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Identification Of Pseudomonas sp.
INTRODUCTION
The classical approach to bacterial identification involves preliminary
microscopic examination of the gram-stained preparation for its categorization
into two broad groups which would later form the basis for the selection of
biochemical tests to be performed to test their identity. The purpose of this
experiment is to characterize and identify Pseudomonas sp. by studied staining
and biochemical methods.
REQUIREMENTS
¾ MR-VP broth
Glass goods: ¾ Tryptone water
¾ Test tubes ¾ Chistensen’s Urea agar
¾ Petri dishes
¾ Conical flask Reagents:
¾ Beaker ¾ Gram staining reagents.
¾ Pipettes ¾ Oxidase reagents
¾ Hanging drop slide ¾ Catalase reagents
¾ Slides and cover slips ¾ Hydrogen peroxidase
Media: Others:
¾ Nutrient broth ¾ Staining tray
¾ Nutrien agar ¾ Inoculating loop and niddle
¾ MacConkey agar ¾ Cotton swabs
¾ Blood agar ¾ Autoclave
¾ Muller Hingtone agar ¾ Incubator
¾ Simmons citrate agar ¾ Microscope
¾ Triple sugar iron agar ¾ Wax marking pencil etc
PROCEDURE
Day-1
1. The unknown bacterial culture is streaked on the surface of dried agar
plates.
2. The plates are incubated at 37oc for 24-48 hours.
Day-2
3. Colony characteristics are observed.
4. Presence of motility observed by Hanging drop method
5. The gram, acid-fast and endospore staining are performed.
6. The biochemical tests are performed.
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INDOLE PRODUCTION TEST
INTRODUCTION
The indole test is a biochemical test performed on bacterial species to
determine the ability of the organism to split indole from the amino acid
tryptophan. This division is performed by a chain of a number of different
intracellular enzymes, a system generally referred to as "tryptophanase."
PRINCIPLE
Indole is generated by reductive deamination from tryptophan via the
intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the
deamination reaction, during which the amine (NH2) group of the tryptophan
molecule is removed. Final products of the reaction are indole, pyruvic acid,
ammonia (NH3) and energy. The indole produced during the reaction is detected
by the addition of Kovac’s reagent (dimethylaminobenzaldehyde) which
produces a cherry-red reagent layer.
TRYPTOPHANASE
Tryptophan Indole + Pyruvic acid +NH3
Indole + Kovac’s reagents Rosindole + H2O
A positive result is shown by the presence of a red or red color in the
surface alcohol layer of the broth. A negative result appears yellow. A variable
result can also occur, showing an orange color as a result.
METHOD
9 Bacterial culture is inoculated in tryptone broth and incubated at 37°C for
48 hours.
9 1 ml Kovac’s reagent is added and gently shaked and observed for a red
color ring in around the interface between the broth and the alcohol
reagent.
INTERPRETATION
Red- color Indole- Positive
Yellow color Indole - Negative
Orange color Indole - Variable
RESULT
The unknown bacterial culture is Indole negative.
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METHYL-RED TEST
PRINCIPLE
The Methyl-Red test tests for the ability to perform mixed-acid fermentation.
MR-VP broth contains glucose, peptone, and a phosphate buffer. Organisms that
perform mixed-acid fermentation produce enough acid to overcome the
buffering capacity of the broth, so a decrease in pH results. Organisms that
perform other kinds of fermentation cannot overcome the buffering capacity of
the broth.
After incubation, the pH indicator Methyl Red is added to the broth. Methyl
Red is red at pH below 4.4 (this would be a positive result) and yellow at pH
above 6.0. An orange color indicates an intermediate pH and would be
considered a negative result.
METHODS
9 Obtained two MR-VP broths in sterile test tubes.
9 Inoculated one broth using aseptic technique. The other broth
uninoculated (this will be a control).
9 Incubated at 370emperature for two days.
9 Broths obtained from the incubator.
9 Added a dropful of Methyl Red to each broth.
9 Observed the color (which should develop within a few minutes).
INTERPRETATION
Red color MR- Positive
Yellow color MR- Negative
Orange color MR- Variable
RESULT
The subjected bacterial culture is MR-negative.
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SIMMON’S CITRATE AGAR TEST
INTRODUCTION
Simmons citrate agar tests the ability of organisms to utilize citrate as a
carbon source. Simmons citrate agar contains sodium citrate as the sole source
of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen,
other nutrients, and the pH indicator bromthymol blue. This test is part of the
IMViC tests and is helpful in differentiating the Enterobacteriaceae.
PRINCIPLE
Organisms which can utilize citrate as their sole carbon source use the
enzyme citrase or citrate-permease to transport the citrate into the cell. These
organisms also convert the ammonium dihydrogen phosphate to ammonia and
ammonium hydroxide, which creates an alkaline environment in the medium. At
pH 7.5 or above, bromthymol blue turns royal blue. At a neutral pH, bromthymol
blue is green, as evidenced by the uninoculated media.
If the medium turns blue, the organism is citrate positive. If there is no
color change, the organism is citrate negative. Some citrate negative organisms
may grow weakly on the surface of the slant, but they will not produce a color
change.
Citric acid Oxaloacetic acid+Acetic acid Pyruvic acid +CO2
CO2 + 2Na+ + H2O Na2CO3
METHOD
9 A Simmon's Citrate agar slants is streaked with the organism and
incubated at 37°C for 48 hours.
INTERPRETATION
Blue Citrate positive
Green Citrate negative
RESULT
The subjected unknown bacterial culture is Citrate positive.
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TRIPLE SUGAR IRON TEST
INTRODUCTION
Triple sugar iron agar (TSI) is a differential medium that contains lactose,
sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH
indicator phenol red. It is used to differentiate enterics based on the ability to
reduce sulfur and ferment carbohydrates.
PRINCIPLE
As with the phenol red fermentation broths, if an organism can ferment any
of the three sugars present in the medium, the medium will turn yellow. If an
organism can only ferment dextrose, the small amount of dextrose in the
medium is used by the organism within the first ten hours of incubation. After
that time, the reaction that produced acid reverts in the aerobic areas of the
slant, and the medium in those areas turns red, indicating alkaline conditions.
The anaerobic areas of the slant, such as the butt, will not revert to an alkaline
state, and they will remain yellow.
METHOD
TSI medium is inoculated with an inoculating needle by stabbing the butt and
streaking the slant and incubated at 37°C for 24 hours.
INTERPRETