Techniques Involved in the Isolation of Autotrophic Bacteria

Aseptic Isolation

• Aseptic isolation means non-sterile or in the absence of microorganisms. Microbiologists must then handle the material in a way that does not add unwanted microorganisms to the material. Aseptic isolation procedures include sterilization, plate streaking, spread plate and pour plating.

Sterilization

• Sterilization kills or removes all living organisms from an experimentation environment by means of heating with steam that is under pressure (autoclave). A growth medium is autoclaved in covered test tubes then poured into sterile Petri dishes. The Petri dish sits in an incubator at a controlled temperature. Mechanical shakers agitate the cultures during growth to permit oxygen to enter the dish.

Plate Streaking

• In this isolation technique, obtaining a sample of bacteria occurs with the help of a metal wire that has a circular loop at one end. A burner flame sterilizes the loop until it is red hot. Collection occurs by dipping the sterile wire loop into a bacteria colony. To identify the bacteria, it is separates from other microbes by streaking the loop across the surface of an agar plate. Three separate steps in the streaking process separate individual cells. When streaking is complete, the plate is incubated at a temperature that permits growth of the isolated bacteria.

Spread Plate

• The spread plate procedure isolates bacteria by placing a sample on a sterile agar surface in a Petri dish. A sterile bent glass rod, shaped like a hockey stick, spreads the sample over the surface of the dish. When evenly distributed and sufficiently diluted, the bacteria grow as discrete colonies.

Pour Plate

• The pour plate technique is useful when counting the number of each colony of bacteria is required. A sample of soil or water is mixed thoroughly with molten agar before it solidifies. The liquid is poured into a Petri dish and allowed to solidify. In this method, bacteria colonies distribute uniformly in the solid medium but the high heat used during the mixing stage has the potential to kill some species.