Handbook of Practicals in Zoology

A HANDBOOK OF
PRACTICALS IN
ZOOLOGY
Dr. C. Varnan Rao M.Sc., Ph.D.
Formerly, Reader & Head Dept. of Zoology,
8t. Xavier's College, Mumbai-400001.
Presently, Professor, Dept. of Biotechnology,
NMAM Institute of Technology, Nltte-57 4110
Dr. Srnita Krishnan M.Sc., Ph.D.
Reader & Head,
Dr. Madhuri Harnbarde M.Sc., Ph.D
Reader In Zoology,
Dept. of Zoology,
8t. Xavier's College,
Mumbai-400001.
Dept. of Zoology,
Mumbai-400001.
Dr. Pushpa Sinkar M Sc Ph D
. "' ..
Senior Lecturer,
Dept. of Zoology,
Mumbai-400001.

Gffimalaya GpublishingGJiouse
MUMBAI NEW DELHI. NAGPUR BANGALURU HYDERABAD CHENNAI
PUNE LUCKNOW AHMEDABAD ERNAKULAM INDORE. BHUBANESWAR
Authors
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CONTENTS
~ C T I C L I
1 - 69
ANIMAL DIVERSITY (INVERTEBRATES) 3 - 36
1. Mounting of formaniferan shells from sand (3).
2. Observation of binary fission and conjugation in Paramecium (4, 5).
3. Observation of V. S. of Grantia and L. S. of Leucosolenia (5, 6).
4. Observation of polymorphism: Obelia colony (6, 7), Obelia medusa (7, 8), Physalia
(8,9), Velella (9), Porpita (9, 10).
5. Observation of types of corals: Fungia (10), Meandrina (10, 11), Madrepora (11),
Tubipora (11, 12), Sea fan (12).
6. Observation of Helminths T. S. ofliver fluke (13), Liver fluke larvae [Miracidium (13,
14),
Redia (14), Cecaria (14, 15)], Scolex of tapeworm (15), Mature proglottid (16),
Cysticercus (16, 17),
7. Observation of Annelids Heteronereis (17) and Trochophore larva (18).
8. Study of crustacean larvae: Nauplius (18,19), Zoea (18, 19), Megalopa (19),
Alima (20), Mysis (20, 21), Phyllosoma (21, 22) and Cypris (22).
9. Study of metamorphosis of insects:
(i) Juvenile and adult of Lepisma (22, 23).
(ii) Different stages in the life history of
Housefly (23, 24), Mosquito (Culex or Anopheles) (23,24), Butterfly, Beetle, Silk
moth (27,28)
10. Types of foot in mollusca: Chiton, Patella, Aplysia, Mytilus, Solen, Cardium, Sepia
(29-30).
11. Types of shells in molluscs: Chiton, Dentalium, Trochus, Placuna, Solen (31, 32),
Internal shell of Sepia, Shell of Nautilus, Sinistral and dextral shells (32, 33).
12. Study of Echinoderm larvae: Bipinnaria (33), Ophiopluteus (34),
Echinopluteus (34), Auricularia (35), Doliolaria (36).
ANIMAL DIVERSITY (CHORDATES): 37 - 55
13. Study of Ascidian tadpole (Retrogressive metamorphosis) (37).
14. Swim bladder in-situ .(Rohu) (37, 38)
15. Study of following organisms with reference to breeding and parental care:
Seahorse, Gouramy, Siamese fighter, Catfish, Tilapia (38, 39)
Caecilian, Midwife toad, Neoteny: Axolotl larva (39, 40).
16. Adaptive radiation in Reptiles: Turtle (40, 51), Tortoise (41, 42), Chameleon (40, 41),_
Phrynosoma (43), Wall lizard (44), Rat snake (44, 45), Sea snake (46), Crocodile or
Gharial (46, 47).
17. Venomous and Non-Venomous snakes: Identification Key (47, 47), Krait (49), Cobra
(49,50),
Russell's viper (50), Saw scaled viper (50,51),
Jaw of any venomous snake to show the fangs and poison apparatus (51).
18. Mammals: Duck billed platypus (52), Kangaroo (52, 53), Pangolin (55, 56),
Bottlenose dolphin (54), Blue whale (55), Sea cow (56).
DEVELOPMENTAL BIOLOGY 56 - 60
19. Study of different types of eggs: Isolecithal (Homolecithal)-Amphioxus or
Mammal (56, 57),
Mesolecithal-Frog or fish or mollusk (57, 58),
Discoidal or Highly telolecithal-hens egg (57, 58).
20. Study of different types of blastulae: Amphioxus, Frog, Mammal (58).
21. Study of gastrulae-Frog (59), Primitive streak (59, 60) and section passing through
primitive streak of chick embryo (60).
PRACTICALS IN ECOLOGY 61 - 69
22. Quantitative estimation of DO of water (61, 62).
23. Quantitative estimation of biochemical oxygen demand in water (1 hr. BOD) (62).
24. Quantitative estimation of free carbon dioxide in water (63, 64).
25. Quantitative estimation of phosphateor phosphones in water (64,65).
26. Quantitative estimation of salinity of water-Argentometric method (65, 66).
27. Determination of total hardness of water (66).
28. To investigate the water content of soil (67).
29. To investigate the organic content of soil (67,68,69).
30. To investigate pH of soil (69).
I PRACTICAL II 71 - 114
1. Study of pH meter-Principle and working (73, 74).
2. Preparation of buffers of different pH using Henderson-Hasselbalch equation
(74,75).
3. Preparation of titration curve using strong acid and strong base with the help of pH
meter (76).
4. Determination of pKa of weak acid (76).
5. Study of colorimeter: Principle and working (77).
(i) Selection offiltez: (78) (ii) Determination of (79).
6. Study of osmosis using RBCs (80, 81).
7. Study of ultra structure from electron micrographs of Mitochondria, ER, Golgi complex,
Nucleus, Lysosomes (82-84).
8. Study of chromosome morphology using temporary squash preparation of onion root
tip (85, 86).
9. Study of polytene chromosome: Temporary squash preparation of salivary gland
chromosome oflarva of Chironomous/DrosophilaIMosquito (87,88).
10. Mimicry and warning colours (89-94).
11. Estimation of blood glucose using glumeter and using glucose estimation kot (God
method (94, 96).
12. Problems based on biotechnology and bioinformatics (97-105)
13. Study of Barr body from buccal epithelium (106, IOn
14. Problems in Genetics with reference to: X-linkage, Multiple alleles, Multiple genes,
and linkage (108-114).
PRACTICAL III 115-161
1. PARASITOLOGY
Types and Identification of: Entamoeba (117), Plasmodium (117,118),
Trypanosoma (118, 119), Leishmania (119, 120),
Taenia whole specimen (120, 121), Ascaris (121), Ancylostoma (121, 122),
Wuchereria (122).
117 - 126
Parasitic Adaptations: Scolex of tapeworm (122), Mature and gravid proglottid of
tapeworm (123-125), T. S. of Ascaris (125, 126).
2. ENTOMOLOGY 127 - 133
2.1 Honeybee: Life history (127), Mounting of mouthparts (128),
2.2 Silk moth: Life history sting apparatus (134,135) and legs (135, 136), Beehive (135,
136).
Harmful insecks: Locust/grasshopper (131), Aphid (131), Rice weevil (131, 132), Flour
e ~ t l e (131, 132).
Entomophagous insect: Dragonfly (132).
Parasitic insect: Ichneumon wasp (13;3)
3. FISHERIES
Fresh water Fishery: Rohu (134), Catla (134), Mrigal (135).
Marine Fishery: Oil sardine (136), Mackerel (136),
Bombay duck (136), Pomfret (137), Shark (137),
Prawn (137,138), Lobster (138), Crab (139),
Edible oyster (139), Pearl oyster (139),
134 - 146
Sepia, Loligo (139, 140), Katelysia (140), Mytilus (141), Dried Bombay duck (141),
Salted mackerel (141).
Fishing Crafts: Dugout canoe (141), Outrigger canoe (142),
Catamaran (142), Masula (142, 143), Satpati (143), Trawler (143, 144).
Fishing gears: Gill net, Dol net, Cast net, Purse net, Rampani net,
Long line (144-146)
4. ANIMAL HUSBANDRY
Poultry: Leghorn (layers) and Broiler (147).
147 - 161
Cattle: Milch breed - Sahiwal, Dual purpose - Hariana, Draught purpose - Killari
(147-148).
Buffalo: Murrah, Jaffarabadi (148, 149).
Goat: Jamunapari, Surti (149, 150).
Sheep: Gaddi, Marwari (150, 151)
Colorimetric estimations:
Colorimetric estimation of protein in Egg-Folin Lowry or Biuret method (152, 154)
Colorimetric estimation of total lipids in yolk (155, 156)
To find adulterants in the milk sample (156, 158)
Colorimetric estimation of total fats in the in different varieties of milk (158, 159)
Extraction of casein from milk and its qualitative test (159, 160)
To measure the density of milk by Lactometer (160, 161)
Panir making (161).
Practical - I
"This page is Intentionally Left Blank"
ANIMAL DIVERSITY (INVERTEBRATES)
1. MOUNTING AND OBSERVATION OF
FORAMINIFERAN SHELLS
These organisms belong to the class Rhizopoda of phylum Protozoa They are manne and
mostly confined to bathyal and abyssal regions. When alive they are free SWimming forms Their
body is generally covered by calcareous, siliceous, gelatinous or chitinous skeleton After the
death of the organism, the shell remains and gets washed off onto the sandy shores The shell
may be single chambered or tubular or monocular or many chambered or multilocular. It differs
from species to species in its architecture. The shell is highly porous through which string like
pseudopodia streams out when the animal is alive. The following specimens could be commonly
seen in the sand sample.
1. Elphidium, 2. Cyclamina, 3. Textularia, 4.Bathysiphon, 5. Saccamina, 6. Saccorhlza,
7. Rhabdamina, 8. Spiroloculina, 9. Turispirillina. Structures of these shells are as shown In the
Fig. 1.1.
ELPHIDIUM CYCLAMINA
BATHYSIPHON SACCAMINA
RHABDAMINA SPIROLOCULINA
f1j

TEXTULARIA
SACCORHIZA
A.

TURISPIRILLINA
Fig. 1.1. Different types of for.aminiferan shells found in sand samples
4 A HANDBOOK OF PRACTICALS IN ZOOLOGY
2. OBSERVATION OF BINARY FISSION AND
CONJUGATION IN PARAMECIUM
(FROM PERMANENT SLIDE)
BINARY FISSION:
This is an asexual mode of reproduction. The micronucleus and macronucleus show
elongation and divide at the median transverse constriction, which divides the body Into two equal
halves. Each half receives a daughter macro and micronucleus and a contractile vacuole. Later
the two daughter cells separate and form two Individual paramecia
MICRONUCLEUS
MACRONUCLUES
Fig. 2.1. Binary fission in Paramecium.
CONJUGATION IN PARAMECIUM:
This IS a sexual mode of reproduction, where two paramecia come In contact with each
other from their oral surfaces The ectoplasm of the oral surface degenerates and a cytoplasmic
bridge IS formed between the two conjugants The macronucleus disappears and the micronucleus
divides twice to give rise to four micronucleus, of which three disappear and one remains The
remaining Single nucleus divides unequally once again to form a large stationary female pronucleus
and a small migratory male pronucleus. The migratory male pronucleus of both paramecia is
exchanged reciprocally through the cytoplasmic bridge After the exchange the male pronucleus
fuses with the stationary female pronucleus-giving rise to zygotic nucleus, which divides into four
each migrating Into a daughter paramecium formed later.
MIGRATORY
MALE PRONUCLEUS
STATIONARY FEMALE
PRONUCLEUS
CONJUGANTS
Fig. 2.2. Conjugation in Paramecium
3. OBSERVATION OF V. S. AND L. S. OF SPONGE
L. S. OF LEUCOSOLENIA:
5
This sponge shows simplest radial form with a tubular body or asconoid structure Each
tube that exists in clusters attached together along their longitudinal aXIs or at their bases IS
perforated by many small openings, called ostia (or incurrent pores) These pores open Into the
interior cavity, the spongocoel (atrium), which in turn opens to the outside through the osculum,
a large opening at the top of the tube. The body wall IS relatively simple covered by epithelial-like
flattened cells, known as the pinacocytes, which together make up the pinacoderm Flagellate
cells called choanocytes line inner body cavity or spongocoel (Fig. 3.1).
V. S. OF GRANTIA:
This sponge shows first stage of body wall folding, hence shows syconoid type of canal
system or syconoid structure. In syconoid structure, the body wall has become "folded" forming
external pockets extending inward from outside, and evaginations, extending outward from the
spongocoel. The many pockets produced by folding do not meet but bypass each other and are
blind. The Choanocytes (flagellated cells) no longer line the spongocoel but are confined to the
evaginations, which are called flagellated or radial canals The corresponding invaglnations from
the pinacoderm Side are known as incurrent canals and are lined by plnacocytes The two canals
are connected by openings called prosopyles, which are equivalent to the pores of asconold
sponges (Fig. 3.2).
DERMAL CORTEX
OSCULLUM
RADIAL CANAL
(FLAGELLATED CHAMBER)
EPITHELIUM
MESENCHYME
SPONGOCOEL
CHOANOCYTES
INTERNAL OSTIA
GASTRAL CORTEX
INCURRENT CANAL
Fig. 3.1. L. S. of Leucosolenia showing
Asconoid canal system.
Fig. 3.2. V. S. Grantia showing
Syconoid canal system.
4. OBSERVATION OF POLYMORPHISM
OBELIA COLONY:
1. The colony consists of hollow root-like tubes, the hydrorhiza, which runs over the surface
of attachment, such as a seaweed, and from these, arise free upright stems, the hydrocauli
which usually end in zooids.
2. The hydranths, or polyps are present at the growing ends of the main branches Each
polyp has a bell-shaped body and is provided with a mouth, which IS surrounded by a
crown of long slender tentacles. It has an investing hydrotheca.
3. The blastostyles occur towards the base of the branches in the axils of the hydranths.
The blastostyle has neither mouth nor tentacles, but is invested by gonotheca.
ANIMAL DIVERSITY (INVERTEBRATES) 7
, 4. The medusae appear like small saucer-shaped bodies along the lateral sides of the
blastostyle. Thus, Obelia colony exhibits trimorphlsm
..:r"I::'J-- POL Y P
~ w ~ e : ? TENTACLES
MEDUSAE BUDS
~ : . . . . . HYDROTHECA
GONOTHECA
BLASTOSTYLE
,1---- PERISARC
":1'1---- COENOSARC
Fig. 4.1. Obelia cOlony.
OBELIA MEDUSA:
The medusa of Obelia has a saucer-shaped body measuring about %" in diameter It IS the
solitary free-swimming sexual zooid of Obelia The chief characteristic features are as follows
1. Presence of an outer convex surface called ex-umbrellar surface and an Inner concave
surface called sub-umbrellar surface.
2. Presence of a short, quadrangular manubrium in the center of the sub-umbrellar surface
bearing the mouth opening at its tip.
3. Presence of large number of short tentacles at the rim of the umbrella or bell provided
with cnidocytes.
4. Presence of radial canals, running from the gastric cavity to the circular canal
5. Presence of four gonads in the radial canals.
6. Presence of statocyst at the bases of eight adradial tentacles
7. Presence of velum (characteristic of most hydromedusae).
SUBUMBRELLAR
TENTACLE
SURFACE
RADIAL CANAL
STATOCYST
VELUM
MANUBRIUM
Fig. 4.2. Medusa of Obelia.
PHYSALlA:
It is commonly called "Portuguese man-of-war." It is a colonial polymorphic, marine, pelagiC
form. The following features characterize the colony:
1. Presence of an enormous cap-shaped pneumatophore or float, which is filled with air.
It is dorsally produce..d into a crest or sail.
2. Presence of a reduced stolon beneath the float bearing several kinds' of zooids:
Gastrozooids, Dactylozooids and Gonozooids.
3. Gastrozooids ar"e provided with mouth without tentacles surrounding it These are nutritive
zooids of the colony.
4. Dactylozooids are the tentacle bearing zooids, which are tactile and protective in nature
and are provided with cnidocytes.
5. Gonozooids bear clusters of medusoids or gonophores and these look like bunches of
grapes. They are the reproductive zooids of the colony.
VELELLA:
It is commonly known as the "little sail" of warm seas. The polymorphic colony has a superficial
resemblance of a single medusa (Fig. 4.4). The important features are as follows.
1. Presence of a rhomboidal float or pneumatophore containing many air chaOlbers
2. Presence of a vertical ridge or sail, which represents the raised portion of the
pneumatophore.
3. Presence of a reduced stem or stolon on the undersurface of the pneumatophore. It
bears a single large gastrozooid in the center which IS surrounded by numerous
blastostyles or gonozooids loaded with clusters of medusae.
4. Presence of a fringe of dactylozooids at the margin containing batteries of nematocysts.
GONOZOOIDS
TENTACLES
BEARING
NEMATOCYSTS
PORPITA:
Fig. 4.3. Physalia.
SAIL (CREST)
.__--1 PNEUMATOPHORE
.. GASTROZOOIDS
SMALLER DACTYLOZOOIDS

DACTYLOZOOIDS
GASTROZOOID
Fig. 4.4. Velella.
It is a flat disc shaped marine pelagic, colonial, polymorphic form (Fig. 4 5). The important
features are as follows:
1. Presence of a large disc shaped circular float or pneumatophore
2. Presence of a shortened and flat stem or coenosarc below the disc. The disc bears the
following zoolds:
(a) Gastrozooid: It is the large nutritive zooid of the colony provided with a mouth and
stomach and is placed in the central region of the disc.
(b) Gonozooids or blastostyles: These occur in clusters around the central gastrozoold
and are provided with medusa.
(c) Dactylozooids: These are also known as tentaculozooids and are long tentacle-like
and provided with nematocysts. The dactylozooids are arranged in a circular manner
around the rim or edge of the disc.
CHAMBERED SAIL
DACTYLOZOOIDS
Fig. 4.5. Porpita.
5. OBSERVATION OF TYPES OF CORALS
FUNGIA
It is commonly known as "Mushroom coral" because of its shape. It IS a solitary 'stony coral'
found in warm seas. It shows following features: (Fig. 5.1).
1. The disc shaped or mushroom shaped corallite's is convex on the upper and concave
on the lower surface.
SEPTA
Fig. 5.1. Fungla.
CONFLUENT
CORALLITES OR
THECAE
Fig 5.2. Meandrina.
2. The thecae are present only on the lower surface
11
3 There are numerous septa on the convex upper surface, which are connected together
by small calcareous rods called synaptlculae
MEANDRINA:
It IS commonly known as "brain coral" because the surface of the corallium appears like
human brain with many convolutions. This coral is formed by the accumulation of several
generations of polyps. The coral shows follOWing features. (Fig. 5.2)
1. The surface of the coral has several long curved grooves running more or less parallel
to each other.
2. The grooves are formed by the confluent corallite or thecae
MADREPORA (ACROPORA):
It is commonly known as "Horn coral" The colony is highly branched like antlers It constitutes
the major part of the coral reef and helps 111 bUilding the coral reef. It IS characterized by
1 The presence of innumerable small elevated cups separated by perforated
coenosteum.
2. The corallium is extremely porous and is of loose construction.
3. Each corallium is a small deep cup consisting of 6 or 12 septa (Sclerosepta) and Without
a columella.
COENOSARC
CORALLITE
Fig. 5.3. Madrepora (Acropora).
TUBIPORA:
Fig. 5.4. Tublpora
OPENING OF
POLYP
VERTICAL
TUBES
PLATFORM
It IS commonly known as "pipe-organ coral". It occurs among marine coral reefs It IS
characterized by.
,6, !-i!\NDBOOK OF PRACTICALS IN ZOOLOGY
1. The presence of long parallel upright polyps enclosed in vertical skeleton tubes arising
from the basal plate.
2. The polyps are united at definite intervals by platforms.
3. The skeleton is dull red to crimson due to iron salts deposition.
GORGONIA:
It is commonly called as "Sea fan". It constitutes submarine gardens in the sea. It is a colonial
form. It is characterized by:
1. The presence of plant like highly branched stem fastened to a substratum by a basal
plate.
2. Supporting axial skeleton contains a horn-like proteinaceous material called gorgonin.
3. The branches on the stem are interconnected by cross connections or septums, which
gives rise to mesh like appearance.
Fig. 5.5. Gorgoma.
STALK
PEDALDISC
6. OBSERVATION OF HELMINTHS
T. S. OF LIVER FLUKE:
A T.8. of liver fluke shows following features: (Fig 6.1)
1. The body wall is covered by a thick cuticle, which bears backwardly directed spines or
spinules.
2. A thin basement membrane lies below the cuticle and IS followed by an outer and Inner
layer of longitudinal muscles.
3. There is no body cavity or coelom. The body space between the body wall and the
internal organs is filled with a loose connective tissue, the mesenchyme or parenchyma
4. The cut sections of gut cecae, lateral nerve cord, vasa deferentia, excretory canals,
ovary, oviduct, uterus and shell glands are seen.
CIRCULAR
MUSCLE
MEHLlS'S GLAND
PARENCHYMA
EXCRETORY
CANAL
LONGITUDINAL VITELLINE DUCT
MUSCLE
Fig. 6.1. T. S. Liver fluke.
MIRACIDIUM LARVA OF LIVER FLUKE:
VITELLINE
GLANDS
Miracidium is a free-swimming larva of liver fluke found in fresh water. It is short lived
and within eight hours of its existence it must come in contact with a pond snail (Limnae truncatula).
The characteristic features are as follows: (Fig. 6.2).
1. The body is conical in shape covered by cilia.
2. Presence of triangular boring apical papilla at the anterior end.
3. Presence of pair of eye spots behind the papilla.
REDIA LARVA OF LIVER FLUKE:
It is the second larva of the liver fluke, which develops within a shapeless sac called sporocyst
in the pulmonary chamber of the Limnae. The structural features are as follows.
1. Body is elongated with a mouth opening at the anterior end, which leads into muscular
pharynx, which further leads Into a blind intestine
2. There is an annular muscular collar just behind the pharynx A birth pore IS present
close to it At the hind part, there IS a pair of stumpy lateral processes
3. In the body cavity, the third type of larva called Cercariae may be seen
APICAL PAPILLA
EYE SPOT
EPIDERMAL PLATE
MOUTH -.lr':,,-
PHARYNX
GERM CELLS
LAPETS
PROCRUSCULA
BIRTH PORE
CERCARIAE
Fig 6 2 Miracidium
Fig 63 Redia
MOUTH
GUT CAE C L U M --IlIlllllV
Fig. 6.4. Cercaria.
ORAL SUCKER
OESOPHAGUS
CYSTOGENOUS
GLAND CELLS
ACETABULLUM
GERM CELLS
CERCARIA LARVA OF LIVER FLUKE:
15
It is the last but one larval stage in the life cycle of liver fluke. It develops Inside redia larva.
Once it completes its development, it escapes from the redia through the birth pore and comes
out of the body of the snail (Limnae). It leads a brief free-swimming life and loses the tall to get it
self encysted. In the cyst form, it attaches itself to the grass or the leaf, which Will be consumed by
the final host-sheep. It is characterized by following features
1. It has a heart shaped body with a long tall, which is shed off In the later part of Its few
hours of life
2. It has two suckers, an anterior oral sucker and a posterior ventral sLIcker
3. Mouth is situated in the center of the oral sucker, which leads into a pharynx, which
further leads into an inverted u-shaped intestine.
SCOLEX OF TAPEWORM:
It is the head of Taenia solium, which is slightly larger than the pinhead It IS adapted to cling
to the inner wall of the intestine of the host. The characteristic features are as follows
1. Presence of rostellum in the anterior region of the scolex.
2. Presence of double circlet of curved, chitinous hooks surrounding the base of the
rostellum
3. Presence of four cup-shaped adhesive suckers on four sides of the scolex
4. A short narrow neck region follows the scolex.
SCOLEX
ROSTELL.A.M
FRONTAL VIEW
OF SCOLEX
Fig. 6.5. Scolex of Tapeworm
MATURE PROGLOTTID OF TAPE WORM:
It is characterized by following features:
1. Presence of a complete set of male and female reproductive organs.
2. The male reproductive 'organ consists of several testes, vasa efferentia, vasa deferens
and cirrus.
3. The female reproductive organ consists of a pair of ovaries, oviduct, vagina, uterus,
shell gland and vitelline gland
4. The male and female genital apertures open into a cup like genital atrium situated on an
elevated genital papilla laterally.
TESTES;
VASA EFFERENTIA
VAS DEFERENS
UTERUS
CIRRUS OR PENIS
COMMON
GONOPORE
FERTILIZED
EGG CAPSULE
SEMINAL
RECEPTACLE
VAGINA
OVARY
OVIDUCT
UTERINE CANAL
VITELLINE VITELLINE MEHLlS'S
DUCT GLAND GLAND
Fig. 6.6. Mature proglottid of tapeworm.
CYSTICERCUS LARVA OF TAPE WORM
It is also called as bladder worm, which develops in the muscle of pig (intermediate
host). It is characterized by following features:
1. Body is bladder like with a characteristic evagination, which protrudes out of bladder.
2. The evagination has suckers and hooks at its tip, which is called proscolex.
3. The proscolex is connected to the bladder by a narrow neck.

