Veterinary Laboratory Techniques Unit: 1 Common laboratory equipment and their functions 1.1 microscope: simple, compound an...
Veterinary Laboratory
Techniques
Unit:
1
Common laboratory equipment and their functions
1.1 microscope: simple, compound and
binocular
1. Simple microscope:
it consists of single lens and single magnifying glass is the example of simple
microscope. With the help of simple microscope the image of an object can be
magnified up to 10x
Fig: simple microscope (source:
https://en.wikipedia.org/wiki/Microscope)
2. Compound microscope:
generally compound microscopes are used in the lab because it consists of
different types of lenses. Compound microscope consists of two types of lenses.
The magnified image of an object by one lens is further magnified by the second
lens due to this the objects can be identified easily. The lenses used in the
compound microscope are known as eye piece and objective lens.
Fig: compound
microscope (source: www.wikipedia.org)
1.
Eyepiece (ocular lens)
2.
Objective turret,
revolver, or revolving nose piece (to hold multiple objective lenses)
3.
Objective lenses
4.
Coarse adjustment
5.
Fine adjustment
6.
Stage (to hold the
specimen)
7.
Light source
8.
Diaphragm and
condenser
9.
Mechanical stage
3. Binocular microscope:
the term binocular refers to “relating to or involving the use of both eyes”.
So binocular microscope provide us the ability to focus both eyes on an object
at the same time so that a single image is seen. This enables judgment of
distance and depth of an image.
1.2 Autoclave:
Autoclave
is used to sterilize the different equipments. The daily usable equipments such
as surgical equipments, rubber gloves etc can be sterilized with the help of
autoclave. This is the most important equipment for sterilization. This
equipment is large and round in shape. It is made up of strong metal and can
tolerate high temperature. Pressure level can be maintained under the
autoclave. It can be used with the help of electricity or fire itself.
Fig:
autoclave (source: www.wikipedia.org)
Principle of autoclave
On
environmental pressure water boils at the temperature of 100 ° Celsius and the
temperature cannot be increased more than 100 ° Celsius. If the water is kept
at more pressure than the temperature of water can be increased for example
ü At
5 lb pressure temperature becomes 110 ° Celsius
ü At
10 lb pressure temperature becomes 115 ° Celsius
ü At
15 lb pressure temperature becomes 121 ° Celsius
ü On
121 ° Celsius all the bacteria along with their spores gets destroyed
At
laboratory autoclave is run on 15 lb pressure for 15 minutes for sterilization
Process to autoclave
ü All
the glassware to be autoclave is kept on a clean paper and autoclave tape is
tied around it.
ü The
packed glassware are kept on autoclave basket and distill water is poured in
the autoclave by covering the heating element.
ü All
the utensils to be autoclaved are inserted inside the autoclave. While placing
the equipments one third part of the space should be kept empty.
ü Close
the lid of the autoclave. Tight the lid with the help of screw clamp. Open the
steam releaser.
ü Switch
on the autoclave. When the water starts the heat the air inside the autoclave
also starts to heat and starts to release out.
ü When
all the air comes out steam starts to come out. After the steam releases for
2-3 minutes close the steam releaser.
ü Now
observe whether the pressure increases or not. When the pressure reaches to 15
lb wait for 15 minutes.
ü After
15 minutes switch off the autoclave, leave the autoclave to cool down, the
pressure meter shows the pressure to zero after that open the lid.
ü All the autoclaved equipment should be air
dried.
Incubator:
It is equipment made up of metal. It is box shaped.
It is double walled. The walls are thick. It consists of a door. For the
maintenance of temperature it also consists of thermostat knob. The temperature
of 37 ° Celsius is maintained. For the placement of Petridis it also contains
racks made up of wires. It also consists of a thermometer for denoting the
temperature. It consists of external metal door and internal glass door.
Fig: incubator (source: www.wikipedia.org)
Procedure
to use incubator:
ü Switch
on the incubator.
ü Roll
the knob and maintain the temperature of 37 ° Celsius. Close the door.
ü Be
careful whether the temperature of incubator increases or not.
Taking
care of incubator:
ü Never
open the door of incubator when the switch is on.
ü Never
use the knob haphazardly.
ü Never
maintain the temperature over 37 °s Celsius.
ü Never
place the edibles and other materials on the incubator.
ü If
any cultures spill over the incubator clean it immediately.
ü Clean
the incubator with distill water time to time.
Hot
air oven:
It is the square box shaped laboratory equipment. It
consists of double layered internal and external wall of steel or aluminum. It
also consists of a door. It consists of a switch for maintaining the
temperature. It also consists of a thermometer. It consists of a hole on both
sides for the entrance and exit of the air. It consists of racks for placing
different materials. On the lower inner side it consists of heating element for
heating purpose. Hot air oven consists of a fan for maintaining the air temperature
inside it. The maximum temperature of 180 ° Celsius can be maintained inside
the hot air oven.
Fig: hot air oven (source: www.wikipedia.org)
Use
of hot air oven:
ü Sterilization
of metals and glasswares can be carried out inside the hot air oven. For
ex.surgicalequipments, glass bottles, pipettes, Petridis etc.
ü Glasswares,
tubes, sterilized equipments can be air dried inside the hot air oven.
ü The
slides containg different bacteria can be fixed by keeping inside the hot air
oven. For ex. Slides to be used for the staining of bacillus anthracis.
Procedure
to use hot air oven:
ü Connect
the plug of hot air oven to the electrical supply and close the door.
ü Switch
on the electrical supply.
ü Maintain
the temperature using the temperature knob. Be careful whether the temperature
increases or not.
ü Keep
the equipment to be kept inside it.
ü After
certain time switch off the electrical supply and open the door when the
temperature goes down.
Taking
care of hot air oven:
ü Always
be sure to perform earthing of the wires to be used on hot air oven.
ü Plastics,
papers, clothes, cottons etc should never be dried on hot air oven.
ü If
water, medias, plastics, etc falls inside the hot air oven they should be
immediately removed and cleaned.
ü Always
close the door before running the hot air oven.
ü Never
keep the edibles, clothes etc inside the hot air oven.
ü Never
set the high temperature and go away for another work.
ü Clean
the hot air oven after the completion of work.
Refrigerator:
This machine is simply used for general household
purpose but on laboratory it has the most important role. The reagents used in
the laboratory are precious and are not available easily on the market so for
preserving the reagents, antisera etc for the longer period of time there is
great importance of refrigerator. Similarly when the sample brought on the lab
cannot be examined immediately on such circumstances there will be the great
importance of refrigerator. The media culture plates, culture bottles, Medias;
vaccines etc can be stored on the refrigerator for long interval of time. The
external part of refrigerator will be plain but the internal part contains
different racks and various small rooms on which the bottles etc can be placed
according to their shapes. The temperature can be maintained from 2-8 ° Celsius
by turning the temperature knob inside it.
Fig: vaccine refrigerator (source:
www.surechill.com)
Cold
chain maintenance:
Laboratories’ samples, precious reagents, vaccines
and other substances that can be degraded by temperature should be kept on
constant temperature. Such substances should not be kept on room temperature or
higher temperature. The reagents and other substances should be kept on
constant low temperature so the refrigerator should always be kept on.
Taking
care of refrigerator:
ü Refrigerator
should be always used by connecting the voltage stabilizer.
ü Always
close the door when the refrigerator is on
ü Unnecessary
low temperature should not be set on refrigerator if done so ice formation may
occur.
ü Temperature
should be maintained according to the nature of storage materials.
ü Laboratory
refrigerator should not be used for household and kitchen purpose.
ü Refrigerator
should be cleaned time to time.
Centrifuge:
Introduction:
Centrifuge is used for the sedimentation of solid
particles from liquid particles. On laboratory centrifuge is used for
separation of plasma from blood, separation of serum from blood, separation of
solid particles from suspension mixture etc.
Fig: centrifuge (source: www.wikipedia.org)
Principle:
Centrifuge works on the principle of centrifugal
force.
Types
of centrifuge:
1.
Hand centrifuge
2.
Electric centrifuge
ü Hand centrifuge:
It must be rotated by hand
manually. They are of 2 types containing 2 buckets and 4 buckets. It can be
placed on table, chair or stool by fixing tightly. It is used on the area where
there is no electrical supply. Normally this type of centrifuge is used while
examining the stool or urine sample. The revolution can be maintained up to
2000 rpm.
ü Electrical centrifuge:
It is used by connecting with
electricity. It contains of electrical motor. A knob is present for controlling
the rotation. It consists of swing rotor and angle rotor.
Parts
of centrifuge:
1. Pillar
2. Arm
3. Bucket
Procedure
of using centrifuge:
ü Open
the lid of centrifuge.
ü Keep
the sample to be centrifuged.
ü All
the tubes of centrifuge must contain equal amount of samples.
