Czarina Mae Q.

Tadeo BSN 1-E

MICROBIOLOGY AND PARASITOLOGY
AUGUST 22, 2021
Exercise 8:
Results and Discussion
1. What are the unique characteristics of lichens?
Lichens may have tiny, leafless branches (fruticose), flat leaf-like structures (foliose),
flakes that lie on the surface like peeling paint (crustose), a powder-like appearance (leprose), or
other growth forms. Lichens are bizarre organisms and no two are alike. Lichens are a complex
life form that is a symbiotic partnership of two separate organisms, a fungus and an alga. The
dominant partner is the fungus, which gives the lichen the majority of its characteristics, from its
thallus shape to its fruiting bodies.
2. Differentiate the three growth patterns of lichens.
 Foliose Lichens
Foliose lichens have two easily distinguishable sides. In other words, there is a top side and there
is a bottom side. They can be very flat, leafy like lettuce, or convoluted and full of ridges and
bumps.
 Fruticose Lichens
Fruticose lichens can be pendant and hair-like, upright and shrubby, or upright and cup-like.
Many fruticose lichens have round branches that have a central core and others are hollow in the
middle. Other fruticose lichens have flat branches that tangle up with each other.
 Crustose Lichens
Crustose lichens are just that, crusts. They form a crust over a surface, like a boulder, the soil, a
car, or your roof shingles. They can come in many bright, vibrant colors like sunny yellow,
orange, and red, as well as grays and greens. Crustose lichens are pressed against their substrate.
Questions for Research:
1. Give examples of green algae and cyanobacteria that maybe found in lichens.
A few lichen fungi (approximately 20) are members of the fungal group Basidiomycota, but the
vast majority are members of the Ascomycota (ascus-forming fungi) and they often produce
conspicuous fungal fruiting bodies (ascocarps) - usually disk-shaped structures
termed apothecia (e.g. Figures E, G). Almost half of the recorded fungi in the world are
ascomycota, and nearly half of these are found only in lichens. Although their spores are
dispersed from the fruiting bodies, these fungi do not seem to have an independent role in nature
because they are extremely slow-growing and generally lack the enzyme systems for degrading
complex polymers.
In contrast to the many thousands of lichen fungi, there are only about 100 photosynthetic
partners. The most common are single-celled green algae of the genus Trebouxia (Figure H),
which are found in many lichens of temperate and arctic/alpine regions, including all species of
the common lichen genus Cladonia. Trebouxiaspecies seldom grow as free-living cells in nature;
instead they seem to be specialised lichen symbionts. Another common photobiont is the
filamentous green algal genus Trentepohlia, especially in Mediterranean and tropical regions.
This and other algal genera of the tropical lichens can be found growing independently in nature.
About 10% of lichens have cyanobacteria (e.g. Nostoc) as the main or only photosynthetic
partner. For example, this is true of many Peltigera species (Figure B). However, some lichens
that contain green algae can also have cyanobacteria in special wart-like structures on the lichen
surface. These structures are termed cephalodia. They are found in about 3-4% of lichen species
and their role is probably to exploit the nitrogen-fixing ability of cyanobacteria. The fact that
lichens can be formed by more than one type of fungus and more than one type of photosynthetic
partner shows us that the lichen symbiosis must have evolved independently on several
occasions.

2. How are lichens classified?

Lichens are classified as fungi and the fungal partners belong to the Ascomycota and
Basidiomycota. Lichens can also be grouped into types based on their morphology. 

3. How do lichens reproduce?

Lichen associations can reproduce in two main ways:
 sexual reproduction and spore production by the fungi, followed by re-association with a
photobiont.
 vegetative, or clonal, reproduction, when both partners disperse together, maintaining a
symbiotic relationship across generations.
4. Enumerate the reasons why lichens are economically important.
Economic importance of lichens is as follows: They are a good pollution indicators. They do not
grow in polluted areas. They grow on rocks and release some chemicals that can disintegrate
rocks and this results in rock weathering.

5. Describe how lichens are used as indicators of air pollution.

Lichens are called very good pollution indicators because these species are susceptible to certain
pollutants. Hence, they do not grow in polluted areas and are found growing well only in non-
polluted areas. Therefore, Lichens are the indicators of pollutants or pollution. Lichens
also absorb sulphur dioxide dissolved in water. Lichens are widely used as environmental
indicators or bio-indicators. If air is very badly polluted with sulphur dioxide there may be no
lichens present, just green algae may be found. If the air is clean, shrubby, hairy and leafy
lichens become abundant.

