North Star Academy College Preparatory High School

Name: ______________________________ Team_____________________

EDUCATION IS FREEDOM! Date__________/Mr. McIntire

AP Biology

Prokaryotic Cells Lab

Lab modified from Biology in the Laboratory, 3rd Ed. By DR Helms, CW Helms, RJ Kosinski and JR Cummings, W.H. Freeman and Co: New York, 1998.

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Introduction

Understanding the nature of cell structure and function is important to an understanding of organisms. All organisms are composed of cells, whether they exist as single cells, colonies of cells, or in multicellular form. Cells are usually very small, and for this reason, a thorough understanding of subcellular structure and function has been possible only through advances in electron microscopy and molecular biology.

There are two general types of cells: prokaryotic and eukaryotic. These two words have their roots in the Greek work karyon (nut), which refers to a cell having a nucleus. The prefix pro- means “before” or “prior to.” Thus, prokaryotic means “before having a nucleus.” Prokaryotic cells do not have a membrane-bound nucleus and their genetic material (DNA) is only loosely confined to a nuclear area within the cell. Bacteria, including the cyanobacteria (formerly known as blue-green algae), are prokaryotes. All other organisms are eukaryotes. The prefix eu- means “true.” The cells of eukaryotes have true, membrane-bound nuclei containing their genetic material.

Prokaryotic and eukaryotic cells also differ in several other ways. Eukaryotic cells are generally larger and contain additional specialized compartments (membrane-bound organelles) in which cell functions such as energy production may occur. Prokaryotic cells lack membrane-bound organelles; their cell functions are carried out in the cytoplasm.

Origin of Cells

Evidence suggests that the first cells were prokaryotic. They were probably anaerobic, living in an atmosphere with little or no oxygen, and heterotrophic, consuming organic materials as a carbon source. But how did these first cells come to be? In the 1930s, Russian scientist A.I. Oparin suggested that molecular aggregates, called coacervates, which can be prepared in the laboratory, may resemble the precursors of the first cells. These aggregates are composed of high-molecular weight substances that, when dissolved in water, aggregate to form viscous droplets.

Although these droplets are not living cells, coacervates and cells having several characteristics in common. They are able to maintain an internal environment that differs from their surroundings and they can selectively absorb substances from their surroundings. Enzymes are included among the high-molecular weight molecules – usually a combination of lipids, polypeptides, polysaccharides, and nucleic acids – used to form coacervates. Enzymes can catalyze reactions, including syntheses, hydrolyses, and electron transfer.

Coacervates are not alive, however, and so they are called protobionts (proto-, “before,” plus bionts, “life”). However, the formation of coacervates may have allowed organic molecules to interact with one another to form new structural entities – the forerunners of cellular organelles.

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Prokaryotes

Present day bacteria are extremely small (approximately 1-2μm in diameter). Most are heterotrophic, depending on preformed food, but some are photosynthetic and make their own food. Morphologically, bacteria are either round (cocci), rod-shaped (bacilli), or spiral-shaped (spirilla). See Figure 1 to observe their shape.

Figure 1: The cells of many familiar genera of bacteria include the (a) rod-shaped bacillus, (b) spherical coccus, and (c) helical spirillum.

You can use the light microscope to study bacteria, but only their external features will be distinguishable. To view them with the light microscope, you must use an oil-immersion lens (100X). Oil has a greater refractive index than air and thus better resolution can be obtained by replacing the later of air between the end of the objective and the specimen with a drop of oil into which the objective is carefully immersed. This eliminates much of the refraction and loss of light that occurs when light passes through air before reaching the dry objective. The working distance of an oil-immersion objective (90X to 100X) is less than 1mm and the objective must also tough the slide to be in focus. Thus, the field of view is much smaller. Light intensity usually needs to be increased when working with oil. Even then, not much more than their basic shapes will be visible. However, you will also note that bacteria are often found in clusters or in chains. Some have one or more flagella. See Figure 2.

Figure 2: A bacterial cell, Proteus mirabilis, showing numerous flagella.

