Laboratory Exercise 11

Physical and Chemical Factors Affecting Growth

Bio 120.1 – Section 3

Jiaren G. Baure
Mia Alexa Ellinor Dagum
Gracechel Ann C. Pormilda

BS in Chemistry IV

November 07, 2018

INTRODUCTION
Growth of microorganisms is defined as the increase in all the chemical constituents
of an organism that results in an increased size of the cell, increase number of cells or both.
Though, microbial growth usually talks about the number of cells that is produced and not the
size of the cells produced. It is of importance to study the growth of microorganisms, as well
as the factors that affect it, so as to understand the biology of an organism. Another reason is
that, through acquiring knowledge of their preferred growth conditions, the development of
pathogenic microorganisms may be controlled (Tortora, et. al, 2013).

Factors that can affect the growth of microorganisms can be divided into two main
categories: physical and chemical factors. Physical factors include temperature, pH, and
osmotic pressure or salt tolerance. One of the chemical factors that can affect the growth of
microorganisms is oxygen requirement (Tortora, et. al, 2013).

Microorganisms usually grow at temperatures between water’s freezing point and the
temperature that causes coagulation of proteins. Bacterial growth generally has a minimum
and maximum temperature growth, having the optimum temperature growth in between
(Kullabs, n.d). Minimum and maximum temperature is the lowest and highest temperature,
respectively, at which a bacterial species will grow. The optimum temperature, on the other
hand, is the temperature in which a bacterial species grows best (Tortora, et. al, 2013).

Similar to temperature, there is only a pH range in which bacterial growth is possible.

Most bacteria grow at pH between 6.5 and 7.5 and only very few species are able to develop
at pH below pH 4 or pH greater than pH 10 (Kullabs, n.d). This is the reason why acidity, and
sometimes alkalinity, is used to preserve foods (Tortora, et. al, 2013).

Water is also essential for bacterial growth. Most bacteria are killed by dry conditions
whereas there are others that are able to tolerate dry conditions for several months (Kullabs,
n.d). High osmotic pressures tend to remove the necessary water from a cell. Different
bacterial species have varying tolerance to salt concentrations. There are bacterial species
that can tolerate medium salt concentrations. But, there are those that are capable of tolerating
high salt concentration.

Based on oxygen requirement, bacteria can be divided into obligate aerobes, obligate
anaerobes, facultative anaerobes, aerotolerant anaerobes, and microaerophiles. Aerobes
require oxygen to develop. Anaerobes die in the presence of oxygen. Facultative anaerobes
can grow with or without oxygen, but growth is best with the presence of oxygen. Aerotolerant
anaerobes grow evenly with or without oxygen. And microaerophiles, grows with presence of
low oxygen concentration (Tortora, et. al, 2013).

1
 Bacteria such as: Escherichia coli, a Gram-negative, facultative anaerobic, rod
shaped, and coliform bacterium of the genus Escherichia; Bacillus subtilis, a Gram-positive,
catalase-positive bacterium, rod shaped, and can form a tough, protective endospore; and
Staphylococcus aureus, a gram-positive, round-shaped bacterium, and it is a usual member
of the microbiota of the body, were used in this exercise.

With the importance of the knowing the effect of various bacterial growth in the body,
environment, and everywhere else, this exercise aims for the student to determine the effects
of several chemical and physical factors on the growth of different microorganisms. It also
aims to classify different bacterial species according to their response to various temperature,
pH, oxygen and salinity/osmolarity.

MATERIALS AND METHODS

A. Materials, Equipment, Samples and/or Organisms Used

1. Alcohol lamp – used to produce an open flame; used in sterile technique

2. Nutrient Agar Plate – used to culture microorganisms
3. Nutrient Agar Slant – used to culture microorganisms
4. Eosin methylene blue (EMB) plates – a selective and differential medium used to
isolate fecal coliforms.
5. Inoculating loop – used to inoculate microorganisms
6. Candle – ignitable wick embedded in wax and provides light
7. Desiccator – a sealable enclosure containing desiccants used for preserving moisture-
sensitive items
8. Cultures of microorganisms
a. E. coli – Gram negative bacteria that are referred to as the most studied free-
living organism
b. B. subtilis – Gram positive bacterium that can form a protective endospore
c. S. aureus – Gram positive bacterium that is cocci-shaped and tends to be
arranged in clusters

2
B. Procedure

In this laboratory exercise the effect of temperature and oxygen availability was performed
on cultures of Escherichia coli and Bacillus subtilis. The effect of pH and osmolarity of the
environment was performed on cultures of Staphylococcus aureus and E. coli. The effect of
oxygen availability was performed on cultures of B. subtilis and E. coli. The activity was
performed aseptically to reduce the risk of contamination.

