wet mounts - the wet mount method and the hanging drop method for motility

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wet mounts - the wet mount method and the hanging drop method for motility in microorganisms. Microbiology Bacteria cells can be seen easily and clearly, when colored by stains, but in most of the staining processes, the cells die and lose their natural shape and size due to heat-fixation as well as due to exposure to chemicals (stains, acid and alcohol). The aim of this experiment is to observe the natural shape, size and arrangement of bacteria in living condition. Motility cannot be observed in wet mount, as the cells are pressed between the slide and the cover slip.

Principle:
In wet mount, a drop of the bacteria suspension is placed on a slide, covered with a cover slip and observed under a compound microscope or preferably under a dark-field or phase-contrast microscope using oil-immersion objective. Materials Required are a glass slide, cover slip, petroleum jelly, immersion oil (cedar wool oil or liquid paraffin), bacteria culture (on solid medium or in liquid suspension), loop and microscope (compound, dark-field or phase- contrast).The slide is dried by wiping with bibulous paper and subsequently moving over flame or keeping in the sun. If the bacteria has been grown on a solid medium (as slant culture or plate culture), a drop of distilled water is put at the center of the slide. A loop is sterilized by heating it over a flame to red hot. It is cooled to room temperature. Then, aseptically a small portion of bacteria culture is scraped by the loop and transferred to the drop of water. A suspension of bacteria is made by gently mixing the loop of bacteria in the water drop, in such a way that, the drop does not spread. If grown in a liquid medium as suspension, aseptically a drop of the bacteria suspension is placed at the center of the slide directly, using a flame-sterilized loop.Petroleum jelly or similar material is applied surrounding the drop, so that, when the cover slip covers the drop, evaporation and effect of air current is minimized. A cover slip is kept in a slanting position near the drop of bacteria suspension with one edge touching the slide.
Slowly the cover slip is lowered, till it touches the surface of the drop. Then, the cover slip is released, so that the drop of bacteria suspension spreads in all directions under the cover slip. Care should be taken, so that no air bubble is formed in the suspension.

The slide is clipped on the stage of a compound microscope and observed using low power and high dry objectives. Then, a drop of immersion oil is put on the cover slip and observed using oil-immersion objective of the microscope. Preferably, a phase- contrast or dark-field microscope should be used for clear observation.

Observations (Under Oil-immersion Objective):
1. Shape of bacteria: Spherical (coccus) Rod-shaped (bacilli) Comma-like (vibrio) Spiral (spirochetes), Pairs diplobacillus/diplococcus) In fours (tetrads) In chains (streptococcus/streptobacillus) Grape-like clusters (staphylococcus) Cuboidal (sarcinae or octet). Size of bacteria can be estimated. Observe under oil-immersion objective.

YouTube url:	https://www.youtube.com/watch?v=BtRT3DSzhWU&ab_channel=ScientistCindy
Created:	11. 2. 2021 14:43:36

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wet mounts - the wet mount method and the hanging drop method for motility