Contributions of Pioneers in Microbiology

Contributions of Anton van Leeuwenhoek, Joseph Lister, Paul Ehrlich, Edward Jenner, Louis Pasteur, Robert Koch, Martinus W. Beijerinck, Sergei N. Winogradsky, Alexander Fleming, Selman A. Waksman

 

Anton van Leeuwenhoek (1632-1723)

Anton van Leeuwenhoek was a Dutch draper, but spent much of his time constructing simple microscopes composed of double convex glass lenses held between two silver plates.  His microscopes could magnify around 50 to 300 times.  With these tools, he was the first to observe and document the world of microbes.  Discovery of "Animalcules": In the 1670s, he began sharing his observations with the Royal Society of London. He described tiny, moving organisms in water, which he called "animalcules." These were the first recorded observations of bacteria and protozoa. He also meticulously documented other microscopic life, including Bacteria in dental plaque and rainwater, Spermatozoa, Blood cells, etc.

Joseph Lister (1827-1912)

Joseph Lister, a British surgeon, is known as the "Father of Modern Surgery". Before his time, post-operative infections often lead to gangrene and sepsis, and were a major cause of death. Surgeons were unaware that invisible germs were the source of these infections.

Inspired by Louis Pasteur's research on fermentation and putrefaction, Lister hypothesized that microorganisms were responsible for surgical infections. He believed that if germs could be prevented from entering a wound, infection could be avoided.  Lister began using carbolic acid (phenol) as a disinfectant. This method was introduced in 1867 and it involved a rigorous antiseptic protocol  of Handwashing where Surgeons and nurses washed their hands with carbolic acid solution, Sterilization of instruments by soaking in the solution, Wound dressing by bandages and dressings which were soaked in carbolic acid to prevent airborne bacteria from entering the wound and adopting an aseptic environment by using a carbolic acid spray to mist the air in the operating room.

These antiseptic techniques dramatically reduced the mortality rates associated with surgery. His work transformed surgery from a dangerous, high-risk procedure into a much safer and effective medical practice.

Paul Ehrlich (1854-1915)

Paul Ehrlich was a German physician and scientist who is considered the "Father of Chemotherapy". His work bridged the gap between chemistry and medicine, leading to the development of the first synthetic drugs.

The "Magic Bullet" Concept: Ehrlich's core idea was to create a "magic bullet"—a chemical compound that could target and kill a specific disease-causing organism without harming the host's healthy cells.  

In his search for a treatment for syphilis, Ehrlich and his team synthesized hundreds of arsenic-based compounds. Their 606^th compound, arsphenamine was marketed as Salvarsan ("the saving arsenic") and became the first effective cure for syphilis, a previously incurable disease.  

Ehrlich also made significant contributions to immunology. He developed the side-chain theory, which laid the groundwork for our modern understanding of immune system and he received the Nobel Prize in Medicine in 1908.

Edward Jenner (1749-1823)

Edward Jenner was an English physician known as the "Father of Immunology" for his work on the smallpox vaccine. Smallpox was a devastating disease with a high mortality rate and often left survivors with severe scarring.

Jenner observed that milkmaids who had been infected with cowpox, a much milder disease, seemed to be immune to smallpox. This led him to the idea that exposure to cowpox could protect a person from smallpox.

In 1796, Jenner took pus from a cowpox lesion on the hand of a milkmaid and inoculated it into the arm of a young boy named James Phipps. The boy developed a mild fever but quickly recovered. A few weeks later, Jenner inoculated the boy with smallpox material, but the boy did not get sick.

Jenner's procedure, which he named vaccination (from the Latin vacca, meaning "cow"), was published in his 1798 paper, An Inquiry into the Causes and Effects of the Variolae Vaccinae. This work laid the foundation for modern immunology and the global campaign that eventually led to the eradication of smallpox in 1980.

Louis Pasteur

Pasteur is known as the father of modern microbiology

In 1849 Pasteur observed that Tartaric acid and racemic acid found in the sediment of fermenting wine have identical chemical composition.  Upon intense inspection using a microscope, Pasteur noticed that these identical molecules have a different spatial arrangement. One was the mirror image of the other.  This discovery led to the branch of science known as ‘stereochemistry’.