Exercise 10
Questions for Research:
1. What is mutation?
A mutation is a change in a DNA sequence. Mutations can result from DNA copying mistakes
made during cell division, exposure to ionizing radiation, exposure to chemicals called
mutagens, or infection by viruses.

2. Describe briefly how mutagens cause mutation. Give specific examples.

Mutagens induce mutations by a variety of mechanisms. Some mutagens mimic normal bases
and are incorporated into DNA, where they can mispair. Others damage bases and either cause
specific mispairing or destroy pairing by causing nonrecognition of bases. Mutagens induce
mutations by a variety of mechanisms. Some mutagens mimic normal bases and are incorporated
into DNA, where they can mispair. Others damage bases and either cause specific mispairing or
destroy pairing by causing nonrecognition of bases.

3. What specific method is used to determine whether an induced mutation causes temporary or
permanent change in bacteria?
The Ames test is a widely employed method that uses bacteria to test whether a given chemical
can cause mutations in the DNA of the test organism. More formally, it is a biological assay to
assess the mutagenic potential of chemical compounds.

4. Identify and explain a method on how to repair DNA damage caused by UV light.
One mechanism of repairing UV-induced pyrimidine dimers is direct reversal of the dimerization
reaction. The process is called photoreactivation because energy derived from visible light is
utilized to break the cyclobutane ring structure. The original pyrimidine bases remain in DNA,
now restored to their normal state. As might be expected from the fact that solar UV irradiation
is a major source of DNA damage for diverse cell types, the repair of pyrimidine dimers
by photoreactivation is common to a variety of prokaryotic and eukaryotic cells,
including E. coli, yeasts, and some species of plants and animals. Curiously, however,
photoreactivation is not universal; many species (including humans) lack this mechanism of
DNA repair.

5. What are the advantages and disadvantages of using bacteria instead of laboratory test animals
in screening mutagens?
The advantages of bacterial systems are their availability, easy cultivation, short time of cell
division, and haploidity. Many DNA damaging agents and/or mutator genes cause mutations that
are readily and clearly observed in changes of phenotype. However, the disadvantages are;
Mutagenic pollution of the natural environment is undoubtedly a serious and general problem.
Reports of various agencies indicate that the presence of mutagenic compounds in different
habitats is a common phenomenon rather than an exception. The list of known mutagenic
chemicals is very long. The Environmental Mutagen Information Center database contains over
20 000 citations to literature on agents that have been tested for mutagenic activity. The
problem of the presence of mutagenic chemicals in natural habitats is very important because
such compounds are capable of inducing serious diseases, including cancer. Moreover, they can
potentially damage the germ line of higher organisms, which may lead to fertility problems and
to negative genetic changes in future generations. Therefore, detection of mutagens in the natural
environment is very important. As chemical mutagens elicit deleterious effects on living
organisms at extremely low concentrations, their detection in natural habitats, at levels that can
be dangerous for animals or humans, may be difficult and complicated.
Exercise 11
Results and Discussion:
1. What are the precautionary measures that must be observed to ensure that the culture media
are free from contamination?

1. Wear gloves, lab-coats and use hoods

2. Use your hood correctly
3. Clean your incubator and water bath regularly
4. Spray EVERYTHING with ethanol or IMS
5. Minimize exposure of cells to non-sterile environments
6. Buy sterile reagents and keep them sterile!
7. Use sterile labware
8. Use filter tips and change them often
9. Check your cells often
10. Bleach your contaminated samples
11. Use good labeling practice
12. Common sense

2. How can it be determined if the prepared medium is not contaminated?

Bacterial contamination is easily detected by visual inspection of the culture within a few days of
it becoming infected;
  Infected cultures usually appear cloudy (i.e., turbid), sometimes with a thin film on the
surface.
 Sudden drops in the pH of the culture medium is also frequently encountered.
 Under a low-power microscope, the bacteria appear as tiny, moving granules between the
cells, and observation under a high-power microscope can resolve the shapes of
individual bacteria.

Questions for Research:

1. Enumerate and discuss the different classifications of culture media according to physical
state, composition, use or purpose.
 BASAL MEDIA. Basal media are those that
may be used for growth (culture) of bacteria that do not need enrichment of the media.
Examples:
Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in
these media.

 ENRICHED MEDIA. The media are enriched

usually by adding blood, serum or egg.
Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in
blood agar media.

 SELECTIVE MEDIA. These media favour the

growth of a particular bacterium by inhibiting the growth of undesired bacteria and allowing
growth of desirable bacteria. Examples: MacConkey agar, Lowenstein-Jensen media, tellurite
media
(Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli).
Antibiotic may be added to a medium for inhibition.

