Combinatorial Chemistry Lab C - Conducting a Kirby-Bauer Analysis

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Objective

Students will test mixtures for antibiotic activity, isolate the individual compound(s) which possess antibiotic properties and screen the mixtures by utilizing techniques used to conduct a Kirby-Bauer Assay.

Big Idea

The Kirby Bauer Assay ability to both identify new potential antimicrobial compounds and identify the presence of antibiotic resistance makes it a valuable research tool.

Introduction

10 minutes

Combinatorial Chemistry is a technique used to synthesize a library of compounds and screen for a desired property. Instead of screening one compound at a time, the compounds are screened more efficiently in mixtures which maximize the use of resources while increasing the possibility of identifying an active compound.  This is a key goal of the pharmaceutical industry and thus a common practice in drug discovery.

This lab protocol was adapted from the original work of Scott Wolkenberg and Andrew Su of The Scripps Research Institute (TSRI) in La Jolla, California. The experiment was originally published in the June 2001 issue of the Journal of Chemical Education (Wolkenberg, Scott E.; Su, Andrew I. J Chem.Educ. 2001 78 784) and implemented in the San Diego area from May 2002 thru 2005.  Through collaboration between the Southern California Biotechnology Center at Miramar College, Junipero Serra High School Biotechnology and Dr. Floyd Romesberg, an expert in the area of antibiotic resistance and professor of chemistry at TSRI, the protocol was revised for implementation in a high school setting.

Revision by: Jasmine Erfe Miramar College/Lab Technician, Ericka Senegar-Mitchell Science in the City/Director; Junipero Serra High School/Science Educator, Sandra Slivka Southern CA Biotechnology Center @Miramar College/Director

Objective(s):

  • Students will successfully perform mathematical calculations required to prepare the media, reagents, and compounds needed for the research and development of a novel drug therapy based on techniques used in the field of Combinatorial Chemistry. (Lab A)
  • Students will identify a drug that kills bacteria by producing libraries of compounds based on the A-B model. (Lab B)
  • Students will test the mixtures for antibiotic activity and then isolate the individual compound(s) which possess antibiotic properties. (Lab C)
  • Students will screen the mixtures by utilizing techniques used to conduct Kirby-Bauer and Ouchterlony tests. (Lab C)

 

NOTE: Instructor's are encouraged to complete the pre-lab activities found in the lesson entitled, "Combinatorial Chemistry - Designing Novel Drug Therapies to Combat Antibiotic Resistance" before completing this laboratory investigation series.

Materials

10 minutes

For each Student Lab Group:

The following supplies should be provided to each student GROUP: 

- 3 LB agar plates

- Nine prepared mixtures (M1, M2, M3, M4, M5, M6, A#B1, A#B2, A#B3)

- 1 cryotube (orange cap) containing 1.0 mL E. coli

- 1 cell spreader

- 15 transfer pipettes (6 will be used to prepare compounds, 9 will be used to transfer compounds onto wells and 1 will be used to transfer the E. coli culture onto the plates)

- 1 sterile wrapped transfer pipette

- 9 eppendorf tubes

- 1 plastic straw, wrapped

- Conical tube rack

- Sharpie Marker

Methods

45 minutes

For each Student Lab Group:

Preparing LB Agar Plates for Kirby-Bauer Assay

1. Using a Sharpie, divide each bottom of the plate into three sections and label each section as shown in the student protocol (Be sure to keep the cover on the plate while doing so). In addition, make sure to uniquely label your group's plates (i.e. initials, class period).
2. Unwrap the straw and tie a knot in one end.  Open the plate slightly and use the straw bore out a well in the middle of each section.  Slowly lift the straw and a circular piece of agar should be in lodged inside the straw. If not, it may help to lift the straw at an angle. Dispose the circular pieces of agar and cover the plates. 
3. While wearing gloves, carefully invert the tube containing 1.0 ml of thawed E. coli several times before opening.

4. Using the sterile wrapped transfer pipette, place 6-7 drops of E. coli on the middle of each plate. Dispose the transfer pipette and E. coli tube in the biohazard waste.

5. Use the cell spreader, to evenly distribute the E. coli culture on the surface of each agar plate. Some of the bacterial culture may get into the wells which will not pose a problem.  It is important that the entire surface of the agar plate is covered with the E. coli culture.

