Overview:
Bushnell-Haas Agar (also known as Bushnell-Haas Medium) is a mineral salts agar formulated as a basal medium for the isolation and enumeration of hydrocarbon-degrading microorganisms. Developed by Bushnell and Haas in 1941, it provides essential inorganic nutrients (nitrogen, phosphorus, sulfur, magnesium, calcium, and iron) but lacks a carbon source, requiring supplementation with hydrocarbons like kerosene, mineral oil, paraffin wax, or gasoline to support growth. This selective setup is ideal for detecting and studying microbial biodegradation of petroleum products, particularly in environmental and industrial microbiology. The medium promotes the growth of bacteria such as Pseudomonas spp. that utilize hydrocarbons as sole carbon and energy sources, with a neutral pH to mimic natural conditions.
Applications:
?? Detection and enumeration of hydrocarbon-degrading bacteria in soil, water, and fuel samples
?? Bioremediation studies for petroleum-contaminated sites, including total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAH)
?? Quality control testing for microbial contamination in aviation fuels, oils, and other hydrocarbons
?? Research on microbial ecology of oil spills and degradation kinetics of volatile or liquid hydrocarbons
Key Features:
?? Minimalist formulation lacks carbon sources, ensuring selective growth only on added hydrocarbons
?? Supports robust growth of Pseudomonas aeruginosa and other degraders upon hydrocarbon supplementation
?? Versatile for overlay methods (liquid hydrocarbons) or inverted plate techniques (volatiles)
?? Validated in ASTM and EPA protocols for fuel microbiology and environmental monitoring
?? Stable pH and precipitate formation (normal white to yellow-orange post-autoclaving) indicate proper preparation
Composition (per liter):
Magnesium Sulfate (MgSO?�7H?O) � 0.20 g
Calcium Chloride (CaCl?�2H?O) � 0.02 g
Monopotassium Phosphate (KH?PO?) � 1.00 g
Dipotassium Phosphate (K?HPO?) � 1.00 g
Ammonium Nitrate (NH?NO?) � 1.00 g
Ferric Chloride (FeCl?�6H?O) � 0.05 g
Agar � 20.00 g
Final pH: 7.0 � 0.2 at 25�C
Preparation:
Suspend 23.27 g of the dehydrated medium in 1 liter of distilled or purified water. Heat with gentle boiling (do not overheat) until completely dissolved, forming a clear solution. A white precipitate may form before sterilization, which turns yellow to orange afterward�this is normal. Dispense into tubes or Petri dishes. Autoclave at 121�C (15 psi) for 15 minutes. Cool to 45�50�C before adding hydrocarbons if needed (e.g., layer 0.5�1 ml liquid hydrocarbon on solidified agar surface). For volatile testing, pour hydrocarbon into the lid of inverted plates.
Storage:
Dehydrated medium: Store in a tightly closed container at 24�C (room temperature), protected from moisture and light; stable until expiry date (typically 3�5 years).
Prepared plates/broth: Store at 2�8�C in sealed containers; use within 2 weeks, or refrigerate inverted to prevent drying. Discard if dehydrated, contaminated, or pH shifts.
?? For laboratory and research use only. Not for diagnostic procedures without proper validation. Not for human or veterinary consumption. Handle with gloves; autoclave waste before disposal. Hydrocarbon supplements may be hazardous�use in fume hood. Not suitable for non-hydrocarbon-utilizing organisms without supplementation.