BIOLOGY UNIT -2
1. Condenser Lens:
○ Focuses light before it hits the specimen.
○ Located near the base of the microscope.
○ May have a built-in light or a concave mirror for external light sources.
2. Eyepiece Lens:
○ The lens you look through at the top of the microscope.
○ Focuses light after it has passed through the specimen.
3. Objective Lenses:
○ Located lower down on the microscope.
○ Attached to a rotating nosepiece for selecting different magnifications.
○ Typically includes at least two lenses:
■ Low-Power Objective Lens: Provides less magnification.
■ High-Power Objective Lens: Provides greater magnification.
4. Focusing Mechanism:
○ Coarse Focus Screw: Moves the stage or objective lenses a larger
distance for initial focusing.
○ Fine Focus Screw: Moves the stage or objective lenses a smaller
distance for precise focusing.
5. Stage:
○ Platform where the specimen is placed.
○ Equipped with clips to hold the specimen in place.
○ Specimens must be thin to allow light to pass through.
6. Glass Slides:
○ Used to support thin specimens.
○ Light must pass through both the specimen and slide.
Types of Preparations:
1. Temporary Preparation:
○ Tissue is placed on a glass slide with a water-based liquid to prevent
drying.
○ Covered with an extremely thin glass coverslip.
2. Permanent Preparation:
○ Water is removed from the tissue and replaced by a firmer substance.
○ Coverslip is secured with resin.
Staining:
● Purpose: Cells are typically translucent, so stains are used to color parts of
the cell.
● Function: Helps identify and differentiate cell structures by reacting with cell
components.
Viewing Cells with a Compound Light Microscope
● Plant Cells: Visible structures include cell walls and chloroplasts.
● Animal (Human) Cells: Visible structures include the nucleus and granules.
● Limitations:
○ Cell membranes are not visible.
○ Limited detail within the cytoplasm.
Practical Considerations
● Core Practical: Includes preparing and viewing cell specimens to understand
cell structures and functions.
● Safety: Wear eye protection when handling glass slides and coverslips.
Preparation of microscope slides
● The key components of an optical microscope are
○ The eyepiece lens
○ The objective lenses
○ The stage
○ The light source
○ The coarse and fine focus
● Other tools that may be used
○ Forceps
○ Scissors
○ Scalpel
○ Coverslip
○ Slides
○ Pipette
○ Staining solution
● Example of Calculating Magnification:
○ If a plant cell with a diameter of 150 µm is photographed and the image
size on the print is 150,000 µm, the magnification is:
Magnification=Size of imageSize of
specimen=150,000 μm150 μm=1,000 times\text{Magnification} =
\frac{\text{Size of image}}{\text{Size of specimen}} = \frac{150,000 \,
\mu\text{m}}{150 \, \mu\text{m}} = 1,000 \text{ times}Magnification=Size
of specimenSize of image=150μm150,000μm=1,000 times
● Further Enlargement:
○ If the image is further enlarged to 300,000 µm, the magnification would
be 2,000 times: Magnification=300,000 μm150 μm=2,000
times\text{Magnification} = \frac{300,000 \, \mu\text{m}}{150 \,
\mu\text{m}} = 2,000 \text{
times}Magnification=150μm300,000μm=2,000 times
○ Doubling the image size does not increase the detail visible beyond the
resolution limit of the microscope.
● Converting Units:
○ Convert units in steps of 1,000 (milli, micro, and nano):
■ 1 m=103 mm=106 μm=109 nm1 \, \text{m} = 10^3 \, \text{mm} =
10^6 \, \mu\text{m} = 10^9 \,
\text{nm}1m=103mm=106μm=109nm
○ Avoid measuring lengths in centimeters (cm) when working with
microscopes.
● Limitation of Light Microscopes:
○ Light microscopes cannot resolve structures thinner than the resolution
limit, such as cell membranes, regardless of how much the image is
magnified.
Key Takeaways
● Magnification is different from resolution; magnification enlarges the image,
while resolution defines the clarity and detail.
● Increasing magnification beyond a certain point does not increase visible
detail if the resolution limit of the microscope is reached.
