Classification of Gram-Classification of Gram-PositivePositive

Gram-positive bacilli

Aerobic Anaerobic

Non spore formingSpore forming Spore forming

Corynebacterium

Listeria

Bacillus Clostridium

Aerobic Spore Forming Aerobic Spore Forming Bacillus sppBacillus spp

Bacillus

PathogenicNon-pathogenic(Anthracoids)

B. anthracis B. cereus e.g. B. subtilis

General Characters of General Characters of Bacillus Bacillus sppspp

Very large Gram positive bacilliVery large Gram positive bacilli 1-1.2 µm in width x 3-5µm in 1-1.2 µm in width x 3-5µm in

lengthlength Arranged in long chainsArranged in long chains Motile Motile exceptexcept B. anthracisB. anthracis Spore forming (outside the host)Spore forming (outside the host) Capsulated (inside the host)Capsulated (inside the host) Non FastidiousNon Fastidious Facultative anaerobicFacultative anaerobic Breakdown glucose by oxidative Breakdown glucose by oxidative

and fermentative i.e. O+/F+and fermentative i.e. O+/F+ Catalase positiveCatalase positive It is found in soil habitatsIt is found in soil habitats

Disease Caused by Disease Caused by B. B. anthracisanthracisAnthraxAnthrax

AnthraxAnthrax is an acute infectious is an acute infectious disease in man & animal caused disease in man & animal caused by the spore-forming by the spore-forming B. anthracisB. anthracis..

Anthrax is Anthrax is zoonotic diseasezoonotic disease Anthrax isAnthrax is occupational disease occupational disease Direct person-to-person spread of Direct person-to-person spread of

anthrax is extremely unlikely to anthrax is extremely unlikely to occur.occur.

Types of AnthraxTypes of Anthrax

Cutanoues Anthrax (Malignant Pustule)Cutanoues Anthrax (Malignant Pustule)– Most common form of the disease to humansMost common form of the disease to humans– It is acquired when the spores from the soil or It is acquired when the spores from the soil or

contaminated animal or carcass infect injured skin or contaminated animal or carcass infect injured skin or mucous membrane usually in face, neck and armmucous membrane usually in face, neck and arm

Pneumonic Anthrax (Woolsorters disease)Pneumonic Anthrax (Woolsorters disease)– It is results most commonly from inhalation of spore-It is results most commonly from inhalation of spore-

containing dust where animal hair or hides are being containing dust where animal hair or hides are being handledhandled

Intestinal AnthraxIntestinal Anthrax– It is analogous to cutaneous anthrax but occurs on the It is analogous to cutaneous anthrax but occurs on the

intestinal mucosaintestinal mucosa– Intestinal anthrax is rare & occurs accidentally among Intestinal anthrax is rare & occurs accidentally among

butchers and in primitive societies eating meat of infected butchers and in primitive societies eating meat of infected animalsanimals

Virulence FactorsVirulence Factors

Poly-D-glutamyl Poly-D-glutamyl CapsuleCapsule– Mediates the invasive stage of the infectionMediates the invasive stage of the infection

Anthrax toxinAnthrax toxin– Mediates the toxigenic stageMediates the toxigenic stage

The toxin consists of three distinct antigenic The toxin consists of three distinct antigenic components, which is thermolabile protein.components, which is thermolabile protein.

Edema Factor (EF): necessary for edema Edema Factor (EF): necessary for edema productionproduction

Protective Antigen (PA): induces protective Protective Antigen (PA): induces protective antitoxic antibodies in guinea pigsantitoxic antibodies in guinea pigs

Lethal Factor (LF): has a lethal effect of Lethal Factor (LF): has a lethal effect of anthrax toxinanthrax toxin

B. cereusB. cereus

B. cereusB. cereus is a normal inhabitant of soil is a normal inhabitant of soil Also isolated from food such as grains and Also isolated from food such as grains and

spicesspices B. cereusB. cereus causes Two Types of food poisoning causes Two Types of food poisoning

– Emetic form or short incubation:Emetic form or short incubation: It is caused by heat stable enterotoxinIt is caused by heat stable enterotoxin NNauseaausea, , vomitingvomiting and and abdominal crampsabdominal cramps Incubation period of 1-6 hrsIncubation period of 1-6 hrs It resembles It resembles S. aureusS. aureus food poisoning food poisoning

– Diarrheal form or long incubation:Diarrheal form or long incubation: It is caused by heat labile enterotoxinIt is caused by heat labile enterotoxin AAbdominal crampsbdominal cramps and and diarrheadiarrhea Incubation period of 8-16 hrsIncubation period of 8-16 hrs Diarrhea may be a small volume or profuse and wateryDiarrhea may be a small volume or profuse and watery It resembles food poisoning caused by It resembles food poisoning caused by Cl. perfringensCl. perfringens In either type, the illness usually lasts In either type, the illness usually lasts < < 24 hrs after 24 hrs after

onsetonset

Differential Differential characteristics of characteristics of B. B.

anthracisanthracis & & B. cereusB. cereusB. anthracisB. anthracisB. cereusB. cereus

HemolysisHemolysisNo No hemolysishemolysis

-hemolysis-hemolysis

MotilityMotilityNon-MotileNon-MotileMotileMotile

Identification of Identification of Bacillus Bacillus Spp.Spp.

