Gram Stain using Bright Field Microscopy

Principles


In bright field microscopy, light passes from the light source through the sample into the eyepiece. All wavelengths of visible light travel through the specimen.   Most of the detail within living cells cannot be seen using bright field microscopy, because there is too little contrast between the cells and the background. Therefore, chemical dyes are used to stain the microbes before viewing them in the microscope.

 

Simple stains indiscriminately bind to the negatively-charged surface of bacterial cells. Some simple stains, such as crystal violet and methylene blue, allow all types of bacteria to be observed under the light microscope.

 

Differential stains act just as their name implies-- they differentiate between different kinds of bacteria. Like simple stains, differential stains improve the contrast so that cells may be seen from the background, but not all cells look the same.

To see a Flash animation explaining this method, click here: GRAM STAIN

Gram Stain

One of the most important differential stains is the Gram stain. For historical reason, the Gram stain is often the first test performed to identify a new bacterium.   While the actual mechanisms are not totally understood, this stain differentiates bacterial cells with an outer lipid layer and thinner cell wall (Gram - type) from those with thicker cell walls and no outer membrane (Gram + type).

Procedure

  1. Fix cells to the slide.
  2. Stain cells with crystal violet. Both cells types take up the crystal violet stain, and all cells look deep purple at this point. Flood sample with a mordant (iodine) which forms an insoluble precipitate with the crystal violet.
  3. Decolorize by rinsing with alcohol. This dissolves the outer membrane (if cells have them) and dehydrates the cell wall.  The crystal violet-iodine complex gets trapped inside bacteria with a thick cell wall, and those cell remains purple (Gram +).  In bacteria with a thin cell wall, the crystal violet-iodine complex leaks out, and the cells become colorless.
  4. Counter stain with safrinin, which stains the Gram (-) cells pink. The end result is purple-colored Gram (+) cells and pink-colored Gram (-) cells.

Advantages

  • Easy and inexpensive
  • Quickly gives you important information about identification, if working with Bacterial cells

Disadvantages

  • Has meaning for prokaryotes within the Bacterial Domain, but does not tell you anything about Eukaryotic or Archeal cell envelope
  • Tells you the Gram reaction and morphology, but does not differentiate between genus or species within the Gram (+) or Gram (-) groups.
  • Cannot view live samples.
 Fluorescent in Situ Hybridization (FISH) method

 

 

 

 

 

Examples

Pseudomonas sp. (Gram negative rods) PHIL          
Streptococcus sp. (Gram positive cocci) PHIL