Microbiology and Man Lab Exam Review
Microbiology and Man Lab Exam Review BSC 242
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This 6 page Bundle was uploaded by Haley Etheridge on Wednesday March 9, 2016. The Bundle belongs to BSC 242 at University of Alabama - Tuscaloosa taught by Dr. Daryl Lam in Spring 2016. Since its upload, it has received 58 views. For similar materials see Microbiology and Man in Biological Sciences at University of Alabama - Tuscaloosa.
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Date Created: 03/09/16
Microbiology Lab Exam #1 Exercise 12: Glo Germ Hand Wash Education System The Glo Germ Hand Wash Education System was developed to train people to wash their hands more effectively. The lotion contains minute plastic particles (artificial germs) that fluoresce when illuminated with ultraviolent (UV) radiation but are invisible with normal lighting. This provides immediate feedback to the washers as to the effectiveness of their hand washing and provides information about where they have to concentrate their efforts in the future. Nontoxic, synthetic fluorescent “germs” Exercise 31: Introduction to the Light Microscope Bright field microscopy produces an image made from light that is transmitted through a specimen. Because most biological specimens are transparent, the contrast between the specimen and the background can be improved with the application of stains to the specimen. Image formation begins with light coming from an internal or an external light source. Light passes through the condenser lens, which concentrates the light and makes illumination of the specimen more uniform. Refraction (bending) of light as it passes through the objective lens from the specimen produces a magnified real image. Total magnification of the specimen can be calculated by using the following formula: o Total Mag. = Mag. By the Objective Lens (4, 10, 40, 100) X Mag. By the Ocular Lens (Always 10) Image clarity is more difficult to maintain as the magnification increases. Clarity of an image is called resolution. The limit of resolution (or resolving power) is an actual measurement of how far apart two points must be for the microscope to view them as being separate. Numerical aperture is a measure of a lens’s ability to “capture” light coming from the specimen and use it to make the image. Using immersion oil between the specimen and the oil objective lens increases its numerical aperture and, in turn, makes its limit of resolution smaller. In darkfield microscopy, a special condenser is used so only the light reflected off the specimen enters the objective. Phrase contrast microscopy uses special optical components to exploit subtle differences in the refractive indices of water and cytoplasmic components to produce contrast. Fluorescence microscopy uses a fluorescent dye that emits fluorescence when illuminated with ultraviolet radiation. Coursefocus adjustment knob – brings image into focus Finefocus adjustment knob – brings the image into sharpest Exercise 14: Common Aseptic Transfers and Inoculation Methods Aseptically – without contamination of yourself, others, the culture, the sterile medium, or the surroundings. A medium that contains living microbes is called a culture. If a culture contains a single species it is said to be a pure culture. Limiting aerosol production is a safety issue and not an issue of keeping pure cultures pure. Minimize the potential of contamination. Be organized. Place all media tubes in a test tube rack when not in use whether they are sterile or not. Take your time. Never hold a tube culture by its cap. Hold the inoculating loop or needle like a pencil in your dominant hand and relax. Adjust your Bunsen burner so its flame has an inner and outer cone. Broths are used to grow microbes when fresh cultures or large numbers of cells are required. Agar slants are generally used to grow stock cultures that can be refrigerated after incubation and maintained for several weeks. Plated media are typically used for obtaining isolation of species, differential testing, and quantifying bacterial densities. In all cases, using these media requires aseptic inoculation in which a portion of an existing pure culture is transferred to a sterile medium to start a new pure culture. Exercise 2 1: Ubiquity of Microorganisms “Ubiquitous in nature” – this means that the organism being considered can be found just about everywhere. Many microorganisms are free living – they do not reside on or in a specific plant or animal host and are not known to cause disease. They are nonpathogenic. Other microorganisms are pathogens and generally are associated with their host. Even many commensal or mutualistic stains inhabiting our bodies are opportunistic pathogens. That is, they are capable of producing a disease