TITLE

Effect of some physical factors influencing the growth of bacteria.

Introduction

According to (Sanders, 2012) aseptic technique is a fundamental and important

laboratory skill in the field of microbiology. Microbiologists use aseptic technique for
a variety of procedures such as transferring cultures, inoculating media, isolation of
pure cultures, and for performing microbiological tests. Proper aseptic technique
prevents contamination of cultures from foreign bacteria inherent in the environment.
For example, airborne microorganisms (including fungi), microbes picked up from the
researcher’s body, the lab bench-top or other surfaces, microbes found in dust, as
well as microbes found on unsterilized glassware and equipment, etc. may
potentially contaminate cultures, thus interfering with the lab results. Using proper
aseptic technique can greatly minimize or even eliminate the risk of contamination. In
addition, aseptic technique is of utmost importance to maintain pure stock cultures
while transferring cultures to new media. Aseptic technique is also essential for
isolation of a single species of microorganism from a mixed culture to obtain a pure
culture. Furthermore, proper aseptic technique prevents microbes used in the
laboratory from accidentally being released into the environment and/ or infecting
people working in the laboratory. This is especially relevant when pathogens are
being handled. 

The effect of some physical factors influencing the growth of bacteria specifically pH
and temperature. pH is a measure of acidity and alkalinity of a solution and
indicates the concentration of hydrogen ions (H+) of any solution. The Hydrogen ions
are protons hence, it is also the measurement of protons in solution. H+ ion
concentration is inversely related to pH scale. Low pH means high H+ concentration
& high pH means low H+ concentration. The pH scale ranges from 0 to 14. Below 7,
the solution is acidic, at 7 it is neutral and above 7, the solution is alkaline. The
hydrogen ion concentration is one of the vital physical requirements for living growth.
pH affects the bacterial growth. The cell needs optimum pH for carrying its
biochemical and molecular process. The microbial cells are evolved and have
developed the mechanism for maintaining the intracellular pH. The research has
found that the bacteria forms biofilm that inhibits entry of extra H+ or uses buffer to
maintain physiological pH. The optimum pH varies from bacterial species to species.
And hence, this parameter has been used for classifying the bacteria (Pansu et al.,
2006).

Temperature effects the growth of bacteria by various ways, the lower temperature
that allows the growth is called minimum temperature and the highest temperature
that allows growth is called maximum temperature. There is no growth below
minimum and above maximum temperature, below minimum temperature cell
membrane solidifies and become stiff to transport nutrients in the cell, hence no
growth occurs. Above maximum temperature, cellular proteins and enzymes
denatures, so the bacterial growth ceases (Glazer et al., 2007).

Sterilization refers to any process that removes, kills, or deactivates all forms
of life (particularly microorganisms such as fungi, bacteria, spores, and unicellular
eukaryotic organisms) and other biological agents such as prions present in or on a
specific surface, object, or fluid. Sterilization can be achieved through various
means, including heat, chemical, irradiation, high pressure, and filtration. Sterilization
is distinct from disinfection, sanitization, and pasteurization, in that those methods
reduce rather than eliminate all forms of life and biological agents present. After
sterilization, an object is referred to as being sterile or aseptic. An autoclave is a
machine used to carry out industrial and scientific processes requiring elevated
temperature and pressure in relation to ambient pressure and temperature.
Autoclaves are used before surgical procedures to perform sterilization and in the
chemical industry to cure coatings and vulcanize rubber and for hydrothermal
synthesis. Industrial autoclaves are used in industrial applications, especially in the
manufacturing of composites(Black, 1993).
AIM
The aim of this practical was to observe the effect of microbial growth under certain
temperatures and different pH levels by learning to apply aseptic techniques

METHOD AND MATERIALS

EXPERIMENT 1: ASEPTIC TECHNIQUE

Three bottles of nutrient agar were provided, one of them was sterilized by
autoclaving. Sterilized nutrient agar was used to pour the plates as it was
demonstrated. Aseptic technique was applied. The agar was allowed to solidify
before the plates were moved. The second bottle was placed in the boiling water
bath, and it was incubated until the medium reached the boiling temperature. It was
then removed from the water bath, and it was poured into the plates as it was
demonstrated before and it was left to solidify. The third medium was not sterilized or
boiled, it was poured into a plate and waited for to solidify before being moved. Once
the plates were solidified, they were inverted, labelled and incubated at 37 degrees
Celsius. Results were then recorded the following day.

