Effects of pH, Temperature, Media Composition, Antimicrobial Agents and UV Light on

Microorganism Growth
Al Jay Mejos, John Warner Carag, Jojiemar De Pano, Allison Vincent Labador, Erika Mari Macapagal
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City

Abstract
Microorganisms are open energy systems wherein the physical and chemical states of the environment affect the metabolic activities inside the cells.
This experiment was aimed to study the effects of a set of environmental factors on an aspect of microorganism performance as a function of its
growth. Five parameters were defined: pH, temperature, growth media composition, presence of antimicrobial agents and their effectiveness in
hindering microorganism growth and exposure to UV light. It was observed in the species used, E. coli, B. subtilis and S. aureus, that they grew best in
slightly alkaline environments and at room temperature. These were deemed their optimum growth pH and temperature, respectively. It was also
found that the presence/absence of nutrients in media greatly affect their growth that media allows for sufficient differentiation of bacteria between
species and that UV, antimicrobial agents are detrimental to their growth.
Introduction wrapped) and 5 min UV (lidless) then 24 hrs sunlight (lid on &
Microorganisms are greatly affected by their environment. inverted). All set-ups were then incubated at 26°C for 24 hours and
Different microbial populations respond differently to varying observed.
conditions. There are certain conditions that favor their growth and Eucalyptus sp.
others that are detrimental for their survival. Understanding their
response to various physical and chemical conditions of their Ethanol
environment, helps us to explain their distribution in nature, and Chloramphenicol
makes it possible for us to control the growth of beneficial bacteria
and cease the growth of the harmful ones. Effect of pH, Control
temperature, culture media or energy source, chemotherapeutic Penicillin
agents like antimicrobials and UV light on different types of bacteria
are to be understood in this experiment. M. oleifera
Fig. 1. Placement of filter paper discs on plate surface.
Materials and Methods Results
Table 1. Microbial growth in varying pH, temperature and UV exposure.
Effect of pH Group 1 Group 2 Group 3
Fourteen test tubes were prepared, seven each for
4 C – minimal 20°C – intermediate 37°C – numerous
Nutrient (NB) and Glucose Yeast Peptone (GYP) broths, each test Temperatures
growth growth growth
tube set-up having a different pH (1, 4, 5, 7, 9, and 11). The pH was
pH (NB &
set using HCl or NaOH. NB tubes were inoculated with 0.1 mL or
GYP)
100 µL of E. coli using a micropipette while GYP tubes were 1 - -
inoculated with S. cerevisiae. Both were incubated for 24 hours and 4 + +
the resulting turbidity evaluated as a function of growth. 5 + +
7 +++ ++++
Effect of Heat Treatment 9 ++++ +++++
Three Nutrient Agar (NA) plates was divided into four 11 +++++ ++
quadrants. Each quadrant was inoculated with E. coli, S. aureus, and B. Growth under 1 colony under
Growth under
subtilis. The last quadrant served as control. One plate was then masking tape and masking tape and
masking tape and
numerous little growth
stored in the locker, refrigerator and incubator together with the UV light growth around
medium growth
around area
tube of GYP inoculated with S. cerevisiae for 24 hours. around area
area surrounding surrounding
surrounding masking
masking tape masking tape
tape (5min)
Use of Differential or Selective Media (1min) (15min)
Mannitol Salt Agar (MSA), MacConkey agar, and Eosin
Methylene Blue (EMB), were each divided into four quadrants. Table II.
Leaving one quadrant as blank, each quadrant was inoculated with a Group 2
different organism using cultures of E. coli, S. aureus, and B. subtilis. It EMB + is a dark metallic color
was incubated and growth was observed after 24 hours. E. coli Theo + Actual +
B. subtilis Theo - Actual +
Effect of Antimicrobials/ Kirby Bauer Disc Diffusion Assay S. aureus Theo - Actual +
Six NA plates, two plates each for E. coli, S. aureus, and B.
