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					            Biology Lab

            Prokaryotes


Start Lab             Complete Report Sheet
                Instructions for Lab
Before you begin your lab read the objectives and the introduction.

Complete the report sheet as you do your lab. Turn your completed report sheet in
to your instructor.

This is a dry lab. You will not have to gather materials and can begin your lab
immediately.




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                         Objectives
In this section you will…
– Identify and give the function of the basic components of a bacterium
– Identify and describe the three major bacterial shapes
– understand the concepts and results of the Gram stain
– Identify bacterial cells and their structures of on prepared slides
– Compare and contrast the three domains




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                         I. Introduction
Living things are made up of cells and these cells are separated into two basic
types: the eukaryotic cells that contain a true nucleus and the prokaryotic cells that
lack a nucleus.

The name prokaryote comes from the Greek meaning “before the nucleus.” they
are the most abundant, widely distributed group of organisms and can be found
almost everywhere: soil, water, air, even in extreme environments. They are the
oldest groups of organisms, with fossil records showing their existence on earth for
nearly 3.5 billion years, while eukaryotes have existed for 1.7 billion years.

There are three domains; Archaea, Bacteria and Eukarya.




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                       I. Introduction
Eukarya domain have eukaryotic cells. Organisms of this domain are mostly
multicellular with internal membrane-bound organelles. Their DNA consists of
multiple chromosomes. Some members have cell walls made of cellulose or chitin.
Their plasma membrane consists of glycerol bonded to straight chained fatty acids.
Members reproduce sexually via meiosis.

Both Archaea and Bacteria have prokaryotic cells. Prokaryotes are generally
single celled organisms with a single circular chromosome. The two domains differ
in the structure of their plasma membrane and cell wall.




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                       I. Introduction
The smaller of the two domains is Archaea. They are prokaryotes whose plasma
membrane consists of glycerol bonded to branched fatty acids and their cell wall
does not contain peptidoglycan. These organisms have adapted to extreme
environments such as high heat (thermophiles) and salt (halophiles). Others grow
in anaerobic conditions and generate methane (methanogens).

The domain Bacteria have a cell wall comprised of peptidoglycan. Their plasma
membranes consists of glycerol bonded to straight fatty acids (similar to
eukaryotes). This lab will focus on Bacteria.




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                         I. Domains
                     Experiment (dry lab)
Compare and contrast the three domains.
Fill in the table with differences and similarities between them.


Characteristic                                           Bacteria Archaea   Eukarya
Is a nucleus present?
Is there one or more chromosomes?
Are membrane-bound organelles present?
Is the domain primarily unicellular or multicellular?
Is there sexual reproduction via meiosis?
Is a plasma membrane present? If so, describe it.
Is there be a cell wall in some of the members?



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                      II. Bacteria
                  Experiment (dry lab)
Some of the structures are always found in bacteria and some are not. The typical
bacterium contains: cytoplasm, DNA, ribosomes, a cell wall, plasma membrane,
pili, flagellum, and capsule.

Found in all bacteria
The cytoplasm is a region within the cell filled with a jelly-like fluid called the
cytosol. The nuclear material is a circular dsDNA (double-stranded DNA) molecule.
The nuclear material is located in the cytosol of the cell in a region called nucleoid.
Ribosomes of bacteria are smaller, less dense than eukaryotic ribosomes but they
serve the same function; to synthesize protein. The cell wall of bacteria is made of
peptidoglycan. The amount of peptidoglycan can vary from species to species. The
plasma membrane lies between the cell wall and the cytoplasm and regulates what
enters and leaves the organism.




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                      II. Bacteria
                  Experiment (dry lab)
Found in some bacteria
Some bacteria form pili (pilus, singular). These fingerlike projections are found on
the surface of the body. Their function is for attachment. Bacteria with pili can
attach to teeth, rocks, roots, each other, etc. Some bacteria can have one or more
flagella (flagellum, singular). Flagella are for locomotion. Some bacteria can form
capsules. The capsule lies outside the cell wall and protects the bacteria from
destruction.




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                      II. Bacteria
                  Experiment (dry lab)
Label a typical bacterium
(cell wall, plasma membrane, cytoplasm, DNA, pilus, flagellum, ribosomes)

                                           A

                                B

                         C


                         D



                                                       G

                                           F
                                  E
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         III. Shapes and Arrangements
               Experiment (dry lab)
One way to classify bacteria is by examining its morphology. Bacteria appear in many
different shapes. We will examine the three basic shapes but note that others exist.

