PARENCHYMA
1. It is a simple
permanent tissue.
2.
It is composed of isodiametric
parenchyma cells with thin cellulosic primary cell walls. Each has a central
vacuole. The nucleus and the cytoplasm occupy a peripheral position.
3.
An easily recognizable feature of
parenchyma tissue is the presence of intercellular space.
4.
Parenchyma cells of leaves contain
chloroplasts and form he chloroplasts and form the chlorenchyma. It exists in
two forms - palisade and spongy. It forms the mesophyll of the leaf tissue.
5.
Aerenchyma, found in some plant parts, contain
large air spaces.
COLLENCHYMA
COLLENCHYMA
1.
It is a simple permanent tissue that
rarely occurs in mono cots but is common in dicots. It is absent in the roots.
It is a living mechanical tissue and provides support to growing organs.
2.
The collenchyma cells are somewhat
elongated and have pointed oblique ends. Their cytoplasm is vacuolated and
active, and has a single peripheral nucleus.
3.
The primary cellulosic cell walls are
characteristically thickened at the comers and other places in an uneven
manner, due to a deposition of cellulose and pectin.
4.
Intercellular spaces are generally
absent.
SCLERENCHYMA
1.
Sclerenchyma is a simple permanent dead
tissue with lignified cell walls. There is no living protoplast in the
component cells.
2.
Sclerenchyma is composed of two types of
elements: fibres and sclereids.
3.
The fibres are elongated with pointed
tips: their cell walls are thick and the lumen is narrow. They appear
pentagonal or hexagonal.
4.
The sclereids, also called sclerotic
cells, have very thick lignified walls. Their lumen is narrow with pore canals
leading to the primary wall.
5.
Sclereids occur singly or in groups, in
different parts of the plants. There are different types of sclereids, such as
brachysclereids, macrosclereids, osteosclereids, astrosclereids, and
trichsclereids.
6. Sclerenchyma, whether fibres or
sclereids, functions as a dead mechanical and protective tissue.
XYLEM
1.
It is a complex permanent tissue
composed of both living and dead cells. The living componenet is the xylem
parenchyma. The dead components are the tracheary elements (tracheids,
tracheae) and xylem fibres.
2.
The tracheids are elongated with pointed
or slanting ends. Their walls are lignified.
3.
The tracheae are tubular cells with
lignified walls.
Lignification occurs in different ways in the tracheary elements - annular,
spiral, reticulate,1.
scalariform and pitted.
2.
The xylem fibres, also called wood fibres, are
elongated fibre - like cells with lignified walls.
3.
Xylem is primarily water - conducting tissue. it also
provides support.
T.S OF YOUNG DICOT ROOT
1.
Phloem is also called bast; it is also composed of
living and dead components are the phloem fibres.
2.
The sieve tubes are tubular structures with perforated
cross walls called sieve plates.
They
possess only cytoplasm but no nucleus.
3.
The companion cells associated with a sieve tube,
possesses dense cytoplasm and nucleus.
4.
The phloem parenchyma is composed of living parenchyma
cells, which are mostly elongated. They are absent in monocots.
5.
The phloem or bast fibres are sclerenchyma fibres
associated with the phloem.
6.
Phloem is a food - conducting tissue.
T.S OF YOUNG DICOT ROOT
1.
Epidermis: it is also called epiblema and
is composed of a single layer of cells, some of them bearing unicellular root
hairs.
2.
Cortex: It is composed of several layers of parenchyma
cells with intercellular spaces. The endodermis, the innermost layer of the
cortex, is composed of barrel - shaped cells with thickenings on their radial
walls forming casparian bands.
St ele: The parenchyma lying inner to the endodermis
is the pericycle It is continuous with the small central pith. The vascular
bundles are embedded in the conjunctive tissue.
4.Vascular bundles: The vascular
bundles are radial and tetrarch with four patches of xylem alternating with
four patches of phloem. The xylem is exarch. A pith is present only in young
roots.
