Name: Date: Chapter 19: Bacteria and Viruses Per: Row: 19-1 Bacteria (Moneran) A. Classifying Prokaryotes 1. Prokaryotes-. 2. Classifying Prokaryotes a. All prokaryotes were once placed in the Kingdom. b. Recently, biologists divided them into : the Eubacteria and the Archaebacteria. 3. - have a cell wall that protects the cell and determines its shape. a. The cell wall contains peptidoglycan. b. Have a cell membrane that surrounds the cytoplasm. c. Some have a second membrane that provides added protection. d. Live in a variety of environments, including: i. ii. iii. Follow the prompts to locate structures in a typical bacterium. Color & label: - the cell membrane yellow - the cell wall blue - the flagella red - the pili orange - the DNA green 1. What does the bacterium use to move? 2. What is the bacterium s genetic material called? 4. - the cells walls do not contain peptidoglycan. a. Have different membrane lipids. b. The DNA sequences of key archaebacterial genes are more like those of eukaryotes than those of eubacteria. c. Many archaebacteria. i. live in oxygen-free environments, such as thick mud and animal digestive tracts. ii. Other archaebacteria live in or in hot springs where water temperatures approach the boiling point. Big Idea/Notes/Questions:
Name: Date: B. Identifying Prokaryotes Prokaryotes are identified by characteristics such as: The The The 1. Shapes a. - rod-shaped prokaryotes b. - spherical prokaryotes c. - spiral and corkscrew-shaped prokaryotes 2. Cell Walls a. Two different types of cell walls are found in eubacteria. A method called tells them apart. Big Idea/Notes/Questions: Color: bacilli (blue), cocci (red), and spirilla (yellow) Per: Row: i. Gram-positive bacteria- with large amounts of peptidoglycan. ii. Gram-negative bacteria- inside an outer lipid layer. 3. - prokaryotes can be identified by whether they move and how they move. C. Metabolic Diversity 1. Prokaryotes are divided into two main groups: a. - get their energy by consuming organic molecules made by other organisms. i. Chemoheterotrophs - prokaryotes that take in organic molecules for both energy and a supply of carbon ii. Photoheterotrophs- prokaryotes that use sunlight for energy, but take in organic compounds as a carbon source b. make their own food from inorganic molecules. i. Photoautotrophs- use light energy to convert carbon dioxide and water to carbon compounds and oxygen. 1. Photosynthesis- use light to make energy ii. Chemoautotrophs- perform chemosynthesis. 1. Chemosynthesis- make organic carbon molecules from carbon dioxide, but do not require light as energy. 2. Releasing Energy - bacteria need a constant supply of energy, which is released by the processes of or both. a. Obligate aerobes- b. Obligate anaerobes- bacteria that live c. Facultative anaerobes- bacteria that can D. Bacteria: Growth and Reproduction 1. - asexual reproduction, where an organism replicates its DNA and divides in half, producing two identical daughter cells. 2. - sexual reproduction, where paramecia and some prokaryotes exchange genetic information a. Hollow bridge forms between two bacterial cells, and genes move from one cell to the other. 3. Spore formation
Name: Date: Per: Row: a. - forms when a bacterium produces a thick internal wall that encloses its DNA and some of its cytoplasm. i. can remain. ii. allow bacteria to survive harsh conditions extreme Draw a picture of each: (pg.475) Binary fission Conjugation Spore formation: endospore E. Importance of Bacteria Bacteria are vital to the living world. Some are producers that capture energy by photosynthesis. Others are decomposers that break down the nutrients in dead matter. Still other bacteria have human uses. 1. a. Bacteria recycle nutrients and maintain equilibrium in the environment. b. Bacteria also help in the treatment of sewage 2. a. Plants need nitrogen gas to be changed chemically to ammonia or other nitrogen compounds, which certain bacteria produce. b. - the process of converting nitrogen gas into a form plants can use c. Many plants have symbiotic relationships with nitrogen-fixing bacteria. 3. Human Uses of Bacteria We depend on bacteria for many things, including: i. ii. iii. iv. v. 19-2 Viruses A. What is a Virus? 1. Viruses are particles of nucleic acid, protein, and in some cases, lipids. a. can reproduce only by. b. differ widely in terms of size and structure. c. enter living cells and use the infected cell to produce more viruses. 2. A typical virus is composed of a. Big Idea/Notes/Questions:
Name: Per: Date: Row: 3. - the virus s protein coat. a. Capsid proteins bind to receptors on the cell surface and trick the cell into allowing it inside. b. Once inside, viral genes are expressed and the cell transcribes and translates them into viral capsid proteins. c. The host cell may makes copies of the virus, and be destroyed. 4. Most viruses are. a. - viruses that infect bacteria Label: surface proteins, head, capsid (2), tail sheath, tail fiber, membrane envelope Circle the genetic material in each virus Color the protein parts of each virus orange 1. What do all three kinds of viruses have in common? B. Viral Infection 1. Once the virus is inside the host cell, two different processes may occur. a. Some viruses replicate immediately, killing the host cell. b. Others replicate, but do not kill the host cell immediately. 2. - a virus enters a cell, makes copies of itself, and causes the cell to burst. a. The bacteriophage injects DNA into a bacterium. b.. c. Host cell starts making messenger RNA from the viral DNA d.. e. Copies of viral DNA and the viral protein coats are made and assembled into new viruses f.. Big Idea/Notes/Questions:
Name: Date: Per: Row: Lytic Cycle Label the bacterial DNA, host bacterium, viral DNA, and virus. Circle the step that shows lysis of the host cell 1. What is one result of a lytic infection? Lysis of the virus OR Lysis of the host cell 3. - a host cell makes copies of the virus indefinitely. a. a virus integrates its DNA into the DNA of the host cell, and the viral genetic information replicates along with the host cell's DNA. b.. c. The bacteriophage DNA forms a circle. d.. e.. f. Eventually, it may become active, remove itself from the host DNA, and begin the production of new viruses. Lysogenic Cycle Circle the viral DNA in each diagram. 1. What happens after the viral DNA is inserted into the bacterial DNA? 2. How does a lysogenic infection help a virus spread? Big Idea/Notes/Questions:
Name: Per: Date: Row: C. Retroviruses 1. Retroviruses-. a. When retroviruses infect cells, they. b. This DNA is inserted into the DNA of the host cell c. A retrovirus genetic information is - from RNA to DNA. 2. The virus that causes. D. Viruses and Living Cells 1. Viruses must. 2. They take advantage of the host s respiration, nutrition, and all other functions of living things 3. Viruses have. 4. After infecting living cells, viruses can reproduce, regulate gene expression, and even evolve. Viruses and Cells Characteristics Virus Cell *Draw a picture *Draw a picture Structure Reproduction DNA or RNA core, capsid cell membrane, cytoplasm; eukaryotes also contain nucleus and organelles Genetic Code Growth & Development Obtain and Use Energy Response to Environment Change Over Time Big Idea/Notes/Questions:
Name: Per: Date: Row: 19-3 Diseases Caused by Bacteria and Viruses A. Bacterial Disease in Humans 1. Bacteria and viruses are. 2. Pathogen-. Bacteria produce disease in one of two general ways. a. Some bacteria damage the cells and tissues of the infected organism directly by breaking down the cells for food. b. Other bacteria release toxins (poisons) that travel throughout the body interfering with the normal activity of the host. B. Preventing Bacterial Disease 1. Many bacterial/viral diseases can be prevented by. 2. A vaccine is a preparation of. a. When injected into the body, a virus may prompt the body s immunity to the disease. 3. Immunity -. 4. If infection occurs,. 5. These drugs include, which are compounds that block the growth and reproduction of bacteria 6. A reason for increased human life expectancy is an. *Use the picture to the right to answer the questions *Be sure to label each disk with the letter of antibiotic used on it. 1. Which antibiotic was most effective against the bacteria in the petri dish? 2. Why did you choose that antibiotic? C. Controlling Bacteria 1. There are various methods used to control bacterial growth, including: 2. - destroys bacteria by subjecting them to great heat. a.prolonged high temperatures kill most bacteria. 3. - chemical solutions that kill pathogenic bacteria. 4. Food Storage and Processing Big Idea/Notes/Questions:
