In previous experiments you learned that microorganisms thrive pretty much
everywhere. It is far too easy to contaminate your lab cultures and
experiments with stray microorganisms from the air, the countertop, or your
tools.
It is also possible to expose your surroundings or yourself to a possible
pathogen. In this lab exercise, you will learn to transfer microbiological
cultures from one medium to a second sterile medium without contamination
of the culture, sterile medium, or the surroundings.
Objectives
After studying and performing this experiment, you should be able to:
• know how to handle microorganisms, tubed media, plated media, and
inoculating tools such as loops, needles, or swabs etc.;
• learn how to transfer bacteria from test tubes or broth and agar; and
• learn how to transfer bacteria from Petri plates.
EXPERIMENT
Aseptic technique is a method that prevents the introduction of unwanted
organisms into an environment. In order to protect sterile broth, media, plates,
slants etc. from contamination we must practice aseptic i.e. sterile techniques
to protect our material from contamination. By using aseptic technique only
sterile surface touches other sterile surface and exposure to the non sterile
environment is minimized.
organisms into an environment. In order to protect sterile broth, media, plates,
slants etc. from contamination we must practice aseptic i.e. sterile techniques
to protect our material from contamination. By using aseptic technique only
sterile surface touches other sterile surface and exposure to the non sterile
environment is minimized.
Though, observing aseptic technique is the most important instruction for any
microbiology experiment, some common circumstances will be discussed in
this practical to make you aware of aseptic techniques.
microbiology experiment, some common circumstances will be discussed in
this practical to make you aware of aseptic techniques.
Sterilization of inoculation loop
The inoculation loop is sterilized by passing it at an angle through the
flame of a gas burner until the entire length of the wire becomes orange or
red hot. In this way all contaminants on the wire are incinerated. Never lay
the loop down once it is sterilized or it may again become contaminated.
Allow the loop to cool a few seconds to avoid killing the inoculum.
flame of a gas burner until the entire length of the wire becomes orange or
red hot. In this way all contaminants on the wire are incinerated. Never lay
the loop down once it is sterilized or it may again become contaminated.
Allow the loop to cool a few seconds to avoid killing the inoculum.
Transferring bacteria from broth culture to fresh broth
Requirements
• Bunsen burner.
• Inoculation needle.
• Trypticase Soy Broth cultures of Bacillus subtilis, Escherichia coli and
Micrococcus luteus and Mycobacterium phlei − referred to as Tubes A.
Micrococcus luteus and Mycobacterium phlei − referred to as Tubes A.
• Sterile Trypticase Soy Broth tubes (4 -one for each microorganism) –
referred to as Tubes B.
referred to as Tubes B.
• Glass Marking pen.
Procedure
1. Turn on the Bunsen burner.
2. Vortex culture suspensions of Bacteria given (Tubes A).
3. Place culture suspensions tube near sterile broth tubes (tubes B). Label
sterile tubes with name of microorganism and date.
sterile tubes with name of microorganism and date.
4. Sterilize the inoculation loop as explained above.
5. While holding inoculation loop between thumb and first two fingers of
right hand, pick up tube A with left hand and open the cap/cotton plug
with last two fingers of right hand.
right hand, pick up tube A with left hand and open the cap/cotton plug
with last two fingers of right hand.
6. Flame the lip of test tube A.
7. Place the sterile loop into culture A and take loopful of culture.
8. While still holding the inoculum in your right hand, pick up tube B
with left hand and open the cap/cotton plug with last two fingers of
right hand.
with left hand and open the cap/cotton plug with last two fingers of
right hand.
9. Flame the lip of test tube B gently
10. Place the loop containing droplet of culture in tube B and gently swirl
it to transfer the microbes into sterile broth..
it to transfer the microbes into sterile broth..
11. Take out the loop and continue to hold it in your hand.
12. Flame the lip of test tube B gently and replace the cap/plug which
should be still in your right hand. Place tube B back in the test tube
rack. Like wise plug the tube A and place in a test tube rack.
should be still in your right hand. Place tube B back in the test tube
rack. Like wise plug the tube A and place in a test tube rack.
13. Sterilize the inoculation loop in flame.
14. Repeat the procedure with all bacterial cultures.
Results
1. Draw and describe the growth seen in each of the four broth cultures
Streaking plating bacteria
a) From broth culture to sterile medium plate
b) From one petridish to fresh sterile medium plate.
Requirements
• Bunsen burner.
• Inoculation needle.
