University of the Philippines, Baguio city

College of Science, Department of Biology

Biology 120: Microbiology
Exercise XI

BACTERIAL ENDOSPORE AND CAPSULES

David, Krisette; Fabros, Jamee ; Relucio, Mike
Department of Biology, College of Science, University of the Philippines, Baguio City

(1)

Abstract

Keywords:
INTRODUCTION
Endospore is a differentiated cell formed within the cells of certain gram positive
bacteria that is extremely resistant to heat as well as to other harmful agents, (Madigan,
2009). It is an adaptive feature in microbes that help them survive in a nutrient deficient
environment. Unlike any other cells, endospore are hard to stain using simple gram stains
since they do not absorb them well. So, it is seen unstained and stand out under the
microscope at as strongly infractile structure, (Madigan, 2009). Differential staining are
essential in determining and visualizing different organism. Similar to other stain
techniques such as the acid-fast staining and Gram staining, a special method should be
used to stain endospore. This is Scaeffer- Fulton method, which uses malachite green as a
stain for such cell coat. It demonstrated the endospre forming within the vegetative cells
and make free spores easy to identify (Hussey et al., 2007).
On the other hand, capsule is a dense, well-defined polysaccharide or protein layer,
known as the glycocalyx, closely surrounding the cell wall, (Madigan, 2009). Glycocalyx
is a general term for any network of polysaccharide or protein coating on the outside of
the cell. In microbes, there are two types of protein coating produced. This includes the
capsule and slime layer which vary according to their organization or distribution.
Capsule is closely associated with cells whereas slime layer is diffused. Compared to
Endospore, this structure can not retain the color of the staining agent. For this reason,
two methods are used to test both gram negative and gram positive bacteria. These
methods are negative capsule staining and positive capsule stain.
This study aims to provide a better understanding on the appropriate methods used to
distinguished two cell coats capsule and endospore (Scaeffer-Fulton method and Negative

staining, respectively). Also, this experiment aims to practiced biology students in

differential staining. Through this, the students will not only be able to familiarize with
the techniques but also will let them visualize the two protein coats which are usually
unstained and unseen in most bacterial specimens that are gram-stained.
MATERIALS AND METHODS
A.Endospore Staining: Scaeffer-Fulton Method
Each member of the group obtained a Fix smear of B. Subtilis. The smear was
flood slide with malachite green and was gently steamed at low flame for about 5
minutes. It was counter-stained with 5% safranin for a minute. Then, it was observed
under IOI for the evidence or appearance of endospore, green in color, and vegetative
cells, red in color.
B. Bacterial Capsule Staining: Negative Staining
A small drop of nigrosin was placed on the edge of a clean slide. Then, a loopful
of E. Coli was mix with the nigrosin solution. Using a new clean slide, the mixture
was runacross the width of the slide. The smear was airdried and covered with
safranin for about 30 secs. Safranin was removed using water. The smear was
examined under IOI for the presence of capsule.
RESULTS AND DISCUSSION
CONCLUSION AND RECOMMENDATION
REFERENCES
Marise, H., & Zayaitz, A. (2007, January 1). Endospore Stain Protocol. Retrieved
November
11,
2014,
from
http://www.microbelibrary.org/component/resource/laboratory-test/3112
Madigan, M.; Martinko, J.; Dunlap, P; Clark, D.; 2009. Brock Biology of Microorganism.
12 ed. Pearson Education, Inc. South Asia Pte Ltd.