PHC 429: PHARMACEUTICAL

MICROBIOLOGY AND PARASITOLOGY

PRACTICAL I
a) INTRODUCTION TO MICROSCOPE
b) ASEPTIC TECHNIQUE AND BACTERIAL CULTURE

NAM
E STUDENT ID

NOR NAJIHAH BINTI OMAR

NUR ARFA HUSNA BINTI ISMAIL

NURUL ‘AQILAH BINTI OTHMAN

NUR AIN NABIHAH BINTI KAMARUDIN

KHAIRUL AYRINA BINTI KHAIRIL ANUAR

PRACTICAL 1a: INTRODUCTION TO MICROSCOPE

INTRODUCTION:

Microscope is an important tool in order to analyze the organisms that are too small to be
seen with naked eye. This small organism is termed microorganism. Although small, some
microorganism can cause serious infections to humans. There are about 500 different
bacterial genera but only 10% of these species may be pathogens so in order to identify these
pathogens, microscope was definitely an important tool to study every aspect of the microbes
including its genetics, physiology, characteristics, interactions and uses. We must know how
to use the microscope properly in order to identify various microorganisms that exist around
us.

OBJECTIVE:

1. To determine all the parts of a compound microscope.

2. Able to use the microscope correctly with the use of oil immersion lens and wet
mounts.
3. Understand how microorganisms can be measured under the light microscope.

METHOD:

Materials

1. Prepared demonstration slides:

Bacteria:

a. Staphylococcus aureus b. Escherichia coli c. Salmonella typhi

d. Streptococcus sp. e. Bacillus subtilis f. Bacillus megaterium

g. Proteus vulgaris h. Clostridium botulinum i. Haemophilus influenzae

Fungi:

j. Rhizopus sp. k. Aspergillus sp. l. Candida albican

m. Penicillium sp. n. Mucor sp.

2. Broth culture of Saccharomyces cerevisiae

A. Technique using microscope.

1. The voltage control dial of microscope was turn to 1. Then the microscope were plug
in and turn on.
2. Slide was put on the slide holder, the slide was centered using stage control and a
drop of immersion oil was dropped onto the observed area.
3. The white striped 100X oil immersion objective was rotated until locked into place.
Give the magnification 1000X.
 4. The slide and objective lens was watched carefully, the oil immersion objective was
lowered until it touched the slide.
5. Fine focus was turn with slow steady until the specimen comes into focus.
6. The light was adjusted to optimum contrast using iris diaphragm lever.
7. Oil immersion objective was wiped using lens paper, the voltage control dial then turn
back to 1 and the microscope off.

B. Observe all the prepared demonstration slides.

All 14 samples were observed under microscope using magnification 4X, 10X, 100X.

C. Prepare a wet mount of baker's yeast Saccharomyces cerevisiae.

1. A small drop of the yeast culture was put on a microscope slide using a pipette and a
cover slip was placed over the drop.
2. The light was reduced for improved contrast using the iris diaphragm lever by moving
the lever almost all the way to the right.
3. The yeast cell was observed using oil immersion microscopy. The diameter of a
typical yeast cell was measured.
4. At the completion of the lab, oil from the oil immersion objective was removed using
lens paper and microscope was put away. Several of the bacteria from each of the four
prepared slides were drew and their approximate size was indicated in micrometers

PRACTICAL 1b: ASEPTIC TECHNIQUE AND BACTERIAL CULTURE

INTRODUCTION:

There are various types of culture media used to promote microbial growth in the laboratory
such as selective media, enrichment media and chemically defined media. These media are
used to observe the microbes present around us such as the microbes from soil, air, water and
volcanoes and humans. The process of transferring the bacterial cultures from one media to
another media requires a proper aseptic technique to prevent contamination of cultures from
foreign bacteria inherent in the environment which can lead to the false results about the
types of bacteria present in certain places. Not only the transfer of bacterial cultures from one
media to the other media needs to be aseptic, the medium in which the transfer takes place
must also be sterile which means the media is free of all living things because the media will
be used to observe organisms in the form of pure culture.

OBJECTIVE:

1. Determine the different types of media available in the lab.

2. Know how to use the aseptic techniques when handling bacterial cultures.
3. Able to differentiate and describe the various colony morphologies grown from their
specimen.

