ANTIBIOTIC INHIBITION OF BACTERIA LAB BAC 2

INTRODUCTION Single-celled organisms were the first life on the planet. Bacteria have adapted and evolved over millions of years, resulting in the numerous varieties of bacteria that exist on the planet today. Most bacteria can be divided into two classes, gram-negative and gram-positive, based on a differential staining process called the Gram stain. The Gram stain separates bacteria based on the ability of the cell wall to retain crystal violet stain when decolorized by an organic solvent like ethanol. The differences in the cell wall also play an important role in the types of antibiotics that will be effective against them. The ability to retain Gram stain is based on the structure of the bacterial cell membrane. Gram-positive bacteria, which retain the Gram stain, have a membrane that is composed of two parts, the cell wall and the cytoplasmic membrane (Fig. 1). The cell wall is composed primarily of peptidoglycan, a complex of linked polysaccharide chains that provide strength and rigidity. This peptidoglycan layer also is responsible for the ability of the cell to retain the Gram stain Figure 1. Cell Wall of a Gram-positive Bacterium

Gram-negative bacteria have a cell wall that consists of an outer membrane, a periplasmic space and a cytoplasmic membrane. The peptidoglycan layer, found in the periplasmic

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Antibiotic Inhibition of Bacteria space, is much smaller, and there is no teichoic acid present. The outer membrane and cytoplasmic membrane are comprised of phospholipids. The outer membrane also contains lipopolysaccharides (LPS) and porins. The porins allow small molecules, like glucose, to diffuse through the outer membrane. A cell wall of this type does not retain the Gram stain. Figure 2. Cell Wall of a Gram- Bacterium

Many microorganisms produce chemicals, or antibiotics, which protect them from bacteria. The first of these defensive chemicals to be isolated was penicillin. In 1928, Alexander Fleming noticed that a mold growing on his bacterial culture produced a clear zone around it, as though the bacterial growth was inhibited right near the mold. Upon further inspection, he determined that the mold Penicillium notatum produced a diffusible chemical, named penicillin, which was lethal to several bacterial species. Since that time, numerous other antibiotics have been discovered and isolated, as is evident by the wide selection of antibiotics available to the medical community. Antibiotics act against bacteria in two different ways. Some are bacteriocidal, and are capable of killing the bacteria. Other antibiotics are bacteriostatic and only inhibit the growth of the bacteria. The effect of bacteriostatic antibiotics is reversible. If an antibiotic is removed before the immune system can eliminate the bacteria, bacterial growth will begin again. Antibiotics are also classified as broad spectrum or narrow spectrum. A

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Antibiotic Inhibition of Bacteria broad-spectrum antibiotic is effective on many different bacteria, while a narrow spectrum drug only attacks a limited variety of pathogens (Table 1). It should be noted that some antibiotics are also used against protozoa, fungi, and viruses. The spectrum of an antibacterial drug is usually determined by its mode of action against the bacteria. For example, penicillin is a bacteriocidal drug that inhibits the synthesis of the cell wall. Penicillin is a narrow spectrum antibiotic because it only affects grampositive bacteria (Table 1). In contrast, tetracyclines inhibit protein Table 1. Spectrum of Commonly Used Antibiotics Drug Primary Effect Chloramphenicol Static Erythromycin Static Penicillin Cidal Streptomycin Cidal Sulfonamides Static Tetracyclines Static

Drug Spectrum Broad (gram +/- ; rickettsia, chlamydia) Narrow (gram +, mycoplasma) Narrow (gram +) Broad (gram +/- ; mycobacteria) Broad (gram +/-) Broad (gram +/- ; rickettsia, chlamydia)

