Course Code: MBIO 207 Course Title: Microbiology Practical- I V Department: Microbiology

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a mission with a
vision
Lab Report
Submitted to
Name:
Designation: Lecturer
Department: Microbiology
Institute: Primeasia University
Submitted by
Name: Md Azizul Haque
Student ID: 193016031
Course Code: MBIO 207
Course Title: MicrobiologyPractical-VII
Department: Microbiology
Date of Submission: 15,03, 2021

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Index
S
I
Name of Experiment Date Page
1 Carbohydrate
Fermentation 27/02/2021 2-5
2 Tripe Sugar Iron Agar
Test 20/02/2021 6-8
3 IMViC Test Part A
06/03/2021 9-13

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Experiment No: 01
Experiment Name: Carbohydrate Fermentation
Experiment Date:31-10-2020
Objectives
 To demonstrate the ability of microorganisms to ferment the
carbohydrate and production of organic acid end products.
 To determine the ability of the microorganism to produce gaseous end
products in fermentation.
Principle
Carbohydrate fermentation is the process by which the microorganism
utilizes to produce energy in the form of ATP, the ultimate energy source of
the organism. Glucose after entering a cell can be catabolized either
aerobically (in the presence of O2), where molecular oxygen serves as the
final electron acceptor (oxidative pathway), or anaerobically (in absence of
O2) in which inorganic ions can serve as the final electron acceptor
(fermentative pathway). The metabolic end products of a carbohydrate
fermentation can either be organic acids (lactic, formic, acetic acid) or
organic acid and gas (hydrogen or carbon dioxide). Fermentative
degradation of the carbohydrates (monosaccharide, disaccharide, and
polysaccharide) by microorganisms under the anaerobic condition is carried
out in the fermentation tube, which comprises of Durham tube for the
detection of the gas production. A fermentation medium is composed of a
basal medium containing a specific carbohydrate (glucose, sucrose, or
cellulose) along with a pH indicator (phenol red, Andrade’s indicator, or
bromocresol). When the organism ferments carbohydrates, organic acid
products (Lactic acid, formic acid, or acetic acid) are obtained which turns
the medium into yellow color with a reduction in the pH (acidic-below pH of
6.8). The change in the pH indicator in the fermentation tube and the gas
production in the Durham tube is indicative of the metabolic reaction with
the production of acid end product and gas. Color change only occurs and is
visible when a sufficient amount of acid is produced, as bacteria may utilize
the peptone producing alkaline by-products. The degradation of peptones in
the broth may result in the production of alkaline end products, which will
change the broth color to pink often at the top of the tube.
Materials Required:
1. Phenol Red Carbohydrate Fermentation Broth.
2. Bacterial culture.

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3. Inoculation loop.
4. Incubator(370 C).
Procedure:
I. Preparation of Carbohydrate Fermentation Broth
1. Weigh and dissolve trypticase, Sodium chloride, and Phenol red in 100 ml distilled water
and transfer into conical flasks.
2. Add 0.5% to 1% of desired carbohydrate into all flasks.
3. Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with
broth.
4. Sterilize at 1150 C for 15 minutes.
5. Important: Do not overheat the Phenol red Carbohydrate fermentation broth. The
overheating will result in breaking down of the molecules and form compounds with a
characteristic color and flavour. The process is known as caramelization of sugar (the
browning of sugar).
6. Transfer the sugar into screw capped tubes or fermentation tubes and label properly.
Ingredients of the Fermentation Broth:
1. Trypticase: 1g
2. Carbohydrate: 0.5g
3. Sodium Chloride: 0.5g
4. Phenol red : 0.0189mg
*Autoclave at 115o C for 15 minutes
II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate
Broth
1. Aseptically inoculate each labeled carbohydrate broth with bacterial culture.(keep
uninoculated tubes as control tubes).
2. Incubate the tubes at 18-24 hours at 37oC.
3. Observe the reaction.
Precautions:
1. After inoculation into a particular sugar, sterilize the loop in order to avoid cross
contamination of the tube with other sugars.
2. Keep uninoculated sugar tubes as control tubes.
3. Do not use the tubes with Durham tubes that are partially filled or with bubbles.
4. Over incubation will help the bacteria to degrade proteins and will result give false positive
results.

