Laboratory Exercises in Microbiology - part 4 pot

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 20. Carbohydrates I: Fermentation andßGalactosidase Activity © The McGraw−Hill Companies, 2002 Review Questions 1. Define fermentation. 2. Do all microorganisms produce the same end product from pyruvate? Explain your answer. 3. What is the purpose of the phenol red or bromcresol purple in the fermentation tube? 4. What is the function of the Durham tube in the fermentation tube? 5. What are some of the metabolic end products produced by the different microorganisms used in this experiment? 6. What is the color of phenol red at an acid pH? 7. What is the function of β-galactosidase? 132 Biochemical Activities of Bacteria Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 21. Carbohydrates II: Triple Sugar Iron Agar Test © The McGraw−Hill Companies, 2002 133 EXERCISE Carbohydrates II: Triple Sugar Iron Agar Test 21 Materials per Student 24- to 48-hour tryptic soy broth cultures of Alcaligenes faecalis (ATCC 8750), Escherichia coli (ATCC 11229), Proteus vulgaris (ATCC 13315), Pseudomonas aeruginosa (ATCC 10145), and Shigella flexneri (ATCC 12661) 5 triple sugar iron agar slants Bunsen burner inoculating needle incubator set at 35°C test-tube rack Learning Objectives Each student should be able to 1. Understand the biochemical reactions involved in the triple sugar iron agar test 2. Differentiate among members of the family Enterobacteriaceae 3. Distinguish between the Enterobacteriaceae and other intestinal bacteria 4. Perform a TSI test Suggested Reading in Textbook 1. Carbohydrate catabolism, section 9.7. 2. The Enterobacteriaceae, section 22.3. Pronunciation Guide Alcaligenes faecalis (al-kah-LIJ-e-neez fee-KAL-iss) Escherichia coli (esh-er-I-ke-a KOH-lee) Proteus vulgaris (PRO-tee-us vul-GA-ris) Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jin- OH-sah) Shigella flexneri (shi-GEL-la flex-ner-i) Why Are the Above Bacteria Used in This Exercise? This exercise will provide the student experience in using the triple sugar iron agar test to differentiate among the members of the family Enterobacteriaceae and between Enterobacte- riaceae and other intestinal bacteria. The authors have cho- sen three common bacteria in the family Enterobacteriaceae: Escherichia coli, Proteus vulgaris, and Shigella flexneri. All three are facultatively anaerobic gram-negative rods. In a TSI tube, E. coli produces an acid butt, an acid or alkaline slant, and no H 2 S, but does produce gas. P. vulgaris pro- duces an acid butt, an acid or alkaline slant, H 2 S, and gas. S. flexneri produces an acid butt, an alkaline slant, no H 2 S, and no gas. For the other intestinal bacteria, the authors have chosen Alcaligenes faecalis and Pseudomonas aeruginosa. Both of these intestinal bacteria are gram-negative aerobic rods. In a TSI tube, A. faecalis produces an alkaline butt, al- kaline slant, H 2 S, and gas; P. aeruginosa, an acid butt, alka- line slant, H 2 S, and gas. Principles As originally described in 1911 by F. F. Russell, the triple sugar iron (TSI) agar test is generally used for the identification of enteric bacteria (Enterobacteri- aceae). It is also used to distinguish the Enterobacteri- aceae from other gram-negative intestinal bacilli by their ability to catabolize glucose, lactose, or sucrose, and to liberate sulfides from ferrous ammonium sulfate or sodium thiosulfate. (See exercise 24 for the biochem- istry of H 2 S production.) TSI agar slants contain a 1% concentration of lactose and sucrose, and a 0.1% glu- cose concentration. The pH indicator, phenol red, is also SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame. Be careful when working with these bacteria, especially Shigella dysenteriae, as they are known pathogens. Keep all cul- ture tubes upright in a test-tube rack or in empty cans. Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 21. Carbohydrates II: Triple Sugar Iron Agar Test © The McGraw−Hill Companies, 2002 incorporated into the medium to detect acid production from carbohydrate fermentation (see exercise 20). Often Kligler Iron Agar (named after I. J. Kligler in 1917), a differential medium similar to TSI, is used to obtain approximately the same information. TSI slants are inoculated by streaking the slant surface using a zig-zag streak pattern and then stab- bing the agar deep with a straight inoculating needle (see figure 14.5). Incubation is for 18 to 24 hours in order to detect the presence of sugar fermentation, gas production, and H 2 S production. The following reac- tions may occur in the TSI tube (figures 21.1–21.3): 1. Yellow butt (A) and red slant (A) due to the fermentation of glucose (phenol red indicator turns yellow due to the persisting acid formation in the butt). The slant remains red (alkaline) (K) because of the limited glucose in the medium and, therefore, limited acid formation, which does not persist. 2. A yellow butt (A) and slant (A) due to the fermentation of lactose and/or sucrose (yellow slant and butt due to the high concentration of these sugars) leading to excessive acid formation in the entire medium. 3. Gas formation noted by splitting of the agar. 4. Gas formation (H 2 S) seen by blackening of the agar. 5. Red butt (K) and slant (K) indicates that none of the sugars were fermented and neither gas nor H 2 S were produced. Table 21.1 gives reactions usually expected from some of the more frequently encountered genera of the Enterobacteriaceae. Figure 21.4 summarizes the 134 Biochemical Activities of Bacteria A = acid, K = alkaline, V = varies between species Slant Butt Gas A K AA +– + – H 2 S Tube a Tube b Figure 21.1 Triple Sugar Iron Reactions (TSI-1) and Their Interpretation. (a) The tube on the left has a yellow butt (acid), red slant (alkaline), H 2 S production as indicated by blackening of the agar, and no gas production. (b) The tube on the right shows no H 2 S formation, a yellow slant (acid), gas production, and an acid butt. Note that the gas on the bottom has lifted the agar. Figure 21.2 Triple Sugar Iron Reactions (TSI-2) and Their Interpretation. (a) The tube on the left has a red butt (alkaline), red slant (alkaline), and no acid or H 2 S production. (b) The tube on the right has a yellow slant (acid), yellow butt (acid), and no gas or H 2 S production. Slant Butt Gas A K AK –– –– H 2 S Tube a Tube b (b)(a) (a) (b) Table 21.1 Results of TSI Reaction TSI Reaction Bacterium Butt Slant H 2 S Gas Enterobacter AA –+ Escherichia A A or K – + Klebsiella AA –+ Citrobacter A K or A V + Proteus vulgaris A A or K + + Edwardsiella AK V+ Morganella AK –+ Serratia A K or A – V Shigella AK –– Salmonella typhi AK +– Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 21. Carbohydrates II: Triple Sugar Iron Agar Test © The McGraw−Hill Companies, 2002 possible reactions and results in TSI for the various bacteria used in this experiment. Procedure First Period 1. Label each of the TSI agar slants with the name of the bacterium to be inoculated. Use one of the tubes as a control. Place your name and date on each tube. 2. Using aseptic technique (see figure 14.3), streak the slant with the appropriate bacterium and then stab the butt. Screw the caps on the tubes but do not tighten! 3. Incubate for only 18 to 24 hours at 35°C for changes in the butt and on the slant. Tubes should be incubated and checked daily for up to seven days in order to observe blackening. Second Period 1. Examine all slant cultures for the color of the slant and butt, and for the presence or absence of blackening within the medium. 2. Record your results in the report for exercise 21. Carbohydrates II: Triple Sugar Iron Agar Test 135 HINTS AND PRECAUTIONS (1) If screw-cap tubes are used, leave the caps loose about b turn after inoculating the tubes to prevent excessive dis- ruption of the agar should large amounts of gas be pro- duced during incubation. (2) Record the butt as acid pro- duction if the black color of FeS masks the color in the butt. (a) (b) (c) Figure 21.3 Triple Sugar Iron Reactions (TSI-3) and Their Interpretation. (a) The tube on the left is an uninoculated control. Notice the red color. (b) The second tube from the left has a yellow slant (acid), yellow butt (acid), gas production at the bottom of the tube, and no H 2 S production. This would indicate a weak lactose fermenter. (c) The third tube from the left has a red slant (alkaline), red butt (alkaline), and the black indicates H 2 S production, but no gas. (d) The tube on the right has a red slant (alkaline), yellow butt (acid), H 2 S production, but no gas production. This would indicate a nonlactose fermenter. Slant Butt Gas – – – – H 2 S Tube a A A + – Tube b K K – + Tube c K A – + Tube d (d) No carbohydrate fermentation or hydrogen sulfide production Glucose fermentation only Example: Alcaligenes faecalis Example: Shigella flexneri glucose, lactose, sucrose → glucose, lactose, sucrose (red slant/red butt) (K; red slant/red butt) glucose → decrease in pH due to acid (red butt) (A; yellow butt) cysteine → cysteine (no black color) Figure 21.4 The Possible Reactions and Results in TSI Agar for the Various Bacteria Used in This Experiment. (continued) Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 21. Carbohydrates II: Triple Sugar Iron Agar Test © The McGraw−Hill Companies, 2002 136 Glucose fermentation only with hydrogen sulfide production Lactose and/or sucrose and glucose fermentation Example: Pseudomonas aeruginosa Example: Escherichia coli glucose → decrease in pH due to acid (red butt) (A; yellow butt) lactose, sucrose → lactose, sucrose (red slant) (K; red slant) lactose and/or sucrose → decrease in pH due to acid (red butt) (A; yellow slant) glucose → decrease in pH due to acid (red butt) (A; yellow butt) cysteine → H 2 S production H 2 Sϩ FeSO 4 → FeS (black color in media) cysteine → cysteine (no black color in media) Lactose and/or sucrose and glucose fermentation with hydrogen sulfide (H 2 S) production Example: Proteus vulgaris lactose and/or sucrose → decrease in pH due to acid (red slant) (A; yellow butt) glucose → decrease in pH due to acid (red butt) (A; yellow butt) cysteine → H 2 S production H 2 Sϩ FeSO 4 → FeS production (black color in media) Figure 21.2 (continued) lactose, sucrose → lactose, sucrose (red slant) (K; red slant) cysteine → cysteine (no black color) Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 21. Carbohydrates II: Triple Sugar Iron Agar Test © The McGraw−Hill Companies, 2002 137 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 21 Carbohydrates II: Triple Sugar Iron Agar Test 1. Complete the following table on the TSI test. Carbohydrate Fermentation H 2 S Production Bacterium Butt Color Slant Color Black H 2 S A. faecalis ______________ __________________ ____________ ____________ E. coli ______________ __________________ ____________ ____________ P. vulgaris ______________ __________________ ____________ ____________ P. aeruginosa ______________ __________________ ____________ ____________ S. flexneri ______________ __________________ ____________ ____________ Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 21. Carbohydrates II: Triple Sugar Iron Agar Test © The McGraw−Hill Companies, 2002 Review Questions 1. For what bacteria would you use the TSI test? 2. Why must TSI test observations be made between 18 to 24 hours after inoculation? 3. Distinguish between an acid and alkaline slant. 4. What is the purpose of thiosulfate in the TSI agar? 5. What is meant by a saccharolytic bacterium? What reaction would it give in a TSI tube? 6. Why is there more lactose and sucrose in TSI agar than glucose? 7. What is the pH indicator in TSI agar? 138 Biochemical Activities of Bacteria Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 22. Carbohydrates III: Starch Hydrolysis © The McGraw−Hill Companies, 2002 Materials per Student 24- to 48-hour tryptic soy agar slant cultures of Bacillus subtilis (ATCC 6051), Escherichia coli (ATCC 11229), and Proteus vulgaris (ATCC 13315) 1 starch agar plate Gram’s iodine (1 g I 2 , 2 g KI, 300 ml distilled water) wax pencil inoculating loop Bunsen burner Pasteur pipette with bulbs incubator set at 35°C Learning Objectives Each student should be able to 1. Understand the biochemistry of starch hydrolysis 2. Perform a starch hydrolysis test Pronunciation Guide Bacillus subtilis (bah-SIL-lus SUB-til-is) Escherichia coli (esh-er-I-ke-a KOH-lee) Proteus vulgaris (PRO-te-us vul-GA-ris) Why Are the Following Bacteria Used in This Exercise? The major objective of this exercise is for the student to gain expertise in performing a starch hydrolysis test. If a bac- terium produces Ȋ-amylase, it can hydrolyze starch; if Ȋ- amylase is not produced, the bacterium will not hydrolyze starch. The three bacteria the authors have chosen vary in their ability to produce Ȋ-amylase. Bacillus subtilis is amy- lase positive; Escherichia