Negative Stain Procedure

To conduct a proper negative stain the following procedure should be followed:

1. Place a very small drop (more than a loop full--less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.

2. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.

3. Use another clean slide to spread the drop of stain containing the organism using the following technique.

4. Rest one end of the clean slide on the center of the slide with the stain.  Tilt the clean slide toward the drop forming an acute angle and draw that slide toward the   drop until it touches the drop and causes it to spread along the edge of the spreader slide.  Maintaining a small acute angle between the slides, push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even, thin smear.

5. Allow the smear to dry without heating.

6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain.

Results of a Negative stain
Lab Procedures