Download Model Answer B.Sc. VISemester LZC 602: Economic Zoology and

Model Answer
B.Sc. VI Semester
LZC 602: Economic Zoology and Biotechniques
SECTION-A (Multiple choice question)
ANSWER 1:
i.
ii.
iii.
iv.
v.
vi.
vii.
viii.
ix.
x.
d
b
d
d
a
b
b
b
d
a
SECTION-B Long answer
ANSWER 2: Sericulture, the art and technique of silk production, is an ancient industry in India
which is the second largest silk producing country in the world. Silk has played a vital role in the
history of human civilisation ever since its discovery 4,000 years ago. It occupies an important
place among all fabrics and is the "queen of textiles". It is traditionally associated with the socioeconomic life of many countries. Silkworm, Bombyx mori being a monophagous insect derives
almost all the nutrients essential for its growth from the mulberry leaf itself for production of
silk. Mulberry silkworm passes from four different life stages i.e. egg, larva, pupa and adult or
moth.
Silkworm eggs kept at 25 0C and 75 % RH for three days in incubation chamber for proper
development of embryo. Further, these eggs transfer into black box for two days to get uniform
hatching. Newly hatch out silkworm larvae known as ant. Young larvae, soon after hatching,
start feeding on mulberry leaves and grow.
At the end of the first instar larvae stop feeding completely and undergoes in moulting for 18 to
24 hours. The period of ecdysis is called the first moult. After moulting they resume feeding,
grow and repeat the moulting process. In common strains of silkworm, this repeated four times,
so it called as tetramoulter. At the end of 5th instar, silkworm larvae start spinning of silk thread
to form cocoon. Larvae spent 23-25 days in larval stage.
1
Climate required for rearing: Temperature and humidity is required for survival of silkworms
is between 23-280C and 65-85%.
Larval and moulting duration
1st instar: 3 days
1st moult: 18-20 hours
2nd instar: 2 days
2nd moult: 18-20 hours
rd
3 instar: 2 days
3rd moult: 18-20 hours
4th instar: 4 days
4th moult: 36-48 hours
th
5 instar: 6 to 8 days
Pupal stage is known as resting, non-feeding or inactive stage. But this is transitional phase, the
larval body & internal organs undergo a complete change into adult moth. The mature larva
passes through a short transitory stage of ‘pre-pupa’ before becoming pupa. Pupa spent 5-6 days
in pupal stage.
Soon after pupal stage, within 6-8 days pupa changes into moth and moths emerging from the
pupa are incapable of flight due to long domestication about >4000 years. Newly emerged
moths do not feed, their body surface covered with scales. Male and female moth copulating and
female lays about 300-400 eggs.
2
ANSWER 3:
Morphological features of egg, larva, pupa and adult
Eggs: The silkworm eggs are tiny. They measure 1- 1.3 mm in length and 0.9 – 1.2 mm in width.
Usually eggs of the European races are larger than those of Japanese or Chinese race. When the
larvae feed on highly nutritious mulberry leaves and the development of pupa is good, the eggs
laid tend to be larger in size. Also the eggs are larger if the external temperature is high during
the period between mounting and emergence of moth.
Silkworm eggs can be divided into hibernating and non-hibernating eggs. In hibernating eggs
the embryo develops only half-way undergoes a stage of dormancy called diapause, and hatches
out the following spring. In non-hibernating eggs, the embryo develops without undergoing
diapause and hatches out in the normal way. Silkworms laying hibernating eggs in spring, which
do not hatch out till the following spring, so producing only generation in a year are called
univoltines. When the first generation of moths lays non – hibernating eggs and the second
generation lays hibernating eggs which hatch out only in the following spring year then such
silkworms are known as bivoltines. Silkworms which lay non–hibernating eggs and so are able
to produce many generations in a year are called multivoltines.
Soon after oviposition the egg is usually pale yellow in colour. The eggs of silkworm which have
yellow blood and yellow cocoons are dark yellow in colour. The race producing white cocoons
lay pale yellow eggs.