ü Place
the centrifuge tube on centrifuge bucket.
ü Switch
on the centrifuge.
ü For
increasing the motion of centrifuge keep 1000/rpm after some time increase to
2000/rpm again after some time increase to 3000/rpm.
ü Rotate
the centrifuge for 10-15 minutes as required.
ü After
15 minutes rotate the knob to zero.
ü Open
the lid only after the centrifuge stops completely.
ü Remove
the centrifuge tubes.
ü Close
the lid after all the centrifuge tubes are taken out.
ü Switch
off the centrifuge.
Taking
care of centrifuge:
ü All
the buckets of centrifuge should be well balanced.
ü Never
spill the liquids, salts etc on the centrifuge.
ü While
increasing the motion at first maintain 1000/rpm, 2000rpm, 3000rpm
respectively.
ü Never
seal the mouth of centrifuge tube.
ü Never
try to stop the rotation of centrifuge with unnecessary force.
Distillation
set/water distiller:
Veterinary laboratory
requires the distilled water regularly for making different standard solution.
Distilled water must be the source of the water. Other sources of water may
contain different chemicals and minerals such as magnesium, sulphate,
carbonates etc. The tests may not provide the accurate results if the source of
water is not clean.
Fig: distillation set
(source: www.wikipedia.org)
Principle:
The water is vaporized
by keeping on a vat and cooled by keeping on condenser. Due to this the pure
water only gets collected
Parts
of distillation set:
ü Water
boiler
ü Condenser
ü Stand
ü Pipe
ü Water
reservoir
ü Heater
Procedure
to use distillation set:
ü At
first place the water up to the half region of water boiler.
ü Allow
the water to pass from the condenser.
ü When
the water starts to exit from the condenser switch on the heater and heat the
water. When the water starts to boil vaporization starts and the water reaches
to the condenser. The tap water cools the condenser continuously. The vapor
cools down and changes into water and get stored at reservoir.
ü While
stopping the distillation set at first switch off the heater.
ü Then
close the water supply.
Taking
care of distillation set:
ü Never
place the large volume of water in the boiler, place nearly half part of the
water in the boiler.
ü Continuous
water supply should be provided in the condenser. Otherwise the vapor goes out
by vaporizing.
ü Check
the water supply whether it is continuous or not.
ü Never
place the dirty water on the boiler.
ü The
boiler should be cleaned regularly.
Water
bath:
Introduction:
It is box shaped equipment made up of metal. It
consists of double layer. Both the inner
and outer part of water bath is made up of steel. The upper part is open and
can be closed with lid. This electrical equipment is insulated. Water can be
heated for different chemical reactions in water bath. It consists of heating
element which helps in heating the water. For the maintaince of water
temperature thermostat is placed. On laboratory the water temperature is
maintained on 56 and 37 ° Celsius. It consists of thermometer for measuring the
temperature.
Fig: water bath (source: www.wikipedia.org)
Procedure
to use:
ü Distilled
water should be placed on water bath.
ü Switch
on the water bath, maintain the temperature as required. Be careful whether the
temperature increases or not.
Taking
care of water bath:
ü Only
distilled water should be placed on water bath. If the water gets dirty new
distilled water must be placed.
ü Run
the water bath only after placing the water inside it otherwise heating element
may get destroyed.
ü Add
water if the water on water bath dries.
ü Never
mix any other materials on the water of water bath.
ü Always
close the lid of water bath.
ü Switch
off the water bath when the works get finished.
ü While
using the water bath at first the temperature should be maintained.
PH
meter:
It is used for measuring the PH of solution on the
lab. Different types of PH meter are available on the market. Some PH meter
runs through battery while some runs with direct current. It consists of
sensitive voltmeter. Ti measures the current influence of measuring electrode
and reference electrode.
Fig: PH meter (source: www.wikipedia.org)
PH
of solution:
All the solution contains the mixture of hydroxide
ion and hydrogen ion. For example some solution contains the equal amount of
hydroxide ion and hydrogen ion which is known as neutral solution. Distilled water ionizes one molecule of
hydrogen ion and one molecule of hydroxide ion. If the solution contains more
amount of hydrogen ion it is called acidic solution and if the solution
contains more amount of hydroxide ion it is called to be alkaline solution. The
value of PH ranges from 0-14. If the PH values goes 6,5,4 it is acidic and if
the PH increases from 7 and goes to 9,10 it is called alkaline solution.
|
Acidity
|
Neutrality
|
Alkalinity
|
|
1,2,3,4,5,6
|
7
|
8,9,10,11,12,13,14
|
The acidity and alkalinity of a solution can be
measured by the help of PH value. If the solution contains low amount of
hydrogen ion it contains more amount of hydroxide ion therefore the solution is
alkaline. If there is low amount of hydroxide ion and more amount of hydrogen
ion the solution is acidic.
PH can be measured by using the PH paper or PH
meter.
PH value of some chemical solution and other
solution
|
Solution
|
PH
|
|
0.1 mol. HCL
|
1
|
|
Lemon
|
2.3
|
|
Apple
|
3.0
|
|
Saliva
|
6.4
|
|
Milk
|
6.4
|
|
Urine
|
7.4
|
|
Sea water
|
8.2
|
|
Lime water
|
12.3
|
|
0.1 mol. NaOH
|
13
|
Measuring
of PH:
ü PH
value is compared with the PH of a standard solution, which can be directly
known from electric amplifier, digitally.
ü The
temperature of standard solution and the sample solution which PH is to be
measured should be equal.
Calibration
of PH meter:
Before using the PH meter it should be checked on
the buffer solution containing the PH of 4, 7 and 9 .
Procedure
to use PH meter:
ü Switch
on the PH meter.
ü Electrode
should be cleaned with distilled water and wiped with tissue paper
ü Dip
the electrode on PH 4 buffer solution and maintain the PH of 4. Clean the
electrode with distilled water.
ü Dip
the electrode on PH 9 buffer solution and maintain the PH of 9. Clean the
electrode with distilled water.
ü Dip
the electrode on the solution which PH is to be measured record the value of PH
seen on PH meter.
ü Clean
the electrode with distilled water after measuring the PH of every solution.
ü After
the completion of measuring wash the electrode with distilled water or dip it
on distilled water.
Taking
care of PH meter:
ü The
electrode of PH meter should be dipped on water.
ü Never
measure the PH of extremely hot solution.
ü Always
keep the electrode of PH meter clean.
ü While
measuring the PH of different solution the electrode should be immediately
cleaned after measuring the PH of one solution.
Colorimeter:
Colorimeter is equipment used in the biochemistry
lab. Nowadays different types of colorimeter are available on the market. The
main purpose of colorimeter is to measure the color present on the liquid. In
biochemistry lab the amount of color produced while performing the reaction
of biochemical
(glucose, urea, uric acid, bilirubin, etc)
mineral (phosphorous, magnesium, calcium, and copper) with different
reagents is measured. After performing the reaction high amount of color
produced represents the high level of biochemicals and low amount of color
represents the low level of biochemical. The advanced form of colorimeter is
spectrophotometer.
Fig: colorimeter (source: www.wikipedia.org)
Principle:
The principle of colorimeter is based on Lambert’s
law and Beer’s law
Parts
of colorimeter:
ü Electric
source
ü Light
source
ü Filter
ü Cuvette
holder
ü Cuvette
ü Photo
cell
ü Galvanometer
ü Scale
Procedure
to use colorimeter:
ü Switch
on the colorimeter 10-15 prior to use.
ü Maintain
the filter and wavelength according to need.
ü Place
the blank solution on cuvette and place it on covette holder. Maintain the OD
on zero by adjusting the coarse and fine adjustment. Maintain the OD of blank
solution to zero for 2 times.
ü Remove
the blank solution from cuvette and place the test solution and observe its OD.
Similarly observe the OD of standard solution.
ü Remove
the solution from cuvette and clean it. Switch off the colorimeter.
Calculation:
Amount of solution= OD
T/OD St *100
ü OD=
Optical Density
ü St=
Standard Solution
ü T=
Test
Taking
care of colorimeter:
ü Always
connect voltage stabilizer with colorimeter.
ü Never
spill the water on colorimeter.
ü Switch
off the colorimeter after performing the test.
ü Colorimeter
should be placed/fixed on the area with absence of light.
ü While
not in use it should be covered with its cover
Weighing
balances:
Weighing balance is the most important equipment of
laboratory. While preparing the reagents, solutions, staining solution etc the
chemical reagents should be measured on exact amount. Weighing balance on lab
should be very sensitive and have the capacity to differentiate the weight of
0.01 gm (10mg).
Different
types of weighing balances used on laboratories:
ü Manually operated balance
ü Electronic balance.
ü Analytical balance.
Manually
operated balance:
It contains pans on both sides. On the one side
weight is placed and on the other side the sample to be measured is placed.
