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.
13Brownian 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 |
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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
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