Rt------_ SUCKE
HETERONEREIS:
SCOLEX
Fig. 6.7. Cysticercus.
CYSTICERCUS
WITH SCOLEX
AND PROGLOTTID
7. OBSERVATION OF ANNELIDS
17
It is a reproductive phase, where posterior part of the Nereis shows some structural
modification. Important characters of heteronereis are as follows:
1. Body is differentiated into anterior non-sexual atoke and posterior sexual epitoke region.
2. The parapodia of the posterior region of the body are large and highly vascu.larized. The
setae on the parapodia are long and oar shaped arranged in a fan like mariner.
ATOKE
(NON-REPRODUCTIVE)
ORDINARY
PARAPODIA
EPITOKE
(SEXUAL REGION)
OAR SHAPED
PARAPODIA
Fig. 7.1. Heteronereis.
TROCHOPHORE LARVA:
It is the larva of nereis having a minute pear shaped body with following characteristics.
1. At the apical end, apical tuft of cilia are present.
2. The alimentary canal shows regional differentiation like mouth, oesophagus, stomach,
Intestine and anus.
3 There IS a double circlet of cilia around the body, one lies above the mouth called pre-
oral ciliated band (prototroch) and the other one lies below the mouth called post-oral
ciliated band (perianal circlet).
4. Between the gut and the body wall is a cavity called blastocoel.
NAUPLlUS:
APICAL TUFT
OF CILIA ------
PRE ORAL
BAND OF CILIA
MOUTH . . . J U ~
ANAL TUFT
OF CILIA ----_e
OESOPHAGUS
STOMACH
............ - INTESTINE
ANUS
Fig. 7.2. Trochophore larva.
8. STUDY OF CRUSTACEAN LARVAE
It is the first larval stage during the development of many crustaceans. Important characters
are as follows:
1. Body IS ovai, unsegmented, which is broad at the anterior end.
2. There are three pairs of appendages, a pair of uniramous antennules, a pair of biramous
antennary and mandibular legs. All legs are provided with sWimming setae
3 There IS a single median eye at the anterior region.
ZOAEA OF CRAB:
It is the first larval stage in the development of crab. The key characters are as follows
1 The body is divided into a distinct unsegmented cephalothorax and a segmented
abdomen
2. The cephalothorax has a long dorsal spine and an anterior rostral spine.
3. The cephalothorax bears a pair of antennule, antenna, a pair of mandibles, two pairs of
maxilla, first two pair of maxillipeds and a pair of sessile large compound eyes
4. The abdomen is six segmented without any appendage except at the caudal region,
which bears forked telson at the tip.
MEGALOPA LARVA:
It is the second larval stage during the development of crab. The key characters are
1. The body is divided into two regions, anterior cephalothorax and posterior abdomen
2 Anterior region protrudes as a median spine and on either side of the spine are present
a pair of antennules, a pair of long antennae and a pair of large stalked compound eyes
,.,--__ ANTENNULE
ANTENNAE
'-__ MANDIBULAR
L.EGS
Fig. 8.1. Nauplius.
THORACIC
APPENDAGES
TELSON
ANTENNULE ROSTRUM
CEPHALOTHORAX --.
THORACIC
LEGS
TELSON
Fig. 8.3. Megalopa larva.
Fig 82. Zoaea
ANTENNULE
. ROSTRAL
SPINE
3. Five pairs of well-developed walking legs are present in the thoracic region of which the
first pair is chelate.
4. The abdomen has six segments and each bears biramous pleopods or swimmerets.
ALIMA LARVA OF SQUILLA:
It is a modified zoaea larva and marks the beginning of life history of squilla. Important
characters are:
1. Body is glassy, transparent and slender, which is divisible into two parts: Cephalothorax
and abdomen.
2. The cephalothorax is covered by a flat carapace, which is protruded anteriorly into rostrum
and posteriorly into posterolateral spines.
3. The cephalothorax anteriorly bears a pair of antennules, a pair of antennae and a pair of
stalked compound eyes.
4. The second maxillipeds are large and chelate form a powerful raptorial (grasping) organ
of the larva.
5. The abdomen is segmented and terminates in a broad armoured telson. The last few
segments of the abdomen bear fairly well developed appendages.
MYSIS LARVA:
ANTENNULE
EYE
ANTENNA
2
nd
MAXILLIPED
CARAPACE
ABDOMEN - - ~ : C 1 I
Fig. 8.4. Alima larva.
It is also called schizopod, which occurs at the end of the metamorphosis of prawn. The
zoaea stage instead of changing into megalopa, moults into mysis. Important features are as
follows.
1. Body is elongate, laterally compressed, and divisible into anterior cephalothorax and
posterior abdomen.
COMPOUND CEPHALOTHORAX
ROSTRUM EYE
ANTENNULE
TELSON
Fig. 8.5. Mysis larva.
2. The cephalothorax is produced anteriorly into a pOinted rostrum and it bears a pair of
stalked compound eyes and thirteen pairs of appendages.
3. The abdomen is six segmented and the last segment is excessively long, which terminates
in a telson. The abdomen bears five pairs of pleopods and a pair of uropods.
PHYLLOSOMA LARVA OF PANULIRUS
It is commonly called as glass crab marks the beginning of the life history of rock lobster
(Panulirus). It is a highly modified mysis stage that is found as the last larval stage during the
metamorphosis of prawn. The main structural features are as follows:
ANTENNULE
3rd THORACIC LIMB
6th THORACIC LIMB
Fig. 8.B. Phyllosoma larva.
1. Body is flat, glassy, leaf-like, transparent and divisible into head, thorax and abdomen.
2. The head bears a pair of antennules, a pair of antennae and a pair of stalked compound
eyes.
3. The thorax bears five pairs of thoracic limbs and the abdomen IS slender and highly
reduced and has no appendages.
CYPRIS LARVA:
It is the larva of sacculina and barnacles. Cypris is characterized by follOWing features
(Fig. 8.7)
1. Body is enclosed in a bivalve shell.
2. A median and two compound eyes are present.
3. Anterior antennae are four jointed with a characteristic disc and the posterior antennae
are absent.
4. Six pairs of biramous thoracic appendages are present.
5. Short abdomen ends in caudal fork.
GUT
ANTENNULE
MOUTH
Fig. 8.7. Cypris larva.
9. STUDY OF INSECT METAMORPHOSIS
JUVENILE AND ADULT OF LEPISMA:
There is not much difference in the external morphology between juvenile and adult of lepisma
except the size. Hence there is no metamorphosis as such therefore they are grouped under
ametabola. As the juvenile grows to adult form it develops the internal organs like gonads, and
other structures and matures into an adult form by increasing the body size
DIFFERENT STAGES IN THE LIFE HISITORY OF:
HOUSE FLY
The life history of housefly consists of three stages, egg, larva, pupa and adult. The eggs are
laid in garbage dumps, which hatch into apodous (leg less) larvae. The larva is commonly known
as maggot. The maggot goes through three instar stages and fully-grown larva measures about 8
to 12 mm. The larva metamorphoses Into a pupa, encased In a brown coloured leathery covertng
called puparium From the pupa emerges the fly or Imago (immature) Since there IS a remarkable
difference between the larva, pupa and adult, which Involves senes of moults. hence the
metamorphosis is called holometabola (Fig. 9.2)
/
NYMPH
MOSQUITO:
WITHOUT METAMORPHOSIS
(AMETABOLA) ~ t
.... . ~
4 ~ V
NYMPH
Fig. 9 1. Lepisma.
The life history consists of egg, larva, pupa and adult. Since there is a remarkable change
during development from larva to adult and the larva goes through many moults during
metamorphosis they are called holometabola. The eggs are laid on the surface of water in clutches
as egg raft (Culex) or individually with floats (Anopheles) Eggs hatch Into larvae, which are
segmented vermiform, provided with respiratory siphon on the 8
th
segment and tracheate gills on
the last segment, by which they respire. Pupae have long anterior breathing tubes or respiratory
trumpets and often hang nearly straight down from the surface From the pupa, the adult hatches
out and the females start feeding on the blood of vertebrates and males feed on plant sap (Fig 9 3).
PUPA
\
/'
0- AIRFlOAT
~ . ~
-, ~ :
EGGS
ADULT
MAGGOT
(FULL GROWN)
~
,It
/ EGGS
Fig. 9.2. Life history of housefly.
LARVA
PUPA
1/
' . .

Fig. 9.3. Life history of mosquito.
(I) LIFE CYCLE OF BUTTERFLY
Southern Bird Wing
India's Largest Butterfly
Chrysalis
LIFE CYCLE
Fig. 9A. Life Cycle of Butterfly.
Caterpil lar
or larva
25
Butterflies belong to group Insecta and lay eggs. They pass through four stages in their life
cycle.
The Egg stage
The eggs of butterfly are tiny and have a variety of shapes. The female butterfly lays the eggs
on the leaves of the plants. After 6-7 days, a worm like creature hatches out of the egg which is light
green or yellow in colour.
The Larva stage (caterpillar stage)
It is tiny worm like known as baby caterpillar but does not undergo moulting (it's the process of
shedding of skin several times before growing into an adult) .
This is the active stage. The larva eats leaves and grows to a tremendous amount. This fully
grown larva is called a caterpillar which has six real legs and a number of extra prolegs.lt is the
young one of a butterfly and does not resemble its parents.
The Pupa stage
This is called the resting stage. The caterpillar first spins a cocoon around its body and also
fastens itself to a plant with a silk thread. The caterpillar which is inside the pupa changes into a
butterfly. Generally pupas camouflage themselves to hide from predators.
The Butterfly stage
In this stage, the baby butterfly emerges from the pupa and soon is transformed into an adult
butterfly.
(II) LIFE CYCLE OF BEETLE
r
4. Adult
~
~
Life Cycle of
a typical
beetle
3. Pupa
~
2. Larvae
~
J
. Fig. 9.5. Life' Cycle of Beetle.
Beetles, like other insects, go through a complete process of metamorphosis in which it goes
through four stages of development.
Eggs
It begins with the female beetle laying hundreds of tiny, oval white or yellow eggs, usually on
a leaf or in rotten wood. (Some female beetles keep their eggs inside of them and give birth to live
larvae). It usually takes from 4-19 days for the eggs to hatch. They then enter into the 'larval stage'.
Larvae
At this stage, they will eat a tremendous amount of food and continue to grow, shedding its
exoskeleton many times while it grows. Most beetles pass through 3-5 stages during the larval
period and some can even have up to 30 stages whereas other beetles can have only 1 stage as
larvae.
Pupa
It then enters into the ' pupal stage' which can take up to 9 months and usually happens over
the winter period. After pupating, an adult emerge, and there you have your beetle.
Adult Beetle
This beetle will then feed, mate and if it is a female, she will lay eggs for the beginning of
another generation.
(III) LIFE HISTORY: SILK MOTH
Life cycle of silkworms (Bombyx mori)
10-120,y'
-+u
silkworm feeding
on mulberry leaves
Emergence
from cocoon
10-12 Days

Cocoon
cut open
Cocoon
Hatching
3-4 Days

2 'h -3 Days
\j
c:aJIj1)
2. Moult
3-4 Days

3. Moult
4Days

4. and final Moult
6-8 Days
j
Fig. 9.6(A). Life History of Silk Moth.
Fig. 9.6(B). Silk moth larvae or Silkworms feeding on mulberry leaves.
27
Life cycle of silk moth . A silkworm starts its life as a tiny egg laid by the female moth. The egg
is just about this size: . The egg, laid in the summer 0r early fall, remains dormant until the warmth
of spring stimulates it to start developing. When silkworms first hatch in the spring, they are tiny -
3 mm or so (about 1/8") - and hairy. They require young tender mulberry leaves during their first
few days. As they grow, they can eat tougher leaves, and late in their development they will eat any
mulberry leaf you can supply.
The larvae advance through five stages of growth, called instars. The silkworm literally outgrows
its skin five times, and molts its outgrown skin. With the first molt the silkworm loses its hairy exterior,
and for the rest of its larval life its skin is soft and smooth.
Silkworms grow rapidly, eventually reaching the size of your ring finger. Then they spin beautiful
oval white or yellow cocoons in which they pupate. After 2-3 weeks, the creamy-white adult moths
emerge from the cocoons. They clamber around, vibrate their wings rapidly, and mate, but they
don't fly or attempt to escape from their container. During the adult phase of the life cycle, the
silkworm moths do not eat or drink. After mating, the female lays a profusion of eggs, and the moths
die. . ,
Males and females look slightly different, and students will be able to tell them apart with a
little practice. The female has a larger abdomen. The male has a much larger pair of antennae,
which look like long rakes or comb-shaped eyebrows, and vibrates its wings rapidly to attract a
female.

10. TYPES OF FOOT IN MOLLUSCA
CHITON:
The foot is flat and occupies the whole ventral surface of the body. The foot also acts as a
sucker to attach to a suitable substratum. The foot is used in creeping on hard surfaces.
PATELLA:
The ventral side of the body is almost occupied by large, broad creeping sole, which acts as
an organ of attachment to rocks.
APLYSIA:
The foot is elongated, muscular and pointed posteriorly used as thrusting organ w ~ n aplysla
is on rocky surface in shallow water.
MYTILUS:
The foot is elongated and tongue shaped with a ventral groove continuous with byssus thread
pit. The foot is used as thrusting organ to move on hard rocky and soft sandy surfaces
SOLEN:
It has a well-developed muscular cylindrical foot at the anterior end used for thrusting and
burrowing in sand.
CARDIUM:
Tongue shaped muscular foot at the posterior end, used for thrusting and burrowing
SEPIA:
The foot is modified into arms or tentacles and siphons, which are present in the head region
They are used for capturing the prey and in mature male, one pair is modified for transfer of
sperms in to female's mantle cavity.
30
A HANDBOOK OF PRACTICALS IN ZOOLOGY
- I ' R ~ - _ FOOT
PATELLA
CHITON
SOLEN
FOOT
MYTILUS
APLYSIA
FOOT
CARDIUM
SEPIA
Fig. 10.1. Types of foot in molluscs.
11. TYPES OF SHELLS IN MOLLUSCS
CHITON:
The shells are flattened oval shaped having 8 plates, which overlap antero-posteriorly. A
surrounding girdle keeps the shells together
DENTALlUM:
It is called 'tusk shell' because of its shape. The shell IS open at both ends with grooves
running down the entire length.
TROCHUS:
It is commonly called as 'top shell' because of top shaped structure. The shell is conical with
horny operculum and a nacreous interior.
PLACUNA:
This is the shell of the windowpane oyster. It is flat, thin and circular having a pearly
appearance. Earlier the Chinese and Portuguese used it for glazing the wmdows.
SOLEN:
It is called 'razor shell' because of its shape. It is thin with sharp edges, open at both ends.
SEPIA:
It is popularly known as Cuttle bone. It lies internally enclosed in a sac in the mantle on the
upper side The shell is characterized by following features.
1 It is stout, broad and oval in shape. The anterior end is broader and rounded, while the .
posterior end is more pOinted and terminates in a projecting spine or rostrum
2. It is entirely composed of fine calcareous laminae enclosing air space.
3. The shell gives internal support to the animal and thus forms a sort of dead endoskeleton.
NAUTILUS:
It is spirally coiled many-chambered shell and the animal occupies the outermost and
largest chamber into which body can be withdrawn.
2. The chambers are separated by a system of septa, which are perforated in the middle
and traversed by narrow tubular vascular structure of the visceral mass, the siphuncules
3. Except the outer chamber, all the chambers are filled with air so that the shell floats and
animal swims easily in water.
SINISTRAL AND DEXTRAL SHELL:
When a gastropod shell is viewed from the ventral side with the mouth of the shell facing the
observer, if the mouth lies to the right of the columella and the shell is coiled clockwise then it IS
called right handed or dextral coiling. On the other hand, if the mouth of the shell lies to the left of
the columella and the shell is coiled anticlockwise then the shell is left handed or sinistral cOiling
32
TROCHUS
CHITON
OYSTER
PROOSTRACUM
'PHRAGMOCONE
EMPTY
SEPIA
SEPTAL NECKS
NAUTILUS
Fig. 11.1. Types of shells in Molluscs.
DEXTRAL
SINISTRAL
Fig. 11.2. Types of shells in Molluscs.
12. STUDY OF ECHINODERM LARVAE
BIPINNARIA:
It is free-swimming first larval stage of the asteroidae. Body is bilaterally symmetrical and
the larva is characterized by following features.
1. The pre-oral band of cilia has transformed into an unpaired median process and a pair
of pre-oral arms.
2 The post oral band of cilia has transformed into an unpaired median arm, and paired
antero-dorsal, postero-dorsal, postero-Iateral, and postoral arms
3. The larval alimentary canal is represented by mouth, oesophagus, stomach, intestine
and anus.
MEDIAN DORSAL ARM
ANTERIOR DORSAL ARM
POSTERO-DORSAL ARM
POST-ORAL ARM

OESOPHAGUS
POSTERO-LATERAL ARM .......... ........... - ANUS
Fig. 12.1. Bipinnaria larva of starfish.
34
OPHIOPLUTEUS:
It is the free-swimming larval stage of ophiuroidae The larva shows following features (Fig
122).
It has four pairs of arms; antero-Iateral, postero-dorsal, postero-Iateral and post-oral
Skeletal rods support all arms Posterolateral arms are often very long while the postero-
dorsal arms are sometimes wanting.
2. It has two pairs of coelomic sacs, the right axohydrocoel, left hydrocoel with lobes and
left and right sornatocoel
3. The larval alimentary canal has mouth, oesophagus, stomach, intestine and anus
MOUTH
OESOPHAGUS
RIGHT STOMATOCOEL
ANTERO LATERAL ARM
POST-ORAL ARM
I ~ ~ - - POSTERO-DORSAL ARM
LEFT HYDROCOEL
LEFT STOMATOCOEL
Fig. 12.2. Ophiopluteus larva of Brittle star.
ECHINOPLUTEUS:
It is the free-swimming larva of echlnoidae. Its characteristic features are as follows (Fig
123).
1. It has six pairs of arms; antero-Iateral, antero-dorsal, postero-dorsal, postero-Iateral, pre-
oral and post-oral. Skeletal rods support the arms. A median posterior proJection, termed
aboral spike IS the characteristic feature of the larva.
2. A pair of Ciliary patches one at each end of the larval body are present.
3. The larval alimentary canal shows mouth, oesophagus, stomach, Intestine and anus
OESOPHAGUS
PREORAL ARM
ANTERO LATERAL ARM
ANTERIOR APAULETTES
I NTESTI N E ----'It''t-T.
POSTERIOR EPAULETTES
POSTERO-LATERAL PROCESS
Fig. 12.3. Echinopluteus larva of Sea urchin
AURICULARIA:
It IS the free-swimming larva of Holothurians The chief structural features are as follows
(Fig 12.4).
PRE ORAL LOOP __ "..--r1I
PHARYNX
HYDROCOEL
ANAL LOOP-....". ...
HYDROPORE
ANUS ___ STOMACH
RIGHT STOMATOCOEL
LEFT STOMATOCOEL
INTESTINE
Fig. 12.4. Auricularia larva of sea cucumber.
1. The continuous ciliated band appears like sinuous curves along the side
2. It has a pre-oral loop in the anterior ventral region above the mouth and post-oral loop or
anal loop in the posterior ventral region encircling the anus.
3. Interior of the larva has curved digestive tract and three coelomic sacs; on the right
hydrocoel and on the left somatocoel.
DOLIOLARIA:
It is the free-swimming larva of the crinoids and holothurians. It is characterized by the follOWing
features (Fig 125):
1. It has an apical sensory plate with tuft of cilia and has four or 5 transverse ciliated rings.
2. Near the anterior end it has a midventral adhesive Pit and a midventral stomodeum.
3. In the anterior half irregular shaped hydrocoel and in the posterior end highly coiled
somatocoel is present.
APICAL TUFT
APICAL PLA c-___ _
CILIATED RINGS
..,.....ot'"- VESTIBULE
CILIATED RINGS
Fig. 12.5. Doliolaria larva of crinoids.
ANIMAL DIVERSITY (CHORDATES) 37
ANIMAL DIVERSITY (CHORDATES)
13. STUDY OF ASCIDIAN TADPOLE
It leads a very short free-swimming life because it does not possess feedmg apparatus It is
characterized by following features:
1. Presence of adhesive discs in the anterior region.
2. Presence of pharyngeal basket
3. Presence of dorsal tubular nerve cord and a notochord restricted to the tail region.
4. Presence of atrial aperture.
5. Presence of muscular tail with dorsal, ventral and caudal tail fin, which helps In swimming.
NERVE CURD
ORAL APERTURE
ADHESIVE PAPILLAE
TAIL
ATRIAL CAVITY
NOTOCHORD
HEART
PHARYNEAL BASKET
ENDOSTYLE
Fig. 13.1. Ascidian tadpole.
14. SWIMBLADDER "IN-SITU
The swim bladder or air bladder is large, air-filled, fibrous, cylindrical, closed sac, situated
just below the vertebral column and above the digestive tube. It is rounded anteriorly and pOinted
posteriorly. Anteriorly the bladder is closely applied to the septum transversum. Posteriorly it extends
beyond the anus and gives off sub-terminally a pair of tubular lateral caeca. The wall of the bladder
is formed of silvery white, elastic fibres, arranged in circular sheets and rendering it non-collapsible
The main function of the bladder is hydrostatic, i.e., by varying the volume of the gas Inside the
bladder the fish is able to swim at different levels in the water.
SEPTUM
TRANSVERSUM AIR BLADDER
DORAL VIEW
Fig. 14.1 Swim bladder In situ (Sciaena).
15. BREEDING AND PARENTAL CARE
SEA HORSE (Hippocampus)
Once the eggs are fertilized, the male collects them in its brood pouch, which IS situated on
the ventral side of its abdomen. The eggs are retained in the brood pouch until they hatch as
young ones In this way male shows the parental care.
SIAMESE FIGHTER
The male builds a nest of raft made of mucous bubbles of air at the surface, which lasts for
a long time. As the female releases 3 to 7 eggs at a time, male catches each of them in its mouth
and coats it with mucous them swims up to his raft and sticks them on the underside of the raft.
This is repeated until all eggs are laid and finally male guards the nest and drives away the female
The young hatch within 24 to 30 hrs.
TILAPIA
They are mouth breeders. The pair digs a pit an inch or two deep and five to twelve inches
across m the sandy bottom scooping up mouthful of sand or small pebbles and spitting them a
little away from the nest. As soon as the nest is ready, the female starts laymg the eggs and male
fertilizes them by releasing the milt over them and later the male and female collect the fertilized
eggs in their mouth. The eggs are retained in the mouth until they hatch Both male and female
guard young fry and whenever there is a threat from the enemy, the parents open their mouth and
the fry take refuge in the parent's mouth.
CATFISH
Marine catfish of the family Ariidae are mouth breeders The male holds the eggs in its mouth
until they hatch. Male continues to shelter the fry in the same way whenever there IS danger For
a month or so, male starves to protect its young one.
RAFT OF MUCOUS BUBBLES
BROOD
POUCH
SEA HORSE
SIAMESE FIGHTER
TILAPIA YOUNG FRY COMING
OUT OF MOUTH
Fig. 15.1. Breeding and parental care in fish.
BREEDING AND PARENTAL CARE IN APMHIBIA
CAECILIAN
EGGS IN THE MOUTH
CATFISH
39
The female lays 10 to 25 large pea sized eggs with a special outgrowth on the membrane,
which helps In adherence of eggs so that they form a cluster The female entwines the spawn and
broods It wetting the eggs abundantly with its body slime. The female takes the eggs to the shallow
water shortly before they hatch and the young one emerges in water.
MIDWIFE TOAD
The European midwife toad Alytes obstetricans lays eggs on ground In the form of 2 strings
in which 20 to 50 ova are arranged quite far from each other. Male grasps the strings of egg with
both the toes of its hind limb, pulls them out and entwines them around itself Male carnes this
burden until the eggs are ready to hatch. The eggs get mOisture from the skin surface of the
carrier When the eggs are ready to hatch into tadpoles the carner male goes to a nearby water
body and releases the string of eggs
NEOTENY IN AXOLOTL:
Axolotl is the larva of tiger salamander Once in a while when the environmental conditions
are not favourable axolotl exhibits a phenomenon called neoteny, where the larva falls to
metamorphose but undergoes sexual maturity and reproduces like an adult to give rise to many
more axolotl. When conditions in the environment return to favourable state, axolotl metamorphoses
into a salamander by losing external gills and tall fin.
CLUTCH OF EGGS
AL YTES OBSTETRICANS
CAECILIAN
Fig. 15.2. Breeding and parental care in Amphibia.
CAUDAL FIN
MOUTH
Fig. 15.3. Axolotl larva of salamander.
16. ADAPTIVE RADIATION IN REPTILES
TURTLE
They are marine in habitat, omnivorous in nature feed on marine algae, fish, molluscs and
corals. The key adaptive characteristics are as follows:
1 Fore limbs 'are modified as large paddles used for swimming.
2. Hind limbs are provided with clawed digits, which are webbed. Used in swimming as
well as for thrusting during locomotion on coral reefs and shallow ocean bed
3. Carapace is convex and smooth and plastron is more or less flat covered by smooth
epidermal horny scales to reduce resistance in water while swimming.
4 Head and neck are not completely retractable like in tortoise because adult turtles hardly
have any predators.
5. Jaws are protected by horny beak like sheath, which guards the mouth and helps in
grasping and tearing the food.
HEAD
FORE LIMB
PADDLE
TORTOISE
TAIL
Fig. 16.1. Turtle.
They are terrestrial in habit, predominantly herbivorous but occaSionally feed on worms and
larvae of insects Important adaptive characteristic features are as follows'
1. Body is enclosed in a bony dome shaped box made of carapace and plastron. The dorsal
carapace is rough and warty covered by horny epidermal sheath and the ventral plastron In more
Dr less flat covered by smooth epidermal sheath.
2. Carapace and plastron are fused laterally and leave openings for paired fore limbs and
the hind limbs, which protrude out through these openings.
HEAD
CLAWED
FORE LIMB
CARAPACE
PLASTRON
Fig. 16 2 TortOise
3. In the front carapace and plastron has the opening for the head, which can be withdrawn
fnto the carapace and plastron when the animal is threatened. Similarly there IS a opening for the
tall In the posterior region of the carapace and plastron, which can be withdrawn.
4 Both fore and hind limbs are used for walking cylindrical in shape, provided With clawed
digits Females use them for digging trenches to deposit eggs and burry them With SOil
5 The head IS Withdrawn Into the bony box by an'S' curvature of the neck
CHAMELEON
It is adapted for arboreal mode of life and is insectivorous in nature Main adaptive features
are as follows:
1. Body is laterally compressed and covered oy tubercles or granules. Animal has the
capacity to change its body colour depending on the substratum on which It lies as a
means of camouflage.
2 The fore limbs and hind limbs are adapted for grasping, In which in the fore limb three
inner fingers are fused completely throughout their length to form one bundle and two
outer fingers form another bundle by fusion Similarly two Inner toes are fused to form
one bundle and three outer toes are fused to form another bundle. It IS an excellent
adaptation for clasping and the phenomenon is called syndactyly
3. Eye are large capable of movement independent of each other and can be focused
Independently on a object.
Fig. 16.3. Chameleon.
4. The tongue is club shaped at Its tiP and sticky in nature, highly protrusslble with spring
action up to a distance of 10 to 15 cm and can be withdrawn Into a sheath In the mouth
at rest.
5. Tail is a long and prehensile, cOiled like a spring DUring locomotion on tree branches, It
is used as a grasping device by cOiling It around the branch.
PHRYNOSOMA
It is commonly called as horned toad because It IS capable of absorbing mOisture or dew
drops fallen on its body through the skin like a toad and horn because It has two long horn like
spines on the head. It is also called horned lizard because of the horns or spines on the head It IS
perfectly adapted for a life in the desert and feeds on insects
1. The body is flat and broad giving a large surface area for mOisture to condense on body
to absorb water droplets trickling down the spines. It meets ItS water through
food and absorption through skin.
2. Body colour is sandy grey and matches well with the desert condition
3. Large and small spines cover body and at the base of each spine the skin has a small
pore through which the water is absorbed into the body, which IS a good adaptation for
desert environment.
4. Behind the eye a glandular reservoir IS present which stores blood coloured obnOXIOUs
flUid, which is squirted out with great force on to the face of the predator when the animal
is threatened by the predator.
EYE
NOSTRIL
MOUTH
HORE LIKE SPINES
.,/
CLAWED DIGITS
Fig 16-4 Phrynosoma
WALL LIZARD (GECKO)
It is commonly called as house lizard because it is found on the walls of the house and it
feeds on insects. The key charactenstic features are as follows'
1. The digits are clawed and are provided with adhesive pads on the ventral side. Each
adhesive pad is expanded laterally and is provided with double series of lamellae, which
indeed work on the principle of vacuum-pressure principle. The lamellae is pressed on
the substratum, then these lamellae are raised so as to create a vacuum between the
lamellae and the substratum thus enabling the lizard to walk very easily on smooth
surfaces and upside down on the ceiling.
2. Another important adaptation is its ability to break off the tail to escape from the enemy
when threatened, which is called autotomy. Later the tail is regenerated without the
vertebrae.
.L.A"' ___ CLAWED DIGITS
.*-DIGITAL PADS
Fig. 16.5. Gecko or wall lizard.
RAT SNAKE (DHAMAN OR PTYAS)
It is the most common non-poisonous snake found in populated areas where rats and chicken
are available in plenty. It grows to a length of about a meter and half. Characterized by following
features:
DORSAL RIDGE
POINTED TAIL
VERTEBRAL SCALES
Fig. 16.6. Rat snake.
1. Perfectly adapted for fossorial and arborial mode of life.
2. Presence of prominent dorsal ridge of the backbone along the mid-dorsal line
3. Head is distinct from the neck.
4. Eyes are large with round pupil
5. Fourth and fifth supra-Iabials are touching the eye.
6. Maxillary teeth are present but poison fangs are absent.
7. Tail is long and prehensile.
SEA SNAKE (HYDROPHIS)
It IS commonly called as sea snake, grows to about a meter in length. Characterized by
following features.
1. Body is long and laterally compressed at the tail region. Tail acts like a paddle for
swimming in sea.
2. Eyes are very small.
3. General body colour is olive green above with yellowish crossbars with white bands
alternating on the ventral side.
4. Carnivorous, mainly feeds on fish.
5. 14 to 18 maxillary teeth are present behind a pair of poison fangs.
6. Poisonous and the venom is neurotoxic.
Fig.16 7. Sea snake.
CROCODILE
LATERALLY
COMPRESSED TAIL
It is commonly called as Mu'ggar of India or marsh crocodile. Characterized by following
features.
1. Body is covered by horny exoskeleton made of thick epidermal shields supported by
dermal bony plates. In the dorsal region of the tail the exoskeletal shields are modified
into spikes.
2. Head is triangular adapted for ploughing through the water while sWimming.
3 Tail is laterally compressed adapted for paddling in the water
4. Toes and fingers are webbed, help In sWimming
5. Carnivorous in nature, feeds on large fish and small aquatic mammals
SPINY SCUTES
CLAWED DIGITS
Fig. 168. Crocodile
\
17. VENOMOUS AND NON-VENOMOUS SNAKES
IDENTIFICATION KEY
SNAKES

