1.Bright-Fiold Light asurement Review Questions 1.Differentiate between the resolving power and magnifying power ofa lens.What is meant by the term parfocal 2.Why is the low-power objective placed in position when the microscope is stored or carried? 3.Why is oil necessary when using thexto100x objective? 4.What is the function of the iris diaphragm?The substage condenser? 5.What is meant by the limit of resolution? 10 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 Review Questions 1. Differentiate between the resolving power and magnifying power of a lens. What is meant by the term “parfocal”? 2. Why is the low-power objective placed in position when the microscope is stored or carried? 3. Why is oil necessary when using the 90× to 100× objective? 4. What is the function of the iris diaphragm? The substage condenser? 5. What is meant by the limit of resolution? 10 Microscopic Techniques
Microscopic Techniques ses in logy.Fift 6.How can you inrease the bulb life of your microscope if its voltage is regulated by a rheostat? 7.In general,at what position should you keep your microscope's substage condenser lens? 8.What are three bacterial shapes you observed? 9.How can you increase the resolution on your microscope? 10.In microbiology,what is the most commonly used objective?Explain your answer. 11.In microbiology,what is the most commonly used ocular?Explain your answer. 12.If 5x instead of 10x oculars were used in your microscope with the same objectives.what magnifications would be achieved? Bright-Field Light Microscope(Basic Microseopy) 11
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 6. How can you increase the bulb life of your microscope if its voltage is regulated by a rheostat? 7. In general, at what position should you keep your microscope’s substage condenser lens? 8. What are three bacterial shapes you observed? 9. How can you increase the resolution on your microscope? 10. In microbiology, what is the most commonly used objective? Explain your answer. 11. In microbiology, what is the most commonly used ocular? Explain your answer. 12. If 5× instead of 10× oculars were used in your microscope with the same objectives, what magnifications would be achieved? Bright-Field Light Microscope (Basic Microscopy) 11
ey-Pc 1.Bright-Field Light surement 13.Why is it necessary to calibrate the ocular micrometer with each objective? 14.In the prepared slides,which organism was the largest? 15.When identifying microorganisms,why should a wet-mount be used when making measurements? 16.What is a stage micrometer? 17.Complete the following for the 10xobjective: a. ocular micrometer divisions= stage micrometer divisions b._ ocular micrometer divisions=1 stage micrometer division= mm c.One ocular micrometer division= stage micrometer divisions= 一mn 18.Complete the following on units of measurement: Unit a.I centimeter 102m eter b.I millimeter mm c. um 10-meter d.I nanometer 10 meter e.I angstrom 10-10 meter 12 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 13. Why is it necessary to calibrate the ocular micrometer with each objective? 14. In the prepared slides, which organism was the largest? 15. When identifying microorganisms, why should a wet-mount be used when making measurements? 16. What is a stage micrometer? 17. Complete the following for the 10 × objective: a. _ ocular micrometer divisions = _ stage micrometer divisions b. _ ocular micrometer divisions = 1 stage micrometer division = _ mm c. One ocular micrometer division = _ stage micrometer divisions = _ mm 18. Complete the following on units of measurement: Unit Abbreviation Value a. 1 centimeter _ 10–2 meter b. 1 millimeter mm _ c. _ m 10–6 meter d. 1 nanometer _ 10–9 meter e. 1 angstrom _ 10–10 meter 12 Microscopic Techniques
Microscopic Techniques 284 EXERCISE The Hanging Drop Slide and Bacterial Motility Pronunciation guide SAFETY PRECAUTIONS Be careful with the Bunsen burner flame Slides an OH-sab) Spirillum volutans (spy-RIL-lum VOL-u-tans) slide Materials per Student Why Are the Above Bacteria Used in This Exercise? small,motile bacillus),Bacillus cereus (ATCC The major objectives of this exercise are to allow students makng hanging drop slides sand observ or phase-contrast microscope lture and vary in size shape.of fa lens paper and lens cleaner per rust.hen mmersion oil tha lum:Ba illus c reus waxen. wax toothpicks tha Bunsen burner oluto.tur ut)is a rigid helical cell (14 Learning Objectives about one helical .is wide Each student should be able to of habitats and ning.S.volutans occurs 2.Die en the three bacterial used in this on the pasis of sie.shape. arrangement.and motility Principles Suggested Reading in Textbook 1.Flagella and Motility,section3.6;see also n aqueous envi Thi figures3.31-3.36 ratis movement is due to Brownian. 13
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 2. The Hanging Drop Slide and Bacterial Motility © The McGraw−Hill Companies, 2002 EXERCISE The Hanging Drop Slide and Bacterial Motility 13 Materials per Student 24- to 48-hour tryptic soy broth cultures of Pseudomonas aeruginosa (ATCC 10145, small, motile bacillus), Bacillus cereus (ATCC 21768, large, motile bacillus), and Spirillum volutans (ATCC 19554, spiral, motile bacterium) microscope or phase-contrast microscope lens paper and lens cleaner immersion oil clean depression slides and coverslips petroleum jelly (Vaseline) inoculating loop toothpicks Bunsen burner Learning Objectives Each student should be able to 1. Make a hanging drop slide in order to observe living bacteria 2. Differentiate between the three bacterial species used in this exercise on the basis of size, shape, arrangement, and motility Suggested Reading in Textbook 1. Flagella and Motility, section 3.6; see also figures 3.31–3.36. Pronunciation Guide Bacillus cereus (bah-SIL-lus SEE-ree-us) Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jinOH-sah) Spirillum