ises in L.Microscopic Techniques h2oesgamuw logy.Fift Review Questions 1.In the phase-contrast microscope.what does the annular diaphragm do? 2.When would you use the phase-contrast microscope? 3.Explain how the phase plate works in a phase-contrast microscope that produces bright objects with respect to the background. 4.Whta to the phase ofdifaihincmtddight inahae-cos 5.What advantage does the phase-contrast microscope have over the ordinary bright-field microscope? 6.What is the difference between a bright-phase-contrast and a dark-phase-contrast microscope? 7.In microscopy.what does the term"phase"mean? 6 Microscopic Technique
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 4. Phase−Contrast Light Microscope © The McGraw−Hill Companies, 2002 Review Questions 1. In the phase-contrast microscope, what does the annular diaphragm do? 2. When would you use the phase-contrast microscope? 3. Explain how the phase plate works in a phase-contrast microscope that produces bright objects with respect to the background. 4. What happens to the phase of diffracted light in comparison to undiffracted light in a phase-contrast microscope? 5. What advantage does the phase-contrast microscope have over the ordinary bright-field microscope? 6. What is the difference between a bright-phase-contrast and a dark-phase-contrast microscope? 7. In microscopy, what does the term “phase” mean? 26 Microscopic Techniques
ey-Pc EXERCISE Fluorescence Microscope SAFETY CONSIDERATIONS Remember that the pressurized mercury vapor arc lam Why Is the Following Bacterium Used in This Exercise? Mycobacteritmn tuberculosis (.a small swellin Gr.-osis, erized by)isa human pathogen that cause reinaly while okn roh the curved,singly and ad Materials per Group of Students fluorescence microscope lens paper and lens cleaner m,such as M. ow-fl re ing imme imporant than what is being observed nberculosis)stained with fluorescent dye Learning Objectives Medical Applications Each student should be able to 1.Understand the principles behind the fluorescence rial antigens int ue smears sections.and fluids as well as the rapid identifica on of many dise se-caus stained with a fluorescent dye creenedforM.haberlosibystai g it with a flu dye th y山 Suggested Reading in Textbook when the men is viewed under the fluorescence microscope. 1. Principles Pronunciation Guide Fluorescence microscopy is based on the principle of Mycobacterium tuberculosis(mi-ko-bak-TE-re-um removal of incident illumination by selective absorp too-ber-ku-LO-sis) tion.whereas light that has been absorbed by the 27
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 5. Fluorescence Microscope © The McGraw−Hill Companies, 2002 27 EXERCISE Fluorescence Microscope Materials per Group of Students fluorescence microscope lens paper and lens cleaner low-fluorescing immersion oil protective glasses that filter UV light prepared slides of known bacteria (M. tuberculosis) stained with fluorescent dye Learning Objectives Each student should be able to 1. Understand the principles behind the fluorescence microscope 2. Correctly use the fluorescence microscope by observing prepared slides of known bacteria stained with a fluorescent dye Suggested Reading in Textbook 1. The Fluorescence Microscope, section 2.2; see also figures 2.12, 2.13. Pronunciation Guide Mycobacterium tuberculosis (mi-ko-bak-TE-re-um too-ber-ku-LO-sis) Why Is the Following Bacterium Used in This Exercise? Mycobacterium tuberculosis (L. tuberculum, a small swelling + Gr. -osis, characterized by) is a human pathogen that causes tuberculosis. It is very slow growing and not readily stained by Gram’s method. The cell is 1 to 4 m in length, straight or slightly curved, occurring singly and in occasional threads. This bacterium can most readily be identified after staining with fluorochromes or specifically labelling it with fluorescent antibodies using complicated immunofluorescence procedures, which are both time consuming and expensive. By using commercially prepared slides, the student is able to immediately examine a pathogenic bacterium, such as M. tuberculosis, and gain expertise in using the fluorescence microscope. In this exercise, microscopic technique is more important than what is being observed. Medical Applications Fluorescence microscopy is commonly used in the clinical laboratory for the rapid detection and identification of bacterial antigens in tissue smears, sections, and fluids, as well as the rapid identification of many disease-causing microorganisms. For example, a sputum specimen can be quickly screened for M. tuberculosis by staining it with a fluorescent dye that binds specifically to M. tuberculosis. Only the stained bacterium of interest will be visible when the specimen is viewed under the fluorescence microscope. Principles Fluorescence microscopy is based on the principle of removal of incident illumination by selective absorption, whereas light that has been absorbed by the SAFETY CONSIDERATIONS Remember that the pressurized mercury vapor arc lamp is potentially explosive. Never attempt to touch the lamp while it is hot. Never expose your eyes to the direct rays of the mercury vapor arc lamp. Severe burns of the retina can result from exposure to the rays. In like manner, removal of either the barrier or exciter filter can cause retinal injury while looking through the microscope. 5
