1.

0 Design

1.1 Research Question:

What is the inhibitory effect of various hydrogen peroxide concentrations (0.02%, 0.04%,
0.06%, 0.08%, and 0.1%) when added to pasteurized milk inoculated with Escherichia coli as
measured by plate count method after 24 hours incubation at 37C? Commented [AA1]: In the criteria it says that research
question should be restated and reffered to
Could you please make sure this is done effectively?
1.2 Introduction: Commented [AA2]: Redo intro:
o The effect not pasteurizing has to the immune
Milk is a beneficial fluid for the body containing a mixture of essential nutrients in different system
- Get it down to 12 pages!!
physio-chemical forms, which consequently makes it a perfect medium for stimulating Criteria:
- Scientific theory is used to describe and explain how the
microbial growth. E. coli, being one of the most common pathogens to be the cause of several chosen IV affects changes in the biological material being
dealt with
foodborne illnesses has especially appeared to be present in raw milk. E. coli causes infection -Explain why the chosen DV is a good measure of change
in the biological matter.
by secreting a particular protein that directs an immune enzyme to manipulate specific immune oI dont know how to explain this
responses. This process inhibits the survival of the infected immune enzyme further promoting
E. coli accumulation in the body.1 In terms of
environmental conditions, the growth and
accumulation of E. coli is stimulated by several
conditions particularly including temperature
and time. The optimum temperature for E. coli
growth is 37oC and the longer they are left to
grow the more it accumulates until it denatures. Commented [AA3]: I dont know what to include about
how time influences growth
Both conditions promote growth especially
when E. coli exists in its exponential phase, the
Figure 1: Growth curve for E. coli
growth period where cell division occurs the
most rapidly.2 The growth curve for E. coli is shown in figure 1.3

The current pasteurization techniques employed by developed countries are known to Commented [AA4]: Do you think I should include a
graph?
effectively eliminate microbial populations in milk, however, in many developing counties this Or should I include a graph on my own (that I measured
from the spectrophotometer)?
is often not the case. To have been brought up in India, a developing country, I know that these
highly qualified methods are usually not endured. When it comes to collecting milk produced
from many small farms, it has to be bulked and transported over considerable distances before

1
("How Pathogenic E. Coli Bacterium Causes Illness")
2
("Bacterial Growth Curve (Theory) : Microbiology Virtual Lab I : Biotechnology And Biomedical Engineering : Amrita Vishwa
Vidyapeetham Virtual Lab")
3
("Bacterial Growth Curve (Theory) : Microbiology Virtual Lab I : Biotechnology And Biomedical Engineering : Amrita Vishwa
Vidyapeetham Virtual Lab")
reaching a centre where it can be pasteurized. 4 There is usually a lack of advanced storage
facilities and inappropriate transportation for preserving locally produced milk. Furthermore,
the environmental conditions such as the high temperature in such countries promote rapid
multiplication of bacterial growth such as E. coli. Since, pasteurization seems to be less or not
practically feasible such areas, other alternate methods to prevent milk contamination have
been explored, including treatment with chemicals. Hydrogen peroxide is well known for its
bactericidal properties and it has been demonstrated that addition of hydrogen peroxide at
optimal concentration and temperature per unit volume of milk could be an alternative
substitute for pasteurization. 5 This is supported by many previous research, one particular
research that investigated the effectiveness of hydrogen peroxide as a preservative in treating
milk for cheese making (performed by Nagmoush (1949))6 found using 0.18% of hydrogen
peroxide significantly reduced bacterial growth. However, the Food and Agriculture Commented [AA5]: I am supposed to include every study
here and then link it in my discussion
7
organisation (FAO) has recommended that the maximum concentration of hydrogen peroxide Please check if this is done effectively.
Im trying to limit the size of the introduction because the
added to milk should be 0.08%. This is because research has found higher concentrations of page limit for this assignment is only 12 pages
hydrogen peroxide to lower the naturally present protein in milk and also decrease the
nutritional value of milk. This experiment is designed to investigate the optimal hydrogen
peroxide concentration required to prevent bacterial growth or reduce its population in milk,
which could provide inferences on the application of this method to prevent contamination of
milk during its transportation from farms to dairy industries in developing countries such as
India. Since E. coli is one of the most common pathogens present in milk, measuring its growth Commented [AA6]: The criteria says if relevant a
diagram could be included
against various concentrations of hydrogen peroxide is considered a good measure to the -DO you think I should include a diagram based on any
theory?
determine the efficacy of hydrogen peroxide as an efficient method in avoiding microbial
populations in milk.

