INTRODUCTION

Gram-positive bacilli are often arranged in pairs or chains with rounded or square ends and usually have
a single endospore. The endospores are generally oval or sometimes round or cylindrical and are very
resistant to adverse conditions. Sporulation is not repressed by exposure to air. Bacillus species was
broadly divided in to three groups based on the morphology of the spore and sporangium.

Group 1 – Gram positive, produce central or terminal, ellipsoidal or cylindrical spores that do not
distend the sporangium. It comprises of two subgroups:

- Large cell subgroup include Bacillus anthracis, Bacillus cereus,

- Small cell subgroup include Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis

B. anthracis is an endospore-forming, rod-shaped bacterium, with a width of 1-1.5µm and a length of 3-

10µm in size. It can be grown in an ordinary nutrient medium under aerobic or anaerobic conditions.

Bacillus cereus is 1 x 3-4µm in size. They present as straight or slightly curved slender bacilli with square
ends singly or in short chains. They are facultative anaerobes, and like other members of the genus
Bacillus can produce protective endospores. Capsules are not formed, but spore and sporangial
morphology are similar to those of B. anthracis. They are motile by means of peritrichous flagella and
exhibit two types of motility including swimming and swarming, depending on the environment and are
resistant to lysis by gamma-phage.

The B. subtilis group are closely related and are not easily distinguishable. Cells of these organisms are
less than 1μm wide, sporangia are not swollen, and spores are ellipsoidal. They are in general mesophilic
with regard to temperature and neutrophilic with respect to pH for growth, while often being tolerant to
higher pH levels.

The Ziehl-Neelson stain: Used in the demonstration of acid-fast bacteria and stain endospores in gram-
positive bacilli. PRINCIPLE: The lipoid capsule of the acid-fast organism takes up carbolfuchsin and resists
decolorization with a dilute acid rinse. The lipoid capsule of the bacteria is of such high molecular weight
that it is waxy at room temperature and successful penetration by the aqueous based staining solutions
is prevented.

EXPLANATION

Gram staining and simple staining techniques may or may not reveal the presence of endospores in a
bacterial sample. ZN stain was used involving the slide is flooded with strong carbol fuchsin solution and
a heat source is used to steam the stain into the cells and the spores. This requires approximately 5
minutes three times and must be done carefully to achieve steaming and not charring of the sample.
This heating step stains the vegetative cells and the endospores. Once the heating period is complete,
the slide was washed gently with tap water, then 2% Nitric acid acted as a decolorizer for the vegetative
cells, but the stain is not released by the endospores and free spores. The 1% methylene blue
counterstain is used on the slide to give color to the vegetative cells. The endospores will have retained
the strong carbol fuchsin, appearing red, and the vegetative cells will be bluish.
B. cereus was β- haemolytic on Blood agar due to complete lysis of red blood cells - Colonial appearance
is similar to that of B. anthracis although B. cereus colonies both cream to white or grey and have a
slight green tinge.

Bacillus subtilis was β- haemolytic on Blood agar - Colonies are large (2 - 7mm) with a frosted-glass
appearance, but may become opaque. Color varies. Variable colonial morphology - some species may
produce mucoid or smooth or raised wrinkly colonies.

Hemolysis was a useful characteristic in differentiating Bacillus cereus and Bacillus subtilis from B.
anthracis which is non-haemolytic.

Nutrient agar provided necessary support for growth of the gram-positive bacilli in terms of water,
mineral salts and nitrogen source.

All the species failed to grow on MacConkey agar since the crystal violet and bile salts halt growth of
Gram-positive bacteria.

Bacillus subtilis and Bacillus cereus were catalase positive due to the appearance of bubbles showing
activity of catalase enzyme catalyzing break down of hydrogen peroxide to oxygen and water.

Bacillus subtilis and Bacillus cereus were lactose negative because they lack lactase enzyme.

Bacillus subtilis and Bacillus cereus all fermented glucose releasing acid but no gas since less carbon
dioxide is produced from these organisms and they are facultative anaerobes and change in pH causes
change in color of the indicator in the medium.

The bacteria all ferment sucrose with more activity for Bacillus subtilis more than Bacillus cereus due to
extracellular hydrolysis of the sugar mediated by periplasmic invertase or the sucrose is taken up by the
bacteria cells and hydrolyzed to glucose and fructose.

All fermented maltose producing acid end products that change color of media.

Bacillus cereus inability to produce acid from mannitol caused no change in color of indicator in media
due to no change in pH while Bacillus subtilis produced acid from mannitol fermentation, caused
change in color of indicator in media due to change in pH of media.

All negative for duicitol producing no acid end products, no pH change hence the color of media remains
the same.