2ma 8e2a Laboratory Report 2 Name Date: Lab Section: The Hanging Drop Slide and Bacterial Motility 1.Examine the hanging drop slide and complete the following table with respect to the size,shape,and motility of the different bacteria. Bacterium Size Shape Type of Motility Cell Arrangement B.cereus P.ceruginosa S.volutans 2.Draw a representative field for each bacterium. Magnification: Magnification:× Magnification:x 15
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 2. The Hanging Drop Slide and Bacterial Motility © The McGraw−Hill Companies, 2002 15 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 2 The Hanging Drop Slide and Bacterial Motility 1. Examine the hanging drop slide and complete the following table with respect to the size, shape, and motility of the different bacteria. Bacterium Size Shape Type of Motility Cell Arrangement B. cereus _ _ _ _ P. aeruginosa _ _ _ _ S. volutans _ _ _ _ 2. Draw a representative field for each bacterium. Magnification: _ Magnification: _ Magnification: _ B. cereus P. aeruginosa S. volutans ×× ×
Me6 taa2nw丁 Review Questions 1.Why are unstained bacteria more difficult to observe than stained bacteria? 2.What are some reasons for making a hanging drop slide? a.Brownian movemen b.flagellar motion c.gliding motion 4.Why do you have to reduce theamount of light with the diaphragm inorderto see bacteria ina hanging drop slide? 5.Can the hanging drop slide be used to examine other microorganisms?Explain which ones. 6.Which of the bacteria exhibited true motility on the slides? 7.How does true motility differ from Brownian movement? 16 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 Review Questions 1. Why are unstained bacteria more difficult to observe than stained bacteria? 2. What are some reasons for making a hanging drop slide? 3. Describe the following types of bacterial movement: a. Brownian movement b. flagellar motion c. gliding motion 4. Why do you have to reduce the amount of light with the diaphragm in order to see bacteria in a hanging drop slide? 5. Can the hanging drop slide be used to examine other microorganisms? Explain which ones. 6. Which of the bacteria exhibited true motility on the slides? 7. How does true motility differ from Brownian movement? 16 Microscopic Techniques
1.Microscopic Techniques w咖 EXERCISE Dark-Field Light Microscope SAFETY CONSIDERATIONS Why Is the Following Bacterium Used in This Exercise? ps are glass.Do not cut yo Treponema denticola(M.L.n.denticola.tooth dweller) a part of the norma the a of the ora Place them in the appro dark-field and are b the us Materials per Group of Students the t-o ation T deuticol dark-field light microscope in length.In a we movements due to two or thre per at e slides and coverslips dent isals able to observe bacterial m tility. Principles Learning Objectives be fitted with Each student should be able to 1.Understand the principles behind dark-field ing power)greater than the objective.The condenser microscopy also contains a dark-fiel a da -ee g the obiective lens.whereas undiffracted light does not.resulting in a bright image against a dark back. Suggested Reading in Textbook )Since light 1.The Dark-Field Microscope,section 2.2:see also than the reverse,dark-field micros py is useful in figures 2.7 and 2.8. observing unstained living microorgani sms.microor ganisms that are difficult to sta an ch Pronunciation Guide Treponema denticola (trep-o-NE-mah dent-A-cola) 17
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 3. Dark−Field Light Microscope © The McGraw−Hill Companies, 2002 17 EXERCISE Dark-Field Light Microscope Materials per Group of Students dark-field light microscope flat toothpicks lens paper and lens cleaner immersion oil slides and coverslips prepared slides of spirochetes (e.g., Treponema denticola), radiolarians, protozoa tweezers Learning Objectives Each student should be able to 1. Understand the principles behind dark-field microscopy 2. Correctly use the dark-field microscope 3. Make a wet-mount and examine it for spirochetes with the dark-field microscope Suggested Reading in Textbook 1. The Dark-Field Microscope, section 2.2; see also figures 2.7 and 2.8. Pronunciation Guide Treponema denticola (trep-o-NE-mah dent-A-cola) Why Is the Following Bacterium Used in This Exercise? Treponema denticola (M.L. n, denticola, tooth dweller) often is a part of the normal microbiota of the oral mucosa; thus, this spirochete is readily available and does not have to be cultured. Most species stain poorly if at all with Gram’s or Giemsa’s methods and are best observed with dark-field or phase-contrast microscopy. Thus, T. denticola is an excellent specimen to observe when practicing the use of a dark-field microscope, and also allows the student to continue practicing the wet-mount preparation. T. denticola is a slender, helical cell, 6 to 16 m in length. In a wetmount, the bacteria show both rotational and translational