Name: Date: Per: Row: a.. b. Refrigerated food stays fresh longer because the bacteria will take longer to multiply. c. can sterilize certain foods. D. Viral Disease in Humans Viruses produce disease by disrupting the body's normal equilibrium. 1. Viruses can attack and destroy certain cells in the body, causing. 2. Other viruses cause infected cells to change patterns of growth and development. 3. Viral diseases. 4. Vaccines are often the best protection against most diseases. a. Most vaccines work only. b.. E. Viral Disease in Plants and Animals 1. Viruses produce serious. 2. Many viruses infect plants and pose a serious threat to many crops. F. Viroids and Prions 1. cause disease in plants. a. that have no surrounding capsids. b. Enter an infected cell and synthesize new viroids. c. Disrupt the cell s metabolism and of the entire plant 2. Prions cause disease in animals. a. Contain only protein -. b. Cause disease by forming protein clumps. These clumps induce normal protein molecules to become prions. i. Eventually, there are so many prions in the. ii.. Big Idea/Notes/Questions:
Name Class Date Chapter 19 Bacteria and Viruses Graphic Organizer Concept Map Using information from the chapter, complete the concept map below. If there is not enough room in the concept map to write your answers, write them on a separate sheet of paper. Prokaryotes are classified according to Chemical nature of cell walls Shapes 1. 2. include include 3. 4. 5. Bacilli Autotrophs Pearson Education, Inc. All rights reserved. 6. include Chemoautotrophs Photoheterotrophs 7. Teaching Resources /Chapter 19 241
Name Class Date Chapter 19 Bacteria and Viruses Chapter Vocabulary Review Crossword Puzzle Use the clues below and on the following page to complete the puzzle. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Across 4. the outer protein coat of a virus 5. particle of nucleic acid and protein that can reproduce only by infecting living cells 6. organism that obtains energy directly from inorganic molecules 7. an infectious particle made of protein rather than DNA or RNA 8. sphere-shaped bacterium 13. type of infection in which the host cell bursts and is destroyed 14. single-celled microorganism that lacks a nucleus 15. disease-causing agent Pearson Education, Inc. All rights reserved. 238 Teaching Resources /Chapter 19
Name Class Date Down 1. rod-shaped bacterium 2. type of spore that can remain dormant until favorable conditions for growth arise 3. type of infection in which a host cell makes copies of the virus indefinitely 6. how bacteria exchange genetic material 9. compound that blocks the growth and reproduction of bacteria 10. Bacteria may reproduce by fission. 11. The process of converting nitrogen into a form that plants can use is fixation. 12. a structure prokaryotes use to propel themselves Pearson Education, Inc. All rights reserved. Completion On the lines provided, complete the following sentences. 16. The larger of the two kingdoms of prokaryotes is the. 17. The may be the ancestors of eukaryotes. 18. A corkscrew-shaped bacterium is called a(an). 19. A whiplike structure used for movement is a(an). 20. A prokaryote that carries out photosynthesis is called a(an). 21. A(An) is photosynthetic, but also requires organic compounds for nutrition. 22. Organisms that require a constant supply of oxygen in order to live are called obligate. 23. Bacteria that are killed by oxygen are called obligate. 24. Bacteria that can survive with or without oxygen are known as anaerobes. 25. Bacteria that attack and digest dead tissue are called. 26. A typical is composed of a core of DNA or RNA surrounded by a protein coat. 27. A virus that infects bacteria is called a(an). 28. A virus that stores its genetic information as RNA is called a(an). 29. A(An) can be used to cure many bacterial diseases. 30. Techniques of rely on extreme temperatures or chemical action to destroy bacteria. Teaching Resources /Chapter 19 239
