Unit 9
Unit 9: Carbohydrate Catabolism
By Karen Bentz, Patricia G. Wilber and Heather Fitzgerald. Copyright Central New Mexico Community College, 2015
Introduction Bacteria require carbohydrates for energy production. Carbohydrates include monosaccharides such as glucose and fructose, and disaccharides such as lactose and sucrose. Polysaccharides such as starch and cellulose, which are synthesized by plants, and glycogen, which is produced in animals are composed of repeating glucose subunits. Bacteria have a variety of pathways to utilize the energy in carbohydrates. Many bacteria produce specific membrane transport proteins to transport a specific kind of carbohydrate into the cell. Then the bacteria use one or several of the energy producing pathways (aerobic respiration, anaerobic respiration or fermentation) to extract energy from the carbohydrate. Some bacteria, such as Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes are capable of using all three of these pathways and are termed facultative anaerobes (see Unit 7 for a review on oxygen usage). In this lab you will be observing the growth of various bacteria on media that requires that the organism use fermentation in order to grow. Remember that Glycolysis provides the ATP for energy, and that the purpose of fermentation reactions is to recycle the NAD+ needed for glycolysis.
You will test a variety of carbohydrates to see which ones various bacterial species can ferment. You will also test to see what types of waste products the bacteria produce as a result of fermentation. Waste products are useful to help identify bacteria to species since different species may use different enzymes and pathways in fermentation. Examples of waste products include organic acids, alcohols, ketones, carbon dioxide and hydrogen sulfide gas. The carbon dioxide can be seen as bubbles of gas in the media. An organic acid waste will lower the pH of the media, while an alcohol waste will not. For this reason a pH indicator is added to the media and depending on the end color indicates whether the pH decreased (acid waste produced) or stayed the same (alcohol waste produced). Enterobacteriaceae and Carbohydrate Fermentation
The Enterobacteriaceae, or enterics for short, are a group of bacteria that can live in the intestines. These bacteria, generally referred to as coliforms, are often normal flora in the intestines and are generally Gram(-), rod shaped and capable of fermentation in the low- oxygen environment in the intestines. Since there is often lactose in the intestines, especially in
Unit 9 Page 1 Unit 9 the intestines of infants, many of the bacterial species that normally live in the intestines have evolved the ability to ferment lactose. We call these normal, beneficial, lactose-fermenting gut bacteria coliforms. Coliforms are beneficial when they are in the intestines, but they SHOULD NOT be in our drinking water supply! So coliforms in the intestines are good, coliforms in the drinking water are bad because that indicates the presence of sewage (the coliforms come out of the gastro-intestinal system….) Non-coliforms are other bacteria, such as Salmonella and Shigella that can live in the intestines but cause disease. If you’ve ever had a severe case of diarrhea, it could have been from ingesting one of these pathogens with your food. Non-coliforms generally do not ferment lactose because they are not normally in the intestines where they might encounter lactose.
Carbohydrate Media
MacConkey’s agar plate MacConkey’s media is both selective and differential. The crystal violet and bile salts in the media inhibit the growth of Gram(+) bacteria by interfering with the formation of their cell walls. Gram(-) bacteria are not inhibited by these two chemicals, and thus will grow (they are selected for) on MacConkey media. The differential ingredient in MacConkey media is lactose. Bacteria that can ferment the lactose will produce an acid waste, which will lower the pH of the media. A pH indicator, neutral red, is absorbed by lactose fermenting bacteria, turning the bacteria a fuchsia color. Gram(-) bacteria that do not ferment lactose will grow on the media, and show clear growth.
Triple Sugar Iron (TSI) agar slant TSI media is only differential. It does not select for Gram(+) or Gram(-) growth, so both types of bacteria are capable of growing on TSI media. TSI tests for an organism’s ability to ferment three different sugars (thus the “Triple Sugar” in the name. The three sugars are differential ingredients and they are glucose (monosaccharide), sucrose and lactose (both disaccharides). Generally a bacterium will use the glucose first for energy production, and then if it has the enzymes sucrase and/or lactase, it will ferment the disaccharides for energy production. The pH indicator phenol red is added to TSI media to determine which sugar(s) are being fermented.
