The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –
1938) who originally devised it in 1882 (but published in 1884), to discriminate between
pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method
of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based
on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into
two fundamental groups according to their stainability and is one of the basic foundations on
which bacterial identification is built. Gram staining is not used to classify archaea, since these
microorganisms give very variable responses.
3 microscope slides (precleaned)
Gram stain reagents (crystal violet, Gram’s iodine, 95%
ethyl alcohol, and safranin)
24 hr. TSB cultures of Stapylococcus epidermidis,
24 hr. TSA slant of Bacillus licheniformis
48 hr. TSA slant of Mycobacterium smegmatis
SMEAR PREPARATIONS: Remember to label the slides.
GRAM STAINING PROCEDURE:
1. Cover with CRYSTAL VIOLET for 20 seconds.
2. Gently rinse off the stain with water and shake off
3. Cover with GRAM’S IODINE for one minute.
4. Pour off the Gram’s iodine.
6. Rinse with water to stop the action of the alcohol.
a) DO NOT make your smears too thick!
b) Be very careful when you decolorize.
c) Be sure your cultures are young, preferably less
than 24 hours old. Older cultures tend to lose the
ability to retain stains.
Observe your smears in the microscope under oil immersion. Draw a few representative organisms from each smear in your lab report.
Gram staining reagents and their functions
Staphylococcus Gram-positive coccus
Pseudomonas Gram-negative rod
Bacillus Gram-positive, spore-forming rod
Mycobacterium Gram-positive, acid-fast rod