Review Questions 1.What is the principle behind dark-field microscopy? 2.When would you use the dark-field microscope? 3.Why is the field dark and the specimen bright when a dark-field microscope is used to examine a specimen? 4.Differentiate between bright-field and dark-field microscopy. 5.What is the function of the Abbe condenser in dark-field microscopy? 6.What is the function of the dark-field stop? 7.In dark-field microscopy.why is a drop of oil placed directly on the condenser lens? 20 Microscopic Techniques
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 3. Dark−Field Light Microscope © The McGraw−Hill Companies, 2002 Review Questions 1. What is the principle behind dark-field microscopy? 2. When would you use the dark-field microscope? 3. Why is the field dark and the specimen bright when a dark-field microscope is used to examine a specimen? 4. Differentiate between bright-field and dark-field microscopy. 5. What is the function of the Abbé condenser in dark-field microscopy? 6. What is the function of the dark-field stop? 7. In dark-field microscopy, why is a drop of oil placed directly on the condenser lens? 20 Microscopic Techniques
Microscopic Techniques h2esaugw EXERCISE Phase-Contrast Light Microscope SAFETY CONSIDERATIONS s and c erslips.Disp Why Are the Following Bacteria and and pond wat Pond Water Used in This Exerise? pond water with your mouth-use the pipettor provide Materials per Group of Students vation ofo indistinct.bac ria an pond water op nd re end End pores are oval or som Pasteur pipette with pipettor verslips pictorial guides of common pond water short chains.with rounded meyCoefloeProtoslo.CaoinaBiolbeical ends.The end the Thus,by ntrast m Supply) tweezers ond water is usually teeming with bacteria and pro prepared g is able to observe the interal structure of protists such as Learning Objectives Each student should be able to 1.Understand the basic principles behind phase Principles contrast mic 2 andhenrapnprentcolor livn microor ar ark-field p ssible t that are present because they do not absor.refect.refract.od sufficient light to contrast with the surrounding envi icroo Suggested Reading in Textbook the absorb.reflect.refract or diffract more light than their environment.The phase-contrast microscope permits the observation of otherwise invisible living. unst: Pronunciation Guide ge4. n the the Bacillus (bah-SIL-lus) hasan uar diaphragm.which produces hollow Clostridium (klos-STRID-ee-um) cone of light:the objective has a glass disk (the phase 21
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 4. Phase−Contrast Light Microscope © The McGraw−Hill Companies, 2002 21 EXERCISE Phase-Contrast Light Microscope Materials per Group of Students pond water phase-contrast light microscope new microscope slides and coverslips Pasteur pipette with pipettor pictorial guides of common pond water microorganisms methyl cellulose (Protoslo, Carolina Biological Supply) tweezers lens paper and lens cleaner prepared slides of Bacillus or Clostridium showing endospores Learning Objectives Each student should be able to 1. Understand the basic principles behind phasecontrast microscopy 2. Correctly use the phase-contrast microscope 3. Make a wet-mount of pond water and observe some of the transparent, colorless microorganisms that are present Suggested Reading in Textbook 1. The Phase-Contrast Microscope, section 2.2; see also figures 2.8 and 2.9. Pronunciation Guide Bacillus (bah-SIL-lus) Clostridium (klos-STRID-ee-um) Why Are the Following Bacteria and Pond Water Used in This Exercise? Most microorganisms and their organelles are colorless and often difficult to see by ordinary bright-field or dark-field microscopy. Phase-contrast microscopy permits the observation of otherwise indistinct, living, unstained bacteria and their associated structures (such as endospores). Bacillus species are rod shaped, often arranged in pairs or chains, with rounded or square ends. Endospores are oval or sometimes cylindrical. Clostridium species are often arranged in pairs or short chains, with rounded or sometimes pointed ends. The endospores often distend the cell. Thus, by using prepared slides of Bacillus and Clostridium, the student gains expertise in using the phase-contrast microscope and in observing specific bacterial structures, such as different endospores. Pond water is usually teeming with bacteria and protists. By using the phase-contrast microscope and slowing down the many microorganisms with Protoslo, the student is able to observe the internal structure of protists such as Paramecium. Principles Certain transparent, colorless living microorganisms and their internal organelles are often impossible to see by ordinary bright-field or dark-field microscopy because they do not absorb, reflect, refract, or diffract sufficient light to contrast with the surrounding environment or the rest of the microorganism. Microorganisms and their organelles are only visible when they absorb, reflect, refract, or diffract more light than their environment. The phase-contrast microscope permits the observation of otherwise invisible living, unstained microorganisms (figure 4.1a–d). In the phase-contrast microscope, the condenser has an annular diaphragm, which produces a hollow cone of light; the objective has a glass disk (the phase SAFETY CONSIDERATIONS Be careful with the glass slides and coverslips. Dispose of the slides and coverslips, the used Pasteur pipettes, and pond water properly when finished. Do not pipette pond water with your mouth—use the pipettor provided. 4
