The document is a project report that studied the effect of UV rays and photoreactivation on the colonial morphology and catalase activity of baker's yeast and wild yeast. UV exposure altered the colonial morphology from smooth to rough and increased the catalase activity in both yeast strains. Photoreactivation restored the colonial morphology and catalase activity back to pre-UV exposure levels. The report analyzed the results and discussed how UV radiation causes mutations in yeast by damaging DNA, but photoreactivation can repair this damage.

1. Project Report
2014
Submitted by:
Amna Jalil (17)
Submitted to:
Dr.Nazia Jamil
[EFFECT OF UV RAYS &
PHOTOREACTIVATION ON THE COLONIAL
MORPHOLOGY AND CATALASE ACTIVITY
OF BAKER’S AND WILD YEAST]

2. Contents
Abstract
Objectives
Introduction
Materials & Methods
Results
Discussion
References

3. Abstract
Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible
light, but longer than X-rays, that is, in the range between 400 nm and 10 nm. UV light causes
mutation in yeast cells and is lethal for its survival. Its main effect is on genes and DNA. In this
study two strains of yeast cells (one wild type isolated from soil and 2nd
one was baker’s yeast)
were taken and mutated with UV light for 1 minute to check the effect of UV on the colonial
morphology of yeast as well as on the catalase activity of yeast cells.Colonial morphology is
affected due to UV exposure and so does catalase activity which is surprisingly increased after
UV exposure. After photoreactivation, colonial morphology is same as it was before the
administration of UV and so does the catalase activity.
Objectives
 To check the effect of UV on the colonial morphology of wild as well as baker’s yeast
 To check the effect of Photoreactivation on the colonial morphology of wild as well as
baker’s yeast
 To check the effect of UV on the catalase activity of wild as well as baker’s yeast
 To check the effect of Photoreactivation on the catalase activity of wild as well as baker’s
yeast
Introduction
Ultraviolet (UV) light is electromagnetic radiation with a wavelength shorter than that of visible
light, but longer than X-rays, that is, in the range between 400 nm and 10 nm. It is so-named
because the spectrum consists of electromagnetic waves with frequencies higher than those that
humans identify as the color violet. These frequencies are invisible to most humans except those
with aphakia. Near-UV is visible to a number of insects and birds.

4. Ultraviolet radiation in our environment is as common as sunlight. It generates genetic diversity
and kills cells.When a DNA molecule is damaged by radiation and the damage is not repaired
before the DNA replicates, the cell is likely to die. When a cell cannot divide to form viable
progeny, we say that it has suffered reproductive death. The cell may still be able to metabolize
and grow, but it cannot divide. If the radiation dose is high enough, cells can be killed outright --
metabolic death -- but other metabolic functions are far more resistant than reproduction.
In single-celled organisms such as yeast, other fungi, bacteria, and algae, mutations are an
important sublethal effect. Radiation also produces sublethal chromosomal changes and
stimulates genetic recombination.
Ultraviolet, this does not break the DNA chain outright, is selectively absorbed by the aromatic
rings of the purine and pyrimidine bases, so its energy, being more concentrated, is as damaging
as ionizing radiation. One particularly unpleasant result is the formation of pyrimidine dimers. In
this reaction, two adjacent pyrimidines in the same chain (T-T, C-C, or T-C) become covalently
bonded together. These dimers disrupt the local structure of the DNA double helix and prevent
normal DNA replication. They are not much better than a double-strand break, as far as the cell
is concerned.

5. Figure: Effect of UV on DNA
Photoreactivation (PR) is the recovery from biological damage caused by UV by simultaneous or
subsequent treatment with light of longer wavelength (PR light). Pyrimidine dimmers have been
shown to be induced in the deoxyribonucleic acid (DNA) of Saccharomyces cerevicae and other
yeasts by ultraviolet (UV) radiation. This damage can be repaired by a dark-repair mechanism
that alters or removes the dimmers Or by a light-repair system (photoreactivation) that
monomers them.
The splitting of the dimers is mediated by photoreactivating enzyme (PR), an enzyme that can be
removed in yeast by a mutation in the PHR1 gene.
Materials & Methods
Apparatus
 Petri plates
 Test tubes
 Test tube stands
 Incubator
 Spreader
 Micropipettes
 Microtips
 UV Illuminator
 Aluminium foil
 Spirit lamp
 Wire loop
 Flask

