EXAMINATION OF LIVING BACTERIA IN FRESH PREPARATIONS

 There are two types of wet mount preparations: Normal Wet Mount Method and Hanging Drop Method
 Wet mounts are used when we need to visualize a living microorganism.
o Example: In order to determine if an organism is motile, the wet mount method should be used.
 Wet mounts need special care so you do not kill the microorganisms.
o Microorganisms die when heated so these slides are not heat-fixed.
o Microorganisms die if exposed to stains so these slides are not stained.
o Bright lights can dry out wet mounts and heat up microorganisms, so work quickly under dim light.
 Normal Wet Mount Method Wet Mount Hanging Drop
o Materials: slide, cover slips, dropper Uses normal slides Uses concave slides
o Procedure: Gently squeeze the bulb to draw the Small sample size is acceptable Larger sample sizes needed
sample into the dropper. Place 3-5 drops in the center Only good for short-term use Acceptable for longer-term use
of the slide. Gently place the coverslip on top
 Hanging Drop Method
o Allows microorganisms more room to move around. It allows us to distinguish true motility from Brownian movement.
 Brownian movement is due to the random movement of particles. It is caused by molecules of the liquid colliding with the
organism. This would not be considered true motility because even if the organisms were not alive, they have that same
motion.
 Representative organism: Gaffkya tetragena, Streptococci (Brownian movement) and Proteus vulgaris, Escherichia coli
(true motility)
o Samples should be prepared as early as possible to see them in motile state, otherwise, they become sluggish. As time increases,
more and more bacteria becomes sluggish.
o Materials: coverslip, depression slide, toothpick, Vaseline/petroleum jelly, paper towels
o Procedure: Use toothpick to put one small dab of Vaseline/petroleum jelly on each of the 4 corners of the coverslip. Use a
dropper to place a small amount of the microorganism on the center of the coverslip. Flip the slide upside down and gently lay
the slide on top of the coverslip. Flip the slide back over right-side-up and view on the microscope.

A. Hanging Drop Method

 Purpose: This method is mainly used to observe motility, binary fission, and other characteristics of bacteria which may
be destroyed on staining.
 Materials: Cultures of Gaffkya tetragena and Proteus vulgaris, concave slides, cover slips, Vaseline, wire loop and burner.
 Procedure:
1. Take a loopful of the bacteria and place it on the center of a clean cover slip. Ring the concavity of the concave slide with a
little Vaseline to prevent evaporation of the bacterial drop.
2. Carefully invert the prepared cover slip and apply it over the concavity making sure the drop of bacterial culture is in the
center and the drop is hanging into the concavity without touching the slides.
3. Study the preparation under low power objective with the diaphragm partly open and the condenser slightly lowered. The
best guide in looking for the preparation is the side of the drop.
4. Raise the low power objective very slightly before switching to the high power objective. Lower the high power objective
very slowly until it almost touches the cover slip. This precaution is taken to prevent breaking of the cover slip or damaging
the objectives when you switch to high power. Look for the slide of the drop again.
5. If a concave slide is not available, the cover slip may be ringed with more Vaseline and inverted over an ordinary slide. All
hanging drop preparation must be sterilized before cleaning.

B. Intravital Staining Of Bacteria

 Purpose: This method aims to stain living cells with a dye so diluted that it exerts no toxic or inhibitory action on the cell so
that bacterial motility would be better appreciated.
 Materials: Cultures of Gaffkya tetragena and Proteus vulgaris, cover slip, concave slides, wire loop, Vaseline, burner, 1:2:00
crystal violet.
 Procedure:
1. Place two or three loopfuls of 1:2:00 aqueous solution of crystal violet (intravital stain) on a cover slip.
2. Take a loopful of the bacteria in broth culture and mix thoroughly with the stain.
3. Invert the prepared cover slip over the concavity of the concave slide ringed with Vaseline just as you did with the hanging
drop preparation.
4. Examine the preparation under low power objective and high power objective. Make colored drawings of the organisms
you have just examined.