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					                                             LAB 2:
                              Introduction to Microbiology;
                                  The Scientific Method:
                                Hypothesis Development

I.     Pre-Lab Preparation


In addition to reading this lab and preparing your lab notebook you should begin to read the paper
“The influence of sex, handedness, and washing on the diversity of hand surface bacteria” (Fierer
et.al., 2008. PNAS 105: 17994-17999) which will introduce us to one approach to studying bacterial
diversity. See section IV. D below for hints about reading this paper. You should also read Ch. 1, p.
18-20.



II. Objectives


Today’s lab will continue your introduction to the study of bacteria, the use of microscopes to observe
them, as well as some of the techniques used to culture and identify them. Also, in order to begin to
understand the environmental distribution of bacteria and the importance of their diversity, we will
work together to design and carry out experiments to examine their distribution. By the end of today’s
lab, you should:


        Be able to explain why bacterial species can be found in every environment
        Be able to explain how bacteria can be cultured and how colony morphology can be
         used to identify bacteria
        Be able to identify the components of a well-written hypothesis
        Be able to begin to design an experiment to understand the diversity and abundance
         of bacteria in the environment
        Be able to explain how discovery-based science and “the’ scientific method are
         similar and how they differ.
        Be able to differentiate between eukaryotic and prokaryotic cells under the
         microscope
        Be able to use microscopy to determine the relative sizes of typical eukaryotic and
         bacterial cells




III.     Safety Considerations




BIO 2 Lab Manual, Fall 2009                                                                  Lab 2, Page 1
        Dispose of all used swabs in the trash. You do not need to treat them as biohazardous waste.
        Always use a clothespin (not your fingers) to run the slides through the flame of the Bunsen
         burner. This will help prevent burns.
        Be cautious around open flames.
        Be careful with the crystal violet stain. If you get it on your clothing, you will never be able to
         get it off!
        Tie back long hair.
        Wash your hands when leaving the laboratory.




IV. Introduction


A. BACTERIA



Bacteria are a group of single-celled microorganisms that, along with the Archae, are classified as
prokaryotes. Prokaryotic cells are structurally much simpler than eukaryotic cells because they have
no internal membrane-bound organelles (i.e. no mitochondria, no chloroplasts, no Golgi body, no
nucleus, etc.). Most prokaryotic cells are also much smaller than eukaryotic cells. A typical bacterial
cell is about 1 micrometer (1 µm) in diameter, while eukaryotic cells range from 10 to 100 um in
diameter. This means that eukaryotic cells have between 1,000 and 1,000,000 times the volume of
prokaryotic cells - an enormous difference indeed!


Although bacteria do not possess organelles (although they do contain vesicles called mesosomes that
can bud off from the plasma membrane), the complex and diverse functions carried out by organelles
are present in other parts of the bacterial cell such as the cell membrane (e.g. cellular respiration,
photosynthesis). Moreover, while bacteria may seem "simple" at first glance, their lack of structural
complexity actually represents a clever and amazingly successful adaptation for survival. The lack of
complex internal structures allows bacterial cells to devote all of their cellular energy to rapid growth
whereas eukaryotic cells devote much of their energy to maintaining cellular complexity. You will be
learning more about the structural and functional differences between prokaryotic and eukaryotic cells
in lecture.


Since bacteria are too small to be seen except with the aid of a microscope, it is usually not
appreciated that they are the most abundant form of life on the planet, both in terms of biomass and
total numbers of species. Bacteria and other prokaryotes are found in every environment on the
planet, including all of the habitats where eukaryotes live, but also in environments considered too
extreme or inhospitable for eukaryotic cells/organisms - such as boiling water or ice. Thus, the outer
limits of life on Earth (hottest, coldest, driest, etc.) are usually defined by the existence of




BIO 2 Lab Manual, Fall 2009                                                                        Lab 2, Page 2
prokaryotes. Although many people associate bacteria with illness, less than 1% of all known bacterial
species are capable of inducing disease. The vast majority of bacterial species are free-living (non-
disease causing) and many bacteria form symbiotic relationships with plants and animals that usually
benefit both organisms. Thus, the lives of plants and animals are dependent upon the activities of
bacterial cells. For plants, bacteria help maintain the pH of the soil and provide important nutrients.
In animals, including humans, skin and mucous membranes are habitats for a variety of bacterial
species. The human body provides multiple habitats for hundreds to thousands of different species of
bacteria and the total number of bacteria exceeds the number of cells in all the tissues and organs
which comprise a human. In this lab, you will be sampling multiple environments for different species
of bacteria and visualizing the differences between prokaryotic and eukaryotic cells under the
microscope.