A. Aseptic Technique

The procedure for aseptically transferring microorganisms is as follows:

1. The inoculating loop was sterilized by passing it at an angle through the flame of a gas
burner until the entire length of the wire becomes orange from the heat. In this way all
contaminants on the wire are incinerated. The loop was allowed to cool for a few seconds to
avoid killing the inoculums

2. The inoculums was aseptically transferred from a broth culture (organisms growing in a
liquid medium) into a liquid media containing and agar media containing Petri dish.

3. The inoculums was aseptically transferred from a Petri dish (organisms growing in a solid
medium) into a liquid media containing tube and agar media containing Petri dish.

B. Forms of Culture Media

1. Broth tubes are tubes containing a liquid medium. After incubation, growth (development
of many cells from a few cells) may be observed as one or a combination of three forms:
pellicle, turbidity and sediment.

2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar-agar. The
medium has been allowed to solidify at an angle in order to get a flat inoculating surface.

3. Stab tubes (deeps) are tubes of hardened agar medium which are inoculated by "stabbing"
the inoculums into the agar.
4. Agar plate is sterile Petri plates that are aseptically filled with a melted sterile agar medium
and allowed to solidify. Plates are much less confining than slants and stabs and are
commonly used in the culturing, separating, and counting of microorganisms.

C. Oxygen Requirements for Microbial Growth

Microorganisms show a great deal of variation in their requirements for gaseous oxygen.
Most can be placed in one of the following groups: obligate aerobes, microaerophiles,
obligate anaerobes, aerotolerant anaerobes and facultative anaerobes.

D. Temperature Requirements

Microorganisms are divided into groups based on their preferred range of temperature:
psychrophiles, mesophiles, thermophiles and hyperthermophiles.

MATERIALS:

Media

 2 Nutrient Broth (NB)

 2 Nutrient Agar Slant Tubes (NAS)
 2 Nutrient Agar Stab Tubes (NAST)
 7 Nutrient Agar Plates (NAP)

Broth cultures of
  Bacillus subtilis
 Escherichia coli
 Micrococcus luteus
 Serratia sp.

METHOD :

1) One NB, one NAS, one NAST and one NAP were aseptically inoculated with B.
subtilis. All tubes were labelled with a marker. This procedure is termed streaking
for isolation and has a diluting effect. The friction of the loop against the agar causes
organisms to fall off the loop. Near the end of the streaking pattern, individual
organisms become separated far enough apart on the agar surface to give rise to
isolated single colonies after incubation.

2) One NB, one NAS, one NAST and one NAP were aseptically inoculated with E. coli.
3) M. luteus was aseptically transferred from the plate given to an empty NAP.

4) Serratia sp was aseptically transferred from the plate given to an empty NAP.

5) All the tubes and plates inoculated with the cultures were incubated at 37°C. The
tubes were placed in a wire basket to keep them upright. The plates was incubated
upside down (lid on the bottom) to prevent condensing water from falling on the
growing colonies and causing them to run together.

6) In order to illustrate that microorganisms are all around us and to demonstrate the
necessity for proper aseptic technique, three NAP was contaminated as follows:

a) First, the lid from the first agar plate was removed and the exposed agar portion was
placed in or out of the building for the duration of today's lab. The lid was replaced;
the plate was labelled “air”, and was incubated at room temperature.

b) Next, the second Petri plate was divided in half using a marker. A sterile cotton swab
was moistening in sterile water. The swab was rubbed over the surface of a toilet
 seat. This swab was used to inoculate half of the second agar plate. The plate was
labelled and incubated at room temperature.

c) Lastly, the third Petri plate was divided in half using a marker pen. A member’s
finger was rubbed over the surface of the half of the third agar plate. The plate was
labelled and incubated at 37°C.

