Activity No.

Gram Stain

Materials
1. Equipment: Bright field microscope, alcohol lamp, staining rack and timer, inoculating loops,
forceps, and dropper.
2. Reagents: Crystal violet ( primary stain), Gram’s iodine ( mordant), acetone alcohol ( decolorizer),
and safranin (counterstain)
3. Consumables: Cedarwood oil, sterile cotton swabs, tongue depressor, distilled water, marker, and
tissue paper.
4. Disposables: Microscope slides and cover slips
5. Organisms: Broth medium with unknown colonies ( labeled as A, B, C, and D) and swab samples (
e.g., throat swabs)

Procedure
1. Prepare a thin smear of the culture ( from broth medium) and swabs (e.g.,throat) to be observed.
Air dry and heat fix the samples.
2. Overlay the smear with crystal violet for 30 to 60 seconds. Rinse the smear ( with indirect stream of
distilled water).
3. Overlay the smear with Gram’s iodine for 30 to 60 seconds. Rinse the water.
4. Continuously drop acetone alcohol within 15 seconds until the purple to purple-blue color is
washed away from the smear. Rinse the smear immediately.
5. Overlay the smear with safranin for 30 to 60 seconds. Rinse the smear.
6. Blot the sides and the back of the slide with tissue paper and allow the slide to air-dry.
7. Examine the slide under oil immersion objective (OIO).

Picture 1.2. Throat swab smear Picture 1.3. Smear stained with Gram stain

TABLE 1-1. Gram-staining color reaction and interpretation

Color of Cell Interpretation

Purple or Purple-blue Gram-positive

Pink Gram-negative
Observation
A. Paste colored pictures of your microscopic observations and write a detailed description of the
following: (1) source of specimen; (2) Gram reaction, morphology, arrangement, and possible
genera or family of bacterial cell; and (3) background materials.

B. Give examples of bacterial genera that cannot be stained or do not stain well with Gram stain and
provide an explanation.
1. Mycoplasma: The absence of a cell wall, which is essential for Gram staining, occurs in
Mycoplasma. In the role of It looks colorless under the crystal violet-iodine complex as a
result of losing the magnifying glass.
2. Treponema: Treponema is a spirochete bacteria. The crystal violet-iodine combination finds
it difficult to adhere to and penetrate its thin, helical shape. Therefore, Treponema is not
successfully stained using the Gram stain technique.
3. Chlamydia: Chlamydia has a distinct cell wall structure that deviates from the usual. Qualities
that are either Gram-positive or Gram-negative. Consequently, Chlamydia cannot be
consistently stained with the conventional method of Gram staininG.