EXPERIMENT 2: EFFECT OF TEMPERATURE

PART ONE

Two cultures were provided, E. coli and B. subtilis. Two test tubes were inoculated
containing nutrient broth with E. coli and the other two with B. subtilis. Two test tubes
were left un-inoculated to serve as a control. All test tubes were labelled and placed
in the boiling water bath and boiled for 5 minutes. Thereafter they were removed
and incubated at 37 degrees Celsius overnight. The results were then recorded the
following day.

PART TWO
Eight prepared plates of nutrient agar were provided, and two bacterial cultures were
provided. Four plates were streaked with the first culture E. coli, four with the second
culture B. Subtilis. Single and triple was practiced as it was demonstrated. The
plates were inverted, labelled and incubate overnight at different temperature: 27 ° C,
30° C, 37° C, 4° C. The results were recorded and interpreted the following day

EXPERIMENT THREE: EFFECT OF pH

Different medias of different pH such as Acidic, Neutral and Basic were provided and
poured in different plates. The cultures (E. coli and B. Subtilis) were used to single
and triple streak the plates of each pH. The plates were incubated at 37 ° C until the
following day.
RESULTS
EXPERIMENT ONE: ASEPTIC TECHNIQUE

Table 1: The effect of sterilization in bacterial growth using three different treated
growth media: Autoclave, Boiled and not sterile media.

Growth Media Plate 1 Plate 2

Autoclaved No growth No growth
Boiled No growth No growth
Not sterilized Growth Growth

EXPERIMENT TWO: EFFECT OF TEMPERATURE

PART ONE
Table 2: The effect of boiling temperature on the growth of E. coli and B. subtilis.

Name of Organisms Test tube 1 Test tube 2

E. coli Growth Growth
B. subtilis Growth Growth

PART TWO
Table 3: The effect of different temperatures on the growth of E. coli and B. subtilis.

Name of organism 4° C 27° C 30° C 37° C

E. coli No growth Growth Growth Growth
B. subtilis No growth Growth Growth Growth

EXPERIMENT 3: EFFECT OF pH
Table 4: The effect of pH on the growth of E. coli and B. subtilis.
Name of organisms Acidic Neutral Basic
Plate 1 Plate 2 Plate 1 Plate 2 Plate 1 Plate 2
E. coli Growth Growth Growth Growth Growth Growth
B. subtilis Growth Growth Growth Growth Growth Growth
DISCUSSION
In experiment one aseptic techniques were applied to nutrient agar. Nutrient agar
was boiled and sterilized by autoclaving, and the last nutrient agar was not boiled
and sterilized. The boiled and autoclaved agar solidified while the agar that was not
boiled or autoclaved did not solidify. Both plate 1 and plate 2 of not sterilized growth
media had growth. Both boiled and autoclaved growth media had no growth on plate
1 and plate 2. According to Reyes (2017) Agar is used as a solidifying agent. The
reason why the not boiled and not sterilized by Autoclaving nutrient agar had
growth was because they were contaminated, as aseptic techniques were applied
thus growth took place. The boiled and sterilized by autoclaving showed no growth
which is what is meant to happen because aseptic techniques had been applied to it
to ensure that no microorganisms were present. This shows that it is important to
apply aseptic techniques to kill any foreign microorganisms present, whether in the
air or in the dish.
 REFFERENCES
Black, J. (1993). Microbiology. Prentice Hall. p. 334.

Glazer, A.N.; Hiroshi N. (2007) Microbial biotechnology: fundamentals of applied

microbiology. Cambridge University Press. pp. 77

Pansu, M.; Jacques G. (2006). pH measurement. Springer

Berlin Heidelberg. pp. 203-308

Sanders, E.R. (2012). Aseptic laboratory techniques: plating

methods. Journal of Visualized Experiments. pp. 405