BISC209/S12: Smear slide

Preparing a bacterial smear slide

Preparing a bacterial smear slide is useful for microscopic examination of dead bacterial cells.
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 it with the loop and do not use additional water. (Skip steps 1 and 2 for smears from broth cultures.)

1. Place a very small loopful of deionized water on three evenly spaced parts of 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).
2. Flame the loop, cool, and touch it to a bacterial colony. Place the now contaminated loop into a drop of water on the slide. Use circular motions to spread the drop over a section of the slide. Make sure that you do not spread the bacteria so far that you mix them into adjacent water drops (if there are 2 or 3 drops on a slide).
3. Reflame the loop.
Repeat step 2 until all the smears desired are made on each slide. It is a good idea to make one smear on each slide a control containing known Gram neg and/or Gram positive bacteria (either a single culture or mixtures of both Gram positive and Gram negative organisms in one smear).
4. Be absolutely positive that 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 passing the slide (smear side up) through a flame 3 times. If you are unfamiliar with this step, ask your instructor to demonstrate the appropriate amount of time that the flame is in contact with the slide. Use a slide holder and avoid burning your fingers.

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: