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EXPERIMENT 7:
Antimicrobial analysis
MEMBERS
MATRIC NUMBER
D20131063718
D20131063722
D20131963734
D20131063745
Objectives
To investigate the degree of antibiotics resistance in a bacterial species using a Kirby-Bauer
test.
ABTRACT
An antimicrobial is an agent that kills microorganisms or inhibits their growth.
Antimicrobial medicines can be grouped according to the microorganisms they act primarily
against. By strict definition, the word antibiotic refers to substances produced by
microorganisms
that
act
against
another
microorganism. In
contrast,
the
term
antimicrobials include all agents that act against all types of microorganisms bacteria
(antibacterial), viruses (antiviral), fungi (antifungal) and protozoa (antiprotozoal). As for this
experiment the antimicrobial agent used is Ampicillin with 100mg/ml followed by Ampicillin
with unknown concentration, bleach, iodine, detergent and alcohol. However, we could said
that all antibiotics are antimicrobials, but not all antimicrobials are antibiotics.
INTRODUCTION
Bacteria have existed long before humans set foot on the earth, and will probably outlive the
human species. They are found on almost every surface available, on our desks, in the food
we eat, and even in and on our bodies. Some bacteria are very helpful, such as E. coli, which
lives in our intestines and helps with digestion and production of Vitamin K. Only a small
number of bacteria are considered harmful and actually cause disease. In this experiment, we
did the antimicrobial analysis. An antimicrobial is an agent that kills microorganism or
inhibits their growth. The main classes of antimicrobial agents are disinfectant, antiseptic and
antibiotic. Disinfectants kill a wide range of microbes on non-living surface or on inanimate
objects to prevent the spread of illness. Many household cleaning agents such as ammonia
and bleach are powerful disinfectants. Antiseptics are applied both on inanimate and animate
object which help in reducing infection. For example in cleaning the cuts and prevent
infection during surgery. Examples of antiseptics include: alcohol, iodine, hydrogen peroxide,
heavy metals such as silver nitrate and detergents. While antibiotic destroy microorganisms
within body. Antibiotics are chemical substances produced by microorganisms that stop the
growth of other microorganisms and are effective in very small doses. Antibiotic production
by microorganisms is common, and most medically valuable antibiotics are produced either
by fungi, especially those in the genus Penicillium, which produces antibiotics such as
penicillin or bacteria in the genus Bacillus, which produce antibiotics such as bacitracin and
polymyxin; or bacteria in the genus Streptomyces, which produce a number of medically
important antibiotics. Antimicrobial agents work in one of two ways: they are either
bacteriostatic or bacteriocidal. A bacteriostatic agent does not kill the bacteria, but inhibits
growth by preventing cell wall formation or interfering with biochemical cellular processes.
A bacteriocidal agent kills bacteria on contact by breaking the protective cell membrane and
lysing the cell, or stopping nucleic acid synthesis, or preventing the bacteria from carrying
out normal chemical reactions. So in the lab we used Ampicillin (unknown concentration),
Ampicilin (100mg/ml), alcohol, iodine, bleach and detergent as an antimicrobial agent. we
will be able to test some common antimicrobials to see how effective they are at stopping
bacterial growth using the zone-of-inhibition method. With this method, a chemical is
applied to a freshly inoculated plate and after incubation, bacterial growth around the
antimicrobial agent is observed. A clear area around the chemical agent (zone-of-inhibition)
indicates that bacteria were killed. The size of the zone is usually proportional to the
effectiveness of the antimicrobial.
MATERIALS
METHODOLOGY:
1. The contents of broth culture of S. aureus is swirled until it is equally
murky throughout.
2. Cotton swab was used to created a lawn of S. aureus on 2 plates of
Mueller-Hinton agar aseptically.
3. Antibiotics disc was picked by using sterile forcep and was placed on
opposites sides of petri dish containing S. aureus. Antibiotics disc
was tap gently to stick them to the agar.
4. Step 1-3 was repeated using plates of E. coli.
5. All the plates was wrapped by using Parafilm and the plate was
placed in incubator at 37C for 24 hours.
6. We checked the presence of antibiotic activity after 24 hours in
incubator. We observed for a clear area, called zone of inhibition
surrounding a paper disc.
7. We measured the diameter (mm) od the zone of inhibition to
determined the affectivity of an antibiotic.
8. The test was repeated by using alcohol, iodine, bleach and
detergent.
Ampicillin
100mg/ml
Ampicillin
unknown
Negative control
Alcohol
Bleach
Detergent
Iodine
Negative control
Alcohol
Ampicilin
unknown
Negative
control
Ampicilin
100mg/ml
Bleach
Detergent
Iodine
Negative
control
Figure 4 : Cultures of E. coli in presence of antimicrobial agent that is
iodine, bleach, detergent and negative control.
Diameter
zone
inhibition
of (Condition of bacteria)
Sensitive (S)
Intermediate (I)
Negative control
Ampicillin
of
Resistance (R)
-
unknown 2.0
Sensitive
concentration
Ampicillin 100mg/ml
1.8
Intermediate
Alcohol
1.7
Intermediate
Iodine
1.5
Intermediate
Bleach
1.6
Intermediate
Detergent
2.0
Sensitive
Diameter
inhibition
zone
of (Condition of bacteria)
Sensitive (S)
Intermediate (I)
Resistance (R)
Negative control
Ampicillin
of
unknown 3.2
Sensitive
concentration
Ampicillin 100mg/ml
3.4
Sensitive
Alcohol
Resistance
Iodine
1.4
Intermediate
Bleach
2.3
Sensitive
Detergent
Resistance
diffusion of the antibiotic through the agar, and the depth of the agar.
