Aseptic techniques lab report PDF

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BIOL 317 Aseptic Technique, Sterilization, and Disinfection

Experiment 2 Submitted by: Grayson McKinney Lab Partners: Madison McNeely, Ryan Yeates, Amy Zonts Thursday, September 19, 2019

ODU Honor Code: I will refrain from any form of academic dishonesty or deception, such as cheating or plagiarism. I am aware that as a member of the academic community it is my responsibility to turn in all suspected violations of the Honor Code. I will report to a hearing if summoned. Grayson McKinney

Introduction To effectively study individual bacteria, it must be done so in the absence of other contaminating bacteria. In order to maintain species in pure culture, aseptic techniques must be followed. Appropriate personal hygiene is the start to an effective aseptic technique. The human skin inhabits multiple, naturally occurring, bacterial populations. Any contamination in the growth medium may potentially ruin the results of the experiment. In this experiment, four different aseptic techniques will be carried out: Autoclaving, moist heat, filtration, and UV light. Autoclaving is an extremely effective method of sterilization for items that are not heat sensitive. Autoclaving will kill all spores by subjecting each item to 121 degrees Celsius at 15psi for 15 minutes. Moist heat sterilization subjects bacteria to a boiling water bath to kill bacteria. Filtration removes bacteria from a liquid culture, and it’s used to sterilize heat sensitive liquids. Bacteria can also be killed by exposure to UV rays. The exposure of radiation damages the DNA of bacteria by causing thymine dimers which cause mutations. Each technique is appropriate for a certain type of bacteria. The bacteria will be observed after each technique is complete and the sterilization ability will be compared. Technique 1: Effect of Moist Heat and Autoclaving Procedure 1. Obtain 4 tubes. 3 of the tubes contain spore strips (Bacillus stearothermophilus). 2. One of the tubes with a strip will be your control and will receive no heat treatment. The fourth sterile tube (with no spore strip) will act as a control of your aseptic pipetting technique. 3. Set one tube aside for autoclaving. Autoclave for 15 minutes. 4. The second tube should be placed into a boiling water bath for 15 minutes. 5. After heating treatments, add 2 ml of Tryptic Soy broth to each of the 4 tubes. 6. Incubate at 56oC until the next lab period. 7. Record your results as presence/absence (+/-) of growth. Materials needed - Test tubes - Bacillus stearothermophilus spore strips - Pipette - Boiling water bath - Hot plate - Tryptic soy broth - 56 degrees Celsius incubator Results (+ = bacterial growth present, - = growth absent)

Tube Control with spore strip

Results +

Control without spore strip

-

Autoclave with spore strip

-

Boiling with spore strip

+

Remarks Growth expected, nothing was present to inhibit growth No growth expected, no spores were present Totally effective in killing all bacteria spores Does not completely sterilize. Not as effective as autoclave.

Discussion All the results observed were the expected results. Growth was expected with the control with the strip because there was nothing present to inhibit bacterial growth. The control without the strip showed no growth because no Bacillus stearothermophilus spores were present. The tubes had no contamination since the control without spores showed no growth. Autoclaving is an extremely effective aseptic technique, so no bacterial growth was expected, and the results showed this. Growth was shown in the tube that was placed in the boiling water. Bacillus stearothermophilus is a thermophilus, so it has a high resistance to heat. This is why boiling was not effective in killing the bacteria that was used. Technique 2: Filtration Procedure 1. Obtain 4 sterile test tubes 2. Prepare 3 tubes containing 2 ml of sterile Tryptic Soy Broth. 3. Add 100l (0.1 ml) of non-sterile glucose solution (5%) to one tube that contains TSB. 4. Filter-sterilize the remaining glucose into the 4th sterile tube (tube without TSB). 5. Add 100l (0.1 ml) of this sterile glucose to another tube of media. 6. Incubate at 37oC until the next lab period (the third tube is a control and only contains TSB). 7. Record your results as presence/absence (+/-) of growth. Materials needed - Sterile test tubes - Tryptic soy broth (TSB) - Non-sterile glucose (5%) (nsg) - Filter (2mm width) - Syringe - Pipette - 37 degrees Celsius incubator

