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 before nucleus) and are differentiated from the eukaryotic (meaning true nucleus) 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. Modied from Johnson (1983).

Property True nucleus Mitosis occurs Meiosis occurs Mitochondria Chloroplasts Flagella Ribosomes Lysosomes Microtubules Prokaryotes absent no no absent absent 13 bers present, but 70S absent rare Eukaryotes present yes yes present present 9 + 2 microtubule arrangement present, but 80S present 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:
1. 2. 3. 4. 5. 6. 7. 8.

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-xed 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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Classication 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 signicantly 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 classication 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 difcult to group them by phyllogenetic relationships.
TABLE 48. Selected

groups of bacteria based on morphological, physiological, or ecological similarities.

Group Characteristics anaerobic, photosynthetic bacteria that do not have chlorophyll a; often reduce H2S. anaerobic, obligate photosynthetic; live in high-sulphur environments (deep within lakes or in hot springs) aerobic photosynthetic bacteria that have chlorophyll a; some are x nitrogen long, coiled cell; several cause diseases in mammals able to oxidize inorganic cmpds. (e.g., NH3, NO2, H2S) for energy source; many are important in nutrient cycling. large number of strains, often difcult to identify almost all are obligate intracellular parasites; Gram-negative rods or cocci many tolerate drying, high salt, lack a cell wall; highly plastic in form and usually grow in laments; buds are smallest free-living cells at 23m Examples Chromatium

Purple sulphur bacteria

Green sulphur bacteria

Chlorobium

Cyanobacteria

Anabaena, Nostoc, Oscillatoria

Spirochaetes

Treponema (Syphilis), Leptospira (Leptospirosis), and Borrelia (Lyme disease) Nitosomonas, Nitrobacter, Thiobacillus

Chemolithotrophs

Facultatively aerobic Gramnegative rods Rickettsias Gram-positive cocci Mycoplasmas

Escherichia (E. coli), Salmonella, Proteus Rickettsia (spotted fever, typhus, trench fever) Staphylococcus, Streptococcus, 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 Grams iodine. Uptake of Grams 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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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 Grams iodine for differentiating bacteria. Most bacteria can be cultured in broths and on agar-lled 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 ll 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.

Description growth is even; even margin uniform growth; sawtooth margin colonies conuent towards bottom but isolated at top thin and spreading; difcult to see tree-like: thick along inoculation line, but spreading outwards delicate and root-like

Form Filiform Echinulate Beaded Effuse Arborescent Rhizoid

TABLE 50. Terms

applied to bacteria cultures grown on petri plates.

Type punctiform (pinpoint), circular, irregular, rhizoid, lamentous entire, undulate, lobate, curled, lamentous at, raised, convex, unbonate, craterform smooth, rough, granular, shiny, dull, wet, mucoid, dry, wrinkled, powdery transparent, translucent, opaque red, orange, etc.

Category Form Margin Elevation Texture Opacity Color

Because each bacterium has particular environmental and physiological requirements, a number of differential growth media have been developed to assist in bacteria identication. Three commonly used differential media are given below along with a description of how each is used:

Mannitol salt agar (MSA) contains mannitol (a sugar), 7.5% NaCl, phenol red (a pH indicator), and nutrients. This
medium excludes organisms that cant tolerate its high salt content along with those that cant 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) contains sheep blood along with other standard nutrients. Certain bacteria are capable of lysing red blood cells to obtain nutrients. These organisms are identied by transparent zones surrounding each colony. Degree of hemolysis is categorized as follows: 1.alpha (a) hemolysis incomplete; medium green. 2.beta (b) hemolysis complete; medium clear. 3.gamma (g) hemolysis nonhemolytic; medium unchanged.

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Eosin-methylene blue agar (EMB) 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 oor. As much as 30% of Anarcticas 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
1. 2.

3. 4. 5. 6. 7.

Label a clean microscope slide with your name and date. Prepare a heat-xed 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 ame several times to x 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 Grams 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 identication 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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4.

5.

6.

First, ame the inoculating loop by holding its end in the ame 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 rst holding the capped tube in your left hand, then removing the cap with the little nger 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 ame. In addition to killing microorganisms, aming sets up outward convection currents that reduce the chance of contamination. Insert the transfer loop in the tube and rst cool the loop tip by touching it to the edge of the medium. Now dip the loop end into the culture. Remove the loop, reame 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

7. 8.

Replace the petri cover. Seal the plate with a strip of Paralm. Invert the plate and incubate in the oven at 30 or 37 C for 23 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.
1. 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 (pyrogenes = pus producing). Gram-positive coccus, spherical to ovoid, 0.61 m 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 Identical to S. pyrogenes except it is not hemolytic. Staphylococcus aureus (aureus = golden). Gram-positive coccus; 0.81 m 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 (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 ora of human intestines.

Bacillus megaterium (megaterium = big beast). Very large Gram-positive rods, oval to pear-shaped, up to 5 m long,
motile. Usually occurs in short, twisted chains. Colony yellow. Usually catalase positive, no growth on mannitol salt agar.

Proteus vulgaris Gram-negative rod; highly motile; swarming colony. Serratia marcescens Gram-negative rod. Colony pinkish. Pseudomonas aeruginosa Gram-negative rod. Colony green-pigmented; grape odor. Beggiatoa sp. lamentous 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 625630 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 570580 nm. Bacteria possessing this pigment are brown to red in color. Cyanobacteria have three basic growth forms: unicellular, colonial, and lamentous. Some lamentous forms have specialized cells for xing nitrogen, called heterocysts. These enlarged, almost clear cells stand out from other cells in the lament. Some lamentous forms produce thick-walled spores, or akinetes, which are capable of withstanding periods of drying, freezing, or burial.

Spirulina sp. corkscrew-shaped lamentous, highly motile. Found in freshwaters with high nutrient inputs. Oscillatoria sp. straight lamentous, highly motile. Common in almost all freshwaters. Anabaena sp. lamentous with cells that look like beads on a chain. Typically possess both heterocysts and akinetes. Nostoc sp. lamentous colony. Identical to Anabaena except laments are embedded in a gelatinous sheath. Colonies may be spherical, ear-shaped, or amorphous.

Merismopedia sp. colonial. Cells in a at sheet.

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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-xed smear. Discard toothpicks in the garbage can when you nished with them. Follow the Grams staining procedure outlined above and observe the slide at 1000X. Sketch and label your observations.

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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 cultures identication number. Using aseptic technique procedures outlined in the text, streak the agar plate with bacteria from the nutrient broth, then incubate for 23 days. Prepare a heat-xed 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 Grams Growth form Colony: Form Margin Elevation Opacity Texture Color Differential Media: MSA SBA EMB Other: Catalase Comments

12

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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 ora and sketch your observations below.

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