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A START-UP MANUAL FOR UNDERGRADUATE RESEARCH STUDENTS IN MICROBIOLOGY
Active Learning from the Very Beginning
Keri Law, Rachel Lamb, and Min-Ken Liao

GROWING BACTERIA

All of the previous sections have prepared you to grow the bacteria you desire. Since this section is quite long, we have broken it up into two main sections, growing bacteria for storage and growing bacteria for experiments.

As with all aspects of microbiology the aseptic technique is vital to your success.

PART 1: GROWING BACTERIA FOR STORAGE- plate-to-plate transfer

This is not for the permanent storage of bacteria, which will be discussed in another section. This is about the accessibility of the bacterial culture. When using bacteria on a daily basis, it is important to have the bacteria easily accessible. Bacteria is most accessible on a plate; therefore you must learn how to streak plates. The ability to effectively and efficiently streak plates is imperative to your success in the microbiology lab.

The first step in growing bacteria is transferring the bacteria into/onto the media which you desire. Most of the time, the microorganisms you buy from a company will be in a small vial, frozen. In order to allow these bacteria room to multiply, they must be transferred onto a media plate. A lab instructor will do this. One small vial of bacteria can last you a lifetime if the proper precautions are taken.

Once the bacteria has been transferred to a plate and grown, you will be able to see many isolated colonies. This is the type of plate you will often encounter. An isolated colony contains millions and millions of bacteria, which can be transferred to other media to be grown in colonies. This is referred to in the 'Growing bacteria for experiments' section. For now, we will just focus on growing bacteria for storage.

Bacteria, like everything else, gets old. It goes through lag, log, and stationary stages before death. We planed to include a graph of bacterial growth here but we changed out mind. In order for you to retain what you've learned better, you should go get your microbiology textbook now. From the index, find "growth curve." Now read the section on bacterial growth curve and write down the definitions of each of the following terms.

lag phase:

log phase:

stationary phase:

death phase:

generation time:

In the death phase, the bacteria will not perform up to the normal standards. In order to prevent using 'dead' bacteria, it is important to transfer the bacteria to a new plate. On this new plate the bacteria will have an opportunity to grow and multiply, starting over from the lag phase. Another reason for transferring bacteria to a new plate is that plates don't last forever. It will dry out and bacteria will eventually die. For these two obvious reasons, it is important to transfer the bacteria to a new plate at least every two months.

1. GATHER all of the things you will need for the transfer. Plate with old bacteria, new plate, metal loop, Bunsen burner, striker.

2. LIGHT the Bunsen burner. Note: place the Bunsen burner FARTHEST away from you so you do not have to reach over the flame during the transfer.

3. FLAME the loop until it turns bright orange.

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4. WAIT until the loop has air cooled ? about 15 seconds.

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5. PICK one isolated colony from the old plate. Note: ONE isolated colony will multiply in to millions and millions of bacteria. DO NOT scrap off an entire section of bacteria from the plate!

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6. PLACE lid back on the old plate ? you will NOT be using it again.

7. STREAK section one of the new plate (see diagram on the wall of the lab)

8. FLAME the loop.

9. WAIT until the loop cools.

10. STREAK section two of the new plate (see diagram on the wall of the lab).

11. FLAME the loop.

12. WAIT.

13. STREAK section three of the plate (see diagram on the wall of the lab).

14. ARE YOU GETTING THE PICTURE??? Follow the diagram. ALWAYS flame the loop before you start a new section ? otherwise OLD bacteria will still be on the loop. THE POINT is to THIN out the bacteria so you can get ISOLATE colonies, not bacteria 'lawns.'

15. PLACE newly streaked plate in the incubator. Note: Check appendix for the correct temperature and time it will take for the bacteria to grow.

16. AFTER incubation, place the plate in the refrigerator to be stored. Most bacteria are unable to grow in cool temperatures.

EXERCISE:

Streak a plate from an isolated colony on a plate. Use above instructions. Incubate. Count the number of isolated colonies. A good plate will have 10 isolated colonies. Continue practicing until you are comfortable transferring bacteria to plates and you are getting at least 10 isolated colonies.

PART 2: GROWING BACTERIA FOR USE IN EXPERIMENTS - plate-to-tube and tube-to-flask transfers.

A. Plate to tube transfer

WHY: A mass of bacteria is needed for most of the microbiology experiments you will be performing. Also, we need bacteria in a specific stage of their growth to ensure the consistency of our experiments and results. Therefore an isolated colony will not work. As mentioned before, an isolated colony has thousands of single bacterium in it. All of these bacteria are capable of multiplying and all of the multiplied bacteria are capable of multiplying also. After all this multiplying you will have a mass of bacteria. In order for the bacteria to multiply, the bacteria need nutrients and space. You must start small though. If one isolated colony were placed into a flask containing 500ml of media, the bacteria would be overwhelmed and die. If you place an isolated colony in a tube containing on only 5ml of media, the bacteria will flourish. Once the bacteria has flourished in the small tube, the entire contents of the tube can be transferred to the 500ml flask. Use the following steps as you transfer bacteria from a plate to a tube.

1. GATHER all the items necessary for a plate to tube transfer ? plate with isolated colonies, tube containing nutrient broth, sterile wooden sticks.

2. PICK an isolated colony off the plate using a sterile wooden stick.

3. PLACE the lid back on the petri dish.

4. REMOVE the cap off the test tube using your pinkie of the hand that is holding the stick.

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5. FLAME the mouth of the test tube.

6. INOCULATE the test tube with the colony on the tip of the stick. Swish the stick in the medium. After you are done, you should see tiny particles of bacteria in the tube.

7. PLACE cap back on the tube.

8. INCUBATE the test tube in a shaking incubator at the correct temperature for the bacteria. Note: The incubator should shake only slightly, to allow oxygen to mix with the growing bacteria. It should NOT be violently shaking, causing the bacteria to lyse against the sides of the tube.

After the correct amount of incubation, the tube should be CLOUDY. If it is NOT cloudy there is a problem. Possible problems are:
- The media was not the right type
- The temperature was wrong
- Not enough bacteria inoculated
- Excess soap residue on the inside of the tube

You should also note that if BEFORE you transfer the bacteria to the tube. If the tube is cloudy, there is a problem. DO NOT use that tube! The media is NOT sterile, and therefore your bacteria will be competing with other bacteria and it will not be optimal growing conditions for the bacteria you desire.

After the tube has been incubated it can be transferred to a larger flask in order to grow more bacteria. The tube CANNOT be kept in the refrigerator until a later date. Most of the time the amount of bacteria harvested from a 10ml tube is not enough for an experiment, therefore you will need to perform a tube to flask transfer.

B. Tube to flask transfer

WHY: A tube to flask transfer is necessary to grown a larger amount of bacteria. It won't work if you inoculate a colony-ful of bacteria from a plate directly to a big flask! The bacteria on a plate are used to growing on solid medium. You need to transfer them to a small amount of liquid medium so they can get used to liquid nutrient. Then they will be ready to grow in a larger amount of liquid medium.

1. GATHER all the supplies necessary for a tube to flask transfer ? tube with bacterial growth, flask with sterile broth, bunsen burner, foam lid for flask, aluminum.

2. LIGHT the bunsen burner.

3. TAKE the lids off of the tube and the flask.

4. FLAME the mouth of the tube.

5. POUR the contents of the tube into the flask.

6. FLAME the mouth of the flask.

7. PLACE the foam lid and aluminum on the flask.

8. INCUBATE in a shaker again, not a violent shaker, just a nice MIXING shaker.