I
I
I -Tail IS flat laterally compressed II - Tallis cylindrical, rounded and not
I
compressed
Sea snakes Terrestrla! snakes
I t
Poisonous Poisonous or non-poisonous
A. Small scales on the
belly and on the back
B. Belly scales not
broad enough to
extend nght across
C. Ventrals are broad and coverthe
entire width of the belly
l
Non-poisonous
I
Small scales on the head
Pit is absent.
Viper

Poisonous
Ventrals not enlarged. Third
supra-labial shield touches
the eye and nose shield.
Poisonous
I I

Non-poisonous
,
Small scales or shields on
the head and a loreal-pit
between the nostril and eye.
I
Pit viper

Poisonous
Ventrals are enlarged,
subcaudals undivided
undivided and bands
and half rings across
the back Fourth
Infra labial is largest
I
Neck with hood
and markings.
Neck without hood,
coral spots on the
belly
Krait
t
P,)isonous
Cobra Coral snake

Poisonous Poisonous

Shields on the head
None of the two prevIous
characters
l
Non-poisonous
NO POISONOUS OR POISONOUS
-
------
CYLINDRICAL TAIL
c: 65S5$wtI'tW?i
LAND SNAKE
FLAT TAIL
SEA SNAKE
(POISONOUS)
- - I - - - ~ I ---rrmrrrrmr----r
i
W/J111 IIlU
SNALL BELLY SCALES
(NON POISONOUS)
SMALL SCALES
I
(POISONOUS) VIPER
I
(POISONOUS)
THIRD SUPRA LABIAL SHIELD
(SIDE VIEW OR COeRA)
~ .
. .
. ..
NARROW BELLY
(NON POISONOUS)
I
(POISONOUS) PIT VIPER
I
(POISONOUS)
I
1 I Ild.l.J
.. ,. .
VERTEBRALS
(KRAIT-DORSAL)
BELLY SCALES OR
VENTRALS
I .
(POISONOUS OR
NON POISONOUS)
I
(NON POISONOUS)
I
INFRA LABIALS
Fig. 17.1. Diagrammatic representation of key to identification of non-poisonous and poisonous snakes
KRAIT (BUNGARUS)
It grows to a length of about 4 to 5 ft. It is nocturnal in habit and feeds on frogs, toads, mice,
rat and small birds etc. It IS a poisonous snake and the venom is neurotoxic. Characteristic features
are as follows.
Dorsal side of the body is bluish black and the ventral Side is pale yellow.
2. White transverse bands are conspicuous only in the posterior part of the body and not In
the anterior.
3. Ventrals are large and hexagonal extend across the belly
4. Third and fourth supra-Iabials are touching the eye.
5. Fourth infra labial is largest.
6. The sub-caudals are entire.
LARGE HEXAGONAL
MID-DORSAL SCALES
DARK AND
WHITE BANDS
LARGE SHIELDS ON HEAD
BROAD VENTRAL
PLATES
Fig. 17 2. Bangarus (Krait).
COBRA
It grows to a length of about 5 to 6 feet found mostly near human habitation It feeds on
frogs, toads, birds, rats, mice and other smaller mammals. Occasionally it also feeds on other
snakes. Highly poisonous and the pOison is neurotoxic. The key characteristic features are as
follows.
1. It is capable of expanding the neck region laterally by erecting the ribs in that region and
thus form a characteristic hood.
2. On the dorsal side of the hood, a characteristic V-shaped spectacle mark IS present,
hence the hood is called binoce/latelbioce/late
3. On the ventral side of the hood two black patches are present.
4. Body is usually wheat coloured or brown.
5 Third supra labial touches the eye and nostril-bearing shield
6. The sub-caudals are double
RUSSEL'S VIPER
It measures up to one and a half meter and the body is pale brown in colour Highly pOisonous
and the poison is haemolytic Feeds mainly on warm-blooded animals like rat, mice and birds
Characterized by following features.
1. Head is triangular and covered with small Imbricate scales.
2 The ventrals are broad and extend across the belly
3. The sub-caudals are divided.
4. Three chains of large diamond shaped grayish black spots are present on the back
SPECTACLE
MARK
EYE
BROAD VENTRALS
Fig 17.3. Cobra (Naja naJa)
SAW SCALED VIPER
SMALL IMBRICATE
SCALES
DIAMOND SHAPED
MARKINGS
Fig 17 4. Russel's Viper
(Vlpera russelli)
It IS called as little Indian Viper. It grows to a length of about 2 ft, found in desert and semi-
desert areas feeds mainly on rats, mice and other desert rodent species. It is poisonous and the
poison In haemolytiC. The characters are as follows
1. The scales are keeled and the scales of back are serrated, which gives rough spiny
surface like saw. Hence the name saw scaled viper
2. Small imbricate scales cover the head and the ventrals are broad and extend across the
belly.
3. The sub-caudals are entire
SMALL IMBRICATE
SCALE S
AND DARK
GRAY WAVY MARKING
Fig. 17.5. Saw-scaled viper (Echis carinata).
POISON APPARATUS OF VENOMOUS SNAKE
51
All venomous snakes have a characteristic pOison apparatus, which comprises of pair of
poison glands on either side of the upper Jaw and their ducts leading to the pOison fangs
The pOison glands are the modified posterior supra lablals gland A capsule of fibrous tissue
covers the pOison gland. A fan-shaped muscle called capito mandlbularls superflclalls covers the
entire poison gland The contraction of this muscle facilitates the pOison to flow from the pOison
gland to the duct and from there to the fangs
The pOison fang is either grooved or is a hollow tubular structure and is perfectly adapted for
injecting the poison into the body of victim like hypodermic syringe needle. The pOison fangs
when not in use are withdrawn into a sheath and folded backward. When the snake opens ItS
mouth to strike a victim, the fangs are erected by withdrawing the sheath and the victim IS struck
with a stab of poison fangs. The force of striking makes the fangs press on to the pOison apparatus
and this leads to ejection of pOison by the contraction of muscle
MAIN VENOM
ACCESSORY
VENOM GLAND
SECONDARY
DUCT
GLAND PRIMARY DUCT
COMPRESSOR
GLANDULAE
MUSCLE
UPPER JAW
-" ..t;.J....,..,,- MAXI LLA
rr-- POISON FANG
LOWER JAW
17 6. POison apparatus of a venomous snake.
18. MAMMALS
DUCK-BILLED PLATYPUS (ORNITHORHYNCHUS)
Found only in Australia and it measures about 45 cm long and the body is fully covered by,
velvety soft brown fur except the snout, which is covered with soft epidermal sheath Aquatic in
habit and it lives in burrows on the banks of rivers and streams. Characters are as follows:
1. Limbs are short with clawed digits The digits are also webbed and the webbmg is
predominant in the forelimb as compared to hind limb Limbs are adapted for digging
and swimming.
2. Tail is large and dorso-ventrally flat, covered by epidermal hair, is used as rudder and
paddle while sWimming.
3. The bill or beak is broad and shaped like duck-bill covered with soft epidermal layer of
the skin with tactile sense organs.
4. Teeth are absent but the bill is covered by horny plate from inside.
5. External ear or pinna is absent.
WOOLY HAIR
NOSTRIL
WEB
CLAWS
Fig 18.1 Duck-billed platypus.
KANGAROO
It is found only in Australia inhabiting semi-desert environment and measures up to 3 meters
from tail tip to head tip. Herbivorous in nature. Characteristic features are as follows:
1. It is bipedal, where hind limbs are long and stout used for jumping and the fore limbs are
short used only for grasping the vegetation and supportmg the body while feedmg on
grass and occasionally used for walking. Both forelimbs and hind limbs are proVided
with clawed digits used in defense.
2. Tail is long, round and thick, which serves as a additional leg during sitting posture and
it IS used for thrusting by pressing on the ground when the kangaroo is Jumping The tail
is also used for balancing while jumping.
3. Body is covered by thick coat of hair light brown to dark brown in colour.
4. Head is. proportionately small compared to the body size but the eyes and ears are
large.
5. Females have a large marsupium or pouch In the abdominal region opening In front in
which the young or the Joe is reared. In the marSUpium, nipples of the mammary gland
are present which secrete different types of milk, which vanes In fat content and thickness
depending on the age of the yo.ung one to be fed. "
FORE LIMB
YOUNG ONE
MARSUPIUM
Fig. 18.2. Kangaroo.
PANGOLIN (SCALY ANT EATER)
Measures up to 2 meters in length from tail tip to head tip. Predominantly found in Africa and
Asia. Nocturnal in habit. Characters are as follows:
1. Body is covered by sharp spiky overlapping epidermal scales and In between the scales
few tufts of hair also protrude out.
2. Snout is elongated well sUited for introducing Into the termite mound hole
3. Eyes and ears are highly reduced.
4. Tongue is long and tubular like a whip, sticky in nature and it is protrusible, and measures
up to 10 inch in length.
5. Teeth are absent.
, 6. Limbs are short and stout with five digits. Fore limbs have well developed claws, which
are used for digging termite mounds.
7. When the animal is threatened, it rolls into a ball, which causes the spiky scales to stand
erect.
SCALES
CLAW
FORE LIMB HIND LIMB
Fig. 18.3. Pangolin
BOTTLE NOSE DOLPHIN
'Found in coastal waters of most tropical, subtropical and temperate regions, weigh about
350 to 450 pounds and grows to a length of about 7 ft Characterized by following features'
1. Dark gray on back and lighter gray on flanks and belly. Skin IS smooth well adapted for
aquatic mode of life.
2. Centrally placed, tall, slender, sickle shaped dorsal fin is present.
3 .. Robust head with distinct short beak like snout 6 inch in length with 23 to 28 teeth in
each row on both upper and lower jaws.
4. Pectoral fins are sickle shaped, which are modified fore limbs and measure about one
fifth of the body length used for balancing.
5. Nostrils or the blowhole is situated on the dorsal position of the head, which IS a pair of
slits.
6. Tail fins or flukes are large, forked and horizontal in position used for propulSion by
beating them up and down vertically.
SNOUT
FLUKE
PECTORAL FIN
Fig 18.4 Bottle nose dolphin (Tursiops aduncus).
BLUE WHALE
It is the largest whale measuring up to 100 ft In length and weighing 120 to 150 tons Mostly
found In Antarctic ocean and prefer cold enVIronment. Characters are as follows
1. Body is torpedo shaped and blue above and yellowish gray below
2. Skin IS smooth and naked and deeply grooved below the throat
3 Doral fin is highly reduced and shifted far back and the tall fin IS called fluke IS hOrizontal
and forked used as rudder and for propulsion In water
4. Fore limbs are modified into paddles used for balancing and swimming
5 Teeth are absent, Instead large hair like baleens hang out from the upper Jaw, which
help in sieVing the plankton from the sea water
FLUKE
SEA COW (DUGONG)
DORSAL FIN
PECTORAL FIN
Fig. 185. Blue whale (Balaenoptera)
BALEEN
It IS commonly called as sea cow because It feeds mainly on sea weeds It grows up to 10ft
in length and weighs about 400 to 600 pounds. Characterized by following features
1 Body is more or less rounded or cylindrical with slightly flat belly. Neck is absent and the
head is blunt in front.
2. Tail fluke is horizontal used for propulsion in water
3. Fore limbs are flattened and paddle shaped, used for swimming. The mother for grasping
the young one uses occasionally fore limbs.
4. The upper lip is considerably large and it project over the lower lip and IS horseshoe
shaped with a fleshy pad overhanging the mouth. The upper lip bears sensory bristles,
which are modified hair
5. Nostrils are crescentic in shape and placed on top of the head.
6. External ear is In the form of a small circular aperture
\
FLUKE
Fig. 18.6. Sea Cow (Dugong dugon).
I DEVELOPMENTAL BIOLOGY I
19. STUDY OF DIFFERENT TYPES OF EGGS
ISOLECITHAL (HOMOLECITHAL)
The egg of placental mammals and amphioxus belong to this category. It is characterized by
following features:
ZONA PELLUCIDA
ANIMAL POLE
(PIGMENTED),
VITELLINE MEMBRANE
Homolecithal egg
(Mammalian)
YOLK GRANULE
SHELL
YOLK
ALBUMEN _-... .... ",
Mesolecithal egg
(Frog)
VEGETAL POLE
(VOLKY)
INNER SHELL
MEMBRANE
OUTER SHELL
MEMBRANE
AIR SPACE
OR KAUP
CHALAZA
Discoidal egg
(Hens egg)
Fig. 19.1. Types of eggs.
DEVELOPMENTAL BIOLOGY 57
1. The cytoplasm is translucent and the nucleus is more or less in the center of the-egg.
2. Yolk granules present in the cytoplasm are uniformly distributed.
3. The animal and vegetal pole of the egg cannot be distingUished from one another
MESOLECITHAL
are:
The egg of frog, fish and certain molluscs belong to this category. The charactenstlc features
1. There is moderate amount of yolk present in the cytoplasm that is either uniformly
dlstnbuted as in fish or highly concentrated in the vegetal hemisphere as in frog
2. In the fish, the nucleus IS not visible due to opacity of the cytoplasm where as in the
case of frog egg the nucleus is excentnc, i.e., more toward animal pole due to the
heaviness of yolk in the vegetal pole.
3. In the frog egg, the animal pole is brown in colour due to presence of pigment and the
vegetal pole IS yellow due to the concentration of yolk.
DISCOIDAL EGG
The eggs of reptiles, birds and egg laying mammals belong to thiS category. The egg is
characterized by following features:
1. The true egg is surrounded by many accessory structures like shell, shell membrane and
albumen since it is laid on land. The entire development of the young one takes place Inside the
shell cavity. Therefore it is called cleldoic egg.
2. There is large amount of yolk present in the cytoplasm occupying the entire volume of
the egg. Therefore, the nucleus with little amount of cytoplasm is placed In the animal pole as a
small circular diSC called bastodisc or germinal disc.
20. STUDY OF DIFFERENT TYPES OF BLASTULAE
COELOBLASTULA OF AMPHIOXUS
It is a typical coeloblastula, which shows the following characteristic features In sagittal section
1. The blastomeres are arranged in a single layer around the enlarged blastocoel or
segmentation cavity giving the appearance of a hollow bali.
2. The micromeres are found at the animal pole and the macromeres are confined to the
vegetal pole.
COELOBLASTULA OF FROG
The blastula of frog is a modified coeloblastula. The sagittal section shows the follOWing
essential features.
1 The blastocoel is excentric and is located in the animal hemisphere.
2. The blastomeres of animal and vegetal hemisphere show remarkable difference in size
due to unequal distribution of yolk. Animal pole blastomeres are small called mlcromeres
and ',(egetal pole blastomeres are large ca!led macromeres.
3. Micromeres form blastoderm of 3 to 4 cell layers thickness over the blastocoel in the
animal pole.
4. Macromeres form a floor of blastocoel of several layers of cells.
BLASTOMERES MICROMERES _ ~ a J ~ ~
BLASTOCOEL
BLASTOCOEL
MACROMERES
AMPHIOXUS
FROG
~ ~ EMBRYONIC KNOB
(INNER CELL MASS)
TROPHOBLAST
BLASTOCOEL
MAMMAL (Rabbit)
Fig. 20.1. Types of Blastulae.
BLASTULA OF MAMMAL
It is called blastocyst or bastodermic vesicle. The median section of blastocyst shows the
following features:
1. It consists of an outer single layer of cells called as trophoderm or trophoblast.
2. Enclosed within the trophoderm is a mass of cells called embryonic disc or knob made
of embryonic stem cells attached to one region of the trophoderm.
3. The free space within the trophoderm is called blastocoel, which is filled with fluid
continuously bathing the embryonic stem cells.
DEVELOPMENTAL BIOLOGY 59
21. STUDY OF GASTRULAE
GASTRULA OF FROG (YOLK PLUG STAGE)
It is a late gastrula stage, where the yolk plug IS clearly vIsible on one side of the egg, where
the yellow coloured yolk laden macromeres are seen In the form of a plug to the blastopore The
sagittal section of this stage shows the following characteristics'
1. The dorsal lip (above the yolk plug) and ventral lip (below the yolk plug) of the blastopore
are visible.
2. A large cavity leading from the blastopore called archenteron is visible, it is lined by
entodermal cells.
3. Outer layer of cells constitute the ectoderm of the gastrula
4. In between ectodermal layer and entodermal layer is present a narrow cavity, which is
the obliterated blastocoel after the growth of entoderm.
NEURAL PLATE AREA
NOTOCHORD AREA
YOLK PLUG
YOLK CELLS

