Laboratory Investigations in Microbiology
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Chapter 5: Differential staining

Differential stains can distinguish bacteria based on their affinity for certain dyes and the ease with which cells can be de-stained. The two differential stains we will observe here are the Gram stain and the acid-fast stain.

One of the most commonly used staining procedures in Microbiology is the Gram Stain (Atlas p. 35 - 38). Named after Christian Gram, this procedure distinguishes among two types of bacteria: Gram-positive and Gram-negative (Atlas Fig. 5.2). Because this stain makes a distinction between bacteria, it is known as a differential stain. The Gram stain procedure involves four major steps (Atlas Fig. 5.1):

1. Stain cells with Crystal violet, a basic dye
2. Fix the crystal violet within cells using iodine ("mordant")
3. Treat the cells with alcohol ("decolorizer"), which removes the crystal violet from some cells
4. Counterstain the cells with a pink dye (safranin)

Gram-positive cells are turned purple with the crystal violet. The iodine fixes the stain in the cells so that it does not easily wash out, and the alcohol acts on the thick peptidoglycan cell wall of Gram-positive cells in such a way as to trap the dye in the cells rather than washing it away. Hence, Gram-positive cells stay purple. The safranin also enters the cells, but the darker crystal violet masks it.

Gram-negative cells also are turned purple by the crystal violet, but the iodine mordant is not sufficient to retain the dye in the cells when they are rinsed with alcohol. Since Gram-negative cells have a very thin layer of peptidoglycan, the crystal violet is rinsed out, and the cells become colorless. Counterstaining with safranin then turns Gram-negative cells pink.

It is, however, important to note that not all bacteria follow this pattern. Some Gram-negative cells (e.g. Deinococcus), when stained, appear purple because they have an unusual cell wall structure that retains the crystal violet like Gram-positive cells do. Some Gram-positive cells may, in an older culture, stain pink or pink + purple ("Gram-variable") even though they have a regular Gram-positive wall. Lastly, the meaning of Gram-positive and Gram-negative is lost when applying this technique to Archaea or eukaryotes - since these organisms do not have peptidoglycan cell walls! Nonetheless, this staining procedure can be used as an identifying trait of virtually any bacterial species.

The Acid-fast stain is used to identify bacteria such as Mycobacterium tuberculosis. Mycobacteria have a tough waxy cell wall that makes them tough to kill. This cell wall also resists destaining with acids, and therefore this procedure can distinguish acid-fast bacteria (which keep the dye) from other bacteria (which become colorless). 

The goals of today's lab exercise are to perform a successful Gram stain and to use this procedure to distinguish various types of 'unknown' bacteria.

Note: This procedure is part of the Laboratory Skills Test

Materials & Methods

Materials per lab group

• Gram stain set (includes Crystal Violet, Iodine, Decolorizer, Safranin)
• Mixed culture of E. coli (Gram-negative rods) and S. aureus (Gram-positive cocci)
• Glass slides, lens paper, immersion oil, paper towels, inoculating loops, clothes pins

Procedures

A. Gram staining

1. Prepare and heat-fix a bacterial smear using the mixture of E. coli and S. aureus
2. Immerse the slide in the Crystal Violet jar (1 minute)
3. Rinse with a gentle stream of water
4. Immerse the slide in the Iodine solution (1 minute)
5. Rinse with a gentle stream of water
6. Briefly dip the slide into the decolorizer (1 - 5 seconds).
7. Immediately rinse with a gentle stream of water
8. Immerse the slide in the safranin solution (2 minutes)
9. Rinse with a gentle stream of water
10. Blot the slide dry with a paper towel
11. View the slide at low power until you have found the stained area. Note: At this point of time you will not be able to distinguish Gram-positive (purple) and Gram-negative (pink) accurately. Often the excess dye left on a smear (e.g. pink dye) masks the color of the cells, which may actually be purple. 
12. Go to oil immersion to determine the shape, arrangement, and color of the cells. Identify cells as Gram-positive or Gram-negative.

B. Acid-fast stain (demonstration slide)

1. Observe the demo slide. Complete the data/review sheet

Data Sheet & Review Questions (printable)

© 2003 - 2015 José de Ondarza, Ph.D.