Group members:

Nkosiyethu Diya 223035689

Onke Khiwa 202206767

Simnikiwe Mlakalaka 202220369

Mabinza Thembisa Asanda 202246831

Tlotitso Mphomela 202101291

Sinobom Ntshentshe 203041391

Anathi Onke Nyikiza 202004878

Group Number: 2(session A)

Course Code: MIC 211

Title of the Practical: Effect of some physical factors (Sterilization, Aseptic Technique,
pH and Temperature) influencing the growth of bacteria.

Date: 20 April 2024

Title:

Effect of some physical factors (Sterilization, Aseptic Technique, pH and Temperature)

influencing the growth of bacteria.

Introduction:

Aseptic technique is a process used to eliminate bacteria and to prevent the transfer of
pathogenic microorganisms to a medium that may results in the development of infection
(Wilson, 2019) The aseptic technique Space is often practiced in laboratories by cleaning and
disinfecting lab surfaces. Aseptic technique is mostly used in laboratories for different
procedures such as transferring cultures, inoculating media, isolating pure culture and other
microbiological tests or practices. The aseptic technique prevents the contamination of
culture from unknown bacteria that are obtained from the environment. These practices
include cleaning and disinfecting the lab surfaces or medium where the experiment will be
conducted on (Sanders, 2012). To decrease contamination from germs, aseptic technique uses
target- specific practices and procedures under adequately controlled settings. In order to
create a pure culture, it is also necessary to isolate a single species of bacterium from a mixed
culture Furthermore, using adequate aseptic practice prevents laboratory-used germs from
unintentionally entering the environment and/or infecting others who work there. Barriers,
patient equipment and preparation, environmental controls, and contact rules are the four
main components of the aseptic approach. Each is critical in preventing infections during a
medical procedure. Bacterial growth is a complicated process that includes various anabolic
(cell constituents and metabolites are synthesized) and catabolic (cell constituents and
metabolites are broken down) events.

Bacterial growth is the term used to describe the rise in bacterial cell count that results from
binary fission. The primary means by which bacterial cells reproduce is through binary
fission. A straightforward method of cell division known as binary fission involves the cell
duplicating its genetic material, lengthening, and splitting into two brand-new daughter cells
that are identical to the parent cell. There are some physical factors influencing the growth of
bacteria i.e the pH levels, temperature, warmth, oxygen and nutrients, moisture, water and
dampness (Hamad, 2012).
The effect of temperature, bacteria have optimal temperatures in which they grow best, and
this optimal temperature range differs between bacterial species. Bacteria can be divided into
groups depending on their optimal temperature range: Psychrophiles are bacteria that prefer
to grow at temperatures below 20°C. Mesophiles are bacteria that prefer temperatures within
25- 40°C. Most pathogenic bacteria prefer to grow at 37°C and are, therefore, mesophiles.
Thermophiles are bacteria that grow at high temperatures ranging from 55-80 °C. The
temperature of a place can have a significant impact on the development of microorganisms.
In warm environments, where the temperature is close to that of the human body, bacteria
grow most rapidly. When food is refrigerated to keep it safe to eat for a longer period of time,
cooler environments tend to limit the growth of microorganisms. Moulds and germs can
breed in boiler rooms, heat-generating equipment rooms, and spaces close to heating vents.
Naturally. the spots that can raise the most worry when considering the health of a building
are those near machinery that generates dampness. By developing solutions using techniques
like optimizing circulation with optimum air ventilation, an environmental consultant can
assist you in preventing potential health risks before they even arise by assessing regions
around equipment with heat as a consequence (Rousk and Baath, 2009). The effect of the pH,
microbial growth can be aided or hindered by the pH of an environment. Microbes frequently
suffer when more base or acidic substances are present in an environment because they prefer
pH values that are neutral. Because of this, using cleaning solutions which are frequently
quite acidic kills bacteria effectively. This indicates that, when carried out frequently,
enhancing cleaning procedures in a company's building can significantly lower the amount of
germs growing and deter future growths. The pH that promotes an organism's maximum rate
of growth is known as the optimum growth pH. The minimum growth pH is the lowest pH
that an organism can tolerate, while the maximum growth pH is the highest pH. These values
can range widely, which is crucial for food preservation and the survival of microbes in the
stomach. For instance, Salmonnella species' optimum development pH ranges from 7,0 to 7,
while its minimal pH is closer to 4,2. pH also plays an important role in bacterial growth.
This is because the pH of a particular environment can affect the activity of enzymes required
for bacterial growth and metabolism. Therefore, the proper pH range is critical for bacterial
growth and survival. Most bacteria thrive in a neutral pH environment, between 6.5 and 7.5.
However, some bacteria have adapted to grow in extremely acidic or basic conditions
Acidophiles are bacteria that thrive in acidic environments with a pH as low as 1.0.
Neutrophiles are bacteria that thrive in neutral environments with a pH between 5 and 8.
Alkaliphiles are bacteria that can grow in basic environments with a pH as high as
11.0(Rousk ,2009).

