ERT107 MICROBIOLOGY FOR BIOPROCESS ENGINEERING Pn Syazni Zainul Kamal PPK Bioprocess

Chapter 2: Microscopy Techniques

CO2 : Ability to demonstrate practices in microscopy, staining, sterilization, isolation and identification of bacteria and fungi

Why need to study microscope?

Microbiology concerned with organisms so small they cannot be seen distinctly with the unaided eye Microbiology concerned with organisms so small they cannot be seen distinctly with the unaided eye Thus, it is important to understand how the microscope works and the way in which specimens are prepared for examination Thus, it is important to understand how the microscope works and the way in which specimens are prepared for examination

Unit of measurement

Microscopy Refer to the use of light or electrons to magnify objects Refer to the use of light or electrons to magnify objects The same general principles guide both light and electron microscopy : The same general principles guide both light and electron microscopy : 1) Wavelength of Radiation 2) Magnification of an image 3) Resolving power of the instrument 4) Contrast in the specimen

Wavelength of Radiation Various forms of radiation differ in wavelength, which is the distance between two corresponding parts of a wave. Various forms of radiation differ in wavelength, which is the distance between two corresponding parts of a wave. The human eye distinguishes different wavelengths of light as different colors. (wvlength of 400nm as violet) The human eye distinguishes different wavelengths of light as different colors. (wvlength of 400nm as violet) In addition to light, moving electrons act as waves with wavelengths dependent upon the voltage of an electron beam. In addition to light, moving electrons act as waves with wavelengths dependent upon the voltage of an electron beam. Electron wavelengths are much smaller than those of visible light, and thus their use results in enhanced microscopy. Electron wavelengths are much smaller than those of visible light, and thus their use results in enhanced microscopy.

Magnification Magnification is the apparent increase in size of an object and is indicated by a number followed by an “X” which is read “times.” Magnification is the apparent increase in size of an object and is indicated by a number followed by an “X” which is read “times.” eg. Cells are magnified 16,000 x or 16,000 times Magnification results when a beam of radiation refracts (bends) as it passes through a lens Magnification results when a beam of radiation refracts (bends) as it passes through a lens Light beam slows as it enters glass, and the beam bends Light beam slows as it enters glass, and the beam bends Light also bends as it leave the glass and reenter the air Light also bends as it leave the glass and reenter the air Light beam spread apart as they travel past the focal point and produce enlarged image Light beam spread apart as they travel past the focal point and produce enlarged image

Resolution Resolution (also called resolving power) is the ability to distinguish between small objects that are close together. Resolution (also called resolving power) is the ability to distinguish between small objects that are close together. The better the resolution, the better the ability to distinguish two objects that are close to one another. The better the resolution, the better the ability to distinguish two objects that are close to one another. Modern microscopes can distinguish between objects as close as 0.2 μm. Modern microscopes can distinguish between objects as close as 0.2 μm.

A principle of microscopy is that resolution distance is dependent on A principle of microscopy is that resolution distance is dependent on the wavelength of the electromagnetic radiation the wavelength of the electromagnetic radiation the numerical aperture of the lens, which is its ability to gather light. the numerical aperture of the lens, which is its ability to gather light. Immersion oil is used to fill the space between the specimen and a lens to reduce light refraction and thus increase the numerical aperture and resolution. Immersion oil is used to fill the space between the specimen and a lens to reduce light refraction and thus increase the numerical aperture and resolution.

Contrast Contrast refers to differences in intensity between two objects, or between an object and its background. Contrast refers to differences in intensity between two objects, or between an object and its background. Since most microorganisms are colorless, they are stained to increase contrast. Since most microorganisms are colorless, they are stained to increase contrast. Polarized light may also be used to enhance contrast. Polarized light may also be used to enhance contrast.

Good contrastPoor contrast

Types of microscope Light microscope (use to look at intact cells) Light microscope (use to look at intact cells) Electron microscope (use to look at internal structure or details of cell surfaces) Electron microscope (use to look at internal structure or details of cell surfaces)

Light microscope Types of light microscope : Types of light microscope : 1) bright-field 2) phase contrast 3) dark-field 4) fluorescence 5) confocal

Bright-Field Microscope

bright-field microscope The most common microscope is bright-field microscope. It forms a against a brighter background. The most common microscope is bright-field microscope. It forms a dark image against a brighter background. There are two basic types of bright-field microscope: There are two basic types of bright-field microscope: 1) Simple microscope (Leeuwenhoek microscope) contain a single magnifying lens and are similar to a magnifying glass. contain a single magnifying lens and are similar to a magnifying glass. Capable of approx 300X magnification Capable of approx 300X magnification 2) Compound microscope use a series of lenses for magnification. use a series of lenses for magnification.

