Bacillus

Bacillus sp.

• GP rod• Spore former• Examples

– B. anthracis– B. cereus– B. subtilis– B. megatarium

• Exoenzymes– Lipase– Amylase– Casease– Gelatinase

Spores

Agar Characteristics

Starch Hydrolysis

• Enzymes– Amylase– Maltase

• Substrate– Starch

• Reaction– Clear zone

Protein Hydrolysis

• Enzymes– Proteases– Caseases

• Substance– Casein

• Reaction– Clear zone

Lipid Hydrolysis

• Enzymes– Lipase

• Substrate– Tributyrin

• Reaction– Clear zone

Bacillus Tri-Plate

Collagen Hydrolysis

• Enzyme– Gelatinase

• Use loop (vs. needle)– Inoculate several times– Stab deep

• Substrate– Gelatin

• Reaction– Clear Zone on Agar– Liquefy Gelatin in tube

MSA• Selective

• Differential

• Substrate– Mannitol– Salt (7.5%)

• pH = phenol red

• Reaction– Growth– Fermentation

Nitrate Reduction

• Inoculate with loop• Incubate • Additional solutions

– Sulfanilic acid (A)– Naphthylamine (B)

• (+) = Red– Nitrate to Nitrite

• No color, add Zinc– (+) = not red– (-) = red

Citrate• Enzyme

– Citrase• Substrate

– Citrate• pH

– Bromthymol blue• Reaction

– Keto acids• Oxaloacetic

– Pyruvate

• Acetic acid– CO2

– CO2 + NaH2CO3– Prussian blue = +

Bile Esculin Agar (BEA)

• Selective– Bile Salts

• Differential– Esculin (CH2O)– Fe-NH3-citrate

• Reaction– Growth in bile– Hydrolyze esculin

• Esculetin-Iron Rxn• Black precipitate

Results

• Exoenzymes– Starch (amylase, maltase)– Lipid (lipase)– Proteins (Casease, protease)– Collagen (Gelatinase)

• Reaction– (+) = clearing– (-) = no clearing

Starch Hydrolysis

Lipid Hydrolysis

Protein Hydrolysis

Gelatin (Collagen) Hydrolysis

BEA and Citrate Results

Bile Esculin Citrate

+ +

Hydrolysis Results Summarycasein lipid

Gelatin Starch