T.S OF YOUNG DICOT STEM
T.S OF MONOCOT STEM
1.
Epidermis: It is composed of a single
layer of cells, some of them bearing unicellular root hairs.
2.
Cortex: It is multilayered and made up of parenchyma.
The endodermal cells are barrel - shaped, and have U - shaped thickenings.
Passage cells are present.
3.
Stele: The layer inner to the endodermis is peri
cycle; it is composed of thin walled cells. The conjunctive tissue lies between
the bundle of xylem and phloem.
4.
Vascular bundles: The vascular bundles are
radial and polyarch. The xylem is exarch. The pith is extensive and is
parenchymatous.
T.S OF YOUNG DICOT STEM
1.
Epidremis: It is composed of a single
layer of tabular living cells covered by a thin cuticle. In some places it
bears multicellular hairs.
2.
Cortex: It is the multilayered ground tissue. it is
composed of a) Hypodermis of a few layers of collenchyma b) A general cortex of
a few layers of parenchyma with resin canals and c) An endodermis or starch
sheath of a single layer of barrel - shaped cells.
3.
Stele: It is the tissue lying internal to the
endodermis. It consists of the pericycle, medullary rays, pith and vascular
budles.
a)
The pericycle is partly parenchymatous and partly
sclerenchyatous. The: sclerenchyma occurs as a bundle cap to each vascular
bundle, and forms the hard bast.
b)
The medullary rays are parenchymatous, occurnng In
between the vascular
bundles.
c)
The pith forms the core and is made up of parenchyma.
d)The vascular bundles are conjoint, collateral and
open. They are arranged in the form of a broken ring. The xylem is endarch. The
cambium lies in between the xylem and phloem.
T.S OF MONOCOT STEM
1.
Epidermis: it is a uniseriate layer
covered by a thick cuticle; hairs are absent.
2.
Ground tissue: It is not differentiated into
endodermis, pericycle and pith. Just inner to the epidermis is a hypodermis
made up of two or three layers of sclerenchyma fibres. The rest of it is made
of thin walled parenchyma cells.
3.
Vascular bundles: There are numerous bundles,
scattered irregularly in the ground tissue. each bundle has a sclerenchymatous
bundle sheath. The bundles are collateral and closed. The xylem consists of
four large vessels arranged in the form of a Y. the phloem consists of sieve
tubes and companion cells. There is no phloem parenchyma.
LEAF
T.S OF MONOCOT LEAF: AN ISOBILATERAL LEAF
HANGING DROP TECHNIQUE
LEAF
1.
Upper and lower epidermis: Both are composed
of a single layer of cells covered by cuticle. Both bear numerous multicellular
hairs or trichomes. The epidermal cells do not contain chloroplasts. The lower
epidermis has numerous stomata, each with a pair of guard cells, opening into a
large respiratory cavity.
2.
Mesophyll: It is the groung tissue of the
leaf divisible into an upper palisade parenchyma and a lower spongy parenchyma.
The former consists of elonhgated cells, closely packed with very narrow
intercellular spaces. The latter consists of spherical or irregular cells,
loosely arranged, and with numerous large intercellular spaces.
3.
Vascular bundles: These are represented by the
midrib and the vems, Each is conjoint, collateral and closed. Each has a bundle sheath of
parenchyma. Xylem lies towards the lower. In the midrib region, just inner to
the epidermis, there is collenchyma tissue, forming bundle sheath extensions.
There may be a few patches of sclerenchyma associated with xylem and phloem.
T.S OF MONOCOT LEAF: AN ISOBILATERAL LEAF
L Upper and lower epidermis: Both are composed
of single layer of cells covered by cuticle. There are no chloroplasts, but
both layers possess stomata. The upper epidermis bears bulliform cells.
2.
Mesophyll: It is not differentiated into
palisade and spongy layers. It is uniformly spongy ( or palisade -like
depending on the plant).
3.
