagent is then
added to each; a measuring pipette is convenient and adequate
for this measurement. The color develops very rapidly and will
be completed by the time thorough mixing and evolution of CO2
are completed. The mixture is then diluted to the mark, mixed,
and read in a photoelectric calorimeter at 500 or 520 mµ. The
photometer is adjusted so as to read 100 per cent transmission
through the blank. The color is very stable and may therefore be
read at convenience. The stability of the color is absolute and
not relative; the density of the blanks as well as of the more
deeply colored solutions remains unchanged with time.
{The deproteinieation procedure given is one suggested to us by Dr.
Somogyi several years ago. It has been well known (Benedict Somogyi
that the ZnSO4-NaOH deproteinization procedure leaves a small
amount of Zn remaining in the filtrate and that this trace apparently
accelerates the reoxidation of cuprous oxide. The extent of this is
small and relatively unimportant in a macro blood sugar
estimation, but decidedly significant in a microdetermination. We had
encountered this difficulty and devised a procedure similar to but more
45
cumbersome than the one Somogyi was using to overcome the same
difficulty. We have found his method a
very useful technique for various purposes, since it has the advantage
of yielding a filtrate practically free of the deproteinizing reagents.
(The high Na2S04 concentration of the reagent gives adequate
protection against reoxidation for most purposes; so that neither the
constricted Folin-Wu tube norcovered tubes are essential.)}
46
Experiment No.:14
Aim:- RBC Count
Introduction
The red blood cell count (RBC cont) is important in diagnostic
hematology. It permits the MCV and MCH values to be calculated.
The manual method of RBC count is time consuming and imprecise.
The reference methods preferred for RBC count is an automated
method
Normal valuesMale : 4.5to6.0 x106 cell/ cu mm (u1).
Female : 4.0 to4.5 x 106 cell/ cu mm (u1).
47
A hemocytometer. The two semi-reflective rectangles are the
counting chambers.
Principle:The blood specimen is diluted 1.200 with the RBC diluting fluid and
cells are counted under high power (40 x Objective) by usine a
counting chmber. The number of cells in undiluted blood are
calculated and reported as the number of red cells per cu mm (ul) of
whole blood .
Specimens
1.Double oxalated or EDTA blood or
2.Capillary blood
(The specimen need not be a fastine sample)
Requirements
1. Microscope
2. Improved Neubauer Chamber
3. RBC pipette
4. RBC diluting fluid
It is prepared as follows
a) Sodium citrated
: 3.0g
b) Formalin
:1.0ml
c) Distilled water to
: 100ml
This solutions is stables at room temperature
(250C ± 50C) for at least one year.
Procedure
1. Mix the anticoagulated blood care fully by swirling the
bulb.
2. In the case of capillary blood the lancet stab should be
Sufficiently deep to allow free flow of blood. It is drawn
quickly in the RBC pipette.
3. Draw blood up to 0.5 mark.The pipette by using cotton or a
gauze.Draw diluting fluid up to 101mark
4. The pipette in rotated rapidly by keeping it horizontal
during mixing.
5. After five minted, by discarding few drops from the pipette
and holding it slightly inclined small volume of the fluid is
introduced under the cover slip which is placed on the
counting chamber.
6. Allow the cells to settle for 2to 3 minutes.
7. Place the counting chamber on the stage of the microscope.
8. Switch to low power (10x) objective. Adjust light and
located the large square in the center with 25 small squares.
48
9. Now switch to high power (40x) objective.
10. The red blood cells in the four corner squares and in the
center squar (marked in the diagram as ‘R’) are counted.
11. Use following formulae for the calculation of red blood
cells.
Total red blood cells per liter of blood
=RBCs/ cumm (u1) x106
Or use following formulaRed cell count (per liter)
=No. of cell counted
Volume counted (u1)
x Dilution x 106
Total red blood cells counted x Dilution
Area counted x Depth of fluid
Where (1) dilution = 1.200 (i.e,200)
(2) Area counted = 80
400
═ 1
Sq. mm.
5
Result
49
Experiment No 15
Aim: WBC Count.
Principle:- The glacial acetic acid lyses the red cells while the gentian
violet slightly stain the nuclei of the leukocyte. The blood specimen is
diluted 1:20 in WBC pipette with the diluting fluid and the cells are
counted under low power of microscope by using a counting chamber.