movements due to two or three periplasmic flagella inserted at each end of the protoplasmic cylinder. Young cells rotate rapidly on their axis. Thus, by using T. denticola, the student is also able to observe bacterial motility. Principles The compound microscope may be fitted with a darkfield condenser that has a numerical aperture (resolving power) greater than the objective. The condenser also contains a dark-field stop. The compound microscope now becomes a dark-field microscope. Light 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 (figures 3.1–3.2). Since light objects against a dark background are seen more clearly by the eye than the reverse, dark-field microscopy is useful in observing unstained living microorganisms, microorganisms that are difficult to stain, and spirochetes (figure 3.2), which are poorly defined by bright-field microscopy. 3 SAFETY CONSIDERATIONS Gently scrape the gum line or gingival sulcus with a flat toothpick so that you obtain a small amount of surface scrapings and not lacerated gum tissue or impacted food. Slides and coverslips are glass. Do not cut yourself when using them. Dispose of any broken glass in the appropriately labeled container. Do not throw used toothpicks in the wastebasket. Place them in the appropriate container for disposal
ark-ficld Mi 、which diplay cbt-field In this da rk-fiel hells is show Procedure 6.Nonpathogenic spirochetes (T.denticola)may be part of the normal microbiota of the oral mucosa 1.Place a drop of immersion oil directly on the dark-field condenser lens. To make a wet unt or th genty scrape you 2.Positionne of the prepared slides so that the control until the oil on the condenser lens just es.Examine w ouches the slide 4 Lock the 10 KIobeitentopsitianFoeuswit focus.Do thes vith the 40x obiective to always clea 5.Use the oil immersion objective lens to observe condense ng a dr on it.()M sure th denser diaphragm is wide open for adequate illumina tion of the specimen. 18 Microscopic Techniques
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 3. Dark−Field Light Microscope © The McGraw−Hill Companies, 2002 18 Microscopic Techniques Procedure 1. Place a drop of immersion oil directly on the dark-field condenser lens. 2. Position one of the prepared slides so that the specimen is directly over the light opening. 3. Raise the dark-field condenser with the height control until the oil on the condenser lens just touches the slide. 4. Lock the 10× objective into position. Focus with the coarse and fine adjustment knobs until the spirochetes come into sharp focus. Do the same with the 40× objective. 5. Use the oil immersion objective lens to observe the spirochetes. Draw several in the space provided in the report for exercise 3. 6. Nonpathogenic spirochetes (T. denticola) may be part of the normal microbiota of the oral mucosa. To make a wet-mount of these, gently scrape your gum line with a flat toothpick. Stir the scrapings into a drop of water on a slide. Gently lower a coverslip (see figure 1.2) to prevent trapping air bubbles. Examine with the dark-field microscope and draw several spirochetes in the space provided in the report for exercise 3. Figure 3.1 Dark-field Microscopy. Dark-field microscopy can best visualize transparent, unstained specimens, which display only low contrast in bright-field. In this dark-field photomicrograph (×100), a mixture of radiolarian shells is shown. Notice their many unique and beautiful shapes. Figure 3.2 Photomicrograph of Treponema pallidum, as Seen with Dark-field Microscopy (×500). HINTS AND PRECAUTIONS (1) It is good practice to always clean the condenser lens before placing a drop of oil on it. (2) Make sure the prepared slide is placed right side up (coverslip up) on the stage. (3) If you have trouble focusing with the oil immersion lens, don’t flounder—ask for help from your instructor. (4) Always make sure that the substage condenser diaphragm is wide open for adequate illumination of the specimen
Microscopie Techniques Laboratory Report 3 Date: Lab Section: Dark-Field Light Microscope 1.Drawing of spirochetes from aprepared slide. Drawing of spirochetes from a wet-mount Magnification:× Magnification:× Genus and species: Genus and species:_ Shape: Shape: 2.Label the following parts of a dark-field microscope.Use the following terms:dark-field stop.specimen. Abbe condenser,and objective. 19
Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition I. Microscopic Techniques 3. Dark−Field Light Microscope © The McGraw−Hill Companies, 2002 Dark-Field Light Microscope 1. Drawing of spirochetes from a prepared slide. Drawing of spirochetes from a wet-mount. Magnification: × _ Magnification: × _ Genus and species: _ Genus and species: _ Shape: _ Shape: _ 2. Label the following parts of a dark-field microscope. Use the following terms: dark-field stop, specimen, Abbé condenser, and objective. 19 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 3