《微生物生物学》课程教学资源（书籍文献）Mcgrawhill - Laboratory Exercises In Microbiology，fifth edition，Harley Prescott

Front Matter Precautions for Laboratories Blood and other body fluids from all patients should be considered infective. 5. 1.All specim situations in which there is n nve,and the Ca secure should be taken when collecting each specimen to be followed. avoid contaminating the outside of the container 6.Laboratory work surfaces should be and of the laboratory form accompanying the y fluids sing blood and body-fluid nd when work activities are completed specimens should wear gloves.Masks and 7.Contaminated materials used in laboratory tests protective eyewear s nated membran of infec ce washed after completion of specimen prcessing 8.Scientific equipment that has been contaminated 3.For routine procedures,such as histologic and with blood or other body fluids should be logic culturing.a 9.All persons should wash their hands after used whenever procedures are conducted that completing laboratory activities and should remove have a high potential for generating droplets ve clothing before Summary of Universal Precautions and Laboratory Safety Procedure

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition Front Matter Summary of Universal Precautions and Laboratory Safety Procedures © The McGraw−Hill Companies, 2002 Precautions for Laboratories Blood and other body fluids from all patients should be considered infective. 1. All specimens of blood and body fluids should be put in a well-constructed container with a secure lid to prevent leaking during transport. Care should be taken when collecting each specimen to avoid contaminating the outside of the container and of the laboratory form accompanying the specimen. 2. All persons processing blood and body-fluid specimens should wear gloves. Masks and protective eyewear should be worn if mucous￾membrane contact with blood or body fluids is anticipated. Gloves should be changed and hands washed after completion of specimen processing. 3. For routine procedures, such as histologic and pathologic studies or microbiologic culturing, a biological safety cabinet is not necessary. However, biological safety cabinets should be used whenever procedures are conducted that have a high potential for generating droplets. These include activities such as blending, sonicating, and vigorous mixing. xiv Summary of Universal Precautions and Laboratory Safety Procedures 4. Mechanical pipetting devices should be used for manipulating all liquids in the laboratory. Mouth pipetting must not be done, 5. Use of needles and syringes should be limited to situations in which there is no alternative, and the recommendations for preventing injuries with needles outlined under universal precautions should be followed. 6. Laboratory work surfaces should be decontaminated with an appropriate chemical germicide after a spill of blood or other body fluids and when work activities are completed. 7. Contaminated materials used in laboratory tests should be decontaminated before reprocessing or be placed in bags and disposed of in accordance with institutional policies for disposal of infective waste. 8. Scientific equipment that has been contaminated with blood or other body fluids should be decontaminated and cleaned before being repaired in the laboratory or transported to the manufacturer. 9. All persons should wash their hands after completing laboratory activities and should remove protective clothing before leaving the laboratory. 10. There should be no eating, drinking, or smoking in the work area

PART ONE Microscopic Techniques of the ing the and must be she conte (French naturalist.174189 to and fluorescence are most commonly used.In fact,the same microscope may of ypes bright-field a cusing the (b)prope scopic observations. Those little nimals were everywhere!He told the Royal now fifty he is an There wasa y.then Antony van Leeuwenhoek(1632-1723) (

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques Introduction © The McGraw−Hill Companies, 2002 1 PART ONE Microscopic Techniques The most important discoveries of the laws, methods and progress of nature have nearly always sprung from the examination of the smallest objects which she contains. Jean Baptiste Pierre Antoine Monet de Lamarck (French naturalist, 1744–1829) Microbiologists employ a variety of light microscopes in their work: bright-field, dark-field, phase-contrast, and fluorescence are most commonly used. In fact, the same microscope may be a combination of types: bright-field and phase-contrast, or phase-contrast and fluorescence. You will use these microscopes and the principles of microscopy ex￾tensively in this course as you study the form, structure, staining characteristics, and motility of different microorgan￾isms. Therefore, proficiency in using the different micro￾scopes is essential to all aspects of microbiology and must be mastered at the very beginning of a microbiology course. The next five exercises have been designed to accomplish this major objective. After completing at least exercise 1, you will, at the minimum, be able to demonstrate the ability to use a bright-field light microscope. This will meet the American Society for Microbiology Core Cur￾riculum skill number 1 (see pp. vi–viii): (a) correctly setting up and focusing the microscope; (b) proper handling, cleaning, and storage of the microscope; (c) correct use of all lenses; and (d) recording micro￾scopic observations. Antony van Leeuwenhoek (1632–1723) Leeuwenhoek was a master at grinding lenses for his micro￾scopes. Working in Delft, Holland, in the mid-1600s, he is considered the greatest early microscopist. Leeuwenhoek was a manic observer, who tried to look at everything with his microscopes. Those little animals were everywhere! He told the Royal Society of finding swarms of those subvisible things in his mouth—of all places: “Although I am now fifty years old,” he wrote, “I have uncommonly well-preserved teeth, because it is my custom every morning to rub my teeth very hard with salt, and after cleaning my teeth with a quill, to rub them vigorously with a cloth. . . .” From his teeth he scraped a bit of white stuff, mixed it with pure rainwater, stuck it in a little tube onto the needle of his microscope, closed the door of his study— As he brought the tube into focus, there was an unbelievable tiny creature, leaping about in the water of the tube. . . . There was a second kind that swam forward a little way, then whirled about suddenly, then tumbled over itself in pretty somersaults. . . . There was a menagerie in his mouth! There were creatures shaped like flexible rods that went to and fro . . . there were spirals that whirled through the water like violently animated corkscrews. —Paul de Kruif Microbe Hunters (1926)