11.1 Laboratory Preliminaries

Before the laboratory exercise began working areas were disinfected with 75%
isopropyl alcohol and Lysol.

11.2 Effect of Temperature

Four nutrient agar slants, in Eppendorf tubes, were inoculated with Escherichia coli.
Another four nutrient agar slants, in Eppendorf tubes, were inoculated with Bacillus subtilis.

The first tube, for each species, was incubated at 4˚C in the refrigerator. The second
tube was incubated at room temperature (28˚C). The third tube was incubated at 35˚C in the
incubator. And the fourth tube was incubated at 55˚C in the oven. All the tubes were incubated
for 24 hours. Then growth after incubation was determined.

11.3 Effect of pH

Several Eppendorf tubes containing pH 3, pH 5, pH 7, pH 9, and pH 10 were inoculated

with Staphylococcus aureus. Another set of Eppendorf tubes containing the same pH levels
were inoculated with Escherichia coli.

The tubes were incubated at 35˚C for 24 hours. Then amount of growth was
determined.

11.4 Effect of Osmolarity of the Environment

Several nutrient agar slants, in Eppendorf tubes, containing 0%, 0.5%, 5%, and 10%
salt concentrations, respectively, were inoculated with Staphylococcus aureus. Another set
containing the same percent of salt concentrations wee inoculated with Escherichia coli.

3
 Several nutrient agar slants, in Eppendorf tubes, containing 0%, 10%, 25%, and 50%
glucose concentrations, respectively, were inoculated with Staphylococcus aureus. Another
set containing the same percent of glucose concentrations wee inoculated with Escherichia
coli.

All the tubes were incubated at 35˚C for 24 hours. The amount of growth was
determined after incubation.

11.5 Effect of Oxygen Availability

Two nutrient agar plates were streaked with Bacillus subtilis. And two Eosin-Methylene
Blue plates were inoculated with Escherichia coli.

One NA plate and one EMB plate was incubated for 24 hours. The other NA plate and
EMB plate was placed and incubated inside the desiccator. Growth was observed after 24
hours.

RESULTS AND DISCUSSION

Factors that can affect the growth of microorganisms are divided into the physical and
chemical factors. The physical factors include temperature, pH, and osmotic pressure.
Chemical factors include sources of carbon, nitrogen, sulfur, phosphorus, oxygen, trace
elements, and organic growth factors.

In this laboratory exercise the effect of all three physical factors; temperature, pH and
osmotic pressure, on different bacterial species was examined. Though, among the chemical
factors, only the effect of oxygen availability was tested.

Here, the optimum temperature and oxygen classification of the Escherichia coli and
Bacillus subtilis cultures were determined. At the same time, the optimum pH and optimum
glucose and salt concentrations of the Staphylococcus aureus and Escherichia coli cultures
were established.

Effect of Temperature
According to temperature microorganisms are divided into three groups. These are the
psychrophiles, mesophiles, and thermophiles. The psychrophiles are cold loving microbes.
They can develop between 0˚C to 25˚C, with an optimum temperature of 15˚C. The
mesophiles develop between 25˚C to 40˚C, and most pathogenic bacteria have an optimum

4
growth temperature of 37˚C. Lastly, the thermophiles can grow at temperatures between 45˚C
to 90˚C, with an optimum growth temperature between 50˚C to 60˚C (Tortora, et. al, 2013).
The rate of chemical reactions and protein structure integrity are influenced by
temperature thus affecting rates of enzymatic activity. At low temperature, enzymes are not
denatured, therefore, every 10°C rise in temperature results in rise of metabolic activity and
growth of microorganisms.
However, enzymes have a range of thermal stability and beyond it, their denaturation
takes place. Thus, high temperature kills microorganism by denaturing enzymes, by inhibiting
transport carrier molecules or by change in membrane integrity. Each microbe shows
characteristic temperature dependence and possesses its own cardinal temperatures.