Structure of silkworm egg a: Micropyle, b: Nucleus, c: Anterior polar
plasm, d: Chorion, e: Viteline membrane, f: Periplasm, g: Yolk
3
Larva: The newly hatched larva is about 3 mm long, black in colour and covered with bristles so
that it looks like a hairy caterpillar. Young larvae, soon after hatching, start feeding on mulberry
leaves and grow. After feeding for a little over three days, they stop feeding. At the end of the
first instar larvae stop feeding completely and after 24 hours they undergo ecdysis (shedding of
outer layer. After moulting they resume feeding, grow and repeat the moulting process. In
common strains of silkworm, this repeated four times.
Mature Larvae: At the end of fifth instar larvae stop feeding and start spinning from the
spinneret. The skin becomes semi-transparent. They are then called mature silkworms.
Head: The head is small, hypognathous (i.e. mouth ventral in position), heavily chitinised and
brown in colour. It bears ocelli and mouth parts. The mouth parts include a pair of strong
mandibles (lower jaw), a pair of lips (upper labrum and lower labium), and a pair of maxillae
(upper jaw) with maxillary and labial palps (appendages to find food). It also bears a pair of
antennae.
Thorax (Pro, Meso and Meta thorax): Each segment carries a pair of legs. Legs are made up of
3 joined segments. The true legs are conical shape with distal claws. Claws not used for crawling
but for holding mulberry leaves.
Abdomen: It is made up of 11 body segments but 9 can be distinguished, last 3 fused together to
form 9th segment. 3-6 and last abdominal segments each bear a pair of legs. Legs are fleshy and
form a sort of disc with a series of hooks inwardly curved.
Sexual marking: Female sexual marking 8th & 9th segments, a pair of milky white spots appear,
the pair of spots known as Ishiwata’s fore glands and hind glands, respectively. Male: a small
milky white body Herold’s gland appears between 8th & 9th segments. Nine pairs of spiracles
located laterally on either side of the body. Spiracles are respiratory pores. Larval skin or
integument consists of cuticle and hypodermis. Cuticle is made up of chitin and protein, covered
with thin layer of wax. Nodules are found all over the body.
4
Pupa: Generally the pupal stage is known as resting or inactive stage. But this is transitional
phase, the larval body & internal organs undergo a complete change into adult moth. The mature
larva passes through a short transitory stage of ‘pre-pupa’ before becoming pupa. During the pre
pupal stage the dissolution of the larval organs takes place and formation of adult organs takes
place. The pupa is white soon after pupation and gradually turns brown to dark. Visible
morphological parts of pupa are a pair of large compound eyes, antennae, wings and legs. During
pupal stage there are ten abdominal segments can be seen and seven pairs of spiracles can be
seen, however the last pair is not functional. Sex markings are predominant. Female has a fine
longitudinal line as the 8th abdominal segment.
Silkworm Pupa
Adult: Moths emerging from the pupa are incapable of flight due to long domestication about
>4000 years. Newly emerged moths do not feed, their body surface covered with scales. They
have compound eyes on either side of head, however their ocelli are absent. Antennae are
conspicuous; thorax consists of pro-meso and metathorax. The mesothorax is the largest one.
They have three pairs of thoracic legs, each leg is composed of five segments. The meso and
meta-thorax bear two pairs of wings. Females are larger and less active than males. At the
caudal end the male moth has a pair of hooks known as harpes. The female has knob like
projection with sensory hairs.
Silkworm moth
5
ANSWER 4:
Morphology of honey bee:
Rearing the bees in artificial hives for the production of honey is known as bee keeping or
apiculture. In India the bee keeping industry was started about 50-60 years ago. In the old age
people give smoke to comb at night for collection of honey. This was a crude method. Therefore
artificial method of bee keeping was adopted.
Apis indica, Apis mellifera, Apis dorsata and Apis florea are very common species used for
apiculture. Apis indica have black stripes on their abdomen and they live close to hilly areas and
are sometimes seen in plains regions also. These are less aggressive and also display less
swarming behavior than any other wild bees such as Apis dorsata and Apis florea and therefore
can be easily used for beekeeping.
Honey is a light brown colour viscous fluid produced by honey bees. It contains 78% sugar, 17%
water and 7% enzymes and minerals. To produce honey, honey bees suck the nectar from the
flower with the help of their proboscis and glossa and collect in comb. During sucking process
some saliva is also get mixed with to the nectar. This collected material is filled in the honey
chamber and dried with the help of fanning the wings. When it is ready the mouth of honey
chamber is closed with the wax. Honey is a very nutritious food with medicinal value.
