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lab manual for microbiology onlineWhile many traditional lab manuals are lengthy and comprehensive, descriptions of the labs in this manual are kept minimal to encourage students to further research the procedures and results on their own. Special thanks go to Sara Selby for editing and photography. Designed to support a course in microbiology, Microbiology: A Laboratory Experience permits a glimpse into both the good and the bad in the microscopic world. The laboratory experiences are designed to engage and support student interest in microbiology as a topic, field of study, and career. The design of the lab manual conforms to the American Society for Microbiology curriculum guidelines and takes a ground-up approach — beginning with an introduction to biosafety and containment practices and how to work with biological hazards. From there the course moves to basic but essential microscopy skills, aseptic technique and culture methods, and builds to include more advanced lab techniques. The exercises incorporate a semester-long investigative laboratory project designed to promote the sense of discovery and encourage student engagement. The curriculum is rigorous but manageable for a single semester and incorporates best practices in biology education. She has written nationally published laboratory textbooks in cell and molecular biology, and is the author of scientific articles published in both scholarly and trade journals. Ahern was named a National Science Foundation American Society for Microbiology (ASM) Biology Scholar in 2008, completing the Biology Scholar Transitions Residency program in 2011. Her goal is to capture student interest in microbiology and science by immersing them in research. Except where otherwise noted, content on this site is licensed under a Creative Commons Attribution 4.0 License. We will email you to let you know after your order. By continuing to browse this site you are agreeing to our use of cookies. Find out more here.
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All shipping options assumes the product is available and that it will take 24 to 48 hours to process your order prior to shipping.By continuing to browse this site you are agreeing to our use of cookies. Agar pours are then added to each plate.Do not store food in areas where microorganisms are stored. 3. Purchase a lab coat and safety glasses, bring them to class, and use them. 4. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals are not allowed) in the Laboratory 5. Keep your workspace free of all unnecessary materials. 6. Disinfect work areas before and after use with 70 ethanol or fresh 10 bleach. Laboratory 7. equipment and work surfaces should be decontaminated with an appropriate disinfectant on a 8. routine basis, and especially after spills, splashes, or other contamination. 9. Label everything clearly 10. Replace caps on reagents, solution bottles, and bacterial cultures. Do not open Petri dishes in the lab unless absolutely necessary. 11. Inoculating loops and needles should be flame st erilized in a Bunsen bur ner before you lay them down. 12. Turn off Bunsen burners when is not in use. Long hair must be restrained if Bunsen burners are in use. 13. Sterilize equipment and materials. 14. Consider everything a biohazard. Do not pour anything down the sink.By inhibiting the growth of microorganisms. Co ntrol of growth usually involves the use of physical or chemical agents which either kill or prevent the growth of microorganisms. Agents which kill cells are called cidal agents; agents which inhibit the growth of cells (without killing them) are r eferred to as static agents. Thus the term bactericidal refers to killing bacteria and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills bacteria, a fungicide kills fungi, and so on. Sterilization is the complete de struction or elimination of all viable organisms (in or on an object being sterilized). There are no degrees of sterilization: an object is either sterile or not. Sterilization procedures involve the use of heat, r adiation or chemicals, or physical removal of cells. Methods of Sterilization Heat: most important and widely used. 1. Incineration: burns organisms and ph ysically destro ys them. Used for needles, inoculating wires, glassware, etc. Kills everything except some endospores. Used for glassware, metal, and objects that won't melt. 4. Pasteurization: s a process of heating a food, which is usually a liquid, to a specific temperature for a predefined length of time and then immediately cooling it after it is removed from the heat. This process slows spoilage due to microbial growth in the food. Unlike sterilization, pasteurization is not intended to kill all micro-organisms in the food. Instead, it aims to reduce the number of viable pathogens so the y are unlikel y to cause disease Irradiation: usually destroys or distorts nucleic a cids. Ultraviolet light is usua lly used (commonly used to sterilize the surfaces of objects), although x-ra ys and microwaves are possibly useful.https://ayurvedia.ch/cse-sata-743-manual Filtration: involves the ph ysical removal (exclusion) of all cells in a liquid or gas, especiall y important to sterilize solutions which would be denatured by heat (e.g. antibiotics, injectable drugs, amino acids, vitamins, etc.) Chemical and gas: Alcohol 70: Antiseptics and disinfectants Formalin:( Formaldehyde)is an organic compound with the formula CH 2 O, used as disinfectants Ethylene oxide gas: This highly reactive gas (C 2 H 4 O) is flammable, toxic, and a strong mucosal irritant, used for contaminated medical tools. Halogens: Chlorine, iodine, and derivatives of these halogens are suitable for use as disinfectants. Chlorine and iodine show a generalized microbicidal effect and also kill spores Irradiation (microwave, UV, x -ray, Gamma radiation ): destroys microorganisms as described under sterilization. Many spoilage organisms are easily killed by irradiation. Figure ? ?: An autoclave is a device used to sterilize equipment and supplies Antiseptics: Microbicidal agents harmless enough to be applied to the skin and mucous membrane. Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe for application to living tissues; they are used on inanimate objects such as tables and floors Preservatives: static agents used to inhibit the growth of microorganisms, most often in foods. If eaten they should be nontoxic The physical form s of the culture media are used: liquid, or broth, media; semisolid media; and solid media. The major difference among these media is that solid (1.5-1.8 agar) and semisolid ( ? 1.0 agar ) media contain a solidifying agent (usually agar), wh ereas a liquid medium does not. Liquid media, such as nutrient broth, tryptic soy broth, or brain-heart infusion broth, can be used to propagate large numbers of microorganisms in fermentation studi es and for various biochemical tests. Semisolid media can also be used in fermentation studies, in determining bacterial motility, and in promoting anaerobic growth. Solid media, such as nutrient agar or blood agar, are used (1) for the surface growth of microorganisms in orde r to observe colony appearance, (2) for pure culture isolations, (3) for storage of cultures, and (4) to observe specific biochemical reactions. Solid media can be poured into either a test tube or Petri plate (dish). If the medium in the test tube is allowed to harden in a slanted position, the tube is designated an agar slant; i f the tube is allowed to harden in an upright position, the tube is designated an agar deep tube and if the agar is poured into a Petri plate, the plate is designated an agar plate Agar: is a gelatinous polymer substance der ived from red algae.Classes of Culture Media ( Purpose) 1. A selective medium contains compounds that inhibit the growth of some microorganisms (antibiotics) but not others. For example, media a re available for the selective isolation of pathogenic strains of E. coli from food products 2. differential medium is one in which an indicator, typically a reactive dye, is added that reveals whether a particular chemical reaction has occurred during growth. Preparing a broth Medium 1. The dehydrated media is weighted and dissolved in a specific amount of water, pH is checked and adjusted according to the manufacturer's instructions.Normal flora on and in our body act as an initial defense against invading pathogens. Normal flora compete with foreign m icroorganisms for nutrients and space, thereb y preventing them from flourishing. In the absence of the normal flora (e.g., due to prolonged antibiotic treatment), opportunistic microorganisms may become establis hed, increasing the risk of developing disease. In the environment, many o rganisms in soil play important geochemical roles in pr ocessing vital elements such as phosphorus and nitrogen, making them a vailable to other living things. Nonetheless, there are organisms in our environment that are harmful: soil also harbors the spores of the tetanus and botulism bacteria. Materials Steril e culture swabs, 9 ml sterile dil ution water in a test tube, nutrient agar plates, nutrient broth and a few sterile tongue depressors for throat swabs. Possible samples: 1. Human body ( Throat, skin, scalp, teeth, ear, under the nails between toes, armpit ) 2. Environment ( Garden soil, bench top (before and after disinfection), sink basin, water tap, 2222222222222222 coin, doorknob, windowsill, air in the lab) Incubator: is a device used to grow and maintain of course microbiological cultures or cell cultures. The incubator maintains optimal temperature, hum idity and other conditions such as the carbon dioxide (CO 2 ) and oxygen content of the atmosphere inside. Swab samples and inoculate plates with your samples. Use a fresh swab for each plate. 3. For pl ates, gentl y, so as not to mess up the agar surface, rub the swab over the entire surface of the Petri dish without going back over areas you have already swabbed. 4. Invert the Petri plates and incubate for 3 to 7 days at room temperature. Observe daily for the appearance of colonies. Figure ? ?: Enumerating soil microorganisms Once a sterile culture medium has been prepared, it is ready to receive an inoculum to start the growth process. This manipulation requires aseptic technique, a series of steps to prevent contamination during manipulations of cultures and sterile cu lture media. A mastery of aseptic technique is required for success in the microbiology l aboratory, and it is one of the first methods lea rned by the novice microbiologist. Airborne contaminants are the most common problem because the dust in laboratory air contains microorganisms. When containers are opened, they must be handled in such a way that contaminant laden air does not enter. Suggested procedure to use to achieve aseptic technique 1. Growth medium and its containers must be sterilized as soon as the medium is prepared. 2. The container must be covered to prevent entrance of microorganisms on dust particle sand aerosols. 