Manually operated balances may contain 2 pans, one pan or may be of sliding
type. This type of balance weighs up to 10 gm.
Electronic
balance:
It runs with electricity. It is small, square, flat
and box like in shape. It contains of a measurement to see whether it is on
balance or not. The height of the measurement can be increased or decreased to
balance the weighing balance. It consists of single pan and the weight is
displayed digitally. It is expensive and can’t be used on the place where there
is no electrical supply.
Fig: electronic balance (source: www.wikipedia.org)
Analytical
balance:
It also runs through electricity and is used to
measure the extremely smaller weight. It also consists of measurement of
balance. It is affected by air also so it is kept inside the glass chamber. It
consists of single pan and weight is displayed on digital screen. Extremely low
weight can be measured.
Fig: digital analytical balance (source: www.wikipedia.org)
Taking
care of weighing balance:
ü While
measuring the weight the samples should not be directly placed on pan. Plastic
or paper pieces should be placed over pan before measuring the sample.
ü It
should be kept on strong surface which does not get destroyed easily.
ü Equipments
such as centrifuge, vortex should not be kept nearby balances. It may alter the
weight.
ü It
should not be kept on sunlight and open area.
ü Liquid
solution, extremely cold samples should not be measured on balances.
ü Always
keep the pans clean
ü Never
weigh the samples more heavy than the capacity of the balance.
ü If
something spills over the balance clean it immediately.
ü Before
measuring the weight take care whether the pans are on balance or not.
ü After
completion of work the weighing balance should be kept covered by cleaning.
.
Unit:
2
General
laboratory procedures
2.1 safety
and first aid in laboratory
Lab technician is responsible for the safety and
first aid on the laboratory. If the technician him/herself is not well known
about the working procedures and the materials used on the lab then it becomes
difficult to perform different types of tests. Lab technician must be well
known about the different types of harmful specimens such
as stool, urine, blood, biological etc. if the technician is not well known
about these things there will be great risk of accident on the lab. If lab
procedures are not followed properly there will be the great risk of injuries,
diseases, fire etc. hence for preventing the possible risk of accidents,
knowledge about the following things should be taken.
1. Infective
biological material
-
Specimen
-
Culture
-
Patient
2. Dangerous
chemicals
3. Dangerous
apparatus
-
Autoclave
-
Centrifuge
-
Sterilizer
-
Microtome and knife
-
Heater
-
Spirit lamp, Gas Bunsen burner
-
Oven
-
Glasswares
4. Sharp
needles
5. Dangerous
gases
-
Hydrogen
-
Carbondioxide
-
Formaldehyde
-
Chlorine
-
Ethylene oxide
-
Natural gases
6. Fire
-
Electricity
-
Wiring
-
Overloading
7. Protective
garments
-
Laboratory coat
-
Apron
-
Gloves
-
Goggles
8. Safety
equipments
-
Fume cupboard
-
Eye wash
-
Emergency shower
-
Mask
-
Tong
-
Fire blanket
9. Radiation
-
Ultra-violet light
-
Radioactive chemicals
10. Personal
behavior
-
Wearing of neck tie
-
Necklace
-
Bracelet
-
Long, open hairs
-
Long nails, nail biting
-
Untidiness
-
Hygiene
11. Danger
from animals on lab
-
Scratching, biting, kicking
-
Zoonotic diseases
12. Disposing
of
-
Infective materials
-
Chemicals
-
Carcass
13. Storage
-
Poisonous chemical
-
Blood, serum, plasma
-
Antisera, antibiotics
14. Security
-
No entrance without permission
Rules to be followed on
lab
After
getting the information on the following things, the lab technician should be
careful for stopping the risk of accidents such as burns from chemicals, cut
from sharp knives, cuts from broken glasses, transmission of diseases from
infective specimen etc by being careful on following things.
ü Wearing
of lab-coat immediately after entering the lab
ü All
the chemicals used on the lab should be well labeled along with the date of
manufacture and expiry date.
ü All
the equipments on the lab should be clean and dry.
ü Managing
and keeping the lab clean.
ü Being
careful while collecting and receiving the samples.
ü Never
put water on acid.
ü Never
eat on lab.
ü Never
suck the pipette with mouth.
ü Never
connect the naked wire on socket.
ü Never
touch the electrical equipments with wet hands.
ü Never
use the lab for other purpose such as gossiping, resting etc. lab should be
only used for practical purpose.
ü Always
read the label and instructions before using the chemicals and storage should
be done as instructed.
ü Never
use the chemicals without label.
ü Never
use the expired chemicals.
ü Never
smell the chemicals, bottles should never be carried by holding on its lid.
ü Check
the leakage of water, gas pipes regularly.
ü First
aid should be available on the lab.
ü Never
work on the lab with an assumption, if there is a doubt consult with senior
technician or refer to the literature.
ü Cover
the body with clothes, wear shoes.
ü Enough
supply of water should be made available on lab.
ü Broken
glass and glasses with cracks should never be used. Dispose them properly.
ü Never
allow the entrance for children and animals on lab.
ü Switch
off the electrical supply and tap after the completion of work.
ü If
some accident occurs immediately inform the senior technician.
ü After
the completion of work infective specimen should be disposed by disinfecting on
Lysol solution.
2.2
CLEANING OF GLASSWARES
Introduction:
Glass-wares need to be cleaned and sterilized even
for those that have been not used or used. It is essential to clean and
sterilize for killing or destroying micro-organisms and to prevent
contamination.
Cleaning
of new glass-wares:
New glass wares also require to be cleaned because
there may be the resistant spores in the packing materials. It is not that the
new glass-wares are ready for direct use due to dirt stains etc.
Materials
required:
ü 5%
HCL
ü Autoclave
ü Hot
air oven
ü Distilled
water
ü Wire
bucket
Procedure:
ü Dip
new glass-wares in 5% HCL solution overnight.
ü Place
into a container having tap water and rinse at least for 2 times then rinse in
warm distilled water.
ü Place
the glass-wares in wire basket and dry in hot air oven and use as required or
after wrapping in craft paper which can be autoclaved.
cleaning
of used glass-wares:
ü After
using glass-wares must be dipped immediately in water.
ü Dip
in 3% Lysol or 1% sodium hypo-chloride solution for overnight.
ü Rinse
the glass-wares in tap water properly.
ü Dip
the glassware in 5% soapy solution. (detergent water) for 1 hour.
ü Using
brush scrub each glass-wares and place in container containing clean water.
ü Wash
in tap water to remove soap.
ü Wash
in warm water.
ü Ultimately
rinse in distilled water.
ü Then
dry at 100ºc and use the dried glass-wares as required or sterilize in hot air
oven at 160°c
for 1 hour.
2.3
Sterilization:
Sterilization is the process of destruction of all
forms of micro-organisms. An instrument is considered sterilized when it is
free from living micro-organisms. The very word sterile, sterilize and
sterilization in microbiological stand point indicate total absence or killing
of all micro-organisms.
Sterilization is most commonly done through heat.
Heats are divided into two groups moist heat and dry heat.
A.
Moist
heat
a. Autoclave:
moist heat in the form of saturated steam under pressure is the most practical
method for sterilization. The laboratory apparatus used with stem under
regulated pressure is called autoclave. Both pressure and temperature for a
definite period of time kills the organisms. Autoclave is an essential
instrument in every diagnostic microbiological laboratory. Cotton, wools,
various cultural media, solutions are sterilized with the instrument. In
autoclave most of the materials are sterilized under 15 lb pressure at 121 °
Celsius temperature.
b. Tyndallization:
tyndallization is also a process of moist heat application. Some materials like
amino acids, sugar solutions etc are required to be heated at 90-100 ° Celsius
temperature on three successive days with interval in between. During the
interval or incubation period resistant spore germinate and on subsequent
exposure to heat the vegetative stage get destroyed. This method of heating at
successive heating is called fractional sterilization or tyndallization.
c. Boiling:
through boiling it is possible to destroy most of the vegetative forms of the
micro-organisms, but spores cannot be destroyed with this method. Boiling water
can be considered as a true method of sterilization. In field condition
surgical instruments are frequently put under boiling water. Through this
method instrument can be brought under disinfection but not to the extent of
sterilization.
d. Pasteurization:
pasteurization is a process by which food and food products (milk,fruits,
juices, wine etc) are protected from putrefaction and fermentation. It involves
a short exposure to heat at a lower temperature than that employed in ordinary sterilization.
In this process, the product is subjected to a controlled heat treatment which
kills micro-organisms of certain types but not all. Millk is commonly
pasteurized then marketed. The temperature selected for pasteurization is based
on a thermal death time of most resistant type of micro organisms to be
destroyed by this process.
Two
methods of pasteurization are used commercially low temperature holding method
(LTH) and high temperature short time method (HTST). In LTH method milk is
exposed to 62.8 ° Celsius for 30 minutes and in case of HTST method milk is
exposed to a temperature of 71.1 ° Celsius for 15 seconds.