 INDICATOR (DIFFERENTIAL) MEDIA. An

indicator is included in the medium. A particular organism causes change in the indicator, e.g.
blood, neutral red, tellurite. Examples: Blood agar and MacConkey agar are indicator media.

 TRANSPORT MEDIA. These media are used when specie-men cannot be cultured soon
after
collection. Examples: Cary-Blair medium, Amies medium, Stuart medium.

 STORAGE MEDIA. Media used for storing

the bacteria for a long period of time. Examples:
Egg saline medium, chalk cooked meat broth

2. Cite examples of different types of culture media.

BASAL MEDIA
Examples:
Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in
these media.

ENRICHED MEDIA.
Examples: Enriched media are blood agar and Lowenstein-Jensen media. Streptococci grow in
blood agar media.

SELECTIVE MEDIA
Examples: MacConkey agar, Lowenstein-Jensen media, tellurite media
(Tellurite inhibits the growth of most of the throat organisms except diphtheria bacilli).
Antibiotic may be added to a medium for inhibition.

INDICATOR (DIFFERENTIAL) MEDIA Examples: Blood agar and MacConkey agar are
indicator media.
TRANSPORT MEDIA.
Examples: Cary-Blair medium, Amies medium, Stuart medium.

STORAGE MEDIA.
Examples:
Egg saline medium, chalk cooked meat broth

3. What are the factors to consider when preparing culture media?

Five Tips for Culture Media Preparation Success
 Reduced growth/recovery rates.
 Atypical colonial morphology.
 Inhibition of target organism.
 Failure to inhibit competing flora.
 Reduced shelf life of prepared medium.

4. What are the proper ways of storing cultire media if they are not in use?
Plates should be stored in the inverted position to prevent moisture from contacting the surface
of the media. Media should never be exposed to sunlight or UV light, since many ingredients,
especially dyes and indicators, are not stable upon light or heat exposure. Media should be stored
in a dark environment.

5. Enumerate the importance of culture media in cultivating microbes.

Culture media is of fundamental importance for most microbiological tests: to obtain pure
cultures, to grow and count microbial cells, and to cultivate and select microorganisms. Without
high-quality media, the possibility of achieving accurate, reproducible, and repeatable
microbiological test results is reduced. Culture media contains the nutrients needed to sustain a
microbe. Culture media can vary in many ingredients allowing the media to select for or against
microbes. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates
as inorganic nitrogen sources in culture media.

Exercise 12:
Results and Discussion:
1. What is the importance of sterilizing the inoculating loop and needle before and after the
inoculation process?
We will be working with many pathogenic species of bacteria in the laboratory. Remember that
bacteria are in the air as well as on the skin, the counter, and all objects and equipment that have
not been sterilized. You first sterilize it to kill off any bacteria on the loop because it could alter
your results. You sterilize it again after picking up the culture to kill off bacteria. Flame-sterilize
the inoculating loop in order to prevent contamination of the bench surface and as a
consideration to others in the lab who may later use the inoculating loops. Place the flask into an
incubator for growth overnight.
The most important tool for transferring cultures is the wire inoculating needle or loop. It can be
quickly sterilized by heating it to red hot in a Bunsen burner flame. Adjust the air inlets of the
burner so that there are a hotter inner cone and the cooler outer flame. A dry needle may be
sterilized by holding it at a 30-degree angle in the outer part of the flame. A wet loop with
bacteria on it should first be held in the inner part of the flame to avoid spattering, and then
heated until red hot in the outer part of the flame. Always flame the loop immediately before and
after use! Allow it to cool before picking up an inoculum of bacteria (or you will kill the
bacteria). Hold the loop or wire handle like a pencil.
2. Explain the significance of aseptic culture technique in growing microorganims.
Proper aseptic technique prevents contamination of cultures from foreign bacteria inherent in the
environment that may potentially contaminate cultures, thus interfering with the lab results.
Using proper aseptic technique can greatly minimize or even eliminate the risk of contamination.
In the microbiology lab we use aseptic technique to: Prevent contamination of the specific
microorganism we are working with. Prevent contamination of the room and personnel with the
microorganism we are working with.

3. What is the purpose of flame-sterilizing the mouth of the test tube and the side of the Petri
diah during inoculation?
Flaming the Mouth of the Test Tube: Passing the mouth of a tube through the flame of a Bunsen
burner creates a convection current which forces air out of the tube. This prevents airborne
contaminants from entering the tube.