6. Dispose of the cell spreader in the biohazard waste.

7. Cover each plate and set aside until ready to use.

 

Transferring the Library Compounds to the LB Agar Plates

1. Using a new, properly labeled transfer pipette, transfer 2 drops of each compound to the corresponding well. For example, use the transfer pipette labeled M1 to take a sample of solution in the tube labeled M1.

2. Then, slightly open the plate containing the well labeled M1 and carefully and slowly add two drops of the solution in the well.

3. Follow the same procedure until all wells have been filled with the appropriate compound. 

Results

15 minutes

DATA ANALYSIS

For each Student Lab Group:

1. Using the table below, record the results of the experiment.  Place a "+" in the table for mixtures that resulted in an inhibition of growth.  Place a "-" in the table for mixtures that resulted in no inhibition of growth.

Mixture

Contents

Result

M1

A1, B1, B2, B3

 

M2

A2, B1, B2, B3

 

M3

A3, B1, B2, B3

 

M4

B1, A1, A2, A3

 

M5

B2, A1, A2, A3

 

M6

B3, A1, A2, A3

 

 

2. Each mixture contains three compounds.  For instance, M1 contains the compounds A1-B1, A1- B2, and A1-B3.  Therefore, in the mixtures which show inhibition of growth (antibiotic activity) there are three compounds.  How can we tell which of the three compounds is the one with the antibiotic activity?  In fact, we have already done the experiments needed to make this determination.

3. The table below enables us to determine all the compounds we made.  The outside of the table shows the starting materials for the reactions we performed:  A1, A2, A3, B1, B2, and B3.  Whenever an A molecule comes in contact with a B molecule they react to form A-B molecules.  We can see this in the table by looking at the inside:  when we trace A1 down in the first column, we see that it forms A1-B1, A1- B2, and A1-B3 molecules.  Fill in the rest of the table in the same way by tracing across and down.

 

A1

A2

A3

B1

A1-B1

A2-B1

A3-B1

B2

A1-B2

 

 

B3

A1-B3

 

 

4. The inside of the table represents the contents of each mixture M1-M6.  For instance, the mixture M1 is represented by the first column in the table (see the shading in the left table).  Likewise, the second and third columns represent M2 and M3.  Mixture M4 is represented by the first row in the table.  Similarly, M5 and M6 are represented by the second and third rows of the table.

5. You can use this table to determine which compound in our active mixtures shows antibiotic activity.  Fill in the table below as you did in part 2.  Shade in the column that corresponds to the mixture M1-M3 that shows antibiotic activity.  Now shade in the row that corresponds to the mixture M4-M6 that shows antibiotic activity.  The position in the table where the shaded row and shaded column intersect is the active compound.

Discussion

30 minutes

In a whole group discussion pose the following inquiries:

1. What is the advantage to working with mixtures? 

2. How many reactions would we need to run in order to make each compound individually? 

3. How many antibiotic activity tests would we need to run in order to screen each compound individually?  Compare these numbers with the number of reactions and antibiotic activity screens we ran in this experiment.


EXTENSION ACTIVITY - KIRBY-BAUER TEST

Determining Class Average Zone Size

1. After 24 hours in the incubator or 48 hours at room temperature check for the presence of antibiotic activity. This is done by looking for a clear area, called a zone of inhibition, surrounding a well. Remember to never open the Petri dish for a better view. Use the agar side to observe and measure any zone of inhibition.

2. Using a ruler, measure the diameter (in millimeters) of any zone of inhibition and record your individual group data on the data table below.
 
3. Now gather class data to determine the average zones of inhibition for each of the different compounds M1 thru M6 which display a zone and record the averages.
 
4. After recording the average class data for the diameters of the zones of inhibition, decide whether your groups sample of E.coli bacteria is susceptible (sensitive) displaying a CLEAR zone of inhibition, unaffected (resistant) showing no observable zone of inhibition or intermediate (somewhere in between) for each of the compounds M1 thru M6. Record your conclusion in the final column of the data table.

MIXTURE

DIAMETER OF ZONE (mm)

CLASS AVERAGE OF ZONE (mm)

S=susceptible(sensitive)

R=resistant(unaffected)

 I=intermediate

 

 

 

 

 

 

 

 

 

Submission of the Final Group Lab Report

Group Lab Report A and Lab Report B are examples of lab reports completed by students. At this point students should need to complete the final sections of the final group lab report: data, results, and conclusion. In order to observe how different student groups approached these sections review pages 4-6 of Lab Report A and pages 5-15 of Lab Report B.