EXERCISE 2

 

Smear Preparation and Gram Stain

 

Supplies Required: Microscope slides, inoculating loop, Bunsen burner, marking pen, crystal violet, Gram's iodine, 95% ethanol, safranin, cultures of Micrococcus luteus, Escherichia coli, Rhodospirillum rubrum, Bacillus megaterium, and Saccharomyces cerevisiae.

 

Protocol:

 

1.         The slides are prewashed and ready to use. Touch the slides by the edges only.

 

2.         Label a slide on one edge with the initials or a code number of the organism you wish to smear. You can easily make two separate smears on one slide.

 

3.         Mix the bacterial suspension and, following the description below, aseptically transfer 2

loops of the culture to the area of the slide you have labeled. Aseptic transfer will be demonstrated by the Laboratory Instructor and is illustrated at:

 

           http://www-micro.msb.le.ac.uk/video/labvid.html

Click on Aseptic Technique.

 

            a.         First loosen the tube cap without removing the top.

            b.         Hold the loop between the thumb and the index finger of the right hand

                        (or left if you are left-handed). You should be holding the loop as if it were

                        a pencil, with the top of the loop pointing away from you.

 

            c.         Insert the loop into the flame while holding the loop handle at about a 60o angle.

                        Flame the entire loop until it is red hot, remove, and allow to cool. This

                        procedure has sterilized the loop.

 

            d.         Using the little finger and ring finger, remove the cap from the tube and briefly

                        flame the lip of the tube. This procedure effectively sterilizes the lip of the tube.

 

            e.         Insert the cooled loop directly into the broth (a short distance), withdraw

                        the loop, reflame the lip of the tube, and replace the cap. Set the tube back

                        in the test tube rack and proceed to smear the organisms on your slide.

                        After you have prepared the smear, reflame your loop.

 

            f.          For a second loopful, repeat procedures c-e and add the contents of this loopful

                        to the first before it begins to dry out.

 

4.         Spread the organisms over an area on the slide about the size of a dime.

 

5.         Allow the slide to dry by evaporation (do NOT speed this step up by heating).

            If this takes more than 5 minutes you've probably got too much liquid in your

            smear or have not spread it out properly.

 

6.         After the slide has air-dried pass the slide over the flame to heat fix the organisms

            to the slide (the slide should become moderately warm to the touch).

Heat serves to coagulate cell proteins (which kills the cells) and dries the cells firmly to the slide - this will prevent their washing away during staining.

 

7.         Place your slide on a staining rack over the sink and flood the smear with crystal violet for 1-2 minutes. Pour off the excess stain and rinse gently with distilled water.

 

8.         Cover the smear with Gram's iodine for 2 minutes. Wash gently with water.

 

9.         Decolorize the smear with 95% ethanol by dripping it over the tilted slide until the drops

run colorless. This is by far the most critical step in the Gram stain. Decolorization should take no more than 15-20 seconds.

If you over-decolorize your smear you may eventually begin removing the crystal-violet iodine complex from Gram positive cells as well. If you need more than 15-20 seconds to properly decolorize, you probably have too thick of a smear. Wash your decolorized smear with water (keep in mind here that if you have smeared a Gram-positive organism, the smear will decolorize some with alcohol but the cells will still appear blue-purple after "decolorization.")

 

10.       Counterstain your smear for 2 minutes with safranin. Rinse with water and blot

            carefully on bibulous paper.

 

View under the oil immersion lens (no cover slip is needed). Gram-positive bacteria are

blue to purple, Gram-negative bacteria, pink to red.