lab. bacillus bacillus · species. under stressful environmental conditions, the bacteria can...

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Lab. Bacillus

Bacillus is a genus of Gram-positive, rod-shaped (bacillus) bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes, or facultative anaerobes (having the ability to be aerobic or anaerobic). They will test positive for the enzyme catalase when there has been oxygen used or present. Ubiquitous in nature, Bacillus includes both free-living (nonparasitic) and parasitic pathogenic species. Under stressful environmental conditions, the bacteria can produce oval endospores that are not true 'spores', but to which the bacteria can reduce themselves and remain in a dormant state for very long periods.

Bacillus anthracis, the agent of anthrax, is an aerobic, spore-forming, nonmotile, large Gram-positive bacterium. Anthrax is a zoonotic disease that occurs most frequently in herbivorous animals (e.g., cattle, sheep, and goats), which acquire endospores from contaminated soil. Human disease is less common and results from consumption or handling of contaminated products or commercial products derived from them, such as wool and hides. Infection can occur in one of three forms:

1. Cutaneous, responsible for >95% of naturally occurring cases, is initiated when the bacterium or spores enter the skin through cuts or abrasions, such as when handling contaminated hides, wool, leather, or hair products (especially goat hair) from infected animals. Skin infection begins as a raised itchy bump or papule that resembles an insect bite.

2.Gastrointestinal anthrax may occur 1 to 7 days following consumption of contaminated undercooked meat from infected animals and is characterized by acute inflammation of the intestinal tract. Initial signs of nausea, loss of appetite, vomiting, and fever are followed by abdominal pain, vomiting of blood, and severe bloody diarrhea.

3. Inhalational anthrax results from the inhalation of B. anthracis spores and can occur following an intentional aerosol release.

Gram stain suspicious colonies from agar plates and blood cultures in a BSC. B. anthracis is a large (1 to 1.5 by 3 to 5um) Gram-positive rod. Vegetative cells seen on Gram-stained smears of clinical specimens often occur in short chains of two to four cells that are encapsulated. Gram stains from colonies grown on BAP appear as long chains of nonencapsulated Gram- positive bacilli. Endospores are not

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commonly seen in direct smears of clinical specimens. If present, the spores are oval and located centrally or subterminally and do not cause swelling of the vegetative cell (see Fig 1).

Fig. 1. Gram stain of blood culture containing B. anthracis

B: Characteristics: B. anthracis grows well on BAP and CHOC, but not on MAC or EMB, colony are round with irregular edges, flat or slightly convex with aground glass appearance. There are often “comma-shaped” projections from the edge of the colony, producing the “Medusa head” shape. The colonies are nonhemolytic on BAP and have a tenacious consistency that when teased with a loop, the growth will stand up like beaten egg whites. Catalase – positive, motility – negative.

Fig. 2. Colonies of B. anthracis on Blood Agar

Specimen collection: Un roof vesicle and aspirate fluid or collect with two sterile swabs.

Gastrointestinal: Stool (> 5 grams...pecan size), collect and transport in a leak proof sealed container Collect blood (late stage of infection) directly into an appropriate blood culture bottle (aerobic and anaerobic),Transport specimens and bottles at room temperature

Inhalational: Sputum, Blood: collect directly into an appropriate blood culture bottle (aerobic and anaerobic), Cerebral Spinal Fluid only if signs of meningitis occur, Transport specimens and bottles at room temperature.

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of the vegetative cell (see Fig 1).

Fig. 1. Gram stain of blood culture containing B. anthracis B. Characteristics: B. anthracis grows well on BAP and CHOC, but not on MAC (or EMB). Colonies are round with irregular edges, flat or slightly convex with a ground glass appearance. There are often “comma-shaped” projections from the edge of the colony, producing the “Medusa head” shape. The colonies are nonhemolytic on BAP and have a tenacious consistency that when teased with a loop, the growth will stand up like beaten egg whites. Catalase – positive, Motility – negative.



