M.

G SCIENCE INSTITUTE(SF)

(AUTONOMOUS)

B.S BIOTECHNOLOGY

PRACTICAL MANUAL

SEMESTER II

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 INDEX

Sr.no. Name of the practical Pg. no.

1 Study of permanent slide of various eukaryotic and prokaryotic cell

2 Observation of algal cells from natural source

3 Simple staining of yeast from various natural source

4 Study of bacteria using differential staining method(Gram staining)

5 Diffierential staining of nucleus from human WBCs

6 Study of different phases of mitosis using onion root tip

7 Observation of cell motility using hanging drop method

8 Microscopic observation of wet mount preparation from fungi

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PRACTICAL 1

AIM : Study of permanent slide of various eukaryotic and prokaryotic cell

Eukaryotic cell

Paramecium(protozoa)

Paramecium is unicellular,shoe sole shaped , ciliated, heterotrophic protozoan found in scum and
in fresh water. They feed on bacteria, algae and yeast. They are nonpathogenic. They are important
as study in genetics research.

Example: Paramecium busaria

Amoeba(protozoa)

Amoeba is a unicellular organism that has the ability to change its shape. They are usually found
in water bodies such as ponds, lakes and slow-moving rivers. Sometimes, these unicellular
organisms can also make their way inside the human body and cause various illnesses. Example:
Entamoebahistolyticum

Plasmodium(protozoa)

Plasmodium is a genus of single-celled parasitic organisms that are responsible for causing
malaria, one of the most serious infectious diseases affecting humans worldwide. These parasites
are transmitted primarily by Anopheles mosquitoes. There are several species of Plasmodium that
infect humans.

Example: Plasmodium falciparum, P. vivex, P. malariae

Spirogayara(algae)

Spirogyra is a genus of freshwater green algae that is often studied under a microscope, and it has
a distinctive appearance due to its spiral chloroplasts. Spirogyra forms long, thread-like structures
called filaments. These filaments can be unbranched and can vary in length. The nucleus is usually
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visible as a small, central, spherical structure within the cells. Spirogyra is a fascinating organism
that helps illustrate the diversity of plant life in aquatic ecosystems. It plays important role in
maintaining lack ecology.

Example : Spirogyra porticalis

Euglena(algae)

Euglena is unicellular microorganisms, that have a flexible body. They possess the characteristic
features of plants and animals. Euglena has plastids and performs photosynthesis in light, but
moves around in search of food using its flagellum at night. They are found in freshwater,
saltwater, marshes and also in moist soil. Euglena has an elongated cell measuring 15-500
micrometres

Normal cell and cancerous cell

• Normal Red Blood Cells (RBCs):

• Shape: Biconcave disc-shaped.

• Size: Uniform in size, about 7–8 micrometers in diameter.
• Color: Pale center (due to the lack of a nucleus and hemoglobin), reddish-pink overall.
• Arrangement: RBCs are usually arranged in a random pattern, but they may overlap
slightly in some areas.
• Nucleus: Absent, since mature RBCs do not contain a nucleus.
• Normal blood has well-organized, mature blood cells performing their specific functions.

• Abnormal Red Blood Cells:

• In certain forms of leukemia or blood cancers (like myelofibrosis), RBCs can appear
abnormal, such as with:
o Anisocytosis (unequal sizes).
o Poikilocytosis (abnormally shaped cells, like sickle-shaped or teardrop cells).
o Hypochromia (pale RBCs due to insufficient hemoglobin).

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• Cancerous blood cells (e.g., in leukemia) often show immature cells, abnormal shapes,
irregular nuclear features, and a disrupted overall appearance. This leads to impaired
function and can result in overcrowding of abnormal cells, disrupting normal blood cell
production.

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 Paramecium Ameoba

Spirogyra Euglena

Normal Red Blood cell Cancerous Red Blood cell

Plasmodium

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Prokaryotic cell

Bacillus:

Bacilli are rod shaped , spore forming Gram positive bacteria. They are relatively big in size (0.5
to 2 micrometers in width and 2 to 5 micrometers in length), occur singly and in short and long
chains. Most Bacillus species are Gram-positive bacteria, meaning they have a thick peptidoglycan
layer in their cell walls that retains the crystal violet dye during Gram staining. The genus Bacillus
encompasses a broad diversity of bacteria, ranging from beneficial species used in industry and
biotechnology to pathogenic species responsible for diseases like anthrax and food poisoning.

