Download The Prokaryotes

EXERCISE 7
The Prokaryotes
Organisms like bacteria, methanogens, and blue-green algae have cells lacking the membrane-bound nucleus found in
protozoans, plants, and animals. Instead their single, circular DNA strand is often concentrated in an unbound nuclear
region, called a nucleoid. Such cells are prokaryotic (meaning Ôbefore nucleusÕ) and are differentiated from the
eukaryotic (meaning Ôtrue nucleusÕ) cells that have a nuclear envelope surrounding a nucleus containing several DNA
molecules. Prokaryotes differ from eukaryotes in many other ways, several are listed in the following table:
TABLE 47. Comparison
of prokaryotic and eukaryotic cells. ModiÞed
from Johnson (1983).
Property
Prokaryotes
Eukaryotes
True nucleus
absent
present
Mitosis occurs
no
yes
Meiosis occurs
no
yes
Mitochondria
absent
present
Chloroplasts
absent
present
Flagella
1Ð3 Þbers
9 + 2 microtubule arrangement
Ribosomes
present, but 70S
present, but 80S
Lysosomes
absent
present
Microtubules
rare
present
Exercise Objectives:
In this lab, we will explore representatives from the major groups of prokaryotes and learn basic techniques in culturing,
staining, isolating bacteria. Upon completing this lab exercise, you should be able to:
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Discuss the major differences between prokaryotic and eukaryotic cells.
Recognize two kingdoms of prokaryotes and identify habitats where Archaea predominate.
Identify several common cyanobacterian genera.
Identify three shapes of bacteria.
Discuss differences between Gram-positive and Gram-negative bacteria.
Prepare a heat-Þxed bacteria smear, use Gram staining procedure, and interpret the microscopical examination.
Prepare a streak plate using aseptic technique.
Identify an unknown bacterium based on microscopical examination, Gram staining, differential agars, colony morphology, etc.
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ClassiÞcation of Prokaryotes
Until recently all prokaryotes were included in the Kingdom Monera. However, researchers studying the genetic composition or microorganisms have recognized a new group, the Archaea, having characteristics signiÞcantly different enough
from the rest of the prokaryotes to warrant separate kingdom status. Prokaryotes, unlike the eukaryote, have few morphological characteristics to separate them, therefore microbiologists use a series of metabolic tests to help in differentiation.
There remains little consensus among biologists as to the phyllogenetic relationships among and formal classiÞcation of
prokaryotes, therefore we will informally group them according to their most distinguishing morphological and metabolic
characteristics.
Kingdom Bacteria
Bacteria are often grouped according to morphological, physiological, or ecological similarities rather because it is much
more difÞcult to group them by phyllogenetic relationships.
TABLE 48. Selected
groups of bacteria based on morphological, physiological, or
ecological similarities.
Group
Characteristics
Examples
Purple sulphur bacteria
anaerobic, photosynthetic bacteria that
do not have chlorophyll a; often reduce
H2S.
Chromatium
Green sulphur bacteria
anaerobic, obligate photosynthetic; live
in high-sulphur environments (deep
within lakes or in hot springs)
Chlorobium
Cyanobacteria
aerobic photosynthetic bacteria that
have chlorophyll a; some are Þx nitrogen
Anabaena, Nostoc, Oscillatoria
Spirochaetes
long, coiled cell; several cause diseases
in mammals
Treponema (Syphilis), Leptospira (Leptospirosis), and Borrelia (Lyme disease)
Chemolithotrophs
able to oxidize inorganic cmpds. (e.g.,
NH3, NO2, H2S) for energy source;
many are important in nutrient cycling.
Nitosomonas, Nitrobacter, Thiobacillus
Facultatively aerobic Gramnegative rods
large number of strains, often difÞcult
to identify
Escherichia (E. coli), Salmonella, Proteus
Rickettsias
almost all are obligate intracellular parasites; Gram-negative rods or cocci
Rickettsia (spotted fever, typhus, trench
fever)
Gram-positive cocci
many tolerate drying, high salt,
Staphylococcus, Streptococcus,
Mycoplasmas
lack a cell wall; highly plastic in form
and usually grow in Þlaments; buds are
smallest free-living cells at 2Ð3µm
Mycoplasma, Ureaplasma
Because bacteria have few morphological characteristics, microbiologists employ a wide variety of tools to separate them.