● Understanding unit conversions is crucial for calculating magnification
accurately.
● Differentiation in Biology:
○ In biology, differentiation refers to the process by which unspecialized
cells, such as stem cells, become specialized into specific cell types
with distinct functions. This process involves changes in cell structure,
function, and gene expression, allowing cells to acquire unique
characteristics that enable them to perform specific roles within an
organism. Differentiation is essential for the development of complex
organisms, as it leads to the formation of various tissues and organs
from a single fertilized egg.
● Stomach: Tissue, Organ, or System?
○ The stomach is classified as an organ. An organ is a collection of
tissues that work together to perform a specific function. In the case of
the stomach, it consists of several types of tissues, including epithelial
tissue, muscle tissue, and connective tissue, all of which work together
to perform its primary functions of digesting food, secreting gastric
juices, and mixing and churning food to aid digestion. The stomach is a
component of the digestive system, which is a group of organs working
together to process food, absorb nutrients, and eliminate waste.
● Function of the Condenser Lens in a Compound Microscope:
○ The condenser lens in a compound microscope is responsible for
focusing and concentrating the light onto the specimen being observed.
By adjusting the amount and focus of light, the condenser lens
enhances the illumination of the specimen, improving the contrast and
resolution of the image. This makes it easier to see fine details and
structures within the sample.
● Why Biologists Stain Tissue for Microscopy:
○ Biologists stain tissue to improve visibility and contrast when viewing
specimens under a microscope. Many biological tissues are
transparent or have very little inherent contrast, making it difficult to
distinguish different structures. Stains bind to specific cellular
components, such as proteins, nucleic acids, or lipids, highlighting
these structures and allowing researchers to observe and differentiate
various cell types, structures, and organelles more clearly. Different
stains can also be used to target specific cellular components, aiding in
the identification and study of different biological processes.
● Avoiding Air Bubbles When Making a Temporary Mount:
○ To avoid trapping air bubbles when making a temporary mount of
tissue for microscopic observation, you can follow these steps:
■ Use a clean slide and cover slip to prevent any impurities that
might trap air.
■ Place a drop of the mounting medium (such as water or
glycerin) on the center of the slide.
■ Gently place the tissue sample into the drop of mounting
medium, ensuring it is fully immersed.
■ Hold the cover slip at an angle and slowly lower it over the
specimen, allowing it to gently come into contact with the
mounting medium.
■ Lower the cover slip gradually from one side to the other to
push out air bubbles as you go.
■ Apply gentle pressure to the cover slip if necessary to remove
any remaining air bubbles by moving them to the edge of the
cover slip.
By following these steps, you can minimize the formation of air bubbles and ensure a
clear view of the specimen under the microscope.
Method
● Preparing a slide using a liquid specimen
○ Add a few drops of the sample to the slide using a pipette
○ Cover the liquid / smear with a coverslip and gently press down to
remove air bubbles
○ Wear gloves to ensure there is no cross-contamination of foreign cells
● Methods of preparing a microscope slide using a solid specimen
○ Take care when using sharp objects and wear gloves to prevent the
stain from dying your skin
○ Use scissors or a scalpel to cut a small sample of the tissue
○ Use forceps to peel away or cut a very thin layer of cells from the tissue
sample to be placed on the slide
■ The tissue needs to be thin so that the light from the microscope
can pass through
○ Apply a stain to make cells more visible
○ Gently place a coverslip on top and press down to remove any air
bubbles
● Some tissue samples need to be treated with chemicals to kill cells or make
the tissue rigid
○ This involves fixing the specimen using the preservative formaldehyde,
dehydrating it using a series of ethanol solutions, impregnating it with
paraffin or resin for support and then cutting thin slices from the
specimen
○ The paraffin is removed from the slices and a stain is applied before
the specimen is mounted and a coverslip is applied.
○ Electron Microscopy Basics:
■ An electron microscope uses a beam of electrons instead of
light to produce magnified images.
■ The electron beam is generated by an electron gun and focused
using electromagnets.
■ Since electrons are not visible to the human eye, the image is
projected onto a fluorescent screen or photographic plate.
