Purpose
The purpose of this laboratory work was to learn laboratory skills through culturing procedures and studying the results of incubation. Two microorganisms (E. coli and S. epidermidis) were used for this work and were inoculated on three different media: broth, slanted agar, and agar plate by the streak-plate method. Based on the results of these inoculations, the differences in culturing the organisms were studied, and the advantages and disadvantages of each inoculation method were discussed.
Inoculation on Broth
Inoculation on broth is the cultivation of microorganisms on non-solid surfaces. The main advantage of this microbiological method is the different concentrations of nutrients and oxygen at different levels of the depth of the beaker: this allows bacteria with different aerotolerant needs to grow. In addition, it allows for a large stock of microorganisms for the studies. In contrast, through the broth, it becomes impossible to determine the purity of the culture grown and separate the bacteria from each other qualitatively. Tryptic Soy Broth (TSB) medium was chosen for inoculation in this study: both E. coli and S. epidermidis were cultured in this broth at 37℃ under constant agitation for 18-24 hours. To prepare TSB, 3 g of TSB powder is dissolved in 100 mL of distilled water while stirring on a magnetic stirrer as standard. Then, 10 mL of medium is placed in a glass test tube and autoclaved at 121℃ for 15 minutes: after that, the test tubes are cooled without direct exposure to sunlight. For cultivation, the broth temperature should be equal to room temperature.
Inoculation on Agar Slant
Inoculation on slant agar is used to establish a stock of bacteria for future studies, but its applicability is also realized for mixture differentiation. The advantages of this method are the creation of sterile conditions — by the aseptic technique — for long-term storage of microorganisms without the possibility of drying out. The key disadvantages of growth on slant agar are less visibility of the culture and, consequently, worse observability of their dynamics, and the inability to obtain large stocks of bacterial cultures. For culturing E. coli and S. epidermidis, Tryptic Soy Agar Slants medium was chosen, whose preparation does not differ from TSB. The same ingredients in the same amount are used to make the TSA medium, but the broth is poured into tubes at an inclination for solidification after autoclaving is completed. It is acceptable to add defibrinated pureblood without bubbles if hemolysis is investigated in a clinical setting. The two pathogens are cultured at 37℃ for 18-24 hours.
Inoculation on Agar Plate by Streak-Plate Method
Finally, cultivation on an agar plate by streak-plate method to isolate individual bacteria on a nutrient medium. The advantage of this method is that separate and independent colonies can grow on a plate; hence it saves laboratory resources and allows more efficient use of the dishes. Consequently, it is possible to distinguish pure colonies. However, disadvantages of this method include the possibility of cross-infection, difficulty in counting concentrations, and the inability to grow obligate organisms using this method (Tankeshwar, 2013). In this work, Tryptic Soy Agar medium was again used to cultivate E. coli and S. epidermidis with the difference that the liquid broth was poured into Petri dishes, and cultivation was performed via the streak-plate method. Consequently, the ingredients for preparation were not different, although the procedure for creating the medium was slightly modified. Incubation conditions are also the same and are 37℃ for 18-24 hours.
Reference
Tankeshwar, A. (2013). Chocolate agar: Composition, preparation, uses. Microbe Online. Web.