1.3 Hypothesis: Operational hypothesis: It is hypothesised that the hydrogen peroxide

possesses bacteriostatic effect and its inhibitory effect could be directly proportional to the
increase in its concentration and decrease in E. coli colonies in milk. Hydrogen peroxide shown
to kill bacteria by oxidizing their cell walls and disrupting their chemical structure preventing
them from further growth. Hence increasing the concentration of hydrogen peroxide will Commented [AA7]: Could you please introduce the
concept of hydrogen peroxide effect on bacteria in the
decrease bacterial growth of E. coli8 introduction. Because everything I talk about in the
assignment should be introduced only in the introduction.
-Should I include the null hypothesis?
-

4
("An Alternative Method Of Milk Treatment") Commented [Ak8R7]:
5
("An Alternative Method Of Milk Treatment")
6
(Nagmoush, 1949) Commented [Ak10]: -Should I include the null
7
("An Alternative Method Of Milk Treatment") hypothesis?
8
(Mentel R)
 1.4 Variables
Variables Impact upon the investigation
Independent variables:
Different concentrations
of hydrogen peroxide
Concentration of hydrogen peroxide
could be directly proportional to the
inhibitory effect on bacterial growth.
The different
Higher concentrations of hydrogen
concentrations of
peroxide might have higher
hydrogen peroxide
bactericidal effects on E. coli.
chosen are 0.02%,
0.04%, 0.06%,
0.08%, and 0.1%.
Dependent variables:
The number of E. coli colonies that have
Inhibitory effect of
grown after treatment with hydrogen
different concentration
peroxide is the ideal parameter to assess the
of hydrogen peroxide
inhibitory effect of hydrogen peroxide on
measured by
bacterial growth. This is also because E.
enumerating the E. coli
coli is one of the most apparent milk borne
colonies
pathogen.
It is important that the E. coli sample
used is in its exponential phase, this is
when its growth rate reaches its maximal
rate where it divides and increases
Control variables: logarithmically. It is essential that the E.
Organism coli cells are not dead so that the effect
Temperature of hydrogen peroxide on it could be
Time determined accurately. Secondly, the
Chemicals amount of E. coli immersed in the agar
Sterility of the materials plates for every trial should be constant
used in order to accurately investigate the
effect of hydrogen peroxide.
Temperature directly influence the
growth of the bacteria. Bacteria such as
E. coli grow and reproduce faster at
higher temperatures and this growth rate
Steps to control and measure the variables
The values ranging from 0.02%-0.08% has
been chosen because this range of hydrogen
peroxide concentration has been found to be
the safest amount to add to the milk. Adding
higher concentration of this has been shown
to disrupt the nutrition value of milk. The
value of 0.1% was used to demonstrate the
extent of bacteria hydrogen peroxide can kill
if it goes beyond the safest amount to add in
milk.
E. coli growth was measured by using the
plate count method in colony forming unit
(CFU). The solution with E. coli sample will
be initially diluted through serial dilution to
reach a concentration of 10-5 in order to
avoid bacteria from excessively covering the
plate so that the bacterial count doesnt
exceed 500, which is statistically acceptable.
A UV/VIS spectrophotometer will be used
to ensure that the E. coli sample used is at its
exponential phase. The spectrophotometer is
a quick method of determining cell growth
rate of an organism. This will be done by
measuring the sample at the optical density
of a sample at 600 nanometres (OD600)
which is the middle of the visible
wavelength, hence easy to produce as a
measure. The rate of growth will be
measured at its maximal phase during
exponential phase which corresponds to 0.5-
0.6 OD6009. Finally, the amount of E. coli Commented [Ak9]: Is this right?
inoculated into all the agar plates was 0.5 ml
using a pipette after serial diluting the
sample.
 decreases as temperature decreases.
Hence it is important to ensure that the
temperature is kept constant throughout
the growth analysis of E. coli.
Time is also another important factor
that affects the growth of E. coli. Since
the environment of the incubator stable
and with-bearing, bacterial growth
Quality of the chemicals and sterility of
the materials are important to ensure
accuracy of the experiment. The agar
plates will be sterile and distilled water
will be used.
Temperature will be maintained by
incubating the culture and the plates in an
incubator at 37oC with in-built thermostat
that eliminates temperature fluctuations.
37oC is within the ideal range of
temperature for bacterial growth.
Time will be noted just after the addition of
hydrogen peroxide and just after the swabbed
plates are incubated. The time the E. coli will
be kept in the incubator after plating will be
for 24 hours.