synthesis, a function common to all living organisms. Tetracyclines are considered broad spectrum, inhibiting the growth of both gram-positive and gram-negative bacteria. Other drugs interfere with nucleic acid synthesis (naladixic acid) or metabolite synthesis (sulfonamides). It is important to know which bacterium is causing an infection so that an antibiotic with the appropriate spectrum can be prescribed. An important point to remember is that many antibiotics are, in effect, poisons for living cells and are not specifically targeted to the bacteria causing an infection. Many of the side effects observed from antibiotic treatment are the result of the toxic effect of the drug on human cells as well as the bacterial cells. With the widespread use of antibiotics, particularly in the United States, a significant problem has arisen. Many species of bacteria are acquiring resistance to antibiotics, rendering standard drug therapy for some infections useless. Two of the most common methods by which bacteria evade a drug are: a) the destruction or inactivation of the drug or b) the prevention of penetration to the target site. Alteration (mutation) of the drug target site and transfer of resistance between bacteria are also means by which bacteria are able to evade antibiotics. Some of this resistance has occurred naturally through spontaneous mutation in bacterial genomes. However, multiple human practices have led to an increase in resistance to antibiotics. Overuse of antibiotics, particularly in the case of colds and flu (which are not affected by these drugs) and in animal feed, gives subpopulations of bacteria a chance to acquire resistance. Likewise, not completing a

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Antibiotic Inhibition of Bacteria prescribed treatment of antibiotics allows exposure to the drug without eradicating the entire population of bacteria. In addition, many subpopulations of bacteria that are resistant to multiple antibiotics are spreading rapidly due to world travel. This laboratory uses a disc diffusion assay to examine the effectiveness of different antibiotics on gram-positive and gram-negative bacteria. A bacterial culture is spread on a nutrient agar plate and lines are drawn on the outside of the plate to create “sectors”. A sterile disc soaked with a particular antibiotic is placed in each sector, and the plates are allowed to grow overnight at 37ºC (human body temperature). After incubation, an even growth of bacteria, or lawn, should cover the plate. The only place where the bacteria do not grow is in a region around the antibiotic discs. This clear region is called the zone of inhibition. Measurement of this zone, in millimeters (mm), gives an indication of the effectiveness of an antibiotic at a given dosage. The larger the zone, the more successful the antibiotic is at inhibiting bacterial growth.

PURPOSE This laboratory exercise will show the effect of different types of antibiotics on both Gram+ (Bacillis cereus) and Gram- (Escherichia coli) bacteria using a disc-diffusion assay. This lab will also introduce sterile technique and the handling of bacterial cultures.

EQUIPMENT/MATERIALS Tryptic soy agar plate β-subtilus culture 95% ethanol sterile antibiotic discs bacterial spreader 1 mL pipets, sterile permanent markers 37ºC incubator

LB agar plate E. coli culture Bunsen burner sterile control discs forceps pipet bulb 400 mL beaker rulers (metric)

SAFETY • •

Always wear an apron and goggles in the lab. This lab requires the use of an open flame. The following precautions should be taken. a. 95% ethanol is used in this lab. Since the ethanol is flammable, it is essential to keep the Bunsen burner and ethanol beaker on opposite ends of the lab bench! b. Keep the workspace clear of flammable items. One copy of the lab procedures and the materials provided for the lab are the only items that should be on the lab bench while the Bunsen burner is on. c. Students with long hair should always pull their hair back to keep it away from the flame.

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Antibiotic Inhibition of Bacteria

PROCEDURE 1.

You will be given 2 bacterial nutrient agar plates. These agar plates are labeled, and are specific for the types of bacteria that will be used in the experiment. Tryptic soy = gram-positive bacteria (Bacillis cereus) LB agar = gram-negative (Escherichia coli or K-12)

2.

A culture of gram-negative (Escherichia coli) and gram-positive (Bacillis cereus) bacteria will be provided to each group. These bacteria must be spread on the appropriate agar plate using sterile technique.

3.