Questions for Research:

1. Cite the uses of agar plate, agar slant, agar deep/butt, and broth culture media.
Agar plates will be used for isolation and some assays, and for short term maintenance of
cultures. Agar slant tubes will be used for long term maintenance of isolates. Broths (liquid
media) will be used to grow isolates for some assays or for the assays themselves. Agar Deep
grows bacteria that requires less Oxygen.

2. Differentiate the streak plate method from the pour plate method.
The main difference between streak plate and pour plate is that in streak plate, the first to be
added is the melted nutrient agar and the second to be added is a loop of bacteria from a slant,
whereas the first to be added in pour plate is the bacterial broth and the second to be added is the
nutrient agar.

3. How can one be sure that the culture media are sterile or free from contamination?
Without removing cap/lid, place them in the incubator and see if they grow anything. It is
much better to store media at room temperature and detect contamination before the medium is
used. Moist heat provided by an autoclave or pressure cooker is an efficient way to sterilize most
materials. Most materials are effectively sterilized by 15 minutes exposure to this temperature.
4. Indicate the correct way of inoculating the following media.
Plate media
A. For Sensitivity testing
How is sensitivity analysis performed?
Sensitivity testing starts with a bacterial sample. Your doctor will get this sample by sampling
the infected area. Your doctor can sample any area that has an infection.
Samples may be taken from:
 blood
 urine
 sputum (spit)
 inside the cervix
 a pus-containing wound
Your doctor will send the sample to a laboratory, where it’ll be spread on a special growing
surface. The grown bacteria is known as a culture, and bacteria in the culture will grow and
multiply. The bacteria will form colonies, or large groups of bacteria, that will each be exposed
to different antibiotics. These colonies can be susceptible, resistant, or intermediate in response
to the antibiotics: Susceptible means they can’t grow if the drug is present. This means the
antibiotic is effective against the bacteria. Resistant means the bacteria can grow even if the drug
is present. This is a sign of an ineffective antibiotic. Intermediate means a higher dose of the
antibiotic is needed to prevent growth.


B. For isolation of colonies

There are two main ways to isolate organisms.
 Streaking for isolation on an agar plate
 The pour plate method
Streaking for isolation on an agar plate involves the successive dilution of organisms until
you have the cells at a low enough density that single cells are physically isolated spatially to
give rise to recognizable individual colonies. In the pour plate method, you dilute your sample
sufficiently before you add it to molten cooled agar and then pour this mixture in a dish. The
isolated cells give rise to individual colonies growing in the agar itself. This technique can be a
little tricky. If the melted agar is too hot you kill all the bacteria. If the melted agar is too cool
you end up with a big lump in your Petri dish. The streaking method yields individual colonies
on the surface of the agar. This technique is much faster and easier to master.
5. What are the precautionary measures to be observed before, during, and after applying the
aseptic culture technique?
General Safety Considerations:
 Access to the lab is limited.
 Wear your lab coat and gloves.
 Tie back long hair.
 Leave all food and drink in your backpack. Do not chew gum in lab.
 The only thing on your lab bench should be the equipment you are working with and your
lab book. Place your backpacks on the floor where you or someone else will not trip over
them.
 Discard contaminated material in the appropriate container. Anything that has been in
contact with microorganisms must be disinfected with a disinfectant such as Cavicide©
or autoclaved.
 Clean up all spills immediately!
 Decontaminate your lab bench with disinfectant such as Cavicide© before and after lab.
 Wash your hands before leaving the lab.

Exercise 13
Questions for Reasearch:
1. What is the significance of describing the cultural or colonial characteristics of bacteria?
Although one might not necessarily see the importance of colonial morphology at first, it really
can be important when identifying the bacterium. Features of the colonies may help to pinpoint
the identity of the bacterium. Different species of bacteria can produce very different colonies.

2. How would you determine whether a colonh is a contaminant or a real bacterial culture?
1. Perform Gram staining and look at the morphology of the bacterial cells, if contaminated more
than one cell type shall be visible.
2. Streak the culture on a suitable agar based medium and observe color and type of cfus.

3. How can you determine whether or not there is bacterial culture growth in nutrient broth?
Bacterial growth in broths is indicated by the development of a cloudy appearance. If the newly
inoculated broth looks cloudy at the start, you will have no way to determine if this is due to
bacterial growth during the incubation period.

4. What are the advantages of using a solid culture medium over a liquid form?
Advantages of solid media:
(a) Bacteria may be identified by studying the colony character,
(b) Mixed bacteria can be separated.
Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most commonly used to
prepare solid media. Agar is polysaccharide extract obtained from seaweed.
5. Enumerate other microorganisms that produce pigments in culture media and identify the

color they produce.