Cell cultures are complex processes by which cells are grown under controlled conditions outside of their natural environment. The cultures found in this laboratory were that of Escherichia Coli. The cultures consisted of controlled Escherichia Coli without FabG and an experimental group of cultures with FabG.
Aseptic technique refers to any procedure performed under sterile conditions. Sterile conditions are critical in culturing bacteria. In the absence of a sterile environment, it cannot be assumed that the bacteria growing in the culture is not contaminated. Aseptic technique requires a sterile working field. This field is established with a disinfectant wash of the desired area, accompanied by a lit burner to eliminate any airborne spores. To maintain aseptic technique while working, it is important to maintain a minimal level of talking, mouth breathing, and coughing. It is also important to alleviate any exposure of the culture to the open air. Aseptic technique also recommends working as close to the flame as possible.
Bacterial growth take place in four different phases (Figure 1.), two of which are more commonly addressed. The log or exponential phase and the stationary phase are more commonly referred to when discussing culturing. The first phase is the lag phase in which bacteria adapt to their new environment. It is the period where bacteria mature and prepare to divide. The second phase is the exponential or logrythmic phase, the period associated with cell doubling.
The doubling time for Escherichia Coli is approximately 30 minutes. The phase following exponential growth is the stationary phase. This phase has no new growth; the cells remain stagnant. This phase usually results from a depletion of nutrients and/or the accumulation of toxins in the environment. The final phase is the death phase in which bacteria cannot survive due to total depletion of nutrients. In order to maintain a healthy culture of E. Coli it is important to subculture. Subculturing is removing a single colony from the original culture of bacteria and streaking a new plate. What this allows is a preservation of the health of the bacteria by keeping the bacteria in the exponential/log phase.
Streaking a plate of bacteria allows for the isolation of a pure strain to be grown on a new plate via colony extraction. This allows the lab to keep the bacteria in the log phase.
Streaking is done using a sterile tool such as an inoculation loop following aseptic technique. The loop is first sterilized in the flame and is then used to grab a single isolated colony from the original plate. The colony is spread in a zig-zag pattern as seen in figure 2. in order to spread the bacteria for maximal growth (taking up ¼ of the new plate). The second step requires another sterilization of the loop, which is then used to spread the new colony on the plate across another ¼ section. This is repeated until all four sides of the plate are streaked, with each new streak drawing from the streak of the one prior. This allows for a difference in growth among the four sections; in each subsequent streak, there is less bacteria than the one before it.
After the streak plates are finished and labeled, a 24-hour incubation period is required at 37˚ C. This incubation period is crucial for controlling growth. After this period, a refrigeration period is necessary to inhibit further growth.