Three basic shapes of bacteria
 – Bacilli (bacillus, singular) are rod-shaped
 – Cocci (coccus, singular) are spherically-shaped
 – Spirilla (spirillum, singular) are spiral-shaped

Arrangements of bacteria
Bacterial cells can appear as single cells or can be found in arrangements. Some are
 – Chains (prefix strepto-)
 – Clusters (prefix staphylo-)
 – Twos (prefix diplo-)
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        III. Shapes and Arrangements
              Experiment (dry lab)
Examine the images below. Record the scientific name of each cell shape on your
report sheet.




          A          B          C         D         E




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        III. Shapes and Arrangements
              Experiment (dry lab)
Examine the images below. Record the scientific name of each cell arrangement and
shape on your report sheet.
 A                                         C




       B
                                       D




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                      IV. Diseases
                   Experiment (dry lab)
What disease might you infer from the name of the following bacteria?

           Vibrio cholerae
           Mycobacterium tuberculosis
           Mycoplasm pneumoniae
           Streptococcus pyogenes
           Neisseria meningitidis
           Mycobacterium leprae
           Clostridium tetani
           Clostridium botulinum
           Bacillus anthracis
           Proprionibacterium acnes
           Salmonella enteritidis

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                   V. Gram Staining
                  Experiment (dry lab)
In 1884 Hans Christian Gram developed a staining procedure, Gram Staining. The
composition of the cell wall of bacteria vary among species. Due to this difference
(diagram on next slide), bacteria can be divided into two groups; Gram positive and
Gram negative.
Gram positive bacteria have thick peptidoglycan cell walls and retain a purple
color when stained with crystal violet. Gram negative bacteria have a thin
peptidoglycan inner wall and outer wall made of carbohydrates, proteins and
lipids. These cells retain the red color when stained with safranin.




                Gram staining bottles                                      Continue
 V. Gram Staining
Experiment (dry lab)




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                   V. Gram Staining
                  Experiment (dry lab)
Watch a 3 minute video of the procedure at
   http://www.youtube.com/watch?v=YvZHHNZ8cdo

Gram stain procedure (abridged)
1. Obtain a slide
2. Add one drop of water and place in a staining tray
3. Sterilize a loop


                                                        Staining tray and slide




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                       V. Gram Staining
                      Experiment (dry lab)
4. Transfer bacteria to your slide and
    mix it with the drop of water on
    your slide
5. Heat-fix the slide by passing the
    slide over an open flame several
    times
                                                                 Touching bacteria with loop




  Heat-fixing slide                      Mixing bacteria with water
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                    V. Gram Staining
                   Experiment (dry lab)
6. Flood smear with crystal violet (purple
    stain) for 1 minute
7. Rinse off the stain
8. Flood smear with iodine for 1 minute
9. Rinse off the iodine

   At this point all bacteria will appear
   purple because of the crystal violet.




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                    V. Gram Staining
                   Experiment (dry lab)
   Next step is most important. It will
   differentiate Gram positive from Gram
   negative bacteria.

10. Decolorize with ethanol. Ethanol must be
    added drop-wise to a slanted slide. Stop
    when first clear drop is seen. Rinse slide
    with water.

   At this point, Gram positive bacteria
   remain purple because they do not
   decolorize but Gram negative bacteria         Decolorizing
   decolorize.


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                    V. Gram Staining
                   Experiment (dry lab)
11. Flood smear with safranin (counterstain)
    for 1 minute. Only decolorized cells will
    stain red.

   At this point Gram negative bacteria stain
   red in color. Gram positive bacteria
   remain purple.




                                                Slide with safranin



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                     V. Gram Staining
                    Experiment (dry lab)
12. Rinse slide off with water and blot slide
    dry between sheets of bibulous paper
13. Slide can be examined

   Approximately, how much time does
   Gram staining take?




                                                Packet of bibulous paper




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                   V. Gram Staining
                  Experiment (dry lab)
Record the Gram reaction and cell shape of A.
              A




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                   V. Gram Staining
                  Experiment (dry lab)
Record the Gram reaction and cell shape of B.




B




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                      VI. Bacteriology
A single bacterium multiples to form a colony. A colony consists of millions of cells
that all arise from one cell. See colonies below.




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                      VI. Bacteriology
Each colony exhibits specific colonial morphology (characteristic size, shape,
consistency, texture and color). Here are some colors of bacterial colonies.




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          VI. Bacteriology Questions
Examination of one colony.
True or false. All cells of a pure colony have the same
   ____color
   ____cell shape
   ____Gram stain reaction
   ____DNA




                                                          End of Lab 

				
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