Sterile Plating Technique. Sterile technique ensures that the only bacteria growing on the experimental plates are the ones from your culture. a. Open just one end of the plastic covering on the sterile 1 mL pipet. Be sure to open the end where the pipet bulb will attach, not the end at the tip of the pipet. As long as the lower portion of the pipet does not come in contact with anything, it is considered sterile. Keeping the plastic wrapper around the pipet, fit the pipet bulb onto the top of the pipet, as shown in Figure 3. b. Find the agar plate labeled “LB”. Label the bottom of this plate Gram –- and/or the name of the bacteria. Uncap the top of the test tube labeled Gram – . Carefully remove the plastic wrapper from the pipet and place the pipet in the test tube without touching anything but the inside of the tube. c. Use the pipet bulb to withdraw approximately 0.4 mL of Gram – bacterial culture (between 0.35 and 0.50 mL is acceptable). Open the top of the petri dish and carefully dispense the culture onto the top of the LB agar. You do not want to splatter the culture all over your work area, because it increases the risk of contamination to your second plate. Replace the cover on the plate. d. Light the Bunsen burner. This can be done safely by slowly turning on the gas until you hear a faint hiss. Stand back, and light the top of the burner as directed by your instructor. If the burner does not light right away, turn off the gas and request assistance from the instructor. Trying to repeatedly light the burner significantly increases your risk of starting a fire on the lab bench! e. Once the burner is lit successfully, remove the bacterial spreader from the ethanol bath. Hold it over the beaker and let any excess ethanol drain off. Then place the spreader over the burner flame briefly, and then remove. This will ignite the ethanol, and as it burns off, the spreader will be sterilized. Hold the spreader still while the ethanol burns.

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Antibiotic Inhibition of Bacteria f. To cool the bacterial spreader, place the bottom edge of the triangle (see Fig. 3) on a portion of the agar plate that does not contain the drop of bacteria. The agar should absorb most of the heat. Press the spreader down a couple more times to ensure it is completely cool (it should stop “hissing” when touched to the plate). Using the bottom edge of the spreader, push the bacterial culture around the surface of the agar until there is a relatively even coating of bacteria. Do not press down hard on the agar plate! They are like very firm Jello™, and will disintegrate with too much pressure. Set the plate aside and let the culture soak into the surface of the agar for 5 min. g. Obtain the Gram+ bacterial culture and the tryptic soy agar plate (labeled TS). Repeat Steps a. – f. for these bacteria. Be sure to use a new sterile pipet and resterilize the bacterial spreader or you will contaminate the second culture with the first.

Figure 3. Equipment for Sterile Plating Technique

pipet bulb bacterial spreader

sterile pipet

95% ethanol

plastic wrapper

oxygen valve

nutrient agar plate

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bacterial culture

Bunsen or Fisher burner

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Antibiotic Inhibition of Bacteria 4.

Once the cultures have soaked into the agar plates, turn the plates over. Using a Sharpie or permanent marker, draw lines on the bottom of the petri dish, dividing it into 4 roughly equal sections. Label the sections: control (C), 1, 2 and 3. Do this for both plates.

5.

Each group will be given three antibiotic discs from the sets shown below. Sterile discs that contain no antibiotic will be provided as well. These are a negative control, which should show that placing a disc on the bacteria does not itself inhibit bacterial growth.

Set 1 – Gram+ C 30 = chloramphenicol (30 µg) E 15 = erythromycin (15 µg) NB 30 = novobiocin (30 µg) P 10 = pencillin (10 IU) S 10 = streptomycin (10 µg) Te 30 = tetracycline (30 µg)

Set 2 – Gram– C 30 = chloramphenicol (30 µg) F/M 300 = nitrofurantoin (300 µg) K 30 = kanamycin (30 µg) NA 30 = naladixic acid (30 µg) SSS 0.25 = triple sulfa (250 µg) Te 30 = tetracycline (30 µg)

6.

Obtain the 3 antibiotic discs, all from either Set 1 or Set 2, a vial of sterile control discs, and the sectioned agar plates which contain the Gram+ and the Gram– bacteria.

7.

Take the forceps provided and dip the tip of them into the ethanol. Let the excess ethanol drain off and sterilize the forceps with flame from the Bunsen burner.

8.

Carefully remove a sterile control disk and place it in the center of the agar in the section marked “control”. It is easiest to do this for both plates at once. You do not need to re-sterilize the forceps between each control disc.