B. DRY
HEAT:
It is done by the following.
i.
Red hot: inoculating wire, foreceps,
spatula etc are sterilized at the blue zone of the Bunsen burner up to heated
red hot.
ii.
Flaming: scalpel, needle, cultural tube,
cotton wool plug, glass slide etc are sterilized by passing the articles
through Bunsen flame without allowing becoming red hot.
iii.
Hot air oven: here heating is done by
electricity above chosen temperature which is maintained by thermostat. Here
sterilization is done at 160 ° Celsius for 1 hour. Materials to be sterilized
through this are all glasswares, surgical instruments, glass syringe etc. Should be taken before sterilization by this
method are.
a. All
glass articles must be wrapped with craft paper.
b. All
articles must be free from moisture.
c. After
the switch is put off, the whole system is allowed to be cool.
Dry
materials in sealed container with petroleum jelly that are impermeable to
microbes can be sterilized by this method.
2.4
Antiseptics:
It is an agent that prevents sepsis i.e.
prevents the growth of infective agents like bacteria, virus, protozoa etc.
these substances are substantially non-toxic for superficial application to
living tissues. Therefore these agents can be applied externally on animals to
kill or prevent the growth of microbial population. The antiseptics belong to a
variety of chemical substances e.g alcohol, phenol, variety of metallic salt,
acridine dyes etc. they are used on the intact skin before surgical operation
and injection. They are applied to broken skin following wounds, burns etc.
they are also used on mucous membrane e.g conjunctiva or bladder to prevent or
treat the superficial infection.
They
are grouped as
a. Oxidizing
agentse.g hydrogen peroxide (H2O2), potassium permagnet (KMNO4), chlorine,
iodine, iodophore etc.
b. Reducing
agents e.g. sulphur dioxide and formaldehyde.
c. Metallic
compounds e.g mercuric chloride and iodide.
d. Acids
and alkalis eg boric acid and various sodas etc.
e. Alcohol
-70% ethyl alcohol
f. Phenol
and cresol.
g. Various
dyes- acriflavin, brilliant green, proflavin.
h. Detergents-
various soap
i.
Cetrimides.
j.
Quaternary ammonium compound.
k. Antimicrobial
agents- they include various antibiotics and chemotherapeutics. They are called
as therapeutic agents.
2.5Disinfectants:
Disinfectants are
agents which are too toxic to be applied to the tissues of the host but which
can be used in destroying contaminating inanimate objects eg. Drains, faecal
matter, building, vehicles, cooking materials, surgical instruments and
apparatus and the rooms or sheds
Qualities of
disinfectants:
a.
The price of it must be affordable. The
requirement of it is in bulk quantity. Therefore the price is one of the
important attributes to a farmer.
b.
The disinfectants should not emit
disaggreable offensive odor. This is relevanat in case of the dairy animals.
Since this odour may taint the milk rendeing milk unacceptable to the consumer.
c.
It should not be harmful to the tissues
or materials upon which it is applied. It must have bactericidal property.
d.
Its action should not be such strong
when applied to the house or premises that the life of the animals is at risk.
While they lick or drink water from such surface or pasture.
e.
While it combines to any chemicals it
should not become inert especially when used to disinfect utensils, or blood.
f.
The irritating property should be in
permissive level. It should not be too much irritating to the mucous membrane
lining the respiratory tract causing damage and inflammation. High
concentration of formaldehyde, chlorine gas may be too irritating to the
mucosa.
g.
There must not be reduction of
disinfecting quality to an appreciable level when used in extreme hot or cold
weather. But, it should preserve all its disinfecting quality in ordinary
temperature.
h.
It must be able to act even when diluted
with water to a considerable extent.
i.
It must mix well with water so that it
remains uniform when applied following dilution with water.
2.7 Collection, storage, labelling and
dispatch of samples to laboratories
For
the diagnosis of disease, different type of samples from different parts of the
body of the animal must be collected. After collecting the sample, the samples
should be stored at appropriate media before sending it to the lab for
examination. While sending the samples to the lab following information must be
provided for the diagnosis of different type of diseases.
Sample
no:
collection
date:
Name
of the owner:
Address:
Species
of animal: breed
of animal: age of
animal:
Sex
of animal:
tag of the animal:
Brief
information about the suspected disease:
Chemicals
used to preserve the samples:
Contact
no of the owner:
|
Disease/Condition
|
Sample type
|
|
1. Vitamin
deficiency
|
Feed
|
|
2. Minerals
deficiency
|
Serum
|
|
3. ketosis
|
Urine
|
|
4. iron
deficiency
|
Blood
|
|
5. F.M.D
|
Exudates from
the wounds of mouth, tongue, feet, scraps from the wound, serum
|
|
6. Pox
|
Nodules, serum
|
|
7. Rabies
|
Tissues from
central nervous system
|
|
8. Ranikhet
disease
|
Central
nervous system, serum, tissue sample, liver and spleen should be sent by
preserving on 50% glycerine.
|
|
9. Marex disease
|
Feathers,
serum, tissue sample
|
|
10. Leukosis
|
Tumors, tissue
sample
|
|
11. Gumboro
|
Serum, bursa,
kidney, tissue sample
|
|
12. Leptospirosis
|
Serum, blood,
urine and tissue sample
|
|
13. Anthrax
|
Blood smear,
blood from ear vein by mixing with anticoagulent
|
|
14. Tetanus
|
Blood smear,
swab from the wounds
|
|
15. Black quarter
|
Blood smear,
swab from the inflamed area
|
|
16. Enterotoxaemia
|
Intestinal
parts
|
|
17. Brucellosis
|
Gastric
contents of aborted fetus, serum from infected animal
|
|
18. Actinobacillosis
|
Swab from
wounds, blood smear
|
|
19. Haemorrhagicsepticaemia
|
Blood smear,
nasal swabs, tissue sample
|
|
20. Actinomycosis
|
Swab of
exudates, pus and tissue sample
|
|
21. Chronic
respiratory disease
|
Nasal swab,
tissue sample
|
|
22. Coryza
|
Nasal swab,
tissue sample
|
|
23. Aflatoxin
|
Feed sample
|
|
24. Coccidiosis
|
Samples from
intestine and caecum
|
|
25. Theileriosis
|
Blood and
lymph node’s smears
|
|
26. Anaplasmosis
|
Blood smear
|
|
27. Internal
parasites
|
Fecal samples
|
|
28. Poison
|
Gastric
contents, blood, serum
|
|
29. Skin diseases
|
Skin scrapping
|
|
30. Fowl cholera
|
Blood smear,
blood swab should be sent on charcoal media
|
|
31. Fowl typhoid
|
Liver should
be sent by preserving on formalin
|
|
32. Pullorum
|
Dead chicks
|
Dispatch of the samples to
laboratories:
Collected
samples should be dispatch to the lab for the diagnosis of bacterial, viral and
fungal diseases. Similarly the isolated bacteria from the cultures should be sent
to the reference labs for examination.
While
sending samples to the lab, information about the samples, sample size,
information about the animals, general information about the symptoms, and
information about the sample collector, address of the sample collector should
also be included. The collected samples should be sent to the lab within 10-24
hours. If it is not possible to send the samples to the lab immediately, the
collected samples should be preserved on the refrigerator at 40℃but should not be
kept at deep fridge. While sending the sample to bacteriological lab the sample
should be preserved at on cooked meat media. While sending the samples to
virological lab, the samples should be kept on virus transport medias. The
samples should be well labeled and kept on icepack or thermocool vials. “SAMPLE
HANDLE WITH CARE” should be written on the external part of the sample
containing vials.
Unit:
3.
Parasitology
3.1 Common internal parasites of livestock and
poultry
The common internal parasites of livestock and
poultry are helminthes. Helminthes can be further classified as following.
|
1.
Nematodes (round worms)
|
Ascaris,
Strongylus, Trichuris, Cooperia, Bunostomum, Tricostrongylus,
Esophagustomumetc
|
|
2.
Trematodes (flat worms)
|
Liverfluke/Fasciola,
Paramphistomeetc
|
|
3.
Cestodes (tape worms)
|
Monezia,
Taenia, Echinococcusetc
|
3.2 Fecal sample collection:
5-10 GM of fecal sample can be directly collected
from the rectum of the animal. The samples may also be collected from the
middle part of the excreted feces. Samples should be kept on the clean and dry
vials. In case of small animals fecal samples can be collected from the rectum
with the help of fingers. The samples should be preserved on ice if there is
delay on dispatch. Samples can be also preserved by adding 2-4 drops of 10%
formalin solution. Sample can also be stored at 4°C on refrigerator. Addition of chemical should not be done
on the suspected samples of lung worm. Consistency of fecal sample should be
observed on lab and following conditions should be included on the report.
