3.

4 Theory
Gram staining is worked based on the potential of bacteria cell wall to retain the crystal
violet dye during solvent treatment. Cell walls for Gram positive microorganism have higher
peptidoglycan and less of lipid possessed than the peptidoglycan of Gram negative
microorganism. In this staining method, the cell of bacteria will be stained by crystal violet. In
addition, iodine was subsequently added as the mordant to form crystal violet-iodine complex so
that the dye will not being remove easily. This step is important to fix the dye. For Gram
negative bacteria, the alcohol that acted as decolorize dissolved the lipid layer of the cell wall
and enhanced the leaching of primary stain which is the crystal violet from the cell wall to
surrounding solvent. Finally, a counterstain of safranin was used to give the pink color for
bacteria. For Gram positive bacteria, the alcohol was role as decolorize but it will dehydrate the
cell walls of the bacteria and closing the pores as the cell wall was shrink. Thus, the diffusion of
violet-iodine complex is blocked and caused the bacteria to remain stained. Same goes to this
bacteria, safranin was used for counterstaining and loosed the stain easily. Therefore they will
appear in blue-violet. The polychromatic nature of Gram stain enable to determine the size and
shape (morphology ) of both Gram positive and Gram negative bacteria.
(www.uphs.upnn.edu/bugdrug/antibiotic-manual/gram/.htm)

3.5 Procedures
A. Preparation of Cell Smear
i) Preparation of the glass microscope slide:
- Clean slides are essential for the preparation of microbial smears. Grease or oil from the fingers
on slides must be removed by washing the slides with soap and water or scouring powders,
followed by a water rinse and a rinse 95% alcohol. After cleaning, the slides were dried and
being placed on laboratory towels until ready for used.
ii) Preparation of smear:

-Thick and dense smears had to be avoided. A thick or dense smear occurs when too much of the
culture is used in its preparation which concentrates a large number of cells on the slides. This
type of preparation might be diminished the amount of light that can pass through and made it
difficult to visualize the morphology of single cell.
iii) Heat Fixation
-Unless fixed on the glass slide, the cell smear might be washed away during staining procedure.
This was avoided by heat fixation, during which the bacterial protein were coagulated and fixed
to the glass surface. Heat fixations were performed by rapid passage of the air dried smear two or
three times over the flame of the Bunsen burner.
B. Gram staining
i) The smears were prepared and being heat-fixed.
ii) The smears were prepared on a slide and being heat-fixed.
iii) The slides were stained as follows:
-The crystal violet was flooded for one minute.
-The excess dye was poured off and being washed gently in tap water and the slides were drained
against a paper towel.
-The smears were exposed to Grams iodine for one minute by washing with iodine, the iodine
was added more and being left on the smear until the minute was over.
-The iodine on the smears was washed with tap water and being drained carefully which was the
smear did not have to be bolted.
-The smears were washed with 95% alcohol for 30 seconds.
-The smears were washed with tap water at the end of the 30 seconds to stop decolorization and
the being drained.
-The smears were counterstained with 0.25% safranin for 30 seconds.

- The smears were bolted dried by using bibulous paper but were not to wipe the slides.
- The slides were being examined using microscope. The cells morphology, grouping, and
relative sizes were draw. A few of the cells for each bacterial species was colored to show the
Gram reaction.
iii) The appearance of the cells in the two smears was compared.

3.6 Apparatus and Chemicals

Apparatus

Chemicals

-Glass slides

-Nutrient agar

-Dark field microscope

-Soap

-Staining tray

-Distilled water

-Inoculum loop

-95% alcohol

-Bunsen burner

-Paper towels

-Petri dish

-Grams iodine
-0.25% safranin
-Crystal violet