Name Class Date Chapter 19 Bacteria and Viruses Enrichment Acid-Fast Bacteria Almost all bacteria can be classified as Gram-positive or Gram-negative by a process called Gram staining. Very few bacteria are Gram-positive. Most bacteria, yeasts, and fungi are Gram-negative. How bacteria respond to the Gram-staining procedure can provide information about the nutritive requirements, cell wall composition, and other traits of the bacteria. When treated with special dyes, Gram-positive bacteria appear deep violet in color. Gram-negative bacteria appear pink. The Gramstaining technique can help doctors identify bacteria and choose the correct antibiotics to treat bacterial infections. Some bacteria, however, are resistant to Gram staining. These bacteria are known as acid-fast bacteria, and are identified by using the acid-fast stain. In this procedure, a sample of unknown bacteria is dyed and then washed with acidified alcohol. This will remove most of the dye. Those bacteria that hold fast to the dye will be strongly stained and readily identifiable as acid-fast bacteria. Because acid-fast bacteria are a major cause of disease, it is particularly important to be able to identify them. The acid-fast bacteria form a homogeneous group composed of the genera Mycobacterium and Nocardia. Mycobacteria are usually rod shaped, and are found in soil, water, and animals. Many species are saprophytic (feed on dead organic matter); others cause diseases such as diphtheria, tuberculosis, and leprosy. Acid-fast bacteria are characterized by their high lipid content. Lipids and waxes make up as much as 40 percent of the dry weight of acid-fast bacteria. These lipids and waxes are the key to testing for acid-fast bacteria. The lipids and waxes absorb dye so it can t be removed with acidified alcohol. This is how acid-fast bacteria hold fast to dye and remain stained while bacteria are washed clean. Evaluation On the lines provided, answer the following questions. 1. How are acid-fast bacteria different from other bacteria? 2. Why is it important to wash the bacteria sample after it has been dyed? Pearson Education, Inc. All rights reserved. 240 Teaching Resources /Chapter 19
Name Class Date Chapter 19 Bacteria and Viruses Controlling Bacterial Growth Introduction Chemical substances that either kill bacteria or inhibit bacterial growth are called antimicrobial agents. Antimicrobial agents are of three basic types: antiseptics, or chemicals used to inhibit the growth of or kill bacteria on living tissues; disinfectants, or chemicals used to inhibit the growth of or kill bacteria on nonliving things; and antibiotics, or compounds that block the growth and reproduction of bacteria. The effectiveness of each type of antimicrobial agent is influenced by many factors. Some of these factors include the environmental conditions in which the agent is applied, the chemical properties of the agent, how long the agent has been stored, and the rate of deterioration of the agent. In this investigation, you will test the effectiveness of disinfectants and antibiotics in inhibiting the growth of bacteria. Problem How can the growth of bacteria be controlled? Pre-Lab Discussion Read the entire investigation. Then, work with a partner to answer the following questions. 1. Why is it important not to open sterile agar plates? To prevent the plates from becoming contaminated by airborne microorganisms. You may want to refer students to Section 19 2 in the textbook for a review of the methods of controlling bacterial growth before performing this investigation. Time required: 50 minutes, plus a 15-minute observation period after 48 hours of incubation 2. Why do you think it is so important to write only near the edges of the petri dish? So that the writing will not obstruct the view of the bacteria growing in the petri dish. 3. Why is it important to use sterile techniques while inoculating the agar plates? Sterile techniques should always be used when working with bacteria to prevent contamination Prentice-Hall, Inc. of the environment and of the bacteria being studied. 4. What is the purpose of the disk soaked in distilled water in each inoculated petri dish? The disk serves as a control. 5. What is the purpose of taping closed the lids of the petri dishes? To keep the lids from coming open and allowing contamination of the inoculated plates. Biology Laboratory Manual A/Chapter 19 153