B. CULTURING BACTERIA ON AGAR PLATES


Individual bacteria are too small to be seen without the aid of a
microscope and, before the work of Robert Koch (1843-1910), were
impossible to isolate as pure cultures. Koch was studying the transmission
of bacteria-borne diseases and, like his colleagues at the time, was
frustrated because he could never grow a pure culture of any bacterial
type. Any attempt to isolate and grow up one type of bacteria always resulted in a mixture of bacterial
cell types, as visualized by observing differences in the appearance and size of the cells by light
microscopy. One day, Koch boiled a potato, cut it in half, and accidentally left the potato out on his
kitchen counter overnight. In the morning, he was amazed to see small "colonies" of different colors
and shapes (morphologies) and immediately realized that bacteria from the air had landed on the
sterile potato surface during the night and had begun to divide. Each colony represented a clonal
population derived from a single cell that had landed on the potato's surface, and Koch was delighted
to see that when he placed a colony in some sterile media and then viewed it under the light
microscope, all the bacterial cells had the same appearance.


Koch's discovery of how to grow up pure cultures of different bacteria eventually led to the
development of the ideal medium for promoting such growth: agar. Agar is an unbranched
polysaccharide obtained from the cell membranes of some species of red algae or seaweed, and it
cannot be digested (liquified) by bacteria. Therefore, it provides an ideal surface for bacterial cell
growth (cell division). Bacterial cell division rates are very fast in comparison to eukaryotic organisms.
Many bacterial species can undergo cell division every 20 minutes. Thus, in 8 hours, a single bacterial
cell can give rise to a population of millions of cells, which cluster together as a colony visible to the
naked eye. An average colony may be composed of between 300,000-5,000,000 identical cells.
Colonies of different species will have different observable or phenotypic characteristics, including size,
shape, height, texture, and color. These are collective known as the colony morphology of the




BIO 2 Lab Manual, Fall 2009                                                                       Lab 2, Page 3
species and can be used to distinguish between different species grown on an agar plate. The different
colony morphologies (phenotypes) are the result of the different genetic makeup of each individual
species.


C. BACTERIAL DIVERSITY—THE FLORA OF THE HUMAN BODY


The human body provides many excellent habitats for bacteria. The human body has a stable
temperature, high moisture content, and certain areas, such as the mouth, represent a large food
resource. It is estimated that 1 quadrillion (1015) bacteria comprised of 500 to 1000 or more different
species live in and on us (in comparison to the 100 trillion cells that make up an individual human).
Bacterial species that are expected to be present under normal circumstances, and do not cause
disease, are termed normal flora. To observe bacterial species of the human mouth, you will both
culture and stain cheek and gum samples. A simple staining procedure will allow you to distinguish
prokaryotic cells from cheek cells. Bacterial species that inhabit the human mouth are able to attach to
epithelial cells of the cheek (buccal cells), the gums, as well as the teeth.


D. STUDYING BACTERIAL DIVERSITY


How do scientists study bacterial diversity? Do you think we know how many species live on the
human body and what all of these species are? In preparation for this lab you should begin to read
the paper “The influence of sex, handedness, and washing on the diversity of hand surface bacteria”
(Fierer et.al., 2008) which will introduce us to one approach to studying bacterial diversity. As we all
experience to some degree in reading scientific papers, you will encounter techniques, terms,
statistical methods, references to previous work, etc. that is unfamiliar or entirely new to you. Have
no fear, it is possible to glean much information from a paper such as this despite that. Do not feel
that you must understand the whole paper—that is asking too much. Instead, begin by reading the
abstract and introduction and see if you can understand the basic approach used here as well as their
major conclusions. Begin to read the remainder of the paper, but do not get bogged down in details.
See if you can extract key information without understanding all of the methods. This is a skill that
will improve as you advance in the sciences!


In addition to reading this paper, review pages 18-20 of Chapter 1. Consider whether or not you find
hypotheses in this work and whether or not you would consider this applying the scientific method or
whether this is more accurately considered to be discovery science. Be sure you understand the basic
similarities and differences between both of these before coming to lab.


V. Things To Do


PART A. WRITING A SCIENTIFIC HYPOTHESIS




BIO 2 Lab Manual, Fall 2009                                                                    Lab 2, Page 4
Note: Parts of the following paragraphs were adapted from http://www.accessexcellence.org/
LC/TL/filson/writhypo.html, accessed 2/2/08.


1.       Working together with you lab partners, read and discuss the following information on writing
         a scientific hypothesis.


Not all Science is Hypothesis Driven


First of all, it is important to understand that not all science is hypothesis-driven. A good deal of
science is observational and descriptive, and this does not make it "bad science." For example,
the study of bio-diversity usually involves looking at a wide variety of specimens and maybe sketching
and recording their unique characteristics. Or a scientist might wish to determine the frequency of
various forms of a gene (alleles) in a population. Neither of these types of scientific studies tests a
specific hypothesis.