The butt of the medium can also be assessed for production of carbon dioxide (CO2) gas, the production of H2S or the breakdown of cysteine (found in peptone, one of the ingredients in the TSI). These later two both produce a black butt.
Unit 9 Page 2 Unit 9
If the bacteria produce CO2 as a waste product of fermentation, bubbles of gas will be seen in the media, or an air space will appear at the bottom of the tube as the gas pushes the media up. One way a black butt is produced in the TSI is if the enzyme thiosulfate reductase, is produced by the bacteria. This enzyme will reduce thiosulfate (an ingredient in the media) into H2S. The 2+ H2S reacts with ferrous sulfate (an ingredient in the media that is a source of iron ions (Fe )) resulting in an iron and sulfur-containing product, which is black. The second way a black butt is produced is by the reduction of cysteine (an amino acid found in peptone, an ingredient of the media) into H2S, pyruvate and ammonia by the enzyme cysteine desulfhydrase.
We cannot distinguish between these two methods of H2S production in this test. You will see these same reactions in the SIM test in the next unit, Unit 10.
NOTE: H2S production actually results from protein catabolism not carbohydrate catabolism test and you will see it again in the SIM test, which is presented in the Protein Tests unit, Unit 10.
Carbohydrate fermentation (Durham) broth: Lactose and Sucrose These media are also only differential. The carbohydrate fermentation media contains a single carbohydrate as the differential material, either lactose or sucrose, plus the pH indicator phenol red. In addition, a small, inverted tube called a Durham tube is placed in the media to collect any carbon dioxide produced as a waste product of fermentation.
Methyl-Red Voges-Proskauer (MR-VP) broth This differential media looks at the organic waste products of fermentation of pyruvate produced by glycolysis. These waste products are generally either organic acids, or an alcohol. The media is basic T-soy broth with glucose to encourage the growth of bacteria that can use fermentation. After the bacteria have grown, reagents are added to complete the tests. Methyl red pH indicator is added to the MR tube to determine whether or not organic acid waste has been produced. Methyl red is red at low pH and yellow at pH greater than six. Barritt’s Reagents VP-A and VP-B are added to the VP tube to determine whether or not an alcohol waste product has been produced.
DAY ONE: Inoculations
Unit 9 Page 3 Unit 9
Triple Sugar Iron Slant https://www.youtube.com/watch?v=jA40qA5oAW4 MR/VP Broth https://www.youtube.com/watch?v=2CKBKg0JylQ Carbohydrate Fermentation https://www.youtube.com/watch?v=9sYQjnAReaE This video includes dextrose and maltose tubes that we no longer use. The video does not emphasize the enzymes used but those are important. Materials
Media (per pair of students)
o 2 MacConkey plates
o 4 TSI slants
o 4 Carbohydrate fermentation (Durham) tubes; 2 lactose and 2 sucrose
o 4 MR-VP
Bacteria Cultures
o Escherichia coli (Ec)
o Proteus vulgaris (Pv)
o Serratia marcescens (Sema)
o Enterococcus faecalis (Ef)
Unit 9 Page 4 Unit 9
Procedures
A. MacConkey Plates Figure 9-1. Two MacConkey Plates Inoculated With All Four Species
Image created by Karen Bentz, 2015 1. Use a marker to draw a line to divide each MacConkey plate in half. Draw on the agar (bottom) side. 2. Label the plate with your personal information, and the initials of the bacteria that you will be putting on the plate. 3. Sterilize and cool a loop. 4. Inoculate each section of the MacConkey plate with a different bacteria. Use a simple back and forth curve (squiggle) to inoculate each section. 5. Be sure to sterilize your loop after every inoculation. 6. Place your inoculated MacConkey plates in the correct location for incubation. 7. Return your re-sterilized loop to the metal canister.
Unit 9 Page 5 Unit 9
B. Triple Sugar Iron (TSI) Tubes Figure 9-2. Inoculate four TSI Tubes, each with a different bacteria. Use a streak and a stab to inoculate each tube.
