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EXPERIMENT
Microscopic Examination of Living
Microorganisms Using a HangingDrop Preparation or a Wet Mount
Learning Objectives
Once you have completed this experiment, you
should know how to
1. Microscopically examine living
microorganisms.
2. Make a hanging-drop preparation or wet mount
to view living microorganisms.
Principle
Bacteria, because of their small size and a refractive
index that closely approximates that of water, do
not lend themselves readily to microscopic
examination in a living, unstained state. Examination of living microorganisms is useful, however, o
do the following:
1. Observe cell activities such as motility and binary fission.
2. Observe the natural sizes and shapes of the
cells, considering that heat fixation (the rapid
passage of the smear over the Bunsen burner
flame) and exposure to chemicals during
staining cause some degree of distortion.
In this experiment you will use individual cultures of Pseudomonas aeruginosa, Bacillus
cereus, Staphylococcus aureus, and Proteus
vulgaris for a hanging-drop preparation or a wet
mount. Hay infusion or pond water may be substituted or used in addition to the above organisms.
Figure 5.1 illustrates several organisms commonly
found in pond water and hay infusions.
Algae
Euglena
Diatoms
Scenedesmus
Chlamydomonas
Volvox
Protozoa
Paramecium
Stylonychia
Amoeba
Vorticella
Heteronema
Figure 5.1 Algae and protozoa commonly found in natural infusions and pond water (drawings are not to scale)
You will observe the preparation(s) microscopically for differences in the sizes and shapes of
the cells, as well as for motility, a self-directed
movement. It is essential to differentiate between
actual motility and Brownian movement, a vibratory movement of the cells due to their bombardment by water molecules in the suspension.
Hanging-drop preparations and wet mounts make
the movement of microorganisms easier to see
because they slow down the movement of water
molecules.
AT THE BENCH
Materials
2. Using aseptic technique, place a loopful of the
culture in the center of a clean coverslip.
3. Place the depression slide, with the concave
surface facing down, over the coverslip so that
the depression covers the drop of culture. Press
the slide gently to form a seal between the slide
and the coverslip.
4. Quickly turn the slide right side up so that the
drop continues to adhere to the inner surface
of the coverslip.
5. For microscopic examination, first focus on the
drop culture under the low-power objective
(10X) and reduce the light source by adjusting
the Abbe condenser. Repeat using the highpower objective (40X).
6. Examine the hanging-drop preparation and
record your observations in the Lab Report.
Cultures
24-hour broth cultures of P. aeruginosa, B. cereus,
S. aureus, and P. vulgaris; and/or hay infusion
broth cultures or pond water. (See Appendix 3 for
the preparation of hay infusion broth.)
Equipment
Bunsen burner, inoculating loop, depression slides,
glass slides, coverslips, microscope, petroleum jelly,
and cotton swabs.
Procedure
Hanging-Drop Preparation
Perform the following steps for each culture provided in this experiment. Steps 1-4 are illustrated
in Figure 5.2.
1. With a cotton swab, apply a ring of petroleum
jelly around the concavity of the depression
slide.
Wet Mount
A wet mount may be substituted for the hangingdrop preparation using a similar procedure:
1. With a cotton swab apply a thin layer of petroleum jelly along the edge of the four sides of
a coverslip.
2. Using aseptic technique, place a loopful of the
culture in the center of a clean coverslip.
3. Place a clean glass slide over the coverslip and
press the slide gently to form a seal between the
slide and the coverslip.
4. Follow Steps 4 and 5 in the hanging-drop
procedure.
5. Examine the wet-mount preparation and
record your observations in the Lab Report.
PROCEDURE
Slide
concavity
Petroleum jelly
ring
Loopful
Of bacterial
Culture -
Coverslip
Depression slide
1. Spread a ring of petroleum jelly around the concavity
of the depression slide.
2.
Place a loopful of the bacterial culture in
the center of the coverslip.
Culture drop
Coverslip
Depression
slide
3.
Petroleum jelly
Hanging-drop preparation
Coverslip ^Culture drop
Lower the depression slide, with the
concavity facing down, onto the
coverslip. Press gently to form a seal.
Figure 5.2 Hanging-drop preparation
4.
Turn the hanging-drop preparation
over so that the culture drop adheres
to the coverslip.
Lab Report
Observations and Results
1. Examine the hanging-drop or wet-mount preparation to determine shape and
motility of the different bacteria present. Record your results in the chart below.
Organisms
Shape
True Motility or Brownian Movement?
S. aureus
P. aeruginosa
B. cereus
P. vulgaris
2. Draw a representative field of each of the above organisms.
S. aureus
B. cereus
P. aeruginosa
P. vulgaris
3. Draw representative fields of pond water and hay infusion if you used them.
Try to identify some of the organisms that you see by referring to Figure
5.1. Note the shape and type of movement in the chart below.
Pond water
Hay infusion
Pond Water
Hay Infusion
Shape
True motility or Brownian movement?
Organism
Review Questions
1. Why are living, unstained bacterial preparations more difficult to observe
than stained preparations?
2. What is the major advantage of using living cell preparations (hanging-drop
or wet mount) rather than stained preparations?
3. How do you distinguish between true motility and Brownian movement?
4. During the microscopic observation of a drop of stagnant pond water,
what criteria would you use to distinguish viable organisms from nonviable
suspended debris?