In addition to thousands of worker adults, a colony normally has a single queen and several
hundred drones during late spring and summer. The social structure of the colony is maintained
by the presence of the queen and workers and depends on an effective system of communication.
The distribution of chemical pheromones among members and communicative dances are
responsible for controlling the activities necessary for colony survival. Labor activities among
worker bees depend primarily on the age of the bee but vary with the needs of the colony.
Reproduction and colony strength depend on the queen, the quantity of food stores, and the size
of the worker force.
Queens, Drones and Worker bees
Honey bee is social insect, which means that they live together in large, well-organized family
groups. Social insects are highly evolved insects that engage in a variety of complex tasks not
practiced by the multitude of solitary insects. Communication, complex nest construction,
environmental control, defence, and division of the labour are just some of the behaviours that
honey bees have developed to exist successfully in social colonies. These fascinating behaviours
6
make social insects in general, and honey bees in particular, among the most fascinating
creatures on earth.
A honey bee colony typically consists of three kinds of adult bees: workers, drones, and a queen.
Several thousand worker bees cooperate in nest building, food collection, and brood rearing.
Each member has a definite task to perform, related to its adult age. But surviving and
reproducing take the combined efforts of the entire colony. Individual bees (workers, drones, and
queens) cannot survive without the support of the colony.
Queens (perfect females)
The queen honeybee is the product of a fertilized egg, as are all females in the hive, however, the
queen receives a special diet throughout larval Queen Bee. That diet consists of royal jelly for
the first 3 days and a modified jelly thereafter, and it takes 16 days to produce a queen from an
egg. The queen bee is the biggest bee in the hive, mostly due to her elongated abdomen (for egg
laying). She is the sole source of replacement bees for the many that die daily from a variety of
causes (old age, predation and disease). And how many eggs the queen lays varies dependent on
her age, health, and available clean and empty cells within the hive. She mates with a variety of
drones during her maiden flights (mating does not occur within the hive), and can store enough
sperm in her spermatheca1 to last her lifetime. Normally there is one queen in a hive but there
can be exceptions and a queen may live 2 or more years.
Drones (Male)
Drones are the male bees and they have no father (being the product of an unfertilized egg). They
are very specialized in that they are defenseless (no stinger), do not forage (early in life are fed
by workers), come equipped with very large eyes and antennae with specialized receptors
enabling them to locate a queen on her maiden flight. Their sole purpose is to impregnate a
maiden queen and if successful their reward is death (upon uncoupling they leave behind part of
their anatomy which causes their demise). In areas with a prolonged winter they are evicted from
the hive as the weather gets colder and they often die of predation. A drone is produced from the
egg after 24 days and can live for approximately eight weeks.
Worker bees (imperfect females or pollen collector)
Worker bees are all females. They spend the first few weeks of their lives tending brood2 then
shift jobs to foraging for food. It takes 21 days for an egg to develop into a worker bee and she
can live, during foraging time 6 to 8 weeks but can overwinter in the hive. Worker bees are fed a
7
larval diet which is deficient in some ways (from that given to the queen larva), and results in the
bees sex organs not being fully developed. There are times though some worker bees may
develop functioning ovaries and lay Worker Honeybee unfertilized eggs (parthenogenesis) which
normally will result in drone bees (it is a telltale sign of a queenless hive). Additionally, there is
evidence from studies done with Apis mellifera (the Cape honeybee) that a worker bee can/may
become a pseudo queen and produce female offspring from diploid eggs also through a form of
parthenogenesis (without mating) called Thelytoky.
Drone
Queen
Worker
ANSWER 5:
Rohu (Labeo rohita) is the largest of the three Indian major carp species used in carp
polyculture. This graceful Indo-Gangetic riverine species is native to the rivers of northern and
central India, as well as those of Pakistan, Bangladesh and Myanmar. In India, it has been
transplanted in almost all rivers, including balmy Andaman where she successfully established
waters. This species has also been introduced in many other countries, such as Sri Lanka, the
former Soviet Union, Japan, China, the Philippines, Malaysia, Nepal and some African countries.