Some techniques used are: bacterium shape, staining, colony morphology, and differential culture media. Three of the
most common bacteria shapes are: cocci (spheres), bacilli (rods), and spirilli (corkscrews). In addition, bacteria may grow
as unicellular units, clusters, or in chains. Bacteria can be separated into two basic groups depending on whether or not
their cell walls can be stained with GramÕs iodine. Uptake of GramÕs stain is fundamentally linked with cell wall structure. The cell walls of all bacteria contain a protein-sugar compound called peptidoglycan (or murein). The cell walls of
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ClassiÞcation of Prokaryotes
Gram-negative bacteria have only small amounts of peptidoglycan, but they have a thick, outer cell membrane made of
lipopolysaccharide (a lipid-sugar complex). Gram-positive bacteria have a thick layer of peptidoglycan but lack the
outer lipopolysaccharide membrane. Crystal violet, safranin, methylene blue, carbol fuchsin, and acid-fast stains are often
used in conjunction with GramÕs iodine for differentiating bacteria.
Most bacteria can be cultured in broths and on agar-Þlled petri plates and slated-tubes (slants) providing they have the
necessary nutrients and environmental conditions. When incubated at optimal temperature, bacterial cultures grow quickly
and within a few days, completely Þll the container. Colony shape, color, opacity, margin, surface texture and elevation,
and even odor can be distinctive between bacteria species. Several morphologies associated with bacteria cultures in slants
and petri dishes are given in the tables below:
TABLE 49. Terms
applied to growth forms of bacteria cultures in slants.
Form
Description
Filiform
growth is even; even margin
Echinulate
uniform growth; sawtooth margin
Beaded
colonies conßuent towards bottom but isolated at top
Effuse
thin and spreading; difÞcult to see
Arborescent
tree-like: thick along inoculation line, but spreading outwards
Rhizoid
delicate and root-like
TABLE 50. Terms
applied to bacteria cultures grown on petri plates.
Category
Type
Form
punctiform (pinpoint), circular, irregular, rhizoid, Þlamentous
Margin
entire, undulate, lobate, curled, Þlamentous
Elevation
ßat, raised, convex, unbonate, craterform
Texture
smooth, rough, granular, shiny, dull, wet, mucoid, dry, wrinkled, powdery
Opacity
transparent, translucent, opaque
Color
red, orange, etc.
Because each bacterium has particular environmental and physiological requirements, a number of differential growth
media have been developed to assist in bacteria identiÞcation. Three commonly used differential media are given below
along with a description of how each is used:
¥ Mannitol salt agar (MSA)Ð contains mannitol (a sugar), 7.5% NaCl, phenol red (a pH indicator), and nutrients. This
medium excludes organisms that canÕt tolerate its high salt content along with those that canÕt metabolize mannitol.
The acid end-products of mannitol fermentation produced by organisms living on this medium change its color from
red to yellow.
¥ Sheep blood agar (SBA)Ð contains sheep blood along with other standard nutrients. Certain bacteria are capable of lysing red blood cells to obtain nutrients. These organisms are identiÞed by transparent zones surrounding each colony.
Degree of hemolysis is categorized as follows:
1.alpha (a) hemolysis Ð incomplete; medium green.
2.beta (b) hemolysis Ð complete; medium clear.
3.gamma (g) hemolysis Ð nonhemolytic; medium unchanged.
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¥ Eosin-methylene blue agar (EMB) Ð contains eosin and methylene blue dye along with other standard nutrients. Eosin
inhibits the growth of many bacteria. Those able to live on this medium take up the dye, which gives the colony a
metallic luster.
Kingdom Archaea
Members of this group resemble bacteria in morphology but have unique cell membrane and cell wall structure. Surprisingly, the Archaea are no more closely related to bacteria than they are to eukaryotes. Archaea represent a widely diverse
group with one thing in common: they all live in extreme environments. Some of the best-known Archaea live in cattle
rumens and termite guts, while others live in hotsprings, geyers, and submarine volcanos. Recently, Archaea have been
isolated from glacial ice and the deep-sea ßoor. As much as 30% of AnarcticaÕs biomass may be archaeans.
Most Archaea are obligate anaerobes, which cannot survive exposure to oxygen, and a large number release methane as a
metabolic by-product.
Procedures:
Gram staining
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Label a clean microscope slide with your name and date.
Prepare a heat-Þxed smear of a bacteria sample as follows: Place a small drop of water on the slide. Transfer a small
bacteria sample to the water drop with either a toothpick, transfer loop, or sterile swab, depending on the type of sample taken. Allow the water-sample mixture to air-dry, then pass the slide through a ßame several times to Þx the bacteria to the slide. DO NOT OVERHEAT THE SLIDE.