6. Reagent
 3% H2O2
Media
 YPD Agar
Sr No Ingredients Amount (g/liter)
1. Peptone 20
2. Dextrose 20
3. Yeast extract 10
4. Agar 15
For YPD Agar Peptone, Yeast extract and agar were dissolved in 700 ml of water and pH was set
at 6.5 and then autoclaved it. Dextrose was separately mixed with 300ml of distilled water and
after adjusting the pH autoclaved it for only 10 minutes and after cooling mixed with the rest of
the media.
 YPD Broth
Sr No Ingredients Amount (g/liter)
1. Peptone 20
2. Dextrose 20
3. Yeast extract 10
For YPD Broth Peptone and Yeast extract were dissolved in 700 ml of water and pH was set at
6.5 and then autoclaved it. Dextrose was separately mixed with 300ml of distilled water and after
adjusting the pH autoclaved it for only 10 minutes and after cooling mixed with the rest of the
media.

7. Yeast Strains
Two yeast strains were used in this experiment.
1. One strain used was of baker’s yeast
2. One strain used was isolated from the soil sample taken from the garden of MMG
Procedure
Collection of the Yeast Sample
 Soil sample was collected from the garden of the MMG department of the University of
the Punjab.
 Baker’s yeast strain was purchased from the market
Spreading on YPD Agar plates
 For soil sample, serial dilution was made and two dilutions (10-3
& 10-5
) were spread on
YPD Agar plates and incubated on 30°C for two days in order to achieve the crowding.
Figure: Crowding of yeast on 10-3 dilution of the soil sample

8.  For Baker’s yeast sample, 0.5g of yeast was taken and mixed in about 10 ml autoclaved
YPD broth and incubated on 37°C for 2-3 hours for enrichment and after that spread on
the YPD agar plates and incubated.
Figure: Crowding of yeast from the baker’s sample
Isolation of Pure Strains
Yeast strains were identified by performing wet mount and then purified by performing streaking
on separate YPD agar plates.
Figure:Streaking of the pure sample from soil

9. Figure:Streaking of the pure sample of Baker’s yeast
Exposure of UV Radiations
YPD broth was poured in two test tubes and autoclaved. Then the single colony was picked from
both types of stains and was dissolved in different test tubes. After that, both test tubes were
exposed to UV rays in UV Illuminator for 1 minute and after the exposure were immediately
spread on YPD agar plates and covered with aluminum foil. After that, these plates were
incubated at 30°C for 48 hours.
Exposure of UV plus 2 min in sunlight
Photoreactivation requires exposure to a relatively intense source of visible light either in the sun
or artificial light. For photoreactivation, inoculated YPD plates after exposure to the UV
radiation, were exposed to the sunlight for 2 minutes and then incubated at 30°C for 48 hours.
Analyzing the Effect of UV & Photoreativation on Colony
Morphology
 To check the effect of UV on colonial morphology, colonial morphology was noted
before and after the UV exposure.
 To check the effect of photoreactivation on colonial morphology, colonial morphology
was noted after the exposure of UV irradiated plates to the intense visible light.

10. Analyzing the effect of UV & Photoreactivation on Catalase
Activity
 To check the effect of catalase activity, catalase test was performed before and after the
UV exposure with the help of 3% H2O2 as a reagent.
 To check the effect of catalase activity, catalase test was performed after the exposure of
UV irradiated plates to the intense visible light with the help of 3% H2O2 as a reagent.
Results
Effect of UV & Photoreactivation on the Colonial Morphology of
Baker’s Yeast Sample
Table: showing the effect of UV exposure & Photoreactivation on the colonial morphology of
Baker’s yeast sample
Property Before UV Exposure
After UV Exposure
(1 min)
After
Photoreactivation
(2 min)
1. Form Circular Circular Circular
2. Elevation Convex Raised Convex
3. Margins Entire Entire Entire
4. Color Cream colored Cream colored Cream colored
5. Surface Smooth & Shiny Rough & Matt Smooth & Shiny