SpecimenSpecimen– Pastular exudates in malignant pustule Pastular exudates in malignant pustule – Sputum in pneumonic anthrax Sputum in pneumonic anthrax – Stool in intestinal anthrax (also in food Stool in intestinal anthrax (also in food

poisoning by poisoning by B. cereusB. cereus)) Stool specimen is emulsified and heated to 80 Stool specimen is emulsified and heated to 80

C to kill non spore forming microorganismC to kill non spore forming microorganism

MorphologyMorphology– Macroscopical (Cultural characteristics)Macroscopical (Cultural characteristics)– Microscopical (Gram Stain, Spore Stain)Microscopical (Gram Stain, Spore Stain)

Identification of Identification of Bacillus Bacillus Spp.Spp.

• Cultural Characteristics• Grow on nutrient Agar

• On ordinary medium• Grow aerobically at 37C with characteristic

mucoid or smooth colonies, which indicates the pathogensity of organism (presence of capsule)

• Rough colonies are relatively avirulent• Stab culture on gelatin medium results in

inverted fire tree appearance.• GrGrowth on Blood Agarowth on Blood Agar

BacillusBacillus species grow well on blood agar species grow well on blood agar showing a double zone of hemolysisshowing a double zone of hemolysis

B. anthracisB. anthracis, , which grows well on blood agar without any hemolytic effect.

Cultural Cultural CharacteristicsCharacteristics

B. cereus B. anthracis

Nutrient Agar

Blood Agar

Identification of Identification of Bacillus Bacillus Spp.Spp.

MorphologyMorphology– MicroscopicalMicroscopical

Stain Stain – Gram StainGram Stain

Gram positive bacilliGram positive bacilli Found in chainsFound in chains Non motile Non motile Capsulated inside the hostCapsulated inside the host Sporulated outside the hostSporulated outside the host Spore is central, oval and non-bulgingSpore is central, oval and non-bulging

Spore Stain ProcedureSpore Stain Procedure

1.1. Make a heat fixed smear of Make a heat fixed smear of BacillusBacillus2.2. Place the slide on the slide rackPlace the slide on the slide rack3.3. Cover the smear with malachite green stainCover the smear with malachite green stain4.4. Apply heat for 3-5 min without boiling and Apply heat for 3-5 min without boiling and

drying of the slidedrying of the slide5.5. Wash the slide gently in running water about 20 Wash the slide gently in running water about 20

SS6.6. Counterstain with safranin for one minuteCounterstain with safranin for one minute7.7. Gently rinse with waterGently rinse with water8.8. Gently blot the slide dry, no rubbing, and let it Gently blot the slide dry, no rubbing, and let it

air dry and examine with oil immersion optics.air dry and examine with oil immersion optics.9.9. Observe red vegetative cells and sporangia, and Observe red vegetative cells and sporangia, and

green endospores and free sporesgreen endospores and free spores

Identification of Identification of Bacillus Bacillus Spp.Spp.

SporeSpore StainStain Bacillus spores are oval & centralBacillus spores are oval & central By spore staining technique (Malachite By spore staining technique (Malachite

green & safranin) , the green & safranin) , the sporespore appears appears greengreen while the while the vegetative cellsvegetative cells appear appear redred. .

Biochemical Tests: Biochemical Tests: 1- Catalase Test1- Catalase Test

All All BacillusBacillus species are catalase positive species are catalase positive (Remember staphylococci are catalase (Remember staphylococci are catalase positive)positive)

Starch HydrolysisStarch Hydrolysis(Amylase Activity)(Amylase Activity)

PrinciplePrinciple– Starch + IodineStarch + Iodine blue colorblue color– Glucose + Iodine Glucose + Iodine No reactionNo reaction

Nutrient Agar containing 1% Starch + M.ONutrient Agar containing 1% Starch + M.O GlucoseGlucose

ProcedureProcedure– Inoculate nutrient agar plate containing 1% Starch with the Inoculate nutrient agar plate containing 1% Starch with the

M.O.M.O.– Incubate the plate at 37 for overnightIncubate the plate at 37 for overnight– After incubation, flood the plate with Iodine solutionAfter incubation, flood the plate with Iodine solution

ResultResult– Activity of amylase is indicated by a clear zone around the

growth while the rest of the plate gives blue color after addition of iodine solution

AmylaseIodine

Appearance of colorless zone around the growth

Practical WorkPractical Work

Gram StainGram Stain Spore StainSpore Stain Catalase TestCatalase Test Starch hydrolysisStarch hydrolysis