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The Microbiology Classroom

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Lab Exercise 2: Aseptic Transfers

Background:

                You have already learned two invaluable aseptic techniques you will practice before and after every lab: handwashing and disinfecting your lab area.  Another technique you will practice throughout the semester is transferring microbes from one media to the next. There are 3 general types of media you will come across: 1) broth culture: microbes are growing in a nutrient rich liquid, 2) plate culture: microbes are growing on a nutrient rich semi-solid medium in a petri dish, and 3) slant culture: microbes are growing on at a slant on a  nutrient rich semi-solid medium in a test tube.   This is called an aseptic transfer.

 Aseptic transfer is a technique used to transfer microbes from one medium to the next without contamination of the culture or exposure of  yourself to the organisms. There are two main reasons you may wish to transfer bacteria cultures: 1) to keep existing microbes alive and reproducing or 2) to isolate a single species of bacteria (pure culture) from a mixed culture ( complex communities consisting of several different species of microorganisms).  Proper use of this technique is essential in this course and must be mastered by every student.  The inoculating loop should always be sterilized via heat before and after every procedure.  Care must be taken not to use the loop too quickly after sterilization (wait 40-60 seconds) to avoid killing the microbes of interest.  Mouths of the test tube broth cultures must be passed before and after the loop enters the culture tube.  Try not to engage in conversation while performing these procedures.  (Seriously, this is true.  You do not want to contaminate your culture from possible microbes in your mouth)

Tips

1) Never leave flame unattended. 

2) Make sure hair is pulled back.

3) Open the petri dish lid at a 45 angle like a clam shell.  Never open the petri dish completely due to possible contamination.

4) Make sure to sterilize or heat your loop before and after procedure.

5) Once a loop is sterile, do not sit it down on your lab table.

6) Allow for your loop to cool before using it .    If you hear a sizzle, you have killed your microbes.  Start over.

7) Never wave inoculating loop into the air while waiting it for to cool.

8) Use both hands; caps are held in the same hand as inoculating loop.  Never place caps on surface counter.

9) Flame test tubes broths or slants before and after using them.

10) Try not to engage in conversation to prevent contamination.

Part I.  Aseptic Transfer from one plate to the next

Materials

Bunsen burner                      tryptic soy yeast agar plate

Striker or matches                culture of a bacteria on a plate

Inoculating loop                   cultures from Lab 1: ubiquity of microbes.

Procedure:

1) Disinfect your lab area and hands.

2)  Find a single colony of bacteria.  Look for colonies the size of a pinpoint (   ) with a round margin.   Circle this colony with a sharpie on the bottom of your plate.

3) Label your new plate with the bacteria name/location of the sample, your initials and today's date.

4)  Light your Bunsen burner. 

a) Make sure your hose is secure over your gas outlet.

b) Have one partner slowly turn on the gas, while the other person holds the igniter a little bit above the barrel.

c) Adjust the height of the flame using the gas supply valve on the Bunsen burner. 

d) Adjust the color of the flame using the air intake valve at the base of the barrel of the Bunsen burner.

5) Sterilize your inoculating loop by placing the tip into the flame until appears red.  Allow the loop to cool for about 40-60 seconds.

6) You can test to see if it is cool enough by testing it on the edge of the agar plate.  If you hear a sizzle or the agar melts, wait a few seconds and test again.    Remember never to lid on the desk; always open dish like a clam shell

7) Using the circled colony you found earlier, gently touch the edge of the colony with your sterile inoculating loop to pick some bacteria up.  Don't worry if you can't see it on the loop.

8) Streak your inoculum across a fresh plate of TSY agar in one continuous streak in a zig-zag like fashion.

9) Sterilize your inoculating loop again by placing the tip into the flame until appears red.  Allow the loop to cool for about 40-60 seconds before putting the loop back down on your desk.

10) Seal the sides of your plate with parafilm.  Place your plate jelly side up in the incubator for 24-48 hours.

Part II. Aseptic Transfer from plate to broth

Bunsen burner                      tryptic soy yeast broth

Striker or matches                plate of unknown bacteria culture

Inoculating loop                   test tube holder

1) Disinfect your lab area and hands.