Vascular bundle: There is a bundle sheath of
parenchyma, the cells containing plastids and starch grains. Patches of
sclerenchyma form bundle sheath extensions in the midrib region. Xylem lies
towards the upper side and phloem towards the lower side, in a vascular bundle.
HANGING DROP TECHNIQUE
Aim:
To
observe motility of bacteria.
Principle:
Due
to surface tension of the medium microorganisms are moving towards the surface
of the hanging drop.
Requirements:
·
12 - Hour - old broth culture, Hanging drop (cavity
)slide, Coverslips, Vaseline, Matchsticks
AIR SAMPLING OF MICRO FLORA
Procedure:
1.
Clean and name a hanging - drop slide and place it on
the table with the depression uppermost.
2.
Spread a little Vaseline or petroleum jelly around the
cavity of the slide.
3.
Clean a cover slip an apply Vaseline on each of the
four comers oft~e covrslip, using
a
match stick.
4.
Place the cover slip on a clean paper with the
Vaseline side up.
5.
Transfer a loopful of culture in the centre of the
coverslip.
6.
Place the depression slide on to the coverslip, with
the cavity facing down so that the depression covers the suspension.
7.
Press the slide gently to form a seal between the
coverslip and the slide.
8.
Lift the preparation and quickly tum the hanging drop
preparation cover slip up so that the culture drop is suspended.
9.
Examine the preparation under low - power objective
with reduced light.
10.
Switch to the high - power objective and examine the preparation again. Observations:
Results:
True
motility will be shown by bacteria that move swiftly across the microscope.
AIR SAMPLING OF MICRO FLORA
Aim: To cultivate air
microflora on agar medium.
Principle: Microorganisms does not grow
in air. Therefore it lacks nutrient needed for metabolism and growth. However
spores are carried in air &
vegetative
cells can be carried on dust particles and water droplets in air. The type and
number of air - borne microorganismsvary tremendouslyin different environments.
Large number of many types of microbes are present in indoor air where humans
are crowded together and building ventilation is poor.
Among the organisms found in air mold spore are
numerous and the predominant genus is Cladosporium. Bacteria commonly
found in air include both aerobic spore formers such as Bacillus subtilis & non - spore formers such as Micrococcus and Sarcina. Protozoa, yeasts & viruses have also been isolated from air. While
coughing, sneezing or even talking humans can expel pathogen along with water
droplets. Requirements: Potato Dextrose Agar media, petriplates.
Procedure: Petriplates containing agar
media are exposed to air for 25 seconds and immediately the lid is closed. The
plates are observed evey day for the growth of microbes.
OBSERVATION OF YEAST CELLS IN TODDY
AIM: To observe Rhizobium in root nodules of Mimosa pudica using Gram staining technique.
OBSERVATION OF YEAST CELLS IN TODDY
Aim: To observe
yeast cells in toddy.
Principle: Yeast cells are commonly employed in many
food industries, such as bread, wine, alcohol & invertase industries. They carry out alcoholic
fermentation in which they convert simple sugars into alcohol releasing carbon
- di - oxide gas. Since toddy is a product of alcoholic fermentation; it
contains large number of yeast cells.
Requirements:
•
Toddy, Cotton blue stain, slides, Cover slip.
Procedure:
1.
A drop of toddy is taken on a clean slide.
2.
a drop of lactophenol is added and mixed well and
stained for 2 minutes.
3.
Coverslip is placed on the slide . slide is observed
under high power of the
microscope..
Observation and Significance:
1.
Yeasts are used in baking industry. The bread loaves
become spongy owing to carbon
- di
- oxide produced during fermentation of the dough.
2.
The brewing industry makes use of yeasts in the
production of alcohols, wines and
beer
3.
When yeast cells are compressed and used in the form
of tablets they become rich
source
of vitamins.
OBSERVATION OF RHIZOBIUM IN ROOT NODULES OF MIMOSA PUDICA |
AIM: To observe Rhizobium in root nodules of Mimosa pudica using Gram staining technique.