There are hundreds of years that the carp is traditionally high in small ponds of the states of
eastern India.
Biology and development of Rohu: Bilaterally symmetrical body, moderately elongated, the
dorsal profile is more curved than the ventral profile; body covered with cycloid scales except
the head scaleless, rather flat nose, projecting beyond the mouth, without side lobe; eyes
dorsolateral position not visible from the exterior of the head; small and lower lips; thick, each
lined lips of a separate internal folds, lobed or entire; a pair of maxillary small barbs hidden in a
lateral groove; no teeth on the jaws; three rows of pharyngeal teeth; upper jaw not reaching the
anterior margin of the eye; single dorsal fin, with three or four non-forked rays and 12-14 rays
8
forked; dorsal fin inserted midway between tip of snout and base of caudal fin; pectoral and
ventral fins inserted laterally; pectoral fins without spine bone; caudal fin deeply forked; lower
lip usually attached to the isthmus by a narrow or wide bridge; 12-16 predorsal scales; separate,
complete and located along the midline of the caudal peduncle lateral line; lateral line scales 40
to 44; six or six and a half rows of scales between the lateral line and the base of the ventral
fin; not truncated snout, without lateral lobe; bluish color on the back, silvery on the sides and
belly.
During its early stages of development rohu prefer zooplankton, mainly composed of rotifers and
cladocerans, phytoplankton constituting an emergency food. Stage large fry, there is a strong
positive selection for all major zooplankton and to some smaller phytoplankton organisms, such
as algae desmids, phytoflagellates and algae spores. As adults, they have a strong positive
selection for most organizations phytoplankton. The juvenile and adult stages rohu is essentially
a herbivore feeding between the two waters, preferring algae and submerged vegetation. In
addition, the presence in his gut decomposed organic matter, sand and silt suggests feeding on
the bottom. A mouth made for snacks, soft lips, sharp edges and the absence of teeth in the oral
pharyngeal region allow the fish to feed on tender aquatic plants that do not require be seizing
and crushing. Modified, thin and hair-like gill rakers, also suggest that this fish feeds on tiny
plankton by filtering water. Ponds and small fingerlings travel in schools mainly for food.
However, this behavior is not observed in adults. Rohu is eurythermic. Below 14 ° C does not
suit him. Its growth is rapid. Under normal growing conditions, it reached a total length of about
35-45 cm and a body weight of 700-800 g. In polyculture, its growth rate is generally higher than
the mrigal but lower than catla.
Production system
Rohu is the main species in carp polyculture systems, together with the other two Indian major
carps viz ., the catla (Catla catla ) and mrigal (Cirrhinus mrigala). Its feeding niche is wider (the
period between the waters to the bottom), this species is usually stocked at relatively higher than
the other two species density. In India, it is also high in carp farming systems made, including
not only the three Indian major carps but the common carp (Cyprinus carpio) and two Chinese
carps viz. , silver carp (Hypophthalmichthys molitrix) and grass carp (Ctenopharyngodon
idellus). Even in this combination to six species, the percentage of rohu is maintained at 35-40
percent as in the system of three species polyculture. In recent years, consumer preference for
9
rohu and most important demand that resulted also encouraged the breeding system of two
species, with catla. This latter type of aquaculture is currently practiced in 100 000 ha of ponds
in the region of Lake Koleru in Andhra Pradesh, India, where more than 70 percent of the stock
consists of rohu. Three Indian major carps, rohu is the most important, are the dominant species
cultured in other countries such as Bangladesh, Pakistan, Laos, Vietnam and Nepal. In all these
countries, silver carp, grass carp and common carp are the most commonly bred in aquaculture
with three Indian major carp species.
Production cycle of Rohu
ANSWER 6:
Instrumentation – Instruments for measuring the absorption of radiation by solutions are known
as colorimeter, absorptiometer or spectrophotometers. The term colorimeter is restricted to the
simpler visual and photoelectric devices for the visible region. The term absorptionmeter
includes the class of colorimeter, but can be applied to other spectral region as well.
Colorimeters having photocells consist of filters are known as filter photometers. Such
10
colorimeters in which filters are replaced by monochromators (i.e. prism or gratings) for
monochromatising the light are known as spectrophotometers.
