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Hanging Drop Method Preparation
Take a clean glass slide and apply a paraffin ring, adhesive-tape ring to make circular concavity. (This action is not required if a glass slide with depression is available).Hold a clean coverslip by its edges and very closely dab vaseline on its corners using a toothpick.Place a loopful of the fresh broth culture to be tested in the center of the all set coverslip. Use a light inoculum (not visibly turbid).Turn the prepared glass slide or concavity slide upside dvery own (concavity down) over the drop on the coverslip so that the vaseline seals the coverslip to the slide about the concavity.Turn the slide over so the coverslip is on top and also allow organisms to “settle” for a minute. The drop can be oboffered hanging from the coverslip over the concavity.
Microscopic ObservationPlace the preparation in the microscopic lense slide holder and align it using the naked eye so an edge of the drop is under the low power goals.Turn the objective to its lowest position utilizing the coarse adjustment and also CLOSE THE DIAPHRAGM.Look via the eyepiece and also raise the objective gradually making use of the coarse adjustment knob until the edge of the drop is observed as an irconstant line crossing the field.Move the slide to make that line (the edge of the drop) pass via the center of the area.Without increasing or lowering the tube, swing the high dry objective right into place (be certain the high dry objective is clean).Observe the slide with the eyeitem and adjust the fine adjustment till the edge of the drop deserve to be viewed as a thick, commonly dark line.Focus the edge of the drop very closely and also look at each side of that line for exceptionally small objects that are the bacteria. The cells will certainly look either favor dark or slightly greenish, very tiny rods or spheres. Remember the high dry objective magnifies a small much less than fifty percent as much as the oil immersion objective.Adjust the light making use of the diaphragm lever to maximize the visibility of the cells.Observe the cells noting their morphology and also grouping and also identify whether true motility can be observed.Brownian movement should be visible on slides of all the organisms, yet tbelow need to also display true motility.Wash the depression slide and after soaking in lysol buckets or discard the all set glass slide.
Result and InterpretationsDirectional purposeful motility is a positive test. Motile organisms readjust position via respect to one an additional. Brownian motion (random jiggling or shaking as a result of molecular bombardment), wbelow the organisms remajor in the very same relative position with respect to each various other, should not be mistaken for true motility.
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Campylobacter and Vibrio cholerae display screen exceptionally active motility (darting motility) which shows up as tiny dots darting in and out of the area.For all organisms negative for motility by initial wet mount, repeat the wet mount after incubation in broth, or test by tube strategy.Incubate at 30°C for nonfermenting, Gram-negative rods (24 h).Incubate enterococci and also Listeria at 30°C for 2 h.Other organisms might be incubated at temperatures optimal for their development, normally 35°C.While examining living organism for the property of energetic locomovement, it is important to identify true motility, whereby the organism move in different directions and also readjust their positions in the area, from eitherPassive drifting of the organisms in the exact same direction in a convectional current in the liquid orBrownian motion, which is an oscillatory activity around a nearly addressed allude possessed by all small bodies suspfinished in liquid and because of irregularities in their bombardments by molecules of water.