Program:

School of Biosciences

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Student Name and ID:

Gan Xin Yue (0328481)

Lai Xue Min (0328475)
Marliah Abdul Ghanie (0331520)
Nicholas Gan Yu Xing 0327633
Nur Syafiqah Bt Husain (0333589)
Teoh Kai Ying (0329574)

Email: marliagan@yahoo.com Contact No: 0168307552

Number and title of experiment:

Practical 4 – Thermal destruction of microorganism

Experiment group Date of experiment: 4th October 2018

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Signed: Date: 18th October 2018

Aim

1. To understand more about heat process testing in order to inactivate microorganisms

in food production by controlling its parameter.

2. To use data obtained to generate survival curves as well as determining the

logarithmic number of survivors and linear line.

3. To calculate D and Z values using data obtained from the experiment.

4. To determine potential and possible sources of laboratory errors that might affect the

result of the experiment.

Introduction

Heat treatment is one of most widely used methods for destruction of spoilage and

pathogenic microorganisms (Moats, 1971) and microbial exposure to heat has two parameters

which are temperature and exposure time. The theory that thermal death of microorganisms

follows the first order kinetics which the rate is proportional to the number of molecules

presents (Moats, 1971). This experiment is using thermal destruction in standard test tubes in

water baths to determine heat destruction of non-pathogenic Escherichia coli and destruction

of microorganism by heat has been described as log-linear in nature (Juneja, Snyder &

Marmer, 1997) which have D-value and Z-value for determination. D-value is the value of a

parameter of sterilization required to reduce the population of a known microorganism by 1-

log or 90% of the population whereas Z-value is defined as the number of degree Celsius or

Kelvin required to change a D-value by one factor of ten, in practical sense, Z-value is a

measure of how susceptible a spore population is to changes in temperature (Sandle, 2013).

Results

Table 1: Dilutions plated at different processing temperature and their colony forming unit.

Time Dilutions Plated & Colonies Calculated Surviving log

(mins) Counted Count CFU/ml (CFU/ml)

0 10^-5 157 1.57 x 10^8 8.19

10 10^-4 189 1.89 x 10^7 7.28

20 10^-2 61 6.1 x 10^4 4.79

30 10^-2 154 1.54 x 10^5 5.19

40 10^-1 74 7.4 x 10^3 3.87

50 10^-2 192 1.92 x 10^5 5.28

60 10^-2 49 4.9 x 10^4 4.69

 Figure 1.0: Line Graph of log of Colony Surviving (CFU/ml) against time from 0 to 60

minutes.

Y = -0.5507x + 7.8157

Substitute y with 6,

6 = -0.5507x + 7.8157

6 - 7.8157 = -0.5507x

-1.8157 = -0.5507x

3.2971 =x

Substitute y with 7,

7 = -0.5507x + 7.8157

7 - 7.8157 = -0.5507x
-0.8157 = -0.5507x

1.4812 =x

D value = 3.2971 - 1.4812

= 1.8159mins

Or,

Using the equation y = -0.5507x + 7.8157,

D value = -1/ slope of equation

D value = -1/ (-0.5507)

= 1.8159 mins

Figure 2.0: Line Graph of log (D) value obtained against Temperature of 54°C to 62°C.
Z value = -1/ (slope of equation)

= -1/(-0.56)

= 1.79°C

Table 2: D values (minutes) at different temperature (°C) and the Z value calculated.

Temperature (°C)

----------------------------------- 54°C 58°C 62°C

D value (minutes) 156.25 1.8159 11.80

Z value (°C) 1.79

Discussions

The thermal destruction curves of non pathogenic Escherichia coli K12 used a heat

process testing depends on a time-temperature relationship and can be evaluated for

microbiological survival (Crespo and Ockerman, 1977). This experiment went with unusual

events that have introduced some experimental errors. The sources of error were the

contamination during spreading plate techniques, inaccuracy of micropipetting skills and

environmental conditions. The experimental errors that could be associated with this type of

experiment is human error. For instance, the spreading plate work does not done near the

flame causing contamination to it. To eliminate this error, the work must always be done near

the flame and prevent talking while spreading plate. Moreover, random error also one of the

experimental error that could associated with the experiment. As during dilution, some

droplets of the culture might left in the micropipette. Thus, this will lead to an inaccurate in

results. The thermal destruction curve (TDC) obtained by plotting the group data on a semi–

log plot with log cell numbers (log CFU/ml) on the logarithmic y-axis and time (in minute)

on the linear x-axis. As can be seen from the graph, the relationship of log cell numbers (log

CFU/ml) versus the time (minutes) is roughly linear. D-value is the time taken to reduce

bacterial density by 90% which also known as decimal reduction time. (Abedon, 2011).The

dose required to inactivate 90% of the initial population in this experiment calculated by

using the equation. Two value of which is 6 and 7 has been substituted respectively into the

Equation 1 which gives 1.8159°C as a final D-value .From the final D-value,a thermal death

curve (TDT) is generated by plotting the log D-values (log D) on the logarithmic y-axis and

heating temperature (in Celsius) on the linear x-axis.As can be seen,from the graph above, the

relationship of the log D-values (log D) versus the temperature is also linear. The Z- value is

defined as number of degrees Celcius required to change a D value by one factor of 10

(Sandle, 2016). The Z –value measured of how susceptible a spore population is to changes
in temperature calculated by using the Equation 2. The slope of equation is substituted and

gives final Z- value which is 1.79 °C.

