EX 3: HANGING DROP METHOD

MATERIALS
1. Cultures of Gaffkya tetragena and Proteus Vulgaris
2. Concave Slides
3. Coverslips
4. Vaseline
PROCEDURE
Apply Vaseline or petroleum jelly on the edges of concavity of the slide to prevent
1 evaporation of the bacterial drop,

Take a loopful of the bacteria suspension and place it on the center of a clean
coverslip. Observe proper aseptic technique. (is done to prevent contamination
from the environment)
2
Proper aseptic technique is done to prevent contamination from the environment

ASEPTIC TECHNIQUE PROCEDURE

1 Hold the tube in one hand and the loop on the other.
2 Flame loop until red hot and remove the cotton plug.
Flame the mouth of the tube and insert the loop.
o Before you insert the wire loop into the tube, wait for 10-15 seconds after
3 heating because the heat will kill the organism

4 After taking a loopful of the bacterial suspension from the tube. You flame the mouth
of the tube again then you cover the tube with a cotton.
5 Taking your loopful of bacterial suspension, You place it on the center of your
coverslip.
6 And then you flame your loop until it’s red hot and place on the rack to cool.

back to the procedure

Carefully invert the prepared coverslip and apply it over the concavity making sure
that the drop of bacterial culture is in the center, and the drop is hanging inro the
concavity without touching the slide. Hence, “hanging drop”.
The hanging drop method is done so we can see the motility of the organism.

3 This is also why we use a concave slide so that there will be a space between the
coverslip and the slide and the drop is allowed to hang.
EX 3: HANGING DROP METHOD

This is also why we use a concave slide so that there will be a space between the
coverslip and the slide and the drop is allowed to hang.
But in the case that a concave slide is not available, an ordinary slide may be used
but ringed with Vaseline or pet jelly so that it forms a well which can elevate the
cover slip and give some space for the drop to hang.

The preparation is the studied under the Low Power Objective or LPO with the
diaphragm partly open. The best guide in locating the preparation is the edge of the
drop.
Why the edge of the drop is focused:
i. Better contrast is obtained due to the difference in refractive
4 index of the drop and the cover slip.
ii. Also, as the drop hangs, it thins toward the edge. This means
the edge would contain a lesser number of bacteria thus give
us a chance to see the motility clearer.
iii. Aerobic bacteria usually vomes toward the edge to gert more
oxygen for respiration.
After studying the preparation under the LPO, the LPO is raised very slightly before
switching to the HPO. This is because we have to prevent breaking the cover slip
or damaging the objective when the switch high power is made. Usually, the raising
of LPO is only done when we are using the old microscopes where the objectives
are the ones being raised. (Usually done on old microscopes but in our lab we use
new microscopes wo raising the LPO slightly before changing to HPO is not
necessary).
5

We will then observe the bacteria and draw the bacteria while taking note of its
6 form, arrangement, and movement. The direction of the movement of the bacteria
should be indicated using arrows.
EX 3: HANGING DROP METHOD

Now, all hanging drop preparations must be immersed in disinfectant or Sodium

7 Hypochlorite or bleach for 10-20 mins to kill the organism before cleaning.
TYPES OF MOVEMENTS EXHIBITED IN THIS ACTIVITY

Gaffyka tetragena Exhibits Brownian motion

Proteus vulgaris Exhibits True Motility

Is a to and fro motion of particles suspended in a liquid.

Is actually not true motility. It is a false motion. It only looks like it’s
moving due to the physical forces like the water molecules.
Representative organism: Gaffkya tetragena.
BROWNIAN
MOTION Discovered by British scientist Robert Brown in 1827. Using
microscope, he noticed pollen grains suspended in water jiggled.
Later determined that the motion was due to the action of collision to
the surrounding molecules
Later determined that the motion was due to the action of collision to
the surrounding molecules
Individual bacteria move across the slide with varying degree of
TRUE MOTILITY rapidity.
Representative organism: Proteus vulgaris
- Proteus vulgaris is peritrichous meaning the flagella is all over
the entire body or is uniformly distributed.
In this slide we can see side-by-side how a motile and non-
motile organism look when they move.

The motile organism would go on any direction while the

non-motile organism would look like they are just jiggling
in place or having a side-to-side motion.

cr: shanyu_twt