DO ALL STAINING OVER A SINK. Cover with crystal violet for 45 s, then rinse with water.
Cover with GramÕs iodine for 45 s, then rinse with water.
Decolorize with 95% ETOH for 15 s.
Counterstain with safranin for 30 s, then rinse with water, and blot dry with a paper towel.
Examine preparation with a microscope.
Gram-positive bacteria retain the crystal violet stain and will appear purple; whereas, Gram-negative bacteria lose crystal
violet but take up safranin and appear pink.
Preparing a bacteria culture
Making a pure culture of bacterial specimens is another fundamental technique of microbiology. In order to maintain a
pure culture, the medium, all utensils, and working surfaces must be kept free from contamination from foreign bacteria
and fungi. The overall procedure for maintaining contamination-free conditions is called aseptic (a = without, and septic
= contamination) technique.
1.
2.
3.
Obtain a sterile agar plate. DO NOT REMOVE THE LID. Using a wax pencil or Sharpie, write your name and date on
the bottom of the plate.
Obtain a bacterium culture from the TA. Record the identiÞcation number of the sample.
Work at the culture station set up in the lab. Before you begin, wash your hands and wipe down the working surface.
Make sure there is a bunsen burner and inoculating loop available.
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Characteristics of Selected Bacteria Species
4.
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6.
First, ßame the inoculating loop by holding its end in the ßame until the loop turns red. This will kill any microorganisms and their spores found on the loop or handle end. DO NOT LAY THE LOOP DOWN OR LET IT TOUCH ANYTHING BEFORE YOU DO THE TRANSFER.
Flame the lip of the culture tube by Þrst holding the capped tube in your left hand, then removing the cap with the little
Þnger of your right hand. DO NOT LAY THE CAP DOWN OR TURN IT OVER. Holding the tube as horizontal as
possible, pass the lip through the ßame. In addition to killing microorganisms, ßaming sets up outward convection currents that reduce the chance of contamination.
Insert the transfer loop in the tube and Þrst cool the loop tip by touching it to the edge of the medium. Now dip the loop
end into the culture. Remove the loop, reßame the tube lip, recap it, and set it aside. Carefully move the loop over to
the petri plate. Open the petri plate just enough to insert the inoculating loop. Gently streak the loop over the medium
in area #1 taking care not to cut into the agar surface. Repeat step 5 and 6 to streak in areas #2, #3, and #4.
FIGURE 38.
Plate streaking pattern.
#1
#4
#2
#3
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8.
Replace the petri cover. Seal the plate with a strip of ParaÞlm. Invert the plate and incubate in the oven at 30 or 37 ûC
for 2Ð3 days.
Record colony morphology.
Catalase test
One simple test to see whether a bacterium possesses the catalase enzyme is to observe the reaction when a dilute hydrogen peroxide (H2O2) solution is added to the culture. Hydrogen peroxide is a naturally occurring substance which is
highly toxic to most organisms. Catalase acts on hydrogen peroxide to release gaseous oxygen thus converting hydrogen
peroxide to water.
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2.
Place a small drop of 3% hydrogen peroxide on your bacterium culture plate. Watch for the appearance of oxygen bubbles.
Record the results.
Characteristics of Selected Bacteria Species
¥ Streptococcus pyrogenes Ð (pyrogenes = pus producing). Gram-positive coccus, spherical to ovoid, 0.6Ð1 µm diameter,
occurs in pairs or short chains. Colonies milky and glossy. Beta hemolytic, catalase negative. Does not grow well on
mannitol salt agar. Found in human mouth, nose, throat, respiratory tract, and some lesions.
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¥ Streptococcus pneumonia Ð Identical to S. pyrogenes except it is not hemolytic.
¥ Staphylococcus aureus Ð (aureus = golden). Gram-positive coccus; 0.8Ð1 µm diameter; occurs singly, in pairs, and
irregular clusters. Colony usually large, low-craterform, glossy, margin entire, yellow-gold. ± hemolytic, catalase positive, grows on mannitol salt agar. Found on warm-blooded animal skin, glands, and mucous membranes.
¥ Escherichia coli Ð (coli = colon). Small Gram-negative rod. Colonies low-craterform, glossy, grey. Metallic purple on
EMB agar, strains from pigs are hemolytic and catalase positive. Part of the natural ßora of human intestines.