The basic components for photoelectric instrument (photometer or spectrophotometer)
are as follow
1. An intense source of radiant energy
2. A filter or monochromator to isolate the wavelength region to be used in irradiating the
coloured solution.
3. A pair of cells or curettes' one for the coloured & othr for the blank or reference solution
4. A photometer, comprising a photoelectric detector which converts radiant energy to
electrical energy and a meter to indicate the resulting electric current.
1. Sources – A powerful, stable & continuous beam of radiation is foremost requirement for
it. The most common source of visible radiation is the incandence tungsten filament lamp
which produced radiation in the range of 3500-8000A°. With the help of transformer or
voltage regulator, the constt. Intensity of beam is maintained for a single beam
instruments.
2. Filters/Monochromators – Lamp produces a constinuous emission of all wave lengths
within visible range. Thus an instrument must have an optical system to select
monochromatic light (i.e. light of specific wavelength). This is done by using filters,
prisms, gratings according to the type of instrument. Filters are madeup of coloured glass
which may be of two type – (1) – Absorption filter, (2) Interference filter. In general,
filters isolate a wider band than a monochromator but some interference. Filter have a
narrow half intensity band width than some monochromators.
 Similarly a monochromator, isolates the narrow band of desired wavelength. It consist of
entrance and exit slits and a dispersing device, either a prism or grating. The material of
11
the construction of prism is glass. The exit beam of a monochromator is usually
contaminated with small amount of radiations which may be or wavelength much different
from the instrument setting. These unwanted radiations known as "spurious radiations" or
"Stray radiation" which may be minimized by coating interior surface with flat black
point. Many modern monochromator contains two dispersing elements i.e. two prism or
two grating or one prism & one grating. This arrangement reduce the amount of stray
radiation.
Slits – The light enters through a slit, a spectrum is obtained by the light passing through a
prism or grating, and by suitably rotating the prism, required wavelength alone is
superimposed on the stationary exit slit. Thus slits are of two type (1) Entrance slit (2) Exit
slits.
Gratings – Replica grating are usually employed since, these are cheaper than prism. The
important drawback with grating is that they give more than one order of diffraction.
Strong radiation, if any may either be eliminated by using two gratings or by using filters
in front of entrance slit.
3. Cuvette  The sample in placed in a tube called cuvette. Cuvette is made-up of colour
corrected fused glass which is unable to give any absorption and only solution gives
absorption cuvette may either be rectangular or cylindrical in shape but rectangular shape
is preferred because when cylindrical cuvette are in use, the entrance & exit sides are
curved and there may be slight devtation from Beer's law. Glass or silica cuvettes are
suitable for absorbance measurement in visible region while quartz cuvette is useful both
in UV-Vis regeion. Although plastic containers have also been used in visible region.
Thickness of cuvette of 1, 2 and 5 cm are quite common.
4. Radiation Detector & Indicators
The intensity of light that passes through the sample depends upon the amount of light
absorbed by the sample. These changes of intensity of light are measured by detectors.
These detectors have low noise level and higher sensitivity even for small change in
intensity. For this purpose, the instrument is equipped with photoelectric devices which
converts radiant energy into electrical signals. There is a photomultiplier tube as a
detector. The photomultiplier tube is a combination of photo emissive cathode and an
internal electron multiplying chain of dynodes. Incident radiation ejects the
photoelectrons from the cathode which are further accelerated by dynodes, and finally
converted it into an electrical signal which is then read by a measuring instrument. This
measuring instrument may be a reflector meter or a digital recorder or digital screen.
12
Instrument Design
Spectrophotometer may employ a single beam or double beam optical system.Single
beam – An absorption measurement with a single beam photometer or spectrophotometer
involves three steps –
(a) The indicator is first adjusted to zero transmittance or infinite observance in which no
radiation strikes the detector
(b) Then the indicator is adjusted to 100% transmittance or zero absorbance, with the cell
filled with solvent (reference sol. Or blank sol.) in the beam of instrument.
(c) Finally the solvent is replaced by the solution under examination and transmittance or
absorbance is noted directly from the indicator scale.
The stability of the source is very imp. For reliable photometric measurement with a single
beam instrument.