Uncontrolled Contamination of either the E. coli
variables:
culture and/or the pasteurized milk
Contamination of would directly affect the results of the
sample while handling.
experiment. Contamination mainly
includes airborne microorganisms that
may themselves grow in the agar plate
and affect the analysis of data that is
meant to calculating only the growth of
E. coli.
The work area will be cleaned with 70 %
ethanol and all materials used for the
inoculation of E. coli culture are sterile.
The agar plate, bacteria and milk will handled
closer to a flaming Bunsen burner.
The agar plates should be tightly sealed using
a sticky tape after the inoculation of solution.
To further prevent contamination, test tube
bottles with screw lids will be used instead of
standard test tubes.

1.5 Apparatus: Commented [Ak11]: Check all the no. of equipment used

- Incubator
- 70 % ethanol for sterilization of work place
- Labels
- 90 mL of Pasteurized milk
- Bunsen burner
- 1 x 50 cm3 measuring cylinder ( 1 cm3)
- Black permanent marker
- 8x 5.0 cm3 sterile pipettes ( 0.5 cm3)
- Safety glasses
- 3% hydrogen peroxide solution
- Laboratory coat
- Pre-prepared nutrient broth with E. coli culture
- 17x Nutrient agar plates (NA)

9
("Bacterial Growth Curve (Theory) : Microbiology Virtual Lab I : Biotechnology And Biomedical Engineering : Amrita Vishwa
Vidyapeetham Virtual Lab")
- Gloves - 2x Inoculation loops
- Sticky tape
- 10x sterile cotton swabs
- Timer
- 10x Test tube bottles with screw seals
- UV-visible spectrometer with cuvettes.
- Scientific calculator

1.6 Method: Commented [AA12]: Include uncertainties

1. Wipe work area with 70 % ethanol, arrange materials gathering the nutrient agar (NA) plate
with pre-prepared nutrient broth with E. coli culture, test tube bottles, hydrogen peroxide
solution, agar plates, sterile pipettes and cotton swabs. Ensure the E. coli culture is not
contaminated by handling it with caution, by wearing laboratory safety equipment (a
laboratory coat, gloves and safety glasses) and working at safe proximity to Bunsen burner.
2. Measure 10 ml of pre-prepared nutrient broth using a plastic pipette with E. coli culture and
add it to a test tube bottle and allow solution to grow for 20 mins at 37 oC in an incubator
and then shaking the solution 10 mins before taking it out (this is done in order to avoid
bacterial settlement on the bottom of the test tube bottle)
3. Measure the absorbance of the E. coli solution at OD600 using a spectrophotometer when
the OD reaches 0.5-0.6. Before inserting the sample of E. coli, calibrate the
spectrophotometer with distilled water.
4. Perform a serial dilution of the E. coli solution to determine the initial concentration of E.
coli in broth solution by spreading them on an agar plate and incubating them at 37oC for
24 hours. Perform this process by taking 1 ml of E. coli solution and adding it to 9 ml of
broth solution using a plastic pipette. This process should be performed 4 more times to get Commented [Ak13]: Im so confused about serial dilution
of ecoli
a dilution of 10-5. Could you please change this?