Materials (per group) Annotations for the preparation of cultures are found after Going Further. glass-marking pencil culture of Escherichia coli B Bunsen burner Read all the 2 sterile nutrient agar plates information on matches chemical safety from 3 disinfectants chosen from the 2 sterile cotton swabs any Materials Safety following: chlorine bleach, Data Sheet that beaker of water household cleaner, household accompanies a test-tube rack chemical. disinfectant, phenol sterile filter-paper disks 3 antibiotic disks chosen from forceps the following: aureomycin, distilled water chloromycetin, penicillin, metric ruler streptomycin, tetracycline, transparent tape terramycin Annotations for preparation of agar plates are found after Going Further. Antibiotic disks are available from biological supply houses. Safety If your immune system has been weakened, you should not be present when this experiment is being performed. Put on safety goggles, a laboratory apron, and plastic gloves. Be careful to avoid breakage when working with glassware. Tie back loose hair when working with flames. Do not reach over an open flame. Always follow your teacher s directions and use special caution when working with bacterial cultures. Wash your hands thoroughly after carrying out this investigation. Return or dispose of all materials according to the instructions of your teacher. Review the meaning of each safety symbol by referring to Safety Symbols on page 8. Procedure Part A. Inoculating a Sterile Nutrient Agar Plate Never culture pathogenic bacteria. However, treat all bacterial cultures as if they are pathogenic. Firmly seal with transparent 1. Put on your laboratory apron. Obtain two sterile nutrient agar plates. Carefully turn over each plate and lay it on your worktable. CAUTION: Be very careful not to open the petri dishes of sterile agar while handling them. 2. With a glass-marking pencil, mark the bottom of each petri dish shown in Figure 1. Draw two lines at right angles to each other so that the dish is divided into four equal areas, or quadrants. Number the quadrants on each dish 1 through 4. Note: Place the numbers near the edges of the dishes. Write your initials Figure 1 near the top center of each dish. Carefully turn the petri dishes right side up. 3. Put on your plastic gloves. Obtain a test tube containing a bacterial culture of Escherichia coli B. Place the test tube in a test-tube rack. CAUTION: Use extreme care when working with bacterial cultures. Avoid spills. If a spill does occur, immediately call your teacher for assistance. Obtain two sterile cotton swabs. Carefully read steps 4 through 8 and study Figure 2 before you proceed. Remove your plastic gloves. tape any bacterial plates that are used for student inspection. For sterilization, use a hightemperature gas flame rather than an alcohol burner or candle flame. Cultures should be killed before disposal. Autoclave or pressure-cook all cultures and contaminated glassware at 15 pounds pressure per square inch (103.4 Pa) for 20 minutes. Disposable plates should be incinerated. Prentice-Hall, Inc. 154 Biology Laboratory Manual A/Chapter 19
Name Class Date A E B F C D G Prentice-Hall, Inc. Figure 2 If a bacterial culture is accidentally spilled, flood the area with a disinfectant solution. Leave the disinfectant on the affected area and any broken glassware for at least 30 minutes before carefully wiping the area with moist paper towels. A clearly marked bottle of disinfectant should be placed on each worktable in case such accidents occur. Make sure students are supervised when using the burner. 4. Put on your safety goggles and light the Bunsen burner. CAUTION: Use extreme care when working with or near an open flame. Tie back loose hair and clothing. 5. Pick up the test tube of E. coli. Remove the cotton plug. Note: Do not let the cotton plug come in contact with any other object. Pass the mouth of the tube back and forth through the burner flame. 6. Insert a sterile cotton swab into the bacterial culture. Note: Shake any excess liquid into the test tube. Remove the cotton swab. Note: Do not let the cotton swab come in contact with any other object. Pass the mouth of the tube back and forth through the burner flame. Replace the cotton plug and return the test tube to the rack. 7. Slightly open a sterile nutrient agar plate. Place the tip of the cotton swab near the top center of the agar. Streak the agar as shown in Figure 3A. Lift the swab off the plate and turn the petri dish 90 to the right. Streak the agar again, as shown in Figure 3B. Close the petri dish. A B Plate turned 90 to right Figure 3 Biology Laboratory Manual A/Chapter 19 155
8. Hold the top of the cotton swab in the flame of the Bunsen burner until it catches fire. Remove the swab from the flame and plunge it into a beaker of water. 9. Repeat steps 5 through 8 for the other sterile nutrient agar plate. Note: Be sure to use a new sterile cotton swab for this transfer. Turn off the Bunsen burner after you have completed the second plate. 10. The nutrient agar plates that you have just streaked, or inoculated, with bacteria will be used in Part B of this investigation. 11. Return the test tube of E. coli to your teacher. Thoroughly wash your hands with soap and water. Part B. Controlling the Spread of Bacteria with Disinfectants and Antibiotics 1. Take one inoculated agar plate of E. coli that you prepared in Part A. 2. Select three disinfectants and three antibiotics, and record these selections in the Data Table. Carefully read steps 3 through 10 and study Figure 4 before you proceed. A Pick up disk with forceps C Place disk in quadrant 1 Forceps Disk B Soak disk in disinfectant D Flame forceps Disinfectant Disk Figure 4 3. Light the Bunsen burner. CAUTION: Use extreme care when working with or near an open flame. Tie back loose hair and clothing. Pass the forceps back and forth through the flame of the Bunsen burner several times. This procedure sterilizes the forceps. Allow the forceps to cool before using. CAUTION: If there is alcohol on the forceps, it will burn brightly and quickly. Stand back from the Bunsen burner when burning alcohol off the forceps. 4. With sterile forceps, pick up a disk of filter paper. Insert the disk into disinfectant 1. Shake off any excess liquid. 5. Slightly open an inoculated agar plate. Position the filter-paper disk in the center of quadrant 1. With the tip of the forceps, gently press the disk against the agar until it sticks. Remove the forceps and close the petri dish. Prentice-Hall, Inc. 156 Biology Laboratory Manual A/Chapter 19
Name Class Date 6. Repeat steps 4 and 5 with the remaining disinfectant-soaked disks in quadrants 2 and 3 of the inoculated plate of E. coli. Note: Remember to sterilize the forceps after each use. 7. In quadrant 4, place a filter-paper disk soaked in distilled water. 8. Repeat steps 4 through 7 with the other inoculated agar plate using antibiotic disks instead of disinfectants. You should have two inoculated agar plates as shown in Figure 5. Disinfectant 1 Disinfectant 2 Antibiotic 1 Antibiotic 2 Disinfectant 3 Distilled water Antibiotic 3 Distilled water E. Coli E. Coli Figure 5 9. With transparent tape, tape the petri dishes closed, as shown in Figure 6. Turn the dishes upside down. Incubate the dishes for 48 hours at 37 C. Tape Prentice-Hall, Inc. Figure 6 Biology Laboratory Manual A/Chapter 19 157
10. Observe the petri dishes after 48 hours. White or cloudy areas of the agar indicate bacterial growth. Notice any clear areas, called zones of inhibition, surrounding the filter-paper disks. A clear area indicates that the disinfectant or antibiotic inhibited bacterial growth. A lightly cloudy area surrounding a disk indicates that bacterial growth was slowed down. Note: You may want to hold the petri dishes to the light to see the zones of inhibition more clearly. 11. With a metric ruler, measure to the nearest millimeter the size of the clear zone surrounding each disk. Record your measurements in the Data Table. If no clear zone is present, record the measurement as 0. 