Introduction:
The use of antimicrobial agents to treat infections began in the early 1900’s, when Paul Ehrlich developed Salvarsan to treat individuals infected with Treponema pallidum, the spirochete that causes syphilis. In 1928, Alexander Fleming later observed that the Penicillium mold growing on his agar plates could inhibit the growth of bacteria: years later penicillin was purified and used to treat many types of infections. Since this time, many other antimicrobial agents have been used to treat a wide variety of bacterial infections. Antibiotic producers include many types of fungi (Penicillium, Cephalosporium) and bacteria (Bacillus, Streptomyces). In addition, many antimicrobial agents currently used to treat infections are either synthetic (made in a laboratory) or semi-synthetic (a modification of a naturally-produced antibiotic). Today there are over 100 different antimicrobials that are used to treat infectious diseases. These include broad-spectrum and narrow-spectrum antimicrobial drugs. Narrow-spectrum drugs are more desirable to use whenever possible because they target the pathogen more specifically and do less damage to the normal microbiota; broad-spectrum drugs are used when the cause of the infection is unknown or when other antibiotics are not effective.


It is important to remember that not all antibiotics are effective at killing all types of bacteria. Bacteria may have intrinsic resistance to a particular antibiotic. For example, gram negative bacteria are intrinsically resistant to vancomycin because the drug cannot penetrate the outer membrane of the gram-negative cell wall. Also, the misuse and overuse of antibiotics has led to the evolution of resistance among bacteria by selecting for individual cells within a population that are not affected by the drug. This acquired resistance can occur in several ways, including through transformation, conjugation and mutation. Antibiotic-resistant bacteria have become a major problem of growing concern in health care, as it is often difficult (or impossible) to treat bacterial infections caused by these microbes (for example, multidrug-resistant Staphylococcus aureus, or MRSA). Therefore, clinical isolates are often tested for their antibiotic susceptibility in a laboratory setting so that health care providers can choose an appropriate drug to treat a particular infection.

There are several ways to determine antibiotic susceptibility in a laboratory setting—one common test is called the Kirby-Bauer method. This method is similar to the filter paper disk method used to test disinfectants, except that it uses filter paper disks impregnated with a known concentration of an antimicrobial compound. It also uses Mueller-Hinton agar, and is often performed with larger (150 mm) petri dishes that allow for the testing of several antibiotics simultaneously. When performing the Kirby-Bauer method, it is important to measure the size of the zones of inhibition and compare them to a set of standardized values established by the Clinical Laboratory Standards Institute (CLSI).

In this lab you will set up experiments to evaluate the effectiveness of several antibiotics.

Recall from the Chemical Control lab: One way to evaluate the effectiveness of antibiotics is to inoculate an agar plate with a lawn of bacteria and add filter paper disks that have been moistened with the disinfectant being tested. This is known as the filter paper disk method, or agar disk diffusion assay. After incubation, plates are observed for the presence of a zone of inhibition (area around a disk where no microbial growth is detected). Generally speaking, the larger the zone, the more effective the disinfectant is against that particular microbe. However, other factors such as the solubility of the test agent and the molecular weight of the disinfectant molecules (which determines the diffusion rate of the disinfectant through the agar) can also affect results.

Figure 1. Filter paper disk method experiment showing zones of inhibition

Procedure: 

Materials for each table: large (150 mm) Mueller-Hinton agar plates, liquid cultures of instructor selected bacteria, sterile cotton swabs, antibiotic disk dispensers with antibiotic discs

Work as a table for this exercise. Use a sterile cotton swab to inoculate the Mueller-Hinton agar plates with a lawn of bacteria for each species listed (one species/plate). Be sure to cover the entire area of the plate. This can best be accomplished by swabbing the entire surface of the plate three times, rotating the plate approximately 60° each time, and finishing the swabbing by going around the entire outer surface of the plate.

After all plates are inoculated, your instructor will show you how to use the disk dispenser to introduce antibiotic disks onto the plates. Be careful to always keep the dispensers in the upright position.