Until the second half of the 19th century, Many scientists and philosophers believed in Spontaneous generation-life could arise spontaneously from non-living matter. Louis Pasteur disproved Spontaneous Generation and proved that all life comes from preexisting life.  Pasteur demonstrated that in sterilised and sealed flasks, no life forms develop, and in sterilised but open flasks, microbes could grow.  Microorganisms from the air contaminate sterile solutions, but air itself does not create microbes.

His studies showed that living cells, yeast, are responsible for forming alcohol from sugar and contaminating microorganisms make the fermentation sour

To prevent contamination, Pasteur heated wine to 50–60 °C, a process now known as pasteurisation. It is used for the preservation of many foods and beverages, particularly milk

Pebrine, or "pepper disease," a disease of silkworms, caused by protozoa, mainly Nosema bombycis and other species.   The silkworm larvae having pebrine are covered in brown dots and are unable to spin thread.  Louis Pasteur identified the cause and developed a method called ‘graining’ when a plague of the disease spread across France.  Graining enabled elimination of contaminated females and the preservation of healthy eggs.  This technique was adopted by silk producers throughout the world.

Pasteur identified that the fermentation process could be arrested by passing air (oxygen) through the fermenting fluid - known today as the Pasteur effect.  He concluded that this is due to a microorganism that could ferment only in the absence of oxygen.  He introduced the terms aerobic and anaerobic to designate organisms that live in the presence or absence of oxygen

In 1879, Pasteur worked with the disease fowl cholera, or chicken cholera which was rampant in chicken coops at the time. When inoculated into a chicken, the bacteria kill the bird - Pasteurella multocida.  Pasteur used a weakened culture of bacteria to inoculate chickens which resulted in immune birds and thus the principle of vaccination with attenuated pathogens was discovered.

In 1879 Pasteur worked with the disease Anthrax caused by Bacillus anthracis.  Anthrax is an infectious disease that affects cattle, sheep, etc that can be transmitted to man.  It was responsible for killing large populations of sheep in France, detrimental to the economy.  Pasteur developed a mild, harmless culture of anthrax bacteria. Used this culture to vaccinate hundreds of livestock, and they became immune to the disease. 

In anthrax or chicken cholera vaccination the causative agents are artificially weakened or attenuated to immunize the animals Pasteur gave these artificially weakened diseases the generic name of "vaccines", in honour of Jenner's discovery

In 1882, Pasteur worked with an epizootic of Swine erysipelas and he developed a vaccine against this bacterial infection.

He carefully studied and discovered several other infectious diseases such as those caused by staphylococcus, streptococcus and pneumococcus.

Pasteur then worked with rabies.  Rabies is a highly contagious, infectious viral disease that attacks the central nervous system, transmitted via the bite of rabid animals.  On 1885, Pasteur administered his rabies vaccine on Joseph Meister, a young man bitten by a rabid dog and saved his life.  To help treat future cases of rabies, the Pasteur Institute was established in 1888 with monetary donations coming from all over the world.

His medical discoveries provided direct support for the germ theory of disease and its application in clinical medicine.  Although Pasteur was not the first to propose the germ theory, his experiments indicated its correctness.  He is regarded as one of the three main founders of bacteriology, together with Ferdinand Cohn and Robert Koch

Robert Koch

Robert Koch was a German physician and microbiologist.

Koch's Postulates: Koch formulated a set of four criteria, known as Koch's Postulates, to establish a causal relationship between a specific microorganism and a disease. These postulates are still used today as a gold standard in microbiology:

1.    The microorganism must be found in abundance in all organisms suffering from the disease, but not in healthy organisms.

2.    The microorganism must be isolated from a diseased organism and grown in a pure culture.

3.    The cultured microorganism should cause disease when introduced into a healthy organism.

4.    The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as identical to the original specific causative agent.

He identified the specific causative agents of tuberculosis, cholera, and anthrax.  Robert Koch discovered the causative agent of Anthrax to be Bacillus anthracis.  He reported the causative agent of the disease tuberculosis to be the slow-growing Mycobacterium tuberculosis.  Koch studied cholera in Egypt and in India and identified the causative agent of cholera to be Vibrio cholerae.

Koch observed the phenomenon of acquired immunity while studying Plasmodium parasites, the cause of malaria

Koch cultured bacteria on the sterile surfaces of cut, boiled potatoes, but without success and tried to solidify regular liquid media by adding gelatin. Gelatin was not an ideal solidifying agent because it was digested by many bacteria and melted when the temperature rose above 28°C. Fannie Hesse, the wife of Walther Hesse, one of Koch’s assistants suggested the use of agar as a solidifying agent. Agar was not attacked by most bacteria and did not melt until reaching a temperature of 100°C.