ler-Pre 41 ned to show budding (000).(d)A W影不 (c) plate)with a thin flm of transparent aterial de 2. duced in the secimen.This phase chanee is scope so over he ligh hole in the specimen as a difference in light intensity plates e retar agm tha producing dark hase-contrast micro or ad oduced by the vance (negative phase plate)the undiffracted light rel- annular diaphragm below the condenser be ative to the centered exactly with the phase plate of the ectve (see nigure 2. are three different annular diaphragms that match Procedure the phase plat tes of the three differer nt phase of pond water.Addad objectives(10x 40 and 90 or 100x).The of the micr or Clostridium may also be used. correct annular diaphragm. 22 Microscopic Techniques
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 4. Phase−Contrast Light Microscope © The McGraw−Hill Companies, 2002 22 Microscopic Techniques plate) with a thin film of transparent material deposited on it, which accentuates phase changes produced in the specimen. This phase change is observed in the specimen as a difference in light intensity. Phase plates may 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-phasecontrast microscopy. Procedure 1. Make a wet-mount of pond water. Add a drop of methyl cellulose (Protoslo) to slow the swimming of the microorganisms. Prepared slides of Bacillus or Clostridium may also be used. 2. Place the slide on the stage of the phase-contrast microscope so that the specimen is over the light hole. 3. Rotate the 10× objective into place. 4. Rotate into position the annular diaphragm that corresponds to the 10× objective. It is absolutely necessary that the cone of light produced by the annular diaphragm below the condenser be centered exactly with the phase plate of the objective (see figure 2.9 in textbook if you do not understand this procedure). Consequently, there are three different annular diaphragms that match the phase plates of the three different phase objectives (10×, 40×, and 90× or 100×). The substage unit beneath the condenser contains a disk that can be rotated in order to position the correct annular diaphragm. Figure 4.1 Some Examples of Microorganisms Seen with Phase-contrast Microscopy. (a) A protozoan, Paramecium caudatum, from pond water stained to show internal structures (×200). (b) A bacterium, Bacillus cereus, stained to show spores (×1,000). (c) A yeast, Saccharomyces cerevisiae, stained to show budding (×1,000). (d) A filamentous green alga, Spirogyra, showing its helical chloroplasts (×200). (a) (b) (c) (d)
Meaanu 8e2a 5.Focus with the 0x objective and observe the anisms HINtS AND PRECAUTIONS 6.Rotate the nosepiece and anular diaphragm into the proper position for observation with thex nole in the stage of th microscop 7.Do the same with the oil immersion lens. 8.In the report for exercise 4.sketch several of the microorganisms that you have observed. Phase-Contrast Light Microscope
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 4. Phase−Contrast Light Microscope © The McGraw−Hill Companies, 2002 Phase-Contrast Light Microscope 23 5. Focus with the 10× objective and observe the microorganisms. 6. Rotate the nosepiece and annular diaphragm into the proper position for observation with the 40× objective. 7. Do the same with the oil immersion lens. 8. In the report for exercise 4, sketch several of the microorganisms that you have observed. 9. If you examined pond water, use the pictorial guides provided by your instructor to assist you in identifying some of the microorganisms present. HINTS AND PRECAUTIONS (1) Make sure the specimen is directly over the light hole in the stage of the microscope. (2) The phase elements must be properly aligned. Misalignment is the major pitfall that beginning students encounter in phasecontrast microscopy. (3) If your microscope is not properly aligned, ask your instructor for help
1.Microscopic Techniques Laboratory Report 4 Name: Date: Lab Section: Phase-Contrast Light Microscope 1.Some typical microorganisms in pond water as seen with the phase-contrast light microscope igh-dry powe 2.Drawings of Bacillus,Clostridiu with the phase-contras microscop Third specime 25
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 4. Phase−Contrast Light Microscope © The McGraw−Hill Companies, 2002 25 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 4 Phase-Contrast Light Microscope 1. Some typical microorganisms in pond water as seen with the phase-contrast light microscope. 2. Drawings of Bacillus, Clostridium, or another bacterium showing endospores as seen with the phase-contrast microscope. Low power High-dry power Oil immersion Bacillus Clostridium Third specimen