7: Environmental Conditions and Micropipetting
- Page ID
- 105494
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- Identify different components of a micropipet and their functions.
- Apply understanding of micropipets to accurately and precisely pipet small volumes.
- Identify important environmental growth conditions, including, temperature, pH, salinity, and oxygen requirements.
- Interpret microbial growth based on variations in environmental conditions.
Getting to Know Your Pipette:
1. Find the following parts on your pipette: 
- Volume adjustment dial
- Tip ejector button
- Plunger button
- Stainless steel micrometer
- Digital volume indicator
- Stainless steel ejector arm (removable)
- Plastic shaft
- Disposable yellow or blue tip
2. Practice holding your pipette correctly, placing a tip on our pipette, and ejected the tip. Do this at least three times.
3. The total volume a pipette can hold is stated on the top of the plunger. We will be working with the following three volume pipettes:
- P-20 0.02 μl – 20 μl
- P-200 20 μl – 200 μl
- P-1000 200 μl – 1000 μl
4. Based on the volume pipette you have the numbers in the digital display have a different meaning.
5. Rotate the volume adjustment knob until the digital indicator reaches the desired volume, then place a disposable tip on the shaft of the pipette (practice all three volumes min, int, max)
6. Press down on the plunger to the First Stop. (You will be able to push past this point, but there is enough resistance to stop the movement if you try to be aware of it.)
7. Hold the pipette vertically and immerse the disposable tip into the sample. Use the colored water and the microcentrifuge tubes provided to you.
8. Allow the plunger button to return slowly to its original position. Do not allow the button to snap up.
9. To dispense the sample: place the tip against the side wall of the receiving tube and push the plunger down to the first stop. Wait 2-3 seconds, then depress the plunger to the second stop in order to expel any residual sample in the tip.
10. While the plunger is still pushed down, remove the pipette from the tube and allow the plunger to slowly return to its original position.
11. Practice until you are ready and then call an instructor for your skills test.
Warning
- Never rotate the volume adjustment knob past the upper or lower range of the pipetman.
- Never lay the pipetman down on its side or hold it horizontally when it contains liquid.
- Never immerse the shaft of the pipetman into the fluid.
Environmental Requirements (Temperature)
How does temperature affect bacterial growth?
Organisms grow best over a certain temperature range, and this range has restrictions. The cardinal temperatures are the range of temperatures over which an organism can grow. Every organism has evolved to live at a particular optimum temperature.
- Minimum: lowest temp where reproduction occurs
- Maximum: highest temp where reproduction occurs
- Optimum: highest rate of reproduction
Organisms are classified based on the temperature ranges they live in:
- Psychrophiles: less than zero
- Psychrotrophs: 0-30°C
- Mesophiles: middle temperatures 15-45°C
- Thermophiles: 40-80°C
- Extreme Thermophiles: above 65°C
WHAT ARE THE CARDINAL TEMPERATURES OF 2 DIFFERENT ORGANISMS?
1. Working as a table you will need 6 Tryptic Soy Broth (TSB) tubes.
2. Label your broth tubes with the bacterial species (2 species) and temperature (room temperature, 37°C, 4°C).
- Escherichia coli (3 broth tubes)
- Bacillus subtilis (3 broth tubes)
3. Mix each broth culture before using by gently tapping on the tube.
4. Using aseptic technique, use a sterile pipette to transfer 20 μl of each organism to appropriate test tube of broth.
Note
If you are not consistent with the volume you inoculate, your results will be undesirable.
5. When your table is done, place your tubes in the appropriate bins for the different temperatures tested.
Environmental Requirements (pH)
How does pH affect bacterial growth?
Hydrogen ions in a solution = pH. Organisms grow best at a specific pH range based, in part, on the environment they have evolved to live in. If bacteria are outside their optimal pH range their proteins can become denatured. Ranges of pH over which an organism can live place them in groups:
- Acidophiles: below pH 5.5
- Neutrophiles: pH 5.5 -8.5
- Alkaliphiles: pH above 8.5
WHAT ARE THE pH RANGES OF 2 DIFFERENT ORGANISMS?