When is it Helpful to Use a Hypothesis?


Other times, however, scientists find it helpful or necessary to develop and test a hypothesis during
their research activities. A hypothesis is a tentative statement that proposes a possible
explanation to some phenomenon or event in which two or more variables may be related.
The key word is variables. That is, you will develop a hypothesis whenever you are performing
a test of how two variables might be related. Usually, a hypothesis is based on some observation
that seems to suggest that two variables may be related - such as noticing that in November many
trees undergo color changes in their leaves as the average daily temperatures are dropping. Are these
two events connected? If so, how?


What is the Difference Between a Hypothesis and a Theory?


A hypothesis should not be confused with a theory. Hypotheses are tentative statements that can be
disproved, and often are. Theories are general explanations with a tremendous amount of predictive
power that are based on a large amount of accumulated data. For example, the Theory of Evolution
applies to all living things and is supported by a wide range of observations that have been carefully
recorded and documented in the hundred and fifty years since the theory was first proposed.
Theories carry much, much more weight than hypotheses.


How Is a Good Hypothesis Written?


First, a good hypothesis should be written as a statement, not a question. Second, it should be an
educated guess, not simply a hunch; it should be based on careful observations and a thorough




BIO 2 Lab Manual, Fall 2009                                                                     Lab 2, Page 5
literature search. Third, it should be testable and straightforward. And finally, it should make a
prediction that can be clearly (and preferably easily) measured and falsified.




      Diagram from http://www.utas.edu.au/sciencelinks/exdesign/HF2C.HTM, accessed 2/2/08


Dependent and Independent Variables


In a hypothesis, a tentative relationship is stated between the variables. For example:


"The frequency of winning the lottery increases with the frequency of buying lottery tickets."


"Exposing plants to low temperatures will result in changes in leaf color."


Notice that these statements contain testable predictions based on whether or not two events are
related. Also notice that both hypotheses contain two variables. One is "independent" and the other is
"dependent." The independent variable is the one that you control. The dependent variable is the one
that you observe and/or measure. In the hypotheses above, the dependent variable is underlined and
the independent variable is emboldened. Also note that, as the scientist, you may not be able to
control the weather ("exposing plants to low temperatures") but you can wait until the conditions are
right and time your experiments so that you do, in essence, control the independent variable.


2.     For each of the hypotheses below, work with your lab partners to circle the independent variable
       and underline the dependent variable. Also assess how well the hypothesis is stated and if it
       could be improved. What’s good about it? What could be better? If it needs improvement, how
       could it be fixed? (Use your lab notebook for your notes and rewrites in this exercise.)


         a.   "The more often people hear leaf blowers, the crazier they get."


         b. "The concentration of E. coli increases in the Stanislaus River downstream of the Pacific
              Systems Sewage Facility after a rainstorm in which at least 2 inches of measurable rainfall
              is recorded."




BIO 2 Lab Manual, Fall 2009                                                                       Lab 2, Page 6
         c.   "Andean red ants become inert when the temperature in the immediate environment drops
               below a certain point."


         d. "What percentage of the human population has type O blood?"


         e. "There are 63 moons orbiting the planet Jupiter when it is at perihelion in its orbit (closet
               to the sun) but only 60 moons orbiting the planet when it is at aphelion in its orbit
               (farthest from the sun)." (Note: The number of moons around Jupiter is measurable when
               a satellite is in orbit around the planet.)


3.    Now, let’s consider Fierer et al. (Answer each of the following in your notebook.)


         a. What is the purpose of this study?
         b.    What are the major conclusions from this study?
         c. Is there a single major hypothesis being tested in this study?
         d. If so, what is this hypothesis?
         e. Are there multiple, less comprehensive hypotheses being tested? If so, what is one of
              these?
         f. Does this study suggest other hypotheses to you that you would like to test, or would you
              like to test one of their hypotheses further?
              --if so, record this in your notebook.


4. Using the Fierer paper as a starting point, develop a clear hypothesis that will examine bacterial
     diversity either on the human body or elsewhere in the environment.
         a. To fit within our upcoming labs be sure that testing your hypothesis can be accomplished
               by comparing the bacteria that are cultured from two sources.
         b. Further, we will culture bacteria from these two sources in this lab and next time we will
               compare the number of colonies and the number of different colony morphologies
               observed.
         c. Also, in later labs we will isolate DNA from one or more colonies and use a DNA test to
               determine if the bacteria bacteria are coliform bacteria (i.e. in the family that includes E.
               coli) and, further, if the colony is, indeed, E. coli.
         d. It is not essential that the testing of your hypothesis requires the DNA test. It should at
               minimum include an analysis of the number of bacteria present and the apparent diversity
               based on colony morphology.
         e. Be sure that your hypothesis meets the criteria for a good hypothesis! Also, consider how
               you will interpret your results. Will this analysis address your hypothesis effectively?
               What problems might you encounter.
         f. Record your hypothesis in your lab notebook and label the dependent and
               independent variables.



