Hanging Drop Preparation And Its Modifications

Microscopy Methods in Microbiology
Microscopy Methods in Microbiology Part 1
Measurements with Optical Microscope
Linear Measurements
Measurement by Eyepiece Graticule
Measurements by Filar Micrometer
Measurements from Photomicrographs
Measurement of Volume or Thickness
Measurements of Area
Electron Microscopic Method for Micrometry
Observation of Microbes in Living State - Introduction
Wet Mount or Wet Film Preparations
Observation of Bacteria Protozoa and Algae
Observation of Fungi
Hanging Drop Preparation and its Modifications

Hanging Drop Preparation and its Modifications

	Hanging Drop Preparation and its Modifications In this technique, a drop of medium containing cells to be observed is allowed to hang in the cavity of slide. The advantage of this preparation over the wet mount preparation is the increased capacity of aeration as the drop is surrounded by an air space. Along with the disadvantages described for the wet mount preparation this technique has an additional interference with the optical path, which is due to the inclusion of an air layer in between the glass slide and the sample. Thus, there are atleast three different refractive indices involved.
The situation becomes still worse by the curvatures of the cavity of the slide and hanging droplet: Hence, really a good microscopy is not possible with this preparation. It has been found to be advantageous to replace cavity slide by a standard flat slide and mount the cover glass containing drop of cells on the platform of a grease. Inspite of all these, this is the best method available for the routine use to observe the motility of bacteria.
This is because, it is relatively easy to make and less time consuming. For the demonstration of motility to students, it is advisable to use the slide which has got two cavities. One can compare the result of sample with the control kept in another cavity side by. It is essential to differentiate true motility from the Brownian movement of bacteria. In true motility, the organism changes its position, while in the Brownian movement, the organism oscillates at its place and does not change the position in the field.
Brownian movement of the organism can be explained on the basis of the, bombardment of small particles suspended in the fluid to the cell. Basically, hanging drop preparation does not differ much from the wet mount preparation. Satisfactory results can be demonstrated by following the below listed steps. Using fine needle or tooth-pick, make a thin layer of petroleum jelly on the periphery of the cavity of the slide.
	This type of sealing material is used primarily to reduce evaporation of liquid and to eliminate air current in the preparation. Place a drop of broth culture onto the centre of the cover glass kept on flat surface. The density of the culture broth should be neither too high nor too low. If broth culture is not available then use the condensation fluid of slope culture. Alternatively, one can transfer carefully a loopful of culture in nutrient broth and allow it to incubate for the sufficient period of time to get the desire culture density. Invert the cavity slide over the cover glass, containing culture drop, in such a manner that the drop comes in the centre of the cavity. Allow cover glass to adhere to jelly and gently turn the slide so that the drops of culture hang in the cavity. Place the slide on microscope stage and carefully focus the edge of the drop so that it appears across the centre of the field.

The amount and the intensity of light should be reduced as described for the wet mount preparation. Turn the high power objective lens into position and focus the edge of the drop. Adjust the amount and intensity of light to obtain sharp view. Observe the behaviour of organisms in the inner side of the drop edge. Certain microorganisms, such as protozoa, move too quickly to be studied. Carboxymethyl cellulose (2%) may be used to slow down the speed of such actively motile organisms. Though, the hanging drop preparation for most purposes other than the observation of motility of bacteria is unsuitable, the early application of this method to study the relationship between the generation time of cells and temperature was made by Barber (8). Later, Allen (9) and Kahn (10) used this method for the estimation of generation time of bacteria growing in milk and to study the mycobacterial life cycle, respectively.

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