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Cyanobacteria are photosynthetic prokaryotes. However, unlike other photosynthetic bacteria, which contain bacteriochlorophyll, cyanobacteria contain chlorophyll a – the same type of chlorophyll found in eukaryotic green algae and plants. The chlorophyll molecules are not located within chloroplasts, as in higher plants, but are found, instead, within photosynthetic thylakoid membranes dispersed throughout the cytoplasm. See Figure 3.

Figure 3: Photosynthetic membranes fold into stacks inside a bacterial cell.

In addition to chlorophyll a, cyanobacteria contain other accessory pigments including the yellow and orange carotenoids and the phycobilins (reddish phycoerythrins and bluish allophycocyanins).

With the aid of the electron microscope, however, you can study these prokaryotic cells more closely. You can even use special staining techniques to learn about their structure. The resolving power of an electron microscope is approximately 1000 times that of a light microscope. This allows us to observe the subcellular structure of bacteria. The most prominent feature within the bacterial cell is the nucleoid region, which may appear as a lighter, fibrous central area. The genetic material of the cell is dispersed throughout this region. The cytoplasm is filled with granular-looking bodies called ribosomes. Both a cell membrane and a thicker cell wall (composed of peptidoglycan; a polymer of amino sugars and other polymers in some species) surround the bacterium. Outside the cell wall there is often a layer of “slime” that forms a polysaccharide capsule. The capsule may help to protect the bacteria against attach by the immune system of a host organism and from dehydration. Many bacteria do not produce capsules, and even those with capsules will not die the capsule is destroyed. See Figure 4.

Figure 4: Convoluted membranes of a large mesosome (arrow) extend throughout this bacterium. The mesosome is continuous with the cell membrane. A thick, slimy capsule surrounds the cell.

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Some bacteria possess mesosomes, whorls of membranous material that extend inward from the cell membrane. The function of mesosomes is not yet certain, but they may play a role in cell division or in various energy-releasing reactions. In photosynthetic bacteria, the cell membrane often folds extensively to form an internal membrane system containing chlorophyll and other pigments.

The chemical nature and physical structure of bacterial cell walls differ among species. This difference is the basis for classifying bacteria into two major groups, gram-positive and gram-negative, based on their staining properties with Gram’s stain, a mixture of iodine and the dye crystal violet. When stained with Gram’s stain and then treated with decoloring solution (alcohol or acetone), gram-positive cells retain the crystal violet color and appear deep purple. Gram-negative cells lose the crystal violet color when washed with alcohol, but are stained red when treated with a second stain, safranin.

They key differences in the nature of the cell wall are shown in Figure 5. Gram-positive bacteria (Figure 4a) have a multilayered, cross-linked network of amino sugars (peptidoglycan) forming a cell wall outside the cell membrane. As many as 20-40 layers of peptidoglycan may be present, making the cell wall about 50nm thick. Polymers of glycerol or ribitol usually extend from the cell membrane into the cell wall. Gram-negative bacteria (Figure 4b) have a much thinner wall of peptideglycan, usually only several layers thick. The cell wall lies outside the cell membrane but is covered by an additional outer membrane of lipid and protein. Under the electron microscope, the cell wall appears as the lighter layer between the two cell membranes, partly filling the periplasmic space.

Figure 5: Electron micrograms of sections through the cell walls of (a) Gram-positive Bacillus polymyxa and (b) Gram-negative Escherchia coli. The wall of a Gram-positive bacteria consists of a homogenous layer of peptidoglycans and polysaccharides seen here as the lower dark band. The upper dark band is a layer of surface proteins. In Gram-negative bacteria, a layer of peptidoglycan (cell wall) is sandwiched between inner and outer cell membranes.

When you go to the doctor’s office and a bacterial infection is suspected, a Gram stain test might be performed to determine whether you are infected with Gram-positive or Gram-negative bacteria. For example, strep infections *from Streptococcus species) can readily be identified in this manner.

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Sterile Technique

Sterile Technique is used to avoid contamination of sterile media and equipment during cell culture. Sterile technique should always be employed when working with live cell cultures, microbiological samples, and reagents/media that will be used for such cultures. The technique involves using flame to kill contaminating organisms, and a general mode of operation that minimizes exposure of sterile media and equipment to contaminants.

