AEROBIC OR FACULTATIVE SPORE-
specimens :
FORMING RODS (BACILLUS SPP.)
Introduction
All are aerobic or facultative anaerobic
Gram (+) or gram-variable spore-forming
bacilli
Widely distributed in nature
2 most important species :
1. Bacillus anthracis (anthrax)
2. Bacillus cereus (food poisoning)
Morphology and General Characteristics
Vegetative cells are straight-sided with
rounded or boxy ends
May occurs singly or in chains
Single spore may be placed centrally or
terminally and can be oval, cylindrical,
rounded
All are motile with few exceptions
Exhibit large flat colonies on non-selective
laboratory media
Often Oxidase (+)
Frequently Beta-hemolytic
Very small colonies, spore formation often
do not occur , resembles nonfermentive
gram (-) bacilli
Strains are strictly aerobes may appear as
nonfermentative gram (-) on KIA and TSI
Agar
Sporulation of atypical strain may stimulate
by subculture into Esculin agar and
facilitated on acidified media such as TSI
Agar
2 test are useful :
1. Susceptibility to vancomycin
2. KOH test
50% sterilized ethanol for 1 hr effective
for selecting Bacillus spp.
Differentiation of Bacillus spp.
a) Cell morphology
b) Spore shape and location
c) Whether obligate aerobic or facultative
anaerobic
d) Action on xylose and arabinose
e) Starch hydrolysis
f) Indole production
g) Nitrate production
h) Presence of protein bodies
i) Susceptibility to gamma phage
j) Capsule production
k) Animal pathogenicity
3 main groups to be encountered in clinical
1. Group 1 cells are 0.9 um wide
2. Group 2 are smaller than G1
3. Group 3 spores cause swelling of the
cell wall
BACILLUS ANTHRACIS
Primary human pathogen in the genus
3 forms of causative agent of anthrax :
1. Cutaneous anthrax infection on the
abrasion of the skin , recognized in the
serosanguineous discharge
2. Pulmonary anthrax woolsorters
disease , found in large number of
sputums, leads to fatal septicemia of
not treated properly
3. Gastrointestinal anthrax
bacilli/spores are swallowed, fatal if not
treated properly
A. Structure and Extracellular Products
Facultative, large, square-ended, nonmotile
rods, ellipsoidal to cylindrical centrally
located spore
Cells occur in long chains giving bamboo
appearance esp. on primary isolation from
infected tissue or discharge
Chains of the virulent forms are usually
surrounded by a capsule, avirulent is
uncapsulated
Encapsulation occurs in enriched media and
when grown on 0.7% sodium bicarbonate
agar under 5% CO2
Sporulation occurs in the soil and on culture
media but not on living tissue unless
exposed to air
Colonies are large, opaque, white to gray,
raised and irregular, curled margin
Nonhemolytic on sheep blood agar
Produce heavy pellicle in broth with little or
no subsurface growth
2 potent exotoxin are produced :
1. Lethal factor (LF)
2. Edema factor (EF)
Both toxins depends on 3rd protein called
PROTECTIVE ANTIGEN which is essential to
enter target cells
B. Identification Methods C. Noncultural Methods of Identification
Extreme caution with BIOSAFETY LEVEL 3 Fluorescent antibody stain for recovery of
35oC is the optimal growth temp encapsulated strains
42-43oC capsule is destroyed and Indirect hemagglutination
organism become avirulent ELISA using the PA component of the toxin
B. anthracis as the capture antigen
presence of large encapsulated gram (+)
rods in the blood BACILLUS CEREUS
grows on Phenylethyl alcohol (PEA) Caused by serious infection:
weak Usually in immunocompromised
catalase (+) host
nonmotile Surgical trauma patients
produces lecithinase Burns
grows on high salt (7% NaCl) IV drug abusers
low pH (6.0) Catheters
Increase CO2 cultures can be cultivated Prosthetic devices
Colonies are nonhemolytic, large, gray to Dialysis patients
white with an irregular margin because of Infections :
outgrowth of long filamentous projection Septicemia
Medusa head described the colony Necrotizing pneumonia
morphology of anthrax bacilli Meningitis
Colonies has a tenacious consistency, with Peritonitis
the appearance of Beaten Egg Whites Wound infection
Characterized by : Osteomyelitis
a. Carbohydrate fermentation : Myonecrosis
Glucose Rabbit skin necrosis is attributed to :
Fermented Enterotoxin
Fructose
Maltose with acid only Hemolysin
Sucrose Phospholipase C (lecithinase)
Arabinose produced by all strains of B. cereus
Xylose
Not Produces Beta-lactamase
Galactose fermented Long been known as an important
Lactose cause of food poisoning
Mannose Short incubation time (emetic form)
b. Gelatin : associated with oriental rice dishes due
Inverted pine tree growth to preformed toxins
Slow liquefaction Long incubation time (diarrheal form)
c. Nitrates : associated with meat and vegetable
Reduce nitrites disease with the toxin formed in vivo
d. Starch : A. Laboratory Identification
Hydrolyzed Colonies are small, shiny, compact to
e. Voges-Proskauer : the large feathery, spreading type ,
Positive lavender colony with beta-hemolysis in
Identification may be use of gamma sheep blood agar
bacteriophage Lysis to gamma phage
Can be determine by injecting each of the Penicillin
resistant
10 mice (2-3 weeks old) with 0.2 mL of Bicarbonate Agar (-)
saline suspension Do not exhibit fluorescence in both cell
Usually dies after 2-5 days after inoculation wall
and organisms are found in the heart, lungs, Lecithinase Provisional
spleen, liver, blood Hemolysin production identification

Basic diagnostic protocols : Beta-hemolytic frosted glass-appearing

1. Gram stain colony containing spore-forming
2. Colony morphology Gram (+) bacilli
3. Catalase test Motile
4. Motility test Able to ferment Salicin
5. Capsule detection
Lecithinase (+)
 OTHER BACILLUS SPP.
There are about 60 spp of the genus Bacillus
B. subtilis
Large, hemolytic on blood agar, and
may be pigmented
Implicated in various infection like
septicemia, pneumonia,
panophthalmitis, wound infection,
peritonitis
B. licheniformis
Implicated in various infection like
septicemia, pneumonia,
panophthalmitis, wound infection,
peritonitis
Other occasionally incriminated:
a. B. brevis
b. B. circulans
c. B. coagulans
d. B. macerans
e. B. megaterium
f. B. pumilus
g. B. sphaericus