Aim:

The aim of this experiment was to observe the effect of some physical factors on microbial
growth effect of temperature, effect of pH and the effect of aseptic techniques on the control
of microbial growth.

Methods and Materials:

Experiment one:

The surface was decontaminated using 70% ethanol disinfectant. Three bottles of nutrient
agar were provided, one of the plates was sterilized by autoclaving. The sterilized agar was
poured in the petri plates. The agar was allowed to solidify before the plates were moved. The
second bottle was placed in boiling water bath and incubated until the medium reached
boiling temperature. The bottle with agar was removed from the water bath and poured in the
plates, the plates were allowed to solidify before being moved. The third nutrient agar that
was not sterilized and not boiled was poured in the plates. After the plates were all solidified,
they were labelled and incubated at 37 degrees Celsius overnight.

Experiment two:

Part one:

The surface was decontaminated with 70% ethanol disinfectant. An inoculating loop was
sterilized by being placed on the flame until it turned red and was let to cool down, two test
tube were individually passed over the flame and the E.coli bacteria was transferred to the
test tubes using the sterilized inoculating loop, the test tube was again passed through the
flame and closed. The inoculating loop was again sterilized by being burnt on flame and was
let to cool down while two test tubes openings were individually sterilized by being passed
through the flame to destroy bacteria that is on the surface and preventing it to enter the test
tube, the inoculating loop was used to transfer the B.subtillis bacteria to the test tubes. Other
two test tubes were not inoculated and served as control. The six tubes were labelled and
placed in boiling water bath and let to boil for 5 minutes. The test tubes were then removed
from the water bath and placed on an incubator at 37 degrees Celsius overnight.

Part two:

The surface was decontaminated using 70% ethanol disinfectant. 20 prepared plates of
nutrient agar, two cultures with E.coli and B.subtillis and 1 yeast culture were provided. The
inoculating loop was sterilized by being burnt on the flame until it turned red and was let to
cool down, using the inoculating loop two plates were individually single streaked with E.coli
and two plates were individually triple streaked with E.coli as well with the loop being
sterilized after each streak was applied on the plate. The inoculating loop was sterilized by
being burnt on the flame and was let to cool down, using the inoculating two plates were
individually single streaked with B.subtillis and two plates were individually triple streaked
with B.subtillis as well, with the loop being sterilized after each streak was applied to the
plate The inoculating loop was sterilized by being burnt on the flame and was let to cool
down, using the inoculating two plates were individually triple streaked with yeast and two
plates were individually single streaked with yeast. The plates were inverted and labelled
with different temperatures namely 27, 30, 37 and 4 degrees Celsius. The plates were placed
in an incubator over night at 27, 30, 37, and 4 degrees Celsius.

Experiment three:

The surface was decontaminated using 70% ethanol disinfectant. Three different pH medias
namely acidic, neutral and basic were provided. The different pH medias were poured in the
four petri dishes per media and allowed to solidify. The inoculating loop was sterilized by
heating until it turned red then cooled down, using inoculating two plates of acid were single
streaked with the E.coli and two plates were triple streaked with the B.subtillis. The
inoculating loop was sterilized by heating until turned red then cooled down, using
inoculating two plates of basic were single streaked with the E.coli and two plates were triple
streaked with the B.subtilis. The inoculating loop was sterilized by heating until turned red
the cooled down, using inoculating two plates of neutral were single streaked with the E.coli
and two plates were triple streaked with the B.subtillis. The plates incubated at 37 degrees
Celsius until the following day.
Results:

Attached

Discussion:

In experiment one the aim was to see the effectiveness of different sterilization methods.
Three nutrient agar were provided during the experiment, one of them was sterilized using
the autoclaving technique and the other one was boiled then one nutrient agar was treated as a
control for the experiment. For the two nutrient agars the sterilized one and the boiled one
there was no bacterial growth for both of them, results reveals that the autoclaving technique
and boiling technique were effective in sterilizing the agar and the aseptic technique was well
performed. The only agar that showed bacterial growth is the one that was treated as a
control. In this agar bacterial growth was evident because the was nothing employed to
hinder the bacterial growth. The two nutrient agar boiled and sterilized solidified and the one
that was used did not solidified and those that are solidified have no growth and those that are
not solidified have growth.