12. Return the petri dishes to your teacher for proper disposal. Thoroughly wash your hands with soap and water. Data Table Effects of Disinfectants and Antibiotics on Growth of E. Coli Disinfectant Zone of inhibition (mm) 1 2 Answers will depend on disinfectants used. 3 4 distilled water 1 Antibiotic Zone of inhibition (mm) 2 Answers will depend on antibiotics used. 3 4 distilled water Analysis and Conclusions 1. Observing Which disinfectant was most effective in preventing the growth of E. coli? Answers will vary depending on the disinfectants used. 2. Observing Which antibiotic was most effective in preventing the growth of E. coli? Answers will vary depending on the antibiotics used. 3. Controlling Variables How do you know that any inhibition you have observed is due to the disinfectants and antibiotics on the disk? There should be no inhibition around the control disks. 4. Analyzing Data Pretend that a serious staphylococcus infection has developed in the locker room of your school s gym. Assume that you are responsible for getting rid of the staph contamination. How would you do so? Use a sterile inoculating loop to collect a sample of suspected sources of the bacteria. Culture the bacteria Prentice-Hall, Inc. with disinfectant disks. Scrub the area with the most effective disinfectant. 158 Biology Laboratory Manual A/Chapter 19
Name Class Date 5. Formulating Hypotheses Scientists have observed that an antibiotic seems to lose its effectiveness against a particular population of bacteria after a prolonged period of time. What do you think is responsible for this phenomenon? The antibiotic kills the susceptible bacteria in the population. The surviving bacteria are immune to the antibiotic and will produce offspring that will inherit this immunity. 6. Formulating Hypotheses Why are the different disinfectants not equally effective against all species of bacteria? Species of bacteria differ from one another and therefore react differently to various disinfectants. 7. Inferring Suppose that your doctor diagnoses your condition as a bacterial infection and prescribes an antibiotic. Your doctor cautions you to take the antibiotic for 10 days even though you may feel fine after a few days. Explain why you should follow your doctor s orders. After a few days, the antibiotic will probably have destroyed most of the bacteria. Thus, you will feel better. However, some bacteria will remain and reproduce. The infection can then begin again. Taking the antibiotic for 10 days helps ensure that all of the bacteria will be destroyed. Going Further Using the procedures presented in this investigation, test other species of bacteria such as B. subtilis, P. vulgaris, and S. lutea for their resistance or sensitivity to various disinfectants and antibiotics. Prentice-Hall, Inc. Pure cultures of Escherichia coli can be obtained from a biological supply house. Bacteria are easily cultured in tubes of nutrient broth. To prepare the nutrient broth, bring 350 ml distilled water to a boil in a 500-mL beaker. Slowly add 3 g dehydrated nutrient broth, stirring constantly. Pour the broth into small test tubes and insert a cotton plug into each tube. Sterilize the tubes of broth for 15 minutes at 15 pounds pressure in an autoclave or pressure cooker. The sterilized tubes can be stored in a refrigerator until they are needed. Transfer bacteria to the tubes of sterile nutrient broth using an inoculating loop and following sterile technique. Store inoculated tubes at room temperature for 2 or 3 days to allow the bacteria to grow. Tubes of bacteria can also be incubated at 37 C for 24 hours and then used immediately. Subcultures and dilutions of the cultures can be made at 2-week intervals. Cultures remain viable stored in a refrigerator for several weeks. To prepare nutrient agar plates, mix 3 g beef extract, 5 g peptone, and 15 g agar in 1 L distilled water. Bring mixture to a boil, stirring constantly to avoid burning. Heat until liquid is clear. Sterilize in containers covered with cotton plugs in autoclave or pressure cooker for 15 minutes at 15 pounds pressure. Recipe makes about 50 petri dishes. Refrigerate. Nutrient agar can also be purchased from biological supply houses. Biology Laboratory Manual A/Chapter 19 159