MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #8: EFFECTS OF CHEMICAL AGENTS AND ANTIBIOTICS ON BACTERIA.

NAME, ID #:_______________________________________________

TA Name __________________________

REVISED: 08/04/99

INTRODUCTION

.
Figure 1. A bacterium that produces an antibiotic that inhibits the growth of another bacteria.  Antibiotics are discovered by looking for microbes that produce something that inhibits other microbes.

We live in a "germ conscious society". We are constantly reminded of dangerous GERMS lurking in the dark corners of our homes, and in the nooks and crannies of our bodies. These GERMS threaten us with all sorts of terrible social consequences such as BAD BREATH, DIRTY TOILETS, WASHING MACHINE ODORS, and UNDERARM FRAGRANCE! A relative of mine was so concerned with this problem that she and her husband could not travel longer than their "clean underwear" would last because she couldn't bring herself to use a washing machine in which someone else's underwear had been washed. Numerous household products are touted for their efficient "germ killing" ability; the claims are such that one wonders how any microbes still survive on this "disinfected-planet". While the above "social problems" may, indeed, injure your social life, they are rarely life-threatening, our real concern should be determining what actions will protect us from seriously harmfully microbes; like those that kill and maim you. In this exercise we will test some of the "germ-killing" products to see just how effective they are. We will also see how the doctors and hospitals determine which antibiotic to use to treat your bacterial infections.

ANTISEPTICS are substances that are commonly applied to the skin, whereas DISINFECTANTS are substances usually employed to kill microbes on inanimate objects (floor, table, sink). For example, alcohol & iodine are antiseptics often applied to the site of an injection, whereas chlorine-bleach is a disinfectant for the laundry, floor, toilet, sewage or the swimming pool. ANTIBIOTICS are chemicals that are produced by the BIOCHEMICAL ACTIVITY of living organisms that KILL OTHER LIVING ORGANISMS. Antibiotics are more specific and limited in the types of organisms that they kill than are antiseptics & disinfectants. For example, a given antibiotic will generally be effective in killing only some bacterial species, whereas antiseptics/disinfectants are lethal to a wide range of microbes.

Since science demands quantification, it is necessary to assess the range and efficacy of various antimicrobial agents. One way of doing this is to place paper disks, soaked with the substance in question, on media covered with the microbe being tested in a petri dish. The petri dish is then incubated and the subsequent growth of the test species around the soaked disks is assessed. If the chemical agent being tested INHIBITS the test microbe there will be a CLEAR ZONE of INHIBITION (ZI) surrounding the disk where no microbial growth has occurred due to the presence of the agent. In general the larger the diameter of the ZI, the more effective the test chemical is. Conversely, if the bacteria grow right up to the disk, the chemical is considered HARMLESS, but it may be lethal to other microbes. This procedure has been standardized in the case of antibiotics and other antiseptics and is the most commonly used method of assessing the efficacy of antibiotics and other antimicrobial agents against microbes. If you develop a bacterial infection the clinician will isolate the bacteria, spread it on an agar plate and place of series of disks soaked in various antibiotics on the plate. Following incubation the plate will be examined for the presence of ZI around the disks. The doctor then decides which antibiotic to use to treat your infection. Newer techniques, utilizing our knowledge of the genes responsible for producing diseases, are coming into usage which will allow the clinician to both identify the infecting bacterium and to determine its antibiotic susceptibility within a few minutes. This will allow the more timely application of the correct treatment measures and will result in the saving of many more lives.

In this exercise, you will employ this simple, but effective procedure to test several antimicrobial agents against some bacteria.

PURPOSE OF LABORATORY:

  1. To illustrate the inhibition of bacteria by a variety of agents.
  2. To demonstrate the quantification of antibiotic efficacy.
  3. To demonstrate how a physician or laboratory technician determines which antibiotic to use against an infectious bacterium.

RELATIONSHIP TO LECTURE MATERIAL

GENERAL INSTRUCTIONS:

  1. Some of the materials used in this exercise are irritants or even toxic if gotten into the mouth or eyes or if left on the skin too long. Please follow the Instructor's directions for handling these materials safely.

PROCEDURE

ANTISEPTICS/DISINFECTANTS/SELECTED MISCELLANEOUS GERM-KILLING PRODUCTS.

  1. Obtain the indicated number of nutrient agar plates and sterile swabs.
  2. Using a sterile cotton swab, spread each of the test bacteria evenly over the surface of the medium as demonstrated by the instructor and allow the excess liquid to soak in. Label the plates with your name and date.
  3. Allow the plates to dry at room temperature for 10 to 15 min, until the excess liquid is gone.
  4. Divide the bottom of the plate into the number of sectors indicated by the instructor and label each sector as to which sample will be placed there.
  5. The instructor will demonstrate how to apply the antiseptics/disinfectants etc. to the paper disks and how to lay the disks on the plate. Incubate in the appropriate incubators.
  6. At the following lab measure the zone-of-inhibition (ZI) diameters and draw the results in the circles below.

EVALUATION OF DISINFECTANTS (NEXT LAB)

  1. Record the ZI of each of the disinfectants used in the table you prepare below.
  2. Evaluate the efficacy of each disinfectant in eliminating microbes.

EFFECT OF ANTIBIOTICS

  1. Read pg. 93-4 in A Photographic Atlas for the Microbiology Laboratory before beginning this procedure.
  2. Obtain the designated plate and swab it with ONE of the bacterial cultures as described above. The Instructor may decide to have 1/2 of the class do one & the other 1/2 the other.
  3. Allow the plates to dry at room temperature for 10 to 15 min, until the excess liquid is gone.
  4. Using forceps sterilized as demonstrated by the instructor, drop the antibiotic disk onto the agar surface as labeled. Alternatively you may use and automatic dispenser to apply the disks. The disks should be 3 cm apart and 2 cm from the edge of the plate. The new (improved versions) also press the disks onto the agar.
  5. Press the disks down with a sterile loop to "plant" them on the agar and to make effective contact.
  6. Incubate the plates upside down at 37oC for approximately 18 hours. The TA will remove them and store them in the cold room until the next lab period.
  7. Measure the diameter of clear ZI around the disks and and draw them in the circle below.
  8. From the chart at the front desk determine the susceptibility of the two bacteria to each of the antibiotics tested. Prepare a chart relating the antibiotic to ZI diameter and susceptibility of the two bacteria tested. Show this to the instructor. Compare your results with those shown in the Atlas and at this site.

DATA TABULATION

Aseptics and Disinfectants:

Draw a table to the right of the circles. In the first column list numbers; in the second column (AGENTS) list the agent tested opposite a number; in the 3rd/4th & 5th/6th columns label the headings with the bacteria and in the 3rd and 5th columns record the diameters of the ZI surrounding each sample next to the appropriate agent; in the 4th and 6th columns rate the efficacy of each agent against the test bacteria using a scale of 0 = no effect to 5+ = very effective.

Antibiotic Susceptibility:

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

Copyright Dr. Ronald E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
SCIENCE HALL, ROOM 440CA
PHONE: 509-335-5108
FAX: 509-335-1907
E-mail address: hurlbert@wsu.edu
Phone: 509-335-5108
Office Hours: Mon. Wed. 2-4 PM