Good afternoon everyone.

This is the continuation of the sixth exercise that deals with the
simple techniques to collect, culture, and visualize microorganisms. Jobeth already explained the
objectives for this exercise and the common materials used in the collection as well as the
visualization of microorganisms. The isolation of microbes from air, incubation and isolation for
pure culture in plates and tubes, and the preparation of bacterial smear for microscopic
examinations were the main topics discussed.
The next subtopic for this exercise is the Preparation of fungi for microscopic
examination.
For the first part, you have the option to choose whether you want the fungal colony you
will examine to come from rotten fruits/vegetables or left-over foods and bread. You can also
utilize the air-exposed NA plate previously discussed, if fungal colonies appeared in it. You can
identify the fungal colony on the agar plate by its fuzzy and powder-like appearance. In teasing
out and picking up few strands of threadlike hyphae of a fungal colony of your interest, you
should use a flame-sterilized inoculating needle instead of an inoculating loop. The reason for
this is that a solid media is more dense over a smaller area so an inoculating needle is used to
retrieve the specimen so that not too much is transferred.
Next, deposit the hyphae on a drop of lactophenol cotton blue (LCB) on a clean glass
slide. Lactophenol cotton blue is a stain that is used to examine fungal elements.
This stain contains phenol, which will kill the organisms, lactic acid which preserves fungal
structures, and cotton blue which stains the chitin found in the fungal cell walls.
A cover slip is used in the preparation of fungi for microscopic examination, unlike
bacterial smears. You should carefully cover the drop with a cover slip. We can observe in the
bottom right picture how the lactophenol cotton blue spreads out towards the edges of the cover
slip. Avoid the formation of bubbles when doing so.
Then, line the 4 sides of the cover slip with natural nail polish to seal it off and prevent
from drying up. Be careful to not pull the brush over the coverslip as it may leave a thin pulled
thread of nail polish. Take note also that the nail polish you will be using should not be too old,
as it will be too solid and hard to handle. The nail polish has to be dry before you put it under the
microscope, as the objective can easily tough the nail polish which will then lead to a very
serious deterioration of the objective. 
Lastly, you can now observe the specimen under the microscope and study fungal
morphology. The fungal elements you will observe will differ depending on the fungal species.
Use the low power objective to locate the object, then use the high power objective to confirm
the presence of fungal structures. Fungi may be detected by the presence of characteristic
structures such as spores and hyphae. The organism depicted in the picture is a Mucor sp.
fungus: a mold often found indoors. This bright field light micrograph shows the release of
spores from a sporangium at the end of a hypha.
The second part of this exercise deals with the microorganisms in pond/canal water. I will be
discussing to you the techniques such as the isolation and microscopic examination of
microorganisms from water sample, the demonstration of true motility of microorganisms, as
well as the comparison of the morphology of representative microorganisms.
For the isolation and microscopic examination of microorganisms from water
sample, you will need a clean glass container filled with either pond or canal water sample just
like in the first picture. Dried leaves should be added on the glass container. The type of leaf was
not specified for this technique. The second picture shows the dried leaves added on the water
sample. For the third step, it should be set aside for several days to promote growth of microbes.
Fourth, after several days, take out 1-2 loopfuls of the pond/canal water in the glass and
then deposit the water at the center of the glass slide using inoculating loop. Be careful on
placing the clean cover slip over the loopful of water on the glass slide. Start from one edge of
the cover slip working towards the opposite edge while simultaneously bringing the cover slip
flat over the water. Make sure that no bubble formations are seen.
Lastly, focus the water drop on the slide under LPO and then HPO. Slowly and
systematically scan the microscopic field and identify the different microorganisms encountered
in the process.
Next topic is about the demonstration of true motility of microorganisms. One characteristic that
is useful in helping to identify an unknown organism is whether or not the organism is motile.
Motility can be defined as the ability of a cell or organism to move of its own accord by
expending energy. Motility confers a bacteria for example, an ability to change direction. This is
important when bacteria require moving away or moving towards repellents or attractants
respectively. It avoids unfavorable conditions of habitat and offers protection. It is important in
the survival and offers to choose favorable environment.
There are two types of motility. The first one is called the true motility which is evident in
flagellated microbial cells. In this type of motility, microbes dart swiftly and then disappear as
quickly as they move about through the liquid medium. We will be utilizing the hanging drop
set-up that I will be discussing later to demonstrate true motility. Another type of motility is the
Brownian movement. A common mistake is to confuse Brownian motion with true motility.
Brownian movement is a continuous vibrating motion caused by invisible molecules striking the
bacteria or the microbe. If the bacteria are truly motile, their movement will be over greater
distances and will be multi-directional, not just back and forth. In this animation, the small blue
circles represent water molecules and other particles that collide with each other. Whereas the
larger green circle in the middle represents a microbial cell. We can observe that the cell moves
very little and stays in place.
Like I said earlier, the hanging drop set-up will be used to demonstrate the true motility of
microorganisms. Hanging drop slides are useful in observing the general shape of living bacteria
and the arrangement of bacterial cells when they associate together.
First, you need to prepare a concave slide. Concave slides, as shown in the picture, have either
one or two round indentations or depressions. The slide might be slightly thicker. Concave slides
are able to hold significantly more liquid compared to regular slides.
Next, the four sides of the cover slip should be lined thinly with Vaseline using a thin applicator.
The role of vaseline for this technique is that it keeps the slide from drying out and creates a seal
that may extend the life of the slide up to a few days. The picture below is just an example of a
cover slip being lined thinly with Vaseline or petroleum jelly.
Using an inoculating loop, take 2-3 loopfuls of the water sample from the glass with dried leaves.
Deposit the water to form a small mound at the center of the cover slip lined with Vaseline. The
picture on the right shows what is being done in this step.
Next, position the concave slide over the cover slip in such a way that the concavity is directly
over the water drop (just like in this picture). Gently press the slide on the slip causing it to
adhere to it. With one quick stroke, invert the slide and slip pair. This should create a drop of
water hanging from the cover slip directly over but not touching the concavity of the slide.
To better visualize what is happening in this set-up, here is another illustration showing how it is
done. This right here is the cover slip lined with petroleum jelly. This thing in the center
represents our water sample from the glass with dried leaves. Then, the slide is positioned over
the cover slip. The slide is then inverted to produce hanging drop of the specimen.
After this step, you can now observe motile organisms swimming about in the hanging drop
under LPO and HPO.

