Observation of Microorganisms under Light Microscope

Observation of Microorganisms under Light Microscope Two general techniques are used to prepare specimens for light -microscope examination 1) Suspend organisms in a liquid -wet-mount or -hanging-drop technique 2) Dry, fix, and stain films or smears of the specimen

The hanging drop and wet mount techniques allow for observation of living organisms. The wet mount tend to dry out quickly under the heat of the microscope light; it is useful for short-term observation only.  The hanging drop is a more complex technique, but it allows for longer-term observation and more reliable observation of motility. These techniques are usually performed without the addition of any stains; therefore, the organisms can be difficult to see.

Wet-mount preparation Wet mount preparations are especially valuable for demonstrating motility in microorganisms. Fresh cultures must be used for maximum motility. No stain is employed since most stains kill the organisms (except vital stains). Wet mount preparations are also useful for giving clear images of fresh specimens under the microscope. Features which may be particulate, such as spores of fungi and ferns etc may be best observed using this technique. A wet mount is made by placing a drop of fluid containing the organisms onto a glass slide and covering the drop with a cover slip. To reduce the rate of evaporation and exclude the effect of air current, the drop may be ringed with petroleum jelly or similar material to provider a seal between the slide and the cover slip.

PREPARATION OF WET MOUNT SLIDE FOR CULTURES:  Place 15 - 20 uL of the culture in the middle of the slide.  FOR COLONIES:  Place a small drop of dH2 O in the center of a slide CULTURES:  The sample of liquid culture is placed on the slide. COLONIES: Sterilely transfer a tiny portion of a single colony to the drop with a loop and suspend (be certain to allow the loop to cool before picking up specimen).  For solid specimens or dry spores, transfer a small portion of the specimen with a scalpel.

PREPARATION OF WET MOUNT SLIDE (contd….) Lower a clean cover slip over the drop as though it were hinged at one side. Finished preparation.  Note that it is essentially transparent, making focusing difficult.

Use of wet preparation: The morphology of spiral bacteria is greatly distorted when these bacteria are dried and stained; they should be examined in living condition. For example, in the examination of serious exudates suspected of containing the spirochete that causes syphilis, the wet preparation are examined by dark-field microscopy. This provides a sharp contrast between the organisms and the dark background.

Hanging Drop Procedure 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, to observe cell activities such as motility and binary fission. And, also to observe the natural sizes and shapes of the cells, since heat fixation and exposure to chemicals during staining cause some degree of distortion.

Hanging Drop Procedure Apply a ring of petroleum jelly around the concavity of the depression slide. Using sterile technique, place a loopful of the mixed culture in the centre of a clean coverslip. Place the depression slide, with the concave surface facing down; cover the coverslip so that the depression covers the drop of culture. Press the slide gently to form seal between the slide and the coverslip. Quickly turn the slide right side up so that the drop continues to adhere to the inner surface of the coverslip. For microscopic examination, first focus in on the drop of culture under the low-power objective with reducd light. Place a drop of immersion oil on the coverslip and use the oil-immersion objective for detailed observation.

Hanging Drop Preparation