9.

Re-sterilize the forceps and choose one of the antibiotic discs. Record which antibiotic you have chosen and record it in the data table for Section 1. Then place that disc in Section 1 on the agar plate. Repeat this procedure with the other plate, placing the same antibiotic disc in Section 1.

10.

Repeat Step 9 for the final two antibiotic discs, being sure to record which antibiotic is in which section. Note: If you forget to record which antibiotic is in which section, don’t worry. Each disc is labeled with the abbreviations shown above. For example, the chloramphenicol disc will have C 30 printed on it.

11.

Let the plates with the antibiotic discs rest on the bench for 5 minutes. During this time, the discs will absorb moisture from the agar and adhere to the plate.

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Antibiotic Inhibition of Bacteria 12.

Turn the plates upside down and place them in a 37ºC incubator for 24 hrs. Each group should label the plates in a way that they can identify them the next class period. The bacteria must grow for 18-24 hrs before the data is collected.

DATA ANALYSIS 1.

Remove the plates from the 37ºC incubator. After incubation, an even growth of bacteria, or lawn, should cover the plate. The only place where the bacteria do not grow is in a region around the antibiotic discs. This is the zone of inhibition.

2.

Using a metric ruler, measure the zone of inhibition (in mm) around each antibiotic disc. Measure the zone for the control disc as well. Do this for the gram-positive and the gram-negative bacteria.

3.

Record the data for the lab group in the “Individual Data” tables provided. Share this data with the rest of the class.

4.

Obtain the data for the antibiotics your lab group did not test from your classmates, and record it in the group data tables provided. This information is necessary to answer the questions at the end of the laboratory.

References: Adapted from “Inhibition of Bacteria: Antibiotics and Antiseptics” (1999) Juniata College – Science in Motion. Additional References: Lansing M. Prescott, John P. Harley and Donald Klein. Microbiology. “Antimicrobial Chemotherapy” Wm. C. Brown Publishers. 1996. 3rd Edition. pp. 656-664. Gerard J. Tortora, Berdell R. Funke and Christine L. Case. Microbiology, An Introduction. “Antimicrobial Drugs” Addison Wesley Longman, Inc. (1998) 6th Edition. pp. 531-538.

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DATA SHEET

Name ________________________ Period _______ Class ___________ Date ___________

ANTIBIOTIC INHIBITION OF BACTERIA DATA TABLES Individual Data Gram-negative (Escherichia coli) Agar Plate Section C 1 2 3

Antibiotic

Zone of Inhibition (mm)

Antibiotic

Zone of Inhibition (mm)

Gram-positive (Bacillus cereus) Agar Plate Section C 1 2 3 Group Data Antibiotic

Average Zone of Inhibition (mm) Gram-negative / Gram-positive

chloramphenicol

/ / / / / / / / / /

kanamycin naladixic acid nitrofurantoin triple sulfa tetracycline erythromycin novobiocin penicillin streptomycin

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QUESTIONS 1.

What does the zone of inhibition indicate about each antibiotic? Which antibiotic is most effective against the E. coli (gram-negative)? the B. cereus (gram-positive)? Which is the least effective?

2.

Each antibiotic disc has a certain low concentration based on doses commonly used to treat disease in humans. If low dose of an antibiotic is proven effective against a certain bacteria, why wouldn’t a higher dose be better?

3.

A common side effect of antibiotic use is intestinal distress and trouble digesting food. Hypothesize why this occurs.

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Antibiotic Inhibition of Bacteria 4.

The following data were obtained using a disc diffusion assay on a gram-negative bacterium. Can you deduce anything from the results of this particular experiment? Why or why not?

5.

Many parents can now request (and receive!) prescriptions from pediatricians over the phone, without the child ever being examined by the doctor. Describe why this is an unwise practice in terms of a) spectrum of antibiotic being used. b) bacterial resistance to antibiotics.

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Antibiotic Zone of Inhibition (mm) Chloramphenicol 8 Kanamycin 12 Naladixic acid 5 Tetracycline 7

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