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    6 views3 pages

    Practical - Hanging Drop Method

    Uploaded by

    ANUREET KAUR

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
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    LaboronExerceo Coon, 212 EXERCISE The Hanging Drop Slide and Bacterial Motility Materials per Student 24-10 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-tee-us) Pseudomonas aeruginosa (s00-do-MO-nas a-tub-in- O-sah) Spirillum volurans (spy-RIL-lum VOL-v-ians) o> The major objectives of this exercise are to allow students to gain expertise in making hanging drop slides and abserv- ing the motility of living bacteria. To accomplish these ob- jectives. the authors have chosen three bacteria that are easy to culture and vary in size, shape, arrangement off gella, 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 um in jength) that exhibits high motility by way of a polar lage! Jum; Bacillus cereus (L. cereus, waxen, wax colored) is & large (3.0 to 5.0 pm in length) rod-shaped and straight bacillus that moves by pevtrchous flagella; and Spivillm volutans (L. voluto, tumble abost) isa rigid helical cell (14 o 60 jam in length) that is highly motile since it contains large bipolar tufts of flagella having a long wavelength and about one helical tum. P. aeruginosa is widely distributed in nature and may be a saproplaytc 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 Why Are the Above Bacteria Used in This Lxerise? Principles Many bacteria show no motion and ate termed non ‘motile. However, in an aqueous environment, these same bacteria appear to be moving erratically. This er- ratic movement is due to Brownian movement. 13 Brownian movement results from the random motion fof the water molecules bombarding the bacteria and ‘causing them to move. ‘True motility (self-propulsion) has been recog- nized in other bacteria and involves several different ‘mechanisms. Bacteria that possess flagella exhibit fla- gellar motion, Helical-shaped spirochetes have axial fibrils (modified flagella that wrap around the bac- terium) 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 2.12). 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 2.15) 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, Tur the hanging drop slide over (ligure 2.1¢) and. place om 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 100x objective. In order to see the bacteria clearly, close the diaphragm as much as possible for increased contrast, Note bacterial 4 Microscopie Techniques LMicmscopieTeeiguee | 2 TheHangng Drop sae | ‘and Batra Mos Tretia tian Copano. 212 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. Figure 21 Preparation of s Hanging Drop Slide Toothpek J vestne tos [ie cncaty : Z, Irscltigoop Dep or beter cure ovr 9 | St ttetecoete

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