Download Antibiotic Inhibition of Bacteria

ANTIBIOTIC INHIBITION OF BACTERIA
STANDARDS
3.2.10B, 3.2.12B Apply process knowledge and
evaluate experimental information
3.3.10B, 3.3.12B Chemical and structural basis of
living organisms
Westminster College
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
which 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 which 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 which consists of an outer membrane, a
periplasmic space and a cytoplasmic membrane. The peptidoglycan layer, found in the
periplasmic space, is much smaller, and there is no teichoic acid present. The outer
membrane and cytoplasmic membrane are comprised of phospholipids. The outer
Westminster College SIM
Page 1
Antibiotic Inhibition
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 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 which inhibits the
synthesis of the cell wall. Penicillin is a narrow spectrum antibiotic because it only
affects gram-positive bacteria (Table 1). In contrast, tetracyclines inhibit protein
Westminster College SIM
Page 2
Antibiotic Inhibition
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, an
important 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. But there are multiple human
practices which 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 prescribed treatment of antibiotics allows
exposure to the drug without eradicating the entire population of bacteria. Also, many
subpopulations of bacteria which 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),
Westminster College SIM
Page 3
Antibiotic Inhibition
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.
GUIDING QUESTIONS
•
•
•
What is the major difference between Gram negative and Gram positive bacteria?
Why are different antibiotics prescribed for different bacterial infections?
Why is it necessary to include a sterile control disc on each bacterial plate?
MATERIALS
Tryptic soy agar plates (2)
E. coli culture
95% ethanol
sterile antibiotic discs
bacterial spreaders (2), sterile
permanent marker
ruler (metric)
Bacillus cereus culture
forceps
1 mL pipets, sterile
sterile control discs
pipet bulb
37ºC incubator
Kimwipes
SAFETY
This lab requires the use of live bacteria. The following precautions should be taken.
a. Wash hands before and after handling the bacterial cultures
b. Use some of the 95% ethanol to wipe down the lab bench after the lab is done.
c. Students with long hair should always pull their hair back to avoid
contamination.
PROCEDURE
1. You will be given 2 tryptic soy agar plates. Use the permanent marker to label them
as follows:
a. Bacillis cereus = Gram-positive bacteria
b. Escherichia coli = Gram-negative bacteria
Remember to write your initials or group name on the plate for identification.
2. A culture of gram-negative (Escherichia coli) and gram-positive (Bacillus 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
Westminster College SIM
Page 4
Antibiotic Inhibition
before pipetting the bacteria, it is considered sterile. Keeping the plastic wrapper
around the pipet, fit the pipet bulb onto the top of the pipet (as shown in Fig. 3).
b. Find the agar plate labeled “E. coli”. 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.
Figure 3. Equipment for Sterile Plating Technique
pipet bulb
bacterial
spreader
sterile pipet
plastic wrapper
Sterile bottom
edge
antibiotic
disc
nutrient agar plate
bacterial culture
antibiotic disc
dispenser
c. Use the pipet bulb to withdraw approximately 0.4 mL of Gram- bacterial
culture (between 0.30 and 0.50 mL is acceptable). Open the top of the petri dish
and carefully dispense the culture onto the top of the agar plate. 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. Carefully unwrap the tinfoil from the end of the blue bacterial spreader. As
long as nothing touches the triangular end of the spreader, everything under the
tinfoil is considered sterile.
Westminster College SIM
Page 5
Antibiotic Inhibition
e. 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.
h. Obtain the Gram+ bacterial culture and the appropriate agar plate (labeled
Bacillus cereus). Repeat Steps a. – e. for these bacteria. Be sure to use a new
sterile pipet and bacterial spreader or you will contaminate the second culture
with the first.
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 6
roughly equal sections. Label the sections: 1-6. Do this for both plates.
5. Sterile discs which contain no antibiotic are provided. These are a negative control
which should show that placing a disc on the bacteria does not, in itself, inhibit bacterial
growth.
6. Take the forceps provided and dip the tip of them into the ethanol. Let the excess
ethanol drain off and wipe the remaining ethanol off with a Kimwipe.
7. Carefully remove a sterile control disk and place it in the center of the agar plate. It is
easiest to do this for both plates at once. You do not need to re-sterilize the forceps
between each control disc.
8. The antibiotics discs come in a cassette that will dispense them in a sterile fashion.
There are a total of 6 different antibiotics, listed below. Choose one of the antibiotics,
hold the disc dispenser over one of the sectors on the E. coli plate, and click the metal bar
once to dispense a single antibiotic disc. Repeat this procedure with the same antibiotic
for the B. cereus plate. Record the antibiotic name in the appropriate sector number on
the Data Sheet.
Antibiotic Disc
kanamycin (30 µg)
pencillin (10 IU)
erythromycin (15 µg)
tetracycline (30 µg)
chloramphenicol (30 µg)
bacitracin (10 IU)
Disc Designation
K 30
P 10
E 15
Te 30
C 30
B 10
9. Repeat step 8 for the five remaining antibiotics.
Westminster College SIM
Page 6
Antibiotic Inhibition
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.
10. 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.
11. 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.
Adapted from: “Inhibition of Bacteria: Antibiotics and Antiseptics” (1999) Juniata
College – Science in Motion.
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.
CREDITS
This lab was adapted and revised by Dr. Stephanie Corrette-Bennett.
Westminster College SIM
Page 7
Antibiotic Inhibition
DATA SHEET
Name: _______________________
Date: _______________________
Individual Data
Gram-negative (Escherichia coli)
Agar Plate Section
C
1
2
3
4
5
6
Antibiotic
None (control)
Zone of Inhibition (mm)
Antibiotic
None (control)
Zone of Inhibition (mm)
Gram-positive (Bacillus cereus)
Agar Plate Section
C
1
2
3
4
5
6
Group Data
Antibiotic
Average Zone of Inhibition (mm)
Gram-negative / Gram-positive
bacitracin (10 IU)
/
/
/
/
/
/
chloramphenicol (30 µg)
erythromycin (15 µg)
kanamycin (30 µg)
pencillin (10 IU)
tetracycline (30 µg)
Westminster College SIM
Page 8
Antibiotic Inhibition
Name: _______________________
Date: _______________________
QUESTIONS AND ANALYSIS
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.
Westminster College SIM
Page 9
Antibiotic Inhibition
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?
Antibiotic
Zone of Inhibition (mm)
Chloramphenicol
8
Kanamycin
12
Penicillin
5
Tetracycline
7
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.
Westminster College SIM
Page 10