○ Vacuum Requirement:
■ A vacuum is necessary to prevent electrons from being
deflected by air molecules. The specimen is housed within a
vacuum chamber in the microscope.
○ Transmission Electron Microscope (TEM):
■ TEM passes an electron beam through a thin specimen section.
■ Specimens are stained with heavy metal ions, making parts of
them electron-opaque and appearing dark in images.
■ TEM is capable of resolving fine details of cell structures,
organelles, and membranes (cell ultrastructure).
○ Transmission Electron Micrograph (TEM Image):
■ The photograph taken using a TEM is called a transmission
electron micrograph.
Answers to the Questions
11. Estimating the Length of a Plant Cell Using a Ruler
If graticules or stage micrometers are unavailable, a student can estimate cell length
using a ruler as follows:
1. Calibrate the Microscope:
○ Place a transparent ruler under the microscope and adjust focus to
measure the diameter of the field of view in millimeters at a specific
magnification.
○ Convert the measurement to micrometers (µm) by multiplying by 1,000.
2. Estimate Cell Length:
○ Observe the plant tissue under the same magnification.
○ Count how many cells fit across the field of view in a straight line.
○ Divide the field of view diameter (in micrometers) by the number of
cells across to estimate the average cell length.
12. Characteristics of Amoeba proteus
a) Evidence that Amoeba proteus is not a Plant:
● Lack of Chloroplasts: It does not contain chloroplasts, which are essential for
photosynthesis in plants.
● Movement and Shape: Amoeba proteus moves using pseudopodia (cell
extensions), unlike plant cells, which are typically rigid and immobile due to
their cell walls.
● Feeding Method: It is a heterotroph, consuming organic matter, while plants
are autotrophs that produce food through photosynthesis.
b) Explanation of 'Eukaryotic' and 'Unicell':
● Eukaryotic: Refers to organisms with cells that have a defined nucleus
enclosed by a membrane and other membrane-bound organelles, like
mitochondria and the endoplasmic reticulum.
● Unicell: Describes organisms consisting of a single cell, capable of
performing all necessary functions of life within that one cell.
13. Advantages of Using a Transmission Electron Microscope
● Higher Resolution: TEMs have a much higher resolution than light
microscopes, allowing for the observation of much finer details of cell
structure.
● Ultrastructure Visualization: TEMs can reveal the detailed ultrastructure of
cells, including organelles and membranes, which are not visible with light
microscopes due to their limited resolution.
Summary
● Electron microscopes provide much greater detail than light microscopes by
using electron beams in a vacuum.
● TEMs specifically allow us to study the ultrastructure of cells in great detail.
● Understanding cell size and structure in laboratory settings can be
accomplished even without precise measuring tools by using creative
methods, such as using a ruler for calibration.
● Amoeba proteus serves as an example of eukaryotic, unicellular organisms
distinct from plants in structure and lifestyle.
Disadvantage of Using a Transmission Electron Microscope
One disadvantage of using a transmission electron microscope (TEM) compared
to a compound light microscope is that TEM requires samples to be prepared in
very thin sections, often less than 100 nanometers thick, and the process is quite
complex. This involves fixing, dehydrating, and embedding the specimen in resin,
followed by cutting it into ultra-thin slices using an ultramicrotome. Moreover,
samples must be placed in a vacuum, and this often requires extensive preparation
that can alter the natural state of the specimen. This complexity makes TEM less
suitable for observing live specimens, unlike a compound light microscope, which
can be used for observing live cells and organisms in their natural state with
relatively simple preparation.
Suggest one disadvantage of using a t ransmission electron microscope
rather than a compound light microscope to study cells. 15 The
resolving power of a light microscope is given as 2 µm and that for a
transmission electron microscope as 5nm. How many times greater is
the resolving power of the electron microscope than the light
microscope? Show your workinResolving Power Calculation
The resolving power of a microscope is defined as the smallest distance between
two points that can still be distinguished as two separate entities. The higher the
resolving power, the finer the details that can be observed.
