Download Unit 14: Review Prokaryotes Lab

Unit 14: Review
Prokaryotes Lab
I. Bacteria
These are the three domains, Bacteria, Archaea, and Eukarya. We examined the Bacteria domain.
Some of the structures below are always found in bacteria and some are not. The typical bacterium
contains: cytoplasm, nuclear material, 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 dsDNA (double-stranded DNA). 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. The plasma membrane lies
between the cell wall and the cytoplasm. This structure regulates what enters and leaves the
organism.
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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Bacteria continued
II. Bacterial Shapes and Arrangements
Labeled typical bacterium
One way to classify bacteria is by examining its shape. Bacteria appear in many
different shapes. We will examine the three basic shapes but note that others exist.
Three basic shapes of bacteria (refer back to unit 7)
• Bacilli (bacillus, singular) are rod-shaped
• Cocci (coccus, singular) are spherically-shaped
• Spirilla (spirillum, singular) are spiral-shaped
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III. Gram Staining
Gram Staining continued
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 double cell walls. A thin peptidoglycan inner wall
and outer wall made of carbohydrates, proteins and lipids. These cells retain the
red color when stained with safranin.
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Gram staining bottles
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Gram Staining continued
Gram stain procedure (abridged)
1. Obtain a slide
2. Add one drop of water and place in a
staining tray
3. Sterilize a loop
Gram Staining continued
4. Transfer bacteria to your slide and mix it
with the drop of water on your slide
Slide
5. Heat-fix the slide by passing the slide
over an open flame several times
Staining
tray
Touching bacteria with loop
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Gram Staining continued
Heat-fixing slide
Gram Staining continued
6. Flood smear with crystal violet (purple
stain) for 1 minute
Next step is most important. It will differentiate
Gram positive from Gram negative bacteria.
7. Rinse off the stain
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.
8. Flood smear with iodine for 1 minute
9. Rinse off the iodine
Mixing bacteria with water
At this point, Gram positive bacteria remain
purple because they do not decolorize but
Gram negative bacteria decolorize.
At this point all bacteria will appear
purple because of the crystal violet.
Decolorizing
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Gram Staining continued
11. Flood smear with safranin (counterstain) for 1
minute. Only decolorized cells will stain red.
Gram Staining continued
12. Rinse slide off with water and blot slide dry
between sheets of bibulous paper
At this point Gram negative bacteria stain red in
color. Gram positive bacteria remain purple.
Packet of bibulous paper
13. Slide can be examined
Slide with safranin
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Gram Staining continued
Gram positive (purple)
•
Bacilli
•
Cocci
•
Spirilla
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Gram negative (red)
IV. Bacteriology
A single bacterium multiples to form a colony. A colony consists of millions of cells that
all arise from one cell. Each colony exhibits specific colonial morphology
(characteristic size, shape, consistency, texture and color).
End of Lab
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