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Results
Bacterial
Species
Lactose
observation
(Color of
medium,
bubble in
ferment
tube.)
Results
(A)(A/G),
or (-)
Dextose
observation
(Color of
medium,
bubble in
ferment
tube.)
Results
(A)(A/G),
or (-)
Sucrose
observation
(Color of
medium,
bubble in
ferment
tube.)
Results
(A)(A/G),
or (-)
S. Aureos Yellow, No
Gas
A Yellow, No
Gas
A Yellow, No Gas A
Psedomonas
spp
Color of
medium Red,
No Gas
(-) Red, No Gas (-) Red , No Gas (-)
Shigella Spp Color of
medium
Yellow, Gas
A/G Color of
medium
Yellow, Gas
A/G Color of
medium Yellow,
Gas
A/G
Salmonella
Speceies
Color of
medium Red,
No Gas
(-) Color of
medium
Yellow ,Gas
A/G Color of
medium Yellow,
No Gas
A
Interpretation
1. Organism ferments the given carbohydrate and produces organic acids
thereby reducing the pH of the medium into acidic condition.
2. Organism ferments the given Carbohydrate and produces organic acids
and gas. Gas production is detected by the presence of small bubbles
in the inverted Durham tubes.
3. The organism cannot utilize the carbohydrate but the organism
continues to grow in the medium using other energy sources in the
medium.

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Experiment No: 02
Experiment Name: Trip SugarIron Agar Test
Experiment Date: -03-2021
Objective
To determine the ability of an organism to ferment glucose, lactose, and sucrose, and their
ability to producehydrogen sulfide.
Principle
The triple sugar- iron agar test employing Triple Sugar Iron Agar is designed to differentiate
among organisms based on the differences in carbohydrate fermentation patterns and
hydrogen sulfide production. Carbohydrate fermentation is indicated by the production of
gas and a change in the colour of the pH indicator from red to yellow.
To facilitate the observation of carbohydrate utilization patterns, TSI Agar contains three
fermentative sugars, lactose and sucrosein 1% concentrations and glucose in 0.1%
concentration. Due to the building of acid during fermentation, the pH falls. The acid base
indicator Phenol red is incorporated for detecting carbohydrate fermentation that is indicated
by the change in color of the carbohydrate medium from orange red to yellow in the
presence of acids. In case of oxidative decarboxylation of peptone, alkaline products are
built and the pH rises. This is indicated by the change in colour of the medium from orange
red to deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the medium
detects the production of hydrogen sulfide and is indicated by the black color in the butt of
the tube.
To facilitate the detection of organisms that only ferment glucose, the glucose concentration
is one-tenth the concentration of lactose or sucrose. The meagre amount of acid production
in the slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to
remain orange red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is
maintained in the butt of the tube since it is under lower oxygen tension.
After depletion of the limited glucose, organisms able to do so will begin to utilize the
lactose or sucrose. To enhance the alkaline condition of the slant, free exchange of air must
be permitted by closing the tube cap loosely.
Media:
TSI Agar
Enzymatic digest of casein (5 g), enzymatic digest of animal tissue (5 g), yeast enriched
peptone (10 g), dextrose (1 g), lactose (10 g) sucrose(10 g), ferric ammonium citrate (0.2 g),
NaCl (5 g), sodium thiosulfate (0.3 g), phenol red (0.025 g), agar (13.5 g), per 1000 mL, pH
7.3.

