Chapter 3 Observing Microorganisms through a Microscope - PDF

Chapter 3 Observing Microorganisms through a Microscope BIO 214: General Microbiology Outline A Brief History of Microscopy Light Microscopy Preparing Bacterial Smears Simple Stain Gram Stain Other Stains Other Types of Light Microscopy Electron Microscopy Bri...

Chapter 3 Observing Microorganisms through a Microscope BIO 214: General Microbiology Outline A Brief History of Microscopy Light Microscopy Preparing Bacterial Smears Simple Stain Gram Stain Other Stains Other Types of Light Microscopy Electron Microscopy Brief History of Microscopy Anton von Leeuwenhoek was the first person to observe microbial cells (late 17th century) A lens-maker, Leeuwenhoek created a lens with minimal imperfections, allowing him to see cells with Lens 300x magnification Location of At the time, other scientists (like Robert Hooke) used specimen on pin compound microscopes (two lenses in sequence) but Specimen- due to their imperfections, these were not powerful positioning screw enough to see microbes Focusing control Stage- positioning screw Microscope replica Light Microscopy Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Body tube Transmits Use of light for observing specimens the image from the objective lens to the Today, the _________________ light microscope is ocular lens the standard in labs across the world Arm Two (or more) lenses in a series Objective lenses Each lens amplifies the magnification Primary lenses that magnify the specimen Total magnification = ocular lens x Stage Holds the microscope slide objective lens in position Most microscopes have 3 or 4 objective lenses Condenser Focuses 4x (scanning) light through specimen 10x (low power) Diaphragm Controls the amount 40x (high power) of light entering the condenser 100x (oil immersion) Illuminator Light source Coarse focusing knob Base Fine focusing knob Terminology ___________________ – enlarging the appearance of an object E.g. 1,000x Magnification can be changed by rotating the nosepiece ___________________ – the ability to distinguish between two objects Limited by the wavelength of light Resolution is lost if magnification increases beyond 1,000x Even if lenses allowed magnification beyond 1,000x, the image would be blurry due to lack of resolution Resolution cannot be adjusted/changed __________ – adjusting the plane of the specimen so the image can be clearly resolved Accomplished by raising/lowering the stage (bring the specimen closer/further) Focus can be adjusted using the coarse and fine focus adjustment knobs Refractive Index All materials have an intrinsic property called ‘___________________________’ Refers to how light bends as it moves through the material Refraction allows us to see a specimen The specimen (e.g. cells) is made of a different material than the surrounding However, refraction can also cause problems Glass and air have different refractive indexes Light moves through the glass slide, then to air, then into the glass objective lens Results in light being lost, leading to lost resolution Immersion oil is used with the 1,000x objective to minimize refraction Oil has the same refractive index as glass so Preparing a Bacterial Smear Most bacteria are colorless  poor contrast under the microscope To improve contrast, bacteria are stained Most stains are positively charged to bind to the bacterial cell surface which has an inherent negative charge However, bacteria can be washed off the slide during the staining process Before staining, the bacteria must be attached the slide  bacterial smear Accomplished adding bacteria to a slide and passing it briefly over a flame  _____________ The heat also kills the bacteria in the process (cells won’t be moving under the microscope) Simple Stain A simple stain involves a single dye so that all cells, despite differences in physiology, all stain the same color Methylene blue is commonly used E. coli stain blue, S. aureus stains blue, P. aeruginosa stains blue Enables observation of ___________________________ (cell shape and arrangement) Cell Morphology Cell morphology refers to the appearance of cells under the microscope Don’t confuse cell morphology with colony morphology (what colonies look like on an agar plate) Two main attributes: Cell shape Spherical  _________________ Rod-shaped  _________________ Other shapes (e.g. curved, spiral) Arrangement Single cells Chains  strepto- Clusters  staphylo- Others arrangements (e.g. diplo-) Gram Stain The Gram stain is a differential stain Different bacteria stain different colors based on their physiology Named after Hans Christian Gram, who developed the technique Based on cell wall composition (thickness of peptidoglycan) Some bacteria have ____ cell wall composed of a single layer of peptidoglycan  Gram negative Meanwhile, others have ____ cell wall composed of a multiple layers of peptidoglycan  Gram positive Gram Stain First, _____________ are stained purple (crystal violet – primary stain) Then, then the purple color is ‘locked’ into ______________________________ cell wall (iodine – mordant) Next, the purple color is washed out of cell with a _____________________ cell wall (alcohol – decolorizer) Finally, _____________ receive a pink stain (safranin – counterstain) But the cells with a thick cell wall are already purple so the pink color is masked KEY Crystal violet Iodine Alcohol Safranin Gram-positive Gram-negative Application of Application of Alcohol wash Application crystal violet iodine (mordant) (decolorization) of safranin (purple dye) (counterstain) Gram Stain gram-positive gram-negative Acid-Fast Stain Some bacteria have a _________ cell wall that does not stain well/consistently by Gram stain The waxy character is due to _____________________ in the cell wall E.g. Mycobacteria spp Another staining procedure must be used Acid-fast stain Primary stain – carbolfuchsin (with heat) Decolorizer – acid alcohol Counterstain – methylene blue Endospore Stain Some bacteria form hardy structures called endospores than allow them to survive harsh conditions E.g. Bacillus spp (including B. anthracis) and Clostridium spp (including C. tetani and C. botulinum) The cell surrounds its DNA will several protective layers The endospore is initially formed within the bacterial cell, eventually the cell breaks down leaving only the endospore behind Once conditions improve, the endospore can reactivate and transition back into a vegetative cell Endospore stain Primary stain – malachite green (with heat) Decolorizer – water Counterstain – safranin Capsule Stain Some bacteria are covered in a gelatinous material (polysaccharide) called a capsule Capsules are common to bacterial pathogens as they function in _________________________ The capsule isn’t recognized as ‘foreign’ so the immune system doesn’t respond Specifically, the capsule allows the bacteria to ‘hide’ from phagocytes that are looking for threats within the body Due to its nature, the capsule doesn’t stain well To view the capsule, we stain everything but the capsule  _____________________ Capsule stain India ink – provides color to the background Counterstain – stains the cell Any (e.g. safranin, crystal violet, methylene blue) Other Types of Light Microscopy Fluorescence microscopy Some molecules have the ability to absorb light at one wavelength and emit at a different wavelength  _____________________ These molecules are engineered into bacterial to aid in detection Confocal microscopy A special type of fluorescence microscopy that captures multiple planes to produce a 3- dimensional image Electron Microscopy Uses electrons instead of photons (light) The shorter wavelength of electrons gives greater resolution Used for images too small to be seen with light microscopes, such as viruses Two types: ____________________ (TEM): Electrons pass through ultrathin sections of a specimen Internal structures can be seen ____________________ (SEM): Electrons scans the surface of an entire specimen External structures and topography can be seen