3. Always sterilize your bench before and after use.( 70 ethanol). 4. Always sterilize your inoculating loop or needle after you complete the transfer. 5. When your cultures are open do not talk. 6. Never leave the tube open any lon ger than the amount of time needed to transfer the culture. 7. Never enter a culture with an inoculating loop that has not been sterilized. 8. Always flam the lips of the tube both before you insert the loop and after transfer the culture 9. Work quickly an all transfers. 10. Never place the cap or plug on the work surface or let it touch anything except the flamed lip and the culture tube. 11. Wash your hands before and after the laboratory session. Do not lay down loop until procedure is co mplete. Work close to Bunsen flame. Opening of Petri dish (solid medium) Open lid for as short a time as possible. Open lid just enough to insert wire loop. Pass neck through a hot Bunsen flame before insertion and after withdrawal of loop to kill any contaminating organisms. Subculturing is the aseptic transfer o f micro-organisms from a culture t o fresh medium b y one of the transfer instruments ( loop, needle, swab, and pipette) Figure ? ?: Microbiological Transfer Instru ments. (a) Inoculating needle, and (b) inoculating loop,(c) and (d) pipette, (e) plastic pump, (F) rubber bulb. Figure ? ?: Good flaming technique. Flaming begins at the handle and slowly continuous towards the tip, placing a loop in blue cone of flame At some point on the streaks, individual cells will be removed from the loop as it g lides along the agar surface and will give rise to separate colonies Again, one assumes that one colony comes from one cell. The ke y principle of this method is that by streaking, a dilution gradient is established on the surface of the plate as cells are deposited on the agar surface. ? Procedure 1. Flame the inoculating loop to red-hot, allow it to cool near burner in air. 2. Hold the your isolated culture ( previous exercise) in left hand near the flame. Open the lid of the plate just enough to insert wire loop. Aseptically withdraw a loopful culture with loop. 3. Place the inoculum on the agar plate at least 1 cm away from sides.After inoculation discrete bacterial colonies can then be f ormed growing both on the agar media and in agar t hen on the isolated colonies can be a separately picked of the isolation plate and transfer to new sterile medium. One advantage of thi s method is that it requires somewhat less skill than that required for a good streak plate; a disadvantage, however, is that it requires more media, tubes, and plates.. Figure ? ?: The Pour-Plate Technique. The original sample is diluted several tim es to decrease or dilute the population sufficiently. 1 ml of each dilution is then dispensed into the bottom of a Petri plate. Agar pours are then add ed to each plate. Isolated cells grow into colonies and can be used to establish pur e cultures In the sp read plate method, you would dilute the bacterial culture in tubes and then transfer the diluted samples to multiple agar plates. In a sa mple that is adequately diluted, the cells will be spread f ar enough apart on the agar that the y will grow into individual colonies. ? Note: students will use the spread plate method later on in this manual Figure ? ?: A comparison between the pou r plate method and the spread plate method Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the final magnification occurs. The objective lenses are identified as the low -power, high-dry, and oil immersion objectives. It is of particular value for examination of stained smears, especially when used at 1000 x total magnification or greater (usually employing an oil immersion lens), but it provides little information about internal cell structure. 2. Dark-Field microscope: The compound microscope may be fitted with a darkfield condenser that has a numerical aperture (resolving power) greater than the objective. L ight passing through the specimen is diffracted and enters the objective lens, whereas undiffracted light does not, resulting in a bright image against a dark background. Since light objects against a dark background are seen more clearly by the eye than the reverse, dark-field microscop y The phase-contrast microscope permits the observation of otherwise invisible living, unstained microorganisms. In the phase-contrast microscope, the condenser h as an annular diaphragm, which produces a hollow cone of light; the objective has a glass disk (the phase plate) with a thin film of transparent material deposited on it, which accentuates phas e changes produced in the speci men. This phase change is observed in the specimen as a difference in light intensity. Phase plates ma y either retard (positive phase plate) the diffracted light relative to the undiffracted light, producing dark-phase-contrast microscopy, or advance (negative phase plate) the undiffracted light relative to the directed light, producing bright-phase contrast microscopy. Dr.Sulaiman Alnaimat 2013 Fluorescent materials are generally of two kinds: one present naturally in cells and the other include the induced one b y stainin g the object with fluorescent dyes or flurochromes. In fluorescent microscope, an object emits light when examined under UV rays. Radiati ons exciting the luminosity do not contribute to image formation. Such objects absorb radiant energy and re lease trapped energy when excited as visible light quickly to return to more stable state. Two kinds of filters are used for filtering harmful rays. Excitation filters, which transmit only the rays o f visible range while blo cking UV radiations the illumina ting beam onl y excluding the radiations, and Barrier filters prevent passing of excited radiations in microscope to protect e yes from UV rays. Th e technique has become very important in medical microbiology, microbial ecology and study of bacterial pathogenesis. The objects are identified after staining with fluorescent dyes or flurochromes or specifically labeled fluorescent antibodies using immunofluorescence procedures. 