state if introduced into a suitable part of the body. Any area, including sites outside the host organism, where a microbe resides and serves as a potential source of infection is called a reservoir. Exercise 3 10: Bacterial Motility: Wet Mount and Hanging Drop Preparations A wet mount preparation is made by placing the specimen in a drop of water on a microscope slide and covering it with a cover glass. Motility often can be observed at low or high dry magnification, but viewing must be done quickly because of drying of the preparation. You should look for independent darting of the cells. A hanging drop preparation allows longer observation of the specimen because it doesn’t dry out as quickly. A thin ring of petroleum jelly is applied around the well of the depression slide. The jelly causes the cover glass to stick to the slide. The petroleum jelly forms as airtight seal that slows drying of the drop, allowing a long period for observation of cell size, shape, binary fission, and motility. Brownian motion – cells will appear to vibrate in place. With true motility, cells with exhibit independent movement over greater distances. Bacterial Structure and Simple Stains (pg. 153) To be visible, the specimen must contrast with the background of the microscope field. Morphologies o Bacterial cells are much smaller than eukaryotic cells and come in a variety of morphologies (shapes) and arrangements. o Cocci (singular coccus) – round; spheres. o Baccilli (singular bacillus) – rods. o Sprilla (singular sprochetes) – flexible spirals. o Virbrios – curved rods. o Pleomorphism – where a variety of cell shapes may be seen in a given sample. Arrangements o Cell arrangement, determined by the number of planes in which division occurs whether the cells separate after division, is also useful in identifying bacteria. o Diplo – pairs (if the two daughter cells remain attached after division) o Strepto – chains (if the cells continue to divide in the same plane and remain attached to form a chain). o If a second division occurs in a plane perpendicular to the first, a tetrad is formed. o A third division plane perpendicular to the other two produces a cubeshaped arrangement of eight cells called a sarcina. o Staphylo – groups (if the division planes are irregular, a cluster of cells is produced). When examining a slide, look for the most common morphology and most complex arrangement. Exercise 34: Simple Stains Stains are solutions consisting of a solvent (water or ethanol) and a colored molecule. Common basic stains include methylene blue, crystal violet, and safranin. Basic stains are applied to bacterial smears that have been heatfixed. Heatfixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent. Basic stains have a positively charges chromagen, which forms an ironic bond with the negatively charged bacterial cell, thus colorizing the cell. Exercise 35: Negative Stains The negative staining technique uses a dye solution in which the chromogen is acidic and carries a negative charge. The negative charge on the bacterial surface repels the negatively charged chromogen, so the cell remains unstained against a colored background. The negative staining technique is used to determine morphology and cellular arrangement in bacteria that are too delicate to withstand heatfixing. A negative stain can also be used because it produces minimal cell shrinkage. Nigrosin stain Acidic stains have a negatively charges chromogen that is repelled by negatively charged cells. Thus, the background is colored and the cell remains transparent. Exercise 36: Gram Stain Differential stains allow a microbiologist to detect differences between organisms or differences between parts of the same organism. The Gram stain is a differential stain in which a decolorization step occurs between the applications of two basic stains. The primary stain is crystal violet. Iodine is added as a mordant to enhance crystal violet staining by forming a crystal violet iodine complex. Decolorization with an alcohol solution follows and is the most critical step in the procedure. Gramnegative cells are decolorized by the solution (that is, they lost the crystal violet stain) whereas Grampositive cells are not. Gramnegative cells can thus be colorized by the counterstain safranin. Grampositive cells appear purple and Gramnegative cells appear reddishpink. Gramnegative cell walls have a higher lipid content (because of the outer membrane) and a thinner peptidoglycan layer than Grampositive cell walls. The alcohol/acetone in the decolorizer extracts the lipid, making the Gramnegative wall more porous and incapable of retaining the crystal violetiodine complex, thereby decolorizing it. It is possible to over decolorize by leaving the alcohol on too long and get reddish Gram positive cells. It is also possible to under decolorize and produce purple Gramnegative cells. Gram Stain Procedure: 1. Heat fix slide 2. Cover the slide: gram +, unknown mixture, gram – 3. Let it dry and then heat fix again 4. Cover slide with crystal violet stain. Let it sit for 1 min and then rinse. 