Double beam Instrument – Double beam instrument is lodged with beam splitting device to
produce two optical beams. One beam passes through the reference cell – while the other
passes through the sample cell. Some instrument have a detector for each beam so that the
ratio o the two photo currents being produced can be measured.
Single beam instruments are used in quantitative analysis while double beam instruments
useful for qualitative analysis. Moreover, double beam device adapted for continuous
monitoring of absorbance, hence all modern recording spectra photometers employ thin
beam.
13
ANSWER 7:
A centrifuge is a piece of equipment, generally driven by an electric motor (or, in some older
models, by hand), that puts an object in rotation around a fixed axis, applying a force
perpendicular to the axis. A centrifuge is also used to separate the components of blood in blood
banks. The centrifuge works using the sedimentation principle, where the centripetal
acceleration causes denser substances to separate out along the radial direction (the bottom of
the tube).
Essentially, the rate of sedimentation is dependent upon the applied centrifugal field (cm
s-2), G, that is determined by the radial distance, r, of the particle from the axis of rotation (in
cm) and the square of the angular velocity, ω, of the rotor (in radians per second):
G = ω 2r
Many different types of centrifuges are commercially available including:
Bench-top or clinical centrifuges:
Some large-volume centrifuge models are quite demanding on space and also generate
considerable amounts of heat and noise, and are therefore often centrally positioned in special
instrument rooms in biochemistry departments. However, the development of small-capacity
bench-top centrifuges for biochemical applications, even in the case of ultracentrifuges, has led
to the introduction of these models in many individual research laboratories. Simple bench-top
centrifuges vary in design and are mainly used to collect small amounts of biological material,
such as blood cells. To prevent denaturation of sensitive protein samples, refrigerated
centrifuges should be employed. Modern refrigerated microfuges are equipped with adapters to
accommodate standardised plastic tubes for the sedimentation of 0.5 to 1.5 cm3 volumes. They
can provide centrifugal fields of approximately 10 000 g and sediment biological samples in
minutes, making microfuges an indispensable separation tool for many biochemical methods.
Large-capacity low-speed preparative centrifuges: In contrast to bench top centrifuges, these
centrifuges have large capacity to accommodate samples that may vary upto 150 ml. They can
provide centrifugal fields of approximately 10 000 g and sediment biological samples in
minutes.
High-speed refrigerated centrifuges: High-speed refrigerated centrifuges are absolutely
essential for the sedimentation of protein precipitates, large intact organelles, cellular debris
derived from tissue homogenisation and microorganisms. The initial bulk separation of cellular
14
elements prior to preparative ultracentrifugation is performed by these kinds of centrifuges.
They operate at maximum centrifugal fields of approximately
100 000 g. Such centrifugal force is not sufficient to sediment smaller microsomal vesicles or
ribosomes, but can be employed to differentially separate nuclei, mitochondria or chloroplasts.
In addition, bulky protein aggregates can be sedimented using high-speed refrigerated
centrifuges. An example is the contractile apparatus released from muscle fibres by
homogenisation, mostly consisting of myosin and actin macromolecules aggregated in
filaments. In order to harvest yeast cells or bacteria from large volumes of culture media, highspeed centrifugation may also be used in a continuous flow mode with zonal rotors. This
approach does not therefore use centrifuge tubes but a continuous flow of medium. As the
medium enters the moving rotor, biological particles are sedimented against the rotor periphery
and excess liquid removed through a special outlet port.
Ultracentrifuge: Ultracentrifugation has decisively advanced the detailed biochemical analysis
of subcellular structures and isolated biomolecules. Preparative ultracentrifugation can be
operated at relative centrifugal fields of up to 900 000 g. In order to minimize excessive rotor
temperatures generated by frictional resistance between the spinning rotor and air, the rotor
chamber is sealed, evacuated and refrigerated. Depending on the type, age and condition of a
particular ultracentrifuge, cooling to the required running temperature and the generation of a
stable vacuum might take a considerable amount of time. To avoid delays during biochemical
procedures involving ultracentrifugation, the cooling and evacuation system of older centrifuge
models should be switched on at least an hour prior to the centrifugation run. On the other hand,
modern ultracentrifuges can be started even without a fully established vacuum and will
proceed in the evacuation of the rotor chamber during the initial acceleration process. For safety
reasons, heavy armour plating encapsulates the ultracentrifuge to prevent injury to the user in
case of uncontrolled rotor movements or dangerous vibrations. A centrifugation run cannot be
initiated without proper closing of the chamber system. To prevent unfavourable fluctuations in
chamber temperature, excessive vibrations or operation of rotors above their maximum rated
speed, newer models of ultracentrifuges contain sophisticated temperature regulation systems,
flexible drive shafts and an over-speed control device. Although slight rotor imbalances can be
absorbed by modern ultracentrifuges, a more severe misbalance of tubes will cause the
centrifuge to switch off automatically.
