Basic Bacteriological Techniques


An experienced microbiologist employs many techniques and skills when handling microorganisms. More so than in any other field of science, the skills of a microbiologist need to be practiced and refined to achieve a level of proficiency. Microscopy is one skill you are already learning and will soon master with further practice. Other skills of the microbiologist allow for the safe handling and manipulation of microorganisms. Isolation and investigation of microorganisms are formidable tasks confronting both novice and experienced microbiologists. The skills employed to achieve these tasks are called aseptic technique.

Aseptic technique reduced the potential spread of bacteria used in laboratory to you or other people. This could happen if a pathogen were allowed to escape the lab on contaminated clothing, books, or other materials. You will be introduced this week to the basic skills of aseptic technique, and will be expected to apply and refine them over the course of the semester.

Aseptic technique is necessary to prevent contamination of pure cultures (a culture containing a single species) which are used routinely for analysis. Extraordinary precautions are needed to prevent contamination because microorganisms, particularly bacteria, are ‘everywhere’. Trillions of bacteria occur in, on and around our bodies, and even slight carelessness can lead to inadvertent contamination. The vast majority of these bacteria are harmless, even beneficial, but analysis of a contaminated culture will yield unpredictable and most likely erroneous results.

In Exercise III of this lab exercise, you will isolate an unknown bacterium, separate it from contaminants, and grow it as a pure culture. This will be the “semester unknown” that your team will maintain and analyze throughout the semester. Remarkably, when bacteria are collected from a site (such as by swabbing a surface) and transferred to standard bacterial culture media, only a small fraction of the cells present will actually grow. Those that do grow are abundant enough, but most species have specialized growth requirements that are not met by standard culture media.

Summary of exercise
I. You will practice making aseptic transfers between culture media.
II. You will practice making a streak plate using a provided mixed bacteria culture
III. You will inoculate a plate of nutrient medium from an environmental site to demonstrate the prevalence of microorganisms in the environment and use streak plating to isolate an unknown bacterial species.
IV. You will examine bacteria cultured on solid media to study the importance of colony morphology in identifying bacterial types.

I. Practicing Aseptic Transfers

The technique that you will use most frequently during the semester is aseptic transfer of bacteria, which you will do whenever you inoculate culture media with an organism. The inoculating loop (shown to the right) is the standard tool of the microbiologist and is used to transfer cells to and from culture media. An important point to remember is that seeing a mass of bacteria on the loop during a transfer is not necessary (although it is reassuring))even a single cell is adequate to start a new culture. An essential rule for any aseptic transfer is to sterilize the inoculating loop BEFORE and AFTER each transfer. Why? The techniques for doing aseptic transfers will be demonstrated in class. The manner in which you hold the tubes, caps and inoculating loop are important. You are encouraged to ask for assistance or a critique while you perform the transfers.


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