l

23 Exercise

Determination of
Bacterial Properties-
Carbohydrate
Fermentation

Objectives

Uponcompletion of thislaboratory exercise,you will be able to:

l. Name three processes by which cells metabolize

carbohydrates to generate energy.

2. Explain the purposes of the indicators used in this exercise.

3. Explain the relationship between enzymes and specific

substrates, and how they are used to identify microorganisms.

4. Perform accurately and observe the results of the procedures

for determining how microorganisms utilize carbohydrates.

139
 > Introduction

One of the ways cells generate energy is to metabolize carbohydrates such as

glucose,lactose, and mannitol. Cells use this energy for active transport, growth,
and the synthesis of organelles. Microbesuse three processes to 'transduce, or
convert, energy from carbohydrates into a form they can use:
(a) aerobic respiration : A seriesof energy-producing
chemicalreactionsinvolving
the transfer of electrons, with oxygen as the final electron acceptor.
..,
(b) anaerobic respiration: A series of energy-producingchemical reactions
involving electron transfer with an inorganic molecule other than oxygen
as the final electron acceptor.Nitrate, sulfate, and carbonate are examplesof
electron acceptors used in anaerobic respiration.

(c) fermentation : The incompletemetabolismof glucoseor other carbohydrates

to form pyruvate. Further metabolismof pyruvate yields other fermentation
products,such as acids,gases,and alcohols.These productsare producedalone
or in combinations, dependingon the organismcarryingout the fermentation
process. Oxygen is not required for fermentation.

PartI - Fermentation
Tubes

Fermentationtubes are often used to determinefecalcontaminationof food,water,

and dairy products. A positive result for fermentation in these tubes is an initial
indication of the presence of enteric bacteria in a specimen.
Fermentation tubes consist of three components:
(1) 0.5% sugar broth, which is the substrate for fermentation. A substrate is
a molecule to which an enzyme hinds in a chemical reaction;

(2) A pH indicator, phenol red. Phenol red is red at a pH of 7 and yellow at

a pH between 6.8 and 5.5. Acidsare produced as a product of fermentation,
causing the phenol red to turn yellow; and

(3) A Durham tube, which is a small inverted test tube within the larger test
tube containing the sugar broth. This tube's function is to collectgas, which
is a product of fermentation.If gas is produced, bubbles appear in the Durham
tube and displace the fluid in the Durham tube.

You will use fermentation tubes in this activity to analyse a microbe's ability
to ferment glucose, lactose, and mannitol.

140
 l

fermentation acids yellow color

Glucose~
Lactose
Mannitol
fermentation acids yellow color and
and gas ~ubbles in the
Durham tube

Figure23.1

Durham Gas
tube (bubble)

Original
Acid
color
(yellow
(red)

No Acid Acid and

fermentation production gas production

Figure23.2

Materials

cultures of Enterobacteraerogenes,Escherichiacoli, Proteus vulgaris,and

Pseudornonasaeruginosa
1 tube of phenol red glucose broth
1 tube of phenol red lactose broth
1 tube of phenol red mannitol broth

Procedure

0 1. Obtain one of the cultures listed above, and label the media with the name
of your culture.

CJ 2. Inoculate each tube with your culture.

0 3. . Incubate the tubes at 37°C for 24-48 hours.

141
 0 4. Afterincubation,examinethe appearanceof the tube.Basedon your observations,
record the results.If the microorganismfermentsthe sugar and producesacids,
the pH of the medium will decrease and the color of the pH indicator will
turn from red to yellow.If gas is produced,bubbleswill appear in the Durham
tube and displace the fluid.

0 5. Observethe fermentation tubes containingthe three other organismsstudied

by other students, and record the results in the table provided.

Results

Complete the table below. Indicate acid and/or gas production with a plus (+).
If no acid or gas is produced, place a minus (-) in the space provided.