15
ANSWER 8:
Staining of paraffin section
The most common method of histological study is to rpeapre thin sections (3-5 micron) from
paraffin embedded tissues. These are then suitably stained and mounted in a medium of proper
refractive index for study and strong. Commonest mountains used are resinous substances of
refractive index close to that of glass. These are soluble in xylol. Hence sections are dehydrated
and cleared inxylol and mounted. Mounting in aqueous mounting media is done directly after
staining for sections which cannot be subjected
to dehydrating and clearing agents. The basic steps in staining and mounting paraffin sections
are as follows:
1.
Deparaffinisation
2.
Hydration
3.
Staining
4.
Dehydration and clearing
5.
Mounting
1. Deparaffinisation
Removal of paraffin is done with xylol. It is essential to remove the wax completely, otherwise
subsequent stages will not be possible. At least 2 to 3 changes in xylol are given for suitable
length of time. Sections of this stage should appear clear and transparent. Presence of any
patches indicates the presence of wax and sections should be kept longer in the xylol.
2. Hydration
Most of the stains used are aqueous or dilute alcoholic solutions. Hence it is essential to bring
the section to what before the stains are applied. The hydration is done with graded alcohol for
higher concentration to lower concentration. Alcohol and acetone are miscible with xylol. First
change is made to absolute alcohol or acetone followed by 90, 70% alcohol and finally distilled
water. Sections now should appear opaque. Presence of any clear areas are indicative of the
presence of xylol. To remove this xylol, sections should be returned to absolute alcohol and
rehydrated.
3. Staining
Various staining procedures are applied from this hydrates stage. The most common stain
applied for histological study is Haemotoxylin and Eosin. Various types of haemotoxylin
16
formulations are used. Certain of the stains use strong chemicals e.g. ammonia. Sections tend to
float off the slides in such stains. This can be prevented by coating the selections by a thin
layers of celloidin. For this sections are returned to absolute alcohol and then dipped in a dilute
solution of celloidin and finally hardened in 70% alcohol.
4.
Dehydration and clearing
Dehydration is done is graded alcohols or acetones from 70% to absolute alcohol or acetone.
Dehydrating alcohol and acetones can remove some of the stains. Time has to be suitably
modified to minimize fading of stains Since alcohol and acetone are miscible in xylol, it is used
for clearing the sections. Any sections from which water has not been completely removed
would give a milky appearance after the first xylol. Such sections should be returned to abs.
alcohol and the process repeated. Mounting is done after
2nd or 3rd xylol.
5. Coverslipping and mounting
Make quite sure that the sections are quite clear. Do not let the section go dry before mounting
1. Hold the slide between the thumb and the forefinger of one hand and wipe with a clean cloth
both ends of the slides. Look for the engraved number to make sure the side the sections is
present.
2. Clean carefully around the section and lay on a clean blotting paper with section uppermost
along with appropriate coverslip which has already been polished.
3. Place a drop of mountant on the slide over coverslip. Amount of mountant should be just
enough. Invert the slide over the coverslip and lower it so that it just adheres to the cover slip
quickly turn the slide over the lay it on a flat surface to allow the mountant to spread. Do not
press or push the slide at all.
4. After the mountant has spread to the edge of the coverslip wipe around it for neatness. If
proper care has been taken there should be no air bubbles. If many are present, slide should be
returned to the xylol to remove the coverslip. It will slip off and remounting is done. No attempt
should be made to pull the coverslip. Slight warming of the slide from below will make the
small air bubbles to escape from the slide of the coverslip.
5. Coverslip should be in the center of the slide with neatly written label on one slide.
17