Mac Conkey + is a pink coloration
subtilis, were inoculated using cotton swab plating. One cotton swab
E. coli Theo + Actual +
per plate was used, dipping the swab in the sample every half the B. subtilis Theo - Actual -
plate. A sterile blank filter paper disc was placed at the center of S. aureus Theo - Actual -
each plate for control. Commercially available antimicrobial discs,
Penicillin and Chloramphenicol, a disc with ethanol, and discs MSA + is a yellow coloration
infused with 20 µL of plant extracts Malunggay (Moringa oleifera) and E. coli Theo - Actual +
Eucalyptus sp, were placed around the blank as shown below (fig. 1). B. subtilis Theo - Actual +
Zones of inhibition as a function of antimicrobial capability of the S. aureus Theo + Actual +
test substances were analyzed after 24-hour incubation.
Table III.
Effect of UV Light Chloramphenicol Penicillin M. Eucalyptus
EtOH Control
(25 units) (10 units) charantia sp.
100 µL 0.1% peptone water-100 µL E. coli sample was EC1 27mm - - - - -
spread on three NA plates using a hockey stick. After the agar EC2 25mm - - - - -
absorbed the liquid, masking tape was suspended across the lidless BS1 28mm - 19mm 16mm - -
Petri plate without touching the surface of the agar. Each of the set- BS2 22mm - 17mm 17mm - -
up was then exposed to 1 min. UV (lidless) then 1 min. sunlight (lid SA1 37mm 49mm 23mm 18mm - -
on & inverted), 1 min. UV (lidless) then 1 min. darkness (lid on and SA2 39mm 52mm 21mm 3mm - -
Discussion
pH
Each microorganism has a specific pH range where it can
grow and reproduce successfully, called the optimum pH. While
most natural environments have a pH value in the range of 5 to 9,
there are a few species that thrive outside this range. Organisms
that live on low pH values are called acidophiles and organisms that
live on high pH values are called alkaliphiles. Most microorganisms
whose pH optimum for growth is between pH 6 to 8 are referred
to as neutrophiles. The optimal growth pH shows the pH of the
extracellular environment only. The intracellular environment must
remain constant or near neutrality in order to prevent the
destruction of macromolecules inside the cell.
In general, fungi are more acid-tolerant than bacteria. In this
experiment, bacteria grown in the NB tube show significant
numbers in the pH range of 7 to 11, while fungi grown in the GYP
tube show significant growth in the pH range of 7 to 9. This
coincides with the theoretical results since both E. coli and S.
cerevisiae are neutrophiles. It can also be seen from our results that
there is limited to no growth of both microorganisms in the
extreme pH levels. S. cerevisiae which is a fungus showed more
growth at acidic pH levels compared to E. coli which exhibited more
growth on basic pH levels.
Temperature
Temperature is one of the most important factors
influencing microbial growth. It can affect living organisms in two
different ways. As the temperature rises, chemical and enzymatic
reactions inside the cell progress at a more rapid rate thus growth is
accelerated. On the other hand, above a certain temperature,
particular proteins may be permanently denatured or damaged.
There are three cardinal temperatures that characterize every
microorganism. The first is called minimum temperature; there is no
growth below this temperature. Second is called the optimum
temperature; this is the temperature where growth is most rapid.
Lastly, the maximum temperature is a temperature above where
growth is not possible. Optimum temperature is always nearer the
maximum temperature than the minimum temperature. The
maximum growth temperature of a given organism most likely
reflects the inactivation of one or more key proteins in the
organism. Minimum temperature of organisms results from freezing
of the cytoplasmic membrane making it unable to function properly
as a transporter of nutrients or proton gradient formation. In the
experiment, only the minimum growth temperature was seen in the
refrigerator setup. The growth in this set up is considerably less
compared to the locker and incubator set up. The incubator set up
exhibited the most growth signifying that the optimum growth
temperature of S. cerevisiae is near 37°C.