G MESODERM
-'""'..-,. AREA
ECTODERM
Fig. 21 1. Gastrula of Frog
GASTRULA OF BIRD (CHICK)-PRIMITIVE STREAK STAGE
After 18 hrs of incubation of hen's egg and cut open, a circular white diSC will be vIsible,
which is nothing but blastodisc or germ center. It measures about 10 mm In diameter At thiS
stage, the blastoderm is in the early primitive streak stage. The key characteristic features are as
follows:
1. The blastoderm shows two general divisions a small clear inner area termed the area
pellucida, and a broad, heavily stained outer marginal area, known as the area opaca
2. There IS a heavily staining, axial thickening of the blastoderm, termed Primitive streak,
which extends in cephalo-caudal direction. The lateral margins are known as the primitive
ridges, and between these lies a sunken primitive groove. The primitive groove terminates
anteriorly in a primitive pit and just in front of it is the node like structure called Hensen's
node.
HENSEN'S NODE
PRIMITIVE PIT
PRIMITIVE GROOVE
PRIMITIVE RIDGE
AREA PELLUCIDA
Fig. 21.2. Gastrula of bird (chick).
SECTION PASSING THROUGH THE PRIMITIVE STREAK
1. In the center, primitive groove with primitive ridge on either side is visible.
2. Dorsally on either side of the primitive ridge extension of many layers of cells IS seen
called ectoderm.
3. On the ventral side also, the pri'mitive streak extends laterally into wing like structures
below the ectodermal layer called entoderm, which is in close proximity to yolk mass.
4. Between the ectoderm and entoderm, a thin layer of cells extending from the pnmitive
streak is visible called mesoderm.
MESODERM
ENDODERM
Fig. 21.3. T. S. of PQmitive streak passing through middle region.
PRACTICALS IN ECOLOGY
22. QUANTITATIVE ESTIMATION OF
DISSOLVED OXYGEN (DO)
61
Aim: To estimate the amount of dissolved oxygen in the given sample of water
Requirements: Winkler's A (40 gm MnS0
4
+ made up to 100 ml in OW), Winkler's B (50 gm
of NaOH + 15 gm of KI + made up to 100 ml in OW), Conc. H
2
S0
4
, Sodium thiosulfate (31 gm/L),
1 % starch solution as indicator.
Principle: Manganous sulfate is added to a known quantity of water and is immediately
followed by addition of alkaline potassium iodide solut'on. Manganous hydroxide precipitates out
and reacts with dissolved oxygen of water and gives nse to tetravalent oxide of Manganese
2Mn
2
+ +
2(OHt
(from Winkler's A) (from Winkler's B)
+ 02
Mn(OH)2
(White precipitate)
2MnO(OH)2
(Manganic oxyhydroxide)
When the resulting solution is acidified with Conc. H
2
S0
4
in the presence of Iodide. the
iodised manganese again reverts to divalent state and iodine equivalent to onginal dissolved oxygen
in water is liberated. The iodine liberated is then titrated with the standardized sodium thiosulfate
solution.
Procedure:
2MnO(OH)2 + 8W + 61-
21 - + 2S 2-
3 2 3
(thlosulphate)
2Mn+2 + 31
3
- + 6H
2
0
61- + S 2-
4 4
(tetrathlonate)
1. To the sample of water In 300 ml BOD bottle add 1 ml Winkler's A rapidly below the level
of water to prevent oxidation. Mix well by closing the stopper of the bottle
2. Add 1 ml Winkler's B below the level of water. Stopper the bottle and mix again. Brown
coloured precipitate is formed which is allowed to settle down.
3. After 5 min. add 1 ml of conc. H
2
S0
4
to dissolve the precipitate Mix we!! and transfer
100 ml into a conical flask and titrate against sodium thiosulfate until straw colour develops
4. Add few drops of 1 % starch and titrate further against sodium thiosulfate to a colourless
or light blue colour end pOint.
Calculations: Since 1 meq (milli equivalent) of 8
2
3 corresponds to 8 mg of 02 Dissolved
oxygen content of sample water IS
1000
= Burette reading (BR) x Normality of Na
2
Sp3 x 8 x 100 mg/L
Bottle correction:
1000
BR x 0.0125 x 8 x 100 mg/L
BR x 1 mg/L
Smce 1 mg of O
2
at NTP has a volume of 0.7 ml
Dissolved oxygen content of sample water
= BR x 0.7 mg/L assuming NTP conditions
Result: The oxygen content of the given sample of water IS ____ mg/L = milL.
23. QUANTITATIVE ESTIMATION OF BIOCHEMICAL
OXYGEN DEMAND IN WATER (BOD)
The reagents required and the procedure involved is the same as explained for DO except
that the water is kept in tightly stoppered BOD bottles m the darkness for 5 days or for 1 hr and
carry out the experiment as explained in DO procedure
Calculation: BOD of the sample water IS
= Mean BR from DO - Mean BR from BOD x 0.7 mg/Llhr or for 5 days ml/Llhr or for 5
days.
24. QUANTITATIVE ESTIMATION OF FREE CARBON
DIOXIDE IN WATER
Aim: To estimate free carbon dioxide In the given sample of water.
Requirements: N/44 NaOH (0.909 gm of NaOH in 1 lit of DW), Phenolphthalein Indicator
(Dissolve 19 phenolphthalein powder in 50% alcohol and neutralize with N/44 NaOH).
Principle: Free carbon dioxide forms weak carbonic acid in the water, which reacts with
sodium hydroxide to form sodium bicarbonate. At pH 8.3, hydrogen is completely displaced and
pink colour is formed with excess of NaOH and phenolphthalein indicator.
pH 83
- - ~ NaHC0
3
+ H
2
0
PRACTICALS IN ECOLOGY 63
Procedure:
1. Take 100 ml of freshly collected sample of water in a conical flask.
2. Add two drops of phenolphthalein indicator and titrate it against NaOH.
3. End point is colourless to pink colour.
Calculation:
[only one W of H
2
C0
3
is displaced at pH 8.3, which turns phenolphthalein pink. 1 meq of
NaOH = 1 meq of H
2
C0
3
= 1 meq of CO
2
or 44 mg of CO
2
]
Since 1 meq of NaOH corresponds to 44 mg of CO
2
, Free CO
2
content of sample water
1000
= BR x Normality of NaOH x 44 x Vol. of sample taken = mg/l
1 44 1000
= BR x- x- x-- mg/l
44 1 100
= BR x 10 mg/l
At NTP, 1 mg of CO
2
has a volume of 0.509 ml
Therefore, Free CO
2
content of sample water
= BR x 10 x 0.509 mill
= BR x 5.09 mill or mg/l
25. ESTIMATION OF INORGANIC PHOSPHATE BY FISKE &
SUBBAROW METHOD
Principle:
Inorganic phosphate reacts with ammonium molybdate under acidic condition to form
phosphomolybdic acid. Addition of reducing agent 1,2,4-aminonaphthosulphonlc aCid (ANSA)
reduces the molybdenum in the phosphomolybdate to give a blue coloured complex, the Intensity
of which is proportional to the amount of phosphate present. The blue coloured complex has an
absorption maxima at 660 nm.
Materials and Reagents:
1. 10N H
2
S0
4
(add 450 ml of conc. H
2
S0
4
slowly whilst cooling to 1,300 ml of distilled
water.
2. 2.5% ammonium molybdate in 5N H
2
S0
4
: Dissolve 25 grams of the salt in about 200 ml
of water, transfer to a litre flask containing 500 ml of 1 ON H 2S0 4. Dilute to 1 lit with water
3. 0.25% 1 ,2,4-aminonaphtholsulphonic acid. Add 0.5 9 of the dry powder to 195 ml of 15%
sodium bisulphate and 5 ml of the 20% sodium sulphite. Stopper and shake until It IS
dissolved __ lf the bisulphate solution is old, more than 5 ml of sulphite may be needed, In
which case add more sulphite, 1 ml at a time shaking well after each addition until complete
64 A tiANDBOOK OF PRACTICALS IN ZOOLOGY
solution is obtained. Store in the cold in a brown bottle. The solution keeps up to four
weeks, and IS more stable the higher the acidity so that no more sulphite should be
added than is required to dissolve the amino-naphtholsulphonic aCid
4. Standard phosphate solution' Dissolve 1.36g of pure potassium dihydrogen phosphate
in water in 30 ml distilled water and make up the volume to 100 mlm a volumetric flask
Add few drops of chloroform, which acts as a preservative. For preparing working standard
dilute 1 ml of stock solution to 100 ml with distilled water in a volumetric flask to give a
phosphorus concentration of 31 mg/ml
Procedure:
Prepare the test tubes as given in the table:
Serial Std. Cone. of Ammonium ANSA DW(ml) Abs. at
No. Phosphorus Phosphorus Molybdate 660 nm
1 0.2 ml 6.2 mg 1.0 ml 0.1 ml 8.7
2 0.4 ml 12.4 mg 1.0 ml 0.1 ml 8.5
3 0.6 ml 18.6 mg 1.0 ml 0.1 ml 8.3
4 0.8 ml 24.8 mg 1.0 ml 0.1 ml 8.1
5 1.0 ml 31.0 mg 1.0 ml 0.1 ml 7.9
6 Blank - 1.0 ml 0.1 ml 8.9
7 U
1
(1.0 ml)
- 1.0 ml 0.1 ml 7.9
8 U
2
(1.0ml)
- 1 0 ml 0.1 ml 7.9
9 U
3
(1.0 ml) - 1.0 ml 0.1 ml 7.9
Construct a .calibration curve on a graph paper, by plotting the concentration of inorganic
phosphorus (6.2 mg to 31 mg) on X-axis and the absorbance on the Y-axis. Compute the
concentration of phosphorus in the sample from calibration curve and by calculation method.
Calculation method:
Conc. of phosphorus =
in unknown sample
RESULTS:
OD of Unknown
x Conc. of Std.
OD of Standard
Amount of phosphorus in unknown solution ________ mg/ml.
26. QUANTITATIVE ESTIMATION OF SALINITY OF WATER
(ARGENTOMETRIC METHOD)
Aim: To determine the salinity in the given sample of water.
65
Requirements: 0.28N silver nitrate, potassium chromate Indicator, burette and water sample.
Principle: Salinity is defined as the total solids in grams that can be obtained from 1 Kg of
seawater when all the carbonates are oxidized, chlorides replace all bromides and Iodides and all
the organic matter has been oxidized. Chloroslty is the total of chlorides, bromides and Iodides all
reported as chlorides ..
In the presence of halides, silver nitrate forms silver halide and potassium chromate In the
absence of any halides will react with silver nitrate to form a red coloured solution of silver chromate
NaCI + AgN0
3
NaN0
3
+ AgCI
Procedure:
1. Take 10 ml of sample water in a conical flask and add two drops of potassium chromate
indicator. Mix the solution.
2. Titrate against 0.28N silver nitrate solution till reddish brown coloured precipitate IS
obtained, which is the end point.
Calculation:
Since 1 meq of AgN0
3
'= 35.5 mg of CI-
Chlorosity equivalent of 1 ml of silver nitrate solution = 0.28 x 0 0355 = 0 0101494
burette reading x 0.0101494 x 1000
Therefore chlorosity (CI/L) = --------------
Volume of sample taken
= CI/L
Salinity (S) = 0 03 (temperature correctionfactor at 30C) + [1 805 x CI/L)]
= S gm/L.
Note: For sea water dilute 1 in 10.with distilled water (1 ml of sea water and 9 ml of DW) to
minimize consumption of silver nitrate and multiply the value by 10 (dilution factor).
27. DETERMINATION OF TOTAL HARDNESS OF WATER
Aim: To determine the hardness of water by EDTA titrimetric method.
Reagents:
1. Buffer solution: Dissolve 16.9 gm of Ammonium chloride in 143 ml conc Ammonium
hydroxide and dilute to 250 ml with distilled water.
2. Eriochrome black T. indicator: 0 5 gm of sodium salt of dye (eriochrome black T)
dissolved in 100 gm of triethanolamine. Store In a brown bottle.
3. Ethylene diamino tetra acetic acid (EDTA) (0.01 M): Weigh 3.723 gm of disodium EDT A
and dissolve In 50 ml of DW and make up the volume to 1000 ml and store In a brown
bottle.
4. Standard Calcium: Weigh 1 gm of calcium carbonate powder into a 500 ml conical
flask. Place a funnel in the neck of the flask and slowly add conc. HCI drop wise and
shake the flask repeatedly until the entire CaC0
3
dissolved. Add 200 ml DW and boil for
a few minutes to expel CO
2
.
Principle:
EDTA forms chelated soluble complex when added to a solution of certain metal cations. If
small amount of dye such as Eriochrome black T or calmagite IS added to an aqueous solution
containing calcium and magnesium ions at pH 100 the solution turns wine red. If EDTA is added
as a titrant, the calcium and magnesium will be complexed. When all of the magnesium and
calcium has been complexed the solution turns wine red to blue, marking the end point of titration.
Procedure:
Take 10 ml of sample in a conical flask
2. Add 1 ml of buffer solution and shake well
3. Add 1 to 2 drops of eriochrome black indicator, shake well to get wine red colour
4. Titrate it against EDTA by adding drop wise from the burette until the colour changes
wine red to blue
Calculation:
1 meq of EDTA = 40.08 mg of Ca
2
+
Since atomic weight of Ca
2
+ is 40.08, 1 ml of 0.01 mol EDTA = 0.4008 mg of Ca
2
+
When standardization is done with Std. Calcium,
Hardness (EDTA) as mg Ca
2
+ IL
=
=
BR x Normality of EDTA x 40.08 x 1000
10 (Volume of sample taken)
BR x 001 x 40.08 x 1000
BR x 40.08 mg of Ca
2
+/Lit.
28. TO INVESTIGATE THE WATER CONTENT OF SOIL
Aim: To determine water content of the soil
Requirement: Aluminum cake cups, Oven set at 150C, desiccator, tongs, and balance
Procedure:
1. Weigh the aluminum cake cup and record the weight (a gm).
2. Add finely broken up soil sample to the aluminum cup and take the weight (b gm)
3. Place the dish with the soil sample in the oven at 110C for 24 hr.
4. Remove the sample from the oven and keep it in the desiccator to cool
5. Weigh the sample when cool and record the mass (c gm)
6. Repeat the procedure 4 and 5 until co!,]sistent weights are recorded
Calculation:
To calculate the percentage of water content:
b-c x 100%
b-a
29. TO INVESTIGATE THE ORGANIC (HUMUS)
CONTENT OF SOIL
Simple method:
Aim: To determine the organic content of the soil.
67
Requirement: Dried soil sample, desiccator, crucible with lid, burner, tripod with heat poof
mat, tongs.
Procedure:
1. Heat the crucible covered with its lid so as to evaporate all traces of mOisture from It
Keep it in the desiccator for cooling. After cooling weigh the crucible and take note of the
weight of empty crucible (a gm).
2. Take some dried SOil sample in the crucible and weigh it Record the weight (b gm)
3. Heat the soil sample In the crucible covered with lid to red heat for 30 min until all the
organic matter is burnt off. Allow it to cool for 15 min.
4. Take the weight of the cooled crucible with sample (c gm).
5. Repeat procedure 3 and 4 until constant weight is recorded. Record the weight.
Calculation:
Calculate the percentage of organic content as follows:
68
b -c x 100%
b-a
Repeat the experiment with soil samples taken from different area Note the difference In
organic content of soil from different areas.
Alternative Titrimetric method (Walkley and Black):
Principle: The organic carbon found in the soil is oxidized by the nascent oxygen produced
from the reaction of K
2
Cr
2
0
7
and H
2
S0
4
, The excess K
2
Cr
2
0
7
, not reduced by organic carbon is
determined by back titration with standard ferrous ammonium sulfate [FeS0
4
(NH
4
)2 S04 6H
2
0]
Reaction:
K
2
Cr
2
0
7
+ 4H
2
S0
4
C (organic carbon) + 20
2FeS0
4
+ H
2
S0
4
+
K
2
S0
4
0 + Cr
2
(S04)3 + 4Hp + 30
CO
2
Fe
2
(S04)3 + H
2
0
Requirements: Burette stand, burette, conical flasks, asbestos sheet, 1 N K
2
Cr 207 - 49 04g1
Lit., 0.5N Ferrous ammonium sulfate - 1969 in 200 ml of DW, heat on a low flame and gradually
add 20 ml of Conc. H
2
S0
4
, raise the volume of clear solution, to 1 liter with DW. Diphenylamine
reagent - 0.5g In a mixture of 20 ml water and 100 ml conc. H
2
S0
4
" Conc. H
2
S0
4
and 85%
Orthophosphoric aCid (Qualigens-commercial).
Procedure:
Take 0.5g of finely powdered air dried soil in 250 ml corning conical flask and add 5 ml of 1 N
K
2
Cr 207 and 10 ml conc. H 2S0 4 slowly by keeping the flask on asbestos sheet SWirl the flask
carefully and keep standing for 30 min. Add 50 ml of DW, 5 ml of 85% orthophosphoric aCid (to
block the interference of oxides of nitrogen and other minerals) Ass 1 ml of diphenylamine reagent
and titrate against ferrous ammonium sulfate till a colour change from blue-violet to parrot green
is obtained Repeat the procedure Without soil (blank)
Calculation:
Weight of the soil taken = 0.5g
Volume of 0.5N ferrous ammonium sulfate required to utilize 5 ml of 1 N K
2
Cr
2
0
7
(blank)
= B ml.
Volume of O.5N ferrous ammonium sulfate required to utilize 5 ml of 1 N K
2
Crp7 (sample)
= C ml
Therefore volume of 1 N K
2
Cr
2
0
7
used for oxidation of organiC carbon is soil
5(8 - C)
= ' ml
B
From the reaction 3, it IS apparent that 4(FeS0
4
) are consumed when one "C" IS OXidized to
CO
2
,
Therefore 1 meq of FeS0
4
= 1 meq of K
2
Cr
2
0
7
= % meq of Cor 12/4 mg of C = 0.003g of C
PRACTICALS IN ECOLOGY 69
5(B -C) 100
Therefore organic carbon of soil = 0.003 x B x 0.5 g%
To determine organic matter content of soil, this value of organic carbon content IS multiplied
by Van Bermmelon factor of 1.724 because organic matter contains average of 58% organic carbon
5(B-C) 100
Therefore organic matter in soil = 0.003 x B x 05 x 1.724g%
30. TO INVESTIGATE THE pH OF SOIL
Aim: To determine the pH of given sample of soil.
Requirement: Test tubes, test tube rack, barium sulfate, BOH universal Indicator and colour
chart, Soil sample, spatula, distilled water and 10 ml and 1 ml pipette.
Procedure:
1. Take a spatula full of soil into a test tube and add a spatula full barium sulfate.
2. Add' 10 ml of distilled water and mix the contents vigorously.
3. To the mixture add 2 ml of BOH pH indicator solution shake the contents vigorously
4. Allow the contents to settle for 5 min.
5. Compare the colour of liquid in the test tube with the colour on the BOH reference colour
chart and read off the corresponding pH.
Note down the pH reading. Repeat the experiment on soil samples from different areas and
record the pH of soil sample from different areas.
000
I Practical - II I
pH AND BUFFERS 73
1. pH METER
1. PRINCIPLE AND MODE OF WORKING
The most convenient and reliable method for measuring the pH of a given solution is by the
use of pH meter, which works on the principle of EMF. (electromotive force). EMF of a concentration
cell formed from a reference electrode, the test solution, and a glass electrode sensitive to hydrogen
ions as shown in the Fig. 1.1.
PH meter
1
--------1 A potentiometer to
measure the EMF
I Ag-AgGI I HGI (0.1 mol/lit) I Glass II I
Glass electrode
Test solution
II' KGI (satd) I H9
2
GI
2
Hg I
Calomel reference electrode
Fig. 1.1. The voltaic cell formed dunng the measurement of pH.
Most pH measurements today are obtained using a single combination electrode (Fig.
1.2). In this electrode, both the reference and the pH-dependent electrode are contained In single
glass or plastic tube Using a pH meter with combination electrode is relatively easy but certain
guidelines must be followed.
1 A pH meter not In use left in a "standby" position for qUite some time Before use check
the level of saturated KCI in the electrode If It IS too low, fill In saturated KCI solution Into
the electrode through the filling hole
2. Use the standard calibration buffers of pH 4.0, 7 0 and 9.0 to caiibrate the Instrument
and make sure the temperature knob is set at correct temperature of the calibration
buffer and the test solution. Be sure the pH is properly set with calibration buffer
3. Lift the electrode gently from the solution. nnse It with distilled water from a wash bottle,
and gently clean the electrode by blotting it with soft tissue. Calibrate the pH meter with
all the three calibration buffers with a accuracy of 0.02 pH unit
4 The bulb of the electrode must be completely covered with solution
5. To measure the pH of different solutions, each time rinse the electrode with distilled
water.
6. If pH measurements of protein solutions are often taken, a protein coat Will form on the
electrode; it can be removed by soaking the electrode in 5% pepsin in'O 1 M HCI for 2 hrs
and rinsing well with distilled water.
7. If the electrode is new, it should be activated for 24 hrs. in 0.1 N HCI by keeping the
electrode in it and then another 24 hrs in distilled water.
8. pH meter should be switched on at least 15 minutes before taking any readings.
74
Ag/AgCI
ELECTRODE
v--FILLING HOLE
..... --SATURATED KCI
FIBER OR CERAMIC
LIQUID JUNCTION
Fig. 1.2. Combined electrode of pH meter.
2. HENDERSON-HASSELBALCH EQUATION
PREPARATION OF BUFFERS OF DIFFERENT pH USING HENDERSON-HASSELBALCH
EQUATION
Principle:
A buffer is a solution that resists change in pH on addition of acid or alkali. A typical buffer
system consists of a mixture of a weak acid (HA) and its salt (SA). The weak acid dissociates as
(1) HA W +A- but extent of ionization is very low. The salt also ionizes as (2) SA S+ + A- but
this is fully ionized. Since the system is in equilibrium, the various concentrations of ionic species
remain constant. If acid (W) is added to this system reaction (1) is driven backward to form HA so
that W concentration remains the same. Notice that the reaction (2) provides the extra A- ions
HENDERSON-HASSELBALCH EQUATION" 75
needed for the equilibrium to be maintained. If on the other hand, a proton acceptor (OH-) ion is
added to the system then W is removed from the system to form water. Therefore, the concept
arises that the ionization of HA, the weak acid which is the source of W ions is controlled by the
presence of a fully ionizing salt. Mathematically an equation can be derived, which is called
Henderson-Hasselbalch equation is as follows:
H K I
Concentration of Salt
p = p + og
a 10 Concentration of Acid
When the concentration of salt and the acid is equal to 1, log10 1 = 0, Therefore pH = pK
a
Ka is the dissociation constant of a weak acid and pK
a
= -log1OKa' When pH = pK
a
maximum
buffer action is observed.
By using Henderson-Hasselbalch equation, it is possible to find out the theoretical pH of a
buffer prepared by mixing the appropriate quantity of acid and its constitutive base or salt at the
same concentration.
Procedure:
Prepare (A): 0.2M solution of acetic acid (11.55 ml In 1000 ml DW)
(8): 0.2M solution of sodium acetate (16.4 gm anhydrous) or 27.2 gm of hydrous
sodium acetate in 1000 ml DW).
Mix the solution of sodium acetate and acetic acid as given in the table and record the pH
from the pH meter. Calculate the pH using Henderson-Hasselbalch equation, where Ka of acetic
acid is 4.76 at 25C.
Test Volume of Volume of pH pH measured from
tubes acetic acid sodium acetate calculated pH meter
in ml in ml
1 47 3
2
;4-1
9
3 28 22
4 15 35
5 10 40
6 6 44
7 5 45
From your results explain, why some solutions are better buffers than others.
3. TITRATION CURVE OF STRONG ACID WITH A
STRONG BASE
Requirement: Hydrochloric acid (0.1 M), Potassium hydroxide (0.1 M), burette, pH meter
Procedure:
1. Pipette 20 ml of 0.1 M Hel into a beaker and measure the pH.
2. Titrate this solution with 0.1 M KOH and measure the pH after the addition of 1 ml of
KOH.
3. Repeat this procedure until you reach the end point. When nearing the end pOint, reduce
the volume of KOH added to give a reasonable change in pH.
4. Plot the titration curve pH against volume of KOH added.
Discuss the result.
4. ,DETERMINATION OF pK
a
OF WEAK ACID
Requirement: 0.1M Oxalic acid (12.6 gm of oxalic acid in 1 lit of OW), 0 1M potassium
hydroxide (5.611 gm in 1 lit of OW), burette and pH meter.
Procedure:
1. Take 20 ml of acid in beaker and measure its pH.
2. To this add 2 ml of alkali, mix it well and measure its pH.
3. Successively add same amount of KOH and measure the pH.
4. Plot a titration curve pH against volume of KOH added.
5. Find out the pH equivalent to the mid point of the each of the two plateaus, which represent
the pK
a
.
6. Repeat the experiment with 0.1 M acetic acid, lactic acid and benzoic acid and find their
pK
a
values in the same manner.
Discuss the result.
COLORIMETER 77
5. STUDY OF COLORIMETER
PRINCIPLE AND WORKING:
The colo'rimeter is an instrument, which helps us determine the extent of absorption of light
of particular wavelength by any solution Here by using different coloured filters one regulates the
range of wavelength that fallon the solution A diagram of the basic arrangement of a tYPical
colorimeter is given In Fig, 5 1.
SLIT FILTER PHOTOCELL
(Q)
I I I
LAMP LENS
SAMPLE
HOLDER
AMPLIFIER
Fig. 5.1. A diagram of a photoelectric colorimeter.
White light from a tungsten lamp passes through a slit, then a condenser lens, to give a
parallel beam, which falls on the solution under investigation contained in an absorptIOn cell or
cuvette, The cuvette is made of glass with the sides facing the beam cut parallel to each other
Beyond the absorption cell is the filter, which is selected to allow maximum transmission of
the colour absorbed. If a blue solution IS under examination, then red is absorbed and a red filter
is selected. The colour of the filter IS therefore, complementary to the colour of the solution under
investigation (Table 5.1). The filter gives narrow transmission bands; which almost Similar to
monochromatic light.
TABLE. 5.1
The relationship between the colour of the solution examined and the filter chosen for
colorimetric analysis.
Colour solution Filter Wave length
Red-orange Blue-blue green 435-500
Blue Red 610-750
Green Red 610-750
Purple Green 440-480
Yellow Violet 435-480
The light transmitted from the cuvette falls on to a photocell, which generates an electrical
current in direct proportion to the intenSity of light falling on it This small electrical signal is Increased
in strength by the amplifier, and the amplified signal passes to a galvanometer or digital readout,
which is calibrated with a logarithmic scale so as to give absorbance reading directly The blank
solution is first put in the colorimeter and the reading is adjusted to zero extinction; this is followed
by the test solution and the extinction is read out directly.
Absorption characteristics: Each compound, depending on its chemical characteristics
absorbs light of only certain wavelength. This is called absorption spectrum. At particular wavelength
the absorption is maximum and it is called absorption maxima.
Laws of absorption: There are two basic laws, which govern the absorption of light by
solutions. Lambert's law states that the amount of light transmitted through a solution depends on
the distance of the light path. Beer's law states that the amount of light absorbed per unit thickness
is proportional to concentration of the absorbing particles.
It can be represented as follows.
10 = intensity of incident light, I = intensity of transmitted light,
1110 = T = Transmittance, 1/10 x 100 = Percent transmission,
I = distance through which light passes, c = concentration of solution.
:. log Iii = log lIT = KCL = optical density (00). If k is a constant called extinction coefficient.
If the Beer's law is obeyed and I is kept constant, then a plot of extinction against concentration
gives a straight line passing through the origin. With the aid of this standard curve the concentration
of an unknown solution can be easily found out.
(i) SELECTION OF FILTER
Aim: To determine the absorbance of different coloured solution against the range of filters
provided with colorimeter.
Principle:
Coloured compounds have their own characteristics absorption spectra and careful selection
of the wavelengths where maximum absorption is found enables a mixture of two-coloured
substance to be analyzed.
Requirements: Colorimeter, methylene blue (10 mg/lit), Potassium dichromate (10 mg/lit),
1: 1 mixture of methylene blue and potaSSium dichromate.
Procedure:
1. Determine the absorbance of each dye individually against the range of filters and note
the optical density (00) (absorbance) or transmittance of each filter.
2. Remember, when the filter is changed each time the colOrimeter should be adjusted for
zero absorbance or 100% transmittance with distilled water in the cuvette for each filter
3. Plot a graph of the absorbance (00) recorded against wavelength and note the
wavelength of maximum transmission or minimum absorbance.
4. Find out which wavelength gives maximum absorbance for each dye.
5. Find out how does mixing of dyes affect the absorption spectrum
Conclude the experiment.
COLORIMETER
(ii) DETERMINATION OF CONCENTRATION
79
Aim: Colorimetric determination of concentration 'Of unknown substance with the help of
absorption curve of substance of known concentration.'
Principle: Coloured compounds show characteristic maximum absorption at a certain
wavelength. A coloured solution of different concentration has different intensity of colour therefore
shows different absorption maximum, which is directly proportional to the concentration of the
dye. By plotting a graph of absorbance against different concentration, it is possible to find out the
concentration of dye of same absorbance in an unknown sample.
Requirements: Colorimeter, Methylene blue (0.1 mg/ml), Unknown sample of methylene
blue (pinch of methylene blue i.e. about the size of pin head in 10 ml distilled water) distilled
water.
Procedure:
1. Prepare 5 test tubes and in each test tube take different volumes, i.e., 0.2, 0.4, 06,0.8,
1.0 ml of 0.1 mg/ml methylene blue solution In each test tube add distilled water so as
make up the final volume to 5 ml!, i.e., 4.8, 4.6, 4.4, 4.2 and 4.0 ml of distilled water. Mix
it well.
'2. Place the filter in its slot in the colorimeter, which gave the maximum absorbance in the
previous experiment (selection of filter).
3. Adjust the colorimeter to zero absorbance or 100% transmittance with distilled water
4. Record the absorbance of each solution from the different test tubes
5. Plot a graph of absorbance against concentration of the dye in each test tube (mg/ml).
6. Find the absorbance of unknown solution and compare It with the graph of absorbance
with known concentration. From thiS, concentration of dye in unknown solution can be
found out. .
7. Find out the concentration of dye in unknown solution by calculation using this formula.
Calculation:
00 of unknown dye X Conc. of standard
Conc. of dye in unknown (mg/ml) = ---------------
.. 00 of standard
Result: Plot a graph of 00 against known concentration of dye. Compare the result With the
answer obtained by calculation method
6. STUDY OF OSMOSIS USING RBC's
1. STUDY OF OSMOSIS USING RBCs BY TEST TUBE METHOD
(IF LARGE AMOUNT OF BLOOD IS AVAILABLE)
Aim: To study the permeability of erythrocyte plasma membrane, specifically In terms of
whether certain substances can induce haemolysis by excessive permeability or alter the
morphology of plasma membrane by excessive loss of cytoplasmic water content due to osmosIs
In the opposite direction.
Requirements: Human, sheep or bovine whole blood, test tubes, 0.065M (hypotonic), 0 145M
(isotonic), 0.35M (hypertonic) NaGI solution, distilled water. Pasteur pipette, stopwatch, microscope
slides, cover slips, microscope.
Principle: When mammalian RBGs are suspended In hypotonic, IsotoniC and hypertonic
salt solution, RBGs behave differently in each solution due to ItS osmotic nature of the plasma
membrane. In hypotonic solution RBGs swell by taking in more water from the medium since the
concentration of salts and organic molecules is higher in the cytoplasm as compared to the medium
ultimately leading to haemolysis of RBGs In isotonic solution. RBGs maintain normal biconcave
shape. RBGs suspended in hypertonic solution undergo shrinkage and show crenated membrane,
i.e., wavy appearance on the membrane due to loss of water from the cytoplasm Into the medium
since the medium is more concentrated with salt Ions compared to the RBG cytoplasm All these
properties emphasize the osmotic property of the plasma membrane.
Procedure:
1. Take four test tubes and in each take 5 ml of NaGI of different concentration and in the
last tube take 5 ml of distilled water, which of course is hypotonic,
2. Take one test tube at a time and add 0.02 ml of whole blood, gently shake it. ThiS IS time
zero and start the stopwatch and"immediately hold the tube against a printed-paper with
bold letters.
3. Looking at the bold letters through the tube filled with RBG suspension In test solution.
watch for the pOint when the image of the bold letters can first be clearly resolved ThiS
is the end pOint. Stop the stopwatch and note the total time taken In seconds for the
Image to resolve through the RBG suspension.
4. Take the reciprocal of that time and record that as the relative rate of penetration Isec.
If the image cannot be discerned within ten minutes, then record the haemolysis time as
> 600 sec and relative penetration rate as < 0.0017/sec. As soon as this step IS completed,
move on to the next.
5. Using a Pasteur pipette, transfer a drop of the test mixture to a microscope slide. Apply
a cover slip and immediately examine the preparation under the microscope. First focus
under low power objective and bring into focus thinly spread out area, turn to high power
and study the appearance of the erythrocytes and sketch it.
Results: Haemolyzed RBGs suspension will have only membrane ghosts or empty shells of
plasma membrane. Grenated RBGs are shrunken and have spiked projections along their margins
OSMOSIS USING RBC'S 81
Normal RBCs are round with smooth margins; viewed from the side they are biconcave. Since
they are thinner in the center than along the margins, these two regions appear slightly different in
brightness or intensity
Tabulate the results against each concentration of salt solution and against distilled water as
well as draw a diagrammatic sketch against each character visualized under the microscope against
different concentrations of salt solution
2. STUDY OF OSMOSIS USING RBC's BY CAVITY SLIDE METHOD
(BLOOD IS OBTAINED BY FINGER PRICK METHOD)
Aim: As given in the previous section
Requirements: 0 85% saline, tooth pick, instead of test tubes cavity slides are used and
rest other requirements are same as given in previous section.
Principle: Same as given in prevIous section.
Procedure:
1. Take four clean cavity slides and in two triple cavity slide take a drop of 0 85% saline in
each cavity.
2. Clean the tip of the middle finger of the left hand with alcohol Allow the tiP to dry USing
a sterile disposable needle, prick the tip of the finger and gently squeeze It to get a drop
of blood. Touch thiS drop of blood to the drop of saline taken In the cavity of the slide
Repeat the same in all SIX cavities of two slides.
3. Take two more clean triple cavity slides, assign each cavity for one concentration of salt
solution and take in it one drop of a particular concentration of salt solution With the
help of Pasteur pipette take the saline mixed drop of blood from the cavity slide and add
it to the cavity where salt solution of known concentration is taken. Mix it With toothpick
Note dowPl this time as zero and start the stopwatch
4 Immediately hold the slide against a paper With bold letters printed, and look through the
cavity of the slide containing the blood suspension Watch for the pOint when the Image
of the bold printed words can be clearly seen ThiS IS the end pOint at which stop the
stopwatch Reco;d the time In seconds
5 Rest of the calculation is the same as given In the previous section.
6 For the observation of structure of RBC under the influence of different concentrations of
salt solution and distilled water, observe the slide under low power and high power
objective of the microscope.
7 Make note of the observations and tabulate the results.
7. STUDY OF ELECTRON MICROGRAPHS OF
CELL ORGAN ELLS
MITOCHONDRIA
1. Membranous, sphencal or elliptical in structure. Outer border consists of double
membranous boundary.
2. Inner membrane is thrown into many involutions or projections called cristae
3. Cristae are spread out in the mitochondrial matrix.
ENDOPLASMIC RETICULUM
There are two types: Rough endoplasmic reticulum (RER) and smooth endoplasmic
reticulum (SER). Both differ from each other by minor differences.
2. The RER consists of large number of membranous channels called cisternae stacked
one over the other in the cytoplasm of the cell.
3. On the membrane of the RER, dark rounded bodies are seen arranged In series all
along the membrane, they are ribosomes or protein synthesizing units, which give rough
appearance for the membrane, hence the name RER
4 The SER consists of large number of membranous channels called cisternae stacked
one over the other in the cytoplasm of the cell and the membrane is smooth, devoid of
ribosomal units, hence the name SER.
GOLGI COMPLEX
1. It is made up of many layered, membranous, elongated channels or cisternae. Some
times, elliptical (condensing vacuoles) or spherical vesicular (transitional vesicles) in nature
and each of them is surrounded by double membranous boundary.
2. Cisternae are interconnected at certain places, hence the Golgi matrix of each cisternae
is in contact with each other.
LYSOSOMES
1. They are spherical, vesicular bodies each is surrounded by double membranous boundary
2. Each vesicle is unique in its own way in its size and the type of matrix enclosed within It.
3. T.he matrix would contain phagocytized material in it, which is yet to be digested or the
digested material. Hence each lysosome looks different as far as its matrix and size is
concerned.
NUCLEUS
1. It is spherical in shape surrounded by double membranous nuclear envelope, which is
discontinuous. Membrane has nuclear pores at definite intervals
2. Matrix is dense, appears dark amorphous in nature. Sometimes depending on the stage
of the cell cycle, the nuclear matrix would contain thread like structure called chromatin,
which is nothing but nucleic aCid and protein.
ELECTRON MICROGRAPHS
MITOCHONDRIA
Fig. 7.1. Electron micrograph of mitochondria.
ROUGH ENDOPLASMIC
RETICULUM
Fig. 7.2. Electron micrograph of endoplasmic reticulum.
GOLGI VESICLES
GOLGI CISTERNAE
Fig. 7.3. Electron micrograph of Golgi complex.
83
84
" -
Fig. 7.4. Electron micrograph of lysosome.
NUCLEAR MEMBRANE
Fig. 7.5. Electron micrograph of Nucleus.
CHROMOSOME MORPHOLOGY 85
8. STUDY OF CHROMOSOME MORPHOLOGY
Aim: To study the morphology of chromosome from the mitotic cell of onion root tip
Requirements: Onion root tip fixed in Carnoy's fixative (Glacial acetic acid - 10 ml , absolute
alcohol - M ml , chloroform- 30 ml) . Onion .root tip should be cut and fixed in fixative before 9.00
AM in order to get many stages of mitosis. Duration of fixing should not be less than 3 to 4 hrs .. 1 N
HCI , acetocarmine or acetoorcein stain, 10% acetic acid.
Procedure:
1. Transfer one or two root tips into a watch glass and wash off the Carnoy's fi xative in
distilled water by giving two to three washes.
2. Decant the water and put a drop or two of 1 N HC!. Allow it to remain in it for 5 min.
3. Wash off the 1 N HCI with distilled water.
4. Add few drops of acetocarmine or acetoorcein stain to the root tip in the watch glass and
allow it to stain for 10 min.
5. Transfer the root tip to a microscope slide, add a drop or two of 45% acetic acid. With
the help of a sharp blade cut out the tip, which is darkly stained from the rest of the
portion of root. Discard the other portion and keep the darkly stained tip.
6. Place a cover slip and gently tap on the cover slip with the blunt end of the needle or
pencil.
7. Blot off the excess of mounting medium (10% acetic acid), apply quick fix on the border
to the cover slip to seal it so as to prevent the slide from drying.
8. Observe under low power objective of the microscope, focus on a thinly spread out area,
turn to the high power objective and locate a metaphase stage polar view to know the
structure of the chromosome and identify other stages of cell cycle to know the stages of
mitosis.
9. Make a diagrammatic sketch of each stage of the mitosis.
MAGNIFIED INTERPHASE NUCLEUS
Fig. 8.1. (a) Interphase.
(b) P R O P H S ~
A D1AGRAM\1ATlC PRESENTATION
' OF PROPHASE NUCLEUS
Fig. 8.1. (b) Prophase of mitosis in onion root tiP preparation
(C) EARLY METAPHASE
ANAPHASE
TELOPHASE
A DlAGRAlVMATIC PRESENTATION
OF METAPHASE (d) METAPHASE
A DlAGRAM\1ATlC PRESENTATION
OF ANAPHASE
A OIAGRAMt.MTlC PRESENTATION
OF TELOPHASE
Fig. 8.1. Metaphase (c, d,), Anaphase (e, f) and Telophase (g , h) of mitosis In onion root tip
preparation.
POL YTHENE CHROMOSOME 87
9. STUDY OF POLYTENE CHROMOSOME
Aim: To study the structure of polytene chromosome from the salivary gland of Chironomous
larva.
Requirements: Chironomous lar-vae, acetocarmine or acetoorcein, 10% acetic acid, 1 N HCI ,
microscope slides, cover slip, 2 fine sharp needles (21 gauge injection needles fixed to a thin
glass rod or broom stick), dissection microscope and microscope.
Procedure:
1. Take a mature chironomous larva on a microscope slide and mount it on a dissection
microscope.
2. Orient the larva in left to right position, place a needle on the head (blackish pigmented)
and the other needle on the body.
3. Pull the head apart from the body. Attached to the head long tubular digestive tract
comes out.
4. Focus through the lens of the dissection microscope near the head region and locate the
bag shaped salivary glands (as shown in the Fig. 9.1) with their ducts attached to the
digestive tract sticking out.
5. Isolate the salivary glands from the digestive tract by gently teasing the duct attached to
the digestive tract.
6. Discard other debris by gently pushing it away from the pair of isolated salivary glands,
blot off excess water.
7. Add a drop or two of 1 N HCI on the salivary gland. Allow it to remain for 5 min.
8. Blot off excess of 1 N HCI and add a drop or two of stain (acetocarmine or acetoorcein) .
Allow it to stain for 15 min. Do not allow the stain to dry.
9. Wash off the excess of stain with 10% acetic acid and mount the salivary glands in 10%
acetic acid.
10. Place the cover slip gently on the glands, blot off the excess of acetic acid, and seal the
border of the cover slip with quick fix.
11. Observe the slide under low power objective of the microscope, locate a cell with properly
spread out chromosome, turn to the high power objective and study the nature of the
chromosome.
Observation: The gland shows linearly arranged globular cells with dense nucleus in the
center of each globular cell . Enclosed within the nucleus is present four independent polytene
chromosomes, each one with definite pattern of dark and light bands and puffs or swollen regions
at certain segments of the chromosome. The centromere cannot be easily distinguished.
Conclusion: Puffs in the salivary gland chromosome indicate gene action, where the DNA
is unwound and transcription and simultaneous translation is going on. Bands indicate inactive
sites of the DNA, which is highly wound and heterochromatized.
SALIVARY DUCT
LUMEN
GLOBULE CELLS
NUCLEUS
Fig. 9.1. Structure of salivary gland of Chironomous larva.
POLYTENE
CHROMOSOMES
BALBIANI RINGS
Fig. 9.2. Structure of polytene chromosomes in the nucleus of globule cells of
salivary gland of Chironomous.
MIMICRY AND WARNING 89
10. MIMICRY AND WARNING COLOURS
MIMICRY
Mimicry is confusing; it involves deception on a grand scale. It is found in many groups of
animals including birds, fish, snakes, amphibians, moths, beetles, bugs, flies and snails, but most
convincing examples are found in butterflies, especially tropical ones. There are two types of
mimicry, namely Batesian and Mullerian.
Batesian mimicry signifies the resemblance of a palatable species to an unpalatable model
and Mullerian mimicry is resemblance between species when both have unpleasant characteristics
to a predator. Sometimes mimicry and warning colouration go hand in hand.
WARNING COLOURATIONS
Sometimes animals are brightly coloured red, yellow or white with bold black stripes, bands
or spots the chances are that it is dangerous or unpalatable to predators. The protective strategy
of warningly coloured animals is that they hardly make any attempt to conceal themselves from
the predators. They are conspicuous by day and predators learn to avoid them because of their
bad taste or painful bite or poisonous.
Certain examples of warning colouration and mimicry are as follows
Bee mimics:
. Bees belong to the hymenoptera and have two pairs of wings; flies belong to the order
dlptera and have only one wing. Yet the resemblance between the bee model and the fly
mimics is striking and deceptive': Many hover flies mimic either honeybee or bumble bee, because
both honey bee and bumble bee have characteristic warning colouration, ie , their abdomen is
yellow and black striped and both inflict painful sting when captured or disturbed. Predators like
birds and lizards avoid honeybees and bumble bees. This offers protection and long survival.
Hence hoverflies, which are palatable, by mimicking honeybees and wasps have deceived the
predators both by warning colours and appearance. Hence, their survival value has increased in
nature.
HOVER FLY
BUMBLE BEE
HOVER FLY
HONEY BEE.:
Fig 10. i. Hoverfly mimicking honey bee and bumble bee.
90
Wasp mimics:
Generally wasps are yellow and black striped with. a narrow abdomen. They inflict a painful
sting when provoked. The characteristic and easily recognized wasp colouration occurs in many
harmless insects including beetles, moths and flies. Most of the wasp-like hoverflies are believed
to be Batesian mimics. -
HOVER FLY
WASP
Fig. 10.2. Moth and Hoverfly mimicking Wasp.
VICEROY
MONARCH
Fig. 10.3. Viceroy (palatable) butterfly mimicking Monarch (unpalatable) butterfly.
Warning colouration:
Among amphibians there are several species, which produce a distasteful or highly poisonous
secretion from their moist skin. In some species the secretion is produced when the animal is
disturbed, while in others the skin appears to carry the secretion at all times. Generally amphibians
Fig. 10.4. Poison tree frog (Dendrobates).
92
. A HANDBOOK OF PRACTICALS IN ZOOLOGY
are coloured green or brown, which is a perfect camouflage, a number of those, which are distasteful
or .poisonous are brightly coloured. Their skin may be brilliant red, blue or yellow or some
combination of these colours, with black often featuring in the pattern. An excellent example is the
South American tree frog Oendrobates histrionics (Fig. 10A). Another example is European fire
salamander SalamandrE! salamandra (Fig. 10.5), which is patterned with irregular patches of black
and vivid yellow. The skin of this salamander produces a milky discharge, which is highly poisonous.
YELLOW PATCH
Fig. 10.5. Tiger salamander.
The caterpillars of the European cinnabar moth (Tyria jacobaceae) are yellow, banded with
black contain in their body chemical compounds derived from the plants on which they feed; they
also synthesize their own poisons compounds. Hence they are unpalatable and rejected by birds.
The fully-grown caterpillar of the European alder moth (Apatele aIm) is similarly banded with black
and yellow stripes, but the young caterpillars look like fresh bird droppings. In this species the
young caterpillar's primary defense is looking like something inedible, while the fully-grown caterpillar
is apparently ,warningly coloured.
MIMICRY AND WARNING
YOUNG CATERPILLAR
RESEMBLING BIRD DROPPING
Fig. 10.6. Young and the adult caterpillar of Alder moth.
93
11. ESTIMATION OF GLUCOSE BY GLUCOSE OXIDASE
METHOD
AIM: To estimate colorimetrically the percent composition of glucose in unknown sample using
standard curve by glucose oxidase method.
PRINCIPLE: Glucose oxidase is found in the growth media of Penicillium notatum. It catalyses
oxidation of to D-glucono-1 ,5-lactone accompanied by formation of hydrogen
peroxide. D-glucono-1 ,S-Iactone is converted to D-gluconic acid. This enzyme is highly specific for
The reaction mixture for estimation of glucose contains perbxidase which utilizes
hydrogen peroxide to oxidize o-toluidine to give a blue coloured product. These reactions are rapid
but the colour is not very stable and the readings are recorded usually after 8 min. The sequence
of events involved in this method is depicted below:
Glucose oxidase
D-glucose + O
2
+ Hp HP2 + D-gluconic acid
Peroxidase
Oxidized toluidine
(Blue coloured product)
REQUIREMENTS:
1. Boiling water bath.
2. 10% Zinc sulfate solution.
3. Isotonic sodium sulfate (93 mM) : Dissolve g of sodium sulfate in 250 ml DW and
make up the volume to 500 ml.
4. Sodium sulfate-zinc sulfate reagent: Take 27.5 ml of 10% zinc sulfate solution and add
isotonic sodium sulfate solution to make up the volume to 500 ml.
5. 0.5N NaOH
6. 0.15M acetate buffer (pH 5.0)
7. Glucose oxidase: 30 IU = 1 Mmol of substrate transformed per min.
8. Peroxidase: 30 international units.
9. O-toluidine: Dissolve 1 g of O-toluidine in 100 ml of absolute alcohol and store in a dark
place in a dark bottle.
10. O-toluidine reagent mixture: 150 ml acetate buffer, 30 IU of glucose oxidase, 30 IU of
peroxidase and 1 ml of O-toluidine solution. Store the mixture in brown bottle and it is
stable for several weeks.
11. Glucose standard (0.1 mg/ml)
Deproteinization of sample:
Take 0.1 ml of blood sample, add 1.8 ml of sodium sulfate-zinc sulfate reagent in a centrifuge
tube and add 0.1 ml of 0.5N NaOH to it.
ESTIMATION OF GLUCOSE 95
Centhfuge at 5000 rpm for 15 min. Take 1 ml of the supernatant dilute It to 20 ml. Take 1 ml
of diluted sample for analysis.
PROCEDURE: Prepare following set of test tubes for standard (Std.) and U 1 U
2
, U
3
(Unknown
samples)
Tube No. Std. Glucose O-Toluidine Distilled
(O.1mg/ml) Reagent water
1 0.2 ml 5ml 0.8 ml
2 0.4 ml 5 ml 0.6 ml
3 0.6 ml 5ml 04 ml
4 0.8 ml 5ml 0.2 ml
5 1.0 ml 5 ml 0.0 ml
Blank 0.0 ml 5ml 10 ml
U
1
. U
2
, U3-
1.0 ml 5ml 0.0 ml
(1 : 20 diluted)
2. Keep all the tubes at 30C for 8 minutes.
3. Read the OD against the blank at 620 nm
OBSERVATIONS:
Tube No. Conc. of glucose Absorbance
1 0.02 mg
2 0.04 mg
3 0.06 mg
4 0.08 mg
5 0.10 mg
U
1
U
2
U
3
GRAPH:
Draw the standard curve/graph by plotting the mg of Std. glucose solution against absorbance
and determine the concentration of unknown glucose in the solution given.
CALCULATIONS:
From the graph, optical density
corresponds to X mg of glucose.
______ of the unknown glucose solution
1 ml of unknown was used for the assay.
1 ml of unknown diluted glucose solution = X mg of glucose.
1QO ml of unknown diluted glucose solution = 100 x X x 20 (dilution factor) mg of glucose.
RESULTS:
Amount of glucose in unknown solution ____ mg% (from graph)
g%
Calculation method:
00 of Unknown
00 of Standard
x Cone Of Std. (0 1 mg/ml) x 20 (dilution factor)
BIOTECHNOLOGY AND BIOINFORMATICS
12. PROBLEMS BASED ON BIOTECHNOLOGY AND
BIOINFORMATICS
97
The table given below is a standard list of one-letter amino aCid code and their three letter
equivalents. The synonymous codons and their depiction In the iUB codes are shown, which Will
be useful in biotechnology and blolnformatics practlcals.
Table. 12.1. Standard list of amino acids and their equivalent one letter code and the
corresponding genetic codon for each amino acid.
Symbol 3-letter Meaning Codons
-- -- .. _----
A Ala Alanine GCU,GCC,GCA,GCG
B Asp, Asn Aspartic, Asparagine GAU,GAC,AAU,AAC
C Cys Cysteine TGU, TGC
D Asp Aspartic GAU, GAC
E Glu Glutamic GAA,GAG
F Phe Phenylalanine UUU, UUC
G Gly Glycine GGU, GGC, GGA, GGG
H HIs Histidine CAU,CAC
I lie Isoleucine AUU,AUC,AUA
K Lys Lysine AAA,AAG
L Leu LeuCine UUG, UUA, CUU, CUC, CUA CUG
M Met Methionine AUG
N Asn Asparagine AAU,AAC
P Pro Proline CCU, CCC, CCA,CCG
Q Gin Glutamine CAA,CAG
R Arg Arginine CGU, CGC, CGA,CGG,AGA,AGG
S Ser Serine UCU, UCC, UCA,UCG,AGU,AGC
T Thr Threonine ACU, ACC, ACA, ACG
V Val Valine GUU. GUC, GUA, GUG
W Trp Tryptophan UGG
X Xxx Unknown
Y Tyr Tyrosine UAU, UAC
Z Glu, Gin Glutamic, Glutamine GAA GAG, CAA,CAG
* Stop/end NonsenselT ermlnator UAA. UAG, UGA
?
Unknown/Deletion
Deletion
Nucleic acid codes:
The input format for the DNA sequence programs is standard I usually has annotations and
sequence with combinations of A, G, C and T (U). The base sequence have characters being one
of the letters A, B, C, D, G, H, K, M, N, 0, R, S, T, U, V, W, X, Y, ? or - Blanks and the numencal
digits If present In the sequence will be ignored This allows GENBANK and EMBL sequence
entries to be read with minimum editing The characters constitute the IUPAC (IUB) nucleic aCid
code plus some slight extensions. They enable Input of nucleic aCid sequences taking full account
of any ambiguities in the sequence
Table 12.2. The meaning of each symbol and its complement for DNA sequence or
coded sequence
Symbol Meaning Meaning Complement
A Adenine A T
C Cytosine C G
G Guanine G C
T/U Thymine/Uracil T A
M Amino A or C K
R Purine A or G Y
W Weak A orT W
S Strong Cor G S
Y Pyrimidine Cor T R
K Keto G or T M
V Not T A or Cor G B
H Not G A or Cor T 0
0 Not C A or G or T H
B Not A Cor G or T V
X/Nt? Unknown G or A or T or C X
-/0 Deletion not G or A or T or C -
Standard Genetic Code
u C A G
U UUU (Phe F) UCU (Ser S) UAU (Tyr Y) UGU (Cys C) U
UUC (Phe F) UCC (Ser S) UAC (Tyr Y) UGC (Cys C) C
UUA (Leu L) UCA (Ser S) UAA (Stop) UGA (Stop) A
UUG (Leu L) UCG (Ser S) UAG (Stop) UGG (Trp W) G
C CUU (Leu L) CCU (Pro P) CAU (His H) CGU (Arg R) U
CUC (Leu L) CCC (Pro P) CAC (His H) CGC (Arg R) C
CUA (Leu L) CCA (Pro P) CAA (Gin Q) CGA (Arg R) A
CUG (Leu L) CCG (Pro P) CAG (Gin Q) CGG (Arg R) G
A AUU (lie I) ACU (Thr T) AAU (Asn N) AGU (Ser S) U
AUC (lie I) ACC (Thr T) AAC (Asn N) AGC (Ser S) C
AUA (lie I) ACA (Thr T) AAA (Lys K) AGA (Arg R) A
AUG (Met I) ACG (Thr T) AAG (Lys K) AGG (Arg R) G
G GUU (Val V) GCU (Ala A) GAU (Asp D) GGU (Gly G) U
GUC (Val V) GCC (Ala A) GAC (Asp 0) GGC (Gly G) C
GUA (Val V) GCA (Ala A) GAA (Glu E) GGA (Gly G) A
GUG (Val V) GCG (Ala A) GAG (Glu E) GGG (Gly G) G
BIOTECHNOLOGY AND BIOINFORMATICS
Table 12.3. Recognition sequences of some restriction enzymes.
(Arrows in the table indicate the site of restriction or cutting)
Restriction enzyme Recognition sequence
J,
8am HI GGATCC
J,
8gllli AGATCT
J,
Eco RI GAATTC
J,
Hae III GGCC
J,
Hind III AAGCTT
J,
Hpa II CCGG
J,
Pst I CTGCAG
J,
Sma CCCGGG
99
Use the information given in table 12.1 and 12.2 wherever applicable to solve these problems.
1. Determine the sequence of both strands of the DNA from which this RNA was transcribed
Indicate the 5' and 3' ends of the DNA and, with an arrow, which strand was transcribed.
5'-C C A U C AUG A C A G A C C C U U G C U A A C G-3'
2. The following DNA fragment was isolated from the beginning of a gene. Determine which
strand is transcribed, indicate the polarity of the two DNA strands, and then give the
sequence of bases in the resultant messenger RNA and its polarity.
CCCTACGCCTTTCAGGTT
GGGATGCGGAAAGTCCAA
GCTACGGATTGCTG
CGATGCCTAACGAC
3. The following DNA fragment represents the beginning of a gene. Determine which strand
is transcribed, and indicate polarity of both strands in the DNA
ATGATTTACATCTACATTTACATT
TACTAAATGTAGATGTAAATGTAA
4. The following sequence of bases in a DNA molecule is transcribed Into RNA
.CCAGGTATAATGCTCCAGTATGGCATGGTACTTCCGG
i
If the T (arrow) is the first base transcribed, determine the sequence and polarity of
bases in the RNA, and identify the Pribnow box and the initiator codon.
5. Given the following end part of a gene, which will be transcribed and then translated into
a penta peptide, provide the base sequence for its messenger RNA. Give the anticodons
on the transfer RNA by making use of wobble rules. What amino acids are Incorporated?
Draw the actual structure of the penta peptide
3'-T ACAA TGGCCCTTTT A TC-5'
5'-ATGTTACCGGGAAAATAG-3'
6. A peptide, fifteen amino acid long, is digested by two methods, and each segment is
sequenced according to the Edman degradation technique The fifteen amino aCids are
denoted by the letter A through 0, with F as the N-terminal amino aCid If the segments
are as follows, what is the sequence of the onglnal peptide?
Method l' CABHLN; FGKI: OEDJM
Method 2 KICA8; JM, FG; HLNOED
7 Part of a DNA strand to be transcribed has the following sequence
3'-TACT AACTT ACGCTCGCCTCA-5'
(a) What IS the sequence of RNA transcribed from this part of the strand?
(b) What sequence of amino acids does the RNA produce?
8. A segment of normal protein and three different mutants appear as follows
Normal
Mutant 1
Mutant 2
___ gly-ala-ser-his-cys-Ieu-phe __ _
gly-ala-ser-hls
gly-ala-ser-Ieu-cys-Ieu-phe __ _
Mutant 3 gly-val-ala-ile-ala-ser . __ _
What IS the probable sequence of bases in the normal and mutants RNA?
9. Give the probable sequence of RNA bases for the following peptide
(a) leu-ser-phe-met-ala, (b) ser-phe-met-ala-Ile, (c) leu-ser-phe-met-ala-Ile
10 Following IS the sequence of different peptldes (represented by symbol) Prepare amino
aCid and codon (RNA) sequence Use the information given In table 12 1 and 12 2 (Each
sequence is one bloinformatlcs problem)
Seq 1 ___ KSKERYKDENGGNYFQLREDWVVDANRETVWKAITCNA
Seq 2 YEGLTTANGXKEYYQDKNGGNFFKLREDWWTANRETVWKAITCGA
Seq 3 KRIYKKIFKEIHSGLSTKNGVKDRYQNMDGDNYFQLREDWWTANRS
TVWKALTCSD
Seq 4 ___ SQRHYKDTDGGNYFQLREDWWTANRHTVWEAITCSA
Seq 5 NVAALKTRYEKRDGQNFYQLREDWWTANRATIWEAITCSA
Seq 6 ___ FSKNIXGQIEELQDEWLLEARYKDNTDNYYELREHWWTENRHTVWE
ALTCEAA
11. From the following sequence of amino acids for different peptides, prepare a sequence
using symbol and give corresponding RNA sequence. Use the information given In
table 12 1 and 122 (Each sequence can be given as one problem)
BIOTECf-jNOLOGY AND BIOINFORMATICS
hls-cys-tyr -ileu-gln-asn-cys-pro-Ieu-g iy( N H 2)
N H 2 -ser -val-ser -g I u-ile-g In-leu-met -h Is-asn-Ieu-g !y-Iys-h Is-Ie u-asn-se r
N H 2 -met -g lu-arg-va I-g I n-trp-Ieu-a rg-Iys-Iys-Iys-g In-leu-va i-h Is-as n-phe
N H
2
-cys-ser -asn-Ieu-ser -th r -cys-val-Ieu-ser -ala-tyr -trp-a rg-asn-Ieu
N H 2-as n-asn-phe-h is-arg-phe-ser -g Iy-met -g Iy-phe-g Iy-pro-g I u-th r -pro
N H 2-g Iy-ile-val-gl u-g I n-cys-cys-h is-Iys-arg-cys-se r -Ile-tyr -asn-Ieu -g I n
N H 2 -arg-th r -th r -g Iy-h is-Ieu-cyc-g Iy-Iys-as p-Ie u-val-asn-a la-Ieu-tyr -lle-a la-cys
N H 2 -h Is-ser -asp-g Iy-th r -phe-th r -ser -g I u-Ieu-ser -arg-Ie u-a rg -as p-ser -a la-a rg
N H 2 -tyr -ala-g I u-g Iy-gly-phe-i le-ser -as p-tyr -ser -lie-ala-met -asp-Ies-Ile-a rg
101
12 A linear DNA molecule of 1000 base pairs long IS digested with follOWing restriction
enzymes producing the following results:
Eco RI 400 bp, 600 bp
8gll1
Eco RI + 8g1 II
250 bp, 750 bp
250, 350, 400 bp
Determine the restriction map.
13. A linear DNA molecule of 5000 base pairs long IS digested with following restriction
enzymes producing the follOWing results
Eco RI 2100 bp, 1400 bp, 1000 bp and 500 bp
Hind III 2500 bp, 1300bp, 1200 bp
Eco RI + Hind III 200 bp, 600 bp, 800 bp, 1000 bp, 500 bp, 1900 bp,
Determine the restriction map.
14. The following is a double helix of DNA. What if any, are potential restriction enzyme
recognition sequences?
5
/
-T AGAA TTCGACGGA TCCGGGGCA TGCAGA TCA-3'
3
/
-ATCTT AAGCTGCCT AGGCCCCGT ACGTCT AGT -5'
For the recognition sequences of some restriction enzymes refer to table 12 3
15. The following segment of DNA is cut four times by the restriction endonuclease Eco RI
at the places shown by arrows Diagram the gel banding that would result from
electrophoresis of the total and partial digests. Note the end labeled segments and regions
where several segments from bands at the same place on the gel.
100 t 300 t 50 t 250 t 150
16. The follOWing figure shows a gel of a total and partial digest of a DNA segment treated
with Hind III Asterisks note end labeled segments Draw the restnctlon map of the Original
segment
bp
800
700
600
500
400
Total digest Partial digest
300
200
100
400
200
100
50
*
750 *
700 *
500 *
400 *
350 *
300 *
250 *
200
100 *
50
17. Restriction maps of a segment of DNA were worked out separately for Bam HI and Taq
I. Two overlays of the maps are possible. The double digest gel is shown in the following
figure (asterisks denote end labels). Which overlay is correct?
Bam HI 100.J.. 300.J.. 50 .J.. 200
Alternative I
Taq I 50 i 250 i 150 i 100 i 100
Bam HI 100 .J.. 300 .J.. 50 .J.. 200
Alternative II
Taq I 100 i 100 i 150 i 250 i 50
bp Double digest gel
200 150
100 *
50 *
0
18. A linear DNA molecule 1000 bp long gives the following size fragments when treated
with these restriction enzymes. Derive a restriction map.
Eco RI 300bp, 700 bp
Bam HI 150bp, 200bp, 250bp, 400bp
Eco RI + Bam HI 50bp, 100 bp, 200bp, 250bp, 400bp
19. A linear DNA molecule is cut with Eco RI yields fragments of 3 kb, 4.2 kb, and 5 kb.
What are the possible restriction maps?
20. You have double-stranded DNA that you radioactively label at the 5' ends. Digestion of
this molecule with either Eco RI or Bam HI yields the following fragments. The numbers
are in kilo bases (kb), and an asterisk indicates the fragments that are labeled.
Eco RI' 2.8, 4.6, 6.2*, 7.4, 8.0*
Bam HI: 6 0*, 10.0*, 13.0
BIOTECHNOLOGY AND BIOINFORMATICS 103
If unlabeled DNA is digested with both enzymes simultaneously, the following fragments
appear: 1.0, 2.0, 2.S, 3.6, 6.0, 6.2, and 7.4. What is the restnctlon map for the two
enzymes?
21. A 12 kb DNA molecule cut with Eco RI yields one 12 kb fragment. When the onglnal
molecule is cut with 8am HI, three fragments of 2 kb, 4.5 kb, and 5.5. kb are produced
When the fragment from Eco RI is treated with 8am HI, four fragments of 2 kb, 25 kb,
3.0 kb, and 4.5 kb are produced. Draw a restriction map.
22. A plasmid 3 kb in length contains a gene for ampicillin resistance and a gene for
tetracycline resistance. The plasmid has a single site for each of the follOWing enzymes:
Eco RI, 8glll, Hind III, Pst I, and Sail. If DNA is cloned into the Eco RI site, resistance to
either antibiotics is not affected. DNA cloned into the 8glll, Hind III, or Sail sites abolishes
tetracycline resistance, and DNA inserted into the Pst I site eliminates ampicillin resistance
If the plasmid is digested completely with enzyme mixes, the follOWing fragments result:
Mixture Fragment size (kb)
Eco RI + Pst I 0.7, 2.3
Eco RI + 8g1 II 0.3, 2.7
Eco RI + Hind III O.OS, 2'.92
Eco RI + Sail 0.S5,2.15
Eco RI + 8gl11 + Pst I 0.3, 0.7, 2.00
Draw a restriction map of the plasmid, and indicate the locations of the resistance genes
and the sites of enzymatic cleavage.
23. A gene has the following Eco RI restriction map (in kilo bases):
1.0 t 0.7 t 2.0
Draw the gel pattern expected from
(a) a mutant that has lost the site between the 1.0 and 0.7 kb fragments.
(b) A mutant that has a new site within the 2.0 kb fragment.
24. A DNA fragment Skb in size is labeled with 32p at the 5' ends. It is then digested with
Eco RI, 8g1 II, or a mixture of both enzymes. The size of the fragments and the labeled
fragments (*) appear as follows. Sizes are in kilo bases.
Eco RI 8g1 II Mix
3.5 * * *
3.0 * * *
2.0 * * *
1.0 * * *
0.5 * * *
(1) E E E
0.5 1.0 3.0 I 35
1.0
I
1 5 2.0 0.5 30
B B B B
(2) E E E
05 1.0 I 3.0 3.5
30
I
0.5 2.0 1.5 1 0
B B B B
25. The following diagram is of a dideoxy sequencing gel What is the sequence of the DNA
under study?
Bp ddCTP ddGTP ddATP ddTTP
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
o
BIOTECHNOLOGY AND BIOINFORMATICS 105
26. Two normal individuals have a child with Down syndrome. RFLP analysis with a probe
from chromosome 21 is performed on all three individuals, and the results of the gels
appear as follows. Based on these results, what can you conclude about the origins of
the number 21 chromosomes?
Mother Father Child
27. Draw the gel pattern derived from the dideoxy sequencing method for a template strand
with the following sequence:
5' -CAGCGAA TGCGGAA-3'
28. A DNA strand with the sequence 3'-GACTATTCCGAAAC-5' is sequenced by the dideoxy
method. If the reaction mixture contains all four radioactive deoxynucleotide triphosphates
plus dideoxythymidine, what size labeled bands do you expect to see on the gel?
13. SEX CHROMATIN
STUDY OF BARR BODY FROM BUCCAL EPITHELIUM
Aim: To observe Barr body or sex chromatin in the somatic cell of a female.
Requirements: 1 %Geimsa stain, 70% alcohol, Johnson's cotton bud or sterile cotton swab,
slides, coverslip, 0.9% saline, microscope. If B.arr body or drumstick is to be identified from the
WBe preparation-sterUe needle for pricking the finger, clean slide, spreader slide and Leishman's
stain (ready made-Qualigens). Buffered distilled water (Na
2
HP0
4
.2Hp- 3.76 gm, NaH
z
P0
4
2.1 .
gm dissolved in 1000 ml OW)
Procedure for preparation of cens from cheek pouch:
1. Ask a girl student to rinse and flush the mouth several times with tap water to get rid off
food particles sticking to the cheek pouch if any.
-2. Take a sterile cotton bud and dip it in 0.9 percent saline to mois!en it.
3. Rub'the cotton bud several times on the inside wall of the cheek pouch.
4. Smear the cotton bud Or:! a clean dry slide and allow the smear to dry.
5. Fix the smear with 70%' alcohol.
6. Stain the smear with Giemsa for 10 min.
7. Wash off the excess of stain with water.
8. Place a coverslip and observe under high power of the microscope.
Observation: Barr body stains bluish-green and is found attached to the nuclear envelope.
Procedure for the preparation of sex. chromatin or drumstick from the WBCs:
BARR BODY I ~ ~ ~ t i i i j ~ I : i ~ ~ ~ NUCLEUS
EPITHELIAL CELL
Fig. 13.1. Barr body or sex chromatin in the cheek pouch cells of a female . .
1. Ask a girl student to clean the fingertip of any finger of left hand with 70% alcohol.
2. Wait for the alcohol to'dry and then prick the area with a sterile needle and squeeze the
tip gently. Blood drop appears.
SEX CHROMATIN 107
3. Touch the blood drop to a clean slide at one corner.
4. Using the spreader slide make a smear of the blood.
5. Allow the smear to dry and fix it in 70% alcohol.
6. Drain off the excess of alcohol and allow the slide to dry.
7. Stain it with Leishman's stain diluted 1: 1 in buffered OW.
8. Wash off the excess of stain in tap water and allow it to dry.
9. Put a drop of oil on thinly spread region and observe under oil immersion lens.
Observation: Observe for a appendix like projection from the nucleus of Neutrophil or
Eosinophil , which is darkly stained than compared to the other part of the nucleus. It is called
drumstick or sex chromatin.
DRUM STICK
WBC
OR SEX CHROMATIN __ -.... ..
NUCLEUS
Fig. 13.2. Drum stick or sex chromatin in WBC of a female.
14. PROBLEMS IN GENETICS
X-LINKED INHERITANCE
1. In Drosophila white-eye is X-linked and is recessive to normal red eye colour. If a white-
eyed male is crossed with red eyed female what will be the eye colour of the offspring.
Show the phenotypes and the genotypes.
2. If a heterozygous red-eyed female Drosophila is crossed with a red eyed male show the
eye colour of the offspring with the genotypes;
3. If a white eyed female Drosophila is crossed with a red-eyed male show eye colour of
the offspring.
4. If a heterozygous red-eyed female Drosophila is crossed with a white eyed male show
the eye colour of the offspring of such a cross.
5. In Drosophila, the recessive gene vermilion and cinnabar have the same phenotypic
effect, a lighter and brighter eye colour than the usual brick-red wild type. Vermilion,
however, is located on the X-chromosome and cinnabar on one of the atutosomes. (a) If
a vermilion male is crossed to a homozygous cinnabar female, what are the genotypes
and phenotypes of their offspring? (b) If these F 1 flies are permitted to breed with each
other, what proportion of each different phenotype will be found in the F 2 ?
6. In Drosophila, the lozenge phenotype, caused by a X-linked recessive allele, is narrow
eyes. Diagram to the F 2 generation a cross of a lozenge male and a homozygous normal
female. Diagram the reciprocal cross.
7. In Drosophila, cut wings are controlled by a recessive X-linked allele (ct), and fuzzy
body is controlled by a recessive autosomal allele (fy) . When a fuzzy female is mated
with a cut male, all the members of the F 1 generation are wild type. What are the
proportions of F 2 phenotypes, by sex?
8. In human beings, red green colour blindness is inherited as an X-linked recessive trait.
A woman with normal vision whose father was colour blind marries a man with normal
vision whose father was also colour blind. This couple has a colour-blind daughter with a
normal complement of chromosomes. Is infidelity is suspected? Explain.
9. Suppose a young lady comes to you_for advice in your capacity as a marriage counselor.
She tells you that her brother has hemophilia, but both of her parents are normal. She
wishes to marry a man who has no history of hemophilia in his family and wants to know
the probability of her children having this disease. What would you tell her and how
would you explain your conclusion?
10. A colour-blind woman marries a man with normal vision. What kind of children would be
expected from such a union?
11. A man sues his wife for divorce on the grounds of infidelity. Both man and wife have
normal eyes, but their baby daughter has coloboma iridis, a fissure in the iris of the eye.
This character is known to be inherited as a sex-linked recessive. If you were the man's
lawyer could you use this fact as evidence? If so, how would you explain the case to the
jury?
PROBLEMS BASED ON GENETICS 109
12. A woman with normal vision marries a man with normal vision and they have a colour-
blind son. Her husband dies and she marries a colour-blind man. Show the types of
children that 'might be expected from this marriage and the proportion of each
13. In the domestic fowl, silver plumage results from a dominant sex-linked gene, gold
plumage from the recessive allele. Show the results of a cross between a rooster with
gold plumage and a hen with silver plumage. If the first generation offspnng are crossed
themselves, what results would be expected in the F 2 ?
14. A barred rooster is crossed to a non-barred hen. One half of the male chicks and one-
half of the female chicks are barred. The same rooster is crossed to a barred hen Would
you expect any-non-barred chicks in the offspring? Explain.
MULTIPLE ALLELIC INHERITANCE
1. In rabbits, full colour, C, id dominant; Himalayan albinism, c
h
, produces white rabbits
with coloured legs, ears, and nose; albinism, c
a
, produces all white rabbits Himalayan is
dominant to albino, but recessive to full colour. What types of offspring would be expected
from a coloured male, heterozygous for Himalayan, crossed with an albino female?
2. Two Himalayan rabbits are crossed. They yield seven Himalayan and five albino rabbits
Could this be an example of 3: 1 ratio? If so, what is the genotype of the parents
3. The following listed in order of dominance, are four alleles in rabbits: C+ coloured, C
Ch
,
chinchilla; c
h
Himalayan; and c, albino. What phenotypes and ratios would be expected
from the following crosses:
(a) C+C+ X cc; (b) c+c X c+c; (c) c+c
ch
X c+C
Ch
; c
ch
X cc; (e) c+c
h
X c+c and (f) chc X cc?
4. In mice, a series of five alleles have been associated with fur colour These alleles are in
order of dominance, AY (homozygous lethal) for yellow fur; AL, agouti with light belly, A +,
agouti; at, black and tan; and a, black. For each of the following crosses, give the coat
colour of the parents and the phenotypic ratios expected among the progeny
(a) AY AL X AYA; (b) AYa X AL at ; (c) at a X AYa; (d) AL at X N N;
(e) ALN X AYA; (f) A at X ata; (g) ata X aa; (h) AY N X A at, and (i) AY at X AYA
5. Apricot, white and coral are eye colours in Drosophila, which are X-linked alleles
Heterozygous coral and white give an eye colour very near apricot. Show the results
from across between two flies with apricot coloured eyes although female is heterozygous
for white and coral. (Heterozygous coral and apricot yields an eye colour Similar to cherry).
6 A man has type A blood and his wife has type B blood. A physician types the blood of
their four children and is amazed to find one of each of the four blood types among
them. He is not familiar with genetics and calls upon you to explain how such a thing
could happen. What would you tell him?
7. A couple preparing for a marriage have their blood typed along with the other required
blood tests. Both are AB. They ask you what types of blood their children may have.
What would you tell them and how would you explain your conclusion?
8. A woman sues a man for the alimony of her child. She has type A blood, her child type
0, and the man type B. Could the man be the father? Explain your answer. Further tests
of the persons reveal that both man and the woman are Rh-negative while the child is
Rh-positive. Would this information be of any value in the case? Explain.
9. A case was brought before a judge in which a woman of blood group 0 presented a
baby of blood group 0, which she claimed as her child, and brought suit against a man
of group AB whom she claimed was the father of the child. What bearing might the blood
type information have on the case?
10. In another case, a woman of blood group AB presented a baby of group 0, which she
claimed as her baby. What bearing might the blood-type information have on the case?
11. A wealthy, elderly couple die together in an accident. Soon a man shows up to claim
their fortune, contending that he is their only son who ran away from home when he was
a boy. Other relatives dispute this claim. Hospital records show that the deceased couple
were blood type AB and 0, respectively. The claimant to the fortune was type O. Do you
think thatthe claimant was an impostor? Explain.
12. A young lady about to be married learns that she was born with erythroblastosis and
greatly perturbed that possibly some of her children have the same condition. Her fiance
refuses to have his blood checked. What could you tell her that might ease her anxiety?
13. Another young lady learns that her fiance was the fifth child In a family In which the
second and fourth children were born with erythroblastosis. She asks you to tell her
what are the chances that she could have any children with erythroblastosis. What would
you tell her?
14. A man of blood group B marries a woman of blood group O. Can they have a baby with
blood group AB? Explain.
15. A couple have brought the baby from the clinic. They think they brought the wrong baby.
The baby is group O. The woman is group 0 and the husband is A. Is it the wrong baby?
Explain.
16. A couple have bro'Ught the baby from the hospital. They think they have brought the right
baby. The baby is group O. The woman is group 0 and the husband is AB. Is it the nght
baby? Explain.
17. A man of blood group A marries a woman with blood group B. Can they have a baby
with blood group O? Explain.
18. A man with blood group 0 marries a woman with blooq group O. Can they have a baby
with blood group A? Explain.
19. A man with blood group A marries a woman with blood group AB. Show the blood groups
of their children.
20. A man with blood group B marries a woman with blood group AB. Can they have a child
with blood group O? Explain.
MULTIPLE GENIC OR POLYGENIC OR QUANTITATIVE INHERITANCE
1. Assume that in man the difference in skin colour between Negro and white is due to two
pairs of genes; that AABB is "black" and aabb is for "white"; and that any three of the
colour producing genes produce "dark" skin; any two "medium" colour and anyone "light".
(a) What will be the skin colour of the offspring from a mating of white with black;' from a
mating of two individuals genotypically like these F 1 offspring?
(b) What are the genotypes and phenotypes produced from the cross between AaBb X
aabb, AaBb X MBB, MBb X aabb, Aabb X MBB, Aabb X aabb.
(c) If people with various degrees of skin pigmentation married only people known to
have genotype aabb, could they have black babies? Explain.
(d) Could a white couple with Negro ancestry have a black baby? Explain.
2. A certain man is rather tall measuring about 6 ft and his wife is of small stature measuring
about 4.5 ft. Out of four children they have a boy and a girl of about average height i e' .