ü Solid, semi-solid, watery or rice gruel etc
ü Presence/absence of the white segments of the
tape worm should also be included on the report.
Shape and color of feces of different animals
|
species
|
Shape
of feces
|
Color
of feces
|
|
1.
Cattle/Buffalo
|
Cake
like
|
Dark
brown to dark green
|
|
2.
Sheep/Goat
|
Pellet
|
Yellowish
brown to black
|
|
3.
Pig
|
cylindrical
|
Dark
yellow
|
|
4.
Horse/Mule
|
Bolus
|
Yellowish
brown to dark green
|
|
5.
Dog,Cat
|
Cylindrical
|
Yellowish
brown
|
3.3 Fecal
sample examination methods
Microscopic fecal examination methods
1.
Direct smear method
2.
Floatation method
a.
Saturated sodium chloride solution method
b.
Saturated sugar solution/zinc sulphate solution
method
3.
Sedimentation method
1.
Direct smear method:
ü 1 drop of normal saline is placed on a clean
glass slide.
ü With the help of spatula, small amount of feces
is kept on the slide with normal saline.
ü Mix well the normal saline and feces and make a
thin smear.
ü Cover the smear with cover slip and examine
under 10 x at first and then at 40 x objective lens.
ü In place of normal saline lugol’s iodine may
also be used. Lugol’s iodine is quite helpful to detect the larva of third
stage and protozoa.
2.
Floatation method:
a.
Saturated sodium chloride solution method:
ü At first 3 GM of feces is measured and mixed
with 42 ml of water and sieved.
ü 15 ml of the sieved solution is kept on
centrifuge tube with round bottom.
ü Centrifuge tube is centrifuged on 1000 RPM for
5 minutes.
ü The clear upper part of the solution is removed
after centrifuge is completed, centrifuge tube is filled with the solution of
sodium chloride and the mouth of centrifuge tube is sealed with cello tape.
ü Centrifuge tube is again centrifuged for 5
minutes on 1000 RPM. Cello tape is kept under the microscopic field and
observed under10 x objective lens.
b.
Saturated sugar solution/zinc sulphate solution
method:
ü At first 3 GM of feces is measured and mixed
with 42 ml of water and sieved.
ü 15 ml of solution is kept on centrifuge tube
and centrifuged on 1000 RPM for 5 minutes.
ü The clear upper part of the solution is removed
after centrifuge is completed, centrifuge tube is filled with the solution
ofsaturated sugar or zinc sulphate and the mouth of centrifuge tube is sealed
with cello tape or cover slip.
ü Let the solution inside tube touch the cello
tape or cover slip.
ü Examine the cello tape or cover slip under
microscope.
3.
Sedimentation method:
ü Prepare the solution of feces similarly as done
in flotation method.
ü Keep the solution on a clean beaker and let it
to sediment, leave it for 15-30 minutes and remove the upper part of the
solution.
ü Fill the beaker with water.
ü Continue the process for 3 times.
ü Again fill the beaker with water.
ü Remove the upper portion of water and keep 2 ml
of sediment on the beaker.
ü Add 3-4 drops of methylene blue on the beaker
containing the sediment.
ü Keep 1-2 drops of the sediment on the glass
slide.
ü Cover it with cover slip.
ü Observe it under 5x, 10x objective lens
3.4 Common external parasites of
livestock and poultry:
The
common external parasites of livestock and poultry are mange/mites. Mites cause
mange on the animal’s body. It can occur on every species of animals. It is a
kind of micro-organism. Different types of mites effect different species of
animals. Identification of mites can only be done with their body structures.
1. Sarcoptic mange:
This type of mange contains pus.
The mites causing this type of mange reside deep inside the skin. It contains
small, undeveloped legs. It contains suckers on pedical.
2. Corioptic mange:
This type of mange is mostly found
in cattle and buffalo. This type of appears on the base of the tail and spreads
throughout the body. This type of mites contains suckers on small pedicles. The
posterior lobe of male is large and contains hairs.
3. Demodectic mange:
This type of mange is seen on
cattle, buffalo, dog, sheep and goats. Adult animals are mostly affected by
this type of mange.
4.
Psoroptic
mange:
Although this mange does not reside
on the depth of the skin but also it affects on wool production of sheep.
3.5
Skin scrapping test
ü At first the skin samples from
the animals suffering from mites should be collected.
ü While collecting the samples the infected
part should be lubricated with Vaseline.
ü After that the infected parts
should be scrubbed with clean scalpel blade until the blood oozes out.
ü The skin samples should be kept
on a clean piece of paper or petri dish.
ü After taking the sample to lab, it
should be kept on test tube.
ü Samples should be mixed with 5 ml
of 10% potassium hydroxide or sodium hydroxide on test tube.
ü The
test tube should be heated on water bath so that the hairs and skin gets
digested.
ü Let
the test tube to cool down.
ü When
the test tube cools, centrifuge it at 1500 RPM for 5 minutes.
ü Discard
the upper portion of solution.
ü Place
1-2 drops of the sediment on clean glass slide, observe it under microscope.
4.1
Types of blood cells
Animal
blood
Fig:
shape wise classification of blood cells (source: www.wikipedia.org)
Blood may be termed as a specialized and circulating
tissue composed of cells suspended in a fluid intercellular substance which
circulates through a closed system of blood vessels (arteries, veins) due to
pumping action of the heart. If the blood is centrifuged it separates into two
distinct fractions, the upper fraction is clear straw-colored fluid called
plasma while less than half of the tube is packed with the so called formed
elements, consisting of red blood cells, white blood cells and platelets. When
the blood is allowed to clot a colorless fluid portion oozes out. This is
serum. It differs from plasma in that the fibrinogen portion is not present in
the fluid portion of the serum.
Blood:
RBC
WBC
PLATELETS
RBC: erythrocytes constitute about 32% of the
total amount of the blood. While viewing under the microscope they appear as
biconcave discs, circular in shape, flexible and possess no nucleus,having lost
it before entering the circulation. It may be noted that the RBC of birds
including chickens, ducks, turkeys etc, fish and reptiles including any of a
group of cold blooded vertebrates that crawl their bellies, possesses a
nucleus, while those of camel erythrocytes are oval in outline and biconvex.
WBC:
WBC
can
be further classified under
1. Granulocytes:
Granulocytes contain granule within the
cytoplasm that stain with common blood stain. Nuclei appear in many
shapes.
Neutrophils:
the
group constitutes the greatest number of all the WBCs. Their number increases
rapidly whenever acute infection is present.
Eosinophils:
granulocyte type that stains with eosin is known as
eosinophil. May be weakly phagocytic. Primary function seems to be the
detoxification of either foreign proteins introduced into the body.
Basophils:
contain
blue staining granules, are also rare in normal blood.
2.
Agranulocytes:
Agranulocytes
usually show few granules.
Monocyte:
the
largest WBC, like neutrophils, are phagocytic. They are functional more for
cases of less acute infections.
Lymphocyte:
variable
in size and appearance. One of the major functions is their response to
antigens (foreign substances) by forming antibodies that circulates in the
blood or in the development of cellular immunity.
Platelets: also called thrombocytes,
are a component of blood whose function (along with the coagulation factors is to stop bleeding by clumping and clotting blood
vessel injuries.Platelets have no cell nucleus they are fragments of cytoplasm that are derived from the megakaryocytes of the bone
marrow, and then enter the circulation. These
unactivated platelets are biconvex discoid (lens-shaped) structures 2–3 µm in greatest diameter. Platelets are found only in mammals,
whereas in other animals (e.g. birds, amphibians) thrombocytes circulate as
intact mononuclear cells.
On a
stained blood smear, platelets appear as dark purple spots, about 20% the
diameter of red blood cells. The smear is used to examine platelets for size,
shape, qualitative number, and clumping. The ratio of platelets to red blood
cells in a healthy adult is 1:10 to 1:20.
The
functions of blood:
1. Blood
carries nutrients made available by the digestive tract to body tissues.
2. It
carries oxygen from the lungs to the tissues.
3. It
carries carbon dioxide from tissues to the lungs.
4. Waste
products from various tissues are carried to the kidneys for excretion.
5. Hormones
are carried from endocrine glands to other organs of the body.
6. Blood
plays an important role in temperature control by transporting heat from deeper
structures to the surface of the body.
7. Water
balance is maintained by the blood.
8. Buffers
such as sodium bicarbonate in the blood help to maintain a constant PH of
tissues and body fluids.
9. The
clotting ability of blood prevents excess loss of blood from injuries.
10. Blood
contains important factors for defense of the body against disease.
