Download Unit 9 - Central New Mexico Community College

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.
Glucose
Glycolysis
2 ADP + 2 P
2 NAD+
2 NADH + 2H+
2 ATP
2 Pyruvate
2 NADH + 2H
Fermentation
2 NAD
Organic Acid Waste
OR
Alcohol Waste
Image created by Karen Bentz, 2016
Unit 9 Page 1
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Unit 9
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 lowoxygen 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, 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.
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Unit 9
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.
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
H2S reacts with ferrous sulfate (an ingredient in the media that is a source of iron ions (Fe2+))
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.
Unit 9 Page 3
Unit 9
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
Video Links:



Videos created by Corrie Andries
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
Sema
Ef
Ec
Ec
Pv
Ec
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.
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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.
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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
Ec
Sucrose
Lactose
Sucrose
Ec
Pv
Pv
E
c
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.
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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.
MR
VP
MR
VP
Ec
Ec
Se
ma
Se
ma
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.
Lactase
Lactose
glucose + galactose
Accessed 8/31/15 from
http://www.microbelibrary.org/library/laboratory-test/2927lactose, but licensed for use by the American Society for
Microbiology, Creative Commons Attribution – Noncommercial –
No Derivatives 4.0 International license.
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.
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Unit 9
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.
Name of Bacteria
Selective Features:
Result: Did it
grow on the
MacConkeys?
Interpretation:
Is it G-?
Unit 9 Page 10
Differential Feature:
If Bacteria Grew,
Result: Did it
turn a fuchsia
color?
Interpretation:
Does it ferment
lactose?
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
Enzymes Involved in TSI Reactions
Sucrase
sucrose
glucose + fructose
Lactase
lactose
glucose + galactose
Thiosulfate reductase
Accessed 2/17/2015 Creative Common copyright, Public Domain.
https://commons.wikimedia.org/wiki/File:TSIagar.JPG
thiosulfate
H2S
Cysteine desulfhydrase
cysteine
H2S, pyruvate, ammonia
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:
Result:
(K/K,
K/A,
or A/A)
Interpretation: What sugars
does this organism
catabolize?
(glucose, sucrose, lactose)
Was CO2
Produced?
Was H2S
Produced?
Is the media
pushed up,
and/or bubbles
seen in tube?
Is there a black
precipitate in
the media?
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
Enzymes Involved in
Carbohydrate Catabolism Reactions
Sucrase
sucrose
glucose + fructose
Lactase
lactose
glucose + galactose
Photo by Karen Bentz
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
Acid (A)
Production?
1. Which bacteria produce the enzyme lactase?
2. Which bacteria produce the enzyme sucrase?
Unit 9 Page 15
Gas (G)
Production?
Sucrose
Acid (A)
Production?
Gas (G)
Production?
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
MR Positive/VP Negative
indicates acid waste products.
MR Negative/VP Positive
indicates alcohol waste products.
Photo provided by Janet Robertson, CNM Microbiology Student, Fall 2016.
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
Result:
Red or not
red?
Interpretation:
Are organic acid
wastes produced?
Voges Proskauer
Result:
Red ring or no
red ring
Interpretation:
Alcohol waste
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
Produce:
Sucrase?
Yes or No
Give ALL the test names and evidence that led to this decision.
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
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
Produce:
Sucrase?
Yes or No
Give ALL the test names and evidence that led to this decision.
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 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
Produce:
Sucrase?
Yes or No
Give ALL the test names and evidence that led to this decision.
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
Produce:
Sucrase?
Yes or No
Give ALL the test names and evidence that led to this decision.
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
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?
7. Organic wastes from fermentation pathways are often either __________________ or
______________________.
Unit 9 Page 22
Unit 9
8. Fill in the blanks with ALL of the letters that are correct regarding the following media.
MacConkey ___________
MR-VP _____________
TSI _______________
Carbohydrate tubes _______________
a.
b.
c.
d.
e.
f.
g.
h.
i.
Lactose, sucrose and glucose are the three sugars in this media.
The pH indicator in this media is phenol red.
The pH indicator in this media is methyl red.
The pH indicator in this media is neutral red.
This media contains an inverted Durham tube for gas collection.
The tubes in this test contain a single sugar, either lactose or sucrose.
This media contains crystal violet and bile salts.
Thiosulfate and ferrous sulfate are ingredients in this media.
Barritt’s reagents VP-A and VP-B are added to this media post-incubation.
Unit 9 Page 23
Unit 9
Table 9-1. Carbohydrate Catabolism Media
Media
Triple Sugar Iron
(TSI)
Inoculate with a
needle; streak and
stab
Ingredients
T-soy agar plus:





Glucose, lactose and
sucrose.
Thiosulfate (sulfur source)
Ferrous sulfate (H2S
indicator)
Peptone (source of
cysteine)
Phenol red pH indicator
What Positive/Negative
Results Look Like
Sugar usage:
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.
Carbohydrate
Fermentation
Tube with Durham
Tube
Inoculate with a
loop.
Methyl RedVoges-Proskauer
(MR-VP)
Inoculate 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
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)
Interpretation of Results


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.
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.
Carbon dioxide is often a byproduct of
fermentation.
A black precipitate means the bacteria
produced the enzyme Thiosulfate reductase or
cysteine desulfhydrase

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.
A positive MR test will be a red color
after the pH indicator is added. A
negative test will be yellow.
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
Of Special Note



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.
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).
Unit 9 Page 24
Always assume that a black precipitate
indicate metabolism of glucose.
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.
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.
Pyruvate
Ethanol
Lactate
Succinate
2,3 Butanediol
Formate
Propionate
Acetyl Co-A
Acetate
Ethanol
Acetoacetyl Co-A
Acetone
Butyryl Co-A
Butyrate
Isopropanol
Butanol
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 25