Download MICROBIAL DIVERSITY AND UBIQUITY

MICROBIAL DIVERSITY AND UBIQUITY
Microorganisms are microscopic organisms that are so small that that they can
only be visualized by the aid of a compound-brightfield microscope. While we generally
cannot see individual microorganisms with the naked eye, they are present in virtually
every habitat known to man. Microorganisms can be prokaryotic—the bacteria or
eukaryotic—the algae, protozoa or fungi. While viruses are acellular they are also
studied in the scope of microbiology because they are small and because they infect cells.
While most bacterial are unicellular they can also exist in colonial or multicellular forms.
In this laboratory exercise you will examine the ubiquity and diversity of various
microbes that are present in the environment or inhabit the human body. Most bacteria
that inhabit the body are harmless or even beneficial to humans, some bacteria are
opportunistic and only become pathogenic when they are present in the wrong place at
the wrong time in the potential host. As microorganisms are so omnipresent, the
following exercises are designed to help you learn the importance of proper asceptic
technique when handling microorganisms.
During the first laboratory exercise you will be examining the examining the presence of
bacteria on inanimate surfaces and on or within the human body; furthermore, you will be
examining the efficacy of various cleansing agents in reducing the amounts of bacteria
associated with these habitats. To this end you will first inoculate a Tryptic Soy Broth
(TSB) plate with a sample from the environment. TSB is rich in nutrients and can
support the growth of a number of bacteria or fungi; therefore you will be able to isolate
microorganisms from your hands or other parts of the body or from the oral cavity, from
your benchtop or from other inanimate objects.
With a wax pen label the bottom of a Petri plate containing TSB-agar with your name,
date, lab section, and source of the inoculum. It is important to label the bottom of your
plates because the lids can sometimes fall off the top of the plate and make identification
of your plate difficult.
Select a place to test for the presence of microorganisms. If you are testing the oral
cavity or an inanimate surface, use the pre-sterilized cotton applicator swabs provided by
your instructor. Remove the swab from the test tube (be careful not to touch the cotton
tip) and immediately swab the source of your inoculum. Using the plates provided
inoculate the specimen onto the plate. To do this partially open the TSB plate and while
continuing to hold the plate by the lid, gently rub the cotton swab along the surface of the
agar. Immediately close the plate. If you are testing for the presence of microbes on the
hands, vigorously rub your hands together and gently rub one or two fingertips along the
surface of the agar.
Now you will test the efficacy of various soaps, chemicals and cleaning agents. If the
source of your inoculum was from the oral cavity, then brush your teeth. Use a fresh
plate and a clean swab to sample the microorganism present after brushing your teeth.
Now rinse out your mouth with your favorite mouthwash, use the same process while
remembering asceptic technique to again sample the microorganisms present in the oral
cavity. If you are sampling from your hands or from the inanimate sources, you can use
similar techniques. For example, you can sample the microorganisms present on your
hands before washing, after washing with your favorite soap or topical cleansing solution
or the antimicrobial soap that is present in the lab, after that you can test the efficacy of
the hand solution (70% alcohol) that we use in the lab.
Make certain that all three plates are labeled clearly and place the plates upside down in
the 37oC incubator (plates are incubated upside down to prevent condensation from
dripping down onto the surface of the agar). The plates will be incubated at that
temperature for 24-48 hours and then placed in the refrigerator so that you can examine
the types and numbers of bacteria or fungi present on the plates at the next lab period.
Second Lab Period—analysis of bacterial colony morphology and the morphology
of individual bacteria: In most cases, the predominant class of microorganisms that you
will find on your plates will be the bacteria. Bacteria have different colony morphologies
and characteristics that are specific to the genus and species of the organism. For
example Enterobacter aerogenes produce large, translucent, off white colonies that
appear moist; the edges of these colonies are somewhat undulating and are raised above
the surface of the plate, Staphylococcus aureus colonies are small, opaque, yellow, shiny
and have regular edges. Enterococcus faecalis colonies are small, opaque, white, shiny
with smooth borders. Bacillus subtilis produces colonies that are large, opaque and
white; the colonies appear dull, rough and dry and have irregular borders. Figure 1
below shows some typical colony morphologies and characteristics.
Figure 1: Typical colony
characteristics and
morphologies. A. form
B. elevation C. margin
Examine your plates for the presence of microorganisms paying close attention to the
types of colonies present and the numbers of colonies that are present on you plate.