1. Working as a table you will need 6 TSB tubes of the following:
- 2 tubes of acidic media (TSB + HCL)
- 2 tubes of neutral media (TSB)
- 2 tubes of basic media (TSB + NaOH)
2. Label your broth tubes with the bacterial species (2 species) and pH (3 pH) = 6 tubes
- Escherichia coli (3 broth tubes)
- Bacillus subtilis (3 broth tubes)
3. Mix the culture before using by gently tapping on the tube.
4. Using aseptic technique, use a sterile pipette to transfer 20 μl of each organism to appropriate test tube of broth.
5. Place all inoculated tubes in the provided racks at the end of your table. When your table is done, place your tubes in the 37°C bin.
Environmental Requirements (Salinity)
How does osmotic pressure affect bacterial growth?
Water is essential to all organisms. The ability to control the movement of water across a membrane is necessary for the survival of all cells. Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a semi-permeable membrane. The movement of water is controlled by the concentration of solutes contained within the water (usually salt). Bacteria can be classified based upon the salinity they can tolerate:
- Halophiles (prefer NaCl concentrations of 3% or higher)
- Extreme halophiles (prefer NaCl concentrations of 15%-25%)
- Xerophile (prefer low salt concentrations)
WHAT ARE THE PREFERRED SALINITY RANGES OF 2 DIFFERENT ORGANISMS?
1. Working as a table you will need 6 TSB broths of the following salinities:
- 2 tubes of NaCl 2.5%
- 2 tubes of NaCl 15%
- 2 tubes of NaCl 25%
2. Label your broth tubes with the bacterial species (2 species) and salinity (3 concentrations) = 6 tubes:
- Escherichia coli (3 broth tubes)
- Bacillus subtilis (3 broth tubes)
3. Mix the culture before using by gently tapping on the tube.
4. Using aseptic technique, use a sterile pipette to transfer 20 μl of each organism to appropriate test tube of broth.
5. Incubate at at 37°C.
Environmental Requirements (Aerotolerance)
What is aerotolerance and how does it affect bacterial growth?
Bacteria can differ dramatically in their ability to utilize oxygen (O2). Under aerobic conditions, oxygen acts as the final electron acceptor for the electron transport chain located in the plasma membrane of prokaryotes. Bacteria use this process to generate ATP, the energy source for most cellular processes. In the absence of oxygen (O2), some bacteria can use alternative metabolic pathways including anaerobic respiration and/or fermentation. During anaerobic respiration, other alternative molecules are used as the final electron acceptor for the electron transport chain such as nitrate (NO3), sulfate (SO4), and carbonate (CO3).
WHAT IS THE AEROTOLERANCE OF TWO DIFFERENT SPECIES OF BACTERIA?
1. Working as a table you will need 2 TSA plates per microbe (one for aerobic conditions, another for anaerobic conditions.
2. You will be using the following two bacterial species:
- Escherichia coli (2 TSA plates)
- Bacillus subtilis (2 TSA plates)
3. Divide both plates into two sections and aseptically transfer each bacteria species to a section, use your loop to draw a circle instead of streaking.
4. One plate will be incubated under anaerobic conditions and one plate will just go directly in the incubator at 37°C (aerobic conditions).
Results:
Observations
Using the method indicated by your professor, quantify the # of bacterial cells/mL for each condition.
| Bacterial Species | Temperature | pH | Salinity | Aerotolerance | |||||||||||
| Escherichia coli |
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| Bacillus subtilis |
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Interpretations:
Briefly summarize the preferred environmental requirements for each of the microorganisms studied today. Your summary should include all of the conditions tested.
LAB ASSIGNMENT
Answer the following questions based on information and findings from this lab.
1. If a given microorganism is a mesophile, how will high temperatures affect it's growth? What about low temperatures?
2. If a given microorganism is an acidophile, how will high pH affect it's growth? What about low ph?
3. What would happen if a microorganism was grown in an isotonic solution? Hypotonic? Hypertonic?
4. Explain each of the oxygen metabolisms discussed in the lab. Be sure to include how growth in the presence of oxygen would affect each type of metabolism.
5. Were there any results that did not align with the expected outcome? In other words, did anything unexpected happen with your experiments? Explain.