L16 - Medical Microbiology Laboratory (Bacillus spp.)

1. Medical Microbiology Laboratory
Gram Positive Bacilli (Rods)
(Bacillus spp.)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. Non-spore formingSpore forming
• Corynebacterium spp.
• Lactobacillus spp.
• Mycobacterium spp.
• Clostridium spp.
• Bacillus spp.
2
GRAM POSITIVE RODS
 Bacterial spores are highly resistant, dormant
structures (i.e. no metabolic activity) formed in response
to adverse environmental conditions.
 They help in the survival of the organisms during
adverse environmental conditions; they do not have a
role in reproduction.

3. 3
GENERAL CHARACTERISTICS
 A large, heterogeneous group.
 Gram-positive rods (at least early in growth), in singles or
chains.
 Aerobic or facultatively anaerobic
 Produces endospores aerobically; spore shape and position
are variable
 Most are catalase-positive
 Most are motile
 Most are soil saprophytes
 Key pathogens are B. anthracis (anthrax) and B. cereus
(food poisoning and opportunistic infections)

4. 4
TAXONOMY
Scientific nameRank
• BacillaceaeFamily
• BacillusGenus
• B. anthracis
• B. cereus
Species
(medically important spp.)

5. 5
Bacillus anthracis
Samples:
 B. anthracis: dermal lesions, sputum, and/or blood
cultures

6. 6
Bacillus anthracis
Microscopic features
 Gram +ve, spore forming rods.
 If cutaneous anthrax is suspected the polychrome Loeffler
methylene blue (capsule stain) smear should be performed.
 Spore stained by special stains (Sudan black B, Schaeffer-Fulton)

7. 7
Bacillus anthracis
Polychrome Loeffler methylene blue procedure:
 Make an evenly spread smear of the specimen on a
slide and allow to air-dry in a safe place.
 Fix the smear by covering it with potassium
permanganate 40 g/L solution for 10 minutes.
 Wash off with water, and stain.
 Examine the smear for chains of large blue-stained rods
surrounded by mauve stained capsules characteristic of
B. anthracis.

8. 8
B. anthracis (culture)
Irregular, round, raised,
dull, opaque, greyish
white colonies with a
frosted glass appearance
Encapsulation test for Bacillus
anthracis. The difference in
appearance of colonies on
bicarbonate agar (smooth colonies
on the left) and rough colonies
(sheep blood agar on the right) is
indicative of capsule.

9. 9
Bacillus cereus
 Specimens: stool, vomitus, food, blood.
 Microscopy: not of much help
 Culture: Blood agar & special MYPA
medium: Mannitol-egg yolk-phenol red-
polymyxin agar, to isolate B. cereus from
feces & other sources.
 Test for toxins: to differentiate from
staphylococcal food poisoning.

10. 10
BIOCHEMICAL TESTS
1. Catalase positive (+ve), (all Bacillus spp. for rapid
differentiation from Clostridium spp.)
2. Gelatin liquefaction, (positive for B. cereus, negative
for B. anthracis).
3. Motility test, (B. cereus is motile, B. anthracis is non-
motile)

11. 11
BIOCHEMICAL TESTS
In this photo, two nitrate positive
tests are on either side of the
negative control.
A gelatinase-positive (+ve) organism
is above, and a gelatinase-negative (-
ve) organism is below.

12. 12
BIOCHEMICAL TESTS

13. 13
B. anthracis
Detection by advanced techniques
 Enzyme Immunoassay (EIA): using purified anthrax toxin antigen
(Ag).
 Ascoli's thermopreciptation test: to demonstrate anthrax Ag in
tissue extracts.
 Polymerase Chain Reaction (PCR): used to detect anthrax
contamination of animal & agricultural products.