coli is amylase negative; and Pro- teus vulgaris is variable; it may be positive or negative. Principles Many bacteria produce enzymes called hydrolases. Hydrolases catalyze the splitting of organic molecules into smaller molecules in the presence of water. This exercise will present the hydrolysis of the carbohy- drate starch. The starch molecule consists of two constituents: amylose, an unbranched glucose polymer (200 to 300 units) and amylopectin, a large branched polymer. Both amylopectin and amylose are rapidly hydrolyzed by certain bacteria, using their ␣-amylases, to yield dextrins, maltose, and glucose, as follows: Starch Ȋ-amylase [Amylose + Amylopectin] (Large polysaccharide) H 2 O Dextrins + Maltose + Glucose (Intermediate (Disaccharide) (Monosaccharide) polysaccharides) Gram’s iodine can be used to indicate the pres- ence of starch. When it contacts starch, it forms a blue to brown complex. Hydrolyzed starch does not pro- duce a color change. If a clear area appears after adding Gram’s iodine to a medium containing starch Carbohydrates III: Starch Hydrolysis 139 EXERCISE Carbohydrates III: Starch Hydrolysis 22 SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame. No mouth pipet- ting. Use caution to avoid dripping bacteria-laden iodine solution out of the plates while making observations. Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 22. Carbohydrates III: Starch Hydrolysis © The McGraw−Hill Companies, 2002 and bacterial growth, Ȋ-amylase has been produced by the bacteria (figure 22.1). If there is no clearing, starch has not been hydrolyzed. Procedure First Period: Starch Hydrolysis Test 1. With a wax pencil, divide a starch agar plate into three straight sections as indicated. Label each with the bacterium to be inoculated. Add your name and date to the plate. 2. Using aseptic technique (see figure 14.3), streak the respective bacteria onto the plate in a straight line within the section. 3. Incubate the plate for 24 to 48 hours at 35°C. B. subtilis E. coli P. vulgaris B. subtilis E. coli P. vulgaris Second Period 1. Place several drops of Gram’s iodine on each of the line streaks on the starch agar plate. If the area around the line of growth is clear, starch has been hydrolyzed, and the test is positive; if it is not clear or the entire medium turns blue, starch has not been hydrolyzed, and the test is negative. 2. If the results are difficult to read, an alternative procedure is to invert the plate (after removing the lid) over a beaker containing iodine crystals. The rising vapor will react with the starch without the interference of the red-brown color of the unreacted iodine. 3. Record your results in the report for exercise 22. 140 Biochemical Activities of Bacteria HINTS AND PRECAUTIONS (1) Carefully adding iodine to only a small part of the growth at one end of the streak does not contaminate the plate, and it may be reincubated and subsequently retested if necessary. (2) Upon addition of iodine, record the presence or absence of blue color immedi- ately. (3) Test bacteria giving a red-violet color with io- dine (partial hydrolysis) should be retested after an ad- ditional incubation period (see no. 1 above). Figure 22.1 Test for Starch Hydrolysis After Adding Gram’s Iodine. (a,c ) Positive hydrolysis. The complete breakdown of all starch is shown by the clear (white) halo. (b) Negative hydrolysis. Starch remains intact—no color change as indicated by the purple to brown color around the streak. α-amylase–producing bacteria No starch remains Starch (a) (b) (c) Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 22. Carbohydrates III: Starch Hydrolysis © The McGraw−Hill Companies, 2002 141 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 22 Carbohydrates III: Starch Hydrolysis 1. In the following plate, sketch the presence or absence of starch hydrolysis. [...]