Microscopy

 

Microscopy is the science of using microscopes to observe objects that are too small to be seen with the naked eye. In microbiology, microscopy is essential for studying microorganisms such as bacteria, fungi, protozoa, and viruses.

Importance of Microscopy in Microbiology

–       Study of morphology (shape, size, arrangement)

–       Identification and classification of microorganisms

–       Observation of stained and unstained specimens

–       Understanding cell structure and function

Microscope-

"Micro" refers to tiny, and "scope" refers to view or look at. Microscopes are tools used to enlarge small objects so that they can be observed and studied. They range from a simple magnifying glass to an expensive electron microscope.

In the field of microbiology, the invention of the microscope is mainly credited to Antonie van Leeuwenhoek (1632–1723). He was the first person to observe and describe microorganisms (which he called “animalcules”). He used a simple microscope with very high magnification (about 200–300×). In 1674–1683, he observed bacteria, protozoa, yeast, and sperm cells.

Note- Zacharias Janssen (c. 1590) → Invented the compound microscope

Antonie van Leeuwenhoek → First to use the microscope to study microorganisms

Classification of microscopes.

Microscopes can be classified based on various properties.  Based on the lens system, microscopes are of two types.

Simple microscope	Compound microscope
1. It has a single lens system.	1. It has two or more lens system.
2. Example:-Magnifying glass.	2. Example:-Compound microscope basically used in the laboratory.
3. Reading small letters, simple observation	3. Studying microorganisms, cells
4.Magnification Low (up to ~10×)	4. High (40×–1000×)

Based on the source of illumination, there are two types of microscope:

Light Microscope	Electron Microscope
1.   It uses light as a source of illumination.	1.   It uses an electron or electron gun as a source of illumination.
2.   Lens used in a glass lens	2.   The lens used is an electromagnetic lens.
3.   Here, a vacuum is not needed.	3.   A vacuum must be created since electrons can travel only in a vacuum.
4.   Resolution is low compared to the electron microscope.	4.   Resolution is very high, capable of viewing particles of size 0.1 to 0.2 nanometers (nm).
5.   Image can be directly seen by the human eye.	5.   Since the human eye can’t see electrons. Electrons are converted into an amazing image by striking a fluorescent screen.

Types of Microscope

Bright Field Microscopy

Bright Field Microscopy is the most basic, commonly used light microscopy technique where light passes directly through a sample, producing a dark image on a bright, illuminated background. It is essential for observing stained, fixed, or high-contrast,, colored samples in biology and materials science.

· Imaging Principle: Transmitted white light passes through the specimen, and the image is formed by the absorption of light in denser, stained areas.

· Applications: Commonly used in microbiology for examining stained bacteria and in cell biology for tissue sections.

Magnification

The microscope produces an enlarged image of the object that is examined through it. This enlargement is known as its magnification and is measured in diameter. Eg: A magnifying lens which gives an image 36 times as large as that of the object is said to have a magnification of 36 diameter or 36X(x=times).

Magnification is the process of enlarging the apparent size of an object. It tells how many times the image of the object is larger than the actual object.

Total Magnification=Magnification of Objective Lens×Magnification of Eyepiece (Ocular Lens)

Example:

• Objective lens = 40×
• Eyepiece = 10×
• Objective lens = 40×

Total Magnification=40×10=400×

Note: High magnification without good resolution gives a blurry image.

Resolution

Resolution (or resolving power) is the ability of a microscope to distinguish two points that are very close together as separate entities. In other words, it measures the clarity or detail of the image.

The ability of a microscope to distinguish two closely spaced objects as separate and distinct entities is called resolution. Eg. The human eye has the resolving power of 0.25mm, which means that two dots placed 0.25mm apart can be distinguished as two dots. If the distance between the two dots is less than 0.25, only one dot will be seen.

•        Resolving Power (R.P.) = λ/2 NA where λ= wavelength of light , N.A=Numerical Aperture

or

Formula for Resolution (d):  d=λ/2 NA

Where:

• d= minimum distance between two distinguishable points (in meters or micrometers)
• λ (lambda) = wavelength of light used (usually 400–700 nm for visible light)
• NA = Numerical Aperture of the objective lens

Interpretation:

The formula RP = λ / (2 x NA) indicates that the resolving power is influenced by both the wavelength of light and the numerical aperture.

The resolving power is inversely proportional to the wavelength of light (λ). As the wavelength decreases, the resolving power increases, allowing for better separation of closely spaced objects.

The resolving power is directly proportional to the numerical aperture (NA). Higher numerical apertures lead to greater resolving power. A larger NA allows the lens to capture more diffracted light, contributing to improved resolution.

• Smaller d → better resolution → can see finer details
• Higher NA or shorter wavelength → better resolving power

 

Malaria

                                                                         Malaria

Malaria is a blood-borne disease caused by the protozoan Plasmodium and is typically transmitted through the bite of an infected Anopheles mosquito. Infected mosquitoes carry the Plasmodium parasites. 4 species of malaria parasites can infect humans: Plasmodium vivax, P. ovale, P. malariae, and P. falciparum.

P. falciparum causes a more severe form of the disease and those who contract this form of malaria have a higher risk of death. Plasmodium falciparum is the most virulent species of Plasmodium in humans.

Habitat of Plasmodium Parasites
In Mosquitoes:
Plasmodium parasites develop in the gut and salivary glands of Anopheles mosquitoes. These mosquitoes require a warm and humid environment for their development, making tropical and subtropical regions ideal.

 
In Humans:
Inside humans, Plasmodium parasites inhabit the liver and red blood cells. The liver stage occurs in hepatocytes (liver cells), while the blood stage takes place within red blood cells.

Geographical Distribution
Tropical and Subtropical Regions: Malaria is predominantly found in tropical and subtropical regions where the climate supports the Anopheles mosquito population. This includes parts of Africa, South Asia, Southeast Asia, and Central and South America.

Morphology

The following are the diagnostic forms of parasites found in humans

Ring form (Early Trophozoite)
This is the young trophozoite found inside RBCs.
The name ring is derived from the morphological appearance of the stage, resembling a ring-like structure.
A small cytoplasmic rim and a chromatin dot (nucleus) are seen

Trophozoite (Mature)
RBC starts enlarging (especially in P. vivax and P. ovale).
Trophozoites are larger and more ameboid in shape.
They feed on hemoglobin, and their morphology includes a central nucleus and pigment granules.
Cytoplasm becomes more prominent, chromatin more condensed.
Pigment (hemozoin) may appear as brown-black granules.

Schizont
As the trophozoites mature, they form schizonts.
These structures contain multiple nuclei and are larger than trophozoites.
Contains multiple merozoites (number varies by species: e.g., P. falciparum: 16–32, P. malariae: 6–12).
They eventually rupture the red blood cell, releasing more merozoites into the bloodstream, which can infect new red blood cells.

Gametocytes (Sexual Forms)
Gametocytes are the sexual stage of the parasite and are infectious to mosquitoes.
P. falciparum: crescent or banana-shaped.
P. vivax, P. ovale, P. malariae: round or oval.
There are two types of gametocytes.
Microgamete: male form
Macrogamete: female form
Gametocytes are infective to mosquitoes.
• Male (microgametocytes) and female (macrogametocytes) gametocytes are taken up by mosquitoes during a blood meal.

Sporozoites
• The sporozoites are the infective form and are infectious to humans
• They are found in infected mosquitoes in the salivary glands of female Anopheles mosquitoes.
• Sporozoites are single-nucleated, sickle-shaped structures with equally pointed ends.
• The peripheral fibres serve as an organ of locomotion.
• These infectious forms are injected into the human host's bloodstream by an infected mosquito. They travel to the liver and invade hepatocytes, initiating the exoerythrocytic cycle.

Ookinete
The male and female gametocytes fuse to form the zygote, which then matures into a motile form called an ookinete.
They are elongated, spindle-like (sausage-shaped).
Ookinete invades the midgut wall of the mosquito to develop into an oocyst.

Oocyst
Once the ookinete successfully penetrates the midgut epithelium, it transforms into a rounded structure known as an oocyst.
They are spherical or oval in shape, can undergo sporogony to produce thousands of sporozoites inside.
When mature, the oocyst ruptures, releasing sporozoites into the mosquito hemocoel, which migrate to the salivary glands for the next transmission.

Life Cycle of Malarial Parasites

The life cycle of Plasmodium begins when an infected female Anopheles mosquito bites a human and injects sporozoites, the infective stage, into the bloodstream along with its saliva. These sporozoites circulate in the blood for about 20–30 minutes, after which they quickly leave the circulation and enter the liver cells (hepatocytes). This marks the beginning of the liver or exo-erythrocytic stage. Inside the liver cells, each sporozoite grows and undergoes repeated asexual division to form a large structure called a schizont, which contains thousands of daughter cells known as merozoites. After several days, the infected liver cells burst open, releasing the merozoites into the bloodstream.

Note-  In infections by P. vivax and P. ovale, some sporozoites do not immediately divide but instead become hypnozoites, dormant forms capable of reactivating after months or years and causing relapse.

Once released into the bloodstream, the merozoites initiate the erythrocytic (blood) stage by invading red blood cells (RBCs). Inside each RBC, the parasite first appears as a delicate ring-shaped trophozoite. The trophozoite feeds on hemoglobin and enlarges, eventually developing into a mature trophozoite, and then undergoes nuclear division to form another schizont filled with merozoites. When the schizont becomes mature, the RBC ruptures, releasing numerous merozoites into circulation, which then infect new RBCs. This cyclic rupture of RBCs, typically every 48–72 hours depending on the Plasmodium species, is responsible for the characteristic bouts of fever, chills, and rigors seen in malaria patients. This blood-stage multiplication continues repeatedly and is responsible for the clinical symptoms of the disease.

During these repeated asexual cycles, some merozoites differentiate into sexual forms called gametocytes. These gametocytes—male (microgametocytes) and female (macrogametocytes)—circulate in the bloodstream but do not cause symptoms.

When another female Anopheles mosquito bites the infected human, it ingests these gametocytes along with the blood meal, beginning the mosquito stage of the life cycle. Inside the mosquito’s gut, the gametocytes quickly mature: the microgametocyte produces several flagellated microgametes, while the macrogametocyte develops into a single macrogamete. Fertilization occurs when a microgamete fuses with a macrogamete to form a zygote, which then elongates into a motile form called an ookinete.

The ookinete penetrates the mosquito’s midgut wall and settles beneath its outer lining, where it develops into an oocyst. The oocyst gradually enlarges and undergoes repeated divisions to produce thousands of sporozoites. When the oocyst matures, it bursts, releasing sporozoites into the mosquito’s body cavity. These sporozoites then migrate to the mosquito’s salivary glands, where they are stored. When the mosquito next bites a human, the sporozoites are injected into the bloodstream, thus completing the cycle and initiating a new infection in another host.