Ec Pv Sema Ef
Image created by Karen Bentz, 2015 1. Label each of your TSI tubes with the initials of one bacterial species as shown in the diagram above. Also include your personal information and date on the tube. 2. Using aseptic technique and a sterile needle, inoculate each TSI tube with a different bacteria. 3. Streak the surface of the slant as shown in Figure 9-2, and then stab the needle to within one cm of the bottom of the tube. Each tube will have a streak and a stab using one species. 4. Be sure to sterilize your needle between each inoculation. 5. Place your four inoculated TSI tubes, with slightly loose caps, in the rack for incubation. 6. Return your re-sterilized needle to the metal canister.
Unit 9 Page 6 Unit 9
C. Carbohydrate Fermentation (Durham) Tubes: Sucrose and Lactose Figure 9-3. Inoculating Two Sets of Carbohydrate Fermentation (Durham) tubes. Each set will have one species of bacteria. The small inverted Durham tube in the media is for carbon dioxide capture. Lactose Sucrose Lactose Sucrose
Ec in Both Tubes Pv in Both Tubes
Image created by Karen Bentz, 2015
1. Label your tubes with the initials of the bacteria, as shown in the diagram above. Be sure that the tube is correctly labeled as either “lactose” or “sucrose”.
2. Using aseptic technique and a sterile loop, pick up a loopful of bacteria and carefully transfer it into the lactose tube.
3. Sterilize your loop, let it cool, and then pick up another loopful of the same bacteria and transfer it into the sucrose tube.
4. Repeat steps two and three to inoculate your other set of carbohydrate fermentation tubes.
5. Re-sterilize your loop and return it to the metal canister.
6. Place the inoculated tubes (caps slightly loose), in the appropriate area for incubation.
Unit 9 Page 7 Unit 9
D. Methyl-Red Voges-Proskauer (MR-VP) Tubes Figure 9-4. Inoculating Two Sets of MR-VP tubes, each with a different bacteria.
Ec in both tubes Sema in both tubes
Image created by Karen Bentz, 2015
1. Label the tubes as shown in Figure 9-4 above. Also add your personal information to each tube. 2. Using aseptic technique and a sterile loop, pick up a loopful of bacteria and carefully transfer it into the MR tube. 3. Sterilize your loop, let it cool, and then pick up another loopful of the same bacteria and transfer it into the VP tube. 4. Repeat steps two and three to inoculate your other set of MR/VP tubes. 5. Re-sterilize your loop, and place the inoculated tubes (caps slightly loose), in the appropriate area for incubation.
Precaution
Leave all of the caps on the carbohydrate usage tubes slightly loose for adequate gas exchange during incubation. Make sure that even though the cap is slightly loose, it is still securely attached to the tube!
Unit 9 Page 8 Unit 9
DAY TWO: Results and Interpretation
Collect the media you inoculated in the previous lab. Observe your results and fill in the information in the tables below. Figure 9-6: MacConkey Plate Results. The bacteria on the left grew, meaning it is Gram(-) but the colonies are clear, meaning the bacteria does not ferment lactose. The bacteria on the right grew, meaning it is Gram(-), and the dark pruple color means the bacteria ferment lactose.
I. MacConkey plates A. Type of Cell Wall Growth on media: The organism is Gram(-). No growth on media: The organism is likely to be Gram(+), but may be a fastidious Gram(-) such as Haemophilus. B. Ability to ferment Lactose Positive test result for lactose fermentation: bacteria absorbs dye and turns hot pink (high lactose fermentation) or light pink (low lactose fermentation). Indicates bacteria produce lactase and live in the intestines. Bacteria is a non-pathogen / coliform. Negative test result for lactose fermentation: bacteria grows, but has clear, white, purplish or yellowish colored growth. Media remains purple. Indicates bacteria does not produce lactase and is a likely pathogen/ non-coliform. In the space below, draw or insert a photograph of the results of your MacConkey test.
Unit 9 Page 9 Unit 9
Name of Bacteria Selective Features: Differential Feature: If Bacteria Grew, Result: Did it Interpretation: Result: Did it Interpretation: grow on the Is it G-? turn a fuchsia Does it ferment MacConkeys? color? lactose?