Gastrointestinal: Stool (> 5 grams...pecan size), collect and transport in a leak proof sealed container �Collect blood (late stage of infection) directly into an appropriate blood culture bottle (aerobic and anaerobic),Transport specimens and bottles at room temperature

Inhalational: Sputum, Blood: collect directly into an appropriate blood culture bottle (aerobic �and anaerobic), Cerebral Spinal Fluid only if signs of meningitis occur, Transport specimens and bottles at room temperature.

Direct testing: Large gram-positive rod (1-1.5 x 3-5 µm) Long chains in liquid culture but may include single cells or short chains, Spores are sub terminal or central.

Bacteria Motility Capsule Spore Pathogenicity for mice

Optimal growth temp.

Reaction with eggs

B. anthracis - + Central high 35 Low

B. cereus + - Central low 30 +

B. mycoides + - Central - 30 +

B. subtilis - - Central - 37 -

The rule out or referral of B. anthracis is accomplished by following the algorithm below and the associated tests.

A. Gram stain suspicious colonies from agar plates and blood cultures in a BSC. B. anthracis is a large (1 to 1.5 by 3 to 5um) Gram-positive rod. Vegetative cells seen on Gram-stained smears of clinical specimens often occur in short chains of two to four cells that are encapsulated. Gram stains from colonies grown on BAP appear as long chains of nonencapsulated Gram-positive bacilli. Endospores are not commonly seen in direct smears of clinical specimens. If present, the spores are oval and located centrally or subterminally and do not cause swelling of the vegetative cell (see Fig 1).


Photo courtesy of Dr. James Rudrick, Michigan Department of Community Health

B. B. anthracis grows well on BAP and CHOC, but not on MAC (or EMB). Colonies are round with irregular edges, flat or slightly convex with a ground glass appearance. There are often “comma-shaped” projections from the edge of the colony, producing the “Medusa head” shape. The colonies are nonhemolytic on BAP and have a tenacious consistency that when teased with a loop, the growth will stand up like beaten egg whites.

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Reaction with eggs










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Slides for Bacterial anthracis

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Reaction with eggs










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Slides for Bacillus anthracis

++ + - Bacillus cereus�

Description and significance Bacillus cereus is a large, 1 x 3-4 µm, Gram-positive, rod-shaped, endospore forming, facultative aerobic bacterium. 16s rRNA comparison reveals Bacillus cereus to be most related to Bacillus anthracis, the cause of anthrax, and Bacillus thuringiensis, an insect pathogen used as pesticide . Although they have similar characteristics, they are distinguishable as B. cereus is most motile, B. thuringiensis produces crystal toxins, and B. anthracis is nonhemolytic. B. cereus is mesophilic, growing optimally at temperatures between 20°C and 40°C, and is capable of adapting to a wide range of environmental conditions. It is distributed widely in nature and is commonly found in the soil as a saprophytic organism. B. cereus is also a contributor to the

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Bacillus anthracis

Colony Morphology . Grows well on Blood Agar (BA); will not grow on MacConkey (MAC) agar . 2-5 mm on BA at 24 h . Flat or slightly convex with irregular borders that have comma-shaped protrusions . Colonies have a ground-glass appearance . Non-hemolytic on BA . Tenacious colonies

Gram Stain . Large gram-positive rod (1-1.5 x 3-5 μm) . Long chains in liquid culture but may include single cells or short chains . Spores are subterminal or central

Additional Information . Can be misidentified as: Bacillus megaterium and other Bacillus spp. . Biosafety Level 2 agent (Use BSL3 for large volume or high titer culture) . Infectious Dose: <10,000 spores . Transmission: Inhalation, ingestion, direct contact with skin . Contagious: No

Acceptable Specimen Types . Swab of vesicular fluid from cutaneous lesion . Sputum (≥ 1 ml) . Whole blood: 5-10ml blood in EDTA, and/or Inoculated blood culture bottle . Stool (> 5g)

24 h growth on BA Comma-shaped colonies

Gram stain

Tenacity on BA

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Bacillus anthracis






Gram stain

Tenacity on BA

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6/06

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Perform all additional work in a certified Class II Biosafety Cabinet

