BIO 220 Microbiology Lab 10

docx

School

Chattahoochee Valley Community College *

*We aren’t endorsed by this school

Course

220

Subject

Biology

Date

May 24, 2024

Type

docx

Pages

Uploaded by CountEaglePerson1041

BIO 220 Microbiology Lab 80 Lab Ten: Assays for Some Microbial Enzymes I. Introduction 1. Metabolic reactions require enzymes. 1. Enzymes are proteins that act as catalysts to speed up a reaction. 2. Enzymes are not changed by the reaction and can be reused. 3. Enzymes are specific; they catalyze specific reactions because they have a specific shape that fits the shape of the substrate-the molecule that they are acting upon in the reaction. 4. Enzymes may be classified as two types. a. Intracellular (endoenzymes) – work inside the cell b. Extracellular (exoenzymes) – work outside the cell 5. A microbe’s physiological properties are determined largely by its enzymes. 6. The activities of the enzymes determine the very nature of the organism. 7. Where an organism can live and how it interacts with other microbes is a direct reflection on the enzymes that it possesses. 8. Since an organism’s genes determine which enzymes it produces, the enzymatic reactions of an organism reflect its taxonomic status. 2. In this lab exercise, we will assay (test for) the presence or absence of specific enzymes that are commonly used to identify a bacterial species: amylase, urease, tryptophanase, gelatinase, and catalase. BIO 220 Microbiology Lab 10 81 II.

Procedures A. Amylase - is an enzyme that breaks down starch, which is a long chain of glucose monomers that is too large to enter a bacterial cell; it is used by some cells to break down starch so the smaller units can enter the cell; a blue-black color when iodine is added is a positive test for the presence of starch and the absence of amylase. 1. Obtain one Petri dish containing starch agar. On the bottom of the dish, divide the plate into 3 equal sections & put your name & your table/station number. 2. Make a single streak of Bacillus subtilis in one section, a single streak of E. coli in another section, and leave the third section uninoculated to be used as the control. Each streak should be about 2-3 cm (1-11⁄2 inches) & near the center of each section. Be sure to label the sections. 3. Incubate the inverted plate for 48 hours at 37°C. B. Urease -is an enzyme that breaks down urea, a major organic waste product, into ammonia and carbon dioxide; a pinkish- fuschia color to the agar slant is a positive indicator for the presence of urease. 1. Obtain a urea agar slant. 2. Inoculate the surface of the urea agar slant with your assigned organism: Tables 1, 5, 9 use: Enterobacter aerogenes Tables 2, 6,10: Escherichia coli Tables 3, 7,11 use: Proteus vulgaris Tables 4, 8, 12 use: Serratia marescens . 3. Label the tube and incubate for 48 hours at 37°C.

BIO 220 Microbiology Lab 10 82 C. Tryptophanase -is an enzyme that breaks down tryptophan into indole, pyruvic acid, and ammonia. Tryptophan is an amino acid. The production of indole is useful in distinguishing E. coli from other enteric/intestinal bacteria. 1. Obtain a tryptone/indole broth tube. 2. Inoculate the tube with your assigned organism. Tables 1, 5, 9 use: Enterobacter aerogenes Tables 2, 6,10: Escherichia coli Tables 3, 7,11 use: Proteus vulgaris Tables 4, 8, 12 use: Serratia marescens . 3. Label the tube and incubate for 48 hours at 37°C. D. Gelatinase -is an enzyme that breaks down gelatin or collagen which is a protein into molecules that can be metabolized for energy and/or used as a carbon source. 1. Obtain a gelatin tube. 2. Inoculate the tube with your assigned organism. Tables 1, 5, 9 use: Enterobacter aerogenes Tables 2, 6,10: Escherichia coli Tables 3, 7,11 use: Proteus vulgaris Tables 4, 8, 12 use: Serratia marescens . 3. Label the tube and incubate for 48 hours at 37°C. E. Catalase – is an enzyme that breaks down hydrogen peroxide into water & oxygen; it is produced by many aerobic microbes to detoxify the hydrogen peroxide that is produced during aerobic respiration; it is detected by the presence of bubbles when catalase.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

hydrogen peroxide is added. Bubbles indicate the release of oxygen from the breakdown of hydrogen peroxide and is a positive test for the presence of 1. Specimens to test: Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus faecalis, Escherichia coli , and Clostridium . 2. Aseptically, pick up some bacterial cells from one of the cultures, using an inoculating loop. 3. Hold the bacterial cells on the loop over an empty Petri dish. 4. Add a drop of hydrogen peroxide to the bacterial cells. BIO 220 Microbiology Lab 10 83 III. 5. Observe to see if bubbles evolve. Bubbles indicate that the organism does produce catalase. Flame the loop. 6. Repeat the procedure for each specimen and record the results. Results A. Data Table A- Catalase - place a check in the box under the microbe’s name if bubbles formed with the addition of hydrogen peroxide. This is a positive test for the presence of catalase. Bacillus subtilis YES Pseudomon a s aeruginosa YES Streptococc us faecalis NO Escherichia coli YES Clostridiu m NO