Rhizobium
is
a gram - negative rods. They infect and live in symbiotic association
with
only the leguminous plants, forming nodules in them and fix atmospheric
nitrogen.
The Gram stain, a differential
stain was developed by DL Hans Christian Gram, a Danish physician, in 1884.
Gram staining is very useful for identifying and classifying bacteria into two
major groups: The gram - positive and gram - negative. In this process,the fixed bacterial smear is
subjected to four different reagents. Crystal Violet (primary stain),
Iodine solution (mordant). alcohol i, decolourizing agent) and safranin (counter stain).
The bacteria which retain the
primary stain (appear dark blue or violet) are called gram - positive, whereas
those that lose the crystal violet and counter stained by safranin (appear red)
are referred to as gram - negative.
Principle: The differences in staining responses to
the gram stain can be related to chemical and physical differences in their
cell walls. The gram - negative bacterial cell wall is thin, complex,
multilayered structure and contains relatively a high lipid contents, in
addition to protein and mucopeptides. The higher amount of lipid is readily
dissolved by alcohol, resulting in the formation of large pores in the cell
wall which do not close appreciably on dehydration of cell wall proteins, thus facilitating
the
leakage of crystal violet - iodine (CV
- I) complex and resulting in the decolorization of the bacterium which later
takes counter stain and appears red. The gram - positive cell walls are thick
and chemically simple, composed mainly of protein and cross - linked
mucopeptides. When treated with alcohol, it causes dehydration and closure of
cell wall pores, therby not allowing the loss of (CV - I) complex and remains
purple.
Requirements:
·
Root nodules, 0.1
HgCb, 70 ethyl alcohol, Crystal violet, Gram's iodine solution, 95 ethyl
alcohol, Safranin, Distilled water, Glass slides, Blottting paper, Spirit lamp,
Microscope
Procedure:
1.
Uproot roots of leguminous plants
2.
Wash the root system in running tap water to remove
adhering soil particles.
3.
Select healthy pink, unbroken and firm root nodules and
wash in water.
4.
Immerse the nodules in 0.1 HgCbfor 5 minutes to surface sterilize these.
5.
Repeatedly wash the nodules in sterile water for 3 - 4
times to get rid off the
sterilizing
agent.
6.
Place the nodules in 70 ethyl alcohol for 3 minutes.
7.
Repeatedly wash the nodule in sterile water.
8.
Crush a nodule with 1 ml of water with a sterile glass
rod.
9.
Make thin smear of bacterial sample.
10. Let the smear air
dry.
11. Heat fix the smear.
12. Cover the smear
with crystal violet for 30 seconds.
13. Wash the slide with
distilled water.
14. Cover the smear
with Gram's iodine solution for 60 seconds.
15. Wash off the
solution with 95 percent ethyl alcohol. add ethyl alcohol drop by drop
until
no more colour flows from the smear.
16. Wash the slide with
distilled water.
17. Apply safranin to
smear for 30 seconds (counter - staining).
18.Wash the slide with distilled
water and air dry. 19. Observe the slide under the microscope.
Observation:
Pink coloured rod shaped Rhizobium is seen.
Significance: Rhizobium leguminosarum is a symbiotic
nitrogen fixing bacteria that lives inside the swollen portions of the roots,
called root nodules, of leguminous plants. When the leguminous plants die or
decay, the fixed nitrogen in the nodules enriches the soil and
increases
the soil fertility.
OBSERVATION OF BACTERIA IN CURDS USING GRAMS STAINING
TECHNIQUE
Aim:
To observe lactobacillus present in curd sample using Gram staining technique.
Principle: Lactobacillus is a kind of bacteria which can convert a sugar into
an alcohol and then into an acid by means of anaerobic respiration. Milk
contains a sugar called lactose, a disaccharide made by glycosidic bonding
between glucose and galactose. When milk is heated to a temperature of 30 - 40
degrees centigrade and a small amount of old curd is added to it, the
lactobacillus in the curd sample gets activated and multiplies. These convert
lactose into lactic acid, which imparts sour taste to the curd.