5. Measure and add 1 ml of bacterial suspension using a pipette and 29 ml of milk using a
measuring cylinder and mix well into each of the 6 test tubes bottles. Label these bottles
according to the different hydrogen peroxide concentrations (0.02%, 0.04%, 0.06%, 0.08%
and 0.1%). Label the last bottle 0%, this acts as a control and should only consist of bacteria
and milk without hydrogen peroxide.
6. Then calculate hydrogen peroxide volume for each concentration for 30 mL milk and
bacteria solution using V1 C1 = V2 C2 (see table 1 for the calculations) for 5 of the test
tube bottles.
7. Mix all the bottles well and keep it at 37 C for 4 hours with periodical shaking at every
hour.
 8. Meanwhile, label 18 agar plates by allocating 3 agar plates for each condition of different
hydrogen peroxide concentrations (0%, 0.02%, 0.04%, 0.06%, 0.08% and 0.1%). Repeat
these conditions as 3 trials in order to limit random errors as much as possible.
9. After 4 hours of incubation, take 0.5 ml from each test tube bottle by pipette and spread
evenly using a cotton swab in the correspondingly labelled agar plates. Then immediately
seal the agar plates with sticky tape.
10. Keep all the test tube bottles as well agar plates in the incubator at 37C for 24 hours.
11. After the agar plates are incubated take them out and calculate the number of bacterial
colonies that grew on each plate using the formula: No. of bacteria = No. of colonies
dilution factor (Table 2).
12. After calculating and recording the number of colonies for each trial clean materials and
clean the workplace with ethanol once again.

Safety and Precautions:

Since hydrogen peroxide could be a highly corrosive chemical, the acid should be handled
extremely carefully. E.coli similarly should be handled with extreme care as it may be
accidentally contaminated with pathogenic bacteria in the environment. Before and after
conducting the experimental work area should be wiped with 70% ethanol. Protective
equipment such as safety glasses, laboratory coat and gloves should be worn when handling
both hydrogen peroxide and E. coli. Since the hydrogen peroxide will be dilute, disposal of it
after the experiment wont cause any environmental hazard, however it must be disposed
safely. After conducting the experiment, all the agar plates that were inoculated with E. coli
should be disposed in a biohazard bag. Commented [AA14]: Please make sure if all this is right
and add any detail if necessary

Table 1. Calculation to determine the volume of different concentrations of H2O2 to be added

Concentration of H2O2 (%) Calculation Volume to be added (ml)* Formula: Commented [AA15]: Include uncertainty
0.02 (0.02 30) 3 0.2
0.04 (0.04 30) 3 0.4 V 1 x C1 = V2 x C2
0.06 (0.06 30) 3 0.6 Commented [AA16]: I am confused about the formula,
0.08 (0.08 30) 3 0.8 V2 2 could you please change what V1, V2 and C1 and C2 means?
V1 =
0.1 (0.1 30) 3 1 1

V1 = Volume of hydrogen peroxide to be drawn from the stock (3% hydrogen peroxide)
V2 = Required final volume (30 ml)
C1 = Concentration of the stock (3 % Hydrogen peroxide)

C2 = Required concentration of hydrogen peroxide (0.02, 0.04, 0.06, 0.08, 0.1 %)

 Table 2 Sample results table:

Number of E. coli colonies on the plate CFU (mL) x 10-4

Trials 1 2 3
0.00
Concentration 0.02
of H2O2 (%) 0.04
0.06
0.08
0.1

Figure 2- Diagram of experimental set up

2.0 Data collection and processing

2.1 Presenting raw data
Calculation of the number of colonies: After incubation, colonies grown on the agar plates
were manually counted and organisms per 0.5 ml of the sample were calculated using the
formula. CFU = No of colonies counted/ (dilution factor volume of culture inoculated). Commented [Ak17]: How to show an example of
concentration
Please could you show an example?
Table 3 Raw data of the number of colonies on the plate at different H2O2 concentrations. I dont remember the dilution factor

