Ex. 6: Hanging Drop Technique

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Presentation transcript:

Ex. 6: Hanging Drop Technique Key concepts: Wet mount technique Motility Bacterial flagella and their arrangement

Motility Determination: Hanging Drop Method Materials needed: Bacterial culture Cover glasses Petroleum Jelly (Vasoline) Depression slides Toothpicks

Step 1: Preparing Cover Glass Using a toothpick, place a small dab of Vaseline in each corner of a clean cover glass. Handle the cover glass by its edges to avoid fingerprints. Aseptically transfer a loopful of a liquid bacterial culture into the center of the cover glass. Do not spread the drop! Alternatively, if you have a solid culture . . . .

Step 2: Attaching Depression Slide Vaseline attaching cover glass to depression slide With the concave side facing down, position the depression slide over the cover glass. Slowly lower the slide, while centering the drop in the depression. Gently press the depression slide against the cover glass just enough to allow the Vaseline to make the attachment. Carefully lower depression slide with depression facing down.

Step 3: Invert Depression Slide Quickly invert depression slide. Drop will now be suspended from the cover glass inside the concave hollow. Suspended drop

Step 4: Examine under Microscope Edge of drop Drop 10x objective To increase contrast ,move condenser all the way down and keep light as low as possible by closing iris diaphragm. With 10x objective and using course adjustment knob, focus on the edge of the hanging drop.

Bacteria With 40x objective, focus on isolated cells near inside edge of drop. Flagella are not visible with light microscope. Motile cells move independently. All other cells seem to vibrate in place (Brownian motion). Observe slide immediately after preparation as motility decreases over time. Also, as drop dries out and recedes, water currents will appear to herd and move bacteria in the same direction – this has nothing to do with motility!

Brownian Motion Discovered by British scientist Robert Brown in 1827. Using microscope, he noticed pollen grains suspended in water jiggled. Later determined to be the action of collisions with surrounding molecules. Temp.   Motion intensity ? Particle size   Motion intensity ?

True Motility Proteus vulgaris: Highly motile

Step 5: Proper Disposal and Cleaning of Wet Mounts Cover glass: dispose of in ….??? Depression slide: spray with H2O2 . Let sit for 1 min. Clean off with Kim wipe. Kim wipe disposed of how? Wipe depression slide off with 70% alcohol to remove all Vasoline. Kim wipe disposed of how?

In-house movie clip on the Hanging Drop Technique the end