Since concave slides are signifcantly more expensive than regular slides. It is therefore
meaningful to reuse them over and over again or an improvised set-up just like the one in the
picture can be equally useful. 2 small pieces of matchsticks thinly coated with Vaseline or
petroleum jelly are aligned, and placed about 1 cm apart at the center of the plain glass slide. The
four edges of the cover slip are also lined with Vaseline.

The next topic for this exercise is the comparison of the morphology of representative
microorganisms. We will be examining the following photomicrographs of prepared slides of
representative microorganisms under LPO and HPO. You should take note of their unique
structures, especially as observed under HPO.

Being able to differentiate bacterial species is important for a host of reasons. Bacterial species,
and even specific strains can be differentiated using a number of molecular techniques. There are
phenotypic differences between groups of bacteria that can be used to differentiate them. This
includes characteristics like their shape, growth in particular nutrients and preference for high or
low oxygen environments. Depending on the characteristic being studied, bacterial species may
be broken down into broad groups, but taken together this information can narrow the possible
identities greatly. One such useful classification is if a bacterium is Gram positive or Gram
negative based on the structure of bacterial cell walls. Gram staining will not tell you the specific
species you are looking at, but it can be a quick way to narrow down greatly the list of potential
candidates and direct follow-up testing where necessary.
Gram positive bacteria have a distinctive purple or bluish appearance when observed under a
light microscope following Gram staining. This is due to retention of the purple crystal violet
stain in the thick peptidoglycan layer of the cell wall.
Clostridium sporogenes is a species of Gram-positive bacteria that belongs to the genus
Clostridium. It is an anaerobic, rod-shaped bacterium and is commonly found in soil.
On the other hand, gram negative bacteria appear a pale reddish or pinkish color when observed
under a light microscope following Gram staining. This is because the structure of their cell wall is
unable to retain the crystal violet stain so are colored only by the safranin counterstain.

Serratia is a genus of Gram-negative, facultatively anaerobic, rod-shaped bacteria

There are three representative microorganisms for Fungi.

First is the Aspergillus Aspergilli can be found throughout nature with their spores being abundant in
air. In addition to largely being saprophytes that obtain their nutrition from dead and decaying matter,
they can also be pathogenic to human beings and animals with some also affecting and damaging
plants.

 They display two distinct morphological stages: the vegetative and reproductive. The vegetative
stage consists of a tangle of slender thread-like structures called hyphae (singular, hypha ),
whereas the reproductive stage can be more conspicuous. The mass of hyphae is a mycelium.
We can easily identify the structures on this picture. Species of Penicillium are recognized by their
dense brush-like spore-bearing structures. The spores of Penicillium are called conidia. This
right here is the phialide which is a flask-shaped projection from the vesicle of the penicillium.

Penicillium is a large and difficult genus encountered almost everywhere, and usually the most
abundant genus of fungi in soils.

Next representative microorganism of fungi is the Rhizopus which is a genus of common

saprophytic fungi on plants and specialized parasites on animals. They are found in a wide
variety of organic substances , including "mature fruits and vegetables" as well as bread. This
part is called the sporangium which is a spherical structure where sporangiospores are
produced.
This is called the rhizoids which are similar in structure to the root hairs found on more complex
vascular plants.

Next are microalgae