The no. of cells in undiluted blood are reported per cu mm(ul) of whole
blood.
Clinical Significance:
Increase in total leukocyte count of more than 10000/cu mm(ul) is
known as leukocytosis and decrease of less than 4000/cu mm(ul) as
leucopenia.
Normal Values:
Adults
At birth
1-3 yrs
4-7 yrs
8-12 yrs
4000-10000/cu mm(ul)
10000-25000/cu mm(ul)
6000-18000/cu mm(ul)
6000-15000/cu mm(ul)
4500-13500/cu mm(ul)
Specimen:
Double oxalated EDTA Blood
Capillary Blood (Specimen need not be a fasting sample)
Requirements:
Microscope
Improved Neubaur’s chamber
WBC Pipette
WBC Diluting Fluid; it is prepared as follows:
a) Glacial acetic acid 2ml
b) 1% w/v Gentain violet 1ml
c) Distilled water
97ml
This solution is stable at room temperature. Thymol may added as
preservative.
Procedure
1. Draw blood upto 0.5 mark of a WBC pipette.
2. Carefully wipe excess blood by using cotton.
3. Draw diluting fliud upto 11 mark.
4. Mix the content in a pipette and after 5 min by discarding few
50
drops,fill the counter chamber and allow the cells to settle for 23 mins.
5.Focus on W-marked areas (each having 16 small squares) by
turning objectives to low power(10X).
6.Count the cells in all 4 W-marked chambers.
Calculations:
No.of White cells/cu mm(ul) of whole blood
=No. of white cells counted X dilution
Area counted X depth of fluid
Where, Dilution=20
Area counted=4X1 sq mm=4sq mm
Depth of fluid = 0.1mm (constant)
Therefore,Whole blood=No. of cells counted X 20
4X 0.1
=No of cell counted X 50
Result
51
Experiment No.16
Aim: Measurement of blood pressure by spygmonameter.
Principle: when an external pressure is appliaed over the artery, the
blood flow through it is obstructed.and the pressure required to cause
occlusion of blood flow indicates the pressure inside the vessel.
Requirements:
Apparatus :
Sphygmonometer
Stethoscope
Procedure :
1.Brachial artery is usually chosen because of convinence.
2.The arm cuff is tied around upper arm above the cubital fossa.
3.The cuff should not be to tight or loose. The cuff is attached to
Sphygmonometer .
4.First the radial pulse is felt. While feeling the pulse, the
pressure is increased in the cuff by inflating air in to it,with
the help of a hand pump.
5.While doing this, the mercury column in the Sphygmonometer
shows the pressure of the cuff.
6.When the radial pulse disappears, the pressure is further
increased by about 20nm Hg.
7.Then ,the pressure in the cuff is slowly reduced by releasing
the valve of the hand pump. This is done by feeling the pulse
and simultaneously watching the mercury column.
8.Pressure is noted when the pulse reappears, this indicates the
systolic pressure.
9.After determine the systolic pressure , the pressure in the cuff
is raised by about 120 mm Hg. Above that level so that, the
brachial artery is occluded due to compression. Now the
stethoscope is placed over the cubita fossa, the pressure of the
cuff is released .
10.While doing so the series of sound are heard in four phases
through the stethoscope .
11.First phase:-While decreasing the pressure from the arm cuff,
the occlusion of the artery is relived and when the blood flows
through the artery the first sound appears suddenly it indicates
the systolic pressure. it slowly becomes loader when the
pressure is reduced.
12.Second phase:- After the taping sound a murmuring sound is
heard when the pressure id further by about 15 mm Hg.
52
13.Third phase:- After murmuring sound ,a very clear and loader
sound is heard. This is of gong type. it is heard while reducing
pressure by another 15mm Hg.
14.Fourth phase:- After the gong type sound ,a mild and muffled
sound is heard till the pressure falls further by 5mm Hg
It disappears afterwards .this disappearance of sound
indicates diastolic pressure.
Result:
53
Experiment No: 17
Aim- Protein estimation by Lowry’s method.