... Reading in Textbook 1 Requirements for Nitrogen, Phosphorus, and Sulfur, section 5 .4 2 Oxidation of Inorganic Molecules, section 9.10 Pronunciation Guide Klebsiella pneumoniae (kleb-se-EL-lah nu-MO-ne-ah) Proteus vulgaris (PRO-tee-us vul-GA-ris) Salmonella typhimurium (sal-mon-EL-ah tie-feeMUR-ee-um) Principles Many proteins are rich in sulfur-containing amino acids such as cysteine When these proteins... Carbohydrates and Intracellular Reserve Polymers, section 9.7, see figure 9.10 2 The Enterobacteriaceae, section 22.3, see table 22.7 Pronunciation Guide Enterobacter aerogenes (en-ter-oh-BAK-ter a-RAHjen-eez) Escherichia coli (esh-er-I-ke-a KOH-lee) Klebsiella oxytoca (kleb-se-EL-lah ok-se-TO-se-ah) Proteus vulgaris (PRO-te-us vul-GA-ris) Salmonella (sal-mon-EL-ah) Shigella (shi-GEL-la) Enterobacteriaceae... culture tubes upright in a test-tube rack or in a can Pronunciation Guide Bacillus subtilis (bah-SIL-lus SUB-til-us) Escherichia coli (esh-er-I-ke-a KOH-lee) Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jinOH-sa) Materials per Student 2 4- to 48 -hour tryptic soy broth cultures of Escherichia coli (ATCC 11229), Bacillus subtilis (ATCC 6051), and Pseudomonas aeruginosa (ATCC 10 145 ) tubes of plate count agar... Enzymes IV: Gelatin Hydrolysis E X E RC I S E 27 Proteins,Amino Acids, and Enzymes IV: Gelatin Hydrolysis SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame No mouth pipetting Keep all culture tubes upright in a test-tube rack Pronunciation Guide Enterobacter aerogenes (en-ter-oh-BAK-ter a-RAHjen-eez) Escherichia coli (esh-er-I-ke-a KOH-lee) Proteus vulgaris (PRO-te-us vul-GA-ris) Materials... vulgaris (ATCC 13315) 4 SIM agar deep tubes Kovacs’ reagent, KEY Indole Test Tablets, or Difco’s SpotTest Indole Reagent Kovacs Bunsen burner inoculating loop and needle 4 MR-VP broth tubes each containing 5 ml of medium methyl red indicator Barritt’s reagent (solutions A and B) or Difco’s SpotTest Voges-Proskauer reagents A and B 4 Simmons citrate agar slants 4 empty test tubes 4- ml pipettes with pipettor... Principles When boiled in water, the connective tissue collagen (which is stringy, insoluble, and indigestible) changes into gelatin, a soluble mixture of polypeptides Certain bacteria are able to hydrolyze gelatin by secreting a proteolytic enzyme called gelatinase The resulting amino acids can then be used as nutrients by the bacteria Since 165 Harley−Prescott: Laboratory Exercises in Microbiology, Fifth... careful when handling the Kovacs’ reagent It contains concentrated hydrochloric acid Keep all culture tubes upright in a test-tube rack or in empty cans Materials per Student 2 4- to 48 -hour tryptic soy broth cultures of Klebsiella pneumoniae (ATCC e13883), Proteus vulgaris (ATCC 13315), and Salmonella typhimurium (ATCC 29631) Bunsen burner inoculating needle test-tube rack 3 SIM (sulfide-indole-motility)... Enterobacter are citrate positive and can be distinguished in the clinical laboratory from the opportunistic pathogen Escherichia coli (urinary tract infections) which is citrate negative Principles The identification of enteric (intestinal) bacteria is of prime importance in determining certain food-borne and waterborne diseases Many of the bacteria that are 1 54 Biochemical Activities of Bacteria All enteric... Student 2 4- to 48 -hour tryptic soy broth cultures of Enterobacter aerogenes (ATCC 13 048 , gel +), Escherichia coli (ATCC 11229, gel – ), and Proteus vulgaris (ATCC 13315, gel +) 4 nutrient gelatin deep tubes Bunsen burner inoculating loop 1-ml pipettes with pipettor refrigerator or ice-water bath test-tube rack incubator set at 35°C 3 KEY Rapid Gelatin Test Strips (KEY Scientific Products, 140 2 Chisholm... ferment glucose, leading to 2,3-butanediol accumulation in the medium The addition of 40 % KOH and a 5% solution of alpha-naphthol in absolute ethanol (Barritt’s reagent) will detect the presence of acetoin—a precursor in the synthesis of 2,3-butanediol In the presence of the reagents and acetoin, a cherry-red color develops Development of a red color in the culture medium 15 minutes following the addition . (al-kah-LIJ-e-neez fee-KAL-iss) Escherichia coli (esh-er-I-ke-a KOH-lee) Proteus vulgaris (PRO-tee-us vul-GA-ris) Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jin- OH-sah) Shigella flexneri (shi-GEL-la. pneumoniae (kleb-se-EL-lah nu-MO-ne-ah) Proteus vulgaris (PRO-tee-us vul-GA-ris) Salmonella typhimurium (sal-mon-EL-ah tie-fee- MUR-ee-um) Why Are the Following Bacteria Used in This Exercise? In this. 9.22. Pronunciation Guide Proteus mirabilis (PRO-te-us meh-RA-bill-iss) Staphylococcus epidermidis (staf-il-oh-KOK-kus e-pee-DER-meh-diss) Why Are the Following Bacteria Used in This Exercise? After this exercise,