Gram Stain


Basic Techniques of Biotechnologies
  Differential Stains
    Gram Stain
    Acid-Fast Stain
    Special Stains
      Staining Bacterial Endospores (Schaeffer-Fulton Method)
      Flagella and Capsule Stains
The simple staining procedure, makes it possible to see bacteria clearly, but it does not distinguish between organisms of similar morphology.

In 1884, a Danish pathologist, Christian Gram, discovered a method of staining bacteria with pararosaniline dyes. Using two dyes in sequence, each of a different color, he found that bacteria fall into two groups. The first group retains the color of the primary dye: crystal violet (these are called gram positive). The second group loses the first dye when washed in a decolorizing solution but then takes on the color of the second dye, a counterstain, such as safranin or carbol fuchsin (these are called gram negative). An iodine solution is used as a mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an insoluble compound) for the first stain.

The exact mechanism of action of this staining technique is not clearly understood. However, it is known that differences in the biochemical composition of bacterial cell walls parallel differences in their Gram-stain reactions. Gram-positive bacterial walls are rich in tightly linked peptidoglycans (protein-sugar complexes) that enable cells to resist decolorization. Gram-negative bacterial walls have a high concentration of lipids (fats) that dissolve in the decolorizer (alcohol, acetone, or a mixture of these) and are washed away with the crystal violet. The decolorizer thus prepares gram-negative organisms for the counterstain.

The Gram stain is one of the most useful tools in the microbiology laboratory and is used universally. In the diagnostic laboratory, it is used not only to study microorganisms in cultures, but it is also applied to smears made directly from clinical specimens. Direct, Gram-stained smears are read promptly to determine the relative numbers and morphology of bacteria in the specimen. This information is valuable to the physician in planning the patient’s treatment before culture results are available. It is also valuable to microbiologists, who can plan their culture procedures based on their knowledge of the bacterial forms they have seen in the specimen.

The numerous modifications of Gram’s original method are based on the concentration of the dyes, length of staining time for each dye, and composition of the decolorizer. Hucker’s modification, to be followed in this exercise, is commonly used today. The choice of decolorizing agent depends on the speed wanted to accomplish this step. The slowest agent, 95% ethyl alcohol, is used in this exercise to permit the student to gain experience with decolorization. Acetone is the fastest decolorizer, while an equal mixture of 95% ethyl alcohol and acetone acts with intermediate speed. The acetone-alcohol combination is probably the most popular in diagnostic laboratories.

Purpose To learn the Gram-stain technique and to understand its value in the study of bacterial
Materials 24-hour agar culture of:
  - Staphylococcus epidermidis
  - Enterococcus faecalis
  - Neisseria sicca
  - Saccharomyces cerevisiae (yeast)
  - Bacillus subtilis
  - Escherichia coli
  - Proteus vulgaris

Specimen of simulated pus from a postoperative wound infection
Hucker’s crystal violet
Gram’s iodine
Ethyl alcohol, 95%
Marking pen or pencil and slide labels

  1. Following the procedures outlined in Simple Stain, steps 1 through 6 (pages 34–35), prepare a fixed smear of each culture and of the simulated clinical specimen. You will have 7 smears labeled Se, Ef, Ns, Sc, Bs, Ec, Pv, and pus. On the underside of each slide, make a code mark so that you can identify the slides after staining.
  2. Stain each smear by the following procedures (this is Hucker’s modification of the Gram stain):
    1. Flood slide with crystal violet. Allow to stand for one minute (check with instructor; time varies with different batches of stain).
    2. Wash off with tap water.
    3. Flood with Gram’s iodine (a mordant). Leave for one minute.
    4. Wash off with tap water.
    5. Decolorize with alcohol (95%) until no more color washes off (usually 10–20 seconds). This is a most critical step. Be careful not to overdecolorize, as many gram-positive organisms may lose the violet stain easily and thus appear to be gram negative after they are counterstained.
    6. Wash off with tap water.
    7. Apply safranin (the counterstain) for one minute.
    8. Wash off with tap water.
    9. Drain and blot gently with bibulous paper. Air dry the slide thoroughly before you examine the preparation under the microscope.
  3. When slides are dry, label them as shown:|

  4. Examine all slides under oil with the oil-immersion objective.
  5. Record observations in table under Results.
  6. Examine Colorplates 1-6 and 8, noting which bacteria are gram positive or gram negative.