RESULT AND OBSERVATION 1A

PREPARED DEMONSTRATION SLIDE
MICROORGANISM (100X) DESCRIPTION

 Gram positive bacteria

 Diameter 0.5-1.25 μm

Streptococcus species
  Gram positive bacteria
 Diameter 1 μm

Staphylococcus aureus

 Gram positive bacteria

 Diameter 0.25-1.0 μm

Bacillus subtilis

 Gram negative bacteria

 Diameter 0.25-1.0 μm

Escherichia coli

WET MOUNT OF BAKER'S YEAST SACCHAROMYCES CEREVISIAE

MICROORGANISM 100X DESCRIPTION

 Yeast
 Diameter of 2-8μm
 Globose, and ellipsoid to elongate in
shape.
 Saccharomyces cerevisiae

DISCUSSION 1A

Microscope is one of the instruments that needed for a microbiologist to discover a

cell in this world. Microscope consists of several components where every single part has its
function. One of the most important parts in the microscope is eyepiece where we can see the
view of the enlarged specimen whether with 4x, 10x, and also 100x magnificent. Next, the
objectives lenses are the closest lenses to the specimen which they can change the
magnificent power by rotating them. Stage is a flat platform to put the slide. While on the
stage, there is a stage clips where the metal clips can holds the slide to make sure it stay in the
place. At the edge of the stage, stage control is there and its function is to move the stage
whether to the left, right, front or back. Other than that, coarse focus knob is to move the
stage up and down quickly to make the specimen views more clear and focus. Besides, fine
focus knob is used same with the coarse focus but it is to make small focus adjustment. Last
but not least, illumination is a light source for a microscope where is located on the base of
the microscope and rheostat light intensity control is to adjust the amount of light passes
through the specimen slide.

Function of oil immersion lenses is to achieve the maximum possible magnification

by immersing both of the objectives lenses and the specimen slide in the oil immersion. This
is because the light rays are do not bend when they passes through the glass to oil immersion
so this allows the maximum amount of lights to enter the objectives lenses. In fact, this action
increasing the numerical aperture of the objectives lenses and it can achieve the
magnification up to 1000x rather than without the oil immersion.

For observation of baker’s yeast Saccharomyces cerevisiae, wet mount was used to
observe the yeast specimen. Basically, the specimen must be drop where usually the
specimen is in liquid form being located between the slide and cover glass. The water
refraction index of the liquid can improve the quality of the image and also can support the
specimen.

Microorganisms are present in almost every location and environment on earth. There
are several types of microorganism such as bacteria, fungi, virus, algae and protozoa.
Bacteria represent a diverse and large group of microorganism that can exist as single cells
organisms. Meanwhile, fungi are widely distributed in air, fomites, normal flora and dust.

Different bacteria have different morphology where its deal with shape, size, margin,
texture, arrangement of bacteria cells and so on. There are 3 kind of shape microorganism
that are called as coccus (spherical), bacillus (rod-shaped) and spirillus (spiral). The
arrangement of cells is based on how the single cocci and bacilli aggregate themselves. When
the bacteria found in pairs it is called diplo, when they are in a long chain form and attached
to each other it is called strepto while when the bacteria are arranged in irregular shape like
grape it is called as staphyl. Cell wall of the bacteria is divided into two which are gram
positive cell wall and gram negative cell wall which can be observe after gram staining
process, gram positive will appear purple in colour while gram negative pink or red. Other
differences between these two cell wall are the thickness of peptidoglycan where gram
positive cell wall have many layer of peptidoglycan forming rigid structure while gram
negative cell wall only have one or very few layers of peptidoglycan so it is more susceptible
to mechanical breakage. Gram negative also consists of lipopolysaccharide (LPS) but not in
gram positive.

Gram positive cell wall are Streptococcus sp., the shape is in a long chain of cocci that
is why the name is strepto and Staphylococcus aureus based on the name staphyl as the
bacteria shape is in irregular cluster of coccus. Next, other specimen that have been observe
was Bacillus subtilis , Bacillus megaterium and Clostridium botulinum which are in a single
form of bacillus. Those bacteria are made of thick layer of peptidoglycan. The other group
which is gram negative cell wall bacteria are Escherichia coli, Salmonella typhi and Proteus
vulgaris are found in a single or pairly shape of bacillus while for Haemophilus influenzae
the shape is like coccobacillus means the shape looks like round, oblong, and tubular shapes
even though they are classically described as being short rods.

