The goal of this experiment is to achieve separation of individual particles species from a mixed culture. This is accomplished through the streak plate isolation technique. The aim is to replicate the streak plate technique on a larger scale, allowing for the visual observation of particles as they are isolated during the experiment. The advantage of using the streaking technique is that it facilitates the identification of different species within a culture once they have been isolated.
This experiment is similar to the agar plate experiments, but on a larger scale and allows for perfecting techniques before using agar plates and specimens. By using materials that mimic those used in streaking on agar plates, this experiment simulates the events and provides instant visualization. The gathering of representative tools for the actual agar plate streaking experiment is also part of this experiment.
The techniques employed will remain the same, but the equipment used will be on a larger scale. Instead of using fire to clean a loop, a particle remover or water will be utilized to clean the utensils. The experiment’s final outcome will demonstrate the separation of particles and variations in their consistency. The lab employed the materials displayed in Picture 1 for the Materials & Methods section. Multicolored sprinkles were utilized in this experiment as a representation of small particles. Due to their diverse colors, these particles formed a mixed culture, rather than a pure culture.
The medium for this lab was made by combining ketchup with sprinkles, creating a culture that was then placed in a square Tupperware container to simulate a culture tube. To streak the culture, a spoon was used and cleaned using water as a particle remover between each streak. A disposable paper plate served as the surface for spreading the culture. The materials mentioned above were collected prior to the lab, and the particles were mixed into the medium as shown in picture 2. Lastly, a clean spoon, acting as a sterile loop for this lab, was used to collect a sample of the mixed culture.
The clean spoon or streaking utensil was gripped in a manner resembling that of a pencil, while the tupperware dish, symbolizing the culture tube, was held at an angle to prevent contamination from airborne microbes. The streaking utensil remained stationary, while the tupperware dish was moved back and forth, and the tip of the utensil was repeatedly dipped into and removed from the mixed culture. The culture obtained on the streaking utensil was then transferred onto the paper plate or spreading surface by using a back and forth motion, as depicted in picture 3. This process resulted in a larger area with a higher concentration of particles.
The streaking utensil was washed with a cup of water, which is the particle remover shown in picture 4. Then, the streaking surface was turned 90 degrees. The second streak was created with a cleansed streaking utensil, starting from the middle of the first streak and moving back and forth, as shown in picture 5. This second streak noticeably reduced the amount of particles from the first streak. Afterwards, the streaking utensil was cleaned with the particle remover from picture 4. Finally, the streaking surface was rotated 90 degrees again.
Using a cleaned streaking utensil, the third streak was made from the middle of the second streak in a back and forth motion as shown in picture 6. This further decreased the consistency and spread out the individual particles. Afterward, the streaking utensil was cleaned using the particle remover as depicted in picture 4. The streaking surface was then rotated 90 degrees. Using a cleaned streaking utensil, the fourth streak was made from the middle of the third streak in a back and forth motion, as seen in picture picture.
7. The particles were further narrowed down in this final streak, enabling the separation of each individual particle. The streaking utensil was cleaned using the particle remover and returned to the utensil storage area. Additionally, the other items gathered for this lab were cleaned and returned to their respective storage areas. Results – The particles collected for the lab were highly concentrated before being mixed into the medium. After the particles were mixed into the medium, their concentration decreased due to the medium filling the spaces between them, allowing for separation.
Once the initial streak was made, the concentration of particles became slightly less intense, enabling them to disperse across the surface. This decreasing concentration of particles occurred with each subsequent streak. In the final streak, depicted in picture 7, the particles were so well-separated that it was possible to identify each color individually. These findings resemble those typically observed in a laboratory when examining a mixed culture under a microscope.
Discussion – The experiment concluded that as the streaking is done, the consistency and concentration levels increase, leading to a more precise identification of the particles. The results supported this conclusion, as the completion of streaks resulted in a decrease in particle concentration and medium consistency, which allowed for the individual identification of colored sprinkles based on their color. Numerous attempts were made before achieving the final results depicted in the pictures below.
The initial mistake was not thoroughly reading the lab instructions before the initial attempt. It became apparent that several items were required to simulate a streaking experiment, but the materials used in the first attempt did not align with the necessary ones. In the first trial, the medium and particles were not mixed together, but rather applied separately to the streaking surface. First, the medium was applied, and then an effort was made to streak the particles through the previously applied medium on the plate.
The experiment yielded unsatisfactory results due to the utilization of incorrect materials and methods. In the second trial, cheerios were substituted for sprinkles, resulting in failure as the particles absorbed water from the medium, leading to their softening and hindering successful streaking. In the third attempt, proper sprinkles were utilized, but an erroneous streaking technique was employed.
The streaking surface was not rotated 90 degrees, resulting in a diagonal back and forth motion that caused some streaks to touch each other. In addition, the first streak was applied without the back and forth motion and with too strong of pressure, causing uneven particle distribution more to one side of the streak.
The third streak does not contain any particles. This experiment could be improved by carefully reviewing instructions before beginning and practicing to improve streaking technique. In the future, more information could be gained by counting the transferred particles in each streak. A log could be created to record the number of particles of each color in each streak.
How does this simulate exercise 1-4 and what are the differences? One of the main differences between this experiment and that completed in lab 1-4 is the utilization of broth culture instead of medium culture. Another distinction is the preference for flaming over particle cleaner. Additionally, a notable difference lies in the aseptic technique; while we did aim to replicate the laboratory setting with actual instruments, there was no use of gloves nor genuine concern that the obtained culture would contaminate the countertop where the experiment took place. Moreover, in contrast to the experiments in 4-1, there was already an existing pure culture, whereas here you are initiating a new pure culture.
References –
Leboffe, Michael J. and Pierce, Burton E. (2012). Breif Microbiology Laboratory Theory and application Second Edition, Common Aseptic Transfers and Inoculation Methods (pp. 26-38). Englewood, CO: Morton Publishing. Streak Plate Methods of Isolation (pp. 39-44).
