BACTERIAL SMEARS AND SIMPLE STAIN

1. Obtain a clean slide. 
   
2. Using aseptic technique, smear a loopful of bacterial sample from a pure culture onto the center of the slide.  (Remember:  you do not need to see any material on the loop; a tiny bit of broth, or a touch from a colony of agar surface contains many bacteria).  If the sample is from a slant, add a tiny drop of water. 
   
3. Smear the sample in a circle on the slide, staying within the center 1/3 of the slide.  Particularly with the moistened sample from the agar slant, care must be taken to see that the bacteria from the colony are separated from each other so they may be observed individually.  This may take rather vigorous action with the loop as you smear onto the slide surface.  If you see dense particles, or a very turbid smear, you have chosen too large a sample. 
   
4. Mark an “X” with a grease pencil to show the side of the slide with the bacteria.  (You would be surprised to know how many students turn the slide over during the procedure and inadvertently stain the wrong side, or try to observe the wrong side with the microscope.  The oil immersion lens will not focus of the bacterial cells if you have placed the slide on the stage smear side down.  If you are not sure, you can scratch the “X” mark to determine if you are on the correct side with staining or observing procedures.  You should not be able to see the smear itself.  If frosted slides are available, you can easily write the name of the organism on the frosted section.) 
   
5. Let the preparation on the slide air dry at room temperature, then using a slide holder to protect your fingers from heat, hold the slide (smear-side away from the heat) near the mouth of the Bacti-Cinerator for a few seconds.  Caution:  A greater length of time would heat the slide to the breaking point.  This procedure is known as “heat-fixing."  It kills the bacteria and causes them to adhere to the slide. 
   
6. Let the slide cool. 
   
7. Put 2-3 drops of a stain on the smear and let it sit for 2 minutes.  Use crystal violet or methylene blue or safranin.  Rinse gently with tap or distilled water. 
   
8. Blot between sheets of bibulous paper.  (CAUTION):  Too much pressure will break the slide;  a “wiping” motion will remove the smear.) 
   
9. Place on microscope and observe.  (If adequately blotted, it is not necessary to use a cover slip, even on oil immersion.) 
   
10. Sketch and label your observations as to morphology and arrangement of cells.