Organism Glucose Lactose Mannitol

Acid/Gas Acid/Gas Acid/Gas

E. aerogenes

E. coli

P. vulgaris

P. aeruginosa

PartII - Oxidative/Fermentative
Useof Glucose

Certainmicrobesfermentglucose.Otherorganismsonly can use glucoseoxidatively

(aerobically), meaning they need oxygen to metabolize glucose. There are still
others that can use glucose both oxidativelyand fermentatively. These different
metabolic properties can be examined using OFglucose medium. This green-
colored medium contains a pH indicator called bromthymolblue, which changes
the color of the mediumto yellowif acidsare producedfrom glucosemetabolism.
Oxidative
_ __ .,. Aerobic
use of glucose: Glucose -- - Yellow color in
Respiration tube without

T
mineral oil only

Fermentative
use of glucose : Glucose .,. Fermentation Yellow color in
tube without
mineral oil . .,

Yellow color in
tube with
mineral oil
Figure 23.3

142
 '
Materials

cultures of Enterobacteraerogenes,Escherichiacoli,Proteusvulgaris,and
Pseudomonas
aeruginosa
2 tubes of OF glucose medium
sterile mineral oil

Procedure

0 1. Obtain the culture you have been assigned to work with, and label 2 tubes
of OF glucose medium with the name of the culture.

0 2. Inoculate each tube with your culture.

0 3. To create an anaerobic environment for fermentation, overlay one tube with

a 1/2 inch of sterile mineral oil. Gentlypour the mineral oil into the
inoculatedtube, so the mineral oil rests on top of the OFglucosemedium.

0 4. Incubate the tubes at 37°C for 24-48 hours.

0 5. Afterincubation,examinethe appearanceof the tubes.Basedon yourobservations,

record the results. If the tubes with and without the mineral oil turn yellow,
the microorganismuses glucose fermentatively.If only the tube without the
mineral oil turns yellow, the microorganism uses glucose oxidatively.

0 6. Observe the tubes containing the three other organisms studied by other
students, and record the results in the table provided.

>-Results
Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observedappearance.

Organism Appearance with Appearance without Interpretation

mineral oil mineral oil

E. aeroge11es

E. coli

P. vulgaris

P. aeruginosa

143
 PartIll - MethylRedTest(MR)

Certain microbes ferment glucose yielding multiple acids, such as formic, acetic,
lactic,and succinicacids.Thisis termed mixed acid fermentation. The production
of multiple acids produces a highly acidic medium, which is hest detected using
the pH indicator methyl red. Methyl red is yellow at pH 6.4 and red at pH
4.4. The mixed acid fermentation test is often used to distinguish between two
closely related enteric organisms,Escherichiacoli and Enterobacteraerogenes,
which are commonly associated with diarrheal illness.

Fermentation methyl red indicator

Glucose formic, acetic, red color
lactic and succinic
acids

Figure23.4

Materials

cultures of Enterobacteraerogenes,Escherichiacoli, Proteus vulgaris,and

Pseudomonasaeruginosa
1 tuhe of MR-VPmedium
methyl red indicator

Procedure

0 1. Obtain the culture you have been assigned to test, and lahel the tube of MR-
VP medium with the name of your culture.

0 2. Inoculate the tube with the culture.

0 3. Incubate the tubes at 37°C for 5 days.

0 4. After incubation, add 5-10 drops of methyl red indicator. Shake the tube
from side to side so that the liquid does not spill out of the test tube. Examine
the tube.

lJ 5. Based on your observations, record the results of your test. lf the medium
is red, the microorganismferments glucoseto produce mixed acids.A yellow
color indicates the organism does not produce mixed acids.

D 6. Observe the tubes containing the other three organisms studied by other
students, and record whether these organisms produce mixed acids.

144
 l

> Results

Completethe table below.Describethe appearance of the tube by writing its color.