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Requirements
 TSIA slant
 Given sample of bacterial cultures (E.coli and Klebsiella)
 Inoculating wire
 Burner
 Incubator.
Procedure of TSI test
1. Take 5-7 ml of the autoclaved TSIA media in a sterile test tube and slant was made by
tilting the media till it solidified.
2. Using a flamed inoculating loop, pick a colony from the bacterial culture.
3. Inoculate the organism in TSIA slant by stabbing in the butt and then with the same
loop streaking on the whole slant surface of the medium
4. Incubate at 37°C for 18-24 hours.
5. Observe for the color change in the slant and butt, gas productionand H2S production.
Result interpretation:
Carbohydrate Fermentation H2 S Production
Bacteria
Species
Butt color
and Reaction
Slant color
and Reaction
Carbohydrate
Fermented
Blackening H2 S
(+) or (-)
S.Aureas Acid, Yellow Acid,Yellow. Lactose/Sucrose (-) (-)
Pseudomonas
spp.
Red, Alkali. Red, Alkali (-) (-) (-)
Klebsiella spp Acid, Yellow Acid, Yellow Lactose/Sucrose (-) (-)
Salmonella Yellow,Acid Red, Alkali Glucose (+) (+)

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*H2S gas productionis indicated by blackening of media
** Gas production is indicated by bubbles in butt
 E. coli gives acid/Acid with gas production without H2S production. This means E.
coli utilize all sugar anaerobically with productionof gas during fermentation.
*** Pseudomonasspp gives alkaline/alkaline (Red/Red)which means it does not utilize any
sugar.

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Experiment No: 03
Experiment Name: IMViC test part A. Indole Test
Experiment Date: -03-2021
Objectives of Indole Test
 To detect the formation of indole from tryptophan by the enzymatic action of
tryptophanese.
 To differentiate members of family Enterobacteriaceae, especially E.
coli from Enterobacter and Klebsiella.
Microorganisms Tested
 Fresh growth of a Gram-negative rod on a medium that does not contain dyes and
contains tryptophan, e.g., BAP or CHOC.
 Anaerobic Gram-positive rods.
 Anaerobic Gram-negative rods.
Principle:
The ability of an organism to split indole from the amino acid tryptophan is due to the
presence of tryptophanase. Tryptophan is an amino acid that undergoes deamination and
hydrolysis in the presence of the enzyme tryptophanase. Reductive deamination of
tryptophan results in the productionof indole via the intermediate molecule indole pyruvic
acid. During the deamination process,the tryptophanase catalyzes the removal of the amino
group (-NH2) from the tryptophan molecule. The enzyme requires pyridoxal phosphate as a
coenzyme. The final products ofthe catalysis reaction are indole, pyruvic acid, ammonium
(NH4
+), and energy. Indole, if present, combines with the aldehyde in the reagent to produce
a pink to red-violet quinoidal compound (benzaldehyde reagent) or a blue to green color
(cinnamaldehyde reagent). The indole combines with Kovac’s reagent (hydrochloric acid
and p-dimethylaminobenzaldehyde in amyl alcohol) solution to form a yellow or cherry red
coloration. The amyl alcohol is water-insoluble and thus, forms a red-colored oily layer at
the top of the broth. In the rapid spot test, indole is detected directly from a colony growing
on a medium rich in tryptophan. The indole combines with the p-
dimethylaminocinnamaldehyde (DMACA) present on the filter paper at an acid pH to
producea blue to the blue-green compound.
Media, Reagents, and Supplies Used
Reagents Used
 For the spottest, 5% p-dimethylaminobenzaldehyde or
1% pdimethylaminocinnamaldehyde is prepared in 10% (v/v) concentrated HCl.
 For the tube method, Kovac’s reagent is used for aerobic organisms, and Ehrlich’s
reagent is used for anaerobes and weak indole producers.
Kovac’s reagent

