Download Chemical Agents that Inhibit Bacterial Growth

Unit 14
Unit 14: Chemical Agents That Inhibit Bacterial Growth
By Karen Bentz, Patricia G. Wilber and Heather Fitzgerald.
Copyright Central New Mexico Community College, 2015
Introduction
There are many chemicals that will kill or inhibit the growth of microorganisms.
An antibiotic generally refers to a chemical that can be used on or inside the human body to
reversibly inhibit the growth of bacteria (bacteriostatic) or irreversibly kill bacteria
(bactericidal). Antibiotics that only inhibit bacterial growth may still be very effective if a
person has a strong enough immune system to eliminate the bacteria that have stopped
growing. Antibiotics are most often produced by soil fungi (Penicillium) and soil bacteria
(Streptomycetes). Bacterial species that normally reside in the soil will have developed natural resistance
to the different antibiotics produced by soil microorganisms. Although bacterial species that normally
reside on or inside the human body (normal flora) may not have been resistant to the antibiotics
produced by soil microorganisms, the continued use of prescription antibiotics can promote the
development of resistance among our normal flora. An even bigger concern is the sharing of genes that
encode for antibiotic resistance between bacteria residing in soil, bacteria that are part of our normal
flora, and bacteria that are disease causing pathogens.
Antibiotics generally work best against specific types of bacteria. For instance, penicillin is most
effective against Gram(+) bacteria, as it does not easily penetrate the Gram(-) outer lipid
membrane. When treating an infected patient, it is important to first determine which
antibiotics the bacterial pathogen is susceptible to. However, it is also important to understand
that the susceptibility of a bacteria to an antibiotic is only one factor that is used in selecting
the most appropriate antibiotic. There are many other factors that are not measured by the
clinical laboratory tests which are just as important. The other factors that impact choice of
antibiotic include: (1) the site of infection and which drugs can penetrate to that site most
effectively; (2) the risk-benefit related to toxicity of the drugs being considered; (3) known
patient allergies or previous adverse events associated with specific antibiotics; (4) current
medications being taken by the patient and potential adverse interactions; (5) and age of the
patient and counter-indications for pediatric or geriatric populations. It is also important to
understand that the susceptibility of a bacterial pathogen to an antibiotic is only one factor that
determines success in treating an infection. Other factors that are just as important include:
(1) the strength of patient’s immune system; (2) variability among patients related to
penetration of antibiotics to site of infection; and (3) the need for surgical intervention to cure
an infection. All the antibiotics in the world will not cure an infection associated with an
abscess, unless that abscess is first opened and drained. Similarly, all the antibiotics in the
world will not effectively treat an infected artificial joint or heart valve, unless the infected
joint/valve is first removed by surgery. Although there are many factors working together to
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Unit 14
cure a bacterial infection, the goal of the clinical microbiology lab is to help the health-care
providers select the most appropriate antibiotic for the patient.
The Kirby-Bauer method is widely used for testing the effects of various antibiotics on bacterial
growth. A bacterial lawn is first created on a large petri plate containing a special agar medium.
Paper discs containing specified amounts of an antibiotic are then put on top of the bacteria.
After incubation if the antibiotic has inhibited the growth of the bacteria there will be a clear
zone around the paper disc. The effect of the antibiotic on bacterial growth can be determined
by measuring the diameter of the circle of inhibition with a metric ruler. This diameter is called
the zone of inhibition. The measurement of the zone is then compared to standards to
determine if the bacteria is sensitive to the antibiotic, or resistant to it. Although the KirbyBauer allows healthcare professionals to determine which antibiotics a pathogen is sensitive to
or resistant to, the test is not quantitative. The results of the Kirby-Bauer only provide a list of
drugs which the healthcare professionals can take to the next step to select the most
appropriate for that particular patient (see above for other factors involved in decision).
Keep in mind that all living organisms, including bacteria and humans, show genetic variation.
Genetic variation is the reason life is able to survive in a constantly changing environment. The
genetic variation in bacteria means that some bacteria will have resistance to a particular
antibiotic. Treatment with that particular antibiotic will select for those bacteria that are
resistant, and they will proliferate. Likewise, in the human population there will be individuals
with genetic variations that make them more susceptible to bacterial infection. Because of
human and bacterial genetic variation, antibiotics are never 100% effective in treating bacterial
infections in humans.
Many cleaning agents have words such as “sanitize”, “germicide”, and “antiseptic” in their
product labeling, but what do these terms really mean as far as microbial growth is concerned?
If a term ends in “cide”, such as viracide, then the agent should kill the specified organism. The
term antiseptic means that the agent prevents bacterial growth, but does not necessarily kill
the organism. The term sanitation refers to any cleaning technique that removes
microorganisms from an object
Chemicals used to inhibit microbial growth outside of the human body are termed disinfectants
or antiseptics. Disinfectants do more to actually kill microorganisms, while antiseptics merely
prevent their growth. Disinfectants are used to clean inanimate surfaces such as floors and
hospital beds. Antiseptics such as mouthwash are safe to use on human skin, but do little to
actually kill microorganisms. When purchasing household cleaners, you might want to read the
label to see what the active ingredient is, to see if it will kill microorganisms or just keep them
from growing.
