Ece Kocabıyık, Wednesday afternoon, group 5

EXERCISE 23-24-26 REPORT

AIM
To understand the effect of pH and UV lights to different microorganisms. And also, to learn what is
the role of thioglycollate medium and how we can use it. And to understand the differences between
results of all three experiments and learn how to make conclusions of them.
RESULTS
-P.vulgaris
-UV EXPOSURE :

Time Number of colonies

Control Carpet like
5 min. 45
10 min. 5
15 min 6
In the control plate, the cells are all over the agar and continusly grew like carpet. In the 5 minutes
plate, there were 45 countable cells but also one side of the plate which is the covered part by the lid of
the plate when uv light was applied covered by cells like carpet structure. In the 10 minute plate, there
were 5 countable cells and also two sides of the plate covered carpet like structure. In the 15 minute
plate, there 6 countable colonies and a little bit carpet like structure in one side of the plate.
-THIOGLYCOLLATE MEDIUM, OYGEN REQUIREMENT :
The microorganis seems to be a facultative organism because there were turbidity on the top of the
tube and also a little below of the top. So,turbidity is mostly in the upper side of tube.
-EFFECT OF PH :
There are no turbidity in the 3 pH tube. 7 and 5 ones are similar and they have most abundance of the
cells. And in 9 pH tube, turbidity can be seen but less than 7 and 5. Also, 7 pH tube has a little bit
turbidity than 5 pH tube. And all of them were yellow.
DISCUSSION
-The optimal growing conditions of this organism is in a facultative anaerobic environment with an
average temperature of about 40 °C and pH 6.0.
-UV EXPOSURE :
In the control plate, we expected to see most abundant colonies and we saw it like that. Colonies were
all over plate and had the carpet like structure. In the 5 minutes plate, there were less colonies than
control group as expected. In the 10 minute plate, there were 5 colonies but also two sides of the plate
covered by carpet like cells because lid of the plate covered the agar when UV light applied.
So,actually, there were more than 5 colonies on the agar. In the 15 minute plate, there were 6
countable cells and less carpet like cells than 10 minute plate. Therefore, there were less colonies but
countable ones are one more extra.
-THIOGLYCOLLATE MEDIUM, OYGEN REQUIREMENT :
We expected to see facultative anaerobic growth on the tube because the organism is facultative
anaerobic. The growth was like in our manuel’s example picture.
-EFFECT OF PH :
P. vulgaris has the optimal pH at around 6. Therefore, we expected to see most turbid ones are 5 and 7.
And the most abundant one is 7 pH tube. Then, 5 pH tube follows it. Also 9 pH tube looked like turbid
but much less than 5 pH one. Finally, there was one turbidity on the 3 pH tube because it too much
acidic to be grow of P.vulgaris so the peptides and amino acids denaturated and cells can not grow. In
the 9 pH tube, some cells grow but other cells are dead so this condition is not proper to P. Vulgaris.
QUESTIONS
Q1: What are the mechanisms of adaptation to acidic/basic environments developed by
microorganisms?
Acidophiles have unique cell membrane structure to stabilize the internal pH of the cell and they have
several mechanisms like passive or active mechanisms. In passive mechanism, some microorganisms
secrete a biofilm to slow down the diffusion of molecules into the cell. Other microorganisms are able
to change their cell membrane to incorporate substances such as fatty acids that protect the cell. Some
of the secrete a buffer to stabilize the pH. In active mechanism, microorganisms pump hydrogen ions
out of their cells at a constantly high rate. By doing this they are able to keep their internal pH at
around 6.5—7.0.
In alkaliphiles cell membrane is important and also compenents of the cytoplasm is important for
stability. They have active and passive mechanisms for regulation. Cytoplasmic pools of polyamines
and low membrane permeability are two elements of passive regulation, whereas sodium ion channels
drive the active regulation. In passive mechanism, some alkaliphiles have cytoplasmic pools of
polyamines which are rich in amino acids with positively charged side groups. Therefore, these cells
are able to buffer their cytoplasm. Moreover, low membrane permeability is another type of passive
mechanism because of preventing fewer protons move in and out of the cell. In active mechanism,
some bacteria have sodium ion channels that actively control the entry of protons through membrane.
Therefore, this is decreasing the overall pH of the cytoplasm.
Q2: What are the UV survival mechanisms of bacteria?
They have efficient DNA repair mechanisms and active defense against UV-induced oxidative stress,
and consequently need to tightly regulate their proteome. Mechanisms include tolerance induction and
SOS response induction. For UV tolerance induction, the UV sensor is an extracellular protein, which
is a UV tolerance-specific extracellular sensing component (ESC). This component is formed by
unstressed cells and on interacting with the stimulus (UV) in the medium. It is this extracellular signal
which interacts with the sensitive organisms and triggers tolerance induction.
Q3: How does atmospheric oxygen induces harm on the microaerophiles and strictly anaerobes?
In microaerophiles, NADH oxidases are used to regulate the oxygen concentration.This family of
enzymes directly reduces molecular oxygen to hydrogen peroxide or water. But they are not efficient
as like in yeasts, enzymes of the pentose phosphate pathway lead to increased sensitivity to oxidative
stress, and the pathway is required for the maintenance of the cellular redox state or like in mammalian
systems, glucose 6-phosphate dehydrogenase areessential in protecting cells against oxidative stres.
Microaerophiles do not have these enzymes. So, they can not tolarate atmospheric oxygen levels.
In strictly anaerobes, they cannot tolerate oxygen because they utilize metabolic schemes built around
enzymes that react with oxidants. The reliance upon low-potential flavoproteins for anaerobic
respiration probably causes substantial superoxide and hydrogen peroxide to be produced when
anaerobes are exposed to air and they died because superoxide anion must be removed from the cell in
order to survive(because of the reaction between superoxide anion and the hydroxyl radical produce
singlet oxygen which when excited is very reactive and also damages the cell.).
REFERENCES
Aron H.,Yonenaka H.H. and Nakada H.I.(1963). GLUCOSAMINE-6-PHOSPHATE
DEGRADATION BY PROTEUS VULGARIS AND ISOLATION OF
PHOSPHOGLUCOSAMINISOMERASE1. Department of Biological Sciences, University of
California, Santa Barbara, California
Bodansky O. and Stewart C.P.Advances in Clinical Chemistry. Academic Press. 355 pages.
Gonzalez-Toril E., Llobet-Brossa E., Casamayor E.O., Armann R. and Amils R.(2003). Microbial
Ecology of an Extreme Acidic Environment. The Tinto River. Appl. Environ. Microbiol. 69:
4853-4865.
Horikoshi, K. (1999). Alkaliphiles : Some Applications of Their Products for
Biotechnology.Microbial. Mol. Biol. Rev. 63(4):735-750.
Imlay JA.(2002). How oxygen damages microbes: oxygen tolerance and obligate anaerobiosis. Microb
Physiol. 46:111-53.

Mobley HLT, Mendz GL, Hazell SL,(2001). Helicobacter pylori: Physiology and Genetics.
Washington (DC)
Rowbury RJ.(2003). UV radiation-induced enterobacterial responses, other processes that influence
UV tolerance and likely environmental significance.Sci Prog. 86(Pt 4):313-32.