4.2 Blood sample collection
methods:
Site of blood
collection differs from one species to another. Before collecting the blood the
animal should be restrained properly. The animals should not be allowed to
move. Prior to collection of blood the area around the vein should be made free
of hairs with the help of scissors or blade. After that application of spirit
should be done. Application of spirit makes easy to locate the vein. In case of
cattle before the collection of blood the jugular vein should be raised by
pressing on jugular furrow. Now fit the needle on the new vacutainer holder.
Pierce the vein with needle. Now draw the blood from the vein slowly.
Different
types of vacutainer for blood collection
ü for
preparation of serum vacutainer without anticoagulant (having red cap)
ü for
hematological tests vacutainer with anticoagulant EDTA (having violet cap)
ü for
biochemical examination vacutainer tube with heparin (having green cap)
|
Animal species
|
Blood
collection site
|
Needle
size(gauge)
|
|
Horse
|
Jugular vein
|
18-19 gauge
|
|
Cattle and buffalo
|
Jugular vein
|
18-19 gauge
|
|
Sheep and goat
|
Jugular vein
|
20 gauge
|
|
Swine
|
Ear vein or anterior venacava
|
20 gauge
|
|
Poultry
|
Wing vein or jugular vein
|
21-22 gauge
|
|
Dog
|
Cephalic or cephanous vein
|
20-22 gauge
|
|
Cat
|
Cephalic or cephanous vein
|
20-22 gauge
|
|
Rabbit
|
Ear vein
|
22-23 gauge
|
|
Mice
|
Orbital sinus
|
Microhematocrit tube
|
4.3
Total count of RBC
Materials
required
ü Neubauer’s
slide counting chamber along with cover slip.
ü RBC
diluting pipette (with red grain)
ü RBC
diluting fluid (Haems fluid) or (Decies fluid)
ü Watch
glass or small Petridish
ü Microscope
Fig: neubauer’s slide (source: www.wikiwand.com)
Fig: neubauer’scounting chamber (source:
www.wikipedia.org)
Procedure
for counting RBC
ü Clean
the counting chamber and cover slip with soft tissue paper and cover the rule
part of neubauers chamber with cover slip.
ü Transfer
the diluting fluid on watch glass or Petridish
ü Mix
well the blood on blood vial
ü Suck
the blood up to 0.5 mark of RBC diluting pipette. Clean the tip of the pipette
with cotton.
ü Suck
the diluting fluid up to 101 mark of the diluting pipette.
ü Mix
well the blood and diluting fluid inside the diluting pipette by keeping it on
the palm.
ü Discard
2-3 drops of solution.
ü Keep
1 drop of the mixed solution on the side of the cover slip (charging)
ü The
fluid get well throughout the counting area of neubauers chamber.
ü If
air bubbles are formed remove the counting chamber and cover slip and repeat
the same process again.
ü Leave
the solution for 2 minutes.
ü At
first observe the diluted blood on 10x on microscope and after that observe on
40x.
ü Count
the RBC present on 5 medium squares (4 of 4 corners and one on the middle)
ü While
counting the RBC touching the right side and down side should not be counted.
ü Total
RBC counted should be multiplied by 10000 and is expressed on cubic ml.
CALCULATION
R= total RBC counted on
5 secondary square chamber
Total RBC present on 1
cu mm or 1 microlitre of blood= R*10,000
4.4
Total count of WBC
Materials
required:
ü WBC
diluting pipette (containing white grain inside)
ü Counting
chamber/ neubauers chamber
ü Cover
slip
ü Microscope
ü WBC
diluting fluid
Procedure
for counting WBC
ü Cover
the rule part of counting chamber with cover slip.
ü Mix
the blood with anticoagulant properly and suck the blood up to 0.5 mark of WBC
diluting pipette.
ü Discard
the extra blood above 0.5 mark
ü Suck
WBC diluting fluid up to 11 marks.
ü Mix
well the blood and WBC diluting fluid by keeping the WBC diluting pipette
within 2 palms.
ü Discard
2-3 drops of fluid and place one small drop of fluid at the edge of cover slip
and let the fluid to spread throughout the rule area and leave it for 1 minute.
ü With
the help of microscope observe it under 10 x power. The condenser of microscope
should be kept down so that the cells can be seen clearly.
ü Count
the WBC on 4 large squares present on 4 corners while counting WBC never count
the WBC on the down and right side.
Calculation
W=
total number of WBC counted on 4 large squares.
WBC
present on 1 cu mm or 1 microlitre blood = W*50
4.5 Differential count of WBC:
Before performing the differential
leukocyte count, a thin smear of blood should be made on glass slide. The smear
should be thin, leveled and should not contain any bubbles.
ü Let
the smear dry on air.
ü Number
the smear for identification
ü Fix
the smear by dipping on methanol solution for 5 minutes.
ü Dip
the smear on the giemsastain solution of 1:10 (1 part of giemsa stain mixed
with 10 parts of water) for 30 minutes.
ü Now
wash the slide with tap water let it dry.
ü Observe
under oil immersion lens (100x)
ü Count
at least 100 blood cells.
ü The
different type of cells visible by this process is eosinophils, basophils,
neutrophils, monocytes, lymphocytes.
ü The
number of cells is denoted in percentage.
Interpretation:
33. Lymphocytosis:
increased number of lymphocytes in blood is known as lymphocytosis.
Lymphocytosis is seen during viral infection, tuberculosis, brucellosis,
hypothyroidism, following vaccination, leukemia, adrenocorticainsufficieny etc.
34. Lymphopenia:
decreased number of lymphocytes in blood is known as lymphopenia. Lymphopenia
is seen during canine distemper, infectious canine hepatitis, corticosteroid
therapy, hypothyroidism, coxiellaburnetti
infection, F.M.D, mucosal disease etc.
35. Neutrophilia:
increased number of neutrophils in blood is known as neutrophilia. Neutrophilia
is seen during septicaemic disease, uremia, gout, coronary thrombosis, pyogenic
infection, acute inflammation, cancer, arthritis, pyometra, post-surgical
operation, pregnancy, calf diphtheria, rheumatic fever etc. the condition in
which the immature neutrophils are present in blood is known as shift to left.
36. Neutropenia:
the condition in which the number of neutrophils decreases is known as
neutropenia. Neutropenia is seen during the infection of diseases such as
pasteurellosis, bovine viral diarrhea, infectious canine hepatitis etc.
37. Eosinophila:
increased number of eosinophil is known as eosinophilia. Eosinophilia is seen
during parasitic infection, skin diseases, anaphylactic reaction etc.
38. Basophilia:
the condition in which the number of basophils increases in blood is known as
basophila. Basophilia is seen during pox, sinusitis, cirrhosis etc.
39. Monocytosis:
increased number of monocytosis in blood is known as monocytosis. Monocytosis
is seen during brucellosis, tuberculosis, carbon tetra chloride poisoning etc.
4.6 Collection of
blood serum
ü For the collection of serum the
blood should be collected on the vacutainer with red cover. The tube should be
Kept on the room temperature for 1-2 hours on inclined position. The blood
should clot. After the blood clots the tube should be kept on the refrigerator
overnight for clot retraction which makes easier for the collection of serum.
ü On
the next day the tube should be taken out from the refrigerator. With the help
Pasteur pipette the serum should be transferred to the serum vial.
ü The
tube should be centrifuged for the further collection of serum. The colour of
serum is slightly yellow.
ü The
vials containing serum should be kept on refrigerator. For longer storage
period the serum may be stored on dip fridge.
4.7Hemoglobin
estimation:
Pic:
sahli’shemoglobinometer (source: www.wikipedia.org)
a. Sahli’shemoglobinometer:
N/10 hydrochloric acid is put in the hemoglobinometer up to the mark 20 below.
Draw fresh or oxalated blood up to 20 c mm. mark of the hemoglobinometer
pipette. Immediately mix it with N/10 hydrochloric acid of the hemoglobinometer.
Mix it properly and allow it to stand in the comparator for 10 minutes for the
conversion of hemoglobin to acid haematin. Now add distilled water drop by drop
with the help of dropper and mix with stirring rod till the color matches well
the fixed colour in the comparator. The mark tallying with the upper level of
diluted acid haematin denotes the level of hemoglobin. This is expressed in
terms g per 100 ml of blood.
Normal level of hemoglobin in
different animals
|
Animal
|
Hemoglobin level (g/100ml)
|
|
Cattle
|
11.3
|
|
buffalo
|
12.9
|
|
horse
|
11.5
|
|
goat
|
10.9
|
|
sheep
|
14.4
|
|
pig
|
11.0
|
|
Dog
|
13.0
|
|
cat
|
12.0
|
Diminished level is observed in
case of anaemia. Increased level is observed in case of polycythaemia and
haemoconcentration.
Unit: 5
Urology
5.1 urine sample collection
While
collecting the urine sample, if the animal is urinating the middle stream of
the urine should be collected, if the animal is not urinating we can give
pressure on urinary bladder to make it urine forcefully. We can also use
catheter to collect the urine.