Principle-The principle of this method is based on the facts that the
Folin-Ciocalteu regents reacts with aromatic residues of proteins and
yields blue color which in turn is read in colorimeter. The different
proteins contain different aromatic residues. Blue color develops
because the alkaline copper reacts with proteins; tyrosin and
tryptophan present in protein reduce phosphomolybdate.(present in
Folin-Ciocalteu reagent)
Requirements1. 1N NaOH Solution.
2. ALKALINE SODIUM CARBONATE Solution-Dissolve
20gm of Na2 CO3 in 100 ml of 0.1N NaoH solution, prepare
fresh.
3.Copper Sulphate-Sodium Potassium Tartarate SolutionDissolve 0.5g/lt of CuSo4.5H2o in 1% OF sod-pot. tartrate
solution, prepare fresh.
4.Alkaline Copper Reagent- Mix 50ml of reagent 2 and 1ml of
reagent 3 only on the day of use.
5.Folin-Ciocalteau Reagent-It is a solution of Sodium tungstate
and Sodium molybdate in phosphoric and hydrochloric acids
(available commercially).Dilute the commercial reagent with an
equal volume of distilled water only on the day of use.
Procedure:a) Transfer1N NaoH solution in a test tube and heat up to 100oC.
b) Suspend 1 ml of protein sample into the above solution for 4-5
minutes.
c) Add 5ml of reagent 4, mix properly and leave this mixture at
room temperature for 10 minutes.
d) Add 0.5 ml of Folin-Ciocaltue reagent rapidly with immediate
mixing.
e) Leave it for 30 minutes; thereafter measure the absorbance of
solution at750 nm in the colorimeter.
54
Protocol:-
Sr
No
Stock of Distille Concentration
protein d water of protein
(ml)
(ml)
Alkaline Fcr
Reagent (ml)
1
0.0
1.0
0
5ml
0.5
2
0.1
0.9
0.02
5ml
0.5
3
0.2
0.8
0.04
5ml
0.5
4
0.3
0.7
0.06
5ml
0.5
5
0.4
0.6
0.08
5ml
0.5
6
0.5
0.5
0.1
5ml
0.5
7
0.6
0.4
0.12
5ml
0.5
8
0.7
0.3
0.14
5ml
0.5
9
0.8
0.2
0.16
5ml
0.5
10
0.9
0.1
0.18
5ml
0.5
11
10
0.0
0.2
5ml
0.5
12
Unknown1 -
-
5ml
0.5
13
Unknown2 -
-
5ml
0.5
O.D
Result- Concentration of protein in unknown sample was found to be
–
Unknown 1 – mg/ml
Unknown 2 – mg/ml
55
Experiment No:-18
Aim:- To determine acid value of fats.
Principle:-During storage, fats may become rancid as a result of
peroxide formation at the double bonds by atmospheric oxygen and
hydrolysis by micro-organisms with the liberation of free acid.The
amount of free acid present therefore gives an indication of the age
and quantity of the fat.
It is suggested that each pair of students choose one lipid and
assay(a) of fresh sample and (b) one that has been exposed to
atmosphere.Results should then be compared with other groups who
have determined other lipids.
The acid value is the number of milligrams of KOH required to
neutralize the free fatty acid present in 1gm of fat.
Materials:1. Olive oil, butter and margarine (use a fresh sample
and one that has stood for several days at room
temperature)- 200gms.
2. Fat solvent (equal volumes of 95% v/v alcohol and
ether neutralized to phenolphthalein)- 6 litres.
3. Phenolphthalein (10gm/litre in alcohol)- 200ml.
4. Potassium hydroxide (0.1 mol/litre)-1 litre.
5. Burettes (5ml and 25ml)-50.
Method:1.Accurately weigh out 10 gm of the test compound and
suspend the melted fat in about 50ml of fat solvent.
2. Add 1ml of phenolphthalein solution, mix thoroughly, and
titrate with 0.1 mol/litre KOH until the faint pink colour
persists for 20-30s.
3. Note the number of milliltres of standard alkali required and
acid value of the fat.
(Note: 0.1 mol/litre KOH contains 5.6gm/litre).
Result:-
56
Experiment No. 19
Aim: Determination of Blood clotting time.
Principle : Blood is collected in a capillary tube after a finger prick
and the stop watch is started. The formation of fibrin string is noted
by breaking the capillary tube at regular intervals. The time is noted
at the first appearance of the fibrin string.
