Portage Learning: Microbiology, Module 3 – MICROSCOPY

Portage Learning: Microbiology, Module 3 – MICROSCOPY micrometer one millionth of a meter nanometer one billionth of a meter resolution and contrast two critical factors that influence our ability to see an object resolution the distance between two objects at which the objects still can be seen as separate greater the closer two objects are to each other the __________________ the resolution requirement contrast the difference in light absorbance between two areas (objects) lower the ________________ the contrast between an object and its background, the harder it will be to see the object bright field microscope simplest form of light, or optical, microscopy/light, most often emitted from a standard halogen bulb, enters the microscope from the base (bottom) and is reflected via mirrors towards the sample condenser before the light reaches the sample, it first passes through a ________________ converging the light beams into a focused area on the sample iris diaphragm controls the amount of light that passes through the sample and into the objective lens objective lens the lens closest to the sample and yields the greatest amount of magnification directly proportional the degree of magnification is ______________________ to the amount of light needed/to image samples clearly at higher magnifications, more light is required ocular lens eyepiece 10x most common power of ocular lens total magnification objective x ocular lens= staining required due to the limitation of resolution on unstained cells, (i.e.) often the flat and transparent regions of a cell may appear invisible under bright field conditions/with various dyes, these regions can become labeled and thus visualized. phase contrast microscope able to visualize certain structures that would otherwise be invisible/ can provide detailed images of live cells without staining./uses specialized condensers and objectives, it amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable/can be used to visualize cell movements dark field microscope can be used to greatly increase the contrast between a specimen and background, resulting in a dark background with bright objects in it/reflects light off of the specimen at an angle/does not permit the visualization of intracellular structures fluorescence microscope takes advantage of fluorescent molecules called fluorophores to visualize cells on a dark background/ energy of the incoming light is in the form of the ultraviolet (UV) spectrum. UV light _____________ excites different fluorophores at varying wavelengths, enabling scientists to use a wide array of colors during imaging GFP, YFP and RFP can be expressed in a cell, nonspecifically illuminating the cell as a whole; linked (coupled) to a normal cellular protein of interest whereby the fluorescent color is indicative of protein movement and localization; or used as tags on molecules or antibodies used to designate the presence (fluorescence detected) or absence (no fluorescence) of a specific protein target confocal (laser scanning) microscope combines the usefulness of fluorescence microscopy with the ability to visualize cells in in 2D or 3D/use lasers to focus on a single plane within an object and with a higher degree of accuracy electron microscope used to visualize incredibly small specimens/ use beams of electrons (rather than light), which have significantly shorter wavelengths than light to increases its resolution capacity to less than 1nm transmission electron microscope (TEM) uses thin slices of a sample, heavily treated and coated in preservatives, placed between the electron beam source and the detector scanning electron microscope uses a beam of electrons but the image is obtained as the electrons reflect off (not through) the surface of the specimen/samples are coated with either gold or palladium to enhance electron reflection/can only be used to generate a detailed three-dimensional 'shell' model of the surface of a specimen STEHM (Scanning Transmission Electron Holography Microscope) uses an electron beam but couples it with a holography technique to study surfaces of proteins and subcellular structures/ has the capacity to magnify subatomic structures up to 20 million times larger than what can be viewed with the naked eye/ has the capacity to resolve 35 pm (a picometer is one-trillionth of a meter, or 10-12) and possibly even smaller stains can be used to examine tissues (muscle or connective), specific types of cells (blood, bacterial, etc) or even organelles within an individual cell (nucleus, ER, etc) gram staining first developed by Hans Christian Gram in 1884 and is still used to this day/ began with the observations that different types of bacteria react differently to various dyes gram-positive bacteria have a thick cell wall with many overlapping strands of peptidoglycan that provides a vital protective barrier to the surround environment crystal violet retained by the thick peptidoglycan cell wall iodine forms a stable complex, effectively trapping the dyes in the cell gram-negative bacteria have a relatively thin peptidoglycan layer followed by an outer membrane composed of lipopolysaccharides (LPS) decolorization wash gram-negative bacteria will initially retain the crystal violet dye; however, by next washing the cells with alcohol, a step referred to as the ________________, the LPS and thin peptidoglycan layers of gram-negative bacteria are unable to retain the dye and the outer membrane is depleted of its color safranin added after decolorization in order to visualize gram-negative bacteria pink by counterstaining with positively charged safranin, gram-negative bacteria appears.... differential stain a generalized term used for any staining technique that separates specimens into further subgroups/most often utilizes at least two dyes tightly adherent cells required to prevent sample loss during the staining and wash steps heat fixation most common method to fix a sample chemical fixation includes the use of paraformaldehyde, ethanol or methanol/this process kills the microorganism and as such, characteristics related to motility (movement) are not possible wet mount small liquid culture containing a microorganism of interest is prepared, added to a slide and then covered with a glass coverslip/ heat fixing is not performed simple staining uses a solution of a positively charged dye such as methylene blue, crystal violet, safranin or fuschin to bind to and stain the negatively charged membrane of the microorganism negative staining the inverse of a simple stain/ stain everything except the microorganism negative charge applying nigrosin (or India ink) to a sample, its _________________ is repelled by the negatively charged membrane resulting in a sharp contrast between the unstained specimen and the now dark background. pathogenic samples a negative stain is only mildly invasive and may not kill the microorganism; as such, a negative stain is contraindicated for ___________________. acid-fast staining a differential stain used to identify bacterial stains showing a high degree of resistance to decolorization mycobacterium tuberculosis the most common use for an acid-fast stain as it is the causative agent of tuberculosis carbolfuchsin acid-fast staining procedure uses the red dye ______________, initially staining all cells red lipid-based (acid-fast) in acid-fast staining, following the decolorization wash step, only cells with a thick ____________ protective membrane remain red giemsa combined with Wright's stain, the resulting combinatorial stain can be applied to blood smears to determine the presence (or absence) of pathogenic bacteria—human (blood) cells appear purple and bacterial cells appear as pink Wright's stain stains blood cells giemsa used for the diagnosis of malaria as well as other blood parasites simple staining simple technique is often used to quickly observe the size, shape and arrangement of cells wet mount used to observe the motility and behavior of an organism