BIOLOGY Volume 2 - A Guide to General Biology - 2004

12. MICROBIOLOGY AND BIOTECHNOLOGY

12.4. Inoculation Methods

To prevent contamination when introducing a small number of microorganisms into a nutrient medium—a process known as inoculation (or seeding)—aseptic techniques must be employed. Inoculation Procedures vary depending on whether the medium is liquid or solid.

12.4.1. Inoculation onto Solid Media

Streak plate method, or dilution streaking

This method is illustrated in Fig. 12.4. It is used to isolate pure bacterial colonies from a mixed population. A wire loop is first sterilized by flaming, as shown in Fig. 12.4,A. The sterilized loop is then used to pick up a thin film of a liquid suspension or a small amount of solid material containing the target microorganisms from a previously grown culture or another source. The loop is gently drawn across The surface of the Agar in a series of streaks. After each set of streaks, the Petri dish is rotated slightly so that the Bacteria from the previous streak are progressively diluted across the new surface, ultimately separating individual bacterial Cells (Fig. 12.4). (Do not expect to see anything visible on the final streaks until the incubation period is complete!) Once mastered, streaking can be performed very rapidly.

This method can be used to isolate bacteria from natural habitats such as soil, milk, and Water. Solid samples, such as soil, are best suspended in a small amount of water or pre-incubated in a liquid medium. Pasteurized milk serves as a safe source for routine laboratory work. Before conducting experiments with bacteria or Fungi, always review safety guidelines and protocols to minimize the risk of culturing harmful organisms.

Class="center">

Fig. 12.4. The streak plate method, or dilution streaking.

Spread plate method

This method is illustrated in Fig. 12.5. It is convenient for inoculating microorganisms from a liquid suspension onto a solid medium and is used to determine the Number of viable cells in a sample following serial dilutions (see Section 12.6.1). It can also be used to produce a uniform bacterial lawn across the agar surface by dense inoculation. This approach is particularly useful when testing The activity of inhibitors, such as Antibiotics or disinfectants, which are placed into wells cut into the agar or applied to filter paper discs laid on the agar surface. The inhibitor diffuses through the agar, creating a growth inhibition zone around the wells or discs, which becomes visible after incubation. The diameter of this zone serves as a measure of the degree of inhibition.

Fig. 12.5. The spread plate method.

Pour plate method

As an alternative to the spread plate method, this technique is used to inoculate cells from a liquid culture and to quantify viable cells. Because the cells are distributed throughout the medium rather than just On the surface, a much higher number of colonies can be counted—up to 1,000 per plate. However, the resulting colonies are significantly smaller in size (Fig. 12.6).

A measured volume (up to 0.5 cm³) of The Cell suspension is added to a suitable volume (approximately 15–20 cm³) of sterilized, molten nutrient agar that has been previously cooled to 45–50 °C in a water bath. Remove the cap and flame the neck of the bottle before adding the cell suspension, as shown in Fig. 12.3, A and B. Thoroughly mix the cell suspension with the nutrient agar by gently swirling the bottle back and forth between your hands (do not shake it) (Fig. 12.6, A). Next, pour the mixture into a sterile Petri dish, as illustrated in Fig. 12.3, C. Label the bottom of the dish and incubate it. After incubation, the plate will appear as shown in Fig. 12.6, B.

Fig. 12.6. The pour plate method.

Stab inoculation

This method is used to culture anaerobic organisms or those that thrive at low oxygen concentrations (microaerophiles). It typically utilizes a test tube containing a solid agar medium. Due to the small surface area and significant depth of the agar compared to a Petri dish, oxygen diffusion into the medium is restricted. Inoculation is performed using a straight wire (without a loop) or a bacteriological needle. A small amount of culture (solid or liquid) is picked up on the tip of the needle, which is then used to stab straight down into the center of the agar (Fig. 12.7). The culture grows radially outward into the agar from the line of the stab.

Fig. 12.7. Stab inoculation.