One of Koch’s assistants, Richard Petri, developed the petri dish (plate), a container for solid culture media. These developments made possible the isolation of pure cultures that contained only one type of bacterium.

Koch also developed media suitable for growing bacteria with meat extracts and protein digests as nutrient sources and resulted in the development of nutrient broth and nutrient agar.

For his research on tuberculosis, Koch received the Nobel Prize in Physiology or Medicine in 1905. The Robert Koch Institute which monitors public health is named in his honor and is located in Berlin and Wernigerode

Martinus W. Beijerinck (1851–1931)

Martinus W. Beijerinck made fundamental contributions to microbial ecology. He is often considered one of the founders of virology and environmental microbiology.

He isolated the aerobic nitrogen fixing bacterium Azotobacter; a root nodule bacterium also capable of fixing nitrogen (Rhizobium); and sulfate reducing bacteria.

Beijerinck proved that a virus causes the tobacco mosaic disease

Beijerinck invented the enrichment culture, a fundamental method of studying microbes from the environment.

Beijerinckia (a genus of bacteria), Beijerinckiaceae (a family of Rhizobiales), and Beijerinck (crater in moon) are named after him.

Sergei N. Winogradsky (1856–1953)

The Russian microbiologist Sergei N. Winogradsky made many contributions to soil microbiology.

He discovered that soil bacteria could oxidize iron, sulfur, and ammonia to obtain energy, and that many bacteria could incorporate CO2 into organic matter much like photosynthetic organisms do. This is chemoautotrophy or chemosynthesis, the process by which organisms derive energy from a number of different inorganic compounds.

Winogradsky also isolated anaerobic nitrogen-fixing soil bacteria and studied the decomposition of cellulose.

Winogradsky discovered the first known form of lithotrophy (using inorganic substrate to obtain energy via aerobic or anaerobic respiration) during his research with Beggiatoa in 1887. He reported that Beggiatoa oxidized hydrogen sulfide (H2S) as an energy source and formed intracellular sulfur droplets.

The Winogradsky column remains an important display of chemoautotrophy and microbial ecology.  This is a simple device for culturing a large diversity of microorganisms.  the device is a column of pond mud and water mixed with a carbon source such as newspaper (containing cellulose), egg-shells (containing calcium carbonate), and a sulfur source such as gypsum (calcium sulfate) or egg yolk. Incubating the column in sunlight for months will results in an aerobic/anaerobic gradient as well as a sulfide gradient and promote the growth of different microorganisms such as bacteria, cyanobacteria, and algae.

Alexander Fleming (1881 - 1955)

Alexander Fleming was a Scottish physician and microbiologist.

Discovery of Penicillin: In 1928, Fleming discovered the first true antibiotic, penicillin. While studying Staphylococcus bacteria, he noticed that a mold, Penicillium notatum, had contaminated one of his petri dishes. He observed that the bacteria colonies around the mold were dying. He correctly identified that the mold was producing a substance that was killing the bacteria, and he named it penicillin.

In 1922, Fleming discovered lysozyme, an enzyme with antibacterial properties found in human tears, saliva, and other body fluids.

He shared the Nobel Prize in Physiology or Medicine with Howard Florey and Ernst Chain in 1945.

Selman A. Waksman (1888–1973)

Selman A. Waksman was a Ukrainian-American biochemist and soil microbiologist.

Waksman and his team systematically screened thousands of soil microorganisms for antibacterial properties. In 1943, they isolated streptomycin from the bacterium Streptomyces griseus. This was the first effective antibiotic against tuberculosis and other Gram-negative bacterial infections.

Waksman's work established a methodical approach to finding antimicrobial agents from soil microbes and lead to the discovery of over a dozen other antibiotics, including neomycin. He also coined the term "antibiotics" to describe these substances produced by microorganisms that inhibit the growth of other microorganisms.

 

Microscopy

Introduction to Microscope

Microscopy is the science of using microscopes to observe objects that are too small to be seen with the naked eye. It plays a vital role in a wide array of fields including biology, medicine, materials science, and chemistry.

The Microscope is an optical instrument consisting of a combination of lenses for making enlarged or magnified images of minute objects. The term is derived from the two Greek words ‘micro’ - small, and ‘scope’ - view.  Since microorganisms are very small and invisible to naked eyes, they must be magnified to be clearly seen. The use of a microscope is absolutely indispensable to study microbiology.