The resolving power is often given as the smallest distance (resolution) that can be
distinguished:
● Resolving power of a light microscope = 2 µm (micrometers)
● Resolving power of a transmission electron microscope = 5 nm (nanometers)
To find how many times greater the resolving power of the TEM is compared to the
light microscope, we first need to convert these units to the same scale:
1 micrometer (µm) = 1,000 nanometers (nm)
Thus, the resolving power of the light microscope in nanometers is:
2 µm=2×1,000 nm=2,000 nm2 \, \text{µm} = 2 \times 1,000 \, \text{nm} = 2,000 \,
\text{nm}2µm=2×1,000nm=2,000nm
Now, we compare the two resolving powers:
Resolving power ratio=Resolving power of light microscopeResolving power of
TEM=2,000 nm5 nm=400\text{Resolving power ratio} = \frac{\text{Resolving power of
light microscope}}{\text{Resolving power of TEM}} = \frac{2,000 \, \text{nm}}{5 \,
\text{nm}} = 400Resolving power ratio=Resolving power of TEMResolving power of
light microscope=5nm2,000nm=400
Therefore, the resolving power of the transmission electron microscope is 400 times
greater than that of the compound light [Link] of Cell
Organization:
● Electron microscopy has revealed two fundamental types of cellular
organization: eukaryotic and prokaryotic.
Eukaryotic Cells:
● Found in animals, fungi, plants, and protoctists.
● Characterized by the presence of a large, obvious nucleus.
● The cytoplasm contains various membrane-bound organelles.
● These cells are called eukaryotic because they have a "true nucleus."
Prokaryotic Cells:
● Found in bacteria.
● Do not have a nucleus; their cytoplasm lacks membrane-bound organelles.
● Named prokaryotic, meaning "before the nucleus."
● Prokaryotic cells are much smaller in size, comparable to individual
mitochondria or chloroplasts found in eukaryotic cells.
Ultrastructure of Eukaryotic Cells:
● Eukaryotic cells are described as a "bag" of organelles, most of which are
made of membranes.
● The fluid surrounding these organelles is called the cytosol, an aqueous
solution containing various chemicals.
● The cell surface membrane encloses the cytosol and organelles, maintaining
the cell's [Link]
● Definition: The nucleus is the most prominent feature in a eukaryotic cell, such as
an epithelial cell. It contains hereditary material and controls cell activities.
● Size: Typically spherical, with a diameter of 10-20 µm.
● Components:
○ Nuclear Envelope: A double membrane that surrounds the nucleus,
continuous with the endoplasmic reticulum, often with ribosomes on the
outer membrane. It controls the entry and exit of materials.
○ Nuclear Pores: Allow large molecules (e.g., mRNA) to pass out of the
nucleus. There are around 3000 pores per nucleus, each 40-100 nm in
diameter.
○ Nucleoplasm: A granular, jelly-like material that makes up most of the
nucleus.
○ Chromosomes: Consist of protein-bound, linear DNA.
○ Nucleolus: A small, spherical region within the nucleoplasm that
manufactures ribosomal RNA and assembles ribosomes. There may be
more than one nucleolus in a nucleus.
● Functions:
○ Acts as the control center of the cell by producing mRNA and tRNA for
protein synthesis.
○ Retains genetic material in the form of DNA and chromosomes.
○ Manufactures ribosomal RNA and ribosomes.
Mitochondrion
● Definition: Mitochondria are rod-shaped organelles (1-10 µm in length) involved in
energy production.
● Components:
○ Double Membrane: Controls the entry and exit of materials. The inner
membrane folds to form cristae.
○ Cristae: Extensions of the inner membrane providing a large surface
area for enzyme attachment, crucial for respiration.
○ Matrix: The remaining part of the mitochondrion, containing proteins,
lipids, ribosomes, and DNA, which allows mitochondria to control the
production of some of their own proteins. Many enzymes involved in
respiration are found here.
● Functions:
○ Sites of aerobic respiration stages (Krebs cycle and oxidative
phosphorylation pathway).
○ Responsible for producing ATP from respiratory substrates like glucose.
○ High number and size in cells with high metabolic activity (e.g., muscle
and epithelial cells), which require a plentiful supply of ATP. Epithelial
cells in the intestines need a lot of ATP for active transport in absorption
processes.
●