1. Bacillus

2. Bacillus sp. GP rod Spore former Examples Exoenzymes B. anthracis
B. cereus
B. subtilis
B. megatarium
Exoenzymes
Lipase
Amylase
Casease
Gelatinase

3. Spores

4. Agar Characteristics

5. Starch Hydrolysis Enzymes Substrate Reaction Amylase Maltase Starch
Clear zone

6. Protein Hydrolysis Enzymes Substance Reaction Proteases Caseases
Casein
Reaction
Clear zone

7. Lipid Hydrolysis Enzymes Substrate Reaction Lipase Tributyrin
Clear zone

8. Bacillus Tri-Plate

9. Collagen Hydrolysis Enzyme Use loop (vs. needle) Substrate Reaction
Gelatinase
Use loop (vs. needle)
Inoculate several times
Stab deep
Substrate
Gelatin
Reaction
Clear Zone on Agar
Liquefy Gelatin in tube

10. MSA Selective Differential Substrate pH = phenol red Reaction Mannitol
Salt (7.5%)
pH = phenol red
Reaction
Growth
Fermentation

11. Nitrate Reduction Inoculate with loop Incubate Additional solutions
Sulfanilic acid (A)
Naphthylamine (B)
(+) = Red
Nitrate to Nitrite
No color, add Zinc
(+) = not red
(-) = red

12. Citrate Enzyme Citrase Substrate Citrate pH Bromthymol blue Reaction
Keto acids
Oxaloacetic
Pyruvate
Acetic acid
CO2
CO2 + NaH2CO3
Prussian blue = +

13. Bile Esculin Agar (BEA)
Selective
Bile Salts
Differential
Esculin (CH2O)
Fe-NH3-citrate
Reaction
Growth in bile
Hydrolyze esculin
Esculetin-Iron Rxn
Black precipitate

14. Results Exoenzymes Reaction Starch (amylase, maltase) Lipid (lipase)
Proteins (Casease, protease)
Collagen (Gelatinase)
Reaction
(+) = clearing
(-) = no clearing

15. Starch Hydrolysis

16. Lipid Hydrolysis

17. Protein Hydrolysis

18. Gelatin (Collagen) Hydrolysis

19. BEA and Citrate Results+ +
Bile Esculin
Citrate

21. Hydrolysis Results Summary
casein
lipid
Gelatin
Starch