Working of lenses

When a ray of light passes from one medium or material to another, refraction occurs, the light ray will bend at the interface.  Refractive index is the measure of how a medium or material slows the velocity of light.  Glass has higher refractive index and air have lower refractive index The direction and magnitude of bending is determined by the refractive indexes of the two media or materials forming the interface. When light passes from air into glass, it is slowed and bent toward the normal, a line perpendicular to the surface and as light leaves glass and returns to air, it accelerates and is bent away from the normal.  As shown in the below image, a prism bends light because of the different refractive indices of glass and air.  

The Bending of Light by a Prism 

(Prescott−Harley−Klein:Microbiology, Fifth Edition)

Lens act like a collection of prisms operating as a unit. When parallel rays of light strike the lens, a convex lens will focus these rays at a specific point termed as the focal point (F). The distance between the centre of the lens and the focal point is called the focal length (f).  Lens strength is related to focal length; a lens with a short focal length will magnify an object more than a weaker lens having a longer focal length.

Lens functions like a collection of prisms 

(Prescott−Harley−Klein:Microbiology, Fifth Edition)

Magnification and resolving power

Magnification and resolving power are important concepts in microscopy and imaging.  Magnification is the process of making an object appear larger than its actual size. Magnification is expressed as the ratio of the object's apparent size to its actual size.  Resolving power or Resolution is the ability to distinguish two objects that are close together. While higher magnification can increase the amount of detail in an image, it doesn't necessarily improve the ability to distinguish fine details. For example, two images may have the same magnification, but one may have a higher resolution and be clearer.

The minimum distance (d) between two objects that reveals them as separate entities is given by the Abbe equation, in which lambda (λ) is the wavelength of light used to illuminate the specimen and n sin θ is the numerical aperture (NA).

With higher resolution, two objects with smaller d could be differentiated as two separate entities.  As d becomes smaller, the resolution increases, and finer detail can be distinguished in the specimen.  Higher resolution is obtained with shortest wavelength light, with light at the blue end (450 to 500 nm) of the visible spectrum.  The wavelength must be shorter than the distance between two objects or they could not be seen clearly.

Numerical aperture

The measure for the resolving powers of a lens is the numerical aperture.  It is n sin θ.  The larger the numerical aperture, then the greater is the resolving power of the lens. Theta is defined as 1/2 the angle of the cone of light entering an objective. Light that strikes the microorganism after passing through a condenser is cone- shaped. When this cone has a narrow angle and tapers to a sharp point, it does not adequately separate images of closely packed objects and thus the resolution will be low. If the cone of light has a very wide angle and spreads out after passing through a specimen, closely packed objects appear widely separated and thus the resolution will be high. 

Numerical Aperture - The angular aperture θ is 1/2 the angle of the cone of light that enters a lens from a specimen, and the numerical aperture is n sin θ. Lens having larger angular and numerical apertures will have higher resolution and its working distance will be smaller (Prescott−Harley−Klein:Microbiology, Fifth Edition)

The angle of the cone of light that enter a lens depends on the refractive index (n) of the medium in which the lens works. The refractive index for air is 1.00. Since sin θ cannot be greater than 1 (the maximum θ is 90° and sin 90° is 1.00), a lens working in air cannot have a numerical aperture greater than 1.00. In order to raise the numerical aperture above 1.00, to achieve higher resolution, the refractive index is to be increased. This could be achieved by using immersion oil, a colorless liquid having the same refractive index as glass (about 1.515). If air is replaced with immersion oil, by adding a drop of oil on the surface of glass slide containing the specimen, light rays that otherwise may not enter the objective due to reflection and refraction will now enter the lens.   So, an increase in numerical aperture and thus increase in resolution results.

Oil immersion objective operating in air and with immersion oil (Prescott−Harley−Klein:Microbiology, Fifth Edition)

Types of Microscopes

A simple microscope consists merely of a single lens or magnifying glass held in a frame, usually adjustable, and will have a stand for holding the object to be viewed and a mirror for reflecting light. A compound microscope consists of two sets of lenses, one known as an objective and the other as an eyepiece.  Compound microscopes give more magnification than simple microscope. 

There are different types of Microscopes. The major being a) Light Microscopes (Optical Microscopes) and b) Electron Microscopes

A. Light Microscope (Optical Microscope):

Light microscopes use visible light and lenses to magnify objects.  Magnification typically ranges from 40x to 1000x.