boy as tall as his father and the girl as tall as her mother. Assuming that environmental
factors were about the same for all, how would you explain these results?
3. One breed of rabbit has ears about 4 inch long and another breed has ears 2 inch long
When the two types are crossed, the offspring have ears about 3 if1ch long. In the second
generation of offspring, however, the ears show a great range of variation from 2 to 4
inch. Out of 1000 rabbits, three have 2-inch ears and five have 4-inch ears. How many
pairs of genes seem to be involved in ear length? Explain.
4. A Holstein bull with solid white colour is mated to a cow with white body colour except
for a small black spotting around the head. About half the calves are ~ o l i white and half
have black spotting over a large part of the body. Explain.
LINKAGE AND CROSSING OVER; CHROMOSOME MAPPING
1. In rabbit's black and short hair are characters resulting from two dominant genes. The
recessive alleles of these genes produce brown and long hair When we mate
homozygous black, short-haired rabbits With brown long-haired rabbits and test cross
the offspring we obtain the following results:
Black short 29
Brown long 33
Black long 35
Brown short 27
From these results, would you conclude that these two genes are located on the same
chromosome? Why? If your answer is yes, what is the percentage of crossing OVCt(
2. In rabbits two recessive genes produce a solid body colour and long hair respectively, In
contrast to a spotted body colour and short hair, which results frolTl the dominant alleles.
The results from a cross between the heterozygous spotted short haired rabbits and
solid long-haired rabbits are as follows:
Spotted short 48
Spotted long 5
Solid short 7
Solid long 40
In terms of crossover units, how far apart these two genes on the chromosome?
3. In rabbits, black is dominant over brown coat colour, but neither of these colours can
show when animal is homozygous for the receive gene for albinism Albinism is said to
be epistemic to the other two genes. The gene for albinism IS nor an allele of the genes
for black or brown. Albino rabbits, which are homozygous for brown (aabb) are crossed
with black rabbits (MBB) The F 1 are all black (AaBb) A test cross of these yields he
following results:
Black 66
Albino 100
Brown 34
Examine these results carefully and determine if these genes are linked. If they are
linked, what is the percentage of crossing over?
4. Let us assume that we have three recessive genes (abc) in a hypothetical animal. When
individuals showing these three recessive characters are mated with the homozygous
dominant and a test cross of their offspring is made, we obtain the following results:
Normal (ABC) 61
Abc 56
aBC 14
AbC 8
Abc 2
Abc 12
aBc 10
abC 3
What is the sequence of these genes on the chromosome? Show how you obtain your
results. How many units apart are these genes?
If a is located at 5.2 on the chromosome, where would you place band c on the
chromosome?
5. The genes for garnet eye and forked bristles are both X-linked characters In Drosophila.
They are 10 units apart on the X-chromosome. Wild type males (GF) are crossed with
garnet, forked females (ggft) and the Flare crossed among themselves. What percentage
of each of the four possible types of offspring would you expect among the males? Among
the females?
If the sex of the parents in the above problem was reversed and garnet, forked males
were crossed with homozygous wild type females, what results would be expected in
the F
2
.
6. In Drosophila the recessive genes sr (stripe) and e (ebony body) are located at 62 and
70 map units respectively, from the left end of the third chromosome. A striped female
(homozygous for e+) was mated with a male with ebony body (Homozygous for sr+)
(a) What kinds of gametes will be produced by the F 1 female and what proportion?
(b) If Fl females are mated with stripe, ebony males, what phenotypes would be expected
and in what proportion?
7. In Drosophila, the gene (vg) for vestigial wing is recessive and is located at 67.0 units
from the left end of the second chromosome. Another gene (cn) for cinnabar eye colour
is also recessive and.is located at 57.0 units from the left end of the second chromosome.
A fully homozygous female with vestigial wings was crossed with a fully homozygous
cinnabar male.
(a) How many different kinds of gametes could the F 1 female produce and in what
proportion?
(b) If the females are mated with cinnabar, vestigial males, what phenotypes would be
expected and in what proportion?
8. In Drosophila, the recessive genes st (scarlet eye), ss (spineless bristles) and e (ebony
body) are located in the same third chromosome in the followmg positions from the left
end of the chromosome: st 44, ss 58 and e 70. Fully heterozygous females with the
~ ~ ~ ~ ~
genotype st+ ss+ e are mated with fully recessive males st S5 e
If many flies are produced and no interference occurs, what phenotypes will be expected
and in what percentages?
9. A cross was made between yellow bodied (y), echinus (ec), white-eyed (w) female yecw
flies and wild males. F1 females were mated with yecw males. The yecw followmg
proportions were obtained when a sample of 1000 flies was counted:
Wild (+ + +) 475
yecw 469
y++ 8
+&W 7
y + w 18
+ ec + 23
++w 0
y ec + 0
Determine the order in which the three loci y, ec, w occur in the chromosome and prepare
a chromosome map.
10. A cross was made between yellow, bar, vermilion female flies and Wild males, and the
F 1 females were crossed with yB+v males. The follOWing results were obtamed when
1000 progeny were counted:
+ + + and y B v
Y + + and + B v
Y + v and + B +
Y B + and + + v
546
244
160
50
Determine the order in which the three loci yBv occur in the chromosome and prepare a
chromosome map.
11. A homozygous claret (ca, claret eye colour), curled (cu, up curved wings), fluted '(fl,
creased wings) fruit fly is crossed with a pure breeding wild-type fly. The F1 females are
test crossed with the following results:
Fluted 4, claret 173, curled 26, fluted claret 24, fluted curled 167, claret. curled 6, fluted,
claret, curled 298, wild type 302.
(a) Are the loci linked
(b) If so give the gene order, map distances and coefficient of co-incidence.
12. In Drosophila, kidney-shaped eye (k), cardinal eye (cd) and ebony body (e) are three
recessive genes. If homozygous kidney, cardinal females are crossed with homozygous
ebony males, the F 1 offspring are all wild type. If heterozygous F 1 females are mated
with kidney, cardinal, ebony males, the following 2000 progeny appear.
Kidney, cardinal 880
Ebony 887
Kidney, ebony 64
Cardinal 67
Kidney 49
Ebony, cardinal 46
Kidney, ebony, cardinal 3
Wild type 4
(a) Determine the chromosomal composition of the F1 females
(b) Derive a map of the three genes.
I Practical - 1111
PARASITOLOGY 117
1. PARASITOLOGY
ENTAMOEBA HISTOL YTICA
It is a protozoan parasite found in the digestive tract of humans. It occurs in the colon feeding
on mucous membrane. It is dimorphic, hence occurs in two forms namely trophozoite and
precystic.
The trophozoite is characterized by:
1. The presence of a thin plasma membrane.
2. Cytoplasm contains the nucleus, food vacuoles and ingested RBC's.
The cystic form is characterized by:
1. Characteristic thick cyst wall as an outer covering is present.
2. Tetranucleate cytoplasm.
Infection occurs in humans due to consumption of contaminated food or water in which the
cysts of the parasite are found.
"-':::-:"'--... PLASMA MEMBRANE
ECTOPLASM
..... _ ENDOPLASM
NUCLEUS
FOOD VACUCLE
TROPHOZOITE
CHROMATOID
BODIES
CYSTWALL - ..........
NUCLEUS
CYST
Fig. 1.1. Entamoeba histolytica.
PLASMODIUM VIV AX
1. It is a protozoan endoparasite found in the red blood corpuscles of humans and causes
malaria.
2. In humans it resides in liver cells and RBC's and reproduces by the asexual method
(Schizogony).
3 Male and female gametocytes are developed inside the human host and these are then
transferred to the female Anopheles mosquito when they feed on the blood of infected
human host, where they go through another part of the life cycle to form Infective
sporozoites.
4. Sporozoites enter human blood through the salivary duct of the female Anopheles
mosquito, when it feeds on the blood of another individual.
5. Blood smear prepared from the blood of infected individual who is showing malaria
symptoms and stained in Leishman's stain would show the presence of plasmodium
parasite in the RBC's in two different forms. (1) Trophozoite and (2) Signet ring.
6. The trophozoite is characterized by irregular shaped body with plenty of chromatin bodies
distributed in the cytoplasm giving a granular appearance. Single darkly staining nucleus
eccentric in position in the cytoplasm of the parasite can be seen.
7. The signet ring stage is early trophozoite stage characterized by ring shaped parasite in
the cytoplasm of RBC. The ring is made of cytoplasm and it encloses a vacuole. There
is a prominent chromatin dot or nucleus placed on the ring.
RBC RBC __
r>\:J'<6f#--- PLASMODIUM SIGNET
RING
TROPHOZOITE SIGNET RING
Fig. 1.2. Plasmodium vivax.
TRYPANOSOMA GAMBIENSE
1. It is a protozoan endoparasite found in the blood of vertebrates, especially warm-blooded
animals and is transmitted to the human being through an intermediate insect host Tsetse
fly (Glossina palpalis).
2. It causes a fatal disease in humans called Gambia fever or sleeping sickness.
3. The parasite is polymorphic and has a slender, spindle shaped body tapering at both
ends.
4. Anterior end bears a flagellum and along the entire body is present an undulating
membrane.
5. The parasite is injected into human body in the trypomastigote form while the tsetse fly
feeds on human blood.
6. On gaining entry into human body, the trypomastigote multiplies locally in the tissue
around the bite site and than- invades the blood stream and lymphatic system'
7 At the final stage of severe infection, the parasite invades the central nervous system
and resides in cerebrospinal fluid of the brain stem leading to sleeping sickness.
INTERMEDIATE
FORM
PARASITOLOGY
LONG SLENDER
FORM
SHORT STUMPY
FORM
RED BLOOD
CELLS
BLOOD SMEAR SHOWING TRYPANOSOMA
FREE
FLAGELLUM
ATTACHED
FLAGELLUM
Fig. 1.3. Trypomastigote of Trypanosoma gambiense.
LEISHMANIA DONOVANI
119
1. It is a protozoan endoparasite found in the reticuloendothelial system of vertebrate host
2. It exists in two forms (a) amastigote (nonflagellate, intracellular) and (b) promastigote
(trypomastigote) found in vertebrate and invertebrate host sand fly (Phlebotomus
argentipus).
3. The parasite exists in macrophages and reticuloendothelial system in the amastlgote
form.
4. The parasite is drawn into the insect when it takes a blood meal from the Infected host
5. Within the insect's gut they multiply and transform into promastigotes, which are flagellate
forms, migrate to the proboscis of the fly from which they may be introduced to a new
host as the insect takes next meal.
6. Introduced into the skin by the bite of an infected sand fly, promastigotes are phagocytized
by local macrophages. Within the macrophage they round up into amastigote forms and
multiply.
7. As the host cell ruptures, aUlastigotes are released to invade new macrophage cells.
8. The repeated cycle of invasion, reproduction and cell rupture leads to tissue destruction
and ulceration on the skin.
LEISHMANIA
.o!!iil!501l\_-- INSIDE THE
AMASTIGOTE
MACROPHAGE
LEISHMANIA
PARASITES
BONE MARROW SMEAR SHOWING
LEISHMANIA PARASITE
Fig. 1.4. Leishmania donovani.
FLAGELLUM
NUCLEUS
PROMASTIGOTE
TAENIA SOLIUM
1. It is commonly called as pork tapeworm measures up to meter or more in length but at
the initial stage it is about 3 cm. It is an endoparasite found in the small intestine of man
and other vertebrates like cow and pig, which act as intermediate hosts.
IMMATURE
PROGLOTIID
PSEUDOSEGMENTATION
Fig. 1.5. Taenia solium (entire worm).
MATURE
PROGLOTIID
PARASITOLOGY 121
2. Body is ribbon shaped, .dorsoventrally flat and pseudosegmented
3. The scolex or head like structure I-Jas four suckers, two rows of hooks on the rostellum.
4. The body segments called proglottids may be fewer than 1000 in the adult worm measure
about 12 x 5 mm and has 7 to 14 uterine branches on each side.
5. The uterus opens out through genital pore.
ASCARIS LUMBRICOIDES
It IS the largest of the intestinal nematode, endoparasite commonly called as roundworm
2. It shows sexual dimorphism The adult female often measuring more than 30 cm and the
male worm is about half this length
3. The male has sharply curved posterior end.
PENIAL SETAE
FEMALE MALE
Fig. 1.6. Ascaris lumbricoides
ANCYLOSTOMA DUODENALE
1. It commonly called as hook worm. Endoparasite in the intestine of humans The male
and female slightly differ in length and the female IS larger (10-13 mm) than male (8-10
mm).
2. Well-developed hook like teeth are present in the buccal capsule, with help of which the
adult worm attaches itself to the intestinal mucosa.
3. It infects the human being by the penetration of skin by the filariform larvae found In the
5011.
4 The larvae enter the circulation, migrate to the lungs, trachea and pharynx, and are
swallowed.
5. Maturation to the adult stage occurs in the intestine, when the worm attaches itself to the
intestinal mucosa.
Fig. 1.7. Ancylostoma duodenale.
WUCHERERIA BANCROFTI
1. It is commonly called a filarial worm The adulLlives in the lymphatic vessels and lymph
glands.
2. The female gives birth to many microfilariae, which either live in lymph or migrate to
blood capillaries.
3. Culex mosquito serves a intermediate host. During its blood meal from an infected host,
the microfilariae are sucked with the blood.
4. Accumulation of living and dead parasites in the lymphatic system results in blockade of
lymphatic system, which may cause excessive growth of connective tissue and enormous
swelling of the infected parts, which is called filariasis or elephantiasis (because arms
and legs swell and look like elephants legs).
Fig. 1.8 Wuchereria bancrofti.
PARASITIC ADAPTATIONS
SCOLEX OF TAPEWORM
PARASITOLOGY
It is perfectly suited for parasitic life because of follOWing features:
123
1. The scolex or head like structure has four suckers, two rows of hooks on the rostellum
2 The hooks on the rostellum help in firm anchorage on intestinal wall
3 The four suckers on the scolex also help In attachment to mucous membrane of Intestinal
wall. They also help In locomotion, where the worm attaches Itself to the intestinal wall
and drags Itself
4. Suckers and hooks on the rostellum of tapeworm prevent the worm from getting washed
off from the intestinal wall.
FRONTAL VIEW OF
SCOLEX
Fig.1 9. Scolex of Tapeworm
MATURE PROGLOTTID OF TAPEWORM
The mature proglottid IS found In the posterior region of the tapeworm, which constitutes few
pseudosegements of the body of the tapeworm followed by graVid proglottid It IS characterized by
following features:
1. Each mature proglottid has a complete set of male and female reproductive organs.
2. Male reproductive organs consist of several testes, vasaefferentia, vas deferens and
cirrus (copullatory organ).
3. The female reproductive organs consist of a pair of ovaries, OViduct, vagina, uterus,
shell gland and vitelline gland.
4 The male and female genital apertures open into common cup-like genital atrium
5 Each mature proglottid will act as a reproductive chamber where fertilization of eggs
takes place In each mature proglottid, which later transforms Into graVid proglottid by
lOOSing all reproductive organs except uterus
6 Each mature proglottid ensures some million eggs are fertilized and transmitted to next
host so that the parasitic life IS continued in the next host
TESTES
VASEFFERENTIA
VAS DEFERENS
CIRRUS OR PENIS
COMMON
GONOPORE
VAGINA
MEHLlS'S
GLAND