The Gram stain, a differential
stain was developed by Dr. Hans Christian Gram, a Danish physician, in 1884.
Gram staining is very useful for identifying and classifying bacteria into two
major groups: The gram - positive and gram - negative.
The differences in staining
responses to the gram stain can be related to chemical and physical differences
in their cell walls. The gram - negative bacterial cell wall is thin, complex,
multilayered structure and contains relatively a high lipid contents, in
addition
to
protein and mucopeptides. The higher amount of lipid is readily dissolved by
alcohol, resulting in the formation of large pores in the cell wall which do
not close appreciably on dehydration of cell wall proteins, thus facilitating
the leakage of crystal violet - iodine (CV - I) complex and resulting in the
decolorization of the bacterium which later takes counter stain and appears
red. The gram - positive cell walls are thick and chemically simple, composed
mainly of protein and cross - linked mucopeptides. When treated with alcohol,
it causes dehydration and closure of cell wall pores, therby not allowing the
loss of (CV - I) complex and remains purple.
Requirements:
·
Curd sample (Bacterial sample), Crystal violet, Gram's
iodine solution, 95 ethyl
alcohol,
Safranin, Distilled water, Glass slides, Blottting paper, Spirit lamp,
Microscope
Procedure:
1.
Take the curd sample and filter it.
2.
Make thin smear of bacterial sample.
3.
Let the smear air dry.
4.
Heat fix the smear.
5.
Cover the smear with crystal violet for 30 seconds.
6.
Wash the slide with distilled water.
7.
Cover the smear with Gram's iodine solution for 60 seconds.
Aim: To study the principle, construction, working and application of various instruments used in Microbiology and biotechnology lab.
HOT AIR OVEN
8.
Wash off the solution with 95 percent ethyl alcohol.
Add ethyl alcohol drop by drop until no more colour flows from the smear.
9.
Wash the slide with distilled water.
10. Apply safranin to
smear for 30 seconds (counter - staining).
11.Wash the slide with distilled
water and air dry. 12. Observe the slide under the microscope.
Observation:
Upon observation, the" Cocci appeared purple in colour, thus it is gram -
positive.
The bacillus appeared pink in colour, thus it is gram - negative.
Significance:
Lactobacillus species are used industrially for the production of
yogurt,
cheese,
sauerkraut, beer and wine.
Aim: To study the principle, construction, working and application of various instruments used in Microbiology and biotechnology lab.
AUTOCLAVE
Principle: The autoclave is an apparatus
in which saturated steam under pressure effects sterilization. The pressure
increases the boiling point of water, there by increasing the temperature to
which water can be heated. Cells are destroyed by the higher temperature and
not by the pressure. Most of the organisms are killed at 121°C (i.e. 15 Ib/in2)
in 15 minutes.
Construction
and working: An autoclave is a double - walled cylindrical metallic
vessel, made of thick stainless steel or copper, one
end of which is open to receive the material to be sterilized. Autoclave lid is
provided with pressure gauge for noting the pressure, steam cock (exhaust
valve) for air exhaustion of the chamber. Autoclave is provided with controls
for adjusting the pressure and temperature and safety valve to avoid
explosions. The articles to be sterilized are kept loosely in the basket,
provided with holes all around for the free circulation of the steam.
Applications: Autoclave is the most
efficient and common instrument used for sterilizing solid and liquid media for
microbial cultures, heat stable liquids - usually the common media ingredients,
heat resistant instruments and equipment, glassware and rubber products.
Scalpels and other sharp metal instruments are damaged by constant flaming and
are usually sterilized by autoclaving.
HOT AIR OVEN
Principle:
An
oven is based on the principle where sterilization is accomplished by dry heat
or hot air.
