MODULE 9

FACTORS AFFECTING MICROBIAL GROWTH

Introduction

Microorganisms have adapted to a variety of extremely variable

environmental conditions. The study of microorganisms from natural
environments requires a variety of physical and chemical growth conditions
for isolation, identification and analysis.

Physical factors include temperature and osmotic pressure, whereas

chemical factors include pH and oxygen requirement, among others.
Temperature is one of the most important factors influencing the activity of
enzymes. There are groups of microorganisms capable of growing in
different temperature ranges. Psychrophile can grow between 0 – 20˚C,
mesophiles can grow in optimum environmental conditions, and
thermophiles can thrive in between 50 to 90˚C. pH is a measure of hydrogen
ion concentration in an organism’s environment. It limits the activity of
enzymes. Microorganisms can also optimize their growth at varying pH
concentrations. Acidophiles can grow below pH 6.5, alkalophiles at pH above
7.5, and neutrophiles can thrive between pH 6.5-7.5. Increasing solute
concentration can affect the amount of water availability and further affects
the survival of microorganisms. However, there are microorganisms capable
of growing in the presence of high solute concentration. Osmophiles require
high solute concentration whereas osmotolerant can grow in higher solute
concentration. Microorganisms are divided into five categories depending on
their need to molecular oxygen. Obligate aerobes use oxygen as the final
electron acceptor while obligate anaerobes generate their energy through
fermentative processes or anaerobic respiration and thus do not require
oxygen as their terminal electron and hydrogen ion acceptor. Both
facultative and aerotolerant anaerobes may or may not require oxygen but
the former prefer the presence of it. However, there are microorganisms
that require low concentration of oxygen (2-10%) and these are
microaerophile.

Learning Objectives
At the end of the activity, the students are expected to:

TLO26. Demonstrate techniques for the classification of microbes based on

temperature, osmotic pressure, pH and oxygen. affecting microbial growth.
TLO27. Classify representative microorganisms based on their responses to
the four growth factors.
Materials
tubes of Nutrient broth (NB) Thioglycollate agar tubes (TA).
Inoculating loop Alcohol lamp
Disinfectant Stove

Culture Media

Bacterial Cultures as indicated

Procedures

A. Microbial Responses to Temperature (see video #1 9:21-17:45

minutes).
1. Label a set of five NB tube with each of the following bacterial
cultures.
a. Pseudomonas flourescens c. Serratia marcescens
b. Escherichia coli d. Bacillus sterothermophilus

2. Inoculate a set five tubes of NB with one bacterial culture. Repeat

the same with the rest of the cultures.
3. Label each set of tubes for each bacterium with the following
temperatures: 5˚C, 25˚C, 38˚C, 42˚C and 55˚C.
4. Incubate the tubes at respective temperatures for 48 hrs.
5. After 48 hrs., observe for the presence of growth by observing
turbidity in each of the tubes and answer items 1-2 in the
worksheet.

Note: It is important that you fill out first item 1 in the

worksheet, then check your answers with the video from
17:46 to 25:28 minutes. The rest of the minutes are post-lab
information.

B. Microbial Responses to Osmotic Pressure (NaCl) (see video #2

and 3).
1. Prepare two sets of three nutrient broth (NB) tubes with the following
NaCl concentration: 1%, 7% and 15% (prepared by addition xg in
100 ml NB respectively). Label.
2. Inoculate one set of NB tubes with Saccharomyces cerevisiae; and
another with the Staphylococcus epidermidis.
3. Incubate the tubes at 37˚C for 48 hrs.
 4. After 48 hrs., observe for the presence of growth by observing
turbidity in each of the tubes. Watch alternative set up below and
answer items 3-5 in the worksheet.

Alternative set up (# 6 video starts at 7:11 minutes till end)

1. Prepare two sets of four nutrient broth (NB) tubes with the following
NaCl concentration: 2%, 5%, 8% and 11% (prepared by addition
specific g in 100 ml NB respectively). Label.
2. Inoculate one set of NB tubes with Staphylococcus aureus and
another with the Escherichia coli.
3. Incubate the tubes at 37˚C for 48 hrs.
4. After 48 hrs., observe for the presence of growth by observing
turbidity in each of the tubes.

C. Microbial Response on pH (see video #2 and 3)

1. Prepare two sets of three nutrient broth (NB) tubes with the following
pH values: pH 5, 7 and 9 (adjusted by adding either HCl or NaOH).
2. Inoculate one set of NB tubes with Saccharomyces cerevisiae; and
another with the Staphylococcus epidermidis.
3. Incubate the tubes at 37˚C for 48 hrs.
4. After 48 hrs., observe for the presence of growth by observing
turbidity in each of the tubes and answer items 6-7 in the worksheet.

Note: This procedure was not shown in video #2, only

introduced. Results are shown in video #3.

Alternative set up (# 6 video up to 7:10 minutes)

1. Prepare three sets of nutrient broth (NB) tubes with the following pH
values: pH 2,4,6,8, and 10 (adjusted by adding either HCl or NaOH).
2. Inoculate one set of NB tubes with Lactobacillus acidophilis; and
another with the Staphylococcus aureus, and last set with alcaligenes
faecalis.
3. Incubate the tubes at 37˚C for 48 hrs.
4. After 48 hrs., observe for the presence of growth by observing
turbidity in each of the tubes and answer items 6-7 in the worksheet.

D. Microbial Response on Oxygen (see video # 4 and 5)

1. Melt 5 Thioglycollate agar tubes (TA).
2. Inoculate each tube with the following organisms: Enterococcus
faecalis, Alcaligenes faecalis, E. coli, Clostridium sporogenes. Label.
3. Incubate at 37˚C for 48 hrs.
 4. Prepare two NA plates and divide each into four quadrants by
drawing lines at the bottom. Label each quadrant with the names of
each test bacteria and inoculate.
5. Incubate one plate in anaerobic jar, and the other in the ordinary
incubator.
6. After 48 hrs., look for the evidence of growth in tube and answer
item 8 in the worksheet.

References:
1. Tabo NA & Tabo HAL. 2020. Laboratory Manual in General
Microbiology. Philippines: Siam Rein Publishing House.
2. https://youtu.be/qGkpw5W25K0 
3. https://youtu.be/l1lf2X1jwQs 
4. https://youtu.be/5ncqtE3XjYg 
5. https://youtu.be/sE7XDTcZbHw 
6. https://youtu.be/AJG18sQd8mU 
7. https://youtu.be/rnRePfAcg3E