UNIVERSITI SAINS MALAYSIA

JIB 221 MICROBIOLOGY

PRACTICAL 2. SIMPLE STAIN AND GRAM STAIN TECHNIQUES

Name: .............................. Matric number: .............................. Group:

OBJECTIVES:
• Prepare a series of smears of different microorganisms and observe using simple
staining techniques
• Prepare a series of smears of different microorganisms and observe using differential
staining techniques (Gram Stain)
• Observe the morphology of various microorganisms both macroscopic (based on
colony shape and texture) and microscopic (shape, size, arrangement and coloration
features)

INTRODUCTION:

One of the main characteristics of bacteria is their morphology, size, shape, arrangement and
structure. This feature can be observed by staining the cells in a certain way
and observing under the microscope.
Simple staining: Microorganisms under natural conditions are quite difficult to see even
when observed using oil immersion. Therefore, a simple dye is placed on the microorganisms
to see their morphology more clearly.
Diffrential staining: Differential staining are used to differentiate or highlight a variety cell
morphology. Gram stain is an example of a differential staining technique.
This staining technique is derived from the name of Christian Gram who discovered it in the
year l884. It is the most commonly used stain in bacteriology and it distinguishes bacteria
into two groups, Gram positive and Gram negative. Gram-positive organisms maintain the
crystal violet dye even when they are decoloured by alcohol. Gram negative organisms are
decoloured by alcohol and reacted with other dyes such as safranin. However, like other
staining techniques, Gram staining results can change. To get reliable results, the cultures

11
used must be between 12-18 hours. Gram staining is a fundamental technique in
microbiology and should be mastered by all students enrolled in this course.

2a. SIMPLE STAIN

MATERIALS:

Microscope
4 clean slides
1 inoculation wire
sterile distilled water
blue methylene solution
plate cultures:
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Candida albicans

PROCEDURE:
1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a smear of
bacteria. Air dry and heat-fixed. (Prepare a heat-fixed smear for each of the above
cultures).
2. Place the slide on the staining rack and flood the smear with the
1% blue methylene solution and let it react for 1 minute.
3. Wash the dye with tap water and dry the slide with absorbent paper.
4. Keep it dry and observe under the 40X objective.
Record your observations.

12
2b GRAM STAIN

MATERIALS:

crystal violet solution

iodine solution
ethanol
safranin solution
sterile distilled water
wire loop
4 clean glass slides
plate culture (24 h):
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Pseudomonas aeruginosa

PROCEDURE:

1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a smear of
bacteria. Air dry and heat-fixed. (Prepare a heat-fixed smear for each of the above cultures)
2. Place the slide on the colouring rack and flood the smear with the crystal violet solution
and let it sit for 1 minute. Wash the dye slowly with tap water.
3. Stain the smear with iodine. Let it sit for 1 minute and then rinse with tap water.
4. Decolourise the smear using 95% alcohol by tapping the slide on the edge of the sink and
dripping alcohol onto the slide until the colour disappears (approximately 30 seconds). Rinse
with tap water immediately.
5. Restain the smear with safranin for 1 minute. Wash the slides, then dry with absorbent
paper. Observe the slides using 40X objective lens with maximum illumination to distinguish
the colours. Record your observations.

13