Type: Process Essays
Sample donated: Marjorie Griffin
Last updated: September 25, 2019
Aseptic technique is analyzed by moist heat, filtration, and ultraviolet experiments. To ensure aseptic technique was present, measures were taken to obtain pure cultures. It was necessary to decontaminate tools and areas of work to prevent contamination of the specimens, minimize exposure by working quickly, and using sterile equipment during the experiments. While pipetting Tryptic Soy broth for the heating treatments, pipets must be opened and closed quickly and not touch the table before use. Test tube caps must also remain with the corresponding tube to prevent cross contamination.
The heat treatments, autoclaving and boiling for all tubes will last for fifteen minutes and be incubated at 56°C until the following lab. During the filtration process pipets must also be carefully handled. To sterilize the glucose, a small filter is added to the end of the syringe.
The filter and syringe opening must not touch anything to ensure sterilization; these filtration tubes will incubate at 37°C until the following lab. Also when opening test tubes, the mouth should be passed through the flame to prevent transfer of any contaminants that may have settled near the top. For the UV test a Tryptic Soy Agar plate is used because microorganisms can easily grow on it due to the supplied nutrients.
When applying bacteria to the agar with the inoculating loop, it must first be sterilized by passing it through the flame until it glows red and allow it to cool. While spreading the bacteria onto the agar plate it should be closed immediately after, not allowing the lid to touch the table or any other area. The plate will be left under the UV exposing sections to the UV for different time lengths. Immediately after the plate will be wrapped in aluminum foil and kept to incubate at room temperature until the following lab. All growth will be analyzed the following lab day.
Results:To test the effectiveness of various sterilization and disinfection techniques three experiments were performed in regards to moist heat, filtration, and ultraviolet light effects. In order to compare and analyze results, controls were used in most cases and bacteria was determined to have grown or not grown after testing.Tube DescriptionGrowth(+) vs No growth(-)Tube 1-Control, spore strip with no heat and 2 mL of Tryptic Soy broth, incubate(+) large amount of growth presentTube 2-Aseptic control, no spore strip with 2 mL Tryptic Soy broth, incubate(-) no growth present, still clearTube 3-Spore strip boiled water bath for 15 minutes, 2 mL Tryptic Soy broth incubate(+) limited growthTube 4-Spore strip autoclaved for 15 minutes, 2 mL Tryptic Soy broth incubate(-) no growth, clearTable 1: Results of effect of moist heat on bacterial spores (Bacillus stearothermophilus) Tube DescriptionGrowth(+) vs No growth(-)Tube 1- 2 mL Tryptic Soy broth and 1 mL non sterile glucose solution(+) growth, cloudy yellowTube 2- 2 mL Tryptic Soy broth and 1mL sterile glucose solution(-) no growth, yellow-clearTube 3- Control only contains 2 mL sterile Tryptic Soy broth(-) no growth, light yellow-clear Tube 4- Control with only sterile glucose solution(-) no growth, clearTable 2: Results of effect of filtration to remove bacteria and spores from culture Time under UVSerratia marcescensMicrococcus luteus0 MinutesCovered plate, pink color, heavy growthFull lawn, opaque light yellow0.5 MinutesScattered coloniesFull lawn, opaque light yellow1 MinutesOne single colony, pinkFull lawn, opaque light yellow3 MinutesNo bacteria presentFull lawn, opaque light yellow5 MinutesNo bacteria presentFull lawn, opaque light yellow7 MinutesTwo colonies, pinkFull lawn, opaque light yellowTable 3: Time visual of effect of ultraviolet (UV) light on two bacteria’s growth over time on Tryptic Soy Agar platesDiscussion:After analyzing results from the differing sterilization and disinfection techniques, all tests proved to be effective in some way to prevent contamination of a culture. The effect of moist heat treatments on Bacillus stearothermophilus survival showed that when placed in the autoclave for 15 minutes before adding the 2 mL of Tryptic Soy broth and incubating, there was no growth of the spores when looked at the following lab. The autoclaving is known to kill all spores and would be most effective due to its high heating under pressure (Rhinehart).
The test tube placed in the boiling water bath for 15 minutes before adding the 2 mL of Tryptic Soy broth and incubating, showed limited growth of spores collected at the bottom of the test tube. The boiling water can only disinfect and is not expected to kill all spores (Rhinehart). The control with no pore strip had no growth and supported that the aseptic pipetting technique was done correctly.
The other control tube with the spore strip and no heat treatments had a large amount of growth as expected. The tube was a cloudy with particles floating throughout. This proves that B. stearothermophilus spores were growing.
In the filtration demonstration, a glucose solution was used to see if the filtration method could removes bacteria from live cultures. This proved to be true when out of the three tubes, the only one to have growth was the test tube containing the Tryptic Soy broth and non-sterile glucose solution. A cloudy white film was present in the tube, while the sterile glucose tube and control remained clear. These results were expected and supported the filtration being done correctly. This test was done because some bacteria must be sterilized in a heat sensitive manner (Weebly).
In the ultraviolet light test Serratia marcescens and Micrococcus luteus are heavily swabbed onto Tryptic Soy Agar plates. These agars are non-selective and promote growth of almost any bacteria (Slonczewski). The plates were then placed under the UV light with each section of the plate for an increasing amount of time. After incubation at room temperature, the S.marcescens had a pink color along the top of the plate with sparse pink colonies decreasing to slightly paler colonies when exposed longer to UV, showing expected results. With M. luteus the strain of bacteria swabbed across the plate must have been resistant to UV light because a lawn of thick opaque yellow bacteria was present. The results for M.
luteus were unexpected as they should have shown that UV light can kill bacteria when they are exposed for a period of time or alter them in some way as shown with color changes (Rhinehart).Overall, almost all results occurred as expected with growth and no growth. The purpose of performing these experiments is to test efficacy was met with the positive results. This proves the autoclave, boiling, filtration, and UV light to be good sources of efficient sterilization and disinfection techniques. The autoclave was the best method of sterilization because it cleared out or killed spores completely, while with boiling it only disinfects, not fully killing spores allowing limited growth.
Filtration would be the best option when sterilizing anything that is heat sensitive and UV is best for surface sterilization. All methods were proven useful, but some are better in various situations and circumstances.