Flu ce micro specimen and re-emitted at an altered wavelength is 5.After the mercury vapor arc lamp has been ransmitted.The light soure must prod that are not effective in 6.Starting with the 10x objective,find and focus the the fluorochrome used.The light fluoresced by the specimen. 7 light.As a n to the mercury vapor ar fluorescence contributes to the intensity of the image .Compareay rig-fed being viewed (figure 5.la.b). microscope with what you see in the fluorescence Procedure 1.Turn on the UV light source at least 30 minutes before using the fluorescence micros NEVER LOOK AT THE UV HINTS AND PRECAUTIONS FILTER UV LIGHT BECAUSE RETINAL BURNS AND BLINDNESS MIGHT RESULT. riod,do not tur the microse Make sure that the proper excitation filter and cope on and of（2R barrier filter are ma for the type o on the condenser the bottom of the slide】 Microscopic Technique
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 5. Fluorescence Microscope © The McGraw−Hill Companies, 2002 28 Microscopic Techniques specimen and re-emitted at an altered wavelength is transmitted. The light source must produce a light beam of appropriate wavelength. An excitation filter removes wavelengths that are not effective in exciting the fluorochrome used. The light fluoresced by the specimen is transmitted through a filter that removes the incident wavelength from the beam of light. As a result, only light that has been produced by specimen fluorescence contributes to the intensity of the image being viewed (figure 5.1a,b). Procedure 1. Turn on the UV light source at least 30 minutes before using the fluorescence microscope. NEVER LOOK AT THE UV LIGHT SOURCE WITHOUT PROTECTIVE GLASSES THAT FILTER UV LIGHT BECAUSE RETINAL BURNS AND BLINDNESS MIGHT RESULT. 2. Make sure that the proper excitation filter and barrier filter are matched for the type of fluorescence expected and are in place. 3. Place a drop of the low-fluorescing immersion oil on the condenser. 4. Place the prepared slide on the stage and position it so that the specimen is over the light opening. Raise the condenser so that the oil just touches the bottom of the slide. 5. After the mercury vapor arc lamp has been warmed up, turn on the regular tungsten filament light source and focus on the specimen. 6. Starting with the 10× objective, find and focus the specimen. 7. After finding the specimen, move to the 90× to 100× objective, switch to the mercury vapor arc and view the specimen. 8. Compare what you see in the bright-field microscope with what you see in the fluorescence microscope by sketching the organisms in the report for exercise 5. Figure 5.1 Fluorescence Microscopy. (a) Escherichia coli stained with DAPI and propidium iodine. These fluorochromes bind DNA (×1,000). The blue cells are viable and the red cells are dead. (b) Giardia lamblia stained with IFA (×1,000). (a) (b) HINTS AND PRECAUTIONS (1) The mercury vapor arc lamp requires about a 30- minute warm-up period. During a normal laboratory period, do not turn the microscope on and off. (2) Make sure the proper filters are in place. If you are in doubt, ask your instructor. (3) Note that there is no diaphragm control on the dark-field condenser. (4) Never use ordinary immersion oil with a fluorescence microscope
1.Microscopic Techniques Laboratory Report 5 Name: Date: Lab Section: Fluorescence Microscope 1.Bacterium as seen with the bright-field microscope.Bacterium as seen with the fluorescence microscope Genus and species Genus and species: Magnification:× Magnification::× Shape: Shape: 2.Label the following parts of a fluorescence microscope.Use the following terms:specimen and fluorochrome heat filter,mercury vapor arc lamp,exciter filter,barrier filter,dark-field condenser. 29
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 5. Fluorescence Microscope © The McGraw−Hill Companies, 2002 29 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 5 Fluorescence Microscope 1. Bacterium as seen with the bright-field microscope. Bacterium as seen with the fluorescence microscope. Genus and species: _ Genus and species: _ Magnification: × _ Magnification: × _ Shape: _ Shape: _ 2. Label the following parts of a fluorescence microscope. Use the following terms: specimen and fluorochrome, heat filter, mercury vapor arc lamp, exciter filter, barrier filter, dark-field condenser. Eyepiece Objective lens Mirror
ises in n logy.Fifth Review Questions 1.What kind of light is used to excite dyes and make microorganisms fluoresce? 2.List two fuorochromes that are used in staining bacteria 3.What is a serious hazard one must guard against when working with mercury vapor are lamps? n) b.barrier filter c.heat filter d.mercury vapor arc lamp 5.When is fluorescence microscopy used in a clinical laboratory? 6.Differentiate between phosphorescence and fluorescence. 7.What advantage is there to using fluorescence procedures in ecological studies?Give several examples 30 Microscopic Technique
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 5. Fluorescence Microscope © The McGraw−Hill Companies, 2002 30 Microscopic Techniques Review Questions 1. What kind of light is used to excite dyes and make microorganisms fluoresce? 2. List two fluorochromes that are used in staining bacteria. 3. What is a serious hazard one must guard against when working with mercury vapor arc lamps? 4. What is the function of each of the following? a. exciter filter b. barrier filter c. heat filter d. mercury vapor arc lamp 5. When is fluorescence microscopy used in a clinical laboratory? 6. Differentiate between phosphorescence and fluorescence. 7. What advantage is there to using fluorescence procedures in ecological studies? Give several examples