For fungi, there are about 100,000 known species of fungi which includes moulds,
yeasts, mushrooms, rusts and smuts. First, fungi of mould type composed of mycelium which
is a branched network of tubes that called hyphae. Hyphae are filaments that their cells are
long, looks like a thread and they connected end-to-end. When moulds reproduce asexually, it
form an asexual spores like what we observed in Aspergillus sp. Meanwhile, for Candida
albicans is a yeast type fungus where it can exhibit sexual reproduction. They can divided by
binary fission or budding where the shape is like a spherical shaped. This shape also can be
seen in the baker’s yeast Saccharomyces cerevisiae.

After observing the prepared slide of rhizophus sp. under a microscope, what have been
observed is that the body of the branching mycelia have sporangiophores that are unbranched
and grouped in tufts. The sporangiophores were observed to be brown in colour, unicellular
as well as round or oval in shape.

Penicillium are known as saprophytic fungus which are organisms that obtains energy from
dead and decaying organic matter. Under a microscope, Penicillium sp has closely-packed
brush like structures called penicilli which produces spores. It can be seen that the branching
were unbranched and have one cluster of phialides that occupy the top of it.

Mucor is a fungus that can be found in soil as well as decaying fruits and vegetables. We
observed that it had a ‘fluffy’ appearance which was green in colour. The Sporangiophores
were observed to be short, erect and taper towards their apices. It also formed short
sympodial branches which are a type of branching pattern where one branch develops more
strongly than others.

CONCLUSION 1A)

In conclusion, identifying all the parts of a compound microscope as well as its functions is
important in order to see clear images of the prepared slide. By using immersion oil, the
prepared slide was observed clearly under the 100X magnification. Thus, the shapes, colour
and forms of the different microorganisms in the prepared slide are able to be observed by
using microscope with correct technique. By using light microscope, it helps the scientist,
researcher to identify and check the present of microorganism that cannot be seen through
naked eye. By using microscope also help to identify various kind bacteria, mould and yeast
present in this earth surface.
RESULTS AND OBSERVATIONS 1B

1. Growth in slant culture and stab culture for pigmentation and purity.
Media Pigmentation Purity Picture
B. Subtilis in Stab Tube Not pigmented High turbidity

E. Coli in Stab Tube Not pigmented High turbidity

B. subtilis in Slant Tube Not pigmented High turbidity

E. Coli in Slant Tube Not pigmented High turbidity

 2. Comparison in morphology of a single colony of B. subtilis, E. coli, M. luteus and
Serratia sp.
Characteristic B. Subtilis E. Coli M. Luteus Serratia sp
s
Shape Irregular Circular Circular Circular

Margin (edge) Lobate Entire Entire Entire

Elevation Flat Convex Flat Convex

Texture/ Rough Shiny Dull Shiny

Appearance
Pigmentation No pigmentation No pigmentation Pigmented Pigmented
(White) (white) (Yellow) (pink)
Optical Translucent Translucent Opaque Translucent
Property
Picture
3. Differences in colony appearances between three ‘contamination plates’.

Area Picture Characteristic

Finger Colony: Moderate colony
Shape: Circular
Pigmentation: White

Toilet Surface Colony: More colony

Shape: Circular
Pigmentation: White

Open air No colony present

DISCUSSION

For the next part, we conduct an aseptic technique and bacterial culture experiment for
Bacillus Subtilis, Escherichia Coli, Micrococcus Luteus and Serratia sp. We inoculate these
bacteria in 4 different media (nutrient broth, nutrient agar slant tubes, nutrient agar stab tubes
and nutrient agar plates) except for M. Luteus and Serratia sp. For experiment with B.
Subtilis, we identify that the microorganism is facultative anaerobe. This is due to the reason
that the microbes grow in the nutrient agar slant tube and nutrient agar stab tube. The growth
in the stab shows that B. Subtilis is an anaerobe as they do not require oxygen to grow. Then,
as it also grows on the surface of the slant, this show that the microbes are also aerobe. So, B.
subtilis grow in both aerobic and anaerobic condition. Next, we also found that the nutrient
broth has changed from clear to turbid. The turbidity shows that the density of the cell present
in the nutrient broth has increase which means that B. Subtilis also grow in the nutrient broth.
The observation on E. Coli also the same as B. Subtilis where it shows the growth in the stab
and slant tube and also the turbidity in the nutrient growth. However, on the agar plates, B.
Subtilis have grown become an irregular, lobate, flat, rough white and translucent single
colony. The morphology of B. Subtilis is different with E. Coli which has a circular, entire,
convex, shiny white and translucent at the nutrient agar plate. This is because both bacteria
have different characteristics which is B. Subtilis is gram-positive bacteria while E. Coli is
gram-negative bacteria. The morphologies differ because of their adaptation. Next, we
inoculate the M. Luteus on the agar plate. The single colony grows on the media as a circular,
entire-edge, flat, dull, pigmented of yellow colour and opaque. This is quite different than the
growth of Serratia sp which has a circular, entire-edge, convex, shiny, pigmented of pink
colour and translucent. These two bacteria have different morphology due to its motility
where Serratia sp. is motile bacteria while M. Luteus is non-motile. So, the difference
morphology of one bacteria is due to its adaptation to make sure that it can functioning well.

