Unit 9

Unit 9: Carbohydrate Catabolism By Karen Bentz, Patricia Wilber and Heather Fitzgerald and Andrea Peterson, 2018Creative Commons Attribution-NonCommercial 4.0 International License.

I. IntroductionBacteria require carbohydrates for energy production. Carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as lactose and sucrose and polysaccharides such as starch, cellulose and glycogen. Bacteria produce different enzymes to utilize the energy in various carbohydrates.

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 during fermentation glycolysis provides the ATP for energy, and that the purpose of fermentation reactions is to recycle the NAD+ needed for glycolysis.

Figure 9-1. The productions of fermentation.

Image created by Karen Bentz, 2016

Enterobacteriaceae and Carbohydrate FermentationThe Enterobacteriaceae, or enterics for short, are a group of bacteria that can live in the intestines. The 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 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, non-pathogenic, lactose-fermenting gut bacteria coliforms. Coliforms SHOULD NOT be in our drinking water supply because that indicates contamination by fecal matter! Non-coliforms are other bacteria, such as Salmonella and Shigella that can live in the intestines, but can be pathogenic. 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.

Unit 9 Page 1

Unit 9

Carbohydrate MediaMacConkey’s agar plate:MacConkey’s media is both selective and differential. The selective materials in the medium are crystal violet and bile salts. These inhibit the growth of Gram(+) bacteria while Gram(-) bacteria are not inhibited and will grow.

The differential ingredient in MacConkey medium is lactose. Bacteria that can produce the enzyme lactase can ferment the lactose and will produce an acid waste, which will lower the pH of the media. A pH indicator, neutral red, turns bright fuchsia in color, due to the lowered pH when lactose is fermented. Gram(-) bacteria that do not ferment lactose will grow on the media, and the growth is clear or slightly purple (due to the crystal violet).

Triple Sugar Iron (TSI) agar slant: The TSI medium is only differential. It does not select for Gram(+) or Gram(-) growth, so both types of bacteria are capable of growing on the TSI.

The TSI tests an organism’s ability to ferment three different sugars (thus the “Triple Sugar” in the name. The three sugars 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. This produces acids, lowering the pH.

The pH indicator phenol red is added to TSI media to determine which sugar(s) are being fermented. The butt and the slant of the tube are assessed independently.

The medium can also be assessed for production of carbon dioxide (CO2) gas, and the production of H2S. Hydrogen sulfide can be produced in two ways. If the enzyme thiosulfate reductase is produced by the bacteria, the enzyme will reduce thiosulfate in the media into H2S. The H2S reacts with ferrous sulfate, also in the media, resulting in a product which is black. Additionally, reduction of cysteine, an ingredient in the media, into H2S, pyruvate and ammonia by the enzyme cysteine desulfhydrase, if produced by the bacteria, can produce black. 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.

Carbohydrate Fermentation (Durham) Tubes: These media are only differential and can detect carbohydrate fermentation and gas production. The medium contains a single carbohydrate as the differential material. Carbohydrate fermentation, as in the other tests, produces acidic wastes. The pH indicator phenol red is used to detect the change in pH. 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.

Unit 9 Page 2

Unit 9

We will use two types of carbohydrate fermentation tubes: lactose tubes (differential material lactose) the sucrose tubes (differential material sucrose) to determine if an organism can produce the enzymes lactase or sucrase, respectively.

Methyl-Red Voges-Proskauer (MR-VP) broth: This differential medium is used to determine organic acid and/or alcohol waste products of 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. There are no enzymes to learn for this medium!

II. DAY ONE Inoculations

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.

Videos created by Corrie Andries.

Materials Media (per pair of students)

2 MacConkey plates 4 TSI slants 4 Carbohydrate fermentation (Durham) tubes; 2 lactose and 2 sucrose 4 MR-VP tubes

Bacteria Cultures Escherichia coli (Ec) Proteus vulgaris (Pv) Serratia marcescens (Sema) Enterococcus faecalis (Ef)

Unit 9 Page 3

Unit 9

ProceduresA. MacConkey PlatesFigure 9-2. 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 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.

B. Triple Sugar Iron (TSI) TubesFigure 9-3. Inoculate four TSI Tubes, each with a different bacterial species.

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 different species.3. Streak the surface of the slant as shown in Figure 9-2, and then stab the needle ¾ of the way into the butt. Each tube will have a streak and a stab using one species.4. Be sure to sterilize your needle between each tube.

Unit 9 Page 4

Unit 9

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.

C. Carbohydrate Fermentation (Durham) TubesFigure 9-4. Inoculate the Sucrose and Lactose Carbohydrate Fermentation (Durham) tubes as shown.

Image created by Karen Bentz, 2015

Each set will have one species of bacteria. The small inverted Durham tube in the media is for carbon dioxide capture.

1. Label your tubes with the initials of the bacterial species, as shown in the diagram above. Be sure that each 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 5

Unit 9

D. Methyl-Red Voges-Proskauer (MR-VP) TubesFigure 9-5. Inoculating two sets of MR-VP tubes, each with a different species.