1.Bright-Field Light EXERCISE Bright-Field Light Microscope and Microscopic Measurement of Organisms SAFETY CONSIDERATIONS Slides and coverslips are glass.Be careful with them.Do Medical Application n t It may result ina shattered or broken slide or lens. Materials per Student Why Are Prepared Slides Used in This Exerise? prepareiofpes of bacteri are be isan el place in using the y the end of the lab dropper with bulb newspaper or cut-out lettere's tweezers Learning Objectives on prepared slides of ious bacteria,fung Each student should be able to 上k也iap wet-mount Principles 4.Uderstand how microorgnisms can be measured The bright-field light microscope is an instrument under the light microscope &m ne o ns. ach ler ay he rotatedin Suggested Reading in Textbook ment with the eyepiece or ocular lens in which the final.The objective ses are icroscope,section2.2:see dcrnii ry,a oil im 2.See tables 2.1 and 34.1 other terms.These terms give either the linear mag

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 2 EXERCISE Bright-Field Light Microscope and Microscopic Measurement of Organisms Materials per Student compound microscope lens paper and lens cleaner immersion oil prepared stained slides of several types of bacteria (rods, cocci, spirilla), fungi, algae, and protozoa glass slides coverslips dropper with bulb newspaper or cut-out letter e’s tweezers ocular micrometer stage micrometer Learning Objectives Each student should be able to 1. Identify all the parts of a compound microscope 2. Know how to correctly use the microscope— especially the oil immersion lens 3. Learn how to make and examine a wet-mount preparation 4. Understand how microorganisms can be measured under the light microscope 5. Calibrate an ocular micrometer 6. Perform some measurements on different microorganisms Suggested Reading in Textbook 1. The Bright-Field Microscope, section 2.2; see also figures 2.3–2.6. 2. See tables 2.1 and 34.1 Medical Application In the clinical laboratory, natural cell size, arrangement and motility are important characteristics in the identification of a bacterial pathogen. Why Are Prepared Slides Used in This Exercise? Because this is a microbiology course and most of the mi￾croorganisms studied are bacteria, this is an excellent place to introduce the student to the three basic bacterial shapes: cocci, rods, and spirilla. By gaining expertise in using the bright-field light microscope, the student should be able to observe these three bacterial shapes by the end of the lab period. In addition, the student will gain an appreciation for the small size and arrangement of procaryotic cell structure. One major objective of this exercise is for the student to understand how microorganisms can be measured under the light microscope and to actually perform some mea￾surements on different microorganisms. By making mea￾surements on prepared slides of various bacteria, fungi, algae, and protozoa, the student will gain an appreciation for the size of different microorganisms discussed through￾out both the lecture and laboratory portions of this course. Principles The bright-field light microscope is an instrument that magnifies images using two lens systems. Initial magnification occurs in the objective lens. Most mi￾croscopes have at least three objective lenses on a ro￾tating base, and each lens may be rotated into align￾ment 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 immer￾sion objectives. Each objective is also designated by other terms. These terms give either the linear magni￾SAFETY CONSIDERATIONS Slides and coverslips are glass. Be careful with them. Do not cut yourself when using them. The coverslips are very thin and easily broken. Dispose of any broken glass in the appropriately labeled container. If your micro￾scope has an automatic stop, do not use it as the stage micrometer is too thick to allow it to function properly. It may result in a shattered or broken slide or lens. 1

Microscopic Technique .in 8 e11Te0i1 Figure 1.2 Pret ion of a Wet-mount Slide.(a)Add a operating i air and with ray ot water to edge of the water.(d)S prevent forming and trapping air imen when in focus and the tip of the objective lens.For example,the low-power objec ve is alsc the millimeter (mm),objective: oil im called the90.100r13m objective.As the magnification increases,the size of the lens at the tip of the objective becomes progres denser and iris diaphragm are required when small The conden viewd ight Prepare a wet-mount as illustrated in figure 1.2.Place the glass slide on the stage of the When the oil immersion lens is used,immersion oil microscope nd sce fimly usng stae cope rely in it Mo the slide until Be the lettere is over the opening in the stage. Th evepiece,or ocular.at the top of the tbe magnifies Vith the low the image formed by the objective lens.As a result,the the tub bjective is withi ation seen by 5.Look into the microscope and slowly raise the ple,when using the x ocular and the 43x objective. tube by tuing the total magnification is 10 x 43 =430 times. view.Once the o t is in y adjustment knob to focus the desired image Open and close the diaphragm,and lower and raise the con nser,noting wha these acton 1.Always carry the microscope with two hands.Place ne is used with the substase o咖h the oper ant away fr nser in its topm st position.The diaphragm lens paper and lens Ibe open and then down unt 7. the oil im the stained bacteria that are provided (figure 1.).The body of he microscope directions for using this lens are as follows:First locate Bright-Field Light Mic and micro pic measurement of oreanisms