Construction
: An
oven consists of an insulated cabinet which is held at constant temperature by
means of an electric heating mechanism and thermostat. It is fitted with a fan
to keep the hot air circulating at a constant temperature and thermometer for
recording the temperature of the oven. For proper circulation of the hot air
the shelves are perforated. For normal sterilization work, the oven should be
operated at 160°C. Applications: Hot air oven is used for
sterilizing the glassware like petridishes, test tubes, pipettes and metal
instruments that can tolerate prolonged heat exposure, oils, powders, waxes
etc.
INCUBATOR
Principle:
incubators are designed to promote the growth of microorganisms by maintaining
a constant temperature within a narrow range.
Construction
: Incubator
consists of an insulated cabinet fitted with a heating element at the bottom.
The temperature of the incubator is maintained at the desired level by an
automatic device called thermostat which cuts the connection off when the
temperature reaches the point for which the thermostat is set, and turns it on
again when the temperature falls slightly below that point. The incubator is
properly ventilated by the perforated shelves. They are provided with double
doors, the inner one made of glass so that the content of the incubator may be
viewed without admitting outside air. Applications: An incubator is
used for incubation (i.e. culturing of microorganisms)
pH
METER
Aim:
To
determine pH of solution using pH meter.
Principle: The pH value or hydrogen ion concentration
is a measure of the acidity or alkalinity of a solution. It is expressed as pH = loglO lI(H+) where
(H+) is hydrogen ion concentration of a solution in moles per litre.
Electric potential developed
between two suitable electrodes has a direct relation to the hydrogen ion
concentration of solution. This property is made use of in measuring the pH
value of the solution using pH meter.
Construction
and working: pH meter consists of glass electrode and reference electrode. The glass
electrode consists of an internal sealed tube with a metallic tip and an
external tube that contains a standard solution. A pH sensitive glass bulb
forms the immersion tip of the electrode. The potential of the glass electrode
is proportional to the pH of the solution in which it is immersed. In addition
to the glass electrode, a pH meter consists of another electrode, the reference
electrode. The reference electrode consists of a metallic internal element -
typically of mercury - mercurous chloride (calomel) or silver - silver chloride
- immersed in an electrolyte, usually a saturated solution of potassium
chloride. The function of electrolyte is to form a conductive salt bridge
between the internal metallic element and the sample solution, a liquid
junction is present in the tip of the outer body of the reference electrode.
This junction consists of an
The pH meter is equipped with
a temperature - compensation circuit for introducing a known potential to
balance out the potential caused by different sample temperatures. The
instrument is also provided which is used to balance the circuit to indicate
the correct pH of the standard used as a reference to measure the pH of a sample
solution.
It should be ensured that the
reference electrode be nearly full with saturated KCl solution. Soak the glass
electrode for several hours before use. The glass electrode terminal is wiped
with clean absorbent tissue or cloth. Glass electrode terminals and reference
electrode terminals are fitted to the pH meter after fixing to the clamp and
electrodes diiped in the distilled water.
After switching the knob 'OB'
the instrument is allowed to warm up for 10 minutes. The meter pointer should
read exactly 7 pH (or 0 mY). Otherwise the 'zero' knob is adjusted to read 7
pH.
The buffer tablet is dissolved
in 100 C.c of distilled water (pH tablet for acidic solution and pH 9 tablet
for basic solution). The standard buffer of known pH is taken in a beaker and
the electrodes are immersed and the temperature compensate is set to room
temperature. The selecter knob is turned to the proper range using 'set buffer'
control, the meter pointer is set to read the pH of the buffer.
The selector knob is kept in
'zero' position. The electrodes are rinsed in distilled water and they are
wiped with absorbent tissue. now the test sample is taken in a clean, beaker
and the electrodes are immersed to this. The selector knob is turned to the
proper range. Noe the meter indicates the pH value of the solution under test.
Applications: pH meter is used to determine
the pH(acidity & alkalinity) of
solutions of unknown pH as well as for setting of pH of various media for
cultivation and testing biochemical activities of microorganisms.
(Continued in next tab-III B.Sc. PRACTICAL NOTES Part 2)
(Continued in next tab-III B.Sc. PRACTICAL NOTES Part 2)