4 Smear Preparation & Simple Stain

As we discussed in learning about brightfield microscopy, the cells must be stained to be viewed. Otherwise, they would be clear against a bright light, and you would not be able to see them. Staining allows for contrast, and allows the bacteria to be studied with respect for its cell shape, size, and arrangement.

The smear preparation is what causes the cells to adhere to the microscope slide so that they are not washed off during the staining process. Heat needs to be applied in order for this adherence to occur. It is this heat that causes cell death during the staining process. A goal in preparing a smear is a thin smear because the thickness can affect several factors. It can determine if you can view individual cells that aren’t stacked on each other. A proper smear can show cell shape, the arrangement of the cells, or any other details regarding the microstructures associated with the cells. Overly thick smears also can affect the staining process by entrapping the stain, keeping it from being removed through the process of decolorizing or rinsing, leading to erroneous results.

Once the smear preparation is finished, it is time to stain. A common staining technique to observe cell shape, arrangement and size is a simple stain using only one stain to colorize the bacteria. Simple stain most often uses a basic stain. A basic stain is a stain that contains color-bearing ions called chromophores that are positively charged. Bacteria themselves have a slightly negative surface. Owing to opposite charges attracting one another, the stain binds to the bacterial cell, causing the bacteria to be colored against a transparent background. Another name for the basic stain is positive stain due to the chromophores having the cationic charge.

MATERIALS

Each student should have:
Blue rack
2 glass slides
Stain bottle rack: Methylene blue (or crystal violet)
Lens paper
Windex (depends on instructor)
Inoculating loop
Wax pencil
Metal slide clip
Bunsen Burner
Striker
Stain tray
1 culture of either Escherichia coli or Staphylococcus epidermidis
Microscope

SMEAR PREPARATION PROCEDURE

From a Slant culture:
MAKE 2 SMEAR PREPS. ONE WILL BE USED AS A BACKUP.

1. Draw a circle with the wax pencil on a slide.
2. Flip the slide over. Write “Up”.
3. Place a small drop of distilled water onto the slide within the wax circle.
4. Using aseptic technique, remove a small amount of bacteria from a slant or an isolated colony on a streak plate, and transfer onto the slide, spreading within the wax circle. (Make sure not to take too much bacteria. You want thin smears.)
5. Air dry the slide. Do not apply heat to a wet slide. Do not blow on the slide.
6. After all water has evaporated and smear is completely dry, place the slide in a metal slide clip and pass it multiple times through the Bunsen Burner flame as demonstrated by your instructor. This is called heat-fixing a smear. It adheres the bacterial cells to the slide. Avoid prolonged heating of the slide as it can distort the cell shape and size.

PROCEDURE OF SIMPLE STAIN

STAIN ONLY ONE SLIDE. You may not need the second slide. This serves as a backup in case you have error in your staining procedure. It saves you time.

1. Place one heat-fixed smear prep on a stain tray.
2. Stain slide with methylene blue or crystal violet for 60 seconds.
3. Rinse slide with distilled water until clear.
4. Blot dry with a paper towel.
5. Examine under the microscope and show your instructor at 1000x magnification, oil immersion.
6. After your instructor’s approval of your staining technique, dispose of your slides in the plastic beaker labeled “Self prepped” in the Discard Area.

REFERENCES

Brown, A. E. (2009). Benson’s Microbiological Applications: Laboratory Manual in General Microbiology. New York: McGraw Hill.

Chess, B. (2015). Laboratory Applications in Microbiology: A Case Study Approach. New York: McGraw Hill. Pommerville, J. (2007). Preparation of a Bacterial Smear and the Simple Stain Technique. In J. Pommerville,

Alcamo’s Laboratory Fundamentals of Microbiology (Chapter 4). Boston: Jones & Bartlett, LLC. Retrieved from http://samples.jbpub.com/9780763795573/95573_ch04.pdf