GRAM STAINING
Aim :
To observe the given bacterial culture using gram staining.
Principle :
The Gram stain, a differential stain was developed by Dr. Hans Christian Gram,
a Danish physician, in 1884 that is why Gram Staining It is a very useful stain for
identifying and classifying bacteria into two major groups the gram-positive and
gram-negatives. In this process, the fixed bacterial smear is subjected to four
different reagents in the order listed crystal violet (primary stain), iodine solution
(mordant), alcohol (decolorizing agent) and safranin (counter stain).The colour
changes that occur in bacterial cells at each stage due to these four reagents in the
Gram staining process. The bacteria which retain the primary stain (appear dark
blue or violet) (i.e. not decolorized when stained with Grams' method) are called
gram-positive, whereas those that lose the crystal violet and counter stained by
safranin (appear red) are referred to as gram-negative. Gram's reaction to some of
the important pathogenic bacteria.
The differences in staining responses to the Gram stain can be related to
chemical and physical differences in their cell walls. The gram-negative bacterial
cell wall is thin, complex, multilayered structure and contains relatively a high
lipid contents, in addition to protein and mucopeptides. The higher amount of
lipid is readily dissolved by alcohol, resulting in the formation of large pores in
the cell wall which do not close appreciably on dehydration of cell-wall proteins,
thus facilitating the leakage of crystal violet-iodine (CV-I) complex and resulting
in the decolorization of the bacterium which later takes the counter stain and
appears red. In contrast, the gram-positive cell walls are thick and chemically
simple, composed mainly of protein and cross-linked mucopeptides. When treated
with alcohol, it causes dehydration and closure of cell wall pores, thereby not
allowing the loss of (CV-I) complex and cells remain purple.
Material Requirements :
24-hour (or less old) cultures of Staphylococcus aureus and Escherichia coli
Gram staining reagents:
Crystal violet
Gram's iodine solution
95 percent ethyl alcohol
Safranin
Staining tray/clothes pin
Wash bottle of distilled water
Droppers
Inoculating loop
Glass slides
Blotting paper/Absorbent paper
Lens paper
Bunsen burner/spirit lamp
Microscope.
Procedure :
1) Make thin smears of Staphylococcus and Escherichia on separate glass
slides.
2) Let the smears air dry.
3) Heat fix the smears.
4) Hold the smears using slide rack or clothes pin.
5) Cover each smear with crystal violet for 30 seconds.
6) Wash each slide with distilled water for a few seconds, using wash bottle.
7) Cover each smear with Gram's iodine solution for 60 seconds.
8) Wash off the iodine solution with 95 per cent ethyl alcohol. Add ethyl
alcohol drop by drop, until no more colour flows from the smear (The
gram-positive bacteria are not affected while all gram-negative bacteria are
completely decolorized).
9) Wash the slides with distilled water and drain.
10) Apply safranin to smears for 30 seconds (counter-staining).
11) Wash with distilled water and blot dry with absorbent paper.
12) Let the stained slides air dry.
Observations :
1) Examine the slides microscopically using oil-immersion objective.
2) 2 Identify the gram reaction of both the cultures and classify them.
3) Make sketches for morphology of the cultures.
4) Describe the morphology and arrangement of the cells.
Results :
Those bacteria that appear purple are referred to as Gram-positive, those
appearing pink are described Gram-negative.
In S.aureus the cocci appear dark purple or blue in colour, thus it is a gram-
positive bacterium whereas in E.coli the rods appear pink and is thus a gram-
negative bacterium. To get reliable results, one should use cultures that are 18 to
24 hours old. Since old cultures of gram-positive bacteria tend to decolorize
more rapidly; thus, they might appear to be gram-negative.