Unit 9 Page 10 Unit 9
II. Triple Sugar Iron (TSI) tubes
Figure 9-7: Triple Sugar Iron (TSI) Results. From left to right: An uninoculated TSI tube, K/K,
A/A+ CO2, K/A + H2S, K/A
Nomenclature for this test:
Slant: top 2/3 of the tube
Butt: bottom 1/3 of the tube
K = red = alKaline pH
A = yellow = Acid pH
K/K = red slant and red butt
K/A = red slant and yellow butt (black might obscure the yellow, but if black, yellow is under there)
A/A = yellow slant and yellow butt (black might obscure the yellow, but if black, yellow is under there)
Unit 9 Page 11 Unit 9
A. Glucose fermentation Positive test result: yellow (A) butt indicates bacteria can ferment glucose. Note: if you have a black butt, assume that the media is yellow underneath. Negative test result: red (K) butt indicates bacteria cannot ferment glucose.
B. Lactose and/or Sucrose fermentation Positive test result: yellow (A) slant indicates bacteria can ferment lactose and/or sucrose. Produces lactase and/or sucrase enzymes. Negative test result: red (K) slant indicates bacteria cannot ferment lactose or sucrose. Does not produce lactase or sucrase enzymes.
C. Carbon dioxide (CO2) production Positive test result: bubbles of gas push the media off of the bottom of the tube. Bubbles may also form between the media and the wall of the test tube, and within the media itself Negative test result: no bubbles of gas form in the media, against the walls of the test tube, or at the bottom of the tube. NOTE: Gas production does not always occur even when it is supposed to. Avoid using gas as a definitive diagnostic characteristic for bacterial identification. Use it only to support other results.
D. Hydrogen sulfide (H2S) production
Positive test result: If the bacteria can produce H2S using the enzyme thiosulfate reductase or the enzyme cysteine desulfhydrase. The hydrogen sulfide will react with the iron ions in the medium, producing a black precipitate in the butt of the media; indicates the bacteria produces the enzyme thiosulfate reductase or cysteine desulfhydrase (we cannot distinguish between the two) and is a possible pathogen Negative test result: no black precipitate forms in the butt of the media, the bacteria does not produce the enzyme thiosulfate reductase. In the space below, draw or insert a photograph of the results of your TSI tests.
Unit 9 Page 12 Unit 9
Name of Bacteria Slant/Butt: Was CO2 Was H2S Produced? Produced? Result: Interpretation: What sugars Is the media Is there a black (K/K, does this organism pushed up, precipitate in K/A, catabolize? and/or bubbles the media? or A/A) (glucose, sucrose, lactose) seen in tube?
1. Which species metabolized glucose?
2. A. Which may have produced the enzyme lactase?
B. Which may have produced the enzyme sucrase?
C. What test or test could you do to distinguish whether the bacteria produced only lactase, only sucrase, or both enzymes??
3. Which species produced thiosulfate reductase or cysteine desulfhydrase?
Unit 9 Page 13 Unit 9
III. Carbohydrate Fermentation (Durham) tubes Figure 9-8: Carbohydrate Fermentation Results: From left to right: negative for fermentation and CO2 , negative for fermentation and CO2 , positive for fermentation, positive for CO2
A. Carbohydrate fermentation Positive test result: Tubes must be lemon yellow to be considered positive for lactose or sucrose fermentation. Positive tests are recorded with an “A” for acid. Negative test result: Orange or red are considered negative for sugar fermentation.
B. CO2 gas production
Positive test result: CO2 gas production in the small Durham tube must be 25% or more to be considered positive for CO2 production. Positive tests are recorded with a “G” for gas. Negative test result: A few bubbles is considered negative.
Unit 9 Page 14 Unit 9
In the space below, draw or insert a photograph of the results of your Carbohydrate Fermentation Tubes.
Name of Bacteria Lactose Sucrose Acid (A) Gas (G) Acid (A) Gas (G) Production? Production? Production? Production?
1. Which bacteria produce the enzyme lactase?
2. Which bacteria produce the enzyme sucrase?
Unit 9 Page 15 Unit 9
IV. Methyl-Red Voges-Proskauer (MR-VP) tubes Day 2 Procedure:
1. As per the directions from Day 1, you should have 4 tubes as follows: Ec MR, Ec VP, SeMa MR, SeMa VP.
2. Add five drops of the Methyl Red reagent to each of the MR tubes and mix thoroughly. These are now ready to observe.
3. Add eight drops of VP-A reagent to each of the VP tubes. Next add four drops of VP-B reagent to each of the VP tubes. Mix the reagents thoroughly in the tubes.
4. Let the VP tubes stand in a rack undisturbed for five minutes before you observe them. Figure 9-9: MR/VP Results
A. MR test result
Positive test result: cherry red media after the methyl red is added. Indicates organism has catabolized glucose and produced organic acid waste. Negative test result: clear to yellow colored media after the methyl red is added. Indicates the organism doesn’t produce organic acid waste, or didn’t catabolize glucose.
Unit 9 Page 16 Unit 9
B. VP test result Positive test result: a red ring at the top of the media. Indicates that the organism catabolized glucose and produces an alcohol waste (2-3 butanediol). Negative test result: no red ring at the top of the media. Indicates the organism doesn’t produce an alcohol waste (2,3 butanediol), or didn’t catabolize glucose. In the space below, draw or insert a photograph of the results of your MR/VP Test
Name of Bacteria Methyl Red Voges Proskauer
Result: Interpretation: Result: Interpretation: Red or not Are organic acid Red ring or Alcohol waste red? wastes produced? no red ring produced?
1. Which of your bacteria performed glycolysis and produced an organic acid waste?
2. Which of your bacteria performed glycolysis and produced an alcohol waste?
Unit 9 Page 17 Unit 9
More Interpretation
Based on ALL of your carbohydrate catabolism results, (MacConkey, TSI, Carb. Tubes and MR/VP) explain what you know about the cell wall structure, enzymes, waste products and pathogenicity of each bacterial species you tested. Bacterial Species: ______
Bacterial Feature: Give ALL the test names and evidence that led to this decision.
Cell wall? Gram(-) or Not Gram(-)
Does This Bacteria Give ALL the test names and evidence that led to this decision. Produce: Sucrase? Yes or No
Lactase? Yes or No Thiosulfate reductase/ cysteine desulfurase? Yes or No
CO2 Waste? Yes or No
H2S Waste? Yes or No
Acid or Alcohol Waste?
Based on Your Results, is this Bacteria a Likely Pathogen? Yes or No
Unit 9 Page 18 Unit 9
Unit 9 Page 19 Unit 9
Bacterial Species: ______
Bacterial Feature: Give ALL the test names and evidence that led to this decision.
Cell wall? Gram(-) or Not Gram(-)
Does This Bacteria Give ALL the test names and evidence that led to this decision. Produce: Sucrase? Yes or No
Lactase? Yes or No Thiosulfate reductase/ cysteine desulfurase? Yes or No
CO2 Waste? Yes or No
H2S Waste? Yes or No
Acid or Alcohol Waste?
Based on Your Results, is this Bacteria a Likely Pathogen? Yes or No
Unit 9 Page 20 Unit 9
Bacterial Species: ______
Bacterial Feature: Give ALL the test names and evidence that led to this decision.
Cell wall? Gram(-) or Not Gram(-)
Does This Bacteria Give ALL the test names and evidence that led to this decision. Produce: Sucrase? Yes or No
Lactase? Yes or No Thiosulfate reductase/ cysteine desulfurase? Yes or No
CO2 Waste? Yes or No
H2S Waste? Yes or No
Acid or Alcohol Waste?
Based on Your Results, is this Bacteria a Likely Pathogen? Yes or No
Unit 9 Page 21 Unit 9
Bacterial Species: ______
Bacterial Feature: Give ALL the test names and evidence that led to this decision.
Cell wall? Gram(-) or Not Gram(-)
Does This Bacteria Give ALL the test names and evidence that led to this decision. Produce: Sucrase? Yes or No
Lactase? Yes or No Thiosulfate reductase/ cysteine desulfurase? Yes or No
CO2 Waste? Yes or No
H2S Waste? Yes or No
Acid or Alcohol Waste?
Based on Your Results, is this Bacteria a Likely Pathogen? Yes or No
Unit 9 Page 22 Unit 9
Post-Activity Questions
1. What is the primary purpose of fermentation reactions?
2. Why are fermentation reactions an advantage to organisms that live in the intestines?
3. Name two gases that can be produced as a consequence of fermentation.
4. Organisms will preferentially catabolize glucose first before fermenting a disaccharide. Why?
5. What are three characteristics of the Enterics?
6. A. What are coliforms?
B. What are non-coliforms?
C. Which of these groups is likely to be pathogenic?
Unit 9 Page 23 Unit 9
7. Organic wastes from fermentation pathways are often either ______or ______.
8. Fill in the blanks with ALL of the letters that are correct regarding the following media.
MacConkey ______
MR-VP ______
TSI ______
Carbohydrate tubes ______
a. Lactose, sucrose and glucose are the three sugars in this media.
b. The pH indicator in this media is phenol red.
c. The pH indicator in this media is methyl red.
d. The pH indicator in this media is neutral red.
e. This media contains an inverted Durham tube for gas collection.
f. The tubes in this test contain a single sugar, either lactose or sucrose.
g. This media contains crystal violet and bile salts.
h. Thiosulfate and ferrous sulfate are ingredients in this media.
i. Barritt’s reagents VP-A and VP-B are added to this media post-incubation.
Unit 9 Page 24 Unit 9
Table 9-1. Carbohydrate Catabolism Media
Media Ingredients What Positive/Negative Interpretation of Results Results Look Like Triple Sugar Iron T-soy agar plus: Sugar usage: Sugar usage: Organisms that ferment lactose are (TSI) Glucose, lactose and Red slant and yellow butt K/A indicates bacteria produce generally normal gut flora, but a Inoculate with a sucrose. (K/A) indicate glucose enzymes for glucose fermentation, yellow slant indicates fermentation of needle; streak and fermentation only but not lactase or sucrase enzymes lactose and/or stab Thiosulfate (sulfur source) sure which disaccharide is being Yellow slant and yellow A/A indicates bacteria produce Ferrous sulfate (H2S fermented without running an indicator) butt, A/A, indicate enzymes for glucose fermentation, additional test. fermentation of glucose, and/or the enzymes lactase, Peptone (source of and fermentation of sucrase cysteine) sucrose and/or lactose K/K indicates no growth, organism Phenol red pH indicator Red slant and red butt (K/K) may be an obligate aerobe and indicate bacteria is not able unable to ferment to ferment glucose Always assume that a black precipitate indicate metabolism of glucose. Carbon dioxide production is indicated Carbon dioxide is often a byproduct of by bubbles or cracks in the media or fermentation. media pushed up from the butt of the tube. A black precipitate means the bacteria
H2S production is indicated by a black produced the enzyme Thiosulfate reductase or color in the media. cysteine desulfhydrase
Carbohydrate Peptone broth with a Media must be lemon AG means fermentation of the Fermentation single sugar such as yellow to be positive for particular sugar with acid and CO2 Tube with Durham sucrose or lactose sugar fermentation with an waste production. Tube acid waste. Orange or red Inoculate with a Small inverted Durham A_ means fermentation of the tube for carbon dioxide indicate no fermentation of particular sugar with acid waste loop. that particular sugar, or no collection production but no CO2 waste. acid waste. Phenol red pH indicator KG means fermentation of the The CO2 level in the Durham particular sugar with alcohol and tube must reach 25% to be CO2 waste production. considered positive.
Unit 9 Page 25 Unit 9
Methyl Red- T-soy broth in tubes for growth of A positive MR test will be a red color MR-VP screens for the end products of glucose Two tubes of broth media are Voges-Proskauer bacteria. after the pH indicator is added. A fermentation. inoculated with a single bacteria. (MR-VP) negative test will be yellow. A positive MR test indicates that After bacterial growth MR reagents Reagents added after bacterial the organism produces organic acid are added to the MR tube, and VP-A Inoculate with a growth: A positive VP test will have a red ring waste. and VP-B reagents are added to the VP loop. Methyl red pH indicator at the top of the tube within 5 minutes tube. of adding the VP-A and VP-B reagents. A positive VP test indicates that the Barritt’s reagent (VP-A organism produces an alcohol and VP-B) waste (2-3 butanediol).
Unit 9 Page 26 Unit 9
Figure 9-10. Acid , Alcohol and Ketone End-Products of Fermentation. Note that pyruvate is the starting point for all. Modified from Leboffe and Pierce. 2011. A Photographic Atlas for Microbiology, 4th edition.
Figure created by Patricia G. Wilber, 2016
The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our dean John Cornish.
Unit 9 Page 27