Number of E. coli colonies on the plate CFU (mL) x 10-4

Trials 1 2 3
0.00% 383.00 390.00 376.00
Concentration 0.02% 284.00 300.00 320.00
of H2O2 (%) 0.04% 183.00 200.00 170.00
0.06% 73.00 55.00 67.00
0.08% 7.00 10.00 8.00
0.1% 1.00 2.00 1.00
Table 4 Observation of E. coli growth formed in the agar plate at different H2O2 concentrations

Concentration
of H2O2 (%) Observations
0.00 This plate had the most number of E. coli colonies. The colonies formed were thick and an
even growth of the bacteria throughout the plate was observed. Some of the colonies were
less pigmented than others but overall this plate had formed huge visible clustered colonies
due to the absence of hydrogen peroxide. This plate was especially challenging to
determine the number of bacterial colonies as this plate appeared to have most growth
compared to others.
0.02 Since, hydrogen peroxide was added, there was notable difference when comparing this to
the plate with no added hydrogen peroxide. There were still many colonies formed
throughout the plate. Colonies were especially clustered around the centre.
0.04 In this plate there were few clusters of colonies formed and were separated out from each
other. Spots of colonies were spaced away from each other.
0.06 There seemed to be a significant decrease in the number of colonies compared to the plate
with 0.04% hydrogen peroxide, upon observation.
0.08 Through observation, this plate was found to have similar amount of colonies to the plate
with the concentration of 0.04% hydrogen peroxide. There seemed to only slightly fewer
colonies. There were no clusters of colonies present and only few spots of colonies were
present that were separated and distanced from each other.
0.10 There were only very few colonies of E. coli formed for this particular concentration of
hydrogen peroxide in each of the trials. The colonies were largely distanced from each
other and were not strongly pigmented.

2.2: Processing Raw Data

The experimental data collected can be summarised by using statistical measurements such as
mean and standard deviation for the number of colonies of E. coli. on the plate with different
concentrations of hydrogen peroxide. The raw data was converted into log values before
performing further calculation:

Table 3 Logarithmic concentration of E. coli on the plate at different hydrogen peroxide concentrations

Logarithmic concentration of E. coli on the plate CFU (mL) x 10-4

Trials 1 2 3
0.00% 6.58 6.59 6.58
Concentration of 0.02% 6.45 6.47 6.51
Hydrogen 0.04% 6.26 6.30 6.23
Peroxide (%) 0.06% 5.86 5.74 5.83
0.08% 4.85 5.00 4.90
0.1% 4.00 4.30 4.00
 1. The mean of a set of data is the sum of all the values divided by the number of values. The
average of each trial was calculated by the addition of the results of each trial divided by
the total number of trials.

The calculation of the mean logarithmic number of E. coli colonies on the plate with 0.02%
hydrogen peroxide is shown below, as an example:

6.45 +6.47 +6.51

0.2% = = 6.48
3

2. Standard deviation is a measure of dispersion which gives an indicatType equation here.ion

of the extent the data values are related to the mean. In this case, it is calculated to show
how far the value of each trial is spread out above and below the mean. While a low
standard deviation suggests that the values are close to the mean, high standard deviation
values indicates that the values are scattered over a broader range of values. Standard
deviation was calculated by Microsoft Excel.

Table 5 Mean and standard deviation values of the logarithmic concentration of E. coli at different
hydrogen peroxide concentrations

Concentration of Hydrogen Mean logarithmic Standard deviation of the

Peroxide (%) concentrations of E. coli at logarithmic concentration of
different hydrogen peroxide E. coli at different hydrogen
concentrations peroxide concentrations
0.00 6.58 0.01
0.02 6.48 0.03
0.04 6.26 0.04
0.06 5.81 0.06
0.08 4.92 0.08
0.10 4.10 0.17

3. The percentage reduction of the mean number of E. coli colonies with regards to different
hydrogen peroxide concentrations was calculated to give a better understanding of the
effect of hydrogen peroxide on the growth of E. coli.
For this the mean of the raw data was calculated rather than using the log values. The same
steps taken to calculate the mean of the log values were used to calculate the mean of raw
data. The table below shows the mean concentration of E. coli at different hydrogen
peroxide concentrations:
 Concentration of Hydrogen Peroxide (%) Mean concentrations of E. coli at different
hydrogen peroxide concentrations
0.00 383.00
0.02 301.33
0.04 184.33
0.06 65.00
0.08 8.33
0.10 1.33

After calculating the mean, the percentage reduction was calculated using the formula
below:
( ) 100
=

A = the mean concentration of E. coli before the addition of hydrogen peroxide (383.00)
B = the mean concentration of E. coli after the addition of a certain concentration of hydrogen
peroxide.