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Unit 14
DAY 1: Inoculation
I. Kirby-Bauer Method for Testing Bacterial Sensitivity to Antibiotics
Materials (per group)
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Lawn of assigned bacteria
1 Mueller-Hinton agar plate
Antibiotic disc
1 swab
various antibiotic discs
1 tweezers
Bacteria
o Escherichia coli (Ec)
o Pseudomonas aeruginosa (Pa)
o Staphylococcus aureus (Sa)
Procedure for Kirby-Bauer Inoculation
1. For the bacterial lawn, choose one bacteria or use the bacteria designated by your
instructor.
2. Label the bottom of your Mueller-Hinton plate with the name of the bacteria that you
will be using
3. Wet the swab in water and use it to pick up bacteria. Create a solid lawn of bacteria on
your plate. Do not leave any gaps, or you may not see the zones of inhibition correctly.
4. Sterilize a pair of tweezers and use it to place one of each type of antibiotic disc onto
the plate. Make sure the discs are spaced as far apart as possible on the plate. Tap each
disc with the tweezers once you have placed it on the plate to make sure the disc sticks
to the plate.
5. Place your plates upside down for incubation. Inverting your plate prevents water from
dripping onto the plate and ruining the zones of inhibition.
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Unit 14
II. Disinfectant and Antiseptic Effects on Bacterial Growth
Materials (per person)
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1 T-soy plate
1 swab
1 tube of sterile water
2 paper discs
1 tweezers
2 cleaning solutions of your choice
Bacteria
o Escherichia coli (Ec)
o Pseudomonas aeruginosa (Pa)
o Staphylococcus aureus (Sa)
Lawn of your choice
of bacteria
Discs soaked in
different chemicals
Procedure for Antiseptic/Disinfectant Inoculation
1. Choose one of the bacteria to use and create a lawn of bacteria on the petri plate.
2. On the bottom of the plate, draw a line down the middle of the plate and write the
chemicals you are going to test on either side of the line.
3. Create your own chemical discs by holding a paper disc with the tweezers and soaking
the disc in the chemical of your choice.
4. Still holding your paper disc with a tweezers, blot off the excess chemical with a Kimwipe and place the disc on your bacterial lawn.
5. Repeat the process of preparing a chemical disc using your second chemical, and put
this disc on the other side of your plate.
6. Invert plates and put into rack for incubation.
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Unit 14
DAY 2: Results and Interpretation
I.
Kirby-Bauer
1. Examine your plate for zones of inhibition, which will be clear areas of no growth around
the disc.
2. Measure the diameter of the zone in millimeters (mm).
3. Compare your zone of inhibition measurement to a standard (Table 14.1)
4. Record your results in Table 14.2 by putting a check by each antibiotic that the bacteria
is sensitive to.
5. Collect results for the other two bacteria from student groups that tested a different
bacteria then you did.
Table 14-1: Antibiotic Zone Diameter Standards (mm)
Drug
Resistant Intermediate Sensitive
Ampicillin (AM)
≤ 11 mm
12-13 mm
≥ 14 mm
Cephalothin (CF)
≤ 14
15 - 17
≥ 18
Erythromycin (E)
≤ 13
14 - 22
≥ 23
Gentamycin (GM)
≤ 12
13 - 14
≥ 15
Penicillin (P)
≤ 28
Streptomycin (S)
≤ 11
12 - 14
≥ 15
Tetracycline (TE)
≤ 14
15 - 18
≥ 19
Bactrim (SXT)
≤ 10
11 - 15
≥ 16
≥ 29
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Unit 14
Table 14-2: Kirby-Bauer Results Showing Antibiotic Sensitivity
(Put a check in the boxes by the antibiotic that the bacteria is SENSITIVE to)
Antibiotic
Pseudomonas
Ampicillin (AM)
Cephalothin (CF)
Erythromycin (E)
Gentamycin (GM)
Penicillin (P)
Streptomycin (S)
Tetracycline (TE)
Bactrim (SXT)
Insert Photos of Results Here:
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E. coli
Staph. aureus
Unit 14
II. Antiseptics/Disinfectants
1. You will not measure a zone of inhibition for the chemical you tested. Instead record
(Table 3) a chemical as effective if there is some inhibition of growth around the disc.
2. Look at other student’s results to determine which cleaners inhibit growth of the
bacteria that you did not test.
Table 14- 3: Effectiveness of Various Chemicals
Bacteria
Chemicals that
Inhibited Growth
Chemicals that
Did Not Inhibit Growth
Pseudomonas aeruginosa
Escherichia coli
Staphylococcus aureus
Post-Activity Questions
1. Which bacteria is resistant to the most antibiotics?
Why might this be so?
2. Which antibiotic provided the largest zone of inhibition on the plate you inoculated?
Is this antibiotic the most potent among those that you tested? Defend your answer
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Unit 14
3. You are caring for a patient that is severely immunocompromised and has a serious infection.
The clinical microbiology lab has performed the Kirby-Bauer test and followed that with a test to
measure potency of those drugs the bacteria is sensitive to. The clinical microbiology lab
reports that the most potent drug is a bacteriostatic drug. Is this the drug that should be
selected to treat the infection? Defend your answer.
4. Which, if any, of the cleaning products that you tested in lab inhibited growth of bacteria?
Look at the label on the cleaning product and record its active ingredient.
5. Why would you not want to use a mouthwash that said “disinfectant” on the label?
6. Do you think long term exposure to household cleaning agents could be detrimental to human
health?
Of those cleaners you tested, which do you think has/have the potential to adversely affect
human health?
Why do you think this is so?
The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms,
our associate dean Linda Martin for many kinds of aid, Mike Jillson and Alex Silage for IT support, and our dean John Cornish .
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