Aseptic Technique & Culturing Microbes

Title: Aseptic Technique & Culturing Microbes

Purpose: To learn how to use the aseptic technique and to become familiar with growing bacteria.

Materials:

• Gloves
• Goggles
• Thermometer
• Candle
• Test-tube-rack
• Blank-Slides
• Cover Slip
• Broth, MRS - 9 mL
• Broth, Nutrient - 5 mL
• Lactobacillus acidophilus
• Stain Solution #1, Crystal Violet
• Swab, Sterile
• Styrofoam cooler
• Face mask

Procedures:

1. I put my mask and gloves on to avoid contaminating the samples and I then disinfected my area with bleach.
2. An incubator was needed, and so I used a Styrofoam cooler I placed aluminum on the inside of it.
3. I cut a small hole on the edge of the cooler and placed the thermometer through it.
4. I have access to the top of a refrigerator and so I placed the incubator on top of the refrigerator. Monitored the temperature for a couple of hours so to make sure it was constant.
5.  I disinfected the area once again to begin generating the microbial cultures, and lit one of the candles.
6. Obtained the one of the Nutrient Broth tubes and labeled it “S. epidermis”
7. Using one of the sterile swabs I rubbed it on my skin to obtain a sample.
8. Took the lid off from the Nutrient Broth and immediately ran the edge of the tube through the flame to sterilize it.
9. I transferred the swab into the tube and once again passed the edges of the test tube through the flame and closed the tube.
10. I placed the tube on the test tube rank into the incubator.
11. Afterwards I obtained the tube of MRS broth and labeled it “L acidophilus”
12. Also obtained a capsule of L acidophilus.
13. I removed the lid from the MRS broth and sterilized the edges with the flame.
14. I immediately transferred the capsule into the MRS broth and once again sterilized it with the flame and closed the tube.
15. I placed the tube in the incubator alongside the Nutrition broth.
16. I observed the organisms after 24 hours and then again after 48 hours.
17. Obtained 4 slides and covers slides for the organisms.
18. Began preparing the wet mount slides, on each of the slides I made a circle.
19. I added a drop of warm water on one of the slides and took and swab and got a sample from the Nutrition broth and placed it on the drop of water and covered it with a cover slide
20. Repeated step 19 for MRS broth and recorded my observations.
21. Began preparing Direct Staining slides. Obtained 2 more slides. I drew a small circle on the slide.
22. I took a sample of the nutrition broth and placed it in the circle and let it dry. I took and passed the slide through a flame 3-4 times.
23. I placed the slide in the staining tray and added a drop of crystal violet to the slide.
24. I let it sit and dry and rinsed the slide without wiping of the sample.
25. Repeated steps 22-24 for MRS.
26. Observed both sample on the microscope.
27. Observed both cultures and stored samples in refrigerator when finished.

Data Tables: The temperature was monitored every 2 hours for 6 hours throughout the day. To make sure it remained constant

Observation:     

• Nutrition Broth Tube – Had a cloudy or turbidity appearance it had white specs in the test tube.
• MRS Broth Tube → it turned a more orangey color and it had sediment growing on the bottom.

Questions

1. What is the difference between a bactericidal and bacteriostatic agent? What is the difference between sterilization and disinfection?

1. The difference is that bactericidal kills bacteria while bacteriostatic prevents the growth of bacteria but doesn’t kill it.

1. List five microbial killing methods, how they work, and what they are used for.

1. Dry heat → It’s a hot air oven used for glassware or any other object that won’t melt due to high temperature, it kills microbes
2. Autoclave → It is good for sterilizing everything it is a stem under pressure, it will destroy heat-labile substances.
3. Filtration → Has to do with the removal of cell in a liquid or gas, sterilizes most solutions that will be destroyed due to heat.
4. Boiling → At about 100C for 30 minutes will kill most germs, longer period is needed to sterilize and kill all germs.
5. Irradiation → Mostly destroys nucleic acids. The ultraviolet light is mostly used to sterilize the surface of an object.

1. What is a pure culture? Why is it important to work with a pure culture?

1. It is something that comes from the same thing or organism. It is important to work with pure culture to get accurate results.

1. What is aseptic technique? Why is it so critical?

1. It prevents any foreign organisms from contaminating the samples and it sterilizes the area.

1. Describe three common forms of growth that you are likely to see in a broth culture.

1. Pellicle → Its when the organism is on top of the broth.
2. Turbidity → When the organism has a cloudy appearance in the broth.
3. Sediment → The organism has white deposits on the bottom of the tube.

1. What is the difference between an aerobe and an anaerobe?

1. Aerobe only grows in the presence of oxygen and anaerobe grows in absence of oxygen.

1. Describe the difference between facultative and obligate.

1. Facultative are organisms that are aerobic or anaerobic. Obligate can only live in one type of environment without oxygen.  

1. Which two types of media did you use in this experiment? Why did you need two types of media instead of only one?

1. The two types of media were nutrient medium and MRS medium. We need two because the L acidophilus can only grow in MRS.

1. Describe your microscopic observations of the cultures

1. The epidermis was shaped in clusters in the wet mount and in the direct stain they looked about the same but the purple color emphasized them a bit more. In the direct stain L. acidophilus was rod shaped and grouped together in a chain and in the direct stain it was emphasized more with the purple.

1.  Define the following terms

1. psychrophile: Are cold loving bacteria, the best temperature for them to grow in is -5 C
2. mesophile: Grow best in moderate temperatures, the optimum growth temperature is between 25 C and 45 C
3. thermophile: Love heat, the best temperature for them to grow is between 45C and 70 C.
4. hyperthermophile: They grow at very high temperatures, the best temperature for them to grow is 70 C to 110 C

1. Which type of organisms did you use in this experiment?

1. S Epidermis and L acidophilus

Conclusion:  I l learned the proper way to sterilize and kill microbes using different treatments. I was able to create an incubator which I had never done out of a Styrofoam cooler. I was able to grow and observe the different types of bacteria growing in the two test tubes.