volutans (spy-RIL-lum VOL-u-tans) Why Are the Above Bacteria Used in This Exercise? The major objectives of this exercise are to allow students to gain expertise in making hanging drop slides and observing the motility of living bacteria. To accomplish these objectives, the authors have chosen three bacteria that are easy to culture and vary in size, shape, arrangement of flagella, and types of motion. Specifically, Pseudomonas aeruginosa (L. aeruginosa, full of copper rust, hence green) is a straight or slightly curved rod (1.5 to 3.0 m in length) that exhibits high motility by way of a polar flagellum; Bacillus cereus (L. cereus, waxen, wax colored) is a large (3.0 to 5.0 m in length) rod-shaped and straight bacillus that moves by peritrichous flagella; and Spirillum volutans (L. voluto, tumble about) is a rigid helical cell (14 to 60 m in length) that is highly motile since it contains large bipolar tufts of flagella having a long wavelength and about one helical turn. P. aeruginosa is widely distributed in nature and may be a saprophytic or opportunistic animal pathogen. B. cereus is found in a wide range of habitats and is a significant cause of food poisoning. S. volutans occurs in stagnant freshwater environments. Principles Many bacteria show no motion and are termed nonmotile. However, in an aqueous environment, these same bacteria appear to be moving erratically. This erratic movement is due to Brownian movement. 2 SAFETY PRECAUTIONS Be careful with the Bunsen burner flame. Slides and coverslips are glass. Do not cut yourself when using them. Dispose of any broken glass in the appropriately labeled container. Discard contaminated depression slides in a container with disinfectant
causing them to move movement True motility (self-propulsion)has been recog- 6.Discard your coverslips and any contaminated nize d in oth vith disi ant solution 1 se terium)that form axial filaments. These spirochetes move in a corkscrew bendi Figure 2.1 Preparation of a Hanging Drop Slide form of gliding motion. over The above types of motility or nonmotility can be erved ove ng penc I in anging drop slid of liv and the they associate together (see coverslip keeps the dure 1.With a toothpick.spread a small ring of Vaseline Move slide to cove 2 After tho the inoculating loop to aseptically place a small drop of one of the center of a spsohhei protrudes into the center of the concavity of the slide(figure 2.le). Pres HINTS AND PRECAUTIONS eover (the (1)Always esure the specimen is on the top side dron is over the light hole breaking the overslip since it is more 5.Examine the drop by first locating its edge under oeoo d its f the slid using on the drop.Switch t reclude the use of the oil immersion obiective wit bacteria clearly.close the diaphragm as much as image into focus by using the coarse adjustment knob possible for increased contrast.Note bacterial 14 Microscopic Techniques
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 2. The Hanging Drop Slide and Bacterial Motility © The McGraw−Hill Companies, 2002 Brownian movement results from the random motion of the water molecules bombarding the bacteria and causing them to move. True motility (self-propulsion) has been recognized in other bacteria and involves several different mechanisms. Bacteria that possess flagella exhibit flagellar motion. Helical-shaped spirochetes have axial fibrils (modified flagella that wrap around the bacterium) that form axial filaments. These spirochetes move in a corkscrew- and bending-type motion. Other bacteria simply slide over moist surfaces in a form of gliding motion. The above types of motility or nonmotility can be observed over a long period in a hanging drop slide. Hanging drop slides are also useful in observing the general shape of living bacteria and the arrangement of bacterial cells when they associate together (see figure 1.3). A ring of Vaseline around the edge of the coverslip keeps the slide from drying out. Procedure 1. With a toothpick, spread a small ring of Vaseline around the concavity of a depression slide (figure 2.1a). Do not use too much Vaseline. 2. After thoroughly mixing one of the cultures, use the inoculating loop to aseptically place a small drop of one of the bacterial suspensions in the center of a coverslip (figure 2.1b). 3. Lower the depression slide, with the concavity facing down, onto the coverslip so that the drop protrudes into the center of the concavity of the slide (figure 2.1c). Press gently to form a seal. 4. Turn the hanging drop slide over (figure 2.1d) and place on the stage of the microscope so that the drop is over the light hole. 5. Examine the drop by first locating its edge under low power and focusing on the drop. Switch to the high-dry objective and then, using immersion oil, to the 90 to 100× objective. In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast. Note bacterial 14 Microscopic Techniques Figure 2.1 Preparation of a Hanging Drop Slide. (d) Turn slide over (c) Coverslip Vaseline (b) (a) Drop of bacterial culture Drop of bacterial culture Inoculating loop Slide concavity Vaseline ring Toothpick Move slide to coverslip HINTS AND PRECAUTIONS (1) Always make sure the specimen is on the top side of the slide. (2) Particular care must be taken to avoid breaking the coverslip since it is more vulnerable when supported only around its edges. (3) With depression slides, the added thickness of the slide and coverslip may preclude the use of the oil immersion objective with some microscopes. (4) If your microscope is equipped with an automatic stop, it may be necessary to bring the image into focus by using the coarse adjustment knob. shape, size, arrangement, and motility. Be careful to distinguish between motility and Brownian movement. 6. Discard your coverslips and any contaminated slides in a container with disinfectant solution. 7. Complete the report for exercise 2