A START-UP MANUAL FOR UNDERGRADUATE RESEARCH STUDENTS IN MICROBIOLOGY

Active Learning from the Very Beginning

Keri Law, Rachel Lamb, and Min-Ken Liao

AUTOCLAVE: LEARNING HOW TO KILL (STERILIZATION)

An autoclave reaches 121 C and 15psi. Therefore it kills nearly any bacteria that could cause contamination. Without the sterilization, NO microbiology experiment would work. To achieve sterilization we have to learn how to use an autoclave.

The use of an autoclave serves three purposes ? to sterilize media, to sterilize glassware and to sterilize biohazardous wastes.

The following instructions are for use of the two autoclaves in Peale Science Center at Hope College.

USING THE AUTOCLAVE TO STERILIZE MEDIA

[images of flasks with liquid media]

The amount of medium you need to prepare depends on what you will be using it for. As we mentioned before, there are two types of media, solid and liquid. All media will be autoclaved and therefore as it comes out of the HOT autoclave all media will be in the liquid state. If the media contains agar, it will harden while it cools. This type of media is used to pour petri plates. The amount of media you makes depends on how many plates you need. Each Petri dish holds about 20-30ml of medium. (More on pouring plates in section 7).

If you are making media in which to grow bacteria you will probably need to aliquot portions of it into tubes. Growing bacteria in liquid media is discussed in depth in section seven, but it is important that you realize you will need different volumes of media (i.e. 10ml tubes, 300ml flasks, 500ml flasks, etc.). Before autoclaving, pour the correct amount of media into the tubes or flasks, which will be utilized in the experiment.

Use the autoclave in the microbiology prep room, 154b.

[images of an autoclave]

1. Turn autoclave ON.

2. Put LABELED flasks, bottles or tubes in the autoclave. Note: If you are autoclaving many flasks or racks of tubes, place all of them in a large tray. This makes it easier to remove. BE SURE the tray you place them in is autoclavable. Also, cover the bottom of the tray with water.

3. Close door tightly.

4. Set STERILIZATION timer. For one liter of liquid use 15 minutes. For each additional liter add five minutes. DO NOT SET DRY TIME.

5. Push LIQUID button.

6. Sign in the logbook.

7. WAIT. The autoclave takes TIME to increase the temperature and pressure. It also takes time to decrease the temperature and pressure after it has sterilized the media. ONLY open the autoclave when you see the orange light which states, COMPLETE.

USING THE AUTOCLAVE TO STERILIZE GLASSWARE OR OTHER LAB UTENSILS

Autoclaving glassware, pipettes, sticks and other general lab supplies will eliminate the amount of contamination. Use the autoclave in the microbiology prep room, 154b.

1. Turn autoclave ON.

2. Put glassware, pipettes, etc. in the autoclave.

3. Set the sterilization timer and DRY timer. For a half load, use 20 minutes for each timer. For a full load, use 30 minutes for each timer.

4. Close door tightly.

5. Push GRAVITY button.

6. Sign in the logbook.

7. WAIT.

8. Move all of the autoclaved lab supplies to 68 C incubator in the lab. Leave them there over night to completely dry them.

USING THE AUTOCLAVE TO STERILIZE TRASH

Biohazard waste treatment is essential in microbiology. Any used plate, kimwipe, or even cotton swab, which has been in contact with living organisms, has the potential to multiply and spread bacteria. Therefore, they must be autoclaved before it can be thrown away in regular trash can.

Use 'kill' autoclave in the greenhouse prep room for trash sterilization.

1. Open the door

2. Remove previous autoclaved waste in the autoclave bin and place it in the trash can.

3. Check the water level in the autoclave. If it is not full, add more until it reaches the brim. (See Picture)

4. Place your biohazard waste bag in the autoclave bin.

5. Close the door.

6. Turn timer to 60 minutes.

7. Sign in logbook.

STERILIZATION EXERCISES: (read through all instructions before you start)

1. Make TWO 100ml batches of Nutrient Broth. (Read section 6 on how to make liquid medium.

2. Take one flask and autoclave it. DO NOT autoclave the other flask.

3. After the autoclaved flask has been cooled, place BOTH flasks in a 37-degree incubator for 3 days. BE SURE the flasks are labeled appropriately.

4. Notice the difference between the two flasks.

The medium that was autoclaved should be clear, whereas the medium, which was not autoclaved, should be cloudy. The cloudiness is due to bacterial contaminants. Remember, the media you make will satisfy the desires of many different bacteria. Since you will only want the specific bacteria you are working with to grow in the media you make, be sure if is VOID of any other bacteria before using it.

Now that you learned how to sterilize your media you need to learn how to KEEP the unwanted bacteria away from the media enter the aseptic technique.