Laboratory 3: Examination of Living Bacteria
Unstained bacteria are difficult to observe because of the lack of contrast between the cells and the surrounding. To see them in an unstained state and also to observe their motility, a hanging-drop or a wet mount technique is used. A wet mount is the technique of placing small amounts of specimen in a droplet of water for viewing with the compound microscope. Motility is an inheritable phenotype and is critical for identification and classification of bacteria. The technique is done by applying petroleum jelly to all sides of a cover glass. Add a water droplet and specimen using an aseptic loop onto the concave side of a depression slide. Put the jelly side of the cover glass over the droplet. View the droplet as it is suspended or "hanging" from the side. It is a "hanging drop" slide because the droplet remains untouched due to the concave shape of the cover glass and it just hangs from the cover glass. Microscopic study of such a wet preparation can provide useful information. Primarily, the method is used to determine whether or not an organism is motile, but it also permits an undistorted view of natural patterns of cell groupings and of individual cell shape. Hanging-drop preparations can be observed for a fairly long time, because the drop does not dry up quickly. Wet-mounted preparations are used primarily to detect microbial motility rapidly. The fluid film is thinner than that of hanging-drop preparations and therefore the preparation tends to dry up more quickly, even when sealed. Although the hanging drop is the classical method for viewing unstained microorganisms, the wet mount is easier to perform and usually provides sufficient information.
To become familiar with method to examine the living bacteria.
1. 24 hours nutrient broth cultures of
a) Staphylococcus aureus
b) E. coli
2. Hollow ground depression slides
3. Inoculating loops
4. Glass slide and cover slips
6. Applicator sticks
A. Hanging Drop Preparation
1. Vaseline is placed at the edges on one surface of the cover slip with the aid of a wooden applicator stick and placed it on the laboratory table so that the Vaseline treated surface faces upwards. 2. The center of the cover slip is placed one drop of the given sample the inoculating loop and gently mix it. 3. A depression slide is inverted so that the depression will cover the suspension, and lowered it onto the prepared cover slip. The slide is press gently. 4. The preparation is lifted and turned it right slide up. 5. The slide is examined under low-power first and be certain to reduce the intensity of the light source. Switched to the high-power objective and examined the preparation again. 6. The method is repeated for the second organism provided. 7. The finding is recorded.
B. Temporary Wet-Mount Technique
The inoculating loop is flamed to redness.
The plug is removed from one of the bacterial culture provided and flamed the lip of the tube. 3.
A loopful of the culture is removed and placed in the center of a clean glass slide. 4.
The lip of the tube is flamed and returned the plug.
The inoculating loop is sterilized.
A cover slip is put over the bacterial suspension and press down gently. 7.
The preparation is examined under low-power first and then under high-power.
Name of bacteria
| Hanging drop technique
| Wet-amount technique
| Staphylococcus aureus
In this experiment, I can determine which one is mobile and non-mobile bacteria by using hanging drop and wet amount techniques. For hanging-drop preparation, when I observed the bacteria under low-power objective, I can prove which organism exhibited vital movement or Brownian movement. When I put Vaseline at the edge on the slides it prevent water drying...
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