In experiment two, part one we were provided with two cultures i.e E. coli and B. Subtilis.
Six test tubes with nutrient broth two of them with B. Subtilis, two of them with E. coli and
the other two with no micro-organism inoculated at 37°C over night were used. There was
growth in the two test tubes containing B.subtilis because the was turbidity and they can
survive at extremely high temperatures. Previous studies have revealed the resistance of
B.subtillis and its ignorance of high temperatures even that of the boiling point they may be
favored and show a great growth (Gilbert et al., 1974). There was growth in the two test tubes
containing E.coli and when inoculated at 37°C because the was also turbidity in it and they
can survive at this certain temperature. According to the previous studies E. coli cannot
survive temperatures above 70°C (Doyle and Schomeni, 2002). There was no growth in the
two test tubes containing nutrient broth which they acted as control.

At 4°C there was no growth in nutrient agar with E. coli and B. subtilis and yeast with triple
streak. B. subtilis have shown a great growth than E. coli and yeast at 27°C in triple streak.
The bacterial growth at 30°C was greater than that at 27°C while B. subtilis always revealed
high growth than E. coli 30°C is the optimum temperature. Yeast at 27°C, 30°C and 37°C
there was a growth but at 37°C yeast was supposed to not have growth. It is known that most
laboratory and industrial yeasts generally grow best between 20-30°C (Walker and John,
1998); At 37°C there was much more growth than previous temperatures in the agar.
Bacteria's growth is influenced by temperature, and they have an ideal temperature at which
they grow best and a temperature at which they cease developing. Varying bacteria have
different temperature preferences, some can thrive at extremely low temperatures, while
others thrive at moderate temperatures, and yet others thrive at extremely high temperatures
(Willey et al 2009).

In experiment three different medias of different pH were used, Microorganisms' occurrence

and distribution are influenced by pH, microbes are classified as acidophiles, neutrophiles, or
alkaliphiles depending on their optimal pH for their growth (Jin et al 2018). From the results
obtained in experiment 3 on the effect of pH on bacterial growth, in acidic media there was
no growth. For basic and neutral there was growth.
References:

1. Anon, 2021a Culture Media Available at https://bio libretexts.org/a/go/page/9162

[Accessed May 21, 20211].

2. Doyle, M.P. and Schoeni, J1., 1984. Survival and growth characteristics of Escherichia coli
associated with hemorrhagic colitis. Applied and Environmental Microbiology, 48(4),
pp.855- 856

3. Gilbert, RJ, Stringer, M.F. and Peace, T.C., 1974. The survival and growth of Bacillus
cereus in boiled and fried rice in relation to outbreaks of food poisoning Epidemiology &
Infection, 73(3), pp.433-444

4. Hamad, S.H., 2012. Factors affecting the growth of microorganisms in food. Progress in
food preservation, pp. 405-427.

5. Rousk, J., Brookes, PC and Baath, E., 2009. Contrasting soil pH effects on fungal and
bacterial growth suggest functional redundancy in carbon mineralization. Applied and
environmental microbiology, 75(6), pp. 1589-1596.

6. Rousk, J. and Baath, E, 2009. Adaptation of soil microbial communities to temperature:

comparison of fungi and bacteria in a laboratory experiment, Global Change Biology, 15(12),
pp.2950-2957 Sanders, E.R, 2012 Aseptic laboratory techniques: plating methods. JoVE
(Journal of Visualized Experiments), (63), p.e3064. Walker GM. Yeast Physiology and
Biotechnology. John Wiley & Sons: England; 1998.

7.Willey, J.M., Sherwood, L. and Woolverton, C.J., 2009. Prescott's principles of

microbiology. Wilson, J., 2019, Infection Control in Clinical Practice Updated Edition.
Elsevier Health Sciences