In the columnlabeled "interpretation" provide a brief explanation for the observed
appearance. •

Organism Appearance Interpretation

E. aerogenes

E. coli

P. vutgaris

P. aeruginosa

PartIV - Voges-Proskauer
Test(VP)

Certain nticrobes ferment glucose to yield alcoholicend products such as ethanol

and 2,3 butanediol. This process is known as alcoholic fermentation. Microbes
with the ability to ferment glucose to produce alcoholicend products are detected
using the Voges-Proskauer test. This test indicatesthe presence of a fermentation
intermediatecalled acetylmethylcarbinol,also known as acetoin.Barritt's reagent
is used to detect this intermediate compound.Barritt's reagent reacts with acetoin
to produce a pink-red color. Like the methyl-red test, the Voges-Proskauertest
is helpful in differentiating Escherichia coli and Enterobacter aerogenes.

Glucose ---- acetylmethylcarbinol 2,3 butanediol

rI,,,,-- Barritt's
re agent
+
ethanol

Pink color

Figure23.5

Materials

cultures of Eulerobacter aerogenes, Escltericltiacoli, Proteus vulgaris, and

Pseudomo1wsaeruginosa
1 tube of MR-VP medium
Barritt's reagent A: alpha-naphthol
Barritt's reagent B:potassiumhydroxide

145
 Procedure

0 1. Obtain the culture you have been assigned to test and label the tube of MR-
VP medium with the name of your culture.

0 2. Inoculate the tube with the culture.

0 3. Incubate the tubes at 37°C for 24-48 hours.

0 4. After incubation,add 18 drops of Barritt's reagent A and 18 drops of Barritt's

reagent B. Shake the tube gently from side to side and allow it to stand
for 15 minutes.

0 5. Examinethe appearance of the tube. Based on your observations,record the

results. If the mediumis pink-red,the microorganismfermentsglucoseto yield
alcoholicend products.A yellowcolor indicatesthe organismdoes not produce
alcoholic end products.

0 6. Observe the tubes containing the other organismsstudied by other students,

and record whether these organisms produce alcoholic end products.

Results

Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observed appearance.

Organism Appearance interpretation

E. aerogenes

E. coli

P. vulgaris

P. aeruginosa

PartV - CitrateUtilization

Certain microbes use citrate as their sole source of carbon. The ability to use
citrate relies on the enzymecitrate permease . Citratepermeasehelps to transport
citrate across the cell membrane into a cell. Once within the cell, citrate enters
the Krebs cycle,causing the production of ATPmolecules.which provide energy,
and the byproduct CO2 •

146
 l

Citrateutilizationis detected using Simmon's citrate agar. This mediumcontains

sodium,along with the pH indicatorbromthymol blue. If an organismmetabolizes
citrate, the CO, produced combines with sodium to form sodium bicarbonate
(Na2CO3), which increases the medium's pH. The medium's color changes from
green to blue. Along with the methyl red and Voges-ProskauerteHs, citrate
utilizationis used in the clinicalidentificationof Escherichiacoli and Enterobacter
aerogenes.
citrate permease
Citrate Pyruvate + CO 2 + excess Na (in medium)
(green)

l
Na 2 C0 3
(increases pH of medium)
blue color

Figure23.6

Materials

cultures of Enterobacteraerogenes,Escherichiacoli,Proteusvulgaris,and
Pseudomonas
aeruginosa
1 Simmon's citrate agar slant

Procedure

0 1. Obtain the culture you have been assigned to test, and label the tube of
Simmon's citrate agar with the name of your culture.

0 2. Inoculate the tube with your culture.

0 3. Incubate the tubes at 37°C for 24-48 hours.

0 4. Afterincubation,observethe appearanceof the tube.Basedon your observations,

record the results. If the medium has changed from green to blue, the
microorganismuses citrate as a sole carbon source. A green color indicates
the organism does not possess citrate permease and therefore does not use
citrate as a carbon source.

0 5. Observe the tubes containing the other organisms studied by other students,
and record whether these organisms use citrate as their sole carbon source.

147
 >--Results
Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observed appearance.

Organism Appearance Interpretation

E. aerogenes

E. coli

P. vulgaris

P. aeruginosa

>--ReviewQuestions
1. Basedon thetestc;perfom1edin the laboratoryexercise,whatare the threepossible
end products of fermentation?

2. Usingphenolred fermentationtubes,howaregaseousendproductsoffem1entation
detected?

3. What test(s) wouid you perform if you wanted to determine if an organism

produces acids upon fermentation?

4. Define the term: mixed acid fermentation.

148
 l

5. What type of fermentation is detected with the Voges-Proskauertest?

6. What is the name of the intermediate detected by Barritt's reagent?

7. Whyare differentindicatorsused for the methylred and Voges-Proskauer

tests?

8. Bacterium "A" is inoculated into oxidative/ fermentative (OF) glucose

medium and only the tube with the mineral oil overlay turns yellow.
Describe the appearance of Bacterium "A" if inoculated into phenol red-
glucose medium.

9. Why can't mixed acid fermentation be detected using phenol red glucose
medium?

10a. Name the enzyme being tested for with Simmon's citrate agar.

10b. What energy producing pathway is detected using Simmon's citrate agar?

149
 11. Explain the color change that results when an organism utilizes citrate in
Simmon'scitrate agar.

>- For Further Thought

1. Design an experiment to determine whether an organism can carry out fermentation or respiration.

..
2. You and your friends decide to make wine. What specific materials would you need and how would you
go about making the wine?

150
 l

Exercise 24
Determination of
Bacterial Properties-
Protein Metabolism

Objectives

Uponcompletion of thislaboratory exercise,you will be able to:

l. Explain why the processes of deamination and

decarboxylation are vital for cellular metabolism.

2. State two cellular components containing amino acids.

3. Explain why gelatinase-producing bacteria can effectively

and rapidly spread throughout the body.

4. Perform accurately the laboratory procedures and observe

and record the results for distinguishing microorganisms based
on protein metabolism.

151
 >- Introduction
Beyond carbohydrate metabolism, protein metabolism is also used to identify
microorganisms.Bacterial cells hydrolyze proteins into amino acids, which are
then synthesized into functional cellular components such •as enzymes and
membrane proteins. Many pathogenic microbes, such as those in the genus
Pseudomonas and Clostridium, produce enzymes that degrade proteins. These
enzymescausetissuedamageduringmicrobialinfections.Protein-degradingenzymes
have been isolated from bacteria and are used to tenderize meat, prepare hides
for tanning, and are added to some detergents to dissolve stains.
There are two further reactionsby whichaminoacidsare brokendown:deamination
and decarboxylation.Deamination is the removalof the amino group (NH2) from
an amino acid resultingin the eliminationof ammonia(NH3) , which is potentially
toxic to cells. The remaining carbon structure is converted into one or more
metabolic intermediates that can be further catabolized to yield ATP.
Decarboxylationis the removal of the carboxyl (COO·) group from an amino
acid.The productsgeneratedare carbondioxideand nitrogen-containingcompounds.
Carbon dioxide can be incorporated into carbohydrate and nitrogen metabolism.
The nitrogen-containingcompounds serve as precursors for synthesizingother
molecules.

PartI - Tryptophan
Hydrolysis
(lndoleProduction)

Tryptophan hydrolysis is an example of a deamination reaction. The enzyme

tryptophanase hydrolyzesthe amino acid tryptophan to produceindole, pyruvic
acid, and ammonia (NHJ Pyruvicacid and ammonia are used by bacterial cells
to satisfy nutritional needs, whereas indole is not. lndole production is detected
using Kovac's reagent. Kovac's reagent reacts with indole to produce a red
compound. Indole production is commonlyused to distinguish Escherichiacoli
and Enterobacteraerogenes, two enteric organisms.

Tryptophan _ t~ry~pt~op_
ha_
n_as_e _

r-
lndole + Pyruvic acid + NH 3

red compound
Kos ac·, ,eage o<

Figure24.1

Materials

cultures of Enterohacter aerogenes, Escherichia coli, Proteus vulgaris, and

Pseudomonasaerup,inosa
1 tube of tryptone broth
Kovac's reagent

152
 l

Procedure

0 1. Obtainthe cultureyou have been assignedto test, and labelthe tube of tryptone
broth with the name of the culture.