Example: bacillus subtilis, Bacillus anthracis , Bacillus megaterium

Short rods:

Short rods refer to bacteria that have a rod-like or cylindrical shape, but are relatively short in
length compared to other rod-shaped bacteria. short rods can be Gram-positive or Gram-negative,
depending on their cell wall structure. Short rods typically range from 1 to 3 micrometers in length,
which is shorter than the usual rod-shaped bacteria, which may be longer. Short rods are an
important group of bacteria that vary in terms of their biological activity, pathogenic potential, and
environmental niches. Some are harmless or even beneficial, while others are pathogenic and cause
diseases in humans and animals.

Example: Escherichia coli,Salmonella

Cocci:

Cocci are gram positive, spherical bacteria. They occur singly, in pairs and in irregular clusters,
resembling bunch of grapes. Some of them from chains. Neisseria is Gram negative cocci. Many
cocci are part of the normal flora of the body and are harmless, others are responsible for serious
infections.

Examples: Staphylococcus aureus, Streptococcus pneumonia

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Filamentous bacteria / actinomycetes:

Actinomycetes are filamentous bacteria. The long filaments may break into smallest fragments
having coccoid or rod like shapes. They are gram positive in nature. They may be pathogenic or
nonpathogenic. Many of them are responsible for increasing soil fertility. Some of the
actinomycetes are antibiotic producers.

Example: Streptomyces gresius

Prokaryotic cell

Bacillus short rod

Coccie Actinomycetes

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PRACTICAL 2

AIM: Observation of algal cells from natural source

In microbiology and botany, studying algae involves examining their morphology, structure, and
types, often using a microscope. This practical is aimed at understanding the characteristics of
algae, such as their cellular structure, types of reproduction, and classification.

Principle:

The study of algae in practicals generally involves examining them under a microscope to identify
different types, structures (unicellular, multicellular, filamentous), and reproductive organs (sexual
or asexual). Algae are important primary producers in ecosystems and their structure varies from
simple unicellular organisms to complex multicellular forms. Microscopic examination is essential
to identify their characteristic features, which include chlorophyll pigments, storage bodies (like
starch), and cellular arrangement.

Materials:

1. Microscope (with 10x, 40x, and 100x objective lenses)

2. Microscope slides and coverslips
3. Water sample containing algae (e.g., pond water, aquarium water, or prepared algal cultures)
4. Inoculating loop (if needed to transfer algae cultures)
5. Dropper or pipette for transferring samples
6. Distilled water (to dilute if necessary)
7. Staining reagents (optional, like iodine or methylene blue, for enhancing visibility)
8. Concave slide (optional, for hanging drop technique to observe motility)

Method:

1. Collection of Algal Sample:

• Collect the algae from a natural water body (pond, river, or aquarium) or use a prepared culture. If
you are using a natural sample, you can collect a small amount of pond water.

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 • You may observe algae from different habitats like freshwater, marine environments, or even from
moist terrestrial surfaces.

2. Preparation of the Slide:

• If the sample is from a water body, use a dropper to place a small drop of the water containing
algae on a clean microscope slide.
• Alternatively, if you have a prepared algal culture, transfer a small portion using an inoculating
loop to the slide.

3. Adding a Coverslip:

• Place a coverslip gently on top of the drop to avoid trapping air bubbles. Be careful not to squish
the sample.

4. Observation Under a Microscope:

• Start by examining the sample under low magnification (10x objective lens) to locate the algae.
• Switch to higher magnifications (40x and 100x) to examine the structure and detailed features of
the algae.
• Look for key features such as:
o Pigments (green, brown, or red pigments)
o Shape (unicellular, multicellular, filamentous)
o Cell wall (made of cellulose, silica, or other compounds)
o Reproductive structures (if visible)
• If required, staining can be done to enhance visibility of certain structures like chloroplasts.

5. Optional: Hanging Drop Method (for motile algae):

• If you suspect the presence of motile algae (e.g., Chlamydomonas), you can use the hanging drop
technique.
• Place a small drop of the algal culture on the center of a coverslip, then invert it and place it over a
concave slide.
• Observe the drop under the microscope to see if any algae are moving.

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6. Record Your Observations:

• Draw the various types of algae seen, labeling their structures (e.g., cell wall, chloroplasts, and
reproductive organs).
• Note the size, shape, and any other distinctive features of the algae under study.

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PRACTICAL 3

AIM: Simple staining of yeast from various natural source

INTRODUCION

One type of staining procedure that can be used is the simple stain, in which only one stain is
used, and all types of bacteria appear as the color of that stain when viewed under the microscope.
Some stains commonly used for simple staining include crystal violet, safranin, and methylene
blue. Simple stains can be used to determine a bacterial species’ morphology and arrangement, but
they do not give any additional information.

PRINCIPLE

In simple staining, the bacterial smear is stained with a single reagent, which produces a distinctive
contrast between the organism and its background. Its principle is based on producing a marked
contrast between the organism and its surroundings by using basic stain. A basic dye consists of a
positive chromophore, which strongly attracts the negative cell components and charged molecules
like nucleic acids and proteins, Basic stains with a positively charged chromogen are preferred
because bacterial nucleic acids and certain cell wall components carry a negative charge that
strongly attracts and binds to the cationic chromogen Thus, a simple staining technique result in a
colored bacterial cell against a colorless background. The purpose of simple staining is to elucidate
the morphology and arrangement of bacterial cells. The most commonly used basic stains are
methylene blue, crystal violet, and carbol fuchsin.

MATERIALS

Clean glass slide, alkaline dye solution(methylene blue),curd sample , Grapes , Banana, Apple,

METHODS

The process of positive simple staining is accomplished in three steps.

• Smear preparation
• Fixation of smear
• Use of stain

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Smear preparation

o Wash slide clean using detergent to remove grease and other impurities. Air dry it.
o Pass the slides through the burner flames 5-7 times and cool it. Mark the area to be
smeared with a glass marker on the opposite side of the glass slide, to the one where
the smear is to be prepared.
o Transfer a loop full of suspension aseptically to the slide with the help of of a
sterilized and cooled nicrome wire. Spread it uniformly with the help of wire loop.
Air dry this smear.

Fixation of smear

• Heat fix the smear by exposing the opposite slide of smeared area to the burner
flame for 5-7times and then cooled it.

Use of stain

• Place the fixed smear slide on a staining rack and flood the smear with basic
staining solution (methylene blue) and allow it to react for 1 minute.
• Remove the stain from the slide gently using flow of water thoroughly.
• Air dry the slide or blot dry it. place a drop of paraffin oil on the smear.
• Observe the slide under oil immersion objectives lens and record the results.

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PRACTICAL 4

AIM: Study of bacteria using differential staining method(Gram staining)

Introduction

Gram Staining is the common, important, and most used differential staining techniques in microbiology,
which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the
bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and
differentiations of microorganisms.

Principle

The Eubacteria possess a distinct cell wall. The basic composition of the bacterial call wall is constituted
by a complex polymer known as murein or peptidoglycan . In the cell wall of Gram positive bacteria, it is
present as a thick and uniform layer. Gram negative bacteria possess little of peptidoglycan. In addition,
their cell wall contains a complex outer membrane mainly comprised of lipids, lipopolysaccharides and
proteins.

Overall electrical charge of the bacterial cell is negative . When crystal violet (primary stain) is applied ,
all types of bacteria get stained with it. Gram’s Iodine serves as an oxidizing mordant and it forms complex
with the stain and cell wall components. Decolorizing like acetone or Alcohol dissolvethe crystal violet –
iodine complex from cell wall Gram negative bacteria but not from that of Gram positive bacteria. This
happenes because the lipids and the lipopolysaccharides of the cell wall in gram negative cells dissolves
readily in organic solvents like Acetone or Alcohol. This allows the decoloizers to extract the primary stain.
On the other hand , Gram positive retain primary stain as they have thick and uniform layer peptidoglycan
layer in the cell wall structure. Therefore,Decolorizers can not remove the primary stain from them . Thus,
such cells do not takeup secondry stain, which is Safranin. Gram negative bacteria which are already
decolorized, take up secondary stain. Gram positive bacteria appear violet in color and Gram negative
bacteria are observed pink in color.

Requirements

Young culture (Gram negative and Gram positive bacteria),crystal violet stain, Gram’s iodine , ethanol or
acetone, safranin stain

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Method

• Prepare the smear from the given bacterial suspension on clean marked glass slide.
• Air dry the smear.
• Heat fix it.
• Pour crystal violet stain(primary stain) on the smear and allow it to act for one to two minutes.
• Drain off the stain and wash the smear gently with water.
• Flood the smear with Gram’s iodine (a mordant) and allow it to act for nearly thirty seconds.
• Drain off Gram’s iodine and wash the smear with water.
• Decolorize the smear with Acetone/Alcohol till violet colorstops oozing out from the smear.
• Allow the decolorizer to evaporate, wash with water , air dry and applying safranin(secondary
stain). Allow it to act for about five minutes.
• Wash the slide with water and dry it. Place a drop of paraffin oil on the smear and observe under
oil immersion lens.
• Record the results.

