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Principles of Microscopy and Staining

Metric System and Properties of Light

Size of Microorganisms

Microorganisms are very small and generally range in size from 10 nanometers to 1 millimeter. In general, microbes are only observable with the aid of a microscope.
The International System of Units (SI), which in French is Systéme Internationale d’Unités, is the system of units used by the global scientific community and is based on seven fundamental units. Each of the seven fundamental units of measurement in the International System of Units is an SI base unit. The SI base unit of length is the meter (m). A meter is approximately the same length as one yard, or three feet. The average height of an adult human (5′7″) is about 1.7 meters. Smaller or larger metric units of length are defined by their relationship in size to the meter. Metric units are based on factors of 10, and prefixes attached to the word meter indicate how many factors of 10 larger or smaller than a meter each unit describes. When discussing the size of microorganisms (microbes), it is necessary to think on a scale much smaller than a meter.

SI Units of Length

Name Abbreviation Decimal Power of 10
Meter m 1.0 100
Centimeter cm 0.01 10–2
Millimeter mm 0.001 10–3
Micrometer µm 0.000001 10–6
Nanometer nm 0.000000001 10–9

The base unit of length is the meter and smaller units are defined based on their relation to the meter.

Microbes are measured on the scale of nanometers or micrometers. The smallest microbes are viruses, such as the influenza or poliovirus. Viruses are between 10 to 500 nanometers in size. The largest viruses are about the same size as the smallest bacteria; bacteria can be 400 nanometers to 5 micrometers in length. A line of 1,000 bacteria cells would be needed to cross the width of a human finger nail. Fungi, such as yeasts, frequently grow to be between 1 and 15 micrometers long. The largest microbes include single-celled parasitic organisms known as protozoa and microscopic algae known as microalgae, and these are only two to four times smaller than what humans could see with their own eyes. Protozoa are approximately 50 micrometers in size, and microalgae are just under 100 micrometers in size. In comparison, a single plant or animal cell is typically about 100 micrometers long, meaning that most single-celled organisms are smaller than individual cells of plants and animals. The small size of viruses, bacteria, and protozoa relative to plant and animal cells allows these organisms to invade and exist intracellularly in plant and animal cells at some point during their life cycle.

Size and Scale of Microorganisms

Microbes include viruses and bacteria that are much smaller than plant and animal cells. Plant and animal cells are approximately 100 micrometers (µm) in length; bacteria and viruses range from around 10 nanometers (nm) to a little over 1 micrometer (µm) in length.
The smallest things that humans can see are about the width of a strand of hair, approximately 100 micrometers wide. Most microbes cannot be directly seen, such as the common bacteria Escherichia coli which is only about 1 µm in length. While individual microbes cannot be seen with the naked eye, bacteria and fungi can in some cases grow in forms that can be seen with the naked eye, such as biofilms (clusters of bacteria that adhere to and grow on exposed surfaces) and mold (type of fungus that grows in long strands made up of many cells instead of growing as single cells). However, the individual bacterial and fungal cells that comprise these structures are still microscopic in size and cannot be distinguished without the aid of a microscope.

Light Wavelength Impacts Microscope Resolution

The wavelength of light directly impacts the resolution of a microscope; long wavelengths result in low resolution and short wavelengths result in high resolution.
Microscopes are scientific instruments that are used to see things that cannot be seen by the naked eye, such as cells, bacteria, and viruses. Microscopes enable direct observation of the properties of of the microbe, including its size, organelles (structures inside cells that have specific tasks), and the molecules that make up its cell wall. Many microscopes use light in order to visualize organisms or small physical structures. Light has unique properties that make it a valuable tool in microscopy. Light behaves like a wave, with peaks and troughs, and also like a particle. When white light is dispersed by a prism, all the colors of the visible spectrum are revealed. A wavelength is the distance between sequential peaks in a wave of light. The wavelengths in the visible spectrum range from 390 to 700 nanometers. Short wavelengths (violet) have a high frequency and more energy, while long wavelengths (red) have low frequency and less energy. The wavelength of light impacts how well the light can be used to clearly see an object under a microscope.

Wavelength of Light

Light can have short or long wavelengths. Wavelengths are measured by the distance between each peak in the wave of light.
The shorter the wavelength of light used by a microscope, the better the resolution, or level of detail of an image, that is seen. Therefore, blue and violet light lead to clearer images than does red light. Images that are blurry are said to have a low resolution as opposed to very crisp and clear images, which have a high resolution. In microscopy, this can be determined by the resolving power of a microscope, or the ability of a microscope to differentiate between two points that are very close to each other.

Because microbes are very small, it is important that microscopes have a high resolving power. If the resolving power of a microscope is too low, it can be difficult to differentiate what is actually being viewed. The resolving power of a microscope is based on its numerical aperture, or measure of the microscope's ability to gather light and resolve details of what is being observed when the object is a specific distance from the lens of the microscope. The numerical aperture of a microscope is unitless because it is a ratio. Numerical aperture values range from 0.025 for a very low-powered microscope in air to 1.5 for high-powered microscopes concentrating light through liquid medium.

Light Can Be Distorted as It Passes through an Object

When objects have different indexes of refraction, the light will bend, resulting in a loss of light and blurry images.
The path of a light wave changes when it hits or moves through an object. Reflection and refraction are two properties that explain what happens to light in these situations. Reflection explains what happens to light at the boundary between two objects. When light hits an object, it can bounce off that object and continue moving at a new angle. When light hits a smooth surface, all waves of light hitting the surface will bounce off it at the same angle. Conversely, when light hits a rough or bumpy surface, light can hit the object and bounce off at many different angles.

Reflection of Light

Light reflects differently off smooth and rough surfaces.
Not all the light that hits an object will be reflected. Instead, some of the light can move into the object that it encounters. Refraction describes the way in which the direction of light bends or changes as it passes from one medium to another, such as when light enters water. Every substance has different properties that impact how fast light is able to move through it. Light bends as it moves between two substances that have a different index of refraction, a measure of how much faster light can travel in a material compared to traveling in a vacuum. For example, waves of light can move more quickly through air than through water. This is why a straw in a glass of water appears to bend at the border between the water and the empty space at the top of the cup even though the straw has not actually been bent. Reflection and refraction are important properties for the function of a microscope. Light waves are reflected off the object being observed and bounce back toward a lens, a curved and transparent object used to focus or disperse light. The lens refracts this light, redirecting the angle of the light's path and focusing it toward a single point, where it can then be seen by the observer. The index of refraction of the lens must be precise so that the light passing through will be directed into the observer's eyes. Otherwise, if the refracted light waves are spread apart, the image will appear blurry.

Reflection and Refraction in Microscopy

The properties of reflection and refraction impact the clarity of an image observed using a microscope.