B.Data Table B- Amylase 1. Flood the surface of the plate with a thin layer of iodine (Gram’s or Lugol’s iodine). 2. Gently swirl the plate to spread the iodine over the entire surface of the plate. 3. Look for a color change in the agar around each streak of growth. 4. If the starch has not been broken down (no amylase), the iodine reacts with the starch and produces a blue-black color. 5. If the starch has been broken down (amylase was produced by the bacterium), there is no starch present and the area around the colony is a clear yellowish-amber color. 6. Record the color in each microbe’s section and indicate which organism produced amylase (has the clear area). Bacillus cereus Contr ol Escherichia coli Color of Agar CLEAR BLACK/BROWN Amylase Presence YES NO BIO 220 Microbiology Lab 10 84 C. Data Table C- Urease 1. After incubation, observe the tubes. The appearance of a red-violet color indicates a positive test (the organism does produce urease). Yellow-orange tubes indicate a negative result.

2. Record the color of each tube and indicate which organism produced urease. Enterobacter aerogenes Escherichia coli Proteus vulgaris Serratia marescens Color PINK PINK PINK PINK Ureas e YES YES YES YES Presence D. Data Table D- Tryptophanase/Indole 1. After incubation, carefully layer 10 drops of Kovac’s reagent directly onto the top of the broth culture tube. If the culture

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

produces tryptophanase, the presence of indole will be detected by the immediate formation of a red layer at the top of the broth. A yellow or brown color is a negative test. 2. Record the color and indicate which organism produced tryptophanase/indole. Enterobacter aerogenes Escherichi a coli Proteus vulgaris Serratia marescens Color Trytophana se Presence BIO 220 Microbiology Lab 10 85 E. Data Table E- Gelatinase 1. After incubation, place the tubes in a refrigerator for 30-60 minutes. 2. After incubation of the tubes in the refrigerator, check them by carefully tilting the tubes. If the medium is liquid in consistency, this is a positive test for gelatin breakdown. If the medium is solid, this indicates the lack of gelatin hydrolysis. 3. Record which organisms produced gelatinase. Enterobacter aerogenes Escherichia coli Proteus vulgaris Serratia marescens Solid or Liquid LIQUID LIQUID LIQUID LIQUID Gelatinase Presence YES YES YES YES IV. Review Questions 1. What do all metabolic reactions require?

__________ ENZYMES _________________ 2. Proteins that act as catalysts are called ____ ENZYMES ________. 3. What term refers to the fact that each enzyme has one type of reaction to control? ________ CATALYST _______________ 4. Name the two types of enzymes: _____ INTRACELLULAR/EXTRACELLUAR ___________________________ 5. Which type of enzyme works inside the cell? ____ ENDOENZYMES ___________________ 6. Which type of enzyme works outside the cell? __________ EXOENZYMES____________ 7. What determines a microbe’s physiological properties? _ENZYMES______________ 8. Describe some things about a microbe that is determined by its enzymes: ACTIVITIES DETERMINE THE NATURE, WHERE IT LIVES AND HOW IT INTERACTS 9. Name some enzymes that microbes may have: AMYLASE, UREASE, TRYPTOPHANASE, GELATINASE, CATALASE 1. What does catalase break down? HYDORPEROXIODE IN WATER BIO 220 Microbiology Lab 10 86 2. Which bacterial culture(s) produced catalase? BACILLUS, PSEUDOMONAS, E.COLI 3. How could you tell that these cultures produced catalase? FORMED BUBBLES WITH HYDROGEN PEROXIDE 4. What is produced when catalase breaks down hydrogen peroxide? AEROBIC MICROBES 5. What does amylase break down? STARCH 6. Which bacterial culture(s) produced amylase? BACILLUS, E. COLI _______________________ 7. How could you tell that these cultures produced amylase? PRESENCE OF BLUE/ BLACK COLOR WHEN IODINE IS ADDED

8. What is urea? ORGANIC WASTE PRODUCT 9. What does urease hydrolyze urea into?_ AMMONIA AND CO2 10. What is a positive test for the presence of urease? PINK IN COLOR ___________________ 11. What is a negative test for the presence of urease? YELLOW IN COLOR 12. What is tryptophan? _ AMINO ACID 13. What does tryptophanase break tryptophan down into? INDOLE, PYRUVIC ACID AND AMMONIA _______________ 14. Which bacterial culture(s) produced tryptophanase? N/A 15. What is the usefulness of indole production? DISTINGUISHING E.COLI 16. Which reagent is added to the tryptone broth tube to test for indole production? KOVAC 17. What is the positive test for indole production (tryptophanase presence)? RED LAYER AT TOP OF BROTH 18. What is the negative test for indole production (tryptophanase presence)? YELLOW BROWN COLOR AT TOP OF BROTH 19. What is gelatin? PROTEIN 20. What does gelatinase break gelatin into? MOLECULES THAT CAN METABOLIZE FOR ENERGY AND USED FOR A CARBON SOURCE 21. What is a positive test for the presence of gelatinase? LIQUID CONSISTENCY OF MEDIUM 22. What is a negative test for gelatinase? _ SOLID MEDIUM 23. Which bacterial culture(s) produced gelatinase? ENTEROBACTER, E. COLI, PROTEUS, SERRATIA

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

BIO 220 Microbiology Lab 10

Related Documents