The calculation of the percentage reduction of mean number of E. coli colonies on the plate
with 0.02% hydrogen peroxide is shown below, as an example:
(383.00301.33) 100
0.02% = 383.00
=
21.32%

Table 6 The percentage reduction of the mean logarithmic number of E. coli colonies with regards to different
hydrogen peroxide concentrations

Concentration of Hydrogen Peroxide (%) Percentage reduction of the mean

concentration of E. coli colonies (%)
0.02 21.32
0.04 51.87
0.06 83.03
0.08 97.83
0.10 99.65
 2.3 Presenting Processed Data:

Figure 3 Bar graph of the mean logarithmic concentration (with standard deviation represented as vertical error bars) and percentage
reduction of E. coli on the plate at different hydrogen peroxide concentrations: 0%, 0.02%, 0.04%, 0.06%, 0.08% and 0.10%

The bar graph in figures 3 uses the data from table 5 and the line graph in the same figure uses
the data from table 6. The X axis of the graph is the different hydrogen peroxide concentrations
(0%, 0.02%, 0.04%, 0.06%, 0.08% and 0.10%). Figure 3 consists of two Y axis, where the bar
graph represents the logarithmic concentration of E. coli, the line graph represents the
percentage reduction of the concentration of E. coli on each of the plates.

3.0 Conclusion and Evaluation:

3.1 Conclusion:

The bar graph in figure 3 shows that there is a negative correlation between the amount of E.
coli colonies formed in the agar plate and the different hydrogen peroxide concentrations. It
can be seen from the results in bar graph that an assumed linear relationship has an R2 value of
0.90 (calculated in MS excel) demonstrating a strong linear correlation. This suggests that as
the concentration of hydrogen peroxide increases the amount of E. coli colonies formed
decreases. The bactericidal effect of hydrogen peroxide is further clearly demonstrated in the
line graph in figure 3. The line graph shows that as the hydrogen peroxide concentration
increases percentage reduction of the E. coli colonies increases.
As expected, hydrogen peroxide had demonstrated its bactericidal effect by inhibiting the
growth of bacteria. Increasing the concentration of hydrogen peroxide, decreases the growth
of E. coli colonies. Highest number of bacterial colonies was observed in the lowest
concentration of hydrogen peroxide. Therefore, the results acquired support the operational
hypothesis that the inhibitory effect of hydrogen peroxide is directly proportional to the
increase in its concentration and decrease in E. coli colonies in milk.

3.1.2 Describe Findings:

From table 5 in the processing raw data section and bar graph in figure 3 it can be seen that in
trials with low hydrogen peroxide concentrations the number of E. coli colonies was relatively
high. For the plate with 0% hydrogen peroxide, the mean logarithmic number of E. coli
colonies was 6.58. In contrast this amount could be compared to the plate with 0.1% hydrogen
peroxide wherein which there was a logarithmic growth of 4.10, suggesting this to be the most
effective concentration of hydrogen peroxide to inhibit bacterial growth compared to other
concentrations used.