5. Electron Microscopy Electron microscopes use electrons instead of visible l ight (photons)to image cells and cell structures. Electromagnets function as lenses in the electron microscope, and the whole system operates in a vacuum. Electron microscopes are fitted with ca meras to allow a photograph, called an electron micrograph, to be taken. a. Transmission Electron Microscopy (TEM) The transmission electron microscope (TEM) is used to examine cells and cell structure at ver y high magnification and resolution. The resolving power of a TEM is much greater than that o f the light microscope, even enabling one to view structures For example, whereas the resolving power of a high-quality li ght microscope is about 0.2 micrometer, the resolving power of a hi gh-quality TEM is about 0.2 nanometer (nm, 1029 m). With such powerful resolution, even individual protein and nucleic acid molecules can be visualized in the transmission electron microscope. Unlike visible light, however, electron beams do not penetrate very well; even a single cell is too thick to reveal its internal contents directl y by TEM. Consequently, special techniques of thin sectioning are ne eded to prepare specimens before observing them. To obtain sufficient contrast, the preparations are treated with stains such as osmic acid, or permanganat e, uranium, lanthanum, or l ead salts. Because these substances are composed of atoms of high atomic weight, they scatter electrons well and thus improve contrast. b. Scanning Electron Microscopy If only the external features of an organism are to be observ ed, thin sections are unnecessary. Intact cells or cell components can be observed directly by TEM with a technique called negative staining. Alternatively, one can image the specimen using a scanning electron microscope (SEM).In scanning electron microscopy, the specimen is coated with a thin film of a heavy metal, such as gold. An electron beam then scans back and forth across the specimen. Electrons scattered from the metal coating are collected and activate a viewing screen to produce an image. In the SEM, even fairly large specimens can be observed, and the depth of field (the portion of the image that remains in sh arp focus) is extremely good. A wide range of magnifications can be obtained with the SEM, from as low as up to about, but only the surfa ce of an object is typically visualized. Note that it has a clamping device, the mechanical stage, whi ch is used for holding and moving the slide around on the stage. Figure ? ??: Electron micrographs. (a) Microgra ph of a thin se ction of a dividing bacterial cell, taken by transmission electron microscop y (TEM). Note the DNA forming the nucleoid. Each hexagonal-shaped molecule is about 25 nanometers (nm) in diameter and consists of two doughnut-shaped rings, a total of 15 nm wide. (c) Scanning electron micrograph of bacterial cells.Note that they are attached to a rotatable nosepiece, which makes it possible to move them into position over a slide. Objectives on most laboratory microscopes have magnifications of 10 ?, 45?, and 100?, designated as low power, high -dry, and oil immersion, respectively. Some microscopes will have a fourth objective for rapid scanning of microscopic fields that is only 4?. C. The third lens system is the condenser, which is located under the stage. It collects and directs the light from the lamp to the slide being studied. The condenser can be moved up and down by a knob under the stage. 5. Focusing Knobs The concentrically arranged c oarse adjustment and fine adjustment knobs on the side of the microscope are used for bringing objects into focus when studying an object on a slide. The ocular lens are 10x magnification. Objective lens are 4x, 10x, 40x, 100x magnification. So once an objective lens is selected, t he total magnification would be g iven by it s product with the 10x magnification of the ocular lens. If the object are closer tog ether than appropriate for your resolution, they blur together, making it impossible to differentiate. ? Refractive index (or index of refraction) n of a substance (optical medium) is a number that describes how lig ht, or any other radiation, propagates through that medium. When light passes from a material of one refractive index to material of another, as from glass to air or from air to glass, it bends. Light of different wavelengths bends at di fferent angles, so that as objects are magnified the images become less and less distinct. Oil imme rsion objective lenses are used to further magnify a specimen. The lens requires more li ght rays to pass through it since it has more mirrors inside, requiring the use of the oil. The oil functions to refract the light rays towards the center of the lens (the normal line of the specimen). By refracting the light towards the center, more rays of light enter the objective, allowing you to see the specimen in a much higher resolution (higher magnification). Do not use paper towels or Kimwipes; they can scratch the lenses. Do not remove the oculars or any other parts from the body of the microscope. 