Microbio ch 3 - Lecture notes 3 PDF

Preview

Summary

unit 3 with hughes...

Description

Yokebed Alvarado Microbiology Chapter 3 Microscopy: the construction and use of microscopes  Began in the 17th century with antony can leeuwenhoek and his little hand held microscopes Culture: to do experiments on microorganisms, microbiologists need to culture (provide suitable conditions for their growth and multiplication in the lab) Properties of light  Radiowaves are at the long wave end of the spectrum  Gamma waves(produced in the nucleus of radioactive atoms) are at the other end. They are smaller than a virus  Light is near the middle of the spectrum

Wavelength: the distance between two peaks (high points) of a wave is a wavelength.  Blue light is the shortest wavelength  Red is the longest  Yellow and green are intermediate  White is a mixture of all wavelengths Intensity: the intensity of a wavelength is the height of the wave When a ray of light strikes an object, three things can happen reflected: the light bounces back. Reflected rays bounce off a smooth surface as a ball would. If a ray strikes the surface straight, it bounces straight back. If a ray stikes at an angle, it strikes back at that same angle.  Transmitted: When a ray of light hits a clear object such as pure water or clear glass it is transmitted undiminished.  Absorbed: if a ray strikes and object and the object is not clear (smoked or colored) some light is absorbed. If all wavelengths of light are absorbed equally, the intensity of

Yokebed Alvarado Microbiology Chapter 3 the light is diminished but the color is unchanged. However, if certain wavelengths are absorbed more than others, the color changes.  A solution of red dye is red bc it absorbs the blue of white light and allows the red to pass through. Refraction: occurs when a ray of light meets at an angle a substance of a different density. When the light ray enters the denser substance (from air to glass of a lens, for ex) it slows down  The amount of bending that occurs depends on the angle and how much the light is slowed down  The greater the angle the greater the slowing, the greater the refraction (the bending) is Refractive index: the speed of light in any material is determined by the materials refractive index [the ratio of the speed of light t raveling in a vacuum to its speed in that particular material]  The higher the density, the higher the refractive index is.  Ex the refractive index of pure water is 1.33 (light travels 1.33 times slower in water than in a vacuum) Diffraction: a process that interferes with the ability of a lens to form a sharp image.  Diffraction occurs when rays of light pass through a small opening or pass by the edge of an opaque object.  Light rays bend when they go through a small opening or around an obstacle. Because of diffraction, a sharp edge of an opaque object casts a fuzzy shadow Microscopy

Convex lens: thicker in the center than at the edge Contrast: refers to a difference in light intensity  Contrast is usually created by light absorption: different amounts of light are absorbed as light rays pass through different regions of the specimen

Yokebed Alvarado Microbiology Chapter 3  Bc bacteria are almost colorless they absorb very little light. To obtin useful images of bacteria and most microorganisms contrast must be increased artificially. One way is to apply stains, dyes that color microbial cells or parts of them Resolution: the second requirement for obtaining a useful image is the ability to distinguish detail within an image  Resolution is measured in terms of resolving power. Three factors determine three resolving power of a microscopeo 1. The size of its objective lens o 2. The wavelength of the light passing though the specimen o 3. The refractive index of the material between the objective lens and the specimen *the third factor that influences resolving power of alight microscope is the refractive index of the material in the space between the objective lens and the specimen.  The greater its refractive index the greater the resolving power Oil immersion lenses: designed to have this space filled with oil, permitting more useful magnification.  Two factors that determine resolving powero Lens size, use of immersion oil o Properties of the lens  A measure of these two factors- numerical aperture(NA). its stamped on the side of a microscopes objective lens  d = Wavelength/ 2NA  d = distance between the two points light microscope: microscopes that use visible light as a source of illumination compound microscope: have two lenses, an objective lens, and an ocular lens condenser: concentrates the light onto the specimen where some of it is absorbed objective lens: after the condenser, the transmitted light enters the objective lens which forms an image of the specimen within the body tube of the microscope. Ocular lens: further magnifies the image within the body tube and projects it to the last lens in the series Wetmount: way to prepare a specimen for microscopic examination  place a drop of the specimen on a slide  then cover with a cover slop stains: are dyes that increase contrast by binding selectively to certain cells or to certain parts of them  they reveal structures that would otherwise be invisible