2)  Find a single colony of bacteria.  Look for colonies the size of a pinpoint (   ) with a round margin.   Circle this colony with a sharpie on the bottom of your plate.

3) Label your new broth test tube with the bacteria name/location of the sample, your initials and today's date.

4)  Light your Bunsen burner. 

a) Make sure your hose is secure over your gas outlet.

b) Have one partner slowly turn on the gas, while the other person holds the igniter a little bit above the barrel.

c) Adjust the height of the flame using the gas supply valve on the Bunsen burner. 

d) Adjust the color of the flame using the air intake valve at the base of the barrel of the Bunsen burner.

5) Sterilize your inoculating loop by placing the tip into the flame until appears red.  Allow the loop to cool for about 40-60 seconds.

6) You can test to see if it is cool enough by testing it on the edge of the agar plate.  If you hear a sizzle or the agar melts, wait a few seconds and test again.    Remember never to lid on the desk; always open dish like a clam shell

7) Using the circled colony you found earlier, gently touch the edge of the colony with your sterile inoculating loop to pick some bacteria up.  Don't worry if you can't see it on the loop.

6) Remove cap with fingers and hold between third and fourth fingers of same hand that holds the loop.

7)  Flame lip or opening of the tube.

8) Insert loop midway into the test tube and gently swirl ( try not to touch sides of tube).

9) Flame lip and replace cap.

10) Flame inoculating loop.

11) Place tube in rack in incubator until next period.

Part II.  Aseptic Transfer from one broth to the next

Materials

Bunsen burner                      tryptic soy yeast broth

Striker or matches                broth culture of a bacteria

Inoculating loop

Procedure:

1) Disinfect your lab area and hands.

2) Using both hands, pick up broth culture and roll between hands to resuspend the cells.

3)  Light your Bunsen burner. 

a) Make sure your hose is secure over your gas outlet.

b) Have one partner slowly turn on the gas, while the other person holds the igniter a little bit above the barrel.

c) Adjust the height of the flame using the gas supply valve on the Bunsen burner. 

d) Adjust the color of the flame using the air intake valve at the base of the barrel of the Bunsen burner.

4) Sterilize your inoculating loop by placing the tip into the flame until appears red.  Allow the loop to cool for about 40-60 seconds.

5) Roll broth culture containing bacteria back and forth in your palms.  Do  not shake up and down.

6) Remove cap with fingers and hold between third and fourth fingers of same hand that holds the loop.

7)  Flame lip or opening of the tube.

8) Insert loop, remove small amount of culture ( try not to touch sides of tube).

9) Flame lip and replace cap.

10) Pick up fresh broth tube, open as before and flame lip.

11) Insert inoculated loop, gently tap to the inside of the tube.

12) Flame lip, replace cap and label newly transferred tube with the bacterial name, your initials and today's date.

13) Flame inoculating loop.

14) Place tube in rack in incubator until next period.

General Tips for Aseptic Technique

1) Never leave flame unattended. 

2) Make sure hair is pulled back.

3) Open the petri dish lid at a 45 angle.  Never open the petri dish completely due to possible contamination.

4) Make sure to sterilize or heat your loop before and after procedure.

5) Once a loop is sterile, do not sit it down on your lab table.

6) Allow for your loop to cool before using it .    If you hear a sizzle, you have killed your microbes.  Start over.

7) Never wave inoculating loop into the air while waiting it for to cool.

8) Use both hands; caps are held in the same hand as inoculating loop.  Never place caps on surface counter.

9) Flame test tubes broths or slants before and after using them.

10) Try not to engage in conversation to prevent contamination.

Results

Part I. Plate to Plate transfer growth                                                             

Part II. Plate to broth transfer growth

Part III. Broth to Broth Transfer

Follow-up questions

1) Describe the 3 general types of media used to grow bacterial cultures.

2) Explain two reasons why you might want to transfer a culture of bacteria from one medium to the next.

3) Why is it important to flame the inoculating loop before and after every procedure?

4) Why must you wait for your inoculating loop to cool before using it?

5) Describe 4 aseptic techniques practiced during this lab and their importance. ( paragraph)