The analysis of the standard deviations suggests that the values are low, this implies that the
data is clustered closely around the mean suggesting that the data is generally reliable. When
analysing all trials from figure 2, the mean number of E. coli colonies decrease with each
condition from 0.0% to 0.1% concentration of hydrogen peroxide. The mean logarithmic
number of colonies formed for these conditions were 6.58, 6.48, 6.26, 5.81, 4.92 and 4.10 and
the mean percentage reduction of the concentration of E. coli were 21.32%, 51.87%, 83.03%,
97.83% and 99.65% respectively. The high decrease in the number of E. coli colonies in the
0.04% and 0.06% hydrogen peroxide concentration with a percentage reduction of 83.03% to
97.83% was surprising as it suggests the huge difference of 31.16% made by increasing the
hydrogen peroxide concentration only by 0.02%. These findings correlate with the observation Commented [Ak18]: [Comment on the qualitative
data:
made of the two plates, where there appeared to be significantly larger sizes as well as number -What impact might the qualitative data of had upon
the findings?
of E. coli colonies in the 0.06% plate compared to the plate with 0.04%. The observation of the -Does it make the measurements likely to be
skewed, i.e. all too large or too small?
plate with 0.1% was the most surprising as there were very few colonies formed with extremely -Does the qualitative data indicate a possible
reason for the natural variation seen in the data?
light pigmentation. The qualitative observation of the plates plays a significant role upon the -Does the qualitative data make it more difficult to
come to a valid conclusion?]
findings as it was noticed that as the concentration of hydrogen peroxide increased, the E. coli
colonies appeared to decrease in pigmentation. This observation of the decrease in
pigmentation in the appearance of the bacterial colonies cannot be demonstrated through
quantitative data hence it is additionally important to consider qualitative data.
 3.1.3 Conclusions based on the data:

The experiment conducted showed that the 0.1% concentration of hydrogen peroxide is the
most effective in inhibiting bacterial growth compared to other concentrations used, the
percentage of bacterial reduction was significantly high, showing 99.65% reduction (as shown
in figure 4), suggesting that this particular concentration inhibits bacterial growth to a large
extent. However, since the FAO10 have recommended that the maximum concentration of
hydrogen peroxide added to milk should be 0.08%, using 0.1% of hydrogen peroxide may not
be the most beneficial in terms of health when added to milk. The concentration of 0.08% still
demonstrates the bactericidal effect of hydrogen peroxide, by largely reducing the bacterial
growth, supported by the 97.83% reduction of bacteria (as shown in figure 4).

Hence from the study conducted, it can be concluded that hydrogen peroxide can be used as a
disinfectant to prevent microbial growth. But only in a minimal concentration to preserve milk
from microbial contamination for a certain period, both during preparation and transportation
process. The hydrogen concentration of 0.08% has been supported to be effective in both
inhibiting bacterial growth to an extent but to also sustain the nutritional value of the milk.

3.1.4 Findings and conclusions compared to literature:

In their research into the effectiveness of hydrogen peroxide as a preservative for milk,
Nagmoush (1949) showed that the bacterial count of milk was significantly reduced by 98.6%
when used 0.18% concentration of H2O2 compared to untreated raw milk11. The percentage
reduction of bacterial growth acquired in this study (98.6%) is slightly lower than the
percentage reduction acquired by the current experiment (99.65%). This might be because the
study conducted by Nagmoush (1949) had allowed the bacteria to grow for 24 hours and 48
hours whereas the current study allowed the bacteria to be incubated for only 24 hours. Time
plays an important factor in bacterial growth; the longer the bacteria are left to grow under
stable conditions the more the colonies of bacteria are developed until growth reaches a plateau.
The addition of extra time in their study allowed the bacteria to grow to a large extent hence
challenging the inhibitory effect of hydrogen peroxide. Limiting the time for bacteria to grow
would possibly have shown a higher percentage reduction of bacteria when hydrogen peroxide

10
("An Alternative Method Of Milk Treatment")
11
(Nagmoush, 1949)
 is added. Despite this difference, the arrived conclusion for both studies correlate to a large
extent hence it can be said that literature supports the conclusion of this current experiment that
hydrogen peroxide largely inhibits growth of bacteria and therefore is an effective method to
treat milk.