Yokebed Alvarado Microbiology Chapter 3 vital stains: stain living cells fixed: killed and attached to slide *microbes can be fixed either by heating or by treating with chemicals *fixation brings difficulties. It often distorts the cells appearance and of course motility and can no longer be studied Basic dyes: dye with positive charges Acidic dyes: dye with negative charges Mordants” intensify staining by increasing a cells affinity for a dye *staining procedures can be groups into three classesSimple stains: use basic dyes to make cells visible. they stain all cells that absorb the dye the same color. Differential stains: used to distinguish between types of microorganisms. Has three steps primary staining: same as simple staining  destining: treatment that removes stains from certain cells  counterstaining: the application of another dye to reveal the cells or parts of cells that have been distained The gram stain: divides bacteria into two groups- the gram positives and gram negatives  gram negatives are pink, and gram positives are a darker purple fast acid stain: detects tuberculosis (,: special stains: Culturing microorganisms Culture: cultivate microorganisms in the laboratory Pure culture: one consisting of a single type of microorganism derived from a single cell.  Such pure cultures exist rarely in nature  Must be obtained artificially Mixed cultures: many microorganisms living together. occur In their natural environment- soil, water, or the human body To obtain a pure culture:  1.All materials must be sterilized to eliminate all microorganisms present  2.One single microbial cell is isolated and cultivated to produce a clone Sterilization: Eliminating all microorganisms  microbiologists use heat, filtrations, and chemicals to sterilize Moist heat: exposure to steam  more effective than dry heat  autoclave: a pressurized container designed to sterilize materials with moist heat. It resembles a larger version of an ordinary home pressure cooker

Yokebed Alvarado Microbiology Chapter 3 o towels, fabrics, glassware, pipettes, and empty flasks can be sterilized in an autoclave Dry heat: heating in an over or by direct exposure to flame Filtration: microbial cells can be removed from liquids or gases by filtrations  it is time consuming and expensive  filtration does not remove most viruses  filters are called membrane filters, they are sheets of uniformly porous nitrocellulose  a liquid is filtered by pouring it onto a membrane filter fitted to a filter flask  then a vacuum is applied to pull the liquid through the filter, leaving the microorganisms behind chemicals: most chemicals cannot be used to sterilize culture media because residues remain that are toxic to microorganisms.  Sodium hypochlorite (household bleach) is commonly added to cultures after experiments to kill them when dealing with dangerous microbes. It kills most microorganisms. Isolation: how to isolate a pure culture  1.seperate a single cell from all others  2.provide it with the nutrients and environment it needs to grow. To isolate a single cell the population must be diluted to that single cell. Dilution is done in one of three ways: the streak plate method: a population of microorganisms is picked up with a a sterile wire inoculation loop. Its diluted by moving the loop back and fort on the surface of an agar solidified medium in a petri dish.  As the loop is streaked back and forth fewer and fewer microorganisms are deposited on the surface.  Then the loop is sterilized in a flame and used to streak the plate again.  Repeating this process several times sufficiently dilutes the microbial population so that individual cells are deposited separately on the agar surface.  The plate Is the incubated until it grows colonies  You cant be sure that the plate is fully a pure culture so you make a second one starting with an isolated colony from the first plate. The second one is probably a pure culture

The pour plate and spread plate method:  Dilutions are made before samples are put on the plate. These dilutions must be enormous.  Its like adding a tablespoon of suspension to a swimming pool.

Yokebed Alvarado Microbiology Chapter 3  The practical solution is to make serial solutions  In the pour plate method, the diluted sample is added to melted agar containing medium mixed and poured into a Petri dish.  The diluted sample is poured onto the surface of agar solidified medium in a plate and spread evenly with a sterile glass rod. Growing a pure culture: if a culture is going to be used it must be propagated (grown)  To grow microorganisms in the lab the nutrients they need must be supplied in the culture medium. The medium could be solid or liquid. Types of culture media: Defined media: is prepared from pure chemicals so its exact chemical composition is known.  Preparing a defined media is time consuming Minimal medium: a defined medium that has just enough ingredients to support growth  The number of ingredients that must be added to a minimal medium varies enormously depending on which microorganism is being grown  Fastidious: organisms need many organic materials as part of the ingredients needed to grow them Complex media: made from extracts of natural materials such as beef blood casein heast and soy beans. Such extracts contain large numbers of components so their precise chemcical composition is not known Broth: a liquid complex medium  Casein is a common component of complex media  Peptone: partially hydrolyzed casein  Casein hydrolysate: completely hydrolyzed casein Selective media: favor the growth of particular microorganisms  They are used to isolate or detect the favored species in a complex mixture of other microorganisms.

Compound light using oil immersion ◦ What is the source of light? ◦ What path does the light travel? ◦ What is unique or special about this type of microscope or the types of samples it can view? Fluorescence ◦ What is the source of light? ◦ What path does the light travel? ◦ What is unique or special about this type of microscope or the types of samples it can view?...