FERTI L1ZED
OOTYPE
Fig. 1.10. Mature proglottid of Tapeworm.
EGG CAPSULE
SEMINAL
RECEPTACLE
OVIDUCT
UTERINE CANAL
GRAVID PROGLOTTID OF TAPEWORM
The gravid proglottid of the tapeworm is the pseudosegment of the body of the tapeworm,
which is well suited for the parasitic mode of life because of follOWing features'
1. It is found in the terminal part of the body of the tapeworm and it IS easily cut off from the
body.
2. The gravid proglottid contains fertile eggs in the uterine chambers and It is covered by
hard cuticle, which is resistant to the action of digestive juices and helps as a carry bag
for millions of fertile eggs.
3. Either the mature proglottid as such or the fertile eggs are passed in the human faeces.
4. Contaminated soil where pig and the cow graze will ingest the fertile eggs, which act as
intermediate host.
5. The fertile egg hatches into onchosphere and develop into cysticercus larva In the animal
tissue.
6. The major function of gravid proglottid is transmission of infection from host to intermediate
host.
PARASITOLOGY
BRANCHES OF
UTERUS
MAIN STEM
CIRRUS
GONOPORE ........
SPERM DUCT
VAGINA
Fig. 1.11. Gravid proglottid of Tapeworm
T.S. OF ASCARIS
125
There IS no difference in the structure of the body wall and gut in the male and female
Ascaris. The difference lies in the cut sections of sex organs. Since the female Ascaris lays a
major role in disease transmission, only the structure of T.S. of female Ascaris IS explained here
The main parasitic adaptations observed are as follows.
1. The body is covered by thick cuticle, which is resistant to digestion by digestive JUices,
which is one of the major adaptation for parasitic mode of life in the intestine of humans
2 Immediately below the cuticle is present epidermis and longitudinal muscle bundles, these
help in contraction and expansion of body for locomotion
3. In the female section of the uterus with eggs can be seen, which are fertile eggs released
Into the intestine from where they escape into the external environment through faeces
lONGITUDINAL
MUSCLE CEllS
. OVIDUCT
UTERUS
CONTAINING
EGGS
PSEUDO COEl
Fig. 1 12. T S of female Ascaris
INTESTINE
4 Internal fertilization and uterine chamber for storing the fertile eggs IS an additional
adaptation, which ensures that the eggs are fertilized before released into the enVIronment
5. Fertile eggs enter human body through contaminated food and water so that the
transmission and parasitic life is continued. The eggs hatch Into larvae In the intestine of
the new host
ENTOMOLOGY 127
2. ENTOMOLOGY
2.1. HONEY BEE - LIFE HISTORY
The queen bee mates once in her life time with a drone and store the sperms in the
spermathecae. She lays a single egg in each cell of the brood chamber in the honeycomb and
while laying the eggs depending on the structure and size of the cell she decides to fertilize it by
releasing the sperms from the spermathecae or lays them without fertilization . Fertilized eggs
develop into sterile females, which become the workers of the colony. Unfertilized eggs develop
by parthenogenesis into males or drones. Eggs laid in larger queen cells hatch into larvae. which
are fed on royal jelly and these develop into queens At the last larval instars i.e. about 5 days
after hatching the celis are sealed and the larvae pupate by spinning a silken cocoon. The pupal
stage of the queen lasts for 7 and half days, the worker 12 days and the drones 14 and half days.
The queen leaves the colony with some workers and drones when the hive is over crowded,
the process is called "swarming" and then re-establish a new colony elsewhere. The virgin queen
first to emerge in the hive stings her sisters queens to death and takes off with a few drones on a
"nuptial" flight. Mating takes place in air and the drone dies after copulation as the posterior part of
its abdomen is torn off.
WORKER /
LARVAE FEED ON LARVAE FEED ON
BEE BREAD ~ Y A E L L Y
PUPA
Fig. 2.1. Life hi story of honeybee.
MOUTHPARTS OF HONEY BEE
The mouthparts are chewing and lapping type. They are modified for collecting nectar and
pollen. Mouthparts consist of labium epipharynx, mandibles and maxillae.
1. The labium lies below the clypeus.
2. The mandibles are smooth and are situated on either side of the labium They are used
for molding the wax and making the honeycomb.
3. The labium has submentum, mentum, paraglossa and glossa with a long labial palp.
4. The glossa has labellum (honey spoon) at its tip.
5. The maxillae fit over the mentum and bear maxillary palps. They are sensory in function.
6. The maxillae and labial palps form a tube enclosing the glossa, which moves up and
down while collecting the nectar.
PALPIFER