Requirement:
Sterile lancet
capillary tubes (1.5 mm diameter and 10 to 15 cm length)
cotton.
70% alcohol
Stop watch
Procedure
1. By using a piece of cotton, apply 70% alcohol to the
patient’s fingertip.
2. Make a deep (1mm) incision with a sterile lancet and start
the stopwatch.
3. Wipe off the first blood drop and collect blood in the
Capillary up to 2/3 of its length.
4. After every half minute. break off about 1 cm of the
capillary to find out whether fibrin string has formed.
5. When the fibrin string appears, stop the stop watch and note
down the time.
Result :
57
Experiment No. 20
Aim:- Assay of hemoglobin by hemoglobinometer.
Principle: - When blood is added to 0.1N HCL, hemoglobin
is converted to brown colour acid hemation. The resulting
colour after dilution is compared with standard brown glass
reference blocks of a Sahli hemoglobinometer.
Clinical Significance:- A decrease in below normal range is
indication of anemia.
Normal Values:
Hb, gm/dl
Male
13-18
Women
12-16.5
Children(upto 1yr)
11-13
Children(10-12yr)
11.5-14.5
Infant
13.5-19.5
Specimen:- Capillary blood or thoroughly mixed ant
coagulated(EDTA or double oxylated) venous blood. The
specimen need not be a fasting sample.
Requirement:-Sahli’s hemoglobinometer;it consist of
a) A std brown glass mounted on a comparator.
b) A graduated tube.
c) Hb pipette 0.02ml.
-0.1N HCL
-Distilled Water
-Pasteur pipette.
Procedure:1. By using Pasteur Pipette :- Add 0.1 N HCL in the tube
upto the lowest mark (20% mark).
2. Draw blood upto 20ul mark in the Hb pipette.Adjust the
blood coloum,carefully without bubble.
58
Result:
59
Experiment No. 21
Aim: Differential Leukocyte count
Principle:-Differential count is useful to identify changes in the
distribution of white cells which may be related to specific types of
disorders. It also gives idea regarding the severity of the disease
and the degree of response of the body.
Normal values
Neutrophilia:
40-75% (mean: 57%)
a) Segmented
2-6% (mean: 3%)
b) Band forms
50-70% (mean: 54%)
Eosinophils
1-4% (mean: 2%)
Basophils
0-1%
Lymphocytes 20-45% (mean: 37%)
Monocytes
2-8% (mean: 6%)
The polychromic staining solutions (Wright, Leishman,
Giemsa) contain methylene blue and eosin. These basic and
acidic dyes induce multiple colours when applied to cells.
Methanol acts as fixative and also as a solvent. The fixative
does not allow any further change in the cells and makes them
adhere to the glass slide. The basic component of white cells is
stained by acidic dye and they are described as eosinophilic or
acidophilic. The acidic components (Eg nucleus in nucleic acid)
take blue to purple shades by the basic dye and they are called
basophilic. The neutral components of the cell are stained by
both the dyes.
Requirements:Microscope
Slides
Glass spreader
Immersion oil
Reagents:
Wright’s Stain:1. Weigh 0.2 gm of powder.
2. Transfer part of it in a clean dry mortar.
3. Add acetone-free methanol (above25ml).
4. Grind the powder by using a pistle.
5. Transfer the ground staining solution to the clean and dry amber
colored bottle by filtering through a filter paper.
6. Repeat the procedure till all the powder and total 100ml of
methanol is used up.
60
7. Stored in an amber colored bottle at room temperature.
8. The stain improves on standing for about a week.
Buffer: pH 7.
1. Sodium dihydrogen phosphate (NaH2PO4.2H2O) 3.76gm
2. Potassium dihydrogen phosphate (KH2PO4) 2.10gm
3. Distilled water 1000ml
Keep at room temperature.
Procedure:
1. A thin smear is prepared by spreading a small drop of blood
evenly on a slide.
Making the film:
-Take a clean, dry (grease free slide).
-Transfer a small drop of blood near the edge of the slide.
-Place the spreader slide at an angle of 300.
-Pull back the spreader until it touches the drop of blood. Let
the blood run along the edge of the spreader.
-Push the spreader forward to the end of the slide with a
smooth movement.
