Hanging Drop Procedure:



 



1. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick.  If too much Vaseline is used, it will be squeezed toward the center and mix with the drop or squeeze out the edges and get on the objective lens of the microscope.

2. Place a loopful of the culture to be tested in the center of the prepared coverslip.

3. Turn the clean concavity slide upside down (concavity down) over the drop on the coverslip so that the Vaseline seals the coverslip to the slide around the concavity.

4. Turn the slide over so the coverslip is on top and the drop can be observed banging from the coverslip over the concavity.

5. Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the drop is under the low power objectives.

6. Turn the objective to its lowest position using the coarse adjustment and CLOSE THE DIAPHRAGM.

7. Look through the eyepiece and raise the objective slowly using the coarse adjustment knob until the edge of the drop is observed as an irregular line crossing the field.

8. Move the slide to make that line (the edge of the drop) pass through the center of the field.

9. Without raising or lowering the tube, swing the high dry objective into position (Be sure the high dry objective is clean).

10. Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the drop can be seen as a thick, usually dark line.

11. Focus the edge of the drop carefully and look at each side of that line for very small objects that are the bacteria.  The cells will look either like dark or slightly greenish, very small rods or spheres.  Remember the high dry objective magnifies a little less than half as much as the oil immersion objective.

12. Adjust the light using the diaphragm lever to maximize the visibility of the cells.

13. Observe the cells noting their morphology and grouping and determine whether true motility can be observed.

14. Brownian movement should be visible on slides of all the organisms, but two should also show true motility.

15. Wash the depression slide and after soaking in lysol buckets.
 

NOTE:  The bacteria are still alive in a hanging drop slide.  Slides made from possible pathogens should be soaked in lysol for 5-10 minutes with the coverslip pulled aside to expose the drop before they are washed.

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Lab Procedures