Identification Of Gram Positive Bacilli

Prepared By: Thamer Hamdan

Classification of Gram-Positive Bacilli

Gram-positive bacilli

Non spore forming

Aerobic

Spore forming

AnaerobicAerobic

1 -Corynebacterium

2 -ListeriaBacillus Clostridium

Aerobic Spore Forming Bacillus spp

Pathogenic Bacillus:-B. anthracis-B. cereus

Non-pathogenic Bacillus:- B. subtilis

General Characters of Bacillus spp

Very large Gram positive bacilliArranged in long chainsMotile except B. anthracisSpore forming (outside the host)Capsulated (inside the host)Non FastidiousFacultative anaerobicCatalase positiveIt is found in soil habitats

1- B. anthracisB. anthracis found in soil; causes anthrax.Transmission through inhalation of spores, breaks in skin/mucous membranes, or ingestion.Direct person-to-person spread of anthrax is extremely unlikely to occur.

2- B. cereus• B. cereus found normally in nature.

Also isolated from food such as grains and spicesTransmission through contaminated medical equipment, or ingestion of contaminated food.B. cereus causes food poisoning

Many other spp. found in the environment; generally considered non-pathogens.

Identification of Bacillus Spp.

Specimens:– Fluid from cutaneous lesions – Sputum in pneumonic anthrax – Stool in intestinal anthrax (also in food poisoning

by B. cereus)

Identification of Bacillus Spp.• Cultural Characteristics:

• Growth on Nutrient AgarGrow aerobically at 37 °C with characteristic

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

Rough colonies are relatively avirulent.

• Growth on Blood AgarBacillus species grow well on blood agar showing

a double zone of hemolysis.B. anthracis, which grows well on blood agar

without any hemolytic effect.

Cultural Characteristics on Blood Agar:

Left: B. cereus Right: B. anthracis

Differential characteristics of B. anthracis & B. cereus

B. cereus B. anthracis

-hemolysis No hemolysis Hemolysis

Motile Non-Motile Motility

Differential Media for B. anthracis

PLET agar:(Polymyxin B - Lysozyme - EDTA – Thallous acetate Agar).

PLET Agar medium is the best selective medium for isolation and cultivation of B.anthracis from environmental specimens, animal products or clinical specimens, inhibiting B.cereus.The colonies are circular, creamy-white with a ground glass texture.

Differential Media for B. cereus 1- Mannitol egg-yolk phenol-red polymyxin agar

(MYPA):MYPA is a selective medium for the isolation of B. cereus from faeces, vomit or food. After overnight incubation at 35-37oC, large flat, dry pinkred colonies surrounded by an area of white precipitate are produced.

2- Polymyxin egg-yolk mannitol bromothymol blue agar (PEMBA):B. cereus produces large (5mm in diameter) and turquoise to peacock blue with a zone of egg yolk precipitation.

Identification of Bacillus Spp.

– Stains:* Gram Stain

• Gram positive bacilli• Found in chains• Spore is central, oval and non-bulging

* Spore Stain

Bacillus spores are oval & central

By spore staining technique (Malachite green & safranin) ,

the spore appears green while the vegetative cells appear red.

Spore Stain Procedure:-1. Make a heat fixed smear of Bacillus2. Place the slide on the slide rack3. Cover the smear with malachite green stain4. Apply heat for 3-5 min without boiling and drying of the slide5. Wash the slide gently in running water about 20 S6. Counterstain with safranin for one minute7. Gently rinse with water8. Gently blot the slide dry, no rubbing, and let it air dry and

examine with oil immersion optics.9. Observe red vegetative cells and sporangia, and green

endospores and free spores

Biochemical Tests: Catalase Test

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

Gelatinase test:B. anthracis produces gelatinase in very small quantities. After inoculation and incubation of the tube which containing gelatin, the tube placed in refrigerator for approximately of 30 minutes. If the tube remains liquid that indicated positive result.

Starch Hydrolysis(Amylase Activity)

Principle– Starch + Iodine blue color– Glucose + Iodine No reaction

Nutrient Agar containing 1% Starch + M.O Glucose

Procedure– Inoculate nutrient agar plate containing 1% Starch with the M.O.– Incubate the plate at 37 for overnight– After incubation, flood the plate with Iodine solution

Result– 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 solutionOrganism that gives (+) results is B. Subtilis & B. Cereus

Amylase IodineAppearance of colorless zone around the growth

Anaerobic Spore Forming Clostridium spp General Characteristics:

Gram positive, straight, thin rod with rounded ends.All species form endospore (drumstick with a large round end).Obligate anaerobe.Fermentative except C. tetaniMotile except C. perfringens.Catalase and Oxidase Negative.

Pathogenic species include:1. C. perfringens gas gangrene & food poisoning

2. C. tetani tetanus

3. C. botulinum botulism

4. C. difficile antibiotic associated diarrhoea

THE END