0 2. Inoculate the tube with your culture.

0 3. Incubate the tube at 37°C for 24-48 hours.

0 4. Afterincubation,add IO drops of Kovac'sreagent to the tube.It is not necessary

to shake the tube in this case, because a color change will appear at the
top of the broth.

0 5. Examinethe appearance of the tube. Based on your observations,record the

results. If the microorganismpossessestryptophanase,indole produced by the
action of this enzyme reacts with Kovac's reagent to form a red ring at the
top of the broth. If no red ring appears, the organism does not contain the
enzyme tryptophanase.

0 6. Observe the tubes containing the other organisms studied by other students,
and record the results in the table.

> Results

Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observed appearance.

Organism Appearance Interpretation

E. aerogenes

E. coli

P. vulgaris

P. aeruginosa

PartII - Phenylalanine
Deamination

The amino acid phenylalanine, in the presence of the enzyme phenylalanine

deaminase (PAD), yieldsthe organicacidphenylpyruvicacidand ammonia.Certain
organismsuse organicacidsin biosyntheticreactions. Phenylpyruvicacidis detected
using ferric chloride (FeCIJ which reacts with the acid to produce a green color.
Bacteria of the genus Proteus are all positive for PAD.Since these bacteria cause
urinary-tract infections. woundinfections,and septicemia(bloodpoisoning),this test
is used to help identify organisms of this genus.

153
 Phenylalanine ___ PA_D__ ..

r
Phenylpyruvic acid + ammonia

,,c,,(oraoge) 1

Green color

Figure24.2

Materials

cultures of Enterobacteraerogenes,Escherichiacoli, Proteus vulgaris, and

Pseudomonasaeruginosa
1 phenylalanineslant
ferric chloride

Procedure

0 1. Obtainthe culture you have been assignedto test, and label the phenylalanine
slant with the name of the culture.

0 2. Inoculate the tube with your culture.

0 3. Incubate the tubes at 3rC for 24-48 hours.

0 4. After incubation, add 1.0 ml of ferric chloride to the growth on the slant.

0 5. Examinethe appearance of the tube and record your results. If the PADhas
deaminated phenylalanine,the product phenylpyruvicacid will react with the
ferric chloride, resulting in a green color. If no green color is visible, the
organism does not contain PADand no deamination has occurred.

U 6. Observe the tubes containing the other organismsstudied by other students,

and record the results in the table.

>-Results
Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observedappearance.

Organism Appearance Interpretation

E. aerogenes

E. coli

P. t·ulgaris

P. aeruginosa

154
 l

PartIll - LysineDecarboxylase

When the amino acidlysine is decarboxylatedby the enzymelysine decarboxylase

{LDC), CO, and cadaverine (an alkaline substance) are produced. Cadaverinewas
named because it is a foul-smellingcompound associated with the putrefaction
of the fleshof cadavers.LDCis activatedunder acidicconditions.Lysinedecarboxylase
broth is used to test for the presence of LDC.This medium contains a pH indicator
called bromcresol purple, which is yellow at an acidic pH and purple at an
alkaline pH. Glucose is also present in the medium. An acidic environment is
generated in the medium when bacteria ferment the glucose. This decrease in
pH stimulates the enzyme LDCto carry out its reaction, thereby generating the
end products CO2 and cadaverine.These products increase the pH of the medium
to the alkaline range, causing the color of the medium to change to purple.
Testing for lysine decarboxylase is commonly performed in clinical labs for
distinguishing bacteria in the family Enterobacteriaceae.

LDC activated at an
Lysine acidic pH (yellow) Cadaverine + CO2

l
purple color at alkaline pH

Figure24.3

Materials

cultures of Enterobacteraerogenes,Escherichiacoli, Proteus vulgaris,and

Pseudomonasaeruginosa
1 tube of lysine decarboxylasebroth
sterile mineral oil

Procedure

CJ 1. Obtain the culture you have been assigned to test, and label the lysine
decarboxylase broth with the name of the culture.

u 2. Inoculate the tube with your culture.

u 3. Overlay the tube with mineral oil (approximately 1/ 2 inch) to create an

anaerobic environment for fermentation of glucose.

u 4. Incubate the tubes at 37° C for 24-48 hours.

l.J 5. Afterincubation,examinethe appearanceof the tube. Basedon your observations

,
record the results. If the microorganismpossesses the enzyme LDC, the tube
will appear purple. If no LDCis present, the tube will appear yellow.