-Dry the blood smear at the room temperature. Adequate
drying is essential to preserve the quality of the film.
Result:
61
Experiment No:-22
Aim:- Estimation of Protein by Biuret Method.
Principle: The proteins are made up of amino acids containing peptide
bonds. Two or more peptide bonds react with copper sulphate and
result in violet colored product. The Cu atoms form complex with four
of nitrogen atoms of peptide chain and develop the color. The number
of peptide bonds present in a sample governs the color intensity of the
sample.
Materials:1. Protein Standard (5mg albumin/ml).Prepare fresh.
2. Biuret reagent.(Dissolve 3gm of copper sulphate
(CuSo4 5H2O) and 9 gm of sodium potassium tartar ate in
500ml of 0.2 mol/litre sodium hydroxide; add 5gm of
potassium iodide and make up to 1 litre with0.2 mol/litre
sodium hydroxide).
3. Water bath at 370C.
Method:1.
2.
3.
4.
Protocol:Regent
1
Std.
protein
solution
Distilled
Water
Biuret
solution
Add 3ml of Biuret reagent .
Add 2 ml of protein solution.
Mix them and warm at 370C for 10 mins.
Cool and read at 540 nm.
2
3
4
5
6
7
8
9
10
Blank
Unknown
0.
1
0.
2
0.
3
0.
4
0.
5
0.
6
0.
7
0.
8
0.
9
1
------
------
3.
9
6
3.
8
6
3.
7
6
3.
6
6
3.
5
6
3.
4
6
3.
3
6
3.
2
6
3.
1
6
4
4
------
6
6
6
Keep all tubes at 370C for 10 min read O.D at 540 nm.
Calculation:Conc. Of unknown = O.D. of unknown
O.D. of Std
χ
Conc. of std.
Result:62
Experiment No. 23
Aim:- Detection of bacterial motility by hanging drop method.
Principle: This technique is meant for microscopic observation of
living bacteria. Due to their small size and close refractive index to
that of water,do not allow them to observe readily under unstained
condition. The motility and binary fission may seen using this
technique.
Requirements:
1. Bacterial culture
2. Microscope
3. Cavity slide
4 .Cover slip
5. Vaseline or petroleum jelly
6. Inoculating needle
7. Bunsen burner
Procedure:
I.
II.
III.
Place a loopful or 1 drop of sample in the center of a cover
slip.
Invert it over the well of a cavity slide in such a way that the
drop does not move or contact the side wall of the well.
Apply Vaseline or petroleum jelly at four corners of cover slip
(to seal the edges).
Observation
Observe the slide under microscope. First focus it under low power
of objective and then under 45x .
Result:
63
Experiment No.24
Aim: Oligodynamic activity test of metals.
Principle: The oligodynamic acton (oligo = Small, dynamic = power)
is effect of small amount of heavy metals on bacteria .This effect is
due to high affinity of heavy metals with the cellular proteins of the
bacteria. The bacterial cells die due to cumulative effects of metal ions
within the cell. The oligodynamic action of different metals on bacteria
can be compared.
Requirements:
1.Nutrient agar tube
2.Culture of E.coli or S.aureus
3.Perti dishes
4.Copper or aluminum coins
5.Incubator
Procedure:
1. Prepare culture tube of nutrient agar or liquefy nutrient agar in
a tube.
2. Cool to 500C and inoculate with E. coli or S. aureus.
3. Pour half of the medium into a sterile petri plate and leave the
other half on water bath.
4. Leave the plates to solidify.
5. Meanwhile take copper and aluminum coins,clean them with
detergent and water. As soon as they are cleaned place them
on the surface of agar.
6. Pour the remaining medium seeded with bacteria over the
metal coins and incubate the plate at 370C for 48 hours.
7. Bacterial growth occurs. The clearing zone may be visible
around the coins.
8. Measure the diameter of clearing zone may be visible around
the coins.
9. Measure the diameter of clearing zone or zone of inhibition
and compare the effect of both the metallic coins on growth of
the bacteria.
Observations:
The clearing zone surrounding the coin indicates no growth,
Narrow zone showing heavy bacterial growth called stimulatory
growth as low amount of heavy metal coins induced bacterial
growth. Rest of the area contains normal growth. Such
phenomenon confirms oligodynamic action.
64
Result:
65