Microbiology Staining Techniques


GRADES: 6-12


TIME NEEDED: 1-2 hours


Microorganisms are not only very small, they are also transparent. Stains and dyes are used to color bacterial cells and increase their contrast so they can be seen more easily with a microscope. Dyes are organic compounds composed of a chromophore group, which gives the dye its color, and an auxochrome, which binds to the bacteria.


Many bacterial dyes are positively charged (cationic) and combine with negatively charged cellular components such as nucleic acids (DNA and RNA) and acidic polysaccharides. Methylene blue, crystal violet, and safranin are cationic dyes. Other bacterial dyes are negatively charged (anionic) and combine with positively charged cellular components, such as proteins. Eosin and acid fuchsin are anionic dyes.


Most staining procedures are performed on fixed cells. Fixing, or heating the dried cells on the slide, kills the bacteria and causes them to stick to the slide.


Simple Stain:


A simple stain consists of a solution of a single dye. Some of the most commonly used dyes are methylene blue, basic fuchsin, and crystal violet. Simple stains allow one to distinguish the shape (morphology) of the bacteria. For example, E. coli and Bacillus Subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may also be observed. Bacilli very greatly in length and diameter. Staphylococcus aureus and Streptococcus pneumoniae are cocci or spherical bacteria. Cocci may occur singularly, in pairs (as in Streptococcus pneumoniae), or in clusters (as in Staphylococcus aureus). R. rubrum is a spirillum or curved bacterium, Spirilla always occur singularly.


Differential Stains:


Differential stains are more complex than simple ones and use more than one stain to differentiate cellular components. They are used to examine structural differences between bacterial groups or to provide contrast to different structures within the same organism.


Gram Stain


The Gram stain procedure uses 3 different stains. These are crystal violet, Gram's iodine, and safranin. The cells are first stained with crystal violet, then Gram's iodine. Following a rinse in alcohol, to de-colorize the cells, the cells are then stained with safranin.

The Gram stain procedure separates almost all bacteria into two large groups: the Gram-positive bacteria that stain blue and the Gram-negative bacteria that stain pink. Bacteria take up the Gram stain differently because they differ in cell wall composition. Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is removed from these cell walls that are then stained with the safranin counterstain.


Streptococcus pneumoniae, Staphylococcus aureus, and Bacillus subtillis are Gram-positive and stain blue. E. coli and R. rubrum are Gram-negative and stain pink.


In this experiment, you will learn the techniques of simple staining (using methylene blue) as well as Gram staining.


Materials Needed


Bacillis subtillis liquid culture and various other bacterial cultures

Sterile loops

Microscops slides


Compound microscope

Sterile water or sterile 0.9% NaCl (sterile saline)

methylene blue stain (Ward's)

gram stain kit (Wards): crystal violet, Gram's iodine; safranin

95% ethanol


Experimental Procedure


A. Wet Mount


The wet mount is a preparation of a culture to observe motility (movement) or structure of microorganisms.

Use a sterile inoculating loop to place a loopful of a motile bacillus culture on a slide. Cover immediately with a coverslip. Do not allow the preparation to dry out. Observe under the microscope. Draw a picture of what you see.


B. Simple Stain


  1. Place a loopful of bacillus culture into a test tube of sterile distilled water to make a suspension of bacterial cells in the water. Place a loopful of this bacterial suspension on a clean slide. Allow the bacteria on the slide to air dry.
  2. Heat fix the cells by passing the slide quickly through the flame of a Bunsen burner two or three times, with the glass surface exposed to the flame. Each pass should only be a second or two. The slide should not be so hot as to be uncomfortable to touch. (NOTE: your instructor will demonstrate this for you!)
  3. Flood the slide with methylene blue stain for 30 seconds.
  4. Rinse the slide with distilled water, blot it dry, and examine it under the microscope.
  5. Draw what you observe.



C. Gram Stain


  1. Transfer a loopful of the bacterial suspension to the surface of a clean glass slide, and spread it over a small area. Allow the slide to air dry. Fix the cells by passing the slide briefly through the Bunsen burner flame.
  2. Flood the slide for one minute with a crystal violet solution. Wash off briefly with tap water (not longer than 5 seconds).
  3. Flood slide with Gram's iodine solution and allow to sit for one minute. Wash off with tap water and drain off excess liquid.
  4. Flood slide with 95% alcohol and pour off immediately. Re-flood with 95% alcohol for 10 seconds and wash off with tap water. Drain off excess liquid. (NOTE: The first flooding with alcohol removes the excess water from the slide, so that alcohol used for decolorization is not diluted)
  5. Flood the slide with safranin solution and allow it to stain for at least one minute. Wash off with tap water. Drain the slide and blot it dry with bibulous paper. DO NOT RUB.
  6. Observe the slide under the microscope. Record what you see. Compare the size, shape and color with each of the 3 different techniques you have used (wet mount, simple stain, Gram stain).


Perform wet mounts, simple stains, and Gram stains on the additional microorganisms (including, for example, bacillus, staphylococcus, and spirillum) that will be provided for you in the lab.




heat fix cells on slide



crystal violet - 1 min



water - 5 seconds



Gram's iodine - 1 min



water - 5 seconds



ethanol - 1 second



ethanol - 10 seconds



safranin - 1 minute



water - 5 seconds



blot on paper