Compound microscope and its parts (Fundamental Principles of Bacteriology : A.J. Salle)

Parts of a Light Microscope:

Eyepiece (ocular lens): The lens you look through, typically 10x magnification.

Objective Lenses: These are multiple lenses that provide different magnifications (usually 4x, 10x, 40x, 100x).

Stage: The flat surface where the slide with the specimen is placed.

Condenser: Focuses the light onto the specimen.

Diaphragm: Controls the amount of light that passes through the sample.

Coarse and Fine Focus Knobs: Adjust the focus of the image.

Illuminator: A light source, usually a lamp, that illuminates the sample.

Types of Light Microscopy:

Bright-Field Microscopy: The most commonly used technique where light passes through the specimen, and the image appears dark against a bright background.

Dark-Field Microscopy: Light is directed at an angle to the sample, which makes the image appear bright against a dark background.

Phase-Contrast Microscopy: Enhances contrasts in transparent specimens and is useful for studying live cells.

Fluorescence Microscopy: Uses ultraviolet (UV) light to excite fluorophores attached to specimen. When the fluorophores emit light, the specimen appears as brightly coloured on a dark background.

B) Electron Microscopes (EM)

Electron microscopes use electrons instead of light to view specimens at much higher magnifications and resolutions than light microscopes.

Transmission Electron Microscope (TEM):

TEM transmits a beam of electrons through a thin sample. The electrons interact with the sample, and the resulting image is projected onto a screen or film and can achieve magnifications up to 10 million times and capable of Resolution at nanometer scale (less than 1 nm).

Scanning Electron Microscope (SEM):

SEM scans a focused electron beam over the surface of the sample. The electrons interact with the surface and produce signals that are used to form an image. Can achieve magnifications up to 1 million times and resolution of about 1-10 nanometers.

C. Scanning Probe Microscopes (SPM)

Scanning Probe Microscopes work by scanning a sharp probe across the surface of the specimen. The most common type is the Atomic Force Microscope (AFM).  This measures the force between the probe and the surface and help for the construction of a high-resolution topographical image.

D. Other Advanced Microscopy Techniques

Confocal Microscopy uses laser light to scan specimens and produce high-resolution 3D images. Confocal microscopes capture optical sections of the sample, which can then be digitally reconstructed to create a 3D image.

Multiphoton Microscopy is a type of fluorescence microscopy that uses multiple photons to excite fluorescent dyes.

Live-Cell Microscopy is used to observe living cells in real-time, often with the help of fluorescent dyes or proteins.

Super-Resolution Microscopy includes techniques like STED (Stimulated Emission Depletion) and PALM (Photo-Activated Localization Microscopy).  These provide resolutions of less than 100 nm.

The Bright-Field Microscope

The ordinary microscope is called a bright-field microscope because it forms a dark image against a brighter background.

The microscope consists of a sturdy metal body or stand composed of a base and an arm to which the remaining parts are attached.

A light source, either a mirror or an electric illuminator, is located in the base.

Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus the image.

The stage is positioned about halfway up the arm and holds microscope slides by either simple slide clips or a mechanical stage clip. A mechanical stage allows the operator to move a slide around smoothly during viewing by use of stage control knobs.

The substage condenser is mounted within or beneath the stage and focuses a cone of light on the slide. Its position often is fixed in simpler microscopes but can be adjusted vertically in advanced models.

The curved upper part of the arm holds the body assembly, to which a nosepiece and one or more eyepieces or oculars are attached. More advanced microscopes have eyepieces for both eyes and are called binocular microscopes.

The nosepiece holds three to five objectives with lenses of differing magnifying power and can be rotated to position any objective beneath the body assembly. Ideally a microscope should be parfocal—that is, the image should remain in focus when objectives are changed. 

The objective lens forms an enlarged real image within the microscope, and the eyepiece lens further magnifies this primary image. The total magnification is calculated by multiplying the objective and eyepiece magnifications together. For example, if a 45 X objective is used with a 10 X eyepiece, the overall magnification of the specimen will be 450 X.

The resolution of a microscope depends upon the numerical aperture of its condenser as well as that of the objective. 

The resolution of a light microscope can be calculated using the Abbe equation. The maximum theoretical resolving power of a microscope with an oil immersion objective (numerical aperture of 1.25) and blue-green light is approximately 0.2 µm.

So, a bright-field microscope can distinguish between two dots which are 0.2 µm apart, the size of a very small bacterium.