Eosin-Methylene Blue Agar Medium
Eosin-methylene blue or EMB agar is both a selective and
differential medium. It is used mainly for the isolation of gram
negative enteric bacteria. Methylene blue is used to inhibit gram
positive bacteria. Eosin on the other hand, responds to the changes
in pH, going from colorless to black in acidic conditions. This agar
also contains lactose and sucrose to be used by lactose fermenting
bacteria as energy sources. In the experiment, E. coli exhibited a
positive result due to a special coloration of black with a green
sheen. B. subtilis and S. aureus gave false positive results in our
experiment. Improper and careless inoculation of these two bacteria
may have caused the wrong results.
MacConkey Agar Medium
MacConkey agar is also a selective and differential
medium. Like the EMB agar it is used mainly for the isolation of gram
negative bacteria. It contains both bile salts and crystal violet dye
which inhibit the growth of gram positive bacteria. It also contains
neutral red dye, peptone, and lactose. It is a differential medium
because it can differentiate gram negative bacteria in to two groups;
those that can ferment lactose and those that cannot. A positive
result for this test is the appearance of pink colonies which signifies
lactose fermentation. Our actual results agree with the theoretical
results since only E. coli reacted positively with the MacConkey agar.
Mannitol Salt Agar
Mannitol Salt Agar or MSA is also a selective and
differential medium. It is mainly used for the isolation of
Staphylococcus. It contains high concentration of NaCl which is
detrimental to other microorganisms except Staphylococcus. It can
differentiate Staphylococcus in two groups with the use of its two
components; mannitol and phenol red. Coagulase-positive
Staphylococci produce yellow colonies with yellow zones, whereas
coagulase-negative Staphylococci produce small pink or red colonies
with no color change to the medium. The results we obtained are
partly correct since our S. aureus colonies showed a yellow
coloration. Unfortunately, we obtained false positive results for both
E. coli and B. subtilis. These results may be attributed to improper
and careless inoculation or errors in media preparation.
UV
Ultraviolet light is a form of electromagnetic radiation and
can affect microbial growth. UV radiation between 220 and 300 nm
in wavelength has sufficient energy to cause modifications or breaks
in DNA, sometimes leading to the destruction of genetic material
and death of the exposed organism. Exposure to UV leads to the
production in DNA of pyrimidine dimmers in which two adjacent
pyrimidine bases (cytosine or thymine) on the same strand of DNA
become covalently bonded to one another in such a way that during
replication, the probability of DNA polymerase misreading the
sequence at this point is greatly increased. UV radiation cannot
penetrate solid, opaque, or light-absorbing surfaces, limiting its use
to disinfection of exposed surfaces.
Penicillin
Penicillin G is active primarily against gram-positive
Bacteria because gram-negative Bacteria are impermeable to the
antibiotic. Transpeptidation process, the cross-linking of two glycan-
linked peptide chains, is important in cell wall synthesis. Penicillin-
binding proteins bind penicillin; they cannot catalyze the
transpeptidase reaction, but the cell wall continues to be formed.
The newly synthesized cell wall is no longer cross-linked and cannot
maintain strength. In addition, the antibiotic-PBP complex stimulates
the release of autolysins that digest the existing cell wall. The result
is a weakened, self-degrading cell wall.
Chloramphenicol
Chloramphenicol and other antimicrobial drugs are used
in individuals who have acquired penicillin allergies. Chloramphenicol
and erythromycin serves as a protein synthesis inhibitor by
increasing error frequencies thereby inhibiting the growth of the
organisms. They reacts w/ 50s portion, inhibiting the formation of
peptide bonds in the growing polypeptide chain. (Madigan, 2006)
References
http://www2.hendrix.edu/biology/CellWeb/Techniques/microspread.html. by
Dr. Mark Sutherland. Date accessed: Sept. 5, 2009
http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Media.htm by Dr.
David D. Fankhauser. Date accessed: Sept. 4, 2009.
Madigan MT & Martinko JM. 2006. Brock Biology Of Microorganisms, 11th ed.
USA: Pearson Prentice Hall, Pearson Education, Inc. 140-147.
http://www.mansfield.ohio-state.edu/~sabedon/biol4022.htm Date
accessed: Sept. 4, 2009.