Lab 7:  Background and protocol

There are two parts to the lab for today.  In the first part, you will learn about the effect of salt concentration on the growth of bacteria.  In the second part, you will see how placing bacteria at certain temperatures for different time periods will affect their growth.

Salt experiment

Bacteria grow under different salt conditions.  High salt concentrations are toxic to some organisms yet others thrive or even require these high salt conditions.  Some bacteria can live in a hypotonic environment.  This is when there is less salt in the environment and more inside the cell.  If there is less salt, then there is more water and water moves into the cell by osmosis.  Under hypertonic conditions, a cell will undergo plasmolysis.  There is more salt in the environment and less water compared to inside the cell so water leaves the cell and the cell shrinks. 

In this laboratory, you will be given agar plates that have either 1% salt, 5% salt, 10% salt or 15% salt.  If an organism can only grow on the 1% or 5% plates only, it is classified as a non halophile.  If the organism can also grow on the 10% plate, it is classified as a facultative halophile.  However, if it can grow on the 15% salt plates, it is called an extreme halophile.

In this experiment, you should work in groups.  What you want to do today is to take 4 plates – a 1% plate, a 5% plate, a 10% plate and a 15% plate. You should divide each plate into 4 quadrants as shown below. 

Then, you will need to get 4 organisms:

E.coli (EC)

Alcaligenes faecalis (AF)

Serratia marcescens (SM)

Staphylococcus aureus (SA)

Each of the 4 organisms will be streaked on the 1%, 5%, 10% and 15% salt plates. Each organism will go in one quadrant of the plate. You will use a loop to do the inoculation but you do not need to streak for isolation.  You just want to know if the organism will grow.  Streak the organisms into each quadrant as shown below.

              1% salt                               5% salt

           + 4 organisms                   + 4 organisms

           10% salt                                    15% salt

      + 4 organisms                           + 4 organisms

You will then put your plates in the 37 degrees C incubator.  In the next lab period, you will record the growth.  You will record your results in the table from the document called lab 7 report sheet.

Temperature experiment

In this part of the experiment, we are looking at the effectiveness of heat in controlling the growth of bacteria.  You will be determining the thermal death time (TDT), which is the time required to kill all bacteria in a liquid culture at a given temperature.

In this experiment, you will take 2 agar plates and divide them into 5 quadrants as shown below.  Your instructor will assign you 2 organisms.  On one plate, you will streak organisms that were heated at 63 degrees C for the indicated time and the other plate, you will streak organisms that were heated at 72 degrees C for the indicated time.

The table below shows which two organisms each group will work with.  These numbers will be assigned by your instructor.  Today, you will streak the plates as explained above.  In the next lab period, you will record the growth in the table from the document called lab 7 report sheet.

                                      63 degrees C                                           

                                       72 degrees C                                          

More specifically, these are the steps that you should follow in this experiment:

1. Get two plates and label them as above.
2. Get your two cultures.
3. Using a loop, streak each culture in the quadrant on the plate that says 0.  Make sure to use the asceptic technique because you will need to open and close this tube many times.
4. Place your cultures at the assigned temperature for 15 seconds.
5. Using a loop, streak each culture in the quadrant on the plate that says 15. 
6. Place your cultures at the assigned temperature for 2 minutes.
7. Using a loop, streak each culture in the quadrant on the plate that says 2 minutes. 
8. Place your cultures at the assigned temperature for 5 minutes.
9. Using a loop, streak each culture in the quadrant on the plate that says 5 minutes. 
10. Place your cultures at the assigned temperature for 15 minutes.
11. Using a loop, streak each culture in the quadrant on the plate that says 15 minutes. 
12. When you are finished, place your plates in the 37 degree C incubator.

In the next lab class, you will record the growth of the bacteria in the table from the document called lab 7 report sheet.

                                       72 degrees C                                          

More specifically, these are the steps that you should follow in this experiment:

1. Get two plates and label them as above.
2. Get your two cultures.
3. Using a loop, streak each culture in the quadrant on the plate that says 0.  Make sure to use the asceptic technique because you will need to open and close this tube many times.
4. Place your cultures at the assigned temperature for 15 seconds.
5. Using a loop, streak each culture in the quadrant on the plate that says 15. 
6. Place your cultures at the assigned temperature for 2 minutes.
7. Using a loop, streak each culture in the quadrant on the plate that says 2 minutes. 
8. Place your cultures at the assigned temperature for 5 minutes.
9. Using a loop, streak each culture in the quadrant on the plate that says 5 minutes. 
10. Place your cultures at the assigned temperature for 15 minutes.
11. Using a loop, streak each culture in the quadrant on the plate that says 15 minutes. 
12. When you are finished, place your plates in the 37 degree C incubator.

In the next lab class, you will record the growth of the bacteria in the table from the document called lab 7 report sheet.