3. Always focus the object by moving the Objective away from the glass slide. Avoid focusing downwards. 4. With coarse adjustment knob move the Objective to be used until it nearly touches the surface upper surface of the mounted specimen. Then focus by moving the coarse knob upwards until the object comes into view. Complete the focusing with Fine adjustment knob. 5. Adjust the mirror and light while using low power Objective to give adequate illumination. 6. While observing unstained objects, the Iris diaphragm should be barel y open to achieve good contrast. Iris diaphragm is fully op en with higher magnification and while viewing stained objects. 7. Always, clean the lenses before and after use with lens paper. Do not to uch the lenses with hands. Leave Objectives with lowest power in working position. 8. Keep the microscope covered when not in use. 9. Observe the slides with both eyes open. Do not incline the microscope. Adjust the stool to use the instrument comfortably. 10. Avoid direct sunlight. North light is advantageous. 11. Place a drop of immersion oil on the illuminated area of slide and shift to 100 X objective. Lower the Objective slowly viewin g from sides until the lens contacts the oil drop and then the surface of slide. 12. Prior you place the microscope in wooden box at the conclusion of each laboratory period turn the nosepiece until the low power objective is in place and lower to the max imum until it reaches stop. To prepare the slide. Place a drop of fluid in the center of the slide. Position sample on liquid, using tweezers. At an angle, place one side of the cover slip against the slide making contact with outer edge of the liquid drop. Lower the cover slowly, avoiding air bubbles. Remove excess water with the paper towel Figure ? ??: Preparation of a wet mount slide To study their properties and to differentiate microorganisms into specific groups for diagnostic purposes biological stains and staining procedures in conjunction with light microscopy have become major tools in microbiology. What are stains and why do they stain cells 1. Stains differentiate microorganisms from their surrounding environment 2. They allow detailed observation of microbial structures at high magnification 3. Certain staining protocols can help to differentiate betwee n different types of micro- organisms. 4. A stain (dye) is a salt composed of two ions, and one will contain the chromogen ( the colored part of the molecule) a. Positive ion (cation). b. Negative ion(anion) 5. Most dyes used are basic dyes with a positive chromogen. The interior of the cell ( cell’s cytoplasm) is negatively charged. ? The negative cytoplasm attracts the positively charged chromogen. The ability of a stain to bind to macromolecular cellular components such as proteins or nucleic acids depends on the electrical charge found on the chromogen portion as well as on the cellular component to be stained. Commonly there are two types of stains - 1. Acidic stains (anionic) - Example: Picric Acid 2. Basic stains (cationic) - Example: Methylene Blue It is used for visualization of morphological shape (cocci, bacilli and spirilli) and arrangement (chains, clusters, pairs and tetrads) of bacter ia. 2. Differential staining is the use o f more than one staining reagent to bring out differences in mi crobial cell types, or to differentiate particular cellular components from the rest of the cell body These cultures must be diluted by placing a loopful of water on the slide in which the cells will be then emulsified. Transfer of cells from the culture requires the use of a sterile inoculating needle. Only the tip of the needle should touch the culture to prevent the transfer of too many cells. Suspension is accomplished by spreading the cells in a circular motion in a drop of water with the needle tip. The finished smear should occupy an area about the size of a nickel and should appear as a semi-transparent, confluent, whitish film. At this point, the smear must be allowed to dry completely. Avoidance of thick, dense smears is absolutely essential. NOTE: Students should use their own isolated bacteria to prepare this smear Heat fixation is performed by the rapid passage of the air dried smear 2-3 times over the flame of the Bunsen burner. \ Figure ? ??: Bacterial Smear Preparation. Some of the most commonl y used dyes for simple staining are methylene blue, basic fuchsin, and crystal violet. All of these dyes work well on bacteria because they have color-bearing ions (chromophores) that are positively charged (cationic). The fact that bacteria are slightly negatively charged produces a pronounced attraction between these cationic chromophores and the organism. Such dy es are classified as basic dyes (methylene blue ). Those dyes that have anionic chromophores a re called acidic dyes. Eosin is such a dye. Exposure times differ for each of above stains: carbon fuchsin requires 15 to 30 seconds, crystal violet 2 to 6 seconds, and methylene blue 1 to 2 minutes. Material Required Cultures: 24-hour nutrient agar slant cultures or nutrient broth of students’ isolated bacteria Reagents: Methylene blue and crystal violet stain Equipment:Bunsen burner,inoculating loop,staining tray, microscope, lens paper and glass slide. A. Direct stain: Procedure 1. Take clean glass slides, wash and dry them. 2. Prepare bacterial smears of both the cultures. All smears must be heat fixed prior to staining. 3.