Antimicrobial Agent Susceptibility Testing and Resistance 1. What is meant by antimicrobial resistance? Susceptibility?
2. Why are pure cultures used for antimicrobial susceptibility testing?
3. Would it be acceptable to use a mixed culture for this test? Why?
4. List three factors that can influence the accuracy of the test.
5. When performing a broth dilution test, why is it necessary to include a growth control tube? A sterility control tube?
References...
Antimicrobial Agent Susceptibility Testing and Resistance 1. What is meant by antimicrobial resistance? Susceptibility?
2. Why are pure cultures used for antimicrobial susceptibility testing?
3. Would it be acceptable to use a mixed culture for this test? Why?
4. List three factors that can influence the accuracy of the test.
5. When performing a broth dilution test, why is it necessary to include a growth control tube? A sterility control tube?
References...
Related textbook solutions
Related questions
yeast population dynamics
Procedure
1. Work in pairs on this lab, so 12 tubes per pair of students. And share a tube rack with one other pair of students
2. Turn on your spectrophotometer. It needs at least 15 minutes to warm up to give you good readings.
3. Add 5 mL of yeast extract solution (YECM) to each of 12 tubes. (The yeast extract provides vitamins and amino acids for yeast growth and will be the same for all cultures). The tubes should be labeled with your initials, treatment, and tube number. Tape or Parafilm down the lids of 3 tubes, and label them âCONTROLâ.
Do not touch the insides of the tubes or lids! Try to keep these as sterile as possible!!
4. Add 50 mL live yeast culture to each of the remaining 9 tubes.
5. Add the varying volumes of sugar and/or ethanol using Table 1 below.
6. Use Parafilm to close the tops of each tube, making sure the Parafilm is tight and no air can get in, and label each tube with the following:
Amount of sugar added (mL) Amount of ethanol added (mL)
Name of your group Tube number
| Table 1: setup yeast tubes (remember, 1 mL = 1000 mL) | ||||
| Tube number | Yeast culture medium? (5 mL) | Live yeast culture? (50 mL) | Sugar added (mL) | Ethanol added (mL) |
| 1 â control | YES | NO | 0 | 0 |
| 2 â control | YES | NO | 0 | 0 |
| 3 - control | YES | NO | 0 | 0 |
| 4 | YES | YES | 0 | 0 |
| 5 | YES | YES | 0.25 | 0 |
| 6 | YES | YES | 0.5 | 0 |
| 7 | YES | YES | 0 | 0.25 |
| 8 | YES | YES | 0.25 | 0.25 |
| 9 | YES | YES | 0.5 | 0.25 |
| 10 | YES | YES | 0 | 0.5 |
| 11 | YES | YES | 0.25 | 0.5 |
| 12 | YES | YES | 0.5 | 0.5 |
Procedure for measuring absorbance (in absorbance units, or AU)
7. Calibrate the spectrophotometer:
Turn on the spectrophotometer and let it warm up for 15 minutes. You will get erroneous results if you donât let it warm up first.
Be sure the spectrophotometer is set to read at the wavelength of 550 nm
With no tube in the spectrophotometer and the lid closed, use the left-hand knob to adjust the reading to 0% Transmittance/push zero button to calibrate
Insert a CONTROL tube (making sure it is clear, without bacterial contamination which would make it cloudy), and use the right-hand knob to readjust the spectrophotometer to 100% Transmittance.
When reading the absorbance, be sure to line up the needle on the spec with its reflection.
8. Immediately before reading any tube, vortex the tube so that the spinning column reaches the bottom of the tube for several seconds. This is critical! The yeast cells are heavy and will tend to sink to the bottom of the tube, so you must vortex the tubes to resuspend them: otherwise, your spectrophotometer readings will be erroneously low. If the vortex is not enough to suspend the pellet of yeast cells at the base of the tube, take a piece of Parafilm and cover the top of the tube, then cover this with your thumb and shake the tube vigorously. The pellet should dislodge and the yeast cells should be easily resuspended after doing this. Use a Kimwipe to wipe down the outside of each tube, to remove fingerprints and other smudges that could affect the absorbance reading. (COULD BE A POTENTIAL ERROR)
9. Record the absorbance (in absorbance units, AU) for the tube on your data sheet.
10. Repeat steps 5 and 6 for every tube.
11. Leave the spectrophotometer turned on for the next user.
Figures you should include are:
Average absorbance vs. time for the no ethanol (0 mL) treatment
Average absorbance vs. time for the 0.25 mL ethanol treatment
Average absorbance vs. time for the 0.50 mL ethanol treatment
Sugar added vs. average carrying capacity (K). Use different symbols to denote each of the three alcohol concentrations
A. After a limit, the increasing concentration of sugar decreases the carrying capacity and growth rate. This is because at higher sugar concentrations, the medium becomes hypertonic and the yeast cells loss water towards the medium.With increasing concentration of the ethanol, the carrying capacity and the growth rate decreases. Why does this happen?
B. Is there any interaction between the effects of adding sugar and alcohol on yeast?
C. why do some cultures not reach K?
D. What are the potential sources of error and assumptions made in this experiment?
E. What do these results mean in a more general (non-yeast) context?
Enzymes Lab Report
- Enzyme Structure and Function
- Define the following:
- enzyme
- substrate
- Explain how enzymes function.
- Go to Lab, Section I, Exercise 1 to participate in an activitythat demonstrates the importance of enzymes. Record the colorintensity of the solution in each test tube at five and tenminutes.
| Time (min) | Tube S1 Potato Extract + Catechol | Tube S2 Potato Extract + Water | Tube S3 Catechol + Water |
0 | |||
5 | |||
10 |
- How can benzoquinone be detected?
- Why were potatoes used in the experiment?
- Enzyme Specificity
- Based on Section II, Exercise 2, as determined by colorintensity, to what extent does catechol oxidase react withhydroquinone?
- What was the role of hydroquinone in the experiment?
- Temperature and Enzyme Activity
- Based on Lab, Section III, Exercise 3 record the relative colorintensity at five-minute intervals to determine the temperaturerange for catechol oxidase.
| Time (min) | Tube 1 4oC | Tube 2 23oC | Tube 3 40oC | Tube 4 60oC | Tube 5 80oC | Tube 6 100oC |
0 | ||||||
5 | ||||||
10 |
- Construct a bar graph of the data for the ten-minute readings.Label axes and provide units. Sign, date and prepare an image ofyour graph and include it with this lab report.
- What is the optimal temperature range for catechol oxidaseactivity?
- pH and Enzyme Activity
- Within what pH range will catechol oxidase catalyze catecholand oxygen to form benzoquinone?
- Define pH.
- What is the role of catechol in the experiment?
- Which test tube contains the most acidic buffer solution?
- Cofactors and Enzyme Activity
- What is a cofactor?
- What cofactor is necessary for catechol oxidase activity?
Summary Questions
- What was the role of catechol oxidase in the experiments?
- What was the purpose of developing a color intensityscale?
- Identify three factors that influence the rate of enzymaticreactions.
- Examine the data in Exercise 3 and suggest a hypothesisexplaining why people with a fever feel tired and listless.
- People who have been suddenly submerged in cold water andremained for long periods of time have survived. Examine the datain Exercise 3 and explain how this is possible.
- How does pH range affect the function and structure of catecholoxidase?
- The stomach maintains a pH of approximately 3 to 5. Do allenzymes function at this pH level? Examine the data in Exercise 4and determine if catechol oxidase would function in the stomach.
- What is enzyme specificity? Give an example.
