Inoculation in the microbiology laboratory is the deliberate process of introducing microorganisms into a suitable growth medium. This technique transfers a small sample of bacteria, fungi, or other microbes from a source culture or environmental sample into a sterile environment. The goal is to provide the microbes with the specific nutrients and conditions they need to multiply for study. This laboratory practice is distinct from the medical act of vaccination, which also uses the term inoculation but involves introducing a pathogen into a host to stimulate an immune response.
Defining Inoculation and Its Goals
Inoculation promotes the growth and propagation of microorganisms under controlled conditions. This culturing process is foundational for microbiological investigations, allowing scientists to generate large quantities of a specific microbe for analysis. The inoculum source can be an existing pure culture, a clinical specimen, or an environmental sample.
The receiving environment is typically a specially formulated culture medium, such as a liquid broth or a solid agar plate or slant. A primary goal is the isolation of a single species from a mixed population to obtain a pure culture. This ensures that subsequent experiments are performed on a genetically uniform population.
Identification is another objective, often involving inoculation into specialized differential or selective media. Observing growth characteristics or metabolic reactions, such as acid production or enzyme activity, helps determine the organism’s identity. Inoculation is also used for the long-term maintenance of stock cultures, preserving them in a viable state.
Essential Techniques for Transfer
The physical method used for inoculation depends on the specific goal of the experiment. For separating individual microbial species from a mixed sample, streak plating is the standard technique. This method uses an inoculating loop to systematically spread and dilute the sample across an agar plate. The goal is to achieve isolated colonies in the final section, where each colony originates from a single cell.
Other techniques are used to count viable microbes or achieve uniform growth. Spread plating involves pipetting a small volume of liquid culture onto the agar surface and spreading it evenly using a sterile glass rod. Pour plating mixes the inoculum directly into the molten agar before it solidifies, resulting in colonies growing both on the surface and embedded within the medium.
When using semi-solid media in a test tube, stabbing is the preferred technique. An inoculating needle is used to puncture the medium down the center. This method is commonly employed to test for bacterial motility or to observe oxygen requirements, as the stab creates an oxygen gradient. Primary tools include the inoculating loop, the inoculating needle for stab cultures, and sterile pipettes for liquid transfers.
The Role of Aseptic Technique
All inoculation procedures must use strict aseptic technique to prevent unwanted contamination. Asepsis means working under conditions that exclude the introduction of foreign microorganisms from the air, work surfaces, or tools into the sterile medium. This technique protects the experimental culture from environmental microbes and safeguards the worker and the laboratory environment from the cultured microbes.
A central component of aseptic technique is using a Bunsen burner flame or micro-incinerator to sterilize metal inoculating loops and needles immediately before and after use. The metal wire is heated until it glows red hot, incinerating any adhering microorganisms. Working near the flame creates a localized zone of rising sterile air that limits airborne contaminants settling on the workspace.
Proper handling of culture vessels is also essential. When transferring from a tube, the cap is removed and held, never placed on the bench. The mouth of glass tubes is quickly passed through the flame just before and after the transfer to sterilize the rim. Petri dish lids are only lifted slightly and used as a shield to minimize exposure of the agar surface to the surrounding air.