BACTERIAL SMEARS AND SIMPLE STAIN

Bacterial cells are usually colorless because cytoplasm, for the most part, is transparent. Since the bacteria are colorless, it is almost essential to add a stain to make the bacteria more visible. Once stained, cell morphology can be observed.

Stains are solutions that contain a solute called a chromophore dissolved in a solvent. A chromophore is the color possessing portion of the solution and is therefore responsible for the stains color. Bacterial cells usually have a negative surface charge, meaning that a positively charged stain is needed to stain the surface of the cell. Most stains are basic and have a positively charged chromophore. When the stain is applied, there is an attraction between the negatively charged cell surface and the positively charged chromophore, leading to the surface of the cell taking on the color of the stain.

A stain that stains the background of a slide is referred to as a negative stain because of its negative charge. Remember that the surface of bacterial cells are negative. When a negative stain is applied, the stain is repelled from the surface of the cell because of the like charges. This causes the background to stained and the cell to remain colorless. This technique is useful for determining cell shape and arrangement. 

STUDENT INSTRUCTIONS
Working in teams of two, make smears of the bacterial species from your pure cultures and some additional specimens.  Follow these steps to make simple stains of the following organisms: Bacillus subtilis, Escherichia coli, Staphylococcus epidermidis, and Rhodospirillum rubrum.

Rhodospirillum rubrum is a unique organism that can be isolated from water and mud. It can growth both aerobically without light, or anaerobically in the presence of light. This is due to the fact that R. rubrum shifts energy metabolism to photosynthesis in the presence of light. It tends to possess a purple-red pigment when grown in light due to photosynthetic pigments, but the pigmentation may be lost if the organism is incubated in the absence of light.

Basic Staining (staining the cell surface)

  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 dry on the slide dryers atop the Bacti-Cinerator. Then, hold the slide (smear-side away from the heat) near the mouth of the Bacti-Cinerator for a few seconds, passing the slide in front of the chamber in sweeping motions to avoid overheating. 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 (1 minute) or methylene blue (1 minute) or safranin (1-2 minutes).  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 using all powers of your microscope, including oil immersion.  (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.

Negative Staining (staining the slide background)

  1. On a clean slide, add a drop of India ink near one end of the slide. Do not let the drop spread or run.

  2. Using proper aseptic technique, sample the culture provided to you .

  3. Mix the sample of the bacteria with the drop of India ink, being careful not to spread out the drop of ink.

  4. Using another clean slide placed at an angle on the original slide, spread the drop out so that it starts to form a thin film on the slide.

  5. Draw the angled slide along the other slide, spreading the mixture of bacteria and stain evenly on the slide. Try to maintain consistent pressure to avoid thick and thin areas. 

  6. Let the slide air dry completely or carefully dry it on the slide dryer atop the Bacti-Cinerator (careful not to over dry, which will cause the ink to crack).

  7. Observe using all powers of your microscope, including oil immersion.