LABIAL PALP __ ...... .c
GLOSSA
(LIGULA) ---...
LABELLUM OR
HONEY SPOON
LABRUM
__ CARDO
STIPES
GALEA
Fig. 2.2. Mouthparts of honeybee.
STING APPARATUS OF HONEY BEE
The sting is made up of three parts, viz., gonapophysis, stylets and poison canal. There are
two stylets, which are articulated along their length. The stylets are .Iaterally covered by plates,
which are named as triangular plate, quadrate plate, and oblong plate. At the tip of the stylet a
pointed lancet is present which is provided with barbs. Attached to the stylet at the posterior
region is a poison sac into which the alkaline gland opens. The three plates act like a spring and
help in inserting the sting. The sting is a modified ovipositor.
ENTOMOLOGY 129
SAC
ACID GLAND
___ ALKALINE GLAND
BASAL ARM
LANCET
__ PALPUS OF STING
BARB S
Fig. 2.3. Sting apparatus of honeybee.
LEGS OF HONEY BEE
There are three pairs of legs performing different functions. Each leg is made up of five parts
namely coxa, trochanter, femur , tibia and tarsus, which terminate with a pair of claws
First pair of legs: It is also called as the prothoracic leg. It has clusters of bristles in a row
on the tibia , forming an eye brush. At the distal end of the tibia a movable spine is present called
velum, which closes over a notch on the tarsus, and it is sailed antenna comb. Long bristles on
the tarsus form the pollen brush, which is used for the removal of pollen.
Second pair of legs: It is also called as mesothoracic leg. It also has a pollen brush on the
tarsus and at the end of the tibia it has a spur or a spine, which is used for removing pollen from
the pollen basket and wax from the abdomen.
Third pair of legs: It is also called the metathoracic leg. It has a large tibia , which contains
the pollen basket whose cavity is filled with bristles to contain the pollen. At the distal end, the
tibia has thick bristles called pectin below which a flat plate called auricle is present Pectin and
auricle constitute the pollen packer. It is also used for removing wax from the abdomen. The outer
surface of tarsus has pollen brush and the inner surface of tarsus has pollen comb. The pollen
comb removes the pollen from the body and fills it in the pollen basket.
HONEYCOMB
The workers seek out a sheltered place and using wax secreted between scales like plates
on the underside of their abdomens they build clusters of cells called combs The combs are
made up of hexagonal cells having 3 main sections the lowest or bottom section containing eggs,
larvae and pupae collectively termed as the brood, the middle one used for storing pollen and the
upper section for storage of honey. The comb hangs vertically with the open ends of the cells
facing the sides. To keep the larvae, which develop within from falling out , cells are constructed
with a slight upward tilt it appears that bees in making honeycombs, use the shape that encloses
the maximum amount of honey and uses the least amount of wax.
COXA
Ji':'Ir-----TIBIA .;;.;
VELLUM OR FIBULA
ANTENNA COMB
TARSUS
PROTHORACIC LEG
MESOTHARACIC LEG
AURICLE
METATARSUS
POLLEN
PULVILUS
TARSUS
METATHARACIC LEG
Fig. 2.4. Legs of Honeybee.
WORKER CELL
QUEEN CELL
DRONE CELLS
Fig. 2.5. Honey comL
TIBIA
ENTOMOLOGY 131
LOCUST (Schistocerca gregaria)
They are the common allies of grasshoppers. The desert locust is migratory in nature and
found in Africa, Arabia, Iran, Palestine, Afghanistan, Pakistan and North-west India. They breed in
large congregation in deserts and migrate to fields of standing crops in large numbers in nymphal
stage by jumping on land with the help of their hind legs. At this stage they do not have wings.
They mature during course of their migration. During their migration whatever vegetation found on
their way is eaten. When they reach the agricultural fields of standing crops they will have grown
and possess fully developed 2 pairs of wings. They can fly miles together in large swarms of
millions in number and they are gregarious in nature feeding on vegetation and crops standing on
their way. They cause great loss to standing crops.
APHID
They are mostly polyphagous insects, which feed on wide range of plants Both nymphs and
adults suck the cell sap from tender shoots or leaves. The affected part turns yellow, curls up or
gets deformed ultimately it dries up. The insects secrete honeydew on which sooty mould grows,
which in turn hinders the photosynthetic activity of the plant. Some aphids also transmit viral
diseases among plants.
Fig. 2.6. Locust (Schistocerca gregaria). Fig 2.7. Aphid .
RICE WEEVIL (Sitophilus oryzae)
They infest stored grains of wheat, barley, sorghum and rice. Heavily infested grains are
powdery and hollow due to the boring activity of the weevil. Weevils consume the kernel of the
grain. Heavily infested grains develop peculiar odour due to development of fungus on the excreta
of the weevils. Weevils have a characteristic proboscis extending from the tip of the head.
FLOUR BEETLE (Tribolium confusum)
They generally infest flour and other grain products. They have a characteristic oblong brown
coloured body, which is more or less flat on the dorsal side. Heavily infested grain products become
un-consumable due to fungal growth on the excreta of these insects. Larval activity of these insects
produces webbing of flour or grain products.
DRAGONFLY
The adult dragonflies catch insects during flight. .The nymphs of the dragonflies are also
called as mosquito hawks as they help in controlling the mosquito population. They catch and eat
their prey in mid air and are good biological control agents. Therefore they are called as
entomophagous insects.
ICHNEUMON WASP
It is solitary in nature with parasite like predatory habits. The female has a pointed ovipositor,
which is used to pierce the host tissues and lay the eggs inside the host's body.
The larvae hatches and begins feeding within the host in such a way that the host continues
its activities in a seemingly normal fashion The host may even pupate but inside the host the
parasite grows and towards the end the adult wasp emerges instead of the host.
Fig. 2.8. Sitophilus oryzae (Rice weevil) Fig. 2.9. Tribolium confusum (Flour beetle)
fOREWING
HINDWING
Fig. 2.10. Dragonfly.
ENTOMOLOGY
133
Fig. 2.11 . Ichneumon wasp.
3. FISHERIES
ROHU (Labeo rohita)
It is a fresh water fish, most common carp in the plains of India. Body is elongated rather
roundish with moderately convex abdomen. Body is bluish or brownish gray above; snout blunt
protruding with abruptly undershot mouth. Attains maximum length of about 80-90 cms. Mostly
cultivated with Catla in fresh water ponds and lakes in the absence of carnivorous fish. They are
bottom feeders hence they have downward stretched lips. Lips are provided with fleshy movable
barbs used as feelers or sensory organs. The fish is fleshy and noted for its delicacy valued next
to Catla (Fi g. 3.1). .
Fig. 3.1. Labeo rohita (Rohu) .
CATLA (Catla catla)
It is a common carp found in fresh water bOdi.es throughout India and it is the largest among
the carp family. It attains a maximum length of about a meter. Mostly cultivated in isolated fresh
water ponds and lakes in the absence of carnivorous fish. They have a characteristic upturned
stretched lip hence they are surface feeders. Body is deep, stout , with broad snout; mouth wide
with continuous transverse fold doubling the lip outwards; dorsal fins with no osseous ray; colour
grayish above; .silvery on sides; fins dark, sometimes black; scales with pink or coppery center
except the ventrals which are whitish. The fish is fleshy and noted for its delicacy and valued very
high in the market It is best for consumption when not more than 61 cm in length (Fig. 3.2) .
Fig. 3.2. Calla calla (Carp) .
FISHERIES 135
MRIGAL (Cirrhina mrigala)
It is found in rivers and tanks allover India. This fish belongs to carp family and resembles
Labeo rohita except that it has a wider mouth and thinner lips. Body silvery, dark gray along back,
sometimes with coppery tinge; pectoral , ventral and anal fins orange tinged with black tips. Breeding
takes place in flooded rivers during July-September. During this time fingerlings are collected in
basket traps for transporting them to interior areas for cultivation. It attains a length of 50 to 65 cm
within 1 yr and weighs approximately 14 to 2.8 kg (Fig. 3.3) .
Fig. 3.3. Mrigal (Cirrhina mrigala) .
OIL SARDINE (Sardinella longiceps)
It is a small pelagic (swimming near the surface) , schooling (moving in large numbers or
shoals) marine fish These fish constitute mainstay of our fishery because they are caught in large
numbers. They have a characteristic bluish back and silvery gray belly. It is of great economic
importance because it is not only used as food, its body oil is used in cosmetics, soaps and
lubricants etc. When in surplus it is salted and sun dried, canned. After the extraction of oil from
the body it is used as manure for cash crops like coconut, areca nut, coffee and tea and it is also
used as a supplement in poultry feed (Fig. 34).
Fig. 3.4. Oil sardine (Sardinella longiceps) .
MACKEREL (Rastrelliger kanagurta)
They are marine, planktophagous, shoaling fish. Fully mature fish attains a length of 18-22
cm and yields good amount of flesh. It is in great demand in fresh condition. Large percentage,
i.e., 40% of the catch is preserved in ice and dispatched to inland areas and 60% catch is salt
cured or pickled or sun dried or smoked. When the catch is abundant , the surplus is converted
into manure. The viscera and gills discarded from the curing and canning plants is used in the
preparation of fish meal or poultry feed, cattle feed and manure. Body is torpedo shaped with a
characteristic bluish-black back and silvery belly. Tail region has 5-7 characteristics detached .
finlets on dorsal and ventral side. There is only one genus and species in India. The chief centers
of fishery are the area between Ratnagiri in Mumbai and Quilon in Kerala (Fig. 3.5).
Fig. 3.5. Mackerel (Rastrelliger kanagurta) .
BOMBAY DUCK (Harpodon neherius):
They are marine in habitat confined to Mumbai coast and northern part of Bay of Bengal. It is
primarily a shoaling fish of the open seas. It comes to inshore of feeding incursions. Fishing season
extends from September to January. 80% of the catch is sun dried. They are also consumed
fresh. Body is long, cylindrical, pinkish in colour, fleshy and less bones. Bones are soft. The fish is
highly carnivorous (Fig. 3.6) .
Fig. 3.6. Bombay duck (Harpodon neherius) .
FISHERIES
POMFRET (Silver pomfret-Stromateus argenteus)
137
These are marine, planktophagous fish. Body is laterally compressed and broad
dorsoventrally. Dorsal side is dark gray and belly is silvery. They are highly priced fish because
they are fleshy and the bones and spines are soft. They are mostly consumed fresh. Occasionally
when they are in surplus, they are dried and salt cured (Fig. 3.7).
Fig. 3.7. Silver Pomfret (Stromateus argenteus) .
SHARK (Scoliodon)
They constitute a considerable part of the commercial catches on both the coast Sharks are
found throughout the year, but the main season of fishery extends from July to March on the west
coast and from May to January on the east coast in waters ranging from 40 to 55 m dept Sharks
are being exceedingly voracious carnivores, follow shoals of other fish and are caught entangled
in the nets. Large sharks are highly priced because of their liver oil. They are consumed fresh as
well as they are salted and sun dried. Shark fin soup is a delicacy, hence sometimes, large sharks
are caught by hooks and lines, and their fins are cut and the fish is dumped back in the ocean to
die in western countries but in India every edible part of the fish is consumed. At present large
shark fishery is banned and they are enlisted in endangered species (Fig. 3.8)
Fig. 3.8. Shark (Scoliodon) .
PRAWN (Pinaeus sp.)
The major prawn fisheries are located on the west coast Fishing is restri cted to shallow
waters near the coast; seldom exceeding 18 m in depth. During the monsoon months of June to
August, the shoals come near the coast. At that time, they are caught by cast nets. Fishing in the
deep sea is carried out with trawl nets from power vessels. They grow up to 20 cm in length.
Mostly consumed fresh and surplus is frozen. It is exported to many countries and major profit for
fishery industry is from prawn exports (Fig. 3.9) .
Fig. 3.9. Prawn (Pinaeus) .
r"LOBSTER (Panuliru.s sp.)
The rock lobster or spiny lobster in which five species belong to the genus Panulirus found
along the Indian coast. They attain a length of 30 cm and are caught by small conical nets, traps
and gill nets along the Mumbai coast, where the fishery lasts from November to March and on the
east coast the fishery lasts from December to April. They are highly priced because of good,
sweet f l e ~ t l They are consumed fresh and frozen and major catch is exported (Fig. 3.10).
Fig. 3.10. Lobster (Panulirus) .
Fig. 3.11 . Crab.
FISHERIES 139
CRAB
Several species are caught in commercial and subsistence fishing in India. In Mumbai , crabs
are collected throughout the year, the peak period being August to October In Madras, the best
season for crab fishery is March to May. In the Southwest coast, particularly Neptunus is caught
from October to April. Crabs are caught in seine-nets and dip-nets, by hooked iron rods and on
lines. They are mostly consumed fresh and a small proportion of crabmeat in brine is exported
after shelling (Fig. 3.11).
EDIBLE OYSTER:
The edible oysters are obtained at all places round the Indian coasts where brackish water
is renewed by tidal flow and the substratum is suitable for their attachment. Important commercial
species are the backwater oyster, Crassostrea madrasensis from the estuaries and backwaters,
the rock oyster C cucul/ata from the intertidal rocky coasts, the disc oyster C discoidae from the
littoral zone of the coastal areas and C gryphoides found in the muddy creeks Majority is consumed
fresh and a small proportion is shelled and the meat in brine is exported (Fig . 3.12)
Fig. 3.12. Edible oyster. Fig. 3.13. Pearl oyster.
PEARL OYSTER
Pearls of high commercial value are obtained from pearl oyster of the genus Pinctada. Several
species occur in Indian waters of which, P. vulgaris is the commonest and most important and is
widely distributed in the Gulf of Kutch, Gulf of Mannar and the Palk bay. They are usually found
on the ridges of rocks or dead corals forming extensive pearl banks or the pars at a depth of 18 to
22 m and a distance of 19 km from the shore. The pearl oyster beds of the east coast are more
extensive and productive. These beds yield excellent quality oriental pearls or the lingha pearls
On the west coast the pearl oysters are fished from the reefs in the Gulf of Kutch to the north of
Halar. Pearl oysters grow up to 89 to 102 mm across in 4-5 years when they are ready for harvesting.
Strict legislative measures are in force forbidding young pearl oyster fishing The pearl beds are
under the control of State Governments. Fishing is conducted by organizing temporary camps
with the help of divers. The fishery may last for about two months during the summer season.
They constitute a major revenue for the Government (Fig. 3.13) .
SEPIA (CUTTLE FISH) AND SQUID (LOLlGO):
Cuttle fish and squid are common on the Indian coasts, though nowhere do they form a
regular fishery, and are incidentally caught in nets all through the year in the normal fishing
operations. About four species each of cuttle fish and squids exist in Indian waters. On the southern
coast , in the region of Palk Bay and the Gulf of M a n n a ~ there is a seasonal fishery for squids from
February to June. In Palk Bay for fishing operations a special seine called o/a va/ai made of strips
of palm leaves tied along the wing-ropes acting as scares to drive the squids into the bag of the
net is used. In the Gulf of ManQar the squids are obtained in shore seines along with fish in small
numbers. Most of the catch is consumed fresh, but some of it is dried for export. At Rameshwaram
coast dried product of squid or cuttle fish is used as bait for long line fishing (Fig. 3.14 and 3.15).
Fig. 3.14. Cuttle fish (Sepia) . Fig. 3.15. Squid (Loligo) .
KATELYSIA (CLAM)
The inflated clam Kate/ysia opima are fished in considerable quantities in some coastal places
where they are mostly consumed by the poor people. They are usually hand picked in shallow
waters at low tides (Fig. 3.16).
Fig. 3.16. Clam. Fig. 3. 17. Sea-MusseL
FISHERIES . 141
MYTILUS (SEA-MUSSELS)
They usually form a thick carped like growth over submerged rocks to which they secure
anchorage by means of their slimy byssus threads. The green sea-mussel has wider distribution
on both the coasts, but is abundant in Cochin and Malabar coast. The green mussel occurs not
only in the coastal waters, but also in the backwaters and bays as in some parts of Orissa and
Chennai States. Fishing on the west coast is usually by hand picking at low tides after southwest
monsoon, but larger specimens from deeper waters are taken by divers. The fishing implements
include an iron chisel or a wooden wedge, sharpened at both ends for dislodging the mussels
from the substratum (Fig. 3.17).
DRIED BOMBAY DUCK
Removal of moisture from fish tissue stops or inhibits bacterial and enzymatic degradation
Sun drying is extremely simple and cheapest way of preservation of fish practiced along both
coasts. Bombay duck is sun dried by hanging them on bamboo or wooden racks or on ropes by
passing the rope or the bamboo through their gill operculum. This helps in uniform drying and
prevents contamination from sand. Sun drying is a slow process and the fish develops a peculiar
cured flavour, which is not acceptable to all .
SALTED MACKEREL
Common salt acts as a preservative and it prevents bacterial and enzymatic degradation
There are two methods of salting, viz. , wet and dry salting. For dry salting fish are gutted, cleaned
in water and then packed in layers in a tub and the dry salt is spri nkled generously in between
each layer. The proportion of salt to fish is 1:3 or 1 :8. The fish are then removed after 24 hrs and
dried in the sun for two to three days.
In the wet salting process, gutted and cleaned fish are packed in large vats containing salt
solution and stirred daily till properly pickled.
FISHING CRAFTS
DUGOUT CANOE
These boats are made by scooping out the inner pith of a large wood or log The bottom part
or the keel is thicker than the sides. These are popular on the Kerala and Konkan coasts. The
large sized ones are called Vanchi or Odam are 10 to 12 m long and used for operation of large
variety of nets. The smaller ones, known as Thonies or Donyare used for gill nets or drift fishing.
Dug out canoes are also seen on the west coast between Colachal and Kathiawar (Fig. 3.18).
Fi g. 3.18. Dugout Canoe.
OUTRIGGER CANOE
They are regular plank built canoe with a narrow keel and planks more spread out. The
usual size is about 15 x 3 m long. They have a single outrigger and are found in Kanara and
Konkan coast. They are locally called Rampani, since they are used for casting of the Rampani
net or shore seine for mackerel fishery (Fig. 3.19).
Fig. 3.19. Outrigger canoe (Rampani boat) .
CATAMARAN
It is a keelless raft constructed by tying together several logs, which are curved and shaped
like a canoe. One end of the craft is shaped into a cone, which rises above the water level.
Catamarans are restricted to the east coast from Orissa to Cape Comorin with a short extension
northwards on the Kerala coast. There are four types of catamarans (a) Coromandel type made of
3-5 longs called Kolamaran; (b) Orissa and Ganjam type is made of 5 logs, which are not tied
together but pegged together with wood; (c) Andhra type is almost like Orissa type but larger ie.
5-7 meters long; (d) Boat catamaran is made up of three logs shaped and fitted into a regular boat
shaped vessel (Fig. 3.20).
-:-- . - -----.
~ ; ~ . . -
-=- -.
... - '
--- - ."----
Fig. 3.20. Catamaran.
MASULA
It is a non-rigid boat constructed by using planks, which are sewn together with coir rope. To
withstand severe knocking of the surf these boats do not have frames and ribs. It is usually 9
meters in length and could be even smaller. It is extensively used in Coromandel coast. There are
several variations of this type. In Orissa, it is called Bar boats and in Andhra it is called Padava or
padagum (Fig. 3.21) .
FISHERIES 143
Fig. 3.21 . Masula boat.
SATPATI
It is a built-up boat popularly called galbat, has a medium-pointed bow, broad beam, straight
keel and high gunwale. It is operated by sails as well as by motor. There is a provision for fitting a
motor and many compartments for fish storage and a compartment for fishing gear (Fig. 3.22).
Fig. 3.22. Satpati boat.
TRAWLER
These are built-up mechanized boats fitted with a diesel engine of very high horse power
They are used for operating trawl nets or boat seine for fishing in open seas and deep seas. They
remain in open seas for a day or two and have the facility for storage of fish and the gears. If it is
a overnight operation far away from the coast, to preserve the fish catch refrigeration facility is
also available. Some trawlers have insulated chambers in which ice blocks are carried, which are
crushed and put in the insulated fish chamber to preserve the catch until they reach the shore
These boats are operated for large catch of fish and for catching big fish like tuna, sharks rays
etc. (Fig. 3.23) .
Fig. 3.23. Trawler.
FISHING GEARS
GILL NET
These are also called driftnets or wall like nets of various sizes and meshes made of hemp
or nylon. They are provided with floaters and sinkers to keep them vertical and straight. Fish of
different sizes are generally trapped in the various meshes through their gill operculum hence
they are called gill nets (Fig. 3.24) .
Fig. 3.24. Drift net or Gill net. Fig. 3.25. Dol net or Bag-net .
FISHERIES
DOL NET
It is a specialized fixed bag-net, which has the
characteristics of both a bag-net and affixed trap. It
is predomjnantly used in Mumbai and Gujarat coast;
it is fixed in the sea by stakes or buoys. These nets
are used in waters where the current is strong and
high enough to keep the net in horizontal expanded
position (Fig. 3.25) .
CAST NET
These are universally used nets and are of two
types, one with string and the other without it. The
net is spread out when thrown. trapping the fish It
is always hand operated. Generally used for catching
surface feeding fish like Pomfret in konkan coast
(Fig. 3.26).
PURSE SEINE
It consists of a long wall of netting provided
with a float line and lead line. Below the lead line is
purse line runs through purse rings connected by
short length of rope to the lead line. The fish are
caught in the purse seine by surrounding them from
side and below thereby preventing their escape
(Fig 327)
RAMPANI NET
It is the biggest shore seine used in India
largely used for mackerel fishing in Konkan and
Fig. 3.27. Purse seine.
145
Fig. 3.26. Cast net
Fig 3.28. Rampani net.
Malabar coasts. This is a wall net of enormous length. Wooden floats and stone sinkers are attached
to the head and footropes respectively to keep the net in vertical position. One extremity of the net
is tied to a stake on the shore, while a boat takes the other end into the sea in a semi-circle and
back to the shore. The two ends are dragged by groups of men on to the shore with shoals of fish
(Fig. 3.28)
LONG LINES
Long lines are generally made of cotton or cat gut, and line fishing is employed in a variety
of ways using baited hooks. For large fishes, Chain hooks, both revolving and non-revolving, are
used, the former being particularly useful in shark fishing. Long lines are also used for catching
large perches (Fig. 3.29).
Fig. 3.29. Long lines.
ANIMAL HUSBANDRY 147
4. ANIMAL HUSBANDRY
LEGHORN
It is one of the most popular breed of the Mediterranean class. It IS the world's number one
egg producer. The breed originated In Italy and there are 12 different varieties, of which three are
very popular. The breed is small, active, and very compact in form It carnes the tall rather low
and has a small head With a well set comb and wattles. It has a relatively long back, prominent
breast and comparatively long shanks Mature birds weigh from 2 to 2 7 kg (Fig 4 1).
Fig. 4.1. Leghorn fowl.
SAHIWAL
Found in Punjab, Delhi, U.P , Bihar and MP It has a deep body, loose skin, short legs,
stumpy horns, and a broad head. General colours are various shades of red, pale red and dark
brown splashed with white. Horns are short and thick, which do not exceed 3 inches Massive
hump is seen in males, along With a voluminous dewlap and pendulous sheath Long whip like tall
almost reaching the ground ,tapering to a good black sWitch Navel flap IS prominent In females
Sheath is pendulous but should not be abnormally loose or long Males weigh about 522 kgs and
females 340 kgs. This is one of the best dairy breeds In India as the average Yield IS 2150 kgs In
300 lactation days (Fig. 4.1).
HARIANA
The breed originated in Punjab, they are now found in Rohtak, Hlssar, Karnal and Deihl The
animal has a proportionate body, arld a compact graceful appearance. Head is carried high, horns
are short, curving upward and Inward and stumpy Colour is white or light gray It has a long
narrow face, flat forehead and a bony prominence in the center of the poll. Small and sharp ears,
skin is of fine texture and close to the body. Sheath is short and navel flap IS absent Udder IS
capacious which extends forward with a well-developed milk vein Legs are moderately long and
lean Pin bones are prominent and far apart in females but close in males Tall is short, thin and
tapering towards the end with a black switch reaching Just below the hocks Bullocks are good
working animals for fast ploughing and road transport. Cows are average milkers with 1400 kg per
lactation (Fig 4.2)
Fig. 4.1. Sahiwal. Fig 42. Hanana
KHILLARI
The breed is well distributed throughout Maharashtra state. It has a compact body with clean-
cut features. The head is massive with small eyes and ears, horns are long and pointed, emerging
close to each other and follow the backward curve of the forehead. Neck IS short and firmly set
The shoulders are tightly muscled .The barrel is long and compact with no loose skin Legs are
clean cut, and straight with black hoofs and digits, which are closely set. Cows are poor milkers.
The breed IS highly valued as fast paced, powerful draught animals (Fig 4.3)
MURRAH
This breed of buffalo originated In Punjab, It is now found In UP, Rajasthan, and other places
The animal has a deep massive frame with a short broad back and a light neck and head. It has
short tightly curled horns, well-developed udders and a long tail with a white switch reaching to
the fetlock. Short massive limbs with good bone, broad hoofs and drooping quarters are the main
characters. Colour is jet black with white markings on the tail, face and extremities. The skin is
soft, smooth with scanty hair. The body of the bulls weighs 550 kg and that of the she buffaloes
weighs 450 kgs. The height at withers on an average is 1.42 m for bulls and 1 32 m for she
buffaloes The average milking capacity is about 1400 to 2000 kg with a butterfat content of 7% In
a lactation of 9 to 10 months The breed IS the most efficient producer of milk not only In India but
In the world (Fig 44)
Fig. 4.3. Kilian Fig 44 Murrah
JAFFRABADI
These animals are seen in the Gir forest where they are bred in large numbers almost solely
for ghee. They have a prominent forehead and heavy horns, which are inclined to droop on each
side of the neck and then turn up at the pOints but not in such a tight curl as in Murrah buffaloes
Body is longer but not so compact. Dewlap and udder are well developed Animals of this breed
are generally black in colour. The Jaffrabadl males on an average weigh 600 kg while the females
weigh 460 kgs. Animals are heavy milkers ranging from 15 to 18 kg per day. The bulls haul heavy
loads as well (Fig. 4.5).
Fig. 4.5. Jaffrabadi.
JAMUNAPARI
It is the biggest and most majestiC breed of goats In India. It is found In UP Agra Mathura
and MP. There is great variation in coat colour but they are generally white or light yellOWish tan
with light brown SiJots on the neck and face, and occasionally patches of tan or biack are found on
the body The typical character of the breed IS a highly convex nose line with a tuft of hair known
as "Roman nose" or parrot appearance The ears are very long, flat and drooping Both sexes are
horned with short thin tall. A tnick growth of hair IS present on the buttocks, known as feathers
The breed has well-developed udders, round in shape with large conical teats. Average daily yield
of milk is 200 kg per lactation. Bone and meat ratio is 1 :3.9 (Fig. 4.6).
SURTI
This goat is distributed in Surat and Baroda and it is a good dairy breed. It IS a medium sized
breed, white in colour with highly developed udders. Ears are medium in size. Both sexes have
small horns directed backward. The breed is unable to walk long distances and has to be stall-
fed They are economic to rear as they can live on leaves or on food waste. The breed IS a good
milk producer yielding 2 kgs milk per day (Fig. 4.7).
Fig. 4.6. Jamunapari. Fig. 4.7. Surti.
GADDI
These sheep are found in Kulu and Kangra valley and in the Chamba and Mandi districts of
Himachal Pradesh. The sheep are small in size but are very sturdy and very good climbers. They
have short tail and ears, horned in the rams but polled in the ewes They are mostly white with
coloured faces. The fleece is relatively fine and lustrous and can grow to 5 inches in length. The
undercoat is used to manufacture high quality Kulu shawls and blankets (Fig. 4.8).
Fig. 4.8. Gaddi
MARWARI
The flock is mostly maintained in Jodhpur, Nagaur, Pali and Barmer districts of Rajasthan.
Marwari sheep are black faced, head is covered with black hair and it has small twisted ears.
Legs are long and thin, body is sturdy and of medium size. The wool is of coarse quality, and
contains heterotype hairy fibres and is thus used for rough carpets and blankets. Live weight of
rams is 35 kgs and ewes are 28 kgs (Fig. 4.9).
Fig. 4.9. Marwari.
~ -
4.1. ESTIMATION OF PROTEIN IN EGG
First Method:
COLORIMETRIC ESTIMATION OF PROTEIN IN TWO DIFFERENT VARIETIES OF EGGS
(Country/Farm)
(The same experiment can be performed with fowl and duck egg)
Since the farm egg and country egg are laid by two different varie:ies of fowl they should
differ in protein concentration. To test this hypotllesis, following experir"ent is performed.
Aim: To determine the protein concentration in two different varieties of eggs by Folin-Lowry
method.
Requirements: Standard protein solution of BSA (bovine serum albumin-O 2 mg/ml), alkaline
copper sulfate reagent (Reagent A-O.5gm of CuS0
4
in 100 ml OW, Reagent 8-1 gm of Na-K
tartarate in 100 ml OW, Reagent C-2 grn of Na
2
C0
3
dissolved in 100 ml of 0.1 N NaOH). MIx 11
proportion of reagent A and B and take 1 ml of this mixture and add it to 50 ml of reagent C to get
final alkaline copper reagent. Calculate the required amount of alkaline copper reagent and plepare
trle rectgent accordingly in the proportion mentioned above and it has to be prepared before
the experiment. Prepare 1:2 dilution of Folin-Ciocaltaeu reagem in D'N 1 In 40 dilution of egg
white of both country and farm egg.
Principle: Egg white is a type of protein, wilen this protein solution is treated with alkaline
CuS0
4
it forms Biuret complex with 20 or more pepIide bonds. On addition of a reugent called
Folin-Ciocaltaeu which contains phosphomolybdic acid it forms a blue coloured complex due to
rp.cluctlon of NlO6 to MO-4 by the amino acids like tyrosine and tryptophan present In the protein
This intense blue Golour is estimated colorimetrically at 620 nrn. IntenSity of colour var,es with the
concentration of protein In the sa,rnple.
Procedure: If a standard graptl is gOIr.g to be providtjd only twq test tube:. Will be needetj W
perform the
If students are estimating protein in the sample by calculation metnod lour test tubes ne:ed to
be provided to p.acil student.
1. In test tubes 1 take 1 ml of standard protein soh/lion (Std.)
2. In test tube 2 and 3 take 0.2 rnl 01' 1 in 50 diluted egg white solution (Unknown).
3. In test tube 4 take 1 ml of distilled water (Blank).
4 To each test tube add 5 ml of alkaline copper sulfate und rwx It well allow the mixture to
remain for 15 min.
5. After 15 min, c.\dd 0 5 ml of 1:2 diluted Folin-Ciocaltaeu reagent to each tube rlllX it well
and walt for 46 min.
6. Read the absorbance of standard and unknown CIt 620 rim ag;: .. lInst blank and tabulah;
thE: readings.
7. Calculate the. concentration of protoin by U:',infi thiS formula.
of th: ... 50 x
Absorbance of Standard x 0.2 (Volume of unknown taken)
::. mg/ml protein
PROTEIN IN EGG 153
Results: Compare the values of protein in the two different varieties of eggs and conclude
your experiment. .
Second Method:
Aim: To estimate colorimetrically the percent composition of protein in egg white by Biuret
method.
Principle: Alkaline CUS0
4
solution reacts with compounds containing two or more peptide
bonds to give a violet coloured complex called Biuret complex. The Intensity of colour IS directly
proportional to the number of peptide bonds present In the protein sample. ThEf reaction IS not
absolutely specific for peptide bonds, since any compound containing two carbonyl (C=O) groups
linked through a nitrogen or carbon atom will give positive result.
Requirements:
1. Biuret reagent: dissolve 3 gm of copper sulfate and 9 gm of sodium potassium tartarate
in 500 ml of 0.2 N NaOH; add 5 gm of potassium iodide and make up the volume to 1 lit
with 0.2N NaOH)
2. Standard protein solution (5mg/ml):
3 Egg white solution made in distilled 'f'ater (1 50 dilution),
4. Test tubes, distilled water
Procedure: Prepare following set of test tubes for standard (Std.), U 1 U
2
, U
3
(Unknown samples)
Tube No. Std. Protein Biuret reagent Distilled
(5mg/ml) water
1 0.4 ml 3ml 16 ml
2 0.8 ml 3 ml - 1 2 ml

3 1.2 ml 3 ml 0.8 ml
4 1.6 ml 3 ml 0.4 ml
5 2.0 ml 3ml 0.0 ml
Blank 0.0 ml 3ml 2.0 ml
U
1
U
2
, U
3
1.0 ml (1 : 50 diluted) 3 ml 1 0 ml
Allow the tubes to stand at room temperature for 15 minutes and then read the 00 against the
blank at 540 nm.
154
OBSERVATIONS:
Tube No.
1
2
..
3
4
5
U
1
U
2
U
3
Graph:
Conc. of protein Absorbance
2.0 mg
4.0 mg
6.0 mg
8.0mg
10.0 mg
Draw the standard curve/graph by plotting the mg of Std. protein solution against absorbance
and determine the concentration of unknown protein in the solution given.
Calculations:
1. From the graph, optical density _______ of the unknown protein solution
corresponds to x mg of protein.
1 ml of unknown was used for the assay.
1 ml of unknown diluted protein solution = x mg of protein.
100 ml of unknown diluted protein solution = 100 x x x 50 (dilution factor) mg of protein
Results:
Amount of protein in unknown solution
Calculatioh method:
_______ mg% (from graph)
-------g%
00 of Unknown
______ x Conc. of Std. (5 mg/ml) x 50 (dilution factor)
00 of Standard
TOTAL LIPIDS IN EGG 155
4.2. ESTIMATION OF TOTAL LIPIDS IN EGG
COLORIMETRIC ESTIMATION OF TOTAL LIPIDS IN TWO DIFFERENT VARIETIES OF EGGS
(Country/Farm)
(The same experiment can be performed with fowl and duck egg)
Since the farm egg and country egg are laid by two different varieties of fowl they should
differ in total lipid concentration in their yolk. To test this hypothesis, following experiment is
performed.
Aim: To determine the total lipid concentration in two different varieties of eggs by Stern and
Shapiro method.
Requirements: Stock standard lipid (700 mg% triolein or tripalmitin or 1 % of commercial
OALOA) prepared in chloroform, Lipid reagent (10% FeCI
3
in 0.1 N HCI acid), 33% HCI (1 volume
of conc. HCI-AR grade + 2 volumes of distilled water, 14% NaOH, 14% Hydroxylamine hydrochloride
prepared freshly in OW, 2: 1 chloroform methanol mixture or 3: 1 ethanol- ether mixture (60-BOOC
bOiling point). Working standard lipid- dilute 1 ml of stock standard in 20 ml of chloroform, where 1
ml of triolein or tripalmitin standard is equal to 35 mg of total lipid, if 1 % Oalda is used 500 mg or
0.5 gm of total lipid. Egg yolk from different varieties of egg.
Principle: Hydroxylamine in alkaline solution reacts with esters of fatty acids to give
hydroxamic acids, which produces red to violet colour with ferric chloride.
Procedure:
For estimating total lipids in the sample by calculation method four test tubes need to be
provided to each student.
1. Take two test tubes of 20 ml capacity and add 0.5 ml of egg yolk from two different
varieties of egg in to two separate tubes to which 9.5 ml of chloroform methanol mixture
Dr ethanol-ether mixture is added.
2. Close the mouth of the test tube with cotton plug and keep the test tubes in a water bath
maintained at 60C for 5 min. Filter the sample through Whitman No.1 filter paper
3. Take 0.1 ml of filtrate and add 0.5 ml of 14% hydroxylamine hydrochlOride, shake well
and add 0.5 ml of 14% NaOH shake again. Allow the tubes to stand at room temperature
for twenty minutes.
4. Add 0.6 ml of 33% HCI and 0.5 ml of 10% FeCI
3
to the tubes and mix well
5. Take a test tube and add 1 ml of working standard, to which add the reagents mentioned
in step 3 and 4 and allow the tubes to remain for twenty minutes.
7. To prepare a blank, take 0.1 ml of chloroform-methanol mixture or ethanol-ether mixture
and add reagents as mentioned in step 3 and 4.
B. Read the absorbance of standard and test sample at 540 nm against the blank.
Calculate the results as follows:
Absorbance of unknown X Conc. of Std.(35 mg or 509,mg)
Absorbance of standard
= mg of lipid / 0 1 ml of filtrate, Therefore for 1 ml of sample whatever the value obtained
should be multiplied by 10
= mg of lipid/ml sample of egg yolk
Results: Compare the values of lipids in the two different varieties of eggs and conclude
your experiment.
4.3.TESTING OF ADULTERATION OF MILK
Milk sold loose in the local market is sometimes adulterated with starch, urea, ammonium
sulfate or glucose to increase the thickness of the milk and sweeten it after it is adulterated with
water. Starch and urea are used to increase the specific gravity of milk so that lactometer fails to
detect the adulteration of milk with water. Following tests can be performed to detect the presence
of starch and urea in milk
DETECTION OF STARCH AS AN ADULTERANT
Since starch reacts with iodine to form a blue coloured complex, Leugol's iodine can be
used as detecting agent.
Requirements: Leugol's iodine, 5% starch solution, Pure sealed packet of milk (Sample 1),
milk adulterated with 5% starch solution (50:50 proportion) (Sample 2)
Procedure:
1. Take two test tubes and in test tube 1 take 2 to 5 ml of sample No.1 and In test tube 2,
sample No.2.
2. To each test tube add few drops of Leugol's iodine and shake the test tube If needed
add few more drops of Leugol's iodine
Results: If the milk turns blue, it indicates that the milk is adulterated with starch If the milk
remains white as it is it is unadulterated.
DETECTION OF UREA IN THE MILK AS AN ADULTERANT
Since urea is highly soluble in water and cools the water, when it IS added to milk it not only
maintains the temperature of the milk cold during transport by acting as a refrigerant and
preservative but also helps in maintaining the specific gravity of milk when adulterated With water.
To test whether the milk is adulterated with urea, following tests can be performed
Requirements: Conc. HCI, 1 % NaN0
2
, Acetate buffer pH 4.6, 2% NaOH, 2% Sodium
hypochlorite, 5% phenol, Pure milk sample (sealed packet) (Sample No.1), milk sample adulterated
with urea (1 to 2% urea solution 7525) (Sample No.2).
Procedure: De-proteinize the milk by adding 10 ml of acetate buffer pH 4.6 to 20 ml of pure
or urea mixed milk. Filter the sample to get the filtrate.
ADULTERATION OF MILK
I. TEST FOR AMMONIA RELEASE:
157
1. Take two test tubes and add 2 to 5 ml filtrate of pure milk (sample No 1) In one test tube
and in other test tube take 2 to 5 ml filtrate of sample No 2
2. To each add 1 ml of 2% NaOH and boil the contents Smell the vapour
Results: If the vapours arising from the test tube smell of ammonia, It Indicates that the
sample is adulterated with urea.
II. TEST FOR NITROGEN RELEASE
urea
1. Take two test tubes and add 2 to 5 ml of filtrate of pure milk (sample No 1) In one test
tube and in other test tube take 2 to 5 ml filtrate of sample No.2
2 To each add 1 ml of Conc HCI and 1 ml of 1% NaN0
2
Shake the contents Observe the
reaction.
Results: If the bubbles of nitrogen arise from the sample, the sample IS adulterated with
III. COLOUR TEST FOR UREA:
(A) 1. Take two test tubes and add 2 to 5 ml of filtrate of pure milk (sample No 1) In one test
urea.
tube and in other test tube take 2 to 5 ml filtrate of sample No.2.
2. To each add 1 ml of 2% NaOH followed by 0 5 ml of 2% sodium hypochlorite solution
and 0.5 ml of 5% phenol solution. Shake the contents Observe the colour reaction The
colour is stable for 12 hours and can detect urea as low as 0 1 %
Result: Blue or bluish green colour develops, which indicates the milk IS adulterated with
(8) TEST FOR UREA
To 2 ml of milk sample, add few drops of bromocresol blue (0 1 % w/v) Indicator reagent
Results: Appearance of dark blue colour is indicative of urea as an adulterant In milk
TEST FOR AMMONIUM SULFATE
The test is very similar to colour test for urea except that there is no need to obtain protein
free filtrate from milk. As in the case of urea a bluish colour forms on heating for 20 seconds The
bluish colour turns deep blue subsequently indicates the presence of ammonium sulfate
TEST FOR GLUCOSE
Requirements: Barfoed's reagent, Phosphomolybdic aCid reagent, pure milk, milk adulterated
with glucose.
Procedure:
1. Take 2 ml of milk in a test tube and add 2 ml of Barfoed's reagent and boil It for 3 to 5
minutes.
2 Cool the tubes and add 1 ml of phosphomolybdic acid reagent.
3. Observe the colour.
Results: Development of blue colour indicates the presence of glucose
IV. TEST FOR HYDROGENATED FAT:
To 2 ml of clarified butter or ghee, add equal proportion of concentrated hydrochloric acid and
a pinch of sucrose. Shake and leave the contents to stand for 5 minutes.
Results: Appearance of maroon colour in the lower acid layer indicates the presence of
hydrogenated fat as adulterant
V. TEST FOR DETERGENT:
To 2 ml of milk sample, add few drops of bromocresol purple (0.1 % w/v) indicator reagent
Results: Development of faint blue colour indicates the presence of detergent
4.4. ESTIMATION OF TOTAL FAT IN MILK
COLORIMETRIC ESTIMATION OF TOTAL FAT IN DIFFERENT VARIETIES OF MILK (Cow,
Buffalo, Sheep or Goat milk)
Milk plays a major role in human nutrition and the m ~ k produced by different farm animals IS
used for human consumption. Therefore, total fat content of the milk consumed by human beings
is of major concern for the proper health. Excessive fat from the milk adds to the body burden,
which will reflect in late age by atherosclerosis or cardiac ailments. Hence it is essential to know
the fat content of the milk produced by different farm mammal. The milk produced by different
farm mammals should contain different levels of total fat in their milk. To test this hypothesis,
following experiment is performed.
Aim: To determine the total lipid concentration in different varieties of milk by Stern and
Shapiro method.
Requirements: Stock standard lipid (700 mg% triolein or tripalmitin or 1 % of commercial
DALDA) prepared in chloroform, Lipid reagent (10% FeCI
3
in 0 1N HCI aCid), 33% HCI (1 volume
of conc. HCI-AR grade + 2 volumes of OW), 14% NaOH, 14% Hydroxylamine hydrochlonde
prepared freshly in OW, 2: 1 chloroform methanol mixture or 3: 1 ethanol- ether mixture (60-80
D
C
boiling point). Working standard lipid- dilute 1 ml of stock standard In 20 ml of chloroform, where 1
ml of triolein or tripalmitin standard is equal to 35 mg of total lipid, if 1 % Dalda is used 500 mg or
0.5 gm of total lipid. Cow and buffalo Milk samples. Sheep and goat milk if available.
Principle: Hydroxylamine in alkaline solution reacts with esters of- fatty acids to give
hydroxamic acids, which produces red to violet colour with ferric chloride.
Procedure:
For estimating total fat in the sample by calculation method four test tubes need to be provided
to each student.
1. Take two test tubes 20 ml capacity and add 1 0 ml of milk of two different vanetJes (cow
and buffalo) in to two test tubes separately to which 9 ml of chloroform methanol mixture
or ethanol-ether mixture is added.
2. Close the mouth of the test tube with cotton plug and keep the test tube in a water bath
maintained at 60C for 5 min. Filter the sample through Whatman No.1 filter paper
CASEIN FROM MILK 159
3. Take 0.1 ml of filtrate and add 0.5 ml of 14% hydroxylamine hydrochloride, shake well
and add 0.5 ml of 14% NaOH shake again. Allow the tubes to stand at room temperature
for twenty minutes.
4. Add 0.6 ml of 33% HCI and 0.5 ml of 10% FeCI
3
and mix well
5. Take a test tube and add 1 ml of working standard, to which add the reagents mentioned
in step 3 and 4 and allow the tubes to remain for twenty minutes.
6. To prepare a blank, take 0.1 ml of chloroform-methanol mixture or ethanol-ether mixture
and add reagents mentioned in step 3 and 4.
7. Read the absorbance of standard and test sample at 540 nm against the blank
Calculate the results as follows:
Absorbance of unknown x Conc. of Std.(35 mg or 500 mg)
. Absorbance of standard
= mg of lipid 10.1 ml of filtrate, therefore for 1 ml, multiply the value obtained with 10
= mg of fat! ml sample of milk
Results: Compare the values of fat in different varieties of milk and conclude your experiment.
4.5. EXTRACTION OF CASEIN FROM MILK
Casein is the main protein in the milk. It is a phosphoprotein, which exists in the four forms
as a, b, g and k casein; each having a different composition. Casein therefore, IS a heterogeneous
mixture of protein. It is nutritionally adequate protein, which contains all the essential amino acids
required for normal growth and development.
Aim: To extract casein from milk and prove its identity by qualitative test.
Requirements: Milk sample, 10% acetic acid (v/v), conc. HN0
3
, NH
4
-molybdate solution,
pH paper
Principle: The isoelectric pH of casein is 4.6. It is therefore preCipitated out from the milk by
adding either acetate buffer of pH 4.6 or dilute acetic acid till the pH reaches to 4 6 The preCipitate
of casein is treated with hot water to remove fat.
Procedure:
1. Take 25 ml of milk in a 250 ml beaker and add 25 ml of distilled water.
2. Add 10% acetic acid drop wise (very carefully) till the pH of the solution reaches 4 6
Check the pH with the pH paper. At this pH casein is completely precipitated from the
milk.
3. Filter the precipitate through Whatman No.1 filter paper.
4. Transfer the precipitate in to the beaker and add 50 ml of hot distilled water
5. Boil the precipitate of casein for 15 min in a boiling water bath, decant the liqUid portion
containing oil droplets.
6. Add some quantity of hot distilled water and decant the supernatant
7. Transfer the entire precipitate on to a rough filter paper and blot off the excess water.
Qualitative test for Casein
Newman's test
Since casein is a phosphoprotein, it gives positive test for phosphate group, which can be
performed as follows.
1. Take a pinch of precipitate of casein in a test tube and add 2 ml of con. HN0
3
, gently
heat the mixture
2. Cool the test tube and add NH
4
-molybdate solution
Results: Canary yellow precipitate indicates the presence of phosphate group Hence casein
IS present
4.6. LACTOMETER
Aim: To determine the specific gravity of milk to know its purity.
Principle: Milk is heavier than water. The specific gravity of cow milk vanes from 1 018 to
1 036 and of buffalo milk from 1 018 to 1 038 It varies with temperature, being lower at higher
temperature and vice versa, but the rate of this variation IS not uniform Specific gravity of milk
gets altered when it is adulterated with water. Therefore specifiC gravity IS determined at 28 8C
by using a lactometer as follows:
Procedure:
1. The sample of the milk is mixed thoroughly and poured into a 250 ml cylinder so that it
fills the cylinder almost to the top or any large container that should give 0 635 cm (0.25
inch) clearance around the widest part of the lactometer and to allow the lactometer to
float clear of the bottom.
2. Lower the lactometer carefully Into the milk (Do not drop It) so that it floats clear of the
Sides of the Jar and IS left In the position for about a minute ,
3 Reading IS taken at the pOint where the main surface of the milk meets the lactometer
stem and not the topmost point of the lactometer. Take the reading from the upper
meniscus
4. Record the temperature of the milk. If the temperature is above or below 15 6C,
correction factor is to be added for calculation.
5. Using lactometer reading, the total solids are ca!culated by the follOWing formula
CLR
Sp. gravity = 1000 + :
Note Standard temperature of specifiC gravity estimation IS 28 8C If the temperature of the
milk IS above thiS temperature, add 0 1 per degree to the lactometer reading and If the temperature
of the milk IS below 28 8C, then subtract 0.1 from the lactometer reading per degree ThiS IS CLR
(corrected lactometer reading).
PANIR MAKING 161
To check whether the lactometer is genuine - It is immersed in distilled water at 288C It
should float at "0" mark if it is genuine.
Take readings on the lactometer for different samples of milk and by diluting the milk with
water to compare the results of non diluted and diluted milk.
4.7. PANIR MAKING
Aim: To prepare cottage cheese from whole milk
Requirements: 1 % Citric acid solution or 1 full size lemon squeezed In 250 ml water, 1 liter
milk, vessel to boil milk, muslin cloth, wooden plank etc.
Principle: When the milk is acidified by adding citric acid or ascorbic aCid, Its pH drops to
4.6, where casein separates from milk.
Procedure:
1. Take 1 liter of milk in vessel and heat it up to 70C.
2 Add citric acid solution to the milk by continuously stirring the milk till light green coloured
water separates from the coagulated milk Leave it for five minutes
3. Collect the coagulum in the muslin cloth and drain off the whey and press In a hoop for
25 minutes or press between two wooden boards by plaCing some weight on It
4. Remove the pressed coagulum, which is the cottage cheese. Place it in chilled 5% salt
solution for 10 minutes. This facilitates removal of excess of water by osmosis as well as
it imparts taste and acts as preservative. It also enhances the firmness of the cheese.
5. Remove the cheese from the muslin cloth and cut Into pieces.
6. Store it in refrigerator at 5-1 OOC. Shelf life is 7 days.
Chemical composition of cottage cheese:
Fat (18-24%), Protein (14-18%), Lactose (4%), Minerals (0.9%), and MOisture (30-34%)
000

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