5.2 general properties of urine
We
cannot store urine at room temperature for a long interval of time because the
physical and chemical properties of urine may alter. If there is the presence
of bacteria in urine their number may rise dramatically. If the bacteria is
capable to breakdown the urea PH of urine may rise. If the urine is alkaline
the cast cells may dissolve and may not be visible while analyzing the urine so
fresh urine at morning time should be collected and analyzed.
5.3 Routine
examination of urine
Examination
of urine can be classified according to
1. Physical
examination
2. Chemical
examination
3. Microscopic
examination
1. Physical
examination: on the basis of physical examination, volume of urine,
color, smell, reaction, PH, specific gravity and transparency are examined
·
Volume: Volume of urine is influenced
by various factors such as; physical condition of animal, water intake,
environmental factors, temperature, feed etc. volume of urine of different
animals are as follows.
|
Species
of animal
|
Volume
of urine/day in litre
|
|
1.
Horse,mule
|
2-11 (Average 4.7)
|
|
2.
Cattle/buffalo
|
8.8-22.6 (Average 14.2)
|
|
3.
Sheep/goat
|
0.5-2 (Average 14.2)
|
|
4.
dog
|
0.5-2 (Average 14.2)
|
|
5.
pig
|
2-6 (Average 14.2)
|
·
Color:
Yellow
color of urine is due to presence of urochrome. Higher the urochrome
concentration the color of urine becomes dark yellow and lower the urochrome
concentration the color of urine becomes faint yellow. Most yellow color is suspected due to diabetes,
pyometra, jaundice or kidney malfunctions. Color of urine is also related to
specific gravity and volume of urine.
Various
color of urine is detected in laboratory. The common color of urine are listed
below
ü
colorless
ü
yellow
ü
dark
yellow
ü
pale
yellow
ü
yellowish
brown
ü
greenish
yellow
ü
greenish
ü
red
and milky color
-
Dark
yellow color is seen in acute nephritis, dehydration and fever.
-
Coffee
color is seen in case of babesiosis, hypo-phosphotaemia
-
Red
color is seen due to presence of hemoglobin, phenothiazine, phenophtalin,
azusulamide etc.
-
Milky
white is due to presence of pus and fat in urine.
-
Green
color is seen due to degradation of urine sample, phenol poisoning and
intravenous application of methylene blue.
-
Orange
color of urine is seen in the case of jaundice where urobilinogen is present.
·
Smell/Odor:
ü
Normally
urine is light aromatic.
ü
Pungent
odor is received due to the presence of ammonia forming bacteria.
ü
Putrid
sample of urine is received in the case of urinary system infection.
ü
Fruit
smell in urine is received in the case of ketosis.
·
PH:
ü
Urine
PH helps to understand the condition of kidneys. PH is analyzed by using litmus
paper, PH meter and reagent strip. If the blue litmus paper turns red the urine
is acidic and if it turns blue the urine sample is alkaline. PH of urine
differs according to animal species, feed and water supplement. PH of
herbivorous animals is alkaline whereas carnivorous animal is acidic.
|
Species
|
PH value
|
|
Horse
|
Alkaline 8.0
|
|
Cattle/Buffalo
|
Alkaline
(7.4-8.4)
|
|
Sheep/Goat
|
Alkaline
(7.2-8.4)
|
|
Pig
|
Alkaline or Acidic
depends upon the feed intake
|
|
Dog
|
Acidic( 6-7)
|
|
Man
|
Acidic (4.6-7)
Average 6
|
·
Specific gravity:
Specific
gravity is detected to know the filtration capacity of kidney. Specific gravity
is analyzed by using urinometer, refractometer and reagent strip.
|
Species of
animals
|
Specific gravity
|
Average
|
|
Horse
|
1.20-1.050
|
1.035
|
|
Cattle/Buffalo
|
1.015-1.045
|
1.035
|
|
Sheep/Goat
|
1.015-1.050
|
1.030
|
|
Dog/Cat
|
1.020-1.045
|
1.025
|
|
pig
|
1.005-1.025
|
1.015
|
·
Transparency:
-
Cattle and buffalo: Normally in the case of cattle
and buffalo fresh urine is clear and transparent.
-
The
urine of horse is thick and cloudy due to the presence of calcium carbonate.
2.
Chemical examination of urine:
The chemical
examination of urine is done to detect the presence of protein,glucose,ketone,
bile, blood etc in urine.
·
Protein test:normally the urine does not
contain protein. Protein in urine is seen during, contraction of muscles, consumption
of diet rich in protein,emotional stress, kidney’s failure and fever
Protein
test can be done
by:
a. Heat
and acetic acid test method
b. Robert’s
test
c. Sulfosalicylic
acid test
d. Reagent
strip test
a.
Heat and acetic acid test method:
ü
Add
2/3 part of urine on 5 ml test tube.
ü
If
the urine sample is alkaline, add some drops of 10% glacial acetic acid and
make the urine acidic.
ü
Heat
the upper part of the test tube.
ü
Add
some drops of 10% acetic acid on urine sample. If the urine gains cloudy color
we can understand that the urine sample contains protein, if the urine shows no
change in color absence of protein can be understood.
ü
In
this method protein and phosphate can be examined.
b. Robert’s
test:
ü
In
this method ,concentrated acid helps to coagulate the urinary protein, as a
result the presence or absence of protein in the urine can be detected.
ü
Place
2 ml of robert’s reagent on a clean test tube.
ü
Add
2 ml of urine sample (drop by drop) with the help of pipette by touching on the
wall of test tube. (if the urine sample is cloudy, it should be centrifuged
before performing the test)
ü
A
ring is formed on the area of mixing of urine and reagent, if the urine
contains protein (albumin) if the urine does not contain protein the ring will
not form.
c. Sulfosalicylic
acid test method:
ü
It
is a trusted method to detect the presence of protein in urine.
ü
If
the urine is alkaline add some acid to the urine and make acidic.
ü
Keep 2 ml of urine sample in a test tube ,add 4-5
drops of 20% sulfosalicylic acid on the test tube with urine sample.
ü
Heat
the test tube, if the urine becomes turbid the sample is declared to be protein
positive.
d. Reagent
strip method:
Protein
in urine can also be detected with the help of multistik, combistiketc
·
Glucose test:
Normally urine does
not contain glucose. In case of cattle/ buffalo, there is less importance of
sugar test. Sugar test has great importance in case of dog. Glucose is seen in
urine in the case of diabetes. To detect the glucose in urine Benedict’s test
is quiet helpful.
Benedict’s
test:
ü
Benedict’s
reagent is easily available at market
ü
Keep
5 ml of benedict’s reagent on test tube.
ü
Add
0.5 ml of urine sample on the test tube and heat the solution for 2 minutes on
Bunsen burner or spirit lamp.
ü
Be
careful that the solution my spill out from the test tube while burning on
Bunsen burner.
ü
Let
the test tube to cool, tally the colour obtained by the solution with the chart
provided on the reagent bottle.
·
Ketone test:
Normally the urine of
cattle/buffalo does not contains ketone bodies. Ketone body is composed of
acetoacitic acid (20%), acetone(2%) and betahydroxy butyric acid (78%). Ketosis
is the diseases caused by the alteration of fat metabolism due to which ketone bodies
are present in urine. This disease is mainly seen on lactating
cattle/buffaloes. To detect the ketone bodies rothera’s test is quiet helpful.
Rothera’s
test:
ü
Keep
4 mg of rothera’s reagent on a clean dry test tube.
ü
Add
some urine to dissolve the reagent powder.
ü
Observe
it after 1 minute.
ü
If
the reagent color change into purple color then the presence of ketone bodies
in urine is declared.
·
Bile salt
The biles present on
urine are urobilinogen, bile salt and bile pigments. Bile pigment is present on
the urine while there is malfunctioning of the liver. Bile salt can be tested
by following way.
Gmelin’s
test:
ü
Take
2 ml of urine sample on clean, dry test tube.
ü
Add
2 ml of concentrated nitric acid on the urine sample.
ü
If
the samples contain bile salt, it changes into greenish blue color.
ü
Bile
salt is present in urine during obstructive jaundice.
·
Blood:
ü
Keep
2 ml of urine on a test tube.
ü
Add
2 ml of benzidine saturated solution.
ü
Mix
1 ml of fresh hydrogen peroxide.
ü
If
the urine contains blood, it changes into blue or green color.
Presence
of blood in urine is known as hematuria, presence of hemoglobin in urine is
known as hemoglobin urea.
3. Microscopic
examination of urine:
Sediment of the urine
is used to examine the microscopic examination. In microscope epithelial cell,
pus cell, crystal, erythrocyte etc can be examined. Spermatozoa , parasites,
fungus and tumor cells can also be examined by this method.
Unit:
6
Microbiology
6.1
preparations of media
For the growth of the bacteria different types of
media should be prepared on the lab.