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. “MR” and “VP” tubes, at this point are exactly the same. The differentiation occurs on Day 2, when you will add some reagents.

2. Use aseptic technique and a sterile loop to 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 6

Unit 9

III. DAY TWO: Results and InterpretationCollect the media you inoculated in the previous lab. Observe your results and fill in the information in the tables below.

MacConkey platesFigure 9-6: MacConkey plate results. The bacterial species 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 fuschia color means the bacteria ferment lactose.

Accessed 8/31/15 from http://www.microbelibrary.org/library/laboratory-test/2927-lactose, but licensed for use by the American Society for Microbiology, Creative Commons Attribution – Noncommercial – No Derivatives 4.0 International license.

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 LactosePositive test result for lactose fermentation: bacteria absorb dye and turn fuchsia (high lactose fermentation). A lighter pink would indicate low lactose fermentation, but we don’t test any species with low lactose fermentation. This color change indicates bacteria produce lactase and live in the intestines. The bacterial species is a non-pathogen/coliform.

Negative test result for lactose fermentation: bacteria grow, but have clear or pale purplish or yellowish colored growth. Media remains purple. This indicates the bacterial species does not produce lactase and is a likely pathogen/non-coliform.

Unit 9 Page 7

Unit 9

Name of Bacteria Selective Features: Differential Feature: If Bacteria Grew,

Result: Did it grow on the MacConkey

plate?

Interpretation:Is it G-?

Result: Did it turn a fuchsia

color?

Interpretation: Does it ferment

lactose?Is it a

pathogen?

Table 9-1. MacConkey Results

In the space below insert a photograph of the results of your MacConkey test.

Unit 9 Page 8

Unit 9

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

Accessed 2/17/2015 Creative Common copyright, Public Domain. https://commons.wikimedia.org/wiki/File:TSIagar.JPG

Terminology for this test: Slant: top 2/3 of the tube

Butt: bottom 1/3 of the tube A = yellow = Acid pH K = red= AlKaline pH

Unit 9 Page 9

Unit 9

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)

A. Glucose fermentation.Analyze the butt.Positive test result: yellow (A) butt indicates bacteria can ferment glucose. If you see 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 fermentationAnalyze the slant.Positive test result: yellow (A) slant indicates bacteria can ferment lactose and/or sucrose. Produces lactase and/or sucrase enzymes. We cannot distinguish which.Negative test result: red (K) slant indicates bacteria cannot ferment lactose or sucrose. Does not produce lactase or sucrase enzymes.

C. Carbon dioxide (CO2) productionAnalyze the butt.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 itselfNegative 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.Analyze the butt.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. This indicates the species produces the enzyme thiosulfate reductase or cysteine desulfhydrase (we cannot distinguish between the two) and is a possible pathogenNegative test result: no black precipitate forms in the butt of the media, the bacteria does not produce the enzyme thiosulfate reductase and/or cysteine desulfhydrase.

Unit 9 Page 10

Unit 9

Table 9-2. TSI Results Name of Bacteria Slant/Butt: Was CO2

Produced?Was H2S Produced?

Result: (K/K, K/A,

or A/A)

Interpretation: What sugars does this organism

catabolize?(glucose, sucrose, lactose)

Is the media pushed up, and/or bubbles seen in tube?

Is there a black precipitate in the media?Is it a pathogen?

In the space below, insert a photograph of the results of your TSI tests.

Unit 9 Page 11

Unit 9

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

Photo by Karen Bentz

A. Carbohydrate fermentation (Durham) tubesPositive 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 productionPositive 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.

Table 9-3. Carbohydrate fermentation (Durham) tube results:Name of Bacteria Lactose Sucrose

Acid (A) Production?

Gas (G) Production?

Acid (A) Production?

Gas (G) Production?

In the space below, insert a photograph of the results of your Carbohydrate fermentation tubes.

Unit 9 Page 12

Unit 9

Methyl-Red Voges-Proskauer (MR-VP) tubesDay 2 Procedure:

1. As per the directions from Day 1, you should have 4 tubes labelled 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 (fifteen when using a Gram(+) organism) minutes before you observe them.

Figure 9-9: MR/VP results

Photo provided by Janet Robertson, CNM Microbiology Student, Fall 2016.

A. MR test result Positive test result: cherry red medium immediately after the methyl red is added. This indicates organism has catabolized glucose and produced organic acid waste.Negative test result: clear to yellow colored medium after the methyl red is added. This indicates the organism doesn’t produce organic acid waste, or didn’t catabolize glucose.

B. VP test resultPositive test result: a red ring at the top of the medium. 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 medium. This indicates the organism doesn’t produce an alcohol waste (2,3 butanediol), or didn’t catabolize glucose.

Unit 9 Page 13

Unit 9

Table 9-4. MR/VP ResultsName of Bacteria Methyl Red Voges Proskauer

Result: Red or not

red?

Interpretation:Are organic acid

wastes produced?

Result: Red ring or no

red ring

Interpretation:Alcohol waste

produced?

In the space below, insert a photograph of the results of your MR/VP Test

Unit 9 Page 14

Unit 9

More InterpretationFill in the table below with the results you collected above. Then use the information to answer the questions.

Table 9.5. Summary of some carbohydrate fermentation results. Address the items in the table and DEFEND YOUR INTERPRETATION using the results you obtained i.e. state how you know.

BacteriaMacConkey TSI Sucrose Lactose MR/

VPGram(-)? Ferment Lactose? Sugar Usage, CO2 and

H2S ProductionEnzymes produced?

Acid/Gas Acid/Gas?

MR/VP

Escherichia coli (Ec)

Proteus vulgaris (Pv)

NA

Serratia marcescens (Sema)

NA NA

Enterococcus faecalis (Ef)

NA NA NA

1. Which species metabolized glucose?

2. Which species produced the enzyme lactase? List each and DEFEND YOUR ANSWERS for each.

3. Which species produced the enzyme sucrase? List each and defend your answers for each.

4. Which species produced the enzyme(s) thiosulfate reductase and/or cysteine desulfhydrase? List each and defend your answers for each.

5. Which of your bacteria performed glycolysis and produced an organic acid waste? List each and defend your answers for each.

6. Which of your bacteria performed glycolysis and produced an alcohol waste? List each and defend your answers for each.

7. Which of your bacteria are possible pathogens? List each and defend your answers for each.

Unit 9 Page 15

Unit 9

Post-Activity Questions Name: _______________________

1. No energy is produced during fermentation so, why do bacteria use 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?

7. Organic wastes from fermentation pathways are often either ______________________ or _____________.

Unit 9 Page 16

Unit 9

Table 9-2. Carbohydrate Catabolism MediaMedia Ingredients What Positive/Negative

Results Look LikeInterpretation of Results Of Special Note

Triple Sugar Iron (TSI) Inoculate with a needle; streak and stab

T-soy agar plus:- Glucose, lactose and sucrose. - Thiosulfate (sulfur source)- Ferrous sulfate (H2S indicator)- Peptone (source of cysteine)- Phenol red pH indicator

Sugar usage:- Red slant and yellow butt (K/A) indicate glucose fermentation only- Yellow slant and yellow butt, A/A, indicate fermentation of glucose, and fermentation of sucrose and/or lactose- Red slant and red butt (K/K) indicate bacteria is not able to ferment glucose

Carbon dioxide production is indicated by bubbles or cracks in the media or media pushed up from the butt of the tube.

H2S production is indicated by a black color in the media.

Sugar usage:- K/A indicates bacteria produce enzymes for glucose fermentation, but not lactase or sucrase enzymes- A/A indicates bacteria produce enzymes for glucose fermentation, and/or the enzymes lactase, sucrase- K/K indicates no growth, organism may be an obligate aerobe and unable to ferment

Carbon dioxide is often a byproduct of fermentation.

A black precipitate means the bacteria produced the enzyme Thiosulfate reductase or cysteine desulfhydrase

Organisms that ferment lactose are generally normal gut flora, but a yellow slant indicates fermentation of lactose and/or sucrose You can’t be sure which disaccharide is being fermented without running an additional test.

Always assume that a black precipitate indicate metabolism of glucose.

Carbohydrate Fermentation Tube with Durham TubeInoculate with a loop.

Peptone broth with a single sugar such as sucrose or lactose

Small inverted Durham tube for carbon dioxide collection

Phenol red pH indicator

Media must be lemon yellow to be positive for sugar fermentation with an acid waste. Orange or red indicate no fermentation of that particular sugar, or no acid waste.

The CO2 level in the Durham tube must reach 25% to be considered positive.

AG means fermentation of the particular sugar with acid and CO2 waste production.

A_ means fermentation of the particular sugar with acid waste production but no CO2 waste.

KG means fermentation of the particular sugar with alcohol and CO2 waste production.

Methyl Red-Voges-Proskauer (MR-VP)

Inoculate with a loop.

T-soy broth in tubes for growth of bacteria.

Reagents added after bacterial growth:- Methyl red pH indicator- Barritt’s reagent (VP-A and VP-B)

A positive MR test will be a red color after the pH indicator is added. A negative test will be yellow.

A positive VP test will have a red ring at the top of the tube within 5 minutes of adding the VP-A and VP-B reagents.

MR-VP screens for the end products of glucose fermentation.

A positive MR test indicates that the organism produces organic acid waste.

A positive VP test indicates that the organism produces an alcohol waste (2-3 butanediol).

Two tubes of broth media are inoculated with a single bacteria. After bacterial growth MR reagents are added to the MR tube, and VP-A and VP-B reagents are added to the VP tube.

MacConkey (review)

Crystal violet and bile saltsLactoseNeutral Red

Growth/no growthFuchsia pink colonies or clear colonies

Growth indicates a Gram(-) organismClear colonies indicate no lactose fermentationFuchsia colonies indicate lactose fermentation.

Most normal intestinal flora ferments lactose

Unit 9 Page 17

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

Unit 9 Page 18