For this exercise, B. subtilis and E. coli broth culture were used as samples. As
observed in Table 1.0 and Figure 1.0, both bacteria grew best at 35°C and they did not grow
at 4°C. An article by Koni, Rusman, and Zuprizal (2017) supports the results. According to
Koni et al. (2017), the optimum temperature for both B. subtilis and E. coli is 37°C and is likely
not to grow in low temperatures. These bacteria can also not tolerate so much heat that they
were not able to grow at 55°C. Since the optimum temperature was 37°C, the microorganisms
are considered to be mesophiles. Most microorganisms fall within this category. Also, almost
all human pathogens are mesophiles as they grow at a fairly constant temperature of 37°C.

Table 1.0 Effect of Temperature in the Growth of Bacteria

Growth at Different Temperatures
Bacteria
4°C 28°C 35°C 55°C
B. subtilis No growth (0) Slight growth (1+) Moderate (2+) Slight growth (1+)
E. coli No growth (0) Moderate (2+) Moderate (2+) No growth (0)

Effect of pH

5
 pH can dramatically affect the growth of microorganisms. Each species of
microorganisms shows specific pH growth range. Microorganisms can be classified as
acidophiles, neutrophils and basophiles (alkalophiles) based on their requirement for particular
pH in their environment. However, most microorganisms grow best at pH between 6.5–7.5,
which is within the range of neutrophils.

The activity of microbial enzymes depends on the change present on the surface of
amino acids. Any change in the environmental pH may either enhance the enzyme activity or
inhibit the activity.

Growing microorganisms produce acidic and basic metabolic waste products. These
wastes often become inhibitory agents as they alter the pH of surrounding environment.

S. aureus can grow in a wide range of pH values ranging from 4.2 to 9.3 with an optimum
pH of 7 to 7.5 (Bergdoll, 1989). While E. coli grown in an optimum pH of 6-7 but ranges 4.4 to
9.0. Also, the limit at the low pH end depends on the acidulant used.

Table 2.0 Effect of pH in the Growth of Bacteria

Growth at Different pH
Bacteria
pH 3 pH 5 pH 7 pH 9 pH 10
E. coli No growth (0) Slight (1+) Abundant (3+) Slight (1+) No growth (0)
S. aureus No growth (0) Moderate (2+) Abundant (3+) Slight (1+) No growth (0)

Effect of Osmolarity of the Environment

It is noted that when the environment is hypertonic to the cell, cellular water draws out
through the plasma membrane via osmosis. This causes the membrane to detach from cell
wall making the cell’s cytoplasm to shrink. This phenomenon is brought about by high salt and
sugar concentration. And it is of high importance since bacterial growth is restrained as the
plasma membrane pulls away from the cell wall (Tortora, et. al, 2013).

Osmolarity in the environment is also one of the factors. It includes the salt and glucose
concentration. For salt concentration, E. coli is a nonhalophile while S. aureus is halotolerant
and can grow in the presence of high NaCl concentrations (Tortora et. al, 2013), such as on
skin surfaces which often have high NaCl concentration of 10% NaCl (Tsai et. al, 2011). This
may suggest that S. aureus may exhibit a better ability to overcome the osmotic shock due to
NaCl, compared to E. coli. It was observed that the optimum growth recorded was in a
decreasing order for both bacteria, except for the 0% concentration where no salt was
consumed. This shows that the higher the NaCl concentration the lesser both bacteria will

6
reach their optimal growth. These decreasing optimal growths as a result of increasing salt
concentration, was also reported by Hajmeer (2001) and Abdulkarim et. al (2009), and they
further suggested that it may be due to hyper osmotic effect on the bacteria.

Table 3.0 Effect of Salt Concentration in the Growth of Bacteria

Growth at Different Salt Concentration

Bacteria
0% 0.5% 5% 10%
E. coli Slight growth (1+) Abundant (3+) Slight growth (1+) No growth (0)
S. aureus Slight growth (1+) Abundant (3+) Moderate (3+) Moderate (2+)

For glucose concentration, the specific growth rate increased with increase in substrate
concentration which in this case is the glucose. A further increase in glucose concentration
also inhibits growth for E. coli (Kazan et. al, 1994). The same relationship is applied for S.
aureus.

The table shows that it was able to generate an abundant growth at 10% glucose
concentration for S. aureus. On the other hand, it was at 25% glucose concentration where E.
coli grew most abundantly. Through this data the optimum glucose concentration for S. aureus
and P. aeruginosa was determined to be at 10% and 25% glucose, respectively.

Table 4.0 Effect of Glucose Concentrations in the Growth of Bacteria

Growth at Different Glucose Concentration

Bacteria
0% 10% 25% 50%
E. coli 1+ 1+ 2+ 1+
S. aureus 1+ 3+ 1+ 1+

Effect of Oxygen Availability

Oxygen is said to be a necessity of life, but there are microorganisms that gets
poisoned and dies with the presence of oxygen. Hence, microorganisms are mainly classified
as obligate aerobes, obligate anaerobes, and facultative anaerobes. Obligate aerobes;
generally, require oxygen to develop, while obligate anaerobes cease to develop with the
presence of oxygen. The facultative anaerobes, on the other hand, are capable of growing
with or without the presence of oxygen.

7
 These varying relationships between microbes especially the bacteria and O2 appear
due to different factors such as protein-inactivation and the effect of toxic oxygen-derivatives.
Bacterial enzymes can be inactivated when interact with oxygen. Nitrogen-fixing enzyme,
nitrogenase, is very sensitive to oxygen and represents a good example of interaction between
enzyme and oxygen.

In this part, both plates of E. coli and B. subtilis were able to grow better when placed
in the incubator than those that were place in the desiccator. Table 5.0 below shows the
summary of results.

Table 5.0 Effect of Oxygen Availability in the Growth of Bacteria

Bacteria In the incubator In the desiccator Interpretation
more growth of less growth of - The bacteria can grow in the
E. coli
bacteria bacteria compared presence or absence of
Small number of oxygen.
More growth of
B. subtilis bacteria were grown - It grows better in the presence
bacteria
in the plate of oxygen

8
 Bacterial growth can be affected by several chemical and physical growth factors.
This includes the effect of temperature, pH, osmolarity of the environment, and oxygen
availability.
Through this exercise it can be concluded that both E. coli and B. subtilis are
mesophiles and facultative anaerobes. It was also found out that S. aureus and E. coli have
an optimum pH concentration of 7, and their optimum salt and glucose concentration were
also able to be determined.
All in all, this exercise showed that different bacterial species generally have varying
reactions and responses to various temperature, pH, salinity or osmolarity, and oxygen
availability.

9
LITERATURE CITED
Abdulkarim, S.M., Fatimah, A.B., and Anderson, J.G. (2009). Effect of salt
concentrations on the growth of heat-stressed and unstressed Escherichia coli. J. Food Agri.
and Environ., 7(3&4): 51-54.
Bergdoll MS. Staphylococcus aureus. In: Doyle MP., editor. (1989). Foodborne
Bacterial Pathogens. New York: Marcel Dekker Inc. 463-523p.

Factors Affecting Bacterial Growth. Retrieved November 2, 2018, from

http://fac.ksu.edu.sa/sites/default/files/7_factors_affecting_bacterial_growth.pdf

Factor Affecting Growth of Microorganisms. Retrieved November 2, 2018, from

https://www.kullabs.com/classes/subjects/units/lessons/notes/note-detail/6797

Garg, M. (2016, September 16). Growth of Microorganisms: 6 Factors. Retrieved

November 2, 2018, from http://www.biologydiscussion.com/microorganisms/growth-of-
microorganisms-6-factors/55161

Koni, T.N.I., Rusman., Hanim C., & Zuprizal. (2017). Effect of pH and Temperature
on Bacillus Subtilis FNCC 0059 Oxalate Decarboxylase Activity. Pakistan Journal of
Biological Sciences, 20, 436-441.

Microbial Growth. Retrieved November 2, 2018, from

https://mymission.lamission.edu/userdata/brownst/docs/Micro20%20Chapter%206.pdf

Tortora, G. J., Funke, B. R., & Case, C. L. (2013). Microbiology: An Introduction

(Eleventh ed.). United States of America: Pearson Education.

Tsai, M., Ohniwa, R.L., Kato, Y., Takeshita, S.L., Ohta, T., Saito, S., Hayashi, H. and
Morikawa, K. (2011). Staphylococcus aureus requires cardiolipin for survival under
conditions of high salinity. BioMed Central Microbiol., 11: 13

10