Group # 3

May 13, 2015

Group members:
Belen, Alexis
Bitera, Christine Danica
Buenaventura, Gelsie Rose
Cantre, Francis Godwin
Castillo, Jamila Anne
Exercise 9
The Staining of Bacterial Cells and Their Structures

Introduction
Staining is a technique used in microscopy to enhance contrast in a microscopic
image. Stains and dyes are frequently used to highlight structures in microbes for
viewing, often with the aid of different microscopes. Stains may be used to define and
examine different types of microbes, various stages of cellular life, and even organelles
within individual cells. Gram stain is a very important differential staining techniques
used in the initial characterization and classification of bacteria in Microbiology. Gram
staining helps to identify bacterial pathogens in specimens and cultures by their Gram
reaction (Gram positive and Gram Negative) and morphology (Cocci/Rod). Endospore
staining demonstrates spore structure in bacteria as well as free spores. Relatively few
species of bacteria produce endospores, so a positive result from endospore staining
methods is an important clue in bacterial identification.

Objectives
1. To learn the technique and acquire skills needed in conducting simple, gram
staining and endospore staining.
2. To differentiate bacterial cells on the basis of their Gram stain.
3. To identify and characterize the endospores of bacterial cells.

Materials
Culture plates/slant plates of the following bacteria:
Gram positive: Bacillus subtilis, Micrococcus luteus
Gram negative: Escherichia coli

Glass slides

Inoculating loop

Small amber bottles with dropper for

reagents

Alcohol lamp

Wooden clothes pin or slide holders

Wash bottles

Large plastic cover for table

Masking tape
detergent

Reagents:
Gram staining reagents:
1.
2.
3.
4.

Crystal violet (primary stain)

Gram iodine (mordant)
95% ethanol (decolorizer)
Safranin (counterstain)

Endospore staining reagents:

1. Malachite green (primary stain)
2. Safranin (counterstain)

Procedure
A. Preparation of Bacterial Smear
The clean glass slides were degreased by rubbing a small piece of cotton with
acetone on the slide surface. This allowed the water to adhere and spread well on the
surface of the slide. The slides were then labeled with masking tape. A loopful of
distilled water was placed on the center of the slide, then using a flame sterilized loop, a
colony of the test bacteria was transferred aseptically on the drop of distilled water. A
bacterial smear was prepared by gently mixing the bacteria with water, forming an oval
shaped smear and was air dried to form a translucent film on the slide. The smear was
then heat fixed by passing the slide over an open flame 3 to 5 times.
B. Hucker Method for Gram Staining
The prepared bacterial smears for either Escherichia coli or Micrococcus luteus was
flooded with crystal violet for 1 minute. The dye on the slide was drained off with a wash
bottle into a 500 ml beaker. The slide was then flooded with gram iodine solution for 1
minute then rinsed and drained again. Afterwards, the smear decolorized with 95%
ethanol for 10 to 15 seconds until no more violet stain ran from the slide. The slide was
rinsed immediately with water then was flooded with the counterstain Safranin for 1
minute. Lastly, the slide was rinsed and dried again and left to dry. The prepared slides
were then observed under OIO.
C. Endospore Staining (Schaeffer Fulton Method)
A prepared smear of Bacillus subtilis was saturated with malachite green and
steamed by holding the glass slide above a small frame for 10 minutes. The dye was
then drained off after 5 minutes and the slide was cooled. The slide was then rinsed

with tap water and counterstained with Safranin for 1 minute. The excess stain was then
drained off after 1 minute and rinsed with tap water and left to dry. The prepared slide
was then observed under OIO.

Data and Results

Escherichia coli (gram staining)

Unknown (gram staining)

Micrococcus luteus (gram staining)

Bacillus subtilis ( Endospore staining)

Analysis of data

Conclusion

References
Bahrami-Hessari, M., Dedeles, G.R., De Jesus, S.M., Papa, D.M.D.C., Dela Cruz, T.E.,
Quinto, E.A. (2014). Laboratory Manual in General Microbiology (8th ed.). Manila,
Philippines.