5. Cover the smear with Gram’s stain for 1 minute. Then rinse. 6. Decolorize with ethanol by allowing it to trickle down the slide until the runoff is clear. Generally, 10 seconds is the maximum time needed for this. 7. Counterstain with safranin stain for 1 minute. Rinse with distilled water. 8. Gently blot dry with bibulous paper. Exercise 15: Streak Plate Methods of Isolation A microbial culture consisting of two or more species is said to be a mixed culture. A pure culture contains only a single species. A commonly used isolation technique is the streak plate. A bacterial sample is streaked over the surface of a plated agar medium. During streaking, the cell density decreases, eventually leading to individual cells being deposited separately on the agar surface. Cells that have been sufficiently isolated will grow into colonies consisting only of the original cell type. Because some colonies form from individual cells and others from pairs, chains, or clusters of cells, the term colonyforming unit (CFU) is a more correct description of the colony origin. A quadrant streak is generally used with samples suspected of high cell density, whereas a simple zigzag pattern may be used for samples containing lower cell densities. Exercise 37: Acid Fast Stains The presence of mycolic acids in the cell walls of acidfast organisms is the cytological basis for the acidfast differential stain. Mycolic acid is a waxy substance that gives acidfast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution. Kinyoun (K) Method is a ‘cold’ stain. The waxy wall of acidfast cells repels typical aqueous stains. The K method uses a slightly more lipidsoluble and concentrated carbolfuschsin as the primary stain. These properties allow the stain to penetrate the acidfast walls without the use of heat but make this method slightly less sensitive than the ZN method. Decolorization with acid alcholol is followed by a contrasting counterstain, such as brilliant green or methylene blue. The acidfast stain is a differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol. Acidfast cells stain reddishpurple; acidfast negative cells stain blue or the color of the counterstain is a different one is used. Exercise 38: Capsule Stain Capsules are composed of mucoid polysaccharides or polypeptides that repel most stains. The capsule stain technique takes advantage of this characteristic by staining around the cells. The capsule remains unstained and appears as a white halo between the cells and the colored background. This technique begins in the negative stain; cells are spread in a film across the slide with an acidic stain and are not heat fixed. Heatfixing causes the cells to shrink, leaving an artificial white halo around them that might be interpreted as a capsule. The capsule stain is a differential stain used to detect cells capable of producing an extracellular capsule. Capsule production increases virulence in some microbes. The acidic stain colorizes the background while the basic stain colorizes the cell, leaving the capsules as unstained, white clearings aroung the cells. Exercise 39: Endospore Stain An endospore is a dormant form of the bacterium that allows it to survive poor environmental conditions. Spores are resistant to heat and chemicals because of a tough outer covering made of the protein keratin. The keratin also resists staining, so extreme measures must be taken to stain the spore. In the SchaefferFulton method, a primary stain of malachite green is forced into the spore by steaming the bacterial emulsion. Alternatively, malachite green can be left on for 15 mins or more to stain the spores. Malachite green is watersoluble and has a low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized with water and counterstained with safranin. Spores may be located in the middle of the cell (central), at the end of the cell (terminal), or between the end and the middle of the cell (subterminal). Spores also may be differentiated based on shapes – either spherical or elliptical (oval) – and size relative to the cell. Upon completion, spores are green, and vegetative and spore mother cells are red. The spore stain is a differential stain used to detect the presence and location of spores in bacterial cells. Reactions to Know S. Epidermidis Gram + (blackpurple) E. Coli Gram – (reddishpink) Mycobacterium Kansasii AcidFast + (reddishpurple) Klebsiella Pneumoniae Capsule + (red background with pink center) Bacillius Subtilis Endospore +
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