Microorganisms Microscopy

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Ways to measure microorganisms

Micrometer and nanometer

Microscopy

The use of light or electrons to magnify small objects

Types of microscopy

Light microscopy, Scanning Electron Microscopy, and Transmission Electron Microscopy

Anton van Leeuwenhoek

A scientist who first observed living microorganisms using a simple homemade microscope by holding toward a light source, studying rainwater, feces, and scrapings from teeth

Light microscopy

The use of visible light and lenses to magnify and view microorganisms

Types of light microscopy

Brightfield, phase-contrast, fluorescence

Compound light microscope

Used in brightfield microscopy, two sets of lenses, light from illuminator passes through condenser which focuses light on specimen then the light passes through the specimen, into the objectives lens then ocular lens

Magnification

Process of enlarging an object in appearance only, total magnification calculated by multiplying ocular lens and objective lens

Resolution (Resolving power)

The ability to tell apart two close points as separate. Light microscope lights only up to 0.2um

Oil immersion

Used to reduce refraction and improve resolution because at 100x light is lost from the refraction

Brightfield Microscopy

A light microscopy method using white light and a bright background. Stains specimens which kills them, can't see viruses

Phase-Contrast Microscopy

View living, unstained cells, uses light microscope with special condenser, can't see viruses

Fluorescence Microscopy

Fluorescent substances absorb UV light and emit visible light, can't see viruses

Fluorochromes

Fluorescent dyes used to stain cells

Fluorescent-antibody technique

A diagnostic method using fluorescent antibodies to detect pathogens, fluorescent antibodies bind to specific pathogens, making them glow. Commonly used to rapidly diagnose infectious diseases

Electron microscopy

A technique that uses a beam of electrons instead of light to see very small microbes and cell structures. Has smaller wavelength to see tiny details

Types of electron microscopy

Transmission and scanning

Transmission Electron Microscopy

Electrons pass through a thin slice of a sample stained with metal dyes, create 2D images of internal structures, can see viruses not living microorganisms. High magnification

Scanning Electron Microscopy

scans the surface of a metal-coated sample with electrons, creating the release of secondary electrons which are collected to produce 3D images of surface structures. Surface view, low magnification, surface details, image of non-living cells and viruses

Steps to prepare specimen for brightfield microscopy

1. Need to go through fixation to stick on the slide; spread thin layer film material on glass slide then dried, then passed through flame to fix. Fixation kills the cells to not wash off. 2. Smear stained by flooding slide with staining solution

Chromophore

Colored part of a stain molecule

Basic vs Acid stains

Basic stain with positively charged chromophore that stains negatively charged bacterial cells. Acidic stain with a negatively charged chromophore that stains the background to make cell more visible

Why do microorganisms need to be stained

They are colorless under brightfield microscope and hard to see

3 main types of staining techniques

Simple stains, differential stains, special stains

Simple Stains

Stain that uses one basic dye to color the entire cell to be able to observe cell shape and basic structure; crystal violet, methylene blue, safranin

Mordant

A chemical that increases dye binding to the cell

Differential Stains

A stain that uses multiple dyes to distinguish different bacteria

Types of differential stains

Gram stain and acid-fast stain

Gram Stain

Classifies bacteria into gram positive and gram negative, reveals cell wall structure and antibiotic susceptibility

Gram Stain process

1. Primary stain - crystal violet then washed off. 2. Covered with mordant - iodine. 3. Washed off with decolorizing agent - alcohol, gram negative is colorless. 4. Slide is stained with counterstain - safranin. Cocci are purple gram positive, and rods are gram negative pink

Why is counterstain needed in gram staining

To see gram negative cells

Acid-fast stain

Classifies cells into acid-fast and non acid-fast bacteria, used to identify mycobacterium species

Acid fast bacteria steps

1. Primary stain - carbofuchsin added then heated to penetrate the waxy cell wall then cooled and rinsed. 2. Decolorizing agent - acid alcohol applied to remove red dye from non-acid fast. 3. Counterstain - methylene blue added which sticks on non-acid

Mycolic acid

Waxy lipids mycobacterium have in their cell wall making it difficult to stain, only in acid fast

Special Stain

Used for visualizing specific structures like capsules, endospores, and flagella

Capsule

polysaccharide layer that increases bacterial harmfulness

Negative staining process

1. Add acidic nigrosin to stain the background. 2. Add safranin basic used to stain cells. Appears as clear halo around the cell

Endospores

Dormant, highly resistant bacterial structures, like bacillus and clostridium. They help bacteria survive in adverse conditions like heat drying or toxic. Normal staining cannot go through its wall

Endospores staining steps

1. Primary stain - malachite green added then heated to go through the wall. 2. Wash with water to remove stain from some cells 3. Counterstain - safranin added.

Flagella

Structures that are used for cellular motility, they are too small to be seen with microscope

Flagella staining steps

1. Used mordant to increase the diameter of it. 2. Stain with carbolfuchsin to see it