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Practical 5

Aim: Diffierential staining of nucleus from human WBCs

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Practical 6

Aim: Study of different phases of mitosis using onion root tip

Introduction

For entities to mature, grow, maintain tissues, repair and synthesize new cells, cell division is
required. Cell division is of two types:

• Mitosis

• Meiosis

Mitosis

In mitosis, the nucleus of the Eukaryotic cells divides into two, subsequently resulting in the
splitting of the parent cells into two daughter cells. Hence, every cell division involves two chief
stages:

• Cytokinesis – Cytoplasm division

• Karyokinesis – Nucleus division

Stages Of Mitosis

The various stages of mitosis are:

1. Prophase

• The process of mitosis is initiated at this stage wherein coiling and thickening of the
chromosomes occurs

• Shrinking and hence the disappearance of the nucleolus and nuclear membrane takes place

• The stage reaches its final state when a cluster of fibres organizes to form the spindle fibres

2. Metaphase

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 • Chromosomes turn thick in this phase. The two chromatids from each of the chromosomes
appear distinct

• Each of the chromosomes is fastened to the spindle fibres located on its controller

• Chromosomes align at the centreline of the cell

3. Anaphase

• Each of the chromatid pair detaches from the centromere and approaches the other end of
the cell through the spindle fibre

• At this stage, compressing of the cell membrane at the centre takes place

4. Telophase

• Chromatids have reached the other end of the cell

• The disappearance of the spindles

• Chromatin fibres are formed as a result of uncoiling of daughter chromosomes

• The appearance of two daughter nuclei at the opposing ends due to the reformation of the
nucleolus and nuclear membrane

• At this phase, splitting of the cell or cytokinesis may also occur

Post mitosis, the next stage is referred to as interphase, which is part of the cell cycle that is non-
dividing and between two consecutive cell divisions. A cell spends most of its life in the interphase.
It comprises the G1, S and G2 stages.

Materials Required

Compound microscope, Acetocarmine stain , Water ,Burner, N/10 Hydrochloric acid, Filter
paper, Coverslip, Aceto alcohol (Glacial acetic acid and Ethanol in the ratio 1:3),Glass Slide,
Onion root peel, Forceps, Blade, Watch glass, Dropper, Needle, Vial

Method

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 • Place an onion on a tile

• With the help of a sharp blade, carefully snip the dry roots of the onion

• Place the bulbs in a beaker containing water to grow the root tips

• It may take around 4 to 6 days for the new roots to grow and appear

• Trim around 3 cm of the newly grown roots and place them in a watch glass

• With the help of forceps, shift it to a vial holding freshly prepared aceto-alcohol i.e., a
mixture of glacial acetic acid and ethanol in the ratio 1:3

• Allow the root tips to remain in the vial for one complete day

• With the help of forceps, pick one root and set in on a new glass slide

• With the help of a dropper, allow one drop of N/10 HCl to come in contact with the tip of
the root. Additionally, add around 2 to 3 drops of the acetocarmine stain

• Heat it lightly on the burner in such a way that the stain does not dry up

• Excessive stain can be carefully treated using filter paper

• The more stained part of the root tip can be trimmed with the help of a blade.

• Discard the lesser stained part while retaining the more stained section

• Add a droplet of water to it

• With the help of a needle, a coverslip can be mounted on it

• Gently tap the coverslip with an unsharpened end of a needle in order for the meristematic
tissue of the root tip present under the coverslip to be squashed properly and to be
straightened out as a fine cell layer

• The onion root tip cells’ slide is now prepared and ready to be examined for different stages
of mitosis

• Observe and study mitosis by placing the slide under the compound microscope. Focus as
desired to obtain a distinct and clear image

Practical 7

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Observation of cell motility using hanging drop method

Inroduction

The Hanging Drop Method is a laboratory technique commonly used to observe microbial motility,
bacterial morphology, and sometimes microbial interactions in a three-dimensional environment. This
method provides a simple, yet effective way to examine live organisms under a microscope in a drop of
liquid suspended from a cover slip.

Requirement

Bacterial culture, cavity slide , cover slips and ‘vaseline’ or grease

Method

• Withdraw the suspension of bacteria from the surface with wireloop and place 1 drop in the center
of clean cover slip.
• Apply grease on the four corners of the cavity slide.
• Place the slide slide on the cover slip and invert rapidly so that the drop hangs on the cover slip.
• First examine under low power and focus the edge of the drop.
• Turn to high power and examine.
• Observe the presence of protozoa as well as bacteria in high power.

PRACTICAL 8

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Aim: Microscopic observation of wet mount preparation from fungi

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