Generally, a microscope is equipped with three or four objectives (4X, 10X, 40X, 100X).  The working distance of an objective lens is the distance between the front surface of the lens and the surface of the specimen when it is in sharp focus. Objectives with large numerical apertures and high resolving power have short working distances.

Normally, Microscopes come with 10 X eyepieces and have an upper limit of about 1,000 X with oil immersion (100 X objective).  A 15 X eyepiece may be used to achieve a magnification of 1,500 X.

The Dark-Field Microscope

The principle of dark-field microscopy is based on scattering of light. Living, unstained cells and organisms can be observed by enhancing the contrast by illuminating them with light at an oblique angle.  Light is directed from an angle, typically using a special condenser to produce a hollow cone of light.  This hollow cone of light is focused on the specimen in such a way that only reflected and refracted rays enter the objective lens and forms an image.  Since most of the direct light does not enter the objective lens, the background remains dark, while the scattered light from the specimen appears bright. This creates a high-contrast image.  So, the field surrounding a specimen appears black, while the object itself is brightly illuminated.

This microscopy is used to identify bacteria like Treponema, the causative agent of syphilis.  A microscope may be converted to dark-field microscope by placing a dark-field stop or light stop or central stop or central aperture underneath the condenser lens system.

The central stop is a round piece of black metal, mounted into an attachment, a slider or the disc of a turret condenser. The stop has to be aligned so that it is exactly placed in the middle of the ray path. 

https://moticeurope.com/

Phase Contrast Microscope

A phase-contrast microscope converts slight differences in refractive index and cell density into easily detectable variations in light intensity and enables to observe living cells. It works by converting phase shifts (differences in the speed of light as it passes through different parts of the specimen) into variations in brightness, so otherwise invisible structures will become visible. When light passes through cells, small phase shifts occur, and these small phase shifts (which are invisible to the human eye) are converted into changes in amplitude.  These changes can be observed as differences in image contrast in a phase-contrast microscope.

·     The condenser of a phase-contrast microscope has an annular stop (an opaque disk with a thin transparent ring) which produces a hollow cone of light.

·        As this cone passes through a specimen, some light rays are bent due to variations in density and refractive index within the specimen.  These rays are retarded by about 1/4 wavelength.

·        Now the rays pass through phase-shift ring or the phase plate.  The deviated rays pass through the plate and undeviated light rays go through the ring in the phase plate (a special optical disk located in the objective).  Now the undeviated light are advanced by 1/4 wavelength.

        So, the deviated and undeviated waves will be about 1/2 wavelength out of phase and interference occurs, the waves combine in such a way that their crests (the highest points) and troughs (the lowest points) align.    This alignment amplifies the overall wave, resulting in a wave with a larger brightness. The undeviated light forms the bright background and the object appears dark and well-defined. 

Deviated and undeviated light rays in dark phase-contrast microscope (Prescott−Harley−Klein:Microbiology, Fifth Edition)

https://microbenotes.com/phase-contrast-microscopy/

Phase-contrast microscopy is useful for studying microbial motility, shape of living cells, endospores and inclusion bodies, etc.

Confocal Microscopy

Confocal microscopy is a powerful tool for visualizing and analysing complex biological and material systems and it provide high-resolution, 3D images. 

A conventional light microscope, uses a mixed wavelength light source and illuminates a large area of the specimen and thus will have a relatively great depth of field. Here, images of bacteria from all levels (above, in, and below the plane of focus) in the field will be visible, and thus image can be fuzzy and crowded.

This problem could be solved by using Confocal Microscope and Fluorescently stained specimens are usually examined.

Conventional light microscopic observation and Confocal scanning laser microscopic observation (Prescott−Harley−Klein:Microbiology, Fifth Edition)

 

Confocal Microscopy

(https://images.app.goo.gl/NkU2EbyRL4cCidHaA)

• Laser Source: Provides the excitation light.
•  Scanning Mirrors (Dichroic Mirror): Control the position of the laser beam on the sample.
• Objective Lens: Focuses the laser beam onto the sample and collects the emitted fluorescence.
• Pinhole Aperture (Emission pinhole): Blocks out-of-focus light.
• Detector: Detects the emitted fluorescence to form the image which will be done by a computer.  Special computer software is used to create high-resolution, 3D images of cell structures and biofilms.

The confocal microscope improves images in two ways.