Bacteria that are resistant to an antibiotic will not show a zone of
inhibition.
Based on the results of this experiment, for S. aureus sp, it shows
that the bacteria is sensitive in antimicrobial agent of ampicillin of
unknown concentration and detergent because diameter zone of inhibition
is longer compared to other antimicrobial agent that is iodine, bleach,
alcohol and ampicillin 100mg/ml. The diameter of zone of inhibition of
ampicillin of unknown concentration and detergent is 2.0mm. From the
result, we could see that zone of inhibition for
ampicillin of unknown
is 1.5mm,
intermediate based on
diameter zone of
C:
Detergent
A: Negative
control
D: Iodine
B: Bleach
Figure 5: Zone of inhibition of Escherichia coli treated with (A) Negative control, (B)
Bleach, (C) Detergent and (D) Iodine.
E: Ampicillin
100mg/ml
G: Negative
control
F: Alcohol
H: Ampicillin
(unknown)
Figure 6: Zone of inhibition of Escherichia coli treated with (E) Ampicillin 100mg/ml, (F)
Alcohol, (G) negative control and (H) Ampicillin (unknown concentration)
Antimicrobial agent
Diameter zone of
Condition of bacteria
inhibition
Negative control
0
Ampicillin (unknown concentration)
3.0
Sensitive
Ampicillin 100mg/ml
3.1
Sensitive
Alcohol
0
Resistance
Iodine
1.0
Intermediate
Bleach
2.4
Sensitive
Detergent
0
Resistance
Table 3: Antimicrobial analysis on Escherichia coli
C: Negative
control
A: Bleach
D: Detergent
B: Iodine
Figure 7: Zone of
inhibition of
Staphylococcus aureus treated with (A) Bleach, (B) Iodine, (C) Negative control and (D)
Detergent.
G: Negative
control
E: Ampicillin
100mg/ml
H: Ampicillin
(unknown)
F: alcohol
Figure 8: Zone of inhibition of Staphylococcus aureus treated with (E) Ampicillin 100mg/ml,
(F) Alcohol, (G) negative control and (H) Ampicillin (unknown concentration)
Antimicrobial agent
Diameter zone of
Condition of bacteria
inhibition
Negative control
0
Ampicillin (unknown concentration)
2.5
Sensitive
Ampicillin 100mg/ml
2.4
Sensitive
Alcohol
0
Resistance
Iodine
1.5
Intermediate
Bleach
1.8
Intermediate
Detergent
2.1
Sensitive
Table 4: Antimicrobial analysis on Staphylococcus aureus
For alcohol and detergent, the diameter of zone of inhibition for both are 0. The
Escherichia coli were resistance toward both antimicrobial agents. The significance of this
may be because of survival of E.coli in the presence of ethanol. For the detergent the bacteria
should be inhibited because the detergent is an antiseptic. For the iodine the diameter of zone
of inhibition are 1.0 which indicate the bacteria is intermediate towards it. As we know the
diameter of size of the zone is usually proportional to the effectiveness of the antimicrobial.
So from the result for antimicrobial analysis for Escherichia coli we can see the strongest
antimicrobial agent is Ampicillin with 100mg/ml followed by Ampicillin with unknown
concentration, bleach, iodine, detergent and alcohol.
As for Staphylococcus aureus, the most sensitive agents is Ampicillin with unknown
concentration which the diameter of zone of inhibition is 2.5 the followed by the Ampicillin
with 100mg/ml which the diameter of inhibit zone is 2.4 which is contradict from the
Escherichia coli.For the bleach, the diameter of zone of inhibition is 2.1 which indicate that
the bacteria are sensitive towards the bleach.
Then, for alcohol, the diameter of zone of inhibition is 0. The Staphylococcus aureus
were resistance the antimicrobial agents. We could said that the Staphylococcus aureus can
survive in the presence of ethanol same as the Escherichia coli. For the detergent which the
diameter of zone of inhibition is 1.8 followed iodine the diameter of zone of inhibition are 1.5
by which indicate the bacteria is intermediate towards it. Thus, from the result, we could see
that antimicrobial analysis for Staphylococcus aureus that the strongest antimicrobial agent is
Ampicillin with unknown concentration followed by Ampicillin with 100mg/ml, detergent ,
iodine ,bleach, and alcohol.
CONCLUSION
From this experiment we could conclude that there is several
antimicrobial agent for the microbes to be able resist them from being
killed. In this experiment we can see that the most effective microbial
agents is ampicillin 110mg/ml while the most least effective agent is
alcohol for the E.coli.
most effective is detergent and the least effective is alcohol. All the
conclusion is based from the observation the result shown. We use the
Kirby Bauer test which is a antibiotic testing (KB testing or disc diffusion
antibiotic sensitivity testing) which uses antibiotic-impregnated wafers to
test whether bacteria are affected by antibiotics. However we can that all
antibiotics are antimicrobial but not all antimicrobial are antibiotics.
REFERENCES
Chemical Methods of Microbial Control. (2016). Classes.midlandstech.edu. Retrieved 23
April 2016, from http://classes.midlandstech.edu/carterp/Courses/bio225/chap07/lecture5.htm
Gerald McDonnell, A. (1999). Antiseptics and Disinfectants: Activity, Action, and
Resistance. Clinical
Microbiology
Reviews, 12(1),
147.
Retrieved
from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC88911/
Ingram
LO,
e.
(2016). Alcohol
NCBI. Ncbi.nlm.nih.gov.
tolerance
Retrieved
http://www.ncbi.nlm.nih.gov/pubmed/7017759
in
23
Escherichia
April
coli.
2016,
PubMed
from