1. What types of experimental errors could be associated with this type of experiment? What

could you do differently to eliminate these errors?

The experimental errors that could be associated with this type of experiment is human error.

For instance, the spreading plate work does not done near the flame causing contamination

to it.To eliminate this error, the work must always be done near the flame and prevent talking

while spreading plate. Moreover, random error also one of the experimental error that could

associated with the experiment. As during dilution, some droplets of the culture might left in

the micropipette. Thus, this will lead to an inaccurate in results.

2. Why would a sealed tube that is completely submerged in the water bath yield more

accurate results than using a test tube?

The tube should have the constant temperature and no temperature gradient between the

samples submerged in the water bath to ensure that the microorganisms in the tube can

reflect their resistance towards thermal. The sealed tube that is completely submerged in the

water bath yield more accurate results than using a test tube because test tube unable to

submerge completely into the water bath.

3. Were your thermal destruction curves straight line? Assuming that the nonlinearity of your

curves was not due to experimental error, what could have caused this variation?
The thermal destruction curves is non linear. The reason which caused the variation are that

cells clumping together, as well as the presence of endospores of different innate resistances

in a given population (Ball & Olson 1957). Moreover, water activity is an environmental

error that caused this variation. It affects the thermal resistance of microorganisms.

According to researchers, microbial growth in food systems in relation to the activity of

water in the system (Syamaladevi et.al 2016).

4. If you wanted to repeat this experiment and look at the amount of injured cell levels, how

could you perform that experiment? Would you need to adjust the dilutions that you plate?

Why or why not?

I might follow the same time and temperature of this experiment to look at the amount of

injured cell. The result that obtained from the experiment is the higher the dilution factor, the

lower the colonies count, hence the dilutions factor of 54°C and 58°C may keep them same. I

will reduce the dilution factors for 62°C as the D value of 62°C higher than the D value of

58°C. The graph of log D should be decrease when temperature increased.

5. Using the D values from your destruction curve, how long would you have to heat the

TSA to assure a 3D destruction? A 5D destruction? What if the pH of your medium was at

pH 4? Would you expect to see differences? Why and why not?

D value is the value of a parameter of sterilisation required to reduce the population of a

known microorganism by 90% of the population. Through D58 values from destruction

curve, the time taken needed to heat the TSA to assure a 3D destruction is 5.45 minutes while
for 5D is 9.08 minutes. In pH 4 creating an acidic environment for the culture as E. coli K12

can survive in an low acidic environment. Thus, there might be no expect to see any

differences.

6. Many people are criticizing the 12D concept, because it is pure extrapolation. Why would

it be?

The sample may contain some amount of microorganisms before apply heat on it. Therefore,

the thermal process usually criticize the 12 D concept or known as 12 log cycle reduction

because it may obtain the needed sterility assurance level and established as a safety (James

2007).
Conclusion

From the experiment, it can be determined that the survival curves as well as determining the

logarithmic number of survivors and linear line can be determined using data obtained from

the experiment. The D values which is the the time at particular temperature required to

reduce known number of microorganism by 90% are determined to be 156.25 at 54°C,

1.8159 at 58°C and 11.80 at 62°C while the Z values which is the change in temperature

required for thermal destruction was determined to be at 1.79 °C. There are some

experimental errors encountered in this experiment which includes contamination during

spreading plate techniques, inaccuracy of micropipetting skills, and environmental conditions

environmental error which causes slight deviation in the results. The experiment has provided

clearer understanding about heat process testing to inactivate microorganisms in food

production by controlling its temperature and exposure time.

References

1. Ball, C.O. and Olson, F.C.W., 1957. Sterilization in food technology. Theory,

practice, and calculations. Sterilization in food technology. Theory, practice, and

calculations.

2. James, PA 2007, ‘Understanding Overkill Sterilization: An End to the Confusion’,

Pharmaceutical Technology, no. 2, viewed 17 October 2018,

<http://www.pharmtech.com/understanding-overkill-sterilization-end-confusion>.

3. Juneja, VK, Snyder, OP Jr & Marmer, BS 1997, ‘Thermal destruction of Escherichia

coli O157:H7 in beef and chicken: determination of D- and z-values’, International

Journal of Food Microbiology, vol. 35, no. 3, pp. 231-237.

4. Moats, WA 1971, ‘Kinetics of Thermal Death of Bacteria’, Journal of Bacteriology,

vol. 105, no. 1, pp. 165-171.

5. Sandle, T 2013, ‘Biological Indicators’, Sterility, Sterilisation and Sterility Assurance

for Pharmaceuticals, pp. 263-278.

6. Syamaladevi, R.M., Tang, J., Villa‐Rojas, R., Sablani, S., Carter, B. and Campbell,

G., 2016. Influence of water activity on thermal resistance of microorganisms in

low‐moisture foods: a review. Comprehensive Reviews in Food Science and Food Safety,

15(2), pp.353-370.