BIO 2 Lab Manual, Fall 2009                                                                        Lab 2, Page 7
         g. Proceed to Part B.


PART B. CULTURING BACTERIA FROM THE ENVIRONMENT


1.       Working individually, based upon the hypothesis you have developed, choose the two
         environmental sources of interest that you would like to sample. Think critically about what
         you choose to sample. Will this selection of samples allow you to test your hypothesis?


2.       Collect two BHI agar plates and one tube of sterile water from the front or side benches. Using
         a wax pencil, write your name, lab section, and sample source on the agar side of the plate
         (not the lid). BHI is “brain heart infusion agar and contains lots of nutrients to promote the
         growth of bacteria that can tolerate oxygen in their environment.


3.      To sample an environmental source, moisten a sterile cotton swab with the sterile water. Press
        out any excess water in the swab on the inside of the tube of sterile water. The swab should be
        moist but not dripping with water. Swab the environmental source for 10 seconds with the
        swab and then gently swipe the swab several times across the surface of the plate, trying to
        cover the entire surface.


4.      Wrap a piece of tape around the plates to secure them together. Then place the plates LID
        SIDE DOWN on the tray at the front of the class for incubation until next lab period. (Placing
        the plates lid side down ensures that any condensation that may form on the lid during
        incubation does not drip down onto the agar.) Your plates will be incubated at room
                                          o                                                                   o
        temperature (approximately 22         C) for several days and then placed at in the refrigerator (4
        C). Make a note of this in your lab notebook. Also note the time that you started the incubation
        of your plates.


5.      Discard the swabs in the trash.


PART C.       VIEWING CHEEK CELLS AND ORAL BACTERIA BY MICROSCOPY


1.     Working individually, obtain a glass slide. Using a black grease pen, draw a circle on the slide
       (as shown below).




BIO 2 Lab Manual, Fall 2009                                                                        Lab 2, Page 8
       Using a sterile cotton swab, scrape the gum line and cheek of the inside of your mouth. Apply
       the collected material to the circle. Discard the used swab in the trash.


2.     Allow the slide to completely air dry. Then, holding the slide with a clothespin (not your
       fingers!), quickly pass the slide through the flame of a Bunsen burner three times. DO NOT
       LEAVE THE SLIDE IN THE FLAME FOR MORE THAN 2 SECONDS AT A TIME!! This procedure will
       “heat fix” the cells to the slide so they do not wash off when dyes and water are applied to the
       slide.


3.     Still holding the slide with the clothespin, take the slide to one of the lab sinks and add a drop of
       crystal violet stain to the circle. Let the dye sit for 1 minute and then thoroughly rinse the slide
       with distilled (dI) water. (Note: dI water comes from the taps with the white caps in the lab.)
       This step MUST be done at the sink! Crystal violet stains clothing permanently.


4.     Lightly pat the slide dry with a Kimwipe.


5.     Your instructor will provide you a handout on how to use the light microscopes in the
       lab. Follow these instructions carefully to observe your slide, using both the 10x and
       40x objective lenses. Bacteria will be seen as small, darkly staining spheres or rod shapes on
       the surface of the buccal (epithelial cheek) cells.


       Be sure to ask your instructor or your TA if you are unable to get a good view of the
       cells on your slide.


6.     For 5 different cheek cells on your slide, count the number of bacterial cells that are attached
       and record your results in your lab notebook. Then calculate the average number of bacteria per
       cheek cell, writing out your calculation in your notebook.


7.       Also in your notebook, draw what you see on your slide. Record the shapes and relative
         sizes of the bacterial and cheek/buccal cells.


8.       Estimate how many bacterial cells could line up end-to-end across the diameter of a
         buccal cell. Estimate the volume difference between a buccal cell and an oral bacterial cell.
         Record your calculations and estimates in your lab notebooks. Did you have to make some
         assumptions to arrive at an estimate? Record these in your lab notebook also.



V. Lab Clean-Up


Before leaving lab today:



BIO 2 Lab Manual, Fall 2009                                                                      Lab 2, Page 9
             Make sure all used swabs are discarded in the trash.
             Discard microscope slides in the broken glass container.
             Tidy up everything at your lab bench for the next lab section.
             Wash your hands.




BIO 2 Lab Manual, Fall 2009                                                    Lab 2, Page 10

				
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