¥ Bacillus megaterium Ð (megaterium = big beast). Very large Gram-positive rods, oval to pear-shaped, up to 5 µm long,
motile. Usually occurs in short, twisted chains. Colony yellow. Usually catalase positive, no growth on mannitol salt
agar.
¥
¥
¥
¥
Proteus vulgaris Ð Gram-negative rod; highly motile; swarming colony.
Serratia marcescens Ð Gram-negative rod. Colony pinkish.
Pseudomonas aeruginosa Ð Gram-negative rod. Colony green-pigmented; grape odor.
Beggiatoa sp. Ð Þlamentous sulphur bacteria
Cyanobacteria
The Cyanobacteria (blue-green algae) have much of the typical prokaryote structure except they also posses photosynthetic membranes with embedded photosynthetic pigments. One of the photosynthetic pigments is chlorophyll a, a form
of chlorophyll which is found in all algae and higher plants. A second, accessory class of pigments is also usually present.
These are biliproteins, or phycobilins. One type of biliprotein common to blue-greens is phycocyanin, a blue pigment,
which absorbs light energy maximally at 625Ð630 nm, towards the red end of the visible spectrum. Cyanobacteria which
have both chlorophyll a and phycocyanin are typically blue-green in color. Other blue-green algae have phycoerythrin, a
red-pigmented biliprotein, in place of phycocyanin and absorbs light energy maximally at 570Ð580 nm. Bacteria possessing this pigment are brown to red in color.
Cyanobacteria have three basic growth forms: unicellular, colonial, and Þlamentous. Some Þlamentous forms have specialized cells for Þxing nitrogen, called heterocysts. These enlarged, almost clear cells stand out from other cells in the Þlament. Some Þlamentous forms produce thick-walled spores, or akinetes, which are capable of withstanding periods of
drying, freezing, or burial.
¥
¥
¥
¥
Spirulina sp. Ð corkscrew-shaped Þlamentous, highly motile. Found in freshwaters with high nutrient inputs.
Oscillatoria sp. Ð straight Þlamentous, highly motile. Common in almost all freshwaters.
Anabaena sp. Ð Þlamentous with cells that look like beads on a chain. Typically possess both heterocysts and akinetes.
Nostoc sp. Ð Þlamentous colony. Identical to Anabaena except Þlaments are embedded in a gelatinous sheath. Colonies
may be spherical, ear-shaped, or amorphous.
¥ Merismopedia sp. Ð colonial. Cells in a ßat sheet.
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Exercises
Exercises
1.
Examine cultures of the following Cyanobacteria: Spirulina, Anabaena, Nostoc, and Merismopedia. Sketch, give a
general dimension, and label any special features of each alga in the space provided below.
2.
Obtain a mixed bacteria slide from the TA and observe at 1000X. Sketch and label size, shape, and any special features
seen in these bacteria in the space provided.
3.
With a clean toothpick, gently scrape tartar from your gingival clefts and prepare a heat-Þxed smear. Discard toothpicks in the garbage can when you Þnished with them. Follow the GramÕs staining procedure outlined above and
observe the slide at 1000X. Sketch and label your observations.
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The Prokaryotes
4.
Obtain a nutrient broth culture and sterile agar plate from the TA. Label the bottom side of the plate with your name,
date, and the broth cultureÕs identiÞcation number. Using aseptic technique procedures outlined in the text, streak the
agar plate with bacteria from the nutrient broth, then incubate for 2Ð3 days. Prepare a heat-Þxed smear of the cultures,
Gram stain, and sketch your observations. Perform a catalase test of your culture. Record the results of the differential
agars on demonstration. Examine and record colony morphologies of the remaining unknowns. Complete the table
below.
TABLE 51. Characteristics
of bacteria unknowns
Unknowns
1
Microscopic:
Shape
GramÕs
Growth form
Colony:
Form
Margin
Elevation
Opacity
Texture
Color
Differential
Media:
MSA
SBA
EMB
Other:
Catalase
Comments
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3
4
5
6
7
8
9
12
Exercises
5.
Match the unknowns with the appropriate bacterium.
¥Streptococcus pyrogenes ______
¥Streptococcus pneumonia ______
¥Staphylococcus aureus ______
¥Escherichia coli ______
¥Bacillus megaterium ______
¥Proteus vulgaris ______
¥Serratia marcescens ______
¥Pseudomonas aeruginosa ______
¥Beggiatoa sp. ______
6.
View the demonstration of termite gut ßora and sketch your observations below.
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