• Trypticase Soy Broth cultures of bacteria (Bacillus subtilis and
Escherichia coli) to be transferred (Tubes A).
Escherichia coli) to be transferred (Tubes A).
• Trypticase Soy Agar plate cultures of bacteria (Bacillus subtilis and
Escherichia coli) to be transferred.
Escherichia coli) to be transferred.
• Sterile petridish having Trypticase Soy Agar medium (4 no. Two for
each bacterium).
each bacterium).
• Glass Marking pen.
Procedure
Removing inoculum from a broth culture
1. Label the plates and tubes.
2. Turn on the Bunsen burner.
3. Loosen the top of the bottle/ Tube containing the inoculum.
4. Hold the loop in the right hand.
5. Flame the loop and allow to cool.
6. Lift the bottle/test tube containing the inoculum with the left hand.
7. Remove the lid/cotton wool plug of the bottle/test tube with the little
finger of the left hand.
finger of the left hand.
8. Flame the neck of the bottle/test tube.
9. Insert the loop into the culture broth and withdraw. At all times, hold
the loop as still as possible.
the loop as still as possible.
10. Flame neck of the bottle/test tube.
11. Replace the lid/cotton wool plug on the bottle/test tube using the little
finger. Place bottle/test tube on bench.
finger. Place bottle/test tube on bench.
Removing inoculum from a plate culture
1. Sterilize the inoculating loop in the flame of a gas burner.
2. Lift the lid of the culture plate slightly and stab the loop into the agar
away from any growth to cool the loop.
away from any growth to cool the loop.
3. Scrape off a small amount of the organisms and close the lid.
Transferring the inoculum into a petri plate
1. Partially lift the lid of the Petri dish containing the solid medium.
2. Hold the charged loop parallel with the surface of the agar; smear the
inoculum backwards and forwards across a small area of the medium
inoculum backwards and forwards across a small area of the medium
3. Remove the loop and close the Petri dish.
4. Flame the loop and allow it to cool. Turn the dish through 90º
anticlockwise.
anticlockwise.
5. With the cooled loop streak the plate from area A across the surface of
the agar in three parallel lines. Make sure that a small amount of
culture is carried over.
the agar in three parallel lines. Make sure that a small amount of
culture is carried over.
6. Remove the loop and close the Petri dish.
7. Flame the loop and allow to cool. Turn the dish through 90º
anticlockwise again and streak from B across the surface of the agar in
three parallel lines.
anticlockwise again and streak from B across the surface of the agar in
three parallel lines.
8. Remove the loop and close the Petri dish.
9. Flame the loop and allow to cool. Turn the dish through 90º
anticlockwise and streak loop across the surface of the agar from C
into the centre of the plate
anticlockwise and streak loop across the surface of the agar from C
into the centre of the plate
10. Remove the loop and close the Petri dish. Flame the loop.
11. Seal and incubate the plates inoculated with Bacillus subtilis and
Escherichia coli at 37°C upside down (lid on the bottom) to prevent
condensing water from falling down on the growing colonies and
causing them to run together in inverted position.
Escherichia coli at 37°C upside down (lid on the bottom) to prevent
condensing water from falling down on the growing colonies and
causing them to run together in inverted position.
Results
Expressing results
Bacterial colonies contain millions of cells and exhibit diverse morphologies;
however, all isolated colonies produced on streak plates arise from a single
bacterial cell. When evaluating colony morphology, use specific terms to
describe the shape, elevation, colony margin shape, and surface texture
(Figure 4.3).
however, all isolated colonies produced on streak plates arise from a single
bacterial cell. When evaluating colony morphology, use specific terms to
describe the shape, elevation, colony margin shape, and surface texture
(Figure 4.3).
Colony size and colour are also useful features that are noted. All
of these characteristics may be useful in the initial identification of unknown
bacteria. Colonies that have different morphologies may be considered to
contain different bacterial species. However, colonies that appear to be similar
in morphology are not always the same bacterial species.
of these characteristics may be useful in the initial identification of unknown
bacteria. Colonies that have different morphologies may be considered to
contain different bacterial species. However, colonies that appear to be similar
in morphology are not always the same bacterial species.
Observations
Obtain your streak plate from the incubator and visually examine the different
regions:
regions:
1. Notice a dilution effect as you move from region to region.
2. Look for isolated individual colonies present.
3. Note different types of colony morphologies present.
4. Measure the size (diameter or length) and record the colony colour.