Transcript Which Concentration of Honey Inhibits Bacterial Growth

HONEY ANTIMICROBIAL
EFFECTS
Alicia Grabiec
Freeport Senior High School
Grade 10
Purpose

To determine the antimicrobial effects of honey
on the Escherichia coli (Gram -) and Staphylococcus
epidermidis (Gram +)
Background Information



Honey has been known for its
healing properties for thousands
of years
Ancient Greeks used it, and so
have many other peoples through
the ages
Up to the second world war,
honey used for its antibacterial
properties in treating wounds
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
Advent of penicillin and other antibiotic
drugs in the twentieth century reduced
honey's medicinal use
Makua honey, like that found in the
antiseptic lotion above, can destroy strains
of antibacterial-resistant bacteria, such as
MRSA
C6H12O6 + H2O + O2 → C6H12O7 + H2O2
This chemical reaction occurs when honey is mixed with
water and oxygen. Its products are glutonic acid and
hydrogen peroxide, both possess antibacterial
properties.
Background Information





Escherichia coli is a large and diverse
group of gram (-) bacteria.
It is found in the intestinal tract of most
animals, including humans.
Estimated to cause infection in more
than 70,000 patients a year in the
United States.
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
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There are many strains of E. coli, most
are non-pathogenic
Pathogenic strains can cause illness
and death in humans

Staphylococcus epidermidis is a spherical
bacterial that is known to cause
infections in wounds.
Gram (+) bacteria
This bacteria is commonly found on the
skin.
It is resistant to antibiotics like penicillin
and methicillin.
Null Hypothesis:

Honey exposure will not significantly reduce the
survivorship of both E. coli and S. epidermidis.
Alternative Hypothesis:

Honey exposure will significantly reduce the
survivorship of both E. coli and S. epidermidis.
Variables
Independent / Manipulated
Concentration of honey being tested
Dependent/ Responding
Bacterial survivorship
0%(control), 1%, 10%, 30%, 50%
Constant/ Controlled
Limitations
Incubated at 37oC
112 sterile petri dishes containing LB Medium
Length of Incubation time (48 hours)
Extended viability of E. coli and
S. epidermidis (established)
Impurities in the honey
Pulse Liquid Exposure Procedure:
1.
2.
3.
4.
5.
Bacteria (E. coli and S.
epidermidis) were grown
overnight in sterile LB media.
A sample of the overnight
culture was added to fresh
media in a sterile sidearm flask.
The culture was placed in an
incubator (37°C) until a density
of 50 Klett spectrophotometer
units was reached. This
represents a cell density of
approximately 108 cells/mL.
The culture was diluted in sterile
dilution fluid to a concentration
of approximately 0.5-0.8x105
cells/mL.
Honey was mixed with the
appropriate amount of SDF to
create honey concentrations of
0%, 1%, 10%, 30%, and 50%.
6.
100 µL of cell culture was then added
to the honey concentrations, yielding
a final volume of 10 mL and a cell
density of approximately 103
cells/mL.
7.
The solutions were vortexed and
allowed to sit at room temperature
for 15 minutes.
8.
After vortexing to evenly suspend the
cells, 100 µL aliquots were removed
from the tubes and spread on LB agar
plates.
9. The plates were incubated at 37°C for
24 hours.
10. The resulting colonies were counted
visually. Each colony was assumed
to have arisen from one cell.
Honey Infused Agar
(Prolonged Exposure) Procedure:
1.
2.
3.
4.
5.
Honey was infused directly into molten agar plates at 0.1, 1, 10% and
allowed to solidify.
Bacteria (E. coli and S. epidermidis) was grown overnight in sterile LB media.
A sample of the overnight culture was added to fresh media in a sterile
sidearm flask.
The culture was placed in an incubator (37°C) until a density of 50 Klett
spectrophotometer units was reached. This represents a cell density of
approximately 0.5-0.8x108 cells/mL.
The culture was diluted in sterile dilution fluid to a concentration of
approximately 105 cells/mL.
6.
The culture was then spread evenly across honey infused agar.
7.
The plates were incubated at 37°C for 24 hours.
Zone of Inhibition Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Bacteria (E. coli and S. epidermidis) was grown overnight in sterile LB
media.
A sample of the overnight culture was added to fresh media in a sterile
sidearm flask.
The culture was placed in an incubator (37°C) until a density of 50 Klett
spectrophotometer units was reached. This represents a cell density of
approximately 0.5-0.8x108 cells/mL.
The culture was diluted in sterile dilution fluid to a concentration of
approximately 105 cells/mL.
The culture was then spread evenly across petri dishes containing sterile LB
Media
Plates were then incubated for one hour at 37°C
50 µL honey aliquots (droplets) were carefully placed in 5 positions on each
plate and repeated for each concentration
The plates were incubated at 37°C for 24 hours.
Determine and record the average zone of inhibition
Pulse Liquid Exposure Data
Pulse Liquid Exposure (E. coli)
Plate
#
1
2
3
4
5
6
7
8
Avg.
Percent of Honey
0%
1%
10%
30%
233
230
154
120
206
210
120
140
211
180
139
100
226
169
127
112
231
163
150
117
209
174
143
121
230
169
147
130
210
167
129
126
219.50 182.75 138.63
120.75
Pulse Liquid Exposure (S. epidermidis)
50%
80
90
83
87
86
84
89
81
85.00
Plate
#
1
2
3
4
5
6
7
8
Avg.
0%
154
163
159
153
157
160
165
143
156.75
Percent of Honey
1% 10% 30%
5
3
2
0
0
0
0
0
0
3
1
0
0
0
0
2
2
0
1
0
0
3
0
1
1.75 0.75
0.38
50%
0
0
0
0
0
0
0
0
0.00
Dunnett Test
0.01 Significance Level
T-critical = 4.17
T > T-critical = Significant Variation, Reject Null Hypothesis
Pulse Liquid Exposure (E. coli)
HONEY
CONCENTRATION
1%
10%
30%
50%
T-VALUE
82.843
83.379
83.578
83.779
INTERPERATION
Significant
Significant
Significant
Significant
Pulse Liquid Exposure (S. Epidermidis)
HONEY
CONCENTRATION
1%
10%
30%
50%
T-VALUE
5.151
11.337
13.824
18.853
INTERPERATION
Significant
Significant
Significant
Significant
Mv  MC
t
2MSE
nh
Honey Infused Agar Data
Honey Infused Agar (S.
epidermidis)
Honey Infused Agar (E.coli)
Plate
#
Percent of Honey
0%
0.10% 1% 10%
1
257
219
3
0
2
222
222
0
0
3
234
206
2
0
4
220
230
1
0
5
213
217
4
1
6
248
212
3
0
7
219
215
0
0
8
254
223
1
0
9
230
221
2
0
10
233
226
1
1
11
237
216
5
0
12
246
215
3
0
Avg. 234.42 218.50 2.08 0.17
Plate
#
1
2
3
4
5
6
7
8
9
10
11
12
Avg.
0%
210
223
190
186
223
221
203
169
173
161
180
155
191.17
Percent of Honey
0.10% 1% 10%
150
0
0
170
0
0
167
0
0
180
1
0
149
0
0
158
1
0
163
0
0
151
0
1
155
1
0
168
0
0
165
0
0
171
0
0
162.25 0.25 0.08
Dunnett Test
0.01 Significance Level
T-critical = 3.58
T > T-critical = Significant Variation, Reject Null Hypothesis
Honey Infused Agar (E. coli)
HONEY
CONCENTRATION
0.01%
1%
10%
T-VALUE
1.412
9.321
9.331
Mv  MC
t
2MSE
nh
INTERPERATION
Insignificant
Significant
Significant
Honey Infused Agar (S. epidermidis)
HONEY
CONCENTRATION
0.01%
1%
10%
T-VALUE
2.133
31.132
31.389
INTERPERATION
Insignificant
Significant
Significant
Zone of Inhibition Data
Zone of Inhibition (mm, S.
epidermidis)
Zone of Inhibition (mm, E.coli)
Plate
#
1
2
3
4
5
Avg.
0%
11.5
12.5
12
13
12.5
12.3
Percent of Honey
1% 10% 30%
8.5
7
3.5
8
6
5
7
6.5
4.5
7.5
5
4.5
7.5
6.5
4
7.7
6.2
4.3
50%
1.5
2
1
1.5
1.5
1.5
Plate
#
1
2
3
4
5
Avg.
0%
11
13.5
12.5
13
13.5
12.7
Percent of Honey
1% 10% 30%
7.5
3.5
1.5
6.5
2.5
1
6
1
1.5
7
2
1
5.5
1.5
1
6.5
2.1
1.2
50%
0.5
1
0.5
0.5
0.3
0.56
Dunnett Test
0.01 Significance Level
T-critical = 4.17
T > T-critical = Significant Variation, Reject Null Hypothesis
Zone of Inhibition (mm, E.coli)
HONEY
CONCENTRATION
1%
10%
30%
50%
T-VALUE
39.49
97.52
73.25
77.32
Mv  MC
t
2MSE
nh
INTERPERATION
Significant
Significant
Significant
Significant
Zone of Inhibition (mm, S. epidermidis)
HONEY
CONCENTRATION
1%
10%
30%
50%
T-VALUE
48.42
94.21
84.21
113.68
INTERPERATION
Significant
Significant
Significant
Significant
Conclusions
In conclusion, my hypothesis was partially supported.



In the pulse liquid exposure to honey experiment, the
different concentrations of honey had a significant
impact in reducing the number of E. coli and S.
epidermidis
In the honey infused agar experiment, 1% and 10%
honey significantly decreased the number of bacteria
while the 0.01% honey had an insignificant impact
in reducing the number of both bacteria
In the zone of inhibition experiment, all the
concentrations of honey tested had a significant
impact in reducing the number of both bacteria
Future Improvements/ Changes
Conduct more trials
 Test a greater variety of honey
concentrations

Future Extensions/ Experiments

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Use a different method to determine the amount
and type of bacteria present
Test different types of honey
Test the effect of honey on its ability to inhibit other
types of bacteria
Test the effect of sugar and salt on their abilities to
inhibit bacterial growth
Test the effects of honey on the bacteria over a
greater period of time