Observe the control plate; this is the plate containing an inoculum from a source
that has not been cleaned. Describe what you observe in the space below—feel free
to draw pictures. (Note: if colonies are not present on your plate, observe the plate
of your neighbor). Pick two colony types and describe them fully using Figure 1 as a
guide. Also describe these colonies in terms of their consistency, that is whether they
are transparent, translucent or opaque.
Observe the plate that contains your inoculum after the first cleaning step (if
applicable). What do you observe?
Observe the plate that contains you inoculum after the second cleaning step (if
applicable). What do you observe?
Now that you have observed the morphology and the characteristics of two different
colonies in some detail, you will apply what you have learned in the previous laboratory
exercise (Use of the Compound Brightfield Microscope) to examine individual bacteria
within these colonies. Bacteria have three typical morphologies: the cocci (spherical
bacteria), the bacilli (rod-shaped bacteria) and the spiral or curved shaped bacteria, such
as the spirillum, the spirochetes and the Vibrio spp. Using the technique that you used in
the microscopy exercise you will prepare a wet mount of bacteria from the two colonies
that you have just studied. (Note:: If the numbers of bacteria on your plate are too
numerous to pick from an individual colony sample bacteria from your neighbor’s plate.)
Place a small drop of sterile media onto a microscope slide. Take a small sample of the
bacteria from the colony using your inoculating loop. It is important that you use
asceptic technique when sampling the bacteria as you will be looking at a pure bacterial
sample. Flame your inoculating loop as shown by the instructor and allow the loop to
cool without waving the loop it the air. When you sample the bacteria make certain that
you can barely see the inoculum on the loop; there are approximately 30,000 bacteria in
such a small sample, if you inoculate with too much, you will not be able to observe
individual bacteria. Mix the bacteria on the loop in the media on the microscope slide
and using the same method that you used in the previous exercise to observe live bacteria
in the hay infusion, place a coverslip over the bacterial suspension. View the bacteria at
450X and at 1000X.
Describe the bacteria in terms of shape under each magnification:
Colony 1:
Colony 2:
Are any of the bacteria motile? Can you distinguish between Brownian motion,
current flow or purposeful bacterial movement as mediated by flagella? How so?
Save your plates for the next week’s exercise where you will use simple and differential
stains to view bacteria!
Demonstrations of microbial diversity: The class of microorganisms that you isolated
from the oral cavity or from your hands most likely did not represent the fungi.
However, you may have found that fungi were present on inanimate surfaces. Pathogenic
fungi can be found in tissues of humans; such organisms might include Candida
albicans which is the causative agent of yeast infections in women or Trichophyton spp.
that cause Athlete’s foot fungus. Today, we will observe three different nonpathogenic
fungi to address the different forms fungi may assume. Aspergillus niger and Penicillium
notatum represent the filamentous fungi, while Saccharomyces cerevisiae represents the
unicellular yeast forms of fungi. The filamentous fungi have long narrow cells that form
hyphae or branching filaments. Hyphae form interwoven mats called mycelia that are
the structural units of the spore bearing structures in fungi. Spores are small structures
that are relatively resistant to adverse conditions of heat, pH etc. Hyphae that are
partially divided by crosswalls are septate, while those that do not have crosswalls are
nonseptate. Most hyphae have more than one nucleus present and are therefore,
multinucleate. The yeast forms of fungi are unicellular and uninucleate. When yeast
reproduce asexually they do not form spores like the filamentous fungi, however with the
aid of a microscope one can observe the presence of small bumps or buds on the surface
of a yeast cell that result from an unequal division of the cytosol after mitosis.
A plate culture of Aspergillus niger, Penicillium notatum and Saccharomyces cerevisiae
have been placed under a dissecting microscope in the back of the lab.
Draw a picture of one or both of the filamentous fungi and the yeast in the space
provided below, noting the presence of the mycelia and the spore forming structures
in the Aspergillus niger and Penicillium notatum species. Note how macroscopically
the Saccharomyces cerevisiae do not have these structures and appear like bacterial
colonies.
Prepared slides of Aspergillus niger and Saccharomyces cerevisiae have been placed
under a Compound Brightfield microscope at a total magnification of 450X; these slides
are also located in the back of the lab.
Locate the spore bearing structures at the ends of the hyphae of the slide containing
Aspergillus niger. Draw a picture of the hyphae and the spores and any nuclei that
are present in these structures, describe the size and shape of the spores.
Observe the slide containing Saccharomyces cerevisiae. Identify the the nucleus that
is present within the cell and buds that are on the surface of the cell. Draw a picture
of what you observe.