Point Illumination:  Illumination of one spot at a time reduces interference from rest of the specimen 

The Pinhole Aperture: This is above the objective lens and it blocks out the out of focus light rays.  So the image will have excellent contrast and resolution.

Types of Confocal Microscopes

•  Laser Scanning Confocal Microscope (LSCM): The most common type, using a single pinhole to block out-of-focus light.
• Spinning Disk Confocal Microscope (SDCM): Uses a rotating disk with multiple pinholes to increase imaging speed.
• Two-Photon Microscopy: Uses a longer wavelength laser to excite fluorophores, reducing photobleaching and increasing imaging depth.
• Stimulated Emission Depletion (STED) Microscopy: A super-resolution technique that can achieve resolutions beyond the diffraction limit. 

Fluorescence Microscope

All the microscopes produce an image from light that passed through a specimen. An object that actually emits light can also be observed, and this is the basis of fluorescence microscopy. Fluorescence is the phenomenon where a substance, after absorbing light energy, emits light of a different color.  Fluorescent molecules absorb radiant energy, become excited and later release much of their trapped energy as light. The light emitted by this excited molecule will have a longer wavelength (or lower energy) than the radiation originally absorbed.

In fluorescence microscopy, the specimen is illuminated with light of a specific wavelength (ultraviolet, violet, or blue light).  Usually, the specimen will be stained with dye molecules, called the fluorophores or fluorochromes.  The light will be absorbed by the fluorophores and they then emit light of a longer wavelength, which is used to form an image.

(https://images.app.goo.gl/o2tGawzgcWn9hRPT7)

•     A mercury vapor arc lamp is used as source and the beam passes through a special infrared filter which limits heat transfer.
•     The light passes through an exciter filter that transmits only the desired wavelength.
•     A darkfield condenser is used to provide a black background
•       The microscope forms an image of the fluorochrome-labelled microorganisms.  A barrier filter is positioned after the objective lens to remove any remaining ultraviolet light, which could damage the viewer’s eyes, or to remove blue and violet light, which would reduce the image contrast

Fluorochromes such as acridine orange, ethidium bromide, calcein-AM and DAPI (diamidino-2- phenylindole) are generally used.

Electron microscopy - TEM and SEM

Electron Microscopy is a technique that uses a beam of electrons to create an image of a specimen. It has a much higher resolution than light microscopy, allowing for the visualization of smaller structures. There are two main types of electron microscopy, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).

The resolution of a light microscope increases with a decrease in the wavelength of the light it uses for illumination. Electron beams behave like radiation and can be focused and is used in an Electron Microscope. Wavelength of electron beams is around 0.005 nm, approximately 100,000 times shorter than that of visible light and thus the resolution is enormously increased.  The transmission electron microscope has a resolution roughly 1,000 times more than light microscope.   

Transmission electron microscope (TEM)

A heated tungsten filament in the electron gun generates a beam of electrons that is then focused on the specimen by the condenser. Since electrons cannot pass through a glass lens, doughnut-shaped electromagnets called magnetic lenses are used to focus the beam. The column containing the lenses and specimen must be under high vacuum to obtain a clear image because otherwise electrons will be deflected by collisions with air molecules. The specimen scatters electrons passing through it, and the beam is focused by magnetic lenses to form an enlarged, visible image of the specimen on a fluorescent screen. A denser region in the specimen scatters more electrons and therefore appears darker in the image and electron-transparent regions will appear brighter.

TEM (Prescott−Harley−Klein:Microbiology, Fifth Edition)

Scanning electron microscope (SEM)

This is used to examine the surfaces of microorganisms. While TEM produces an image from radiation that has passed through a specimen, the SEM produce an image from electrons emitted by an object’s surface.

The SEM scans a narrow, tapered electron beam back and forth over the specimen. When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary electrons, and these are trapped by a special detector. Secondary electrons entering the detector strike a scintillator causing it to emit light flashes.  These light flashes are converted to an electrical current and amplified by a photomultiplier. The signal is sent to a cathode-ray tube and produces an image.

SEM(Prescott−Harley−Klein:Microbiology, Fifth Edition)

Feature	TEM	SEM
Image Formation	Transmission of electrons through the specimen	Scanning of electrons across the surface of the specimen
Image Type	2D projection of the internal structure	3D image of the surface topography
Specimen Preparation	Requires thin sections of the specimen	Can be used on bulk samples
Resolution	Higher resolution	Lower resolution than TEM

 

Specimen Preparation for TEM and SEM

Specimen preparation is a crucial step in electron microscopy, as it directly impacts the quality of the images obtained. The specific techniques used vary depending on the type of electron microscope (TEM or SEM) and the nature of the sample.

Specimen Preparation for TEM

Since electrons are quite easily absorbed and scattered by solid matter, only extremely thin slices of a microbial specimen can be viewed in TEM. The specimen must be around 20 to 100 nm thick (This is about 1⁄50 to 1⁄10 the diameter of a typical bacterium) and this thin specimen must be able to maintain its structure when bombarded with electrons under high vacuum. 

Common method for preparing specimen 

1.    Fixation:

o   Chemical fixation: Using chemicals like glutaraldehyde and osmium tetroxide to preserve the sample's structure.

o   Cryofixation: Rapid freezing the sample to preserve its native state.

2.    Dehydration: Removing water from the sample using a graded series of ethanol or acetone.

3.    Embedding: Infiltrating the sample with a resin (e.g., epoxy resin) and polymerizing it to provide support.

4.    Sectioning: Using an ultramicrotome (with a glass or diamond knife) to cut thin sections (50-100 nm) of the embedded sample.

5.    Staining: Enhancing contrast by staining the sections with heavy metal salts (e.g., uranyl acetate, lead citrate).  The lead and uranium ions bind to cell structures and make them more electron opaque, thus increasing contrast in the material.

6.    The stained thin sections are then mounted on tiny copper grids and viewed.

Negative staining - The specimen is spread out in a thin film with either phosphotungstic acid or uranyl acetate. Heavy metals render the background dark, whereas the specimen appears bright. Negative staining is an excellent way to study the structure of viruses, bacterial gas vacuoles, etc.

Shadowing  - A microorganism also can be viewed after shadowing with metal. It is coated with a thin film of platinum or other heavy metal by evaporation at an angle of about 45° from horizontal.  Sothe metal strikes the microorganism on only one side. The area coated with metal scatters electrons and appears light in photographs, whereas the uncoated side and the shadow region created by the object is dark. This technique is useful in studying virus morphology, bacterial flagella, and plasmids.

Freeze-etching procedure – To study shape of organelles within microorganisms, specimens are prepared by the freeze-etching procedure.  Cells are rapidly frozen in liquid nitrogen and then warmed to -100°C in a vacuum chamber. A knife that has been precooled with liquid nitrogen (-196°C) is used to fracture the frozen cells.  Since the frozen cells are very brittle, they break along lines of weakness, usually down the middle of internal membranes. The specimen is left in the high vacuum for a minute so that some of the ice will sublimate away and uncover more structural detail. Finally, the exposed surfaces are shadowed and coated with layers of platinum and carbon to form a replica of the surface. Then specimen to be removed chemically and this replica is studied in the TEM to obtain a three-dimensional view of intracellular structure. Freeze-etching minimizes the danger of forming artifacts because the cells are frozen quickly rather than being subjected to chemical fixation, dehydration, and plastic embedding.

Specimen Preparation for SEM

Specimen preparation is easy, and in some cases air-dried material can be examined directly. Sometimes, microorganisms must first be fixed, dehydrated, and dried to preserve surface structure and prevent collapse of the cells when they are exposed to the SEM high vacuum. Before viewing, dried samples are mounted and coated with a thin layer of metal to prevent the buildup of an electrical charge on the surface and to give a better image.

 

Labelling and storage of slides

Labelling and storage of slides is crucial to ensure the integrity and traceability of prepared specimen slides.

Labelling of electron microscopic slides is essential to distinguish each slide, to obtain details about the sample and for durable storage.  Label Content may include sample ID (initials, date, etc), type of specimen (e.g., tissue, cell, organism), fixation method, name of embedding resin, section thickness, any specific treatments or stains used, etc.  Label may be directly done on the slide using a diamond scribe or a very fine-tipped, permanent marker designed for EM slides or may attach a label where special, chemically resistant labels designed for EM are adhered strongly to the slide.

Storage of EM Slides may be done using containers such as slide boxes or desiccators.  Specialized slide boxes designed for EM slides will have individual slots to protect each slide.  Desiccators are used for long-term storage where a drying agent is used to minimize humidity.

The slides are kept in a dry environment to prevent damage to the sample and label. Generally, room temperature is suitable for EM slide storage and slides are stored under dark since some EM stains can fade over time when stored under light.