When working with cultures of living organisms, it is extremely important to maintain the environments in which cells are cultured and manipulated as free of other organisms as possible. This requires that exposure of containers of sterilized culture media to outside air should be minimized, and that flame is used to "re-sterilize" container lids and rims. This means passing rims and lids through the flame produced by a bunsen burner in order to kill microorganisms coming in contact with those surfaces. Sterile technique, in general, is a learned state-of-being, or mantra, where every utilization of any sterile material comes with the caveat of taking every precaution to ensure it remains as free of contaminants as possible for as long as possible. Heat is an excellent means of killing microorganisms, and the Bunsen burner is the sterile technician’s best friend.

Objectives

1. Distinguish between the FOUR types of bacteria using the light microscope and oil immersion microscopy.

2. Describe the subcellular structure of a typical bacterium from a transmission electron micrograph.

3. Compare the structures of Gram-positive and Gram-negative bacteria and identify whether E. coli and S. aureus are Gram-negative or Gram-positive using Gram staining.

Materials

Microscope

Prokaryotic slides Sterile gloves Glass slides Coverslips

Bacterial cultures Crystal violet Iodine solution Alcohol

Safranin stain Sodium bicarbonate Sterile loops Bunsen burners

Safety Note: Long hair must be tied back when working with Bunsen burners. Take care not to burn yourself, and You MUST wear latex gloves during the lab when working with live bacteria (Parts 3-4). Dispose of gloves in the wastebasket and wash your hands thoroughly before leaving the lab. Clean all work surfaces with 70% ethanol to sterilize all surfaces of bacteria.

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Part 1: Observing Bacteria Using the Light Microscope

Procedure

1. Observe a prepared slide of Eschericia coli on demonstration. Describe its shape in Table 1, including a representative drawing.

2. Observe a prepared slide of Staphylococcus aureus on demonstration. Describe its shape in Table 1, including a representative drawing.

3. Observe a prepared slide of Spirillim voluntans on demonstration. Describe its shape in Table 1, including a representative drawing.

4. Observe a cyanobacterial species available in the laboratory. Observe the specimen using the 40X objective. Use the electron micrograph (Figure 6) below to assist you in labeling the parts of the cell you have drawn.

Table 1:

Staphylococcus aureus Spirillum voluntans

Oil Immersion Oil Immersion

Spirillim voluntans Cyanobacteria

Oil Immersion 40X

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Part 2: Observing Bacteria Using the Transmission Electron Microscope (TEM)

Procedure

1. Figure 7 is a transmission electron microgram of an Escherichia coli cell that is in the process of dividing. The two new cells have not yet separated completely. The cell wall of E. coli is defined on its inner surface by the cell membrane and on its outer surface by a second, outer membrane. The cell wall exists in the light-colored area between the two membranes; this area is known as the periplasmic space.

Figure 7: Transmission electron micrograms of Gram-negative Escherchia coli actively undergoing binary fission to divide the cytoplasm of these two cells. Replication of the prokaryotic chromosome preceded cell division.

2. Identify the labeled structures in Figure 7.

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Part 3: Using Gram Stain to Study Bacterial Cell Walls

Procedure

1. On the laboratory bench you will find two agar-slant cultures of bacteria. The bacteria are growing on the surface of the nutrient agar; if you look carefully, you can see a “slimy” coating of bacteria growing on the agar. Your challenge is to determine whether the two strains of bacteria, Escherchia coli and Staphylococcus areus are Gram-positive or Gram-negative.

2. Using what you know from your reading about the cell wall structure of the two genera of bacteria (consult the introduction for clarification), form a hypothesis about the Gram staining properties of these two species.

3. Obtain a clean glass slide. Do not touch the surface of the slide with your fingers.

4. Label one end of the slide “E” and the other “S” using a washable overhead marker.

5. Sterilize a wire inoculating loop by flaming it briefly.

6. Using the sterile loop, place two small groups of tap water on the slide approximately 2cm apart between the “E” and “S” labels.

