What is a hanging drop preparation?

Answer 1

Direct demonstration of V.cholerae is not recommended for general purposes. However, darting motility and its inhibition of specific antisera, especially after brief inoculation in broth, has proven valuable in experienced hands for presumptive diagnosis. The method is as follows: Prepare a hanging drop from alkaline peptone water which has been inoculated with faeces and incubated at 37oC for 4-6 hours. Examine it under high power dry lens of microscope. Darting motility characteristic of vibrios is suggestive of diagnosis.

Answer 2

The Hanging Drop Procedure

The direct examination of live microorganisms can be extremely useful in determining size, and shape relationships, motility, and reactions to various chemicals or immune sera. Two methods are generally used for this type of study; hanging drop and wet mount. Both methods maintain the natural shape of microorganisms and reduce the distorted effects that can occur when specimens are dried and fixed. Because the majority of microorganisms are not too different in either color or refractive index from the fluid in which they are suspended, low light intensity is suggested (closed iris).

Purpose: To observe live culture motility using the Hanging Drop method.

To know the difference between true motility (vital movement) and

Brownian motion.

Materials: 24 hour (37oC) nutrient broth culture of Bacillus subtilis

3 day old culture of Saccharomyces cerevisiae ( yeast suspension)

Microscope slides (concave), coverslips, washers (spacers)

Inoculating loop and Inoculating needle, Immersion oil Procedure: Hanging Drop See Diagram 6

• 1. Aseptically transfer a loop full of yeast (Saccharomyces) suspension to the center of a new glass coverslip.
  2. Aseptically transfer a loop full of the Bacillusculture to the same glass coverslip. Together, they should make a small drop.
  3. Obtain a concave slide and place it face-up on the table in front of you. Place a yellow washer on the slide so that it surrounds the depression in the slide.
  4. Carefully flip the coverslip containing the microorganisms over and place it on top of the washer being careful not to let the culture drop touch the washer.
  5. Observe the edge of the drop with the iris closed down. You may go all the way to oil immersion magnification.
  6. Try to observe both bacteria and yeast. Look for motility (vital movement) as well as Brownian motion.
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