You will notice that each disk that is introduced to the plate is stamped with a letter and a number—these indicate the type of antibiotic as well as the concentration. For example, P10 is an abbreviation for 10 units of penicillin; PIP-100 is an abbreviation for 100 mcg (micrograms) of piperacillin.

Return to your lab table, and use your inoculation loop or needle to gently push down on the disks to ensure that they are adhered to the agar surface.

NOTE: Flame your loop in between plates to avoid cross-contamination.

Incubate the Mueller-Hinton agar plates (inverted) until the next lab period.

For each plate, measure zones of inhibition for 3-4 antibiotics as follows:

With a metric ruler, measure the diameter of the zone of inhibition. Express the values in millimeters (mm). It is best to choose some small, medium-sized, and large zones for comparison.

Use the charts provided by your instructor to look up the interpretation for each of the zones you measured. Record measurements and interpretations 

                           in today's lab
  we will assess the growth of four different bacterial species            exposed to incubation under different temperatures.
 question:
    what is the optimal growth temperature for each of these bacterial species?
 hypothesis:
    your educated guess using this information..
   
  1. M. luteus is a mesophile
  2. S. marcescens is a psychrotolerant mesophile 
  3. E. coli is a mesophile
​  4. B. stearothermophilus is a thermophile

what is the purpose of the disc diffusion method?

review question:

What was the purpose of the lab where we exposed 4 different bacterial strains to 4 different temperatures? 

the agar plate we use is covered by a lawn of bacteria.
a lawn of bacteria completely covers the surface of an agar plate.
it is usually created by spreading a suspension of bacteria evenly over the plate using a sterile spreader. 

autoclave uses
moist heat sterilization:
steam generated under pressure kills microbes

15 lbs per inch *2

​121.5 degrees C

 disc diffusion method
(Kirby-Bauer method)
 tests the effectiveness of disinfectants and antiseptics by placing paper discs soaked in the test agent on an agar plate inoculated with bacteria.

a zone of inhibition
around the disc indicates the agent's effectiveness

Procedure: 

Materials for each table: large (150 mm) Mueller-Hinton agar plates, liquid cultures of instructor selected bacteria, sterile cotton swabs, antibiotic disk dispensers with antibiotic discs

Work as a table for this exercise. Use a sterile cotton swab to inoculate the Mueller-Hinton agar plates with a lawn of bacteria for each species listed (one species/plate). Be sure to cover the entire area of the plate. This can best be accomplished by swabbing the entire surface of the plate three times, rotating the plate approximately 60° each time, and finishing the swabbing by going around the entire outer surface of the plate.

After all plates are inoculated, your instructor will show you how to use the disk dispenser to introduce antibiotic disks onto the plates. Be careful to always keep the dispensers in the upright position.

You will notice that each disk that is introduced to the plate is stamped with a letter and a number—these indicate the type of antibiotic as well as the concentration. For example, P10 is an abbreviation for 10 units of penicillin; PIP-100 is an abbreviation for 100 mcg (micrograms) of piperacillin.

Return to your lab table, and use your inoculation loop or needle to gently push down on the disks to ensure that they are adhered to the agar surface.

NOTE: Flame your loop in between plates to avoid cross-contamination.

Incubate the Mueller-Hinton agar plates (inverted) until the next lab period.

For each plate, measure zones of inhibition for 3-4 antibiotics as follows:

With a metric ruler, measure the diameter of the zone of inhibition. Express the values in millimeters (mm). It is best to choose some small, medium-sized, and large zones for comparison.

Use the charts provided by your instructor to look up the interpretation for each of the zones you measured. Record measurements and interpretations 

 1. inoculation: an agar plate is inoculated with a lawn of the target bacteria. 
2. agent application: each disc is soaked with test disinfectant or antiseptic. 
3. disc placement: sterile filter paper discs are placed on the agar surface. 
4. incubation: the plate is incubated
5. zone of inhibition: a clear zone (zone of inhibition) around the disc indicates that the antimicrobial agent has inhibited the growth of the bacteria. 

what is the meant by the zone of inhibition? how does this zone relate to the effectiveness of a disinfectant/antiseptic?

microbial control

​using antibiotics