Methods of Isolation of Bacteria

1. The Streak Plate Method

The streak plate method is perhaps the most common and widely used technique for isolating bacteria. It is primarily a physical dilution method performed on solid agar media. The principle involves dragging a sterile loop (or needle) carrying the bacteria across the surface of the agar plate. As the loop passes over the agar, it deposits bacteria. By streaking in successive phases, the number of organisms deposited decreases mechanically.

The Procedure:

• The agar plate is divided into four sectors, usually labeled I, II, III, and IV.
• A sterile inoculating loop is dipped into the mixed culture.
• The loop is used to streak Sector I, covering approximately 1/4 of the plate. The loop is then re-sterilized.
• After cooling, the loop is passed through the end of the initial streak in Sector I once or twice to pick up a small number of bacteria and then used to streak Sector II.
• This process is repeated for Sector III and Sector IV, always sterilizing the loop between sectors to ensure mechanical dilution.

In the final sector (IV), the bacteria are dilute enough that individual cells are deposited far apart. During incubation, these single cells multiply to form discrete, isolated colonies. This method is quick and does not require specialized equipment beyond a Bunsen burner and a loop.

2. The Pour Plate Method

The pour plate method is useful for quantifying the number of viable bacteria (colony forming units or CFUs) in a sample and also for isolating bacteria. Unlike the streak plate method, which grows bacteria on the surface, the pour plate method embeds bacteria within the agar.

The Procedure:

• The sample is serially diluted to reduce the bacterial concentration.
• Specific volumes (usually 0.1 ml or 1.0 ml) of the diluted sample are placed into sterile, empty Petri dishes.
• Molten agar (cooled to approximately 45C to prevent killing the bacteria) is poured into the Petri dish and mixed gently with the inoculum.
• The agar is allowed to solidify, and the plate is incubated in an inverted position to prevent condensation from dripping onto the colonies.

Bacteria will grow both within the agar and on the surface. Colonies that form deeper within the agar are often smaller and more lens-shaped due to the lack of oxygen, whereas surface colonies are larger and more distinct. This method is excellent for aerobic and facultative anaerobic bacteria and allows for total counts of microbial population.

3. The Spread Plate Method

The spread plate method is similar to the pour plate in that it uses quantitative dilution, but the bacteria are spread on the surface of solidified agar rather than embedded within it. This method is preferred if one wishes to observe the colony morphology on the surface or if the bacteria are sensitive to the heat of molten agar.

The Procedure:

• A small volume (usually 0.1 ml) of the diluted sample is placed on the surface of a solidified agar plate.
• A sterile L-shaped glass rod (hockey stick) or a bent metal rod is used to spread the inoculum evenly over the entire surface of the agar.
• The rod is sterilized by dipping in alcohol and flaming, then cooled before touching the bacteria.
• The plate is incubated. Colonies will grow only on the surface, where oxygen is readily available.

This method produces surface colonies that are easy to pick for sub-culturing. It is the method of choice for isolating strict aerobes, as the oxygen availability on the surface is higher than within the agar depths.

4. Enrichment Culture Method

The enrichment culture method is used when the target bacterium is present in very low numbers within a mixed population. Instead of physically separating colonies immediately, this method creates an environment in the liquid medium that selectively promotes the growth of the desired bacterium while inhibiting others.

The Principle:

Specific nutrients or environmental conditions (pH, temperature, oxygen levels) are manipulated to favor the target organism. For example, if one wishes to isolate a bacterium that can degrade cellulose, a medium containing only cellulose as the carbon source would be used. Only bacteria capable of producing cellulase will grow.

The Procedure:

• The mixed sample is inoculated into a selective liquid medium (broth).
• The culture is incubated for a period (e.g., 24 hours).
• During this time, the target organism multiplies, increasing its numbers relative to the non-target organisms.
• After enrichment, a small aliquot is taken from the broth and streaked onto a solid plate (using the streak plate method) to obtain isolated colonies.

5. Single Cell Isolation

Single cell isolation is a more direct, albeit more technically demanding, method. It involves picking a single bacterial cell from a suspension and transferring it to a culture medium to ensure the resulting colony is pure.

Methods used include:

• Micromanipulator: A mechanical device allows the microbiologist to view individual cells under a microscope and use a micro-needle (micropipette) to pick up a single cell and transfer it to a sterile drop of medium. While precise, this requires expensive equipment and significant skill.
• Serial Dilution to Extinction: This statistical method involves diluting a sample so much that, statistically, only one viable cell is present per tube. The tubes are incubated, and growth in a single tube indicates it likely started from one cell.

While the streak plate is the standard, single cell isolation guarantees at the cellular level that the culture is pure, which is crucial for taxonomic studies or when cloning specific genes.