7. Resterilize the loop. Cool it for several seconds. With the tip of the loop, remove a small amount of growth from the surface fo the E. coli agar-slant culture. To maintain sterile conditions, be sure to flame the neck of the test tube after opening it and before closing it.

8. Mix the bacterial cells on the tip of the inoculating loop with the drop of water nearest the “E” label. With a circular motion, spread the spot out to the size of a dime or until it has a pale, milky appearance.

9.10. Immediately after making the smear, hold the loop above the flame to dry the inoculum, then

flame the loop. (If you plunge the loop into the flame before it is dry, you will splatter the bacteria all over yourself and your lab bench).

11. Your partner should now repeat the above steps 7-9 using the agar-slant culture of S. aureus. Place the S. aureus bacterial cells in the drop of water nearest the “S” label.

12. Allows the smears to air dry. Hold the slide by the end using forceps to “heat fix” it by passing the slide through the Bunsen burner flame (right side up) two or three times. After heat fixing, the slide should not be too hot to handle. If it is, you have heated too much.

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13. Place your slide across the edges of a small petri dish. Place this dish inside a larger Petri dish.

14. Flood the slide drop by drop with Gram’s crystal violet solution using a Pasteur pipette. Add two drops of 5% sodium bicarbonate buffer to the slide. Stain for 1 minute.

15. Pour off excess stain into the dish provided and gently wash the slide with water using a dropper or squirt bottle. Do not allow the dropper to touch the smear.

16. Flood the smears with Gram’s iodine solution and allow it to react for 1 minute. Pour off the excess and let stand for 1 minute.

17. Wash off the iodine solution as in step 14 and gently blot the slide with kimwipes.

18. Hold the slide at an angle and apply Gram’s alcohol drop by drop until the violet color no longer appears in the washes from the smears. This should take only 10-15 seconds. Do not overdo this step.

19. Quickly rinse off the alcohol with water and blot.

20. Floor the slide with safranin and allow it to stain for 1 minute.

21. Wash the slide gently with water. Drain the excess water onto a paper towel and allow the slide to air dry. When the slide is dry, observe it using an oil-immersion objective. Ask your instructor for assistance if necessary. You will only use one of the oil immersion microscopes on the demonstration table.

22. Record your results in the space provided below:a. Which bacterial species showed a positive test with Gram’s stain?

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b. Which bacterial species showed a negative test with Gram’s stain?________________________________________________________________________

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23. Determine whether E. coli and S. aureus are Gram-negative or Gram-positive bacteria. a. E. coli:

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b. S. aureus: ________________________________________________________________________

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c. Do your results support your hypothesis? Explain.________________________________________________________________________

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d. Explain any discrepencies.________________________________________________________________________

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Name: ______________________________ Team_____________________

EDUCATION IS FREEDOM! Date__________/Mr. McIntire

AP Biology

Prokaryotic Cells Lab Analysis Questions

1. How can different species of bacteria be identified?

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2. What are the different types of nutritional categories of bacteria? Describe each.

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3. Describe the evidence that could be used to determine if a specimen under the microscope was prokaryotic or eukaryotic? Give an example from one of the three parts of this lab. __________________________________________________________________________________

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4. Describe the structure of cyanobacteria. What organelles are present?

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5. How did the magnifications of the bacterial species in Part 1 vary? What can be inferred about the size of the specimen based on the magnification?__________________________________________________________________________________

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6. Can you see the cell wall in each bacterial species in Part 1?__________________________________________________________________________________

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7. Sometimes the cell wall is coated with a gelatinous layer called the capsule. Is it visible in the bacterial species observed in Part 1? Which group(s) show(s) it?__________________________________________________________________________________

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8. Can you see a nucleus in each cell? Why? Is there any other well defined cellular inclusion inside each cell. __________________________________________________________________________________

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9. Inside each cell, is the cytoplasm clear or granular. Explain this observation; what do you think these granules might be, why are they located in the cytoplasm, and what is their function in the prokaryotic cell?__________________________________________________________________________________

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10. Do colonies of bacteria represent “multicellular organisms”? Why or why not? __________________________________________________________________________________

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