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 fication or the focal length. The latter is about equal to or greater than the working distance between the specimen when in focus and the tip of the objective lens. For example, the low-power objective is also called the 10×, or 16 millimeter (mm), objective; the high-dry is called the 40×, or 4 mm, objective; and the oil immersion is called the 90×, 100×, or 1.8 mm objective. As the magnification increases, the size of the lens at the tip of the objective becomes progres￾sively smaller and admits less light. This is one of the reasons that changes in position of the substage con￾denser and iris diaphragm are required when using different objectives if the specimens viewed are to be seen distinctly. The condenser focuses the light on a small area above the stage, and the iris diaphragm con￾trols the amount of light that enters the condenser. When the oil immersion lens is used, immersion oil fills the space between the objective and the specimen. Because immersion oil has the same refractive index as glass, the loss of light is minimized (figure 1.1). The eyepiece, or ocular, at the top of the tube magnifies the image formed by the objective lens. As a result, the total magnification seen by the observer is obtained by multiplying the magnification of the objective lens by the magnification of the ocular, or eyepiece. For exam￾ple, when using the 10× ocular and the 43× objective, total magnification is 10 × 43 = 430 times. Procedure for Basic Microscopy:Proper Use of the Microscope 1. Always carry the microscope with two hands. Place it on the desk with the open part away from you. 2. Clean all of the microscope’s lenses only with lens paper and lens cleaner if necessary. 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. Cut a lowercase e from a newspaper or other printed page. Prepare a wet-mount as illustrated in figure 1.2. Place the glass slide on the stage of the microscope and secure it firmly using stage clips. If your microscope has a mechanical stage device, place the slide securely in it. Move the slide until the letter e is over the opening in the stage. 4. With the low-power objective in position, lower the tube until the tip of the objective is within 5 mm of the slide. Be sure that you lower the tube while looking at the microscope from the side. 5. Look into the microscope and slowly raise the tube by turning the coarse adjustment knob counterclockwise until the object comes into view. Once the object is in view, use the fine adjustment knob to focus the desired image. 6. Open and close the diaphragm, and lower and raise the condenser, noting what effect these actions have on the appearance of the object being viewed. Usually the microscope is used with the substage condenser in its topmost position. The diaphragm should be open and then closed down until just a slight increase in contrast is observed (table 1.1). 7. Use the oil immersion lens to examine the stained bacteria that are provided (figure 1.3a–d). The directions for using this lens are as follows: First locate Bright-Field Light Microscope and Microscopic Measurement of Organisms 3 Figure 1.1 The Oil Immersion Objective. An oil immersion objective lens operating in air and with immersion oil. Light rays that must pass through air are bent (refracted), and many do not enter the objective lens. The immersion oil prevents the loss of light rays. Figure 1.2 Preparation of a Wet-mount Slide. (a) Add a drop of water to a slide. (b) Place the specimen (letter e) in the water. (c) Place the edge of a coverslip on the slide so that it touches the edge of the water. (d) Slowly lower the coverslip to prevent forming and trapping air bubbles. (a) (c) (d) (b) Air Oil Cover glass Slide

1.Microscopic Techniques Bright-Field Light s of the the fine adjustment.An altemate procedure is to get the focus very sharp under high power,then move the olving nose unol you ar all dm oil in the center of the illuminated area on the slide.Continue revolving the be mmer of the bacteria in the spaces provided. Microscopic Techniques

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 1. Bright−Field Light Microscope and Microscopic Measurement of Organisms © The McGraw−Hill Companies, 2002 4 Microscopic Techniques Figure 1.3 Examples of Bacterial Shapes as Seen with the Bright-field Light Microscope. (a) Staphylococcus aureus cocci; singular, coccus (×1,000). (b) Bacillus subtilis rods or bacilli; singular, bacillus (×1,000). (c) A single, large spirillum; plural, spiralla (Spirillum volutans; ×1,000). (d) Numerous, small spirilla (Rhodospirillum rubrum; ×1,000). (a) (b) (c) (d) the stained area with the low-power objective and then turn the oil immersion lens into the oil and focus with the fine adjustment. An alternate procedure is to get the focus very sharp under high power, then move the revolving nosepiece until you are halfway between the high-power and oil immersion objectives. Place a small drop of immersion oil in the center of the illuminated area on the slide. Continue revolving the nosepiece until the oil immersion objective clicks into place. The lens will now be immersed in oil. Sharpen the focus with the fine adjustment knob. Draw a few of the bacteria in the spaces provided