Compound microscopes Compound microscopes use a series of lenses for magnification use a series of lenses for magnification view both stained & unstained sample view both stained & unstained sample has 4 objective lenses : has 4 objective lenses : - Scanning objective lens (4X), - low power objective lens (10X), - high power lens or high dry objective lens (40X), - oil immersion objective lens (100X) parfocal microscopes remain in focus when objectives are changed parfocal microscopes remain in focus when objectives are changed

Light from illuminated specimen focused by objective lens (enlarged image) 10 times (10X) Ocular lens further magnify the image, typically another 10 times (10X) multiplying the objective lens & ocular lens magnifications together Total magnification = multiplying the objective lens & ocular lens magnifications together Eg. Total magnification using Eg. Total magnification using 10X ocular lens and a 10X low power objective lens = 100X

Microscope resolution ability of a lens to separate or distinguish small objects that are close together ability of a lens to separate or distinguish small objects that are close together wavelength of light used is major factor in resolution wavelength of light used is major factor in resolution shorter wavelength  greater resolution

Immersion oil is used to fill the space between the specimen and a lens to reduce light refraction and thus increase the numerical aperture and resolution.

working distance — distance between the front surface of lens and surface of cover glass or specimen when it is in sharp focus

Dark-Field Microscope

Dark-field microscope Dark-field microscope utilize a dark-field stop in the condenser that prevents light from directly entering the objective lens. utilize a dark-field stop in the condenser that prevents light from directly entering the objective lens. Only light rays scattered by the specimen enter the objective lens and are seen Only light rays scattered by the specimen enter the objective lens and are seen the specimen appears light against a dark background the specimen appears light against a dark background used to observe living, unstained preparations used to observe living, unstained preparations has been used to observe internal structures in eukaryotic microorganisms has been used to observe internal structures in eukaryotic microorganisms has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis

ath Dark-field stop underneath condenser Condenser produces a hollow cone of light so that the only light entering the objective come from specimen

Phase-Contrast Microscope

Increase contrast between cell & surrounding medium Increase contrast between cell & surrounding medium Cell differ in refractive index from their surrounding, hence it bend some light ray that pass through them Cell differ in refractive index from their surrounding, hence it bend some light ray that pass through them Condenser with anular stop (opaque disk with thin transparent ring) Condenser with anular stop (opaque disk with thin transparent ring) Light passing through unstained cell are retarded Light passing through unstained cell are retarded This effect is amplified by phase ring, leading to the formation of dark image on light background This effect is amplified by phase ring, leading to the formation of dark image on light background

excellent way to observe living cells excellent way to observe living cells studying microbial motility studying microbial motility detecting bacterial structures such as endospores and inclusion bodies that have refractive indices different from that of water detecting bacterial structures such as endospores and inclusion bodies that have refractive indices different from that of water

The Differential Interference Contrast Microscope (DIC) creates image by detecting differences in refractive indices and thickness of different parts of specimen creates image by detecting differences in refractive indices and thickness of different parts of specimen excellent way to observe living cells excellent way to observe living cells live, unstained cells appear brightly colored and three-dimensional live, unstained cells appear brightly colored and three-dimensional cell walls, endospores, granules, vacuoles, and nuclei are clearly visible cell walls, endospores, granules, vacuoles, and nuclei are clearly visible

Fluorescence Microscope

Fluorescence microscope developed by O. Shimomuram, M. Chalfie, and R. Tsien developed by O. Shimomuram, M. Chalfie, and R. Tsien Since UV light has a shorter wavelength than visible light, resolution is increased. Since UV light has a shorter wavelength than visible light, resolution is increased. Contrast is improved because fluorescing structures are visible against a black background Contrast is improved because fluorescing structures are visible against a black background Cells stained with fluorescent dyes (fluorochromes) and bombarded with UV light, they emit visible light and show up as bright orange, green, yellow or other colour against black background Cells stained with fluorescent dyes (fluorochromes) and bombarded with UV light, they emit visible light and show up as bright orange, green, yellow or other colour against black background has applications in medical microbiology and microbial ecology studies has applications in medical microbiology and microbial ecology studies

Confocal Microscopy

confocal scanning laser microscopy (CLSM) creates sharp, composite 3-D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface confocal scanning laser microscopy (CLSM) creates sharp, composite 3-D image of specimens by using laser beam, aperture to eliminate stray light, and computer interface numerous applications including study of biofilms numerous applications including study of biofilms