11. Figure: Colony morphology of Baker’s yeast before UV exposure
Figure: Colony morphology of Baker’s yeast after UV exposure
Figure: Colony morphology of Baker’s yeast after photoreactivation

12. Effect of UV & Photoreactivation on the Colonial Morphology of
Wild type Yeast Sample
Table showing the effect of UV exposure & Photoreactivation on the colonial morphology of
wild type yeast sample
Property
Before UV
Exposure
After UV Exposure
(1 min)
After
Photoreactivation
1. Form Circular Circular Circular
2. Elevation Convex Raised Convex
3. Margins Entire Entire Entire
4. Color Cream colored Cream colored Cream colored
5. Surface Smooth & Shiny Rough & Matt Smooth & Shiny
Figure: Colony morphology of wild yeast before UV exposure

13. Figure: Colony morphology of wild yeast after UV exposure
Figure: Colony morphology of wild yeast after photoreactivation
Effect of UV on the Catalase Activity of Baker’s Yeast Sample
Figure: Catalase activity of Baker’s yeast before UV exposure (Positive)

14. Figure: Catalase activity of Baker’s yeast after UV exposure (increased)
Figure: Catalase activity of Baker’s yeast after photoreactivation
Effect of UV on the Catalase Activity of Wild Yeast Sample
Figure: Catalase activity of wild yeast before UV exposure (Positive)

15. Figure: Catalase activity of wild yeast after UV exposure (increased)
Figure: Catalase activity of wild yeast after photoreactivation
Discussion
It is well known that Ultraviolet radiations are lethal for yeast cells. UV radiations cause
mutations in yeast cells. A certain amount of mutational changes in the genome occurs as a
natural process, though the probability is low. These radiations affect the cell growth rate as well
as survival rate. These radiations also affect the colonial and cellular morphology of yeast cells.
UV radiations also affect the enzymatic activity of yeast cells.
After the UV exposure of 1 minute, In case of colonial morphology of baker’s yeast the form
remains same i.e., circular, elevation is changed from convex to raised. Margins of colonies are
not much affected and remain entire. Color of colonies is also not changed after UV exposure.
Surface of colonies is affected and changed from smooth & shiny to matt and rough. Size of the
colonies is not much changed after the treatment with UV.
After the UV exposure of 1 minute, the results of the colonial morphology of the wild yeast
strain were similar to those with the baker’s yeast strain.

16. In both strains of yeast survival rate also decreases and the effect of UV is mainly on the genetic
level i.e., on genes.
For the analysis of enzymatic activity of catalase, catalase test was performed both before and
after the UV exposure and results were noted. Both yeast strains were catalase positive and after
the exposure of UV, their catalase activity increases. The effect of UV irradiation on the catalase
activity of an aqueous yeast suspension was divisible into 4 periods. First, the period during
which the cells lost their ability to form colonies, but during which no change in catalase activity
was noted. Second, the period during which a considerable rise in catalase activity occurred.
Third, a rather long period during which irradiation led to no diminution in the catalase activity
of the maximally active suspension. Fourth, the period of photoinactivation of the intracellular
enzyme, which was quite similar to that of the crystalline enzyme in vitro. So, by this analysis,
both samples were in second period.
As long as photoreactivation is concerned, colony morphology was same as it was before the
exposure of UV and so does the catalase activity of both the strains of yeasts.
References
Aldous Jg, Stewart Dkr. The effect of ultraviolet radiation upon enzymatic activity and viability of the
yeast cell.Can J Med Sci. 1952 Dec;30(6):561–570.
E. Tomkinson, S. Wei, Z.Y. You. Nucleotide excision repair in yeast: recent progress and implications.
1998. Nucleic Acids Mol. Biol., 12:125-139.
Kaplan Jg.The alteration of intracellular enzymes. II. The relation between the surface and the biological
activities of altering agents. J Gen Physiol. 1954 Nov 20;38(2):197–211.