For the contaminated agar plates, we tested 3 different things which are air, swab of
toilet walls and finger. For the first agar plate, we identify that there is no growth when we
exposed the agar plate to the outside of the laboratory. This is because not all bacteria can
grow on the nutrient agar plate. So, the microorganism in the air maybe unable to grow on the
agar plate. Besides, we put the agar plate in a short duration which may be the reason why
there is no growth. Second, we swab the toilet wall and use it to inoculate the microorganism
on the agar plate. As a result, there is a lot of colony present on the agar plate. This clearly
shows that toilet has too much microbes because when people defecate, the stool excreted
may contain a lot of bacteria. Thus, a lot of bacteria residue can be found in the toilet. We
incubate the plate at room temperature because we want to maintain the temperature that the
bacteria can grow. Lastly, we inoculate the finger from our group member on the third agar
plate. The result shows that there is a moderate growth of colony on the plate. This shows
that the hand was not clean because the student may not wash her hands before start the lab
session. Another possible reason is that the student maybe touched a lot of thing before
entering the lab for example touching the stairs railing or elevator railing. Thus, we have a
growth of bacteria. Our hands already consist of resident and transient microorganism. So if
the student has washed her hands, the growth of the bacteria may also occur because of the
resident microorganism at her hands.

CONCLUSION 1B)

To conclude, we were able to identify the various types of media. Nutrient agar was used as a
main form of media as it supports the growth of various types of bacteria and
microorganisms. We were also able to infer that using the nutrient slant tubes was more
advantageous than using stab tubes as in slant tubes; the surface area of agar nutrients was
bigger than stab tubes. This allowed more microorganisms to grow. Besides, we also
understood the aseptic techniques for working with bacterial cultures as well as the technique
used in for inoculation of microorganisms. The different colony morphologies grown from
their specimen could be identified and described as well. By conducting the experiment, we
were able to identify the different environmental conditions required for the microorganisms
to grow at an optimum rate. Thus, we managed to prepare and identify single colonies of the
microorganisms as well as its shape, appearance, optical properties and the optimum
temperature of growth for different microorganisms.
REVIEW QUESTIONS

1. Why is oil necessary when using the 90X to 100X objective?

It is a natural phenomenon when light bends as it passes through different mediums.
In this case, light travels from glass to air. When light passes through the two
mediums, light is refracted. Light of different wavelengths bend at different angles.
Thus, when magnification increases, the images will be less clear. By reducing the
amount of light refraction, more light that passes through the microscope slide will be
directed through the objective lens. Immersion oil has the same refractive index as the
glass slide. Therefore, more light will be directed through the objective lens and
produces a clearer image.

2. In the prepared slides, which organism was the largest?

According to our observation, the largest organism in the prepared slides was Mucor
sp.

3. When identifying microorganisms, why should a wet mound is used when making
measurements?
A wet mound is when the specimen is placed in drop of water or any liquid which is
held in place between the slide and the cover slip due to surface tension. The
refractive index of the water improves the image quality. The image will be more
clear and easier to observe. This method is commonly used to measure as well as view
microscopic microorganisms and its movement.

REFERENCES:

https://drfungus.org/knowledge-base/penicillium-species/

https://mycology.adelaide.edu.au/descriptions/zygomycetes/mucor/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958177/