155
 0 6. Observethe tubes containing other organismsstudied by other students, and
record the results in the table.

> Results

Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observedappearance.

Organism Appearance Interpretation

E. aerogenes

E. coli

P. vulgaris

P. aeruginosa

PartIV - UreaHydrolysis

Microbesthat contain the enzymeurease detoxifyurea, a waste productof protein

metabolism.These microbes hydrolyze urea to form carbon dioxide, water, and
ammonia. Urea broth is used to test for urease activity. This medium contains
urea and the pH indicator phenol red. If urease is present, ammonia is produced,
increasing the pH of the medium and causing the color of the medium to change
from orange to hot pink/ magenta.No color changeindicatesthe absenceof urease.
The urea hydrolysis test is used in clinicallaboratories to identify organisms of
the genus Proteus, which often cause urinary-tract infections. Urease-producing
Proteus causes the formation of renal stones and kidney damage by increasing
the amount of ammonia (elevating the pH) in urine.

Urea ----- urease

• CO 2 + H2 O +

r
(orange broth)

hot pink/magenta color

Figure24.4

156
 l

Materials
cultures of Enterobacteraerogenes,Escherichiacoli,Proteusvulgaris,and
Pseudomonas
aeruginosa
1 tube of urea broth

Procedure

0 1. Obtain the culture you have been assigned to test, and label the urea broth
with the name of the culture.
0 2. Inoculate the tube with your culture.
0 3. Incubate the tubes at 37°C for 24-48 hours.
0 4. Afterincubation,examinethe appearanceof the tube.Basedon your observations,
record the results. A positive result for urease production is associatedwith
the color hot pink/ magenta. No color change indicates no urease is produced
by the microbe.
0 5. Observe the tubes containing other organisms studied by other students, and
record the results in the table.

>-Results
Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observed appearance.

Organism Appearance Interpretation

E. aerogenes

E. coli

P. vulgaris

P. aerugiuosa

PartV - GelatinHydrolysis
Certain microbescontain the enzymegelatinase, whichhas the abilityto hydrolyze
gelatininto aminoacids.Microbesare tested for gelatinaseactivitybecauseorganisms
that can degradegelatinare also able to degrade collagen.Therefore, they can spread
rapidly throughout the collagen-containingconnective tissue in the human body.
Manyspeciesof Clostridimn producecollagenaseand causeprogressivetissuedamage.
Youcan determinewhether a microbecontainsgelatinaseby inoculatingit into tubes
of gelatin.Gelatinliquefiesat the incubationtemperatureof 37°C.Gelatinaseactivity
also causes gelatin to liquefy.Jf, after incubation, a culture that contains gelatin is
refrigerated,the culture willsolidify.If the culture containsgelatinase,however, the
culturewillremainliquiddespitethe lowrefrigerationtemperature, becausethe gelatin
has been hydrolyzed into amino acids.

157
 Gelatin -+
-----'g'-el_at_ina_s_e

r
Amino acids

,.fri,.,,,.,
liquid

Figure24.5

Materials

cultures of Enterobacteraerogenes,Escherichiacoli,Proteusvulgaris,and
Pseudomonas
aeruginosa
1 tube of gelatin

. '
Procedure

0 1. Obtainthe culture you have been assignedto test, and label the tube of gelatin
with the name of the culture.

0 2. Inoculate the tube with your culture.

0 3. Incubate the tubes at 37 ° C for 24-48 hours.

0 4. After incubation, refrigerate the tubes for 5-10 minutes.