*Motility: Non-Motile(use semi-solid media rather than wet mount; 2,3,5-triphenyltetrazolium chloride indicator)*Catalase: Positive

*Motility and Catalase: Appearances of test results are not agent- specific. Photos represent typical reactions

Continue laboratoryidentification procedures

Immediately notify Wadsworth CenterBiodefense/Bacteriology Laboratories

if within the 5 boroughs of NYC, please call (212) 447-1091

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Bacillus anthracis






Gram stain

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Slides for Bacillus anthracis

++ + - Bacillus cereus�

Description and significance Bacillus cereus is a large, 1 x 3-4 µm, Gram-positive, rod-shaped, endospore forming, facultative aerobic bacterium. 16s rRNA comparison reveals Bacillus cereus to be most related to Bacillus anthracis, the cause of anthrax, and Bacillus thuringiensis, an insect pathogen used as pesticide . Although they have similar characteristics, they are distinguishable as B. cereus is most motile, B. thuringiensis produces crystal toxins, and B. anthracis is nonhemolytic. B. cereus is mesophilic, growing optimally at temperatures between 20°C and 40°C, and is capable of adapting to a wide range of environmental conditions. It is distributed widely in nature and is commonly found in the soil as a saprophytic organism. B. cereus is also a contributor to the

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Bacillus anthracis






Gram stain

Tenacity on BA

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6/06

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Bacillus anthracis






Gram stain

Tenacity on BA

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Bacillus cereus Description and significance

Bacillus cereus is a large, 1 x 3-4 µm, Gram-positive, rod-shaped, endospore forming, facultative aerobic bacterium. 16s rRNA comparison reveals Bacillus cereus to be most related to Bacillus anthracis, the cause of anthrax, and Bacillus thuringiensis, an insect pathogen used as pesticide . Although they have similar characteristics, they are distinguishable as B. cereus is most motile, B. thuringiensis produces crystal toxins, and B. anthracis is nonhemolytic.

B. cereus is mesophilic, growing optimally at temperatures between 20°C and 40°C, and is capable of adapting to a wide range of environmental conditions. It is distributed widely in nature and is commonly found in the soil as a saprophytic organism. B. cereus is also a contributor to the microflora of insects, deriving nutrients from its host, and is found in the rhizosphere of some plants. As a soil bacterium, B. cereus can spread easily to many types of foods such as plants, eggs, meat, and dairy products, and is known for causing 2-5 % of food-borne intoxications due to its secretion of emetic toxins and enterotoxins. Food poisoning occurs when food is left without refrigeration for several hours before it is served. Remaining spores of contaminated food from heat treatment grow well after cooling and are the source of food poisoning.

In addition, Bacillus cereus is an opportunistic human pathogen and is occasionally associated with infections, causing periodontal diseases and other more serious infections.�Bacillus cereus causes two types of food poisoning in humans including diarrhoeal syndrome and emetic syndrome. Food poisoning results from its production of enterotoxins in the gastrointestinal tract.

References��

[1] "Bacillus cereus." NCBI website. Accessed on August 18, 2007.��

[2] Vilain, S., Luo, Y., Hildreth, M., and Brozel, V. “Analysis of the Life Cycle of the Soil Saprophyte Bacillus cereus in Liquid Soil Extract and in Soil.” Applied Environmental Microbiology. 2006. Volume 72(7). p. 4970–4977.

�[3] DelVecchio, V., Connolly, J., Alefantis, T., Walz, A., Quan, M., Patra, G., Ashton, J., Whittington, J., Chafin, R., Liang, X., Grewal, P., Khan, A., and Mujer C. “Proteomic Profiling and Identification of Immunodominant Spore Antigens of

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Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.” Applied Environmental Microbiology. 2006. Volume 72(9). p. 6355–6363.��

[4] “Bacillus cereus.” United States Food and Drug Administration, Center for food safety and applied nutrition (FDA). Accessed August 18, 2007.

A. Prof. Dr. Zainab Al-Mahdi

lab. bacillus bacillus · species. under stressful environmental conditions, the bacteria can...

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