Results Tube Filtered glucose control

Results -

TSB control

-

TSB nsg

-

TSB sg

+

Remarks No growth expected. There were no bacteria present. No growth expected. There were no bacteria present. Growth expected, the glucose was unfiltered No growth expected. Filtered glucose should have prevented growth

Discussion The results of the two control tubes were expected. No bacteria were present in either tube, so no bacteria were available to grow. The absence of bacteria in the controls showed that there was no contamination. The tubes with TSB and glucose showed conflicting results to what was expected. The tube with TSB and non-sterilized glucose showed no growth, nut growth was expected. Unfiltered glucose should have produced bacteria. The tube with TSB and sterilized glucose showed growth, but no growth was expected. Filtered glucose should have prevented bacterial growth. The reason for the unexpected results was most likely due to mislabeling the TSB and glucose tubes. Since the expected results would have been met if the labels were switched, and there was no contamination. Technique 3: Effect of UV light Procedure 1. Inoculate the entire surface of a Tryptic Soy Agar plate with a bacterial culture using a sterile swab (make sure to mix culture first to resuspend all cells). 2. On the back of the plate divide the plate into 6 sections, labeled as 0, 0.5, 1, 3, 5, and 7 minutes. (As shown in figure). 0 min

0.5 min

1 min

3. At the UV light statio minute section of the pla the 5 minute section is now

3 min 5 min 7 min

6” card cover all but the 7 minutes move the card down so move the card to expose the 3

minute section, and repeat after a further 2 minutes to expose the one minute section. After 30 seconds move the card so that only the 0 minute exposure section is still covered and expose for a final 30 seconds. 4. Close the plates - protect from light while repeating with other organisms. 5. Wrap both plates together completely in aluminum foil and incubate at room temperature placing plates in the drawers located at your bench until the next lab period. 6. Record your observations - growth or no growth or relative amount of growth compared to unexposed organisms. Also, note any changes in the bacteria’s appearance. Materials Needed - Tryptic soy agar (TSA) plate - Swabs - Broth culture of Serratia marcescens - Broth culture of Micrococcus luteus - UV light station - 4”x6” card - Aluminum foil Results Microorganism S. marcescens (S) S. marcescens (S) S. marcescens (S) S. marcescens (S) S. marcescens (S) S. marcescens (S)

Time M. luteus (M) M. luteus (M) M. luteus (M) M. luteus (M) M. luteus (M) M. luteus (M)

0 min 0.5 min 1 min 3 min 5 min 7 min

Growth S+ S+ S+ SSS-

M+ M+ M+ M+ M+ M+

Discussion Bacteria are killed by exposure to electromagnetic radiation with wavelengths below 300nm. In this experiment UV light was able to kill bacteria, but not the bacteria we expected. S. marcescens started to show a decrease in growth at around three minutes exposure to UV light. However, this was not the expected result. S. marcescens can repair its own DNA, so the UV light should not have influenced bacterial growth. The plate with M. luteus showed growth throughout all seven minutes of UV light exposure. M. luteus cannon repair its own DNA, so the UV light should have caused thymine dimers which would cause mutations that stop bacterial growth. These results, again, were most likely caused by the mislabeling of our TSA plates since the two expected results were obtained, but in the wrong plate.

Conclusion In this experiment, different aseptic techniques were used to test their ability to kill bacteria. The results showed that different aseptic techniques are more effective in killing certain types of bacteria than others. The importance of properly and accurately labeling samples also became evident during this experiment. Mislabeling can lead to incorrected data, even if the experiment was carries out correctly.

References

Boca, B. M., Pretorius, E., Gochin, R., Chapoullie, R., & Apostolides, Z. (2002). An Overview of the Validation Approach for Moist Heat Sterilization, Part I. Pharmaceutical Technology, 26(9), 62–70. Coté, R. J. (1998). Aseptic Technique for Cell Culture. Current Protocols in Cell Biology, 00(1), 1.3 1–1.3 10. doi: 10.1002/0471143030.cb0103s00 General Microbiology Laboratory Manual, (2019), Department of biological sciences at Old Dominion University, 24-28 Sabnis, R. B., Bhattu, A., & Vijaykumar, M. (2014). Sterilization of endoscopic instruments. Current Opinion in Urology, 24(2), 195–202....