Most of the Medias are found prepared in market
1. Nutrient
broth: weight the required amount of the media dissolve it with mild heat,
after the media gets dissolved keep it on test tube or bottle and seal it. Keep
it on autoclave and maintain the temperature of 121 ° C for 15-20 minutes.
After that take the media out and refrigerate it at 2-8 ° C.
2. Nutrient
agar: weigh the required amount of the media and dissolve it with the help of
mild heat, maintain the PH of 7.4 and autoclave it at 121 °C for 15-20 minutes.
After it gets cooled Keep about 20 ml of media on sterilized Petri Dish and
cover it. Leave the media for about 15-20 minutes on room temperature and let
it to fix. It can be stored for several months at the temperature of 2-8 ° C.
3. MacConkey’s
agar: while performing the culture,
lactose fermenting bacteria produce pink colony on macConkey’s agar whereas non
lactose fermenting bacteria show no change in color. Required amount of media
is weighed and autoclaved. About 20 ml of media is taken out from autoclave and
kept on Petri Dish and covered. Media can be prepared on sterilized tube or
vial.
4. Blood
agar: This is red colored and mostly used for the growth of gram positive
lactose fermenting bacteria. Hemolysis is seen during bacterial growth on blood
agar. Required amount of media is weighed, heated and dissolved. It is
autoclaved at 121 °C for 15-20 minutes. It is brought out from autoclave and
cooled at 45-50 ° C. This task can also be performed at water bath. Sterilized
blood of sheep/rabbit (5-10%) of total media is added and cooled slowly. The
added blood should not contain any air bubbles. Wait for 15-20 minutes and let
it fix at room temperature. The surface of media in Petri Dish should be
leveled to 4 mm and refrigerated. To analyze the contamination it should be
kept on room temperature for 1 day.
5. Chocolate agar: it is chocolate colored agar.
This media is used for the growth of delicate bacteria. While preparing this
media blood agar is hemolysed and heated until chocolate color appears. It is
used for the growth of gonococcus, nemococcus
and hemophilusbacteria.
Reference: veterinary
prayogshalagyan by Dr. jibaccha shah
6.2 sample collection for
bacteriology
6.3 inoculations of media from
various types of specimen
Generally 2 methods of inoculation
of media are done.
1. Plate
culture
2. Testube
culture
1. Plate culture:
Solid state of media like nutrient agar,blood agar and macConkey agar prepared
in Petri Dish are incubated at 37 °C for 10 minutes to dry the moisture content
on the Petri Dish
Inoculating method:
ü Heat
the inoculating loop on the Bunsen burner until gets red.
ü Dip
the inoculating loop on the sample and stab the inoculating loop (3-4 lines) on
the media as shown in the figure.
ü Again
heat the inoculating loop till it gets red let it cool and again stab the
inoculating loop (3-4lines) on the media from another corner as shown in the
figure.
ü Again
heat the inoculating loop till it gets red let it cool and again stab the
inoculating loop (3-4lines) on the media from another corner as shown in the
figure.
ü Label
the Medias and incubate it at 37 °C on incubator for 24-48 hours.
ü After
24-48 hours examine the bacterial colonies present on the Medias. If the sample
is of lungs, liver, heart or any tissues cut it with the help of sterilized
scissors. The liquid obtained while cutting the samples should be again
inoculated with the help of inoculating loop.
Inoculation of culture on test tube
1.
Slope
method
2.
Inoculation
on broth
1.
Slope
method(inoculating on slope media):
In this
method straight inoculating needle is used.
ü Heat
the straight inoculating needle on the Bunsen burner until it gets red
ü When
the needle gets cool stab a vertical line on the slope media.
ü Inoculate
zigzag line with the help of inoculating loop on the vertical line stabbed by
the inoculating wire.
2.
Inoculation
on broth (liquid) media
ü If
the sample to be inoculated is on liquid state then inoculation should be done
with the help of sterilized Pasteur pipette on broth or other liquid media.
ü If
Pasteur pipette is not available then inoculating loop should be used for
inoculation.
ü Inoculating
loop should be heated at first and let to cool down.
ü While
using inoculating loop, at first the inoculating loop should be dipped on the
sample and the sample should be transferred to broth.
ü In
case of tissue sample, it should be cut into small pieces and a piece of tissue
should be kept on broth.
ü After
performing inoculation the media should be kept at 37 °C on incubator for 24-48
hours.
6.4
Examination of culture
We can view the growth of the bacterial
colonies on culture plates with naked eyes. The structures of the colonies also
help on distinguishing the bacteria.
1.
Size
The study of the bacterial colonies can be done by following criteria.
2.
Shape
3.
Smell
4.
Color
5.
Consistency
6.
Opacity
etc
1. Size:
observe the size of the colonies, colonies are measured on millimeter. The
classification of colonies can be done according to their size as follows.
ü Tiny
colonies= 0.1 millimeter
ü Small
colonies= 0.5-1 millimeter
ü Medium
colonies= 1-2 millimeter
ü Large
colonies=2-3 millimeter
The colony of streptococcus
bacteria is very small.
2. Shape:
ü The
shapes of the colony should be observed. The general shapes of the colonies are
round, irregular, lobate, rizoidetc
ü Observe
the part of the colonies whether they are elevated, flat, raised, convex etc
ü The
margin of the colonies should be examined whether it is filamentous or lobate
margin.
ü The
surface of the colonies should be examined whether it is smooth, rough or
papillae shaped.
The shape of the colony of bacillus bacteria is
large and slightly dry.
3. Smell:
The
smell of the some bacteria is of unique type. Proteus bacteria have ammonical smell, the growth of this bacteria
occurs all over the plate which is known as swarming growth.
4. Color:
The
general color of the bacterial colonies is red, white, green, yellow etc. if
the bacterial colonies acquire pink color on MacConkey’s agar , we can
understand that bacteria is lactose fermenting bacteria. Staphylococcus epidermis comprises of white color whereas staphylococcus aureus comprises of
slightly yellow colonies. Micrococcus
comprises of yellow or pink colonies likewise on MacConkey’s agar E.coli comprise of pink colony.
5. Consistency:
Examine
whether the colonies are sticky or non sticky.
6. Opacity:
Bacterial
colonies are transparent, translucent or opaque. On blood agar some bacterial
colonies show hemolysis. Streptococcus
aureus create hemolysis on blood agar. On blood agar Streptococcus pyogens create hemolysis around the colonies.
6.5 Gram’s staining methods
This method was founded
by the scientist named Gram on 1884 for staining the bacteria. The bacteria are
classified according to the color obtained by them while reacting with gram’s
stain. All Bacteria are classified under 2 groups
1. Gram
positive bacteria
2. Gram
negative bacteria
Principle:
This
principle is based upon the presence of lipid. Gram positive bacteria contains
low lipid and they have contains small pores on their cell walls due to which
decoloriser does not get entrance and the bacteria take the stain of the
crystal violet, as a result gram positive bacteria obtain blue color while
reacting with gram stain.
Gram negative
bacteria contains high lipid and the cell wall of the gram negative bacteria
contains large pores. Because of large pores on cell wall, decoloriser
(acetone) enters into the cell wall and removes the color of crystal violet and
after while the cell wall takes the color of counter stain (safranin). Due to
this reason gram negative bacterium gives red color on gram staining.
The PH of gram positive bacteria is (2-3) and the PH
of gram negative bacteria is (4-5)
6.6 Examination of milk by CMT
CMT is
done on laboratory to test whether the milk sample is infected with the
bacteria causing mastitis or not.
CMT reagent
can be prepared on the lab by mixing the following reagents
|
Name of the reagent
|
Amount of the reagent
|
|
Sodium hydroxide
|
1.5 gm
|
|
Teepol
|
0.5 ml
|
|
Bromothylene blue
|
0.01 ml
|
|
Distilled water
|
100 ml
|
Test method:
ü The
plastic paddles containing four cups which is known as CMT paddle is used to
test the milk by this method.
ü Place
3 ml of milk on each cups
ü Mix
3 ml of reagent on each cup. Move the paddle on circular motion and watch the
result after 20 seconds.
ü If
the tested sample is Mastitis positive then the mixture of milk and reagent
become thick, viscous.
ü This
method can be applied on Petri Dish also.
The report of CMT on lab should be made by following
the rules listed below
(-) negative= if there is no change in the mixture
(0-2, 00,000 somatic cell range)
(+) trace= if the mixture contains 1-2 granules (2,
00,000-4, 00,000 somatic cell range)
(++) weak positive= if the mixture is viscous (4,
00,000-12, 00,000 somatic cell range)
(+++) distinct positive= if the mixture is highly
viscous (12, 00,000-50, 00,000 somatic cell range)
(++++) severe positive= if the somatic cell range is
more than 50, 00,000
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