3.2 Evaluation

Source of error Suggested Improvements

The solution composed of E. coli, milk and different To improve this source of error thorough measurement of the
hydrogen peroxide concentrations were added solution being added to the agar plate must be sure. This will
accordingly to agar plates. Although it was attempted decrease the error from the difference in amount of bacteria and
to keep theThe evaluation
amount should
of solution be in this
constant, structure:hydrogen peroxide added. Instead of using a standard pipette, to
the specific
amount may differ. This difference in amount of ensure accuracy a micropipette could be used to add the solution
Equipment errors
bacteria and concentration of hydrogen peroxide may to the agar plates in order to lower the range of uncertainty. Also,
Piece of Uncertainty Smallest % % error > 5%? suggested improvement
cause significant difference in amount of bacteria another step that could be taken to improve this error is to mix the
equipment amount error possible effect on
formed in the
andplate.
usageThis error was due to using a 5 solution well before
measured adding it to the agar plate. This will ensure
data and
magnitude
cm3 pipette when measuring only 0.5 cm3 of the that the bacteria and the of
hydrogen peroxide is evenly mixed
weakness/error?
solution. within the solution and hence be able to plate similar amount of
Another limitation regarding the amount of solution bacteria on each plate including the respective concentration of
added to the plates was the use of cotton swabs for hydrogen peroxide. Finally, instead of using cotton swabs as a
inoculation. The cotton swabs could have absorbed the method of inoculation, a glass rod or an inoculating loop could be
solution. This could cause an error in equally used instead so the solution isnt absorbed.
distributing the solution in each variable.
When transforming the solutions of the different Although there were already steps taken to ensure contamination
Evaluation of experimental errors
variables onto the agar plate, it was exposed to open by the air is limited, further contamination could be reduced by
weakness/source of error possible effect on data and suggested improvement
air and could have possibly been contaminated. magnitude
This considering more improvements. The most crucial thing is to
of weakness/error
Independent
suggests that variable
bacteria from air may have been clean the lab station with a disinfectant. Before conducting the
[Appropriate range
transformed in the agar plate. When analysing the current experiment, the work place was wiped with 70% ethanol.
chosen?]
number of E. coli
[Manipulated
colonies, theeffectively?]
contaminant may have But using other equally or more concentrated disinfectant is
Dependent variable
also been calculated together and affect the result of recommended. Furthermore, when transferring solution, both the
[Method of measurement
the research. effective?] test tube containing the solution and the agar plate must be open
[Precise enough equipment for the minimum amount of time possible to limit airborne
used?]
Control variables contaminants from settling in the agar plate. To further improve
[Was each control variable this, all windows and door should be closed to decrease draughts
and sudden motions that may disturb the air flow since wind
carries such contaminants. To reduce this error, a flaming Bunsen
burner was close by when handling bacteria; this draws the air
currents upwards reducing the wind.
 effectively controlled or
not?]
Uncontrolled errors
[Did std dev / qualitative
data indicate any impact of
biological variation?]
[Was effective monitoring
carried out to minimize the
impact of these variables?]
[Could these errors be
controlled?]
Qualitative Data
[Observations might well be
used implicitly in other
sections]
[How might have the
observations made affected
the investigation?]

References:

"An Alternative Method of Milk Treatment". Fao.org. N.p., 2017. (Accessed: 9 April 2017):
http://www.fao.org/docrep/v6200t/v6200T0t.htm

"Bacterial Growth Curve (Theory) : Microbiology Virtual Lab I : Biotechnology And

Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab". Vlab.amrita.edu. N.p.,
2017. (Accessed: 9 April 2017):

"Growth". Academic.pgcc.edu. N.p., 2017.(Accessed: 20 April 2017):

http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

"How Pathogenic E. Coli Bacterium Causes Illness". ScienceDaily. N.p., 2017. (Accessed: 12
April 2017):
https://www.sciencedaily.com/releases/2011/03/110314111222.htm

Mentel' R, et al. "[Virus Inactivation By Hydrogen Peroxide]. - Pubmed -

NCBI". Ncbi.nlm.nih.gov. N.p., 2017. (Accessed: 20 April 2017):
https://www.ncbi.nlm.nih.gov/m/pubmed/203115/
Nagmoush, Mounir Ramzi, "A Comparative Study of Hydrogen Peroxide in Treating Milk for
Cheddar Cheese Making" (1949). All Graduate Theses and Dissertations. Paper
4828.(Accessed: 11 April 2017)