BISC209: Preparing a bacterial smear slide

Preparing a bacterial smear slide

Preparing a bacterial smear slide is useful for microscopic examination of dead bacterial cells.
1. Place a very small loopful of deionized water on the slide (you can use the deionized water bottle on your bench-remove the cover and dip your loop in since sterility is not required for this step).
Note: If the bacterial smear is being made from a broth, simply place a loop or two of the broth culture on the slide and spread, do not use additional water.
2. Flame the loop, cool, and touch it to the colony or slant growth. Place the now contaminated loop into the drop. Use large circular motions to spread the drop to about the size of a quarter over the slide (if you are making one smear per slide). 3. Reflame the loop.
4. Be sure all the liquid on the slide has evaporated before proceeding to heat fixation (or you will explode the cells in the next step).
5. Heat fix (to kill and attach organisms to slide) by rapidly passing the slide (smear side up) through a flame 3 times. Use a slide holder and avoid contact with hot glass.

With practice it is more efficient to put multiple samples on one slide. If you are making multiple smears on one slide, use a smaller drop of water or less liquid culture for each, space them so they do not mix, and spread to about the size of a nickle rather than a quarter.
When staining multiple smears on one slide you must be careful to apply the staining reagents so they cover all smears evenly and for the same amount of time. Label the slide with a graphite pencil in the ground glass section on the far left of the slide since the reagents used in staining (such as the decolorizer in the Gram stain) can remove your labels if you use pen or wax pencil. By convention, labels (top to bottom) match smears (left to right).
An example of a multiple smear labeled slide:

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