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Ingredients Concentration
p-dimethylaminobenzaldehydre 50 gram
Hydrochloric acid 250 ml
Amyl Alcohol 750 ml
 The medium used with this reagent is either broth containing tryptophan, motility-
indole-ornithine agar, or sulfide-indole-motility agar (SIM).
Ehrlich’s reagent
Ingredients Concentration
p-dimethylaminobenzaldehydre 1 gram
Hydrochloric acid 20 ml
Ethyl alcohol 95 ml
 The medium used with Ehrlich’s reagent is heart infusion or anaerobic medium
with tryptophan.
Supplies Used
 Sterile loop, swab, or stick for inoculation
 Filter paper (optional)
Procedureof Indole Test
Tube Test
 The broth medium or the agar medium in the tube is stabbed with the colony taken from
an 18-24-hour culture.
 For the liquid medium, a small portion of the inoculated broth is taken in a separate
tube.
 The tubes are then incubated at 37°C for 24 hours.
 For Kovac’s reagent, three drops ofKovac’s’ reagent are added down the side of the
tube, and the color change is observed at the meniscus.

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 For Ehrlich’s reagent, 0.5 ml of xylene is added to the tube and inverted to mix well.
Further, six drops ofEhrlich’s indole reagent are added down the side of the tube, and
the color is observed below the xylene layer.
ResultInterpretationof Indole Test
Tube Test
 The formation of pink-red coloration (cherry red ring) in the reagent at the point of
contact between the reagent and the medium indicates a positive result.
 The absenceof color or the appearance of slightly yellow color indicates a negative
result.
BacterialSpecies Color of reagent layer Tryprophan Hydrolysis
(+) or (-)
K/ehsiel/a spp No change Negative
S. Aureus No change Negative
Psedumonas No Change Negative
Bacillus No Change Negative
Protes, E. Coli are indole positive.

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Uses of Indole Test
 The indole test is used to test an organism’s ability to utilize tryptophan and
produce indole.
 The test is used to differentiate members of the Enterobacteriaceae family as a
part of the IMViC test.
 The test also differentiates Proteus mirabilis from other Proteus species.
 This test differentiates indole-positive E. coli from indole-
negative Enterobacter and Klebsiella.
 The test further differentiates K. pneumoniae(indole negative) from K.
oxytoca (indole positive) and Citrobacter freundii (indole negative)
from Citrobacter koseri (indole positive).
Limitationsof Indole Test
 Detectable indole will diffuse to colonies within 5 mm of a 2- to 3-mm colony,
giving false-positive results.
 Media that contain dyes (e.g., EMB, MAC) shouldn’t be used.
 The growth medium must contain an adequate amount of tryptophan. Do not
use Mueller-Hinton agar for the test, because tryptophan is destroyed during
the acid hydrolysis of casein.
 Only the cinnamaldehyde reagent can be used for spot testing of anaerobic
microorganisms. It is a more sensitive reagent, but it is less stable.
 Do not use a plate with a nitrate disk to perform the indole test, as nitrate can
interfere with the spot indole test by inducing false-negative results.
 If the rapid indole test is negative, the isolate could be positive in the more
sensitive tube test. Extraction with xylene is the most sensitive test. Xylene
substitutes are less sensitive.
 For fastidious Gram-negative rods, such as C. hominis, a heavy inoculum and
extraction are necessary.

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References and Sources
 Biochemical Tests forthe Identification of Aerobic Bacteria. (2016). Clinical
MicrobiologyProcedures Handbook, 3.17.1.1–
3.17.48.3.DOI:10.1128/9781555818814.ch3.17.1
 Maria P. MacWilliams. 2009.Indole testprotocol.
 Kovac’s Indole ReagentR008. HiMedia Laboratories.
 5% – https://asm.org/getattachment/200d3f34-c75e-4072-a7e6-
df912c792f62/indole-test-protocol-3202.pdf
 3% – https://microbiologyinfo.com/indole-test-principle-reagents-procedure-
result-interpretation-and-limitations/
 2% – https://himedialabs.com/TD/R008.pdf
 2% – http://universe84a.com/indole-test-introduction/
 1% – https://en.wikipedia.org/wiki/Indole_test
 1% –
https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/IndoleTestRgnts.htm