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Environmental Influences: Osmotic Pressure

Background and Introduction:

Osmotic pressure is the force water exerts on the semi-permeable membrane (plasma membrane) surrounding the cell.  Water moves across the plasma membrane in response to an unequal distribution of dissolved solutes in the environment.  When a microorganism is placed into a hypotonic environment/medium in which the concentration of solute is lower outside the cell than inside the cell, water flows into the cell.  If this influx of water is uncontrolled, the cell may eventually burst.  Most bacteria, algae and fungi have rigid cell walls that allow them to tolerate and even enjoy a somewhat hypotonic environment.  Many microorganisms keep the solute concentration slightly higher in their protoplasm by synthesizing solutes that are compatible with their metabolism and growth, thus keeping their membrane pushed tightly against the cell wall.  By contrast, when microorganisms are placed into a hypertonic environment where the concentration of solute is higher outside the cell, water is lost from the cell resulting in dehydration, shrinkage of the plasma membrane and eventual death.  This is a process known as plasmolysis. 

As can be seen, the osmotic concentration of the environment affects the water that is available to a microorganism.  The greater the solute concentration of the environment, the less readily available the water.  Some prokaryotes can maintain the availability of water in environments with high solute concentrations (hypertonic environments) by increasing the solute concentration within the cell.  Microorganisms that can do this and thus tolerate hypertonic environments are osmotolerant.  Osmotolerant bacteria, such as Staphylococcus aureus can grow in a wide variety of environments with varying osmotic pressures.  In fact this bacteria can be cultured in media containing sodium chloride (NaCl) concentrations as high as 3M. 

Some bacteria specifically require an environment with a high concentration of sodium chloride.  These organisms are called halophiles. 

Procedure 

Osmotic Pressure Procedure (NaCl)

All four organisms will be tested for growth on media containing varying concentrations of NaCl: 0.5%, 7.5%, 20%, and 25%. 

Test Organisms: 

Saccharomyces cerevisiae 
Staphylococcus aureus 
Escherichia coli 
Halobacterium salinarium 

Collect 1 of each of the following plates: 
TSA + 0.5% NaCl 
TSA + 7.5% NaCl (=Mannitol Salt Agar, Red) 
TSA + 20% NaCl 
TSA + 25% NaCl 
2. On the bottom of the plates, use a marker to divide them into 4 quadrants as shown below. 
Label each quadrant with one of the test microorganisms. Also label the bottom of the plates with your initials, lab section, and the NaCl concentration of the plate.  There will be a total of 4 plates. 

diagram of plate setup

3. Follow the procedure for Inoculating organisms into four quadrants. 
4. Repeat the procedure for all four organisms.  Streak them in their appropriately labeled quadrants. 
5. Repeat the inoculation procedure, as described in the video, for the remaining 3 plates.  
6. Invert and incubate the plates at 37˚C for 24 - 48 hours. 

 

Osmotic Pressure Procedure (Sucrose): 

All four organisms will be tested for growth on medium containing varying concentrations of sucrose: 0.5%, 10%, 25%, and 50%. 


Test Organisms: 
Saccharomyces cerevisiae 
Staphylococcus aureus 
Escherichia coli 
Halobacterium salinarium 


1. Collect 1 of each of the following plates: 
TSA + 0.5% sucrose 
TSA + 10% sucrose 
TSA + 25% sucrose 
TSA + 50% sucrose 
2. On the bottom of the plates, use a marker to divide them into 4 quadrants as shown below. 
Label each quadrant with one of the test microorganisms. Also label the bottom of the plates with your initials, lab section, and the sucrose concentration of the plate.  There will be a total of 4 plates. 

diagram of plate setup

3. Follow the procedure for Inoculating organisms into four quadrants. 
4. Repeat the procedure for all four organisms.  Streak them in their appropriately labeled quadrants. 
5. Repeat the inoculation procedure, as described in the video, for the remaining 3 plates.  
6. Invert and incubate the plates at 37˚C for 24 - 48 hours. 

Results

NaCl

Observe and compare the growth of each organism on 0.5% , 7.5%, 20%, 25% NaCl

growth plate at 0.5 NaClgrowth plate at 7.5 NaClgrowth plate at 20 NaClgrowth plate at 25 NaCl

Sucrose

Observe and compare the growth of each organism on 0.5% , 10%, 20%, 50% Sucrose.

growth plate at 0.5 Sucrosegrowth plate at 10 Sucrosegrowth plate at 20 Sucrosegrowth plate at 50 Sucrose

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