0 5. After refrigeration, examine the appearance of the tube. Based on your

observations, record the results. Tilt the tube to see if the medium is liquid.
If the culture is liquid, gelatinase is produced by the organism,causinggelatin
hydrolysis. If the medium appears solid, no gelatinase is present.

0 6. Observethe tubes containing other organismsstudied by other students, and

record the results in the table.

> Results

Completethe tablebelow.Indicatewhetheror notyourtubesolidifiedafterrefrigeration.

In the column labeled "interpretation" provide a brief explanation of your results.

Organism Appearance Interpretation

E. aerogenes

[. coli

P. rnlgaris

p aemginosa

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PartVI - HydrogenSulfide(H2S) Production

Desulfuraseenzymesremove sulfur from the sulfur-containingamino acids,cysteine

and methionine, releasing gaseous hydrogen sulfide (H.,S).Kligler's iron agar
is used to detect hydrogen sulfide (H2S) production. GaseousH1S /eacts with the
iron in the medium, causing a black precipitate to form.
H ,S is produced by the anaerobic organismsfound in sewage-pipesystems.These
- J Sulfuricacid corrodes the
bacteria oxidize H-,S to produce sulfuric acid (H,SO
concrete piping of sewage systems, which can lead to sewage contamination in
the surrounding area.
Desulfurase
Cysteine/ Methionine H2 S (gas)

},--'""
Black precipitate

Figure24.6
Materials

cultures of Enterobacteraerogenes,Escherichiacoli, Proteusvulgaris,and

Pseiutomonas
aeruginosa
1 Kligler'siron agar slant

Procedure

0 1. Obtain the culture you have been assigned to test, and label the Kligler's
iron agar slant with the name of the culture.

0 2. Inoculate the tube with your culture by stabbing the medium with the
inoculating needle. Then, streak the slant with the inoculating loop.

0 3. Incubate the tubes at 37°C for 24-48 hours.

0 4. Afterincubation, examine the appearanceof the tube and record your results.
If a black precipitate is present at the bottom of the tube, then H ,S has been
produced by the organism. ·

lJ 5. Observe the tubes containingother organisms studied by other students, and

record the results in the table.

159
 > Results

Complete the table below. Describe the appearance of the tube by writing its
color. In the column labeled "interpretation" provide a brief explanation for the
observedappearance. •

Organism Appearance Interpretation

E. aerogenes

E. coli

P. vulgaris

P. aernginosa

> ReviewQuestions

1. Explainthe differencebetweendeaminationand decarboxylationofaminoacids.

2a. Name the products produced as a result of tryptophan hydrolysis.

2b. Of the products listed in the answer to the above question, which one reacts
with Kovac's reagent?

3a. Name the medium used to test for phenylalaninedeaminase (PAD).

160
 l

3b. Describethe appearance of a positive phenylalanine deaminase test.

3c. Name the reagent used in the PADtest and the product it detects.

4. Explainwhy a negativelysinedecarboxylasetest appearsyellowand a positive

testappearspurple.Includein youranswerchangesin pHtakingplace.
decarboxylase

5a. Name the indicator in urea broth.

5b. What are the products generated as a result of urea hydrolysis?

5c. Is a positive urease test due to an increase or decrease in pH? Basedon your
observations, how do you know this?

5d. Ureaseproducingbacteriaarecommoncausesofurinarytractinfections.
Explainwhy.

161
 6. Youare told a particular microorganismis a "flesh-eatingbacterium" because
it degrades collagen.Wouldyou expect this organism to be able to degrade
gelatin?Explainyour answer.

7a. Name the medium used to test for H2S production.

7b. Describethe appearance of a H2S-producingorganismin this medium.

7c. How does this reaction take place?

> For FurtherThought

I. Throughout this laboratory, you have made continual use of indicator chemicalsto detect specificmetabolic
products. What assumptionsabout indicators are you makingwhen you use them? Could these assumptions
influence your results?

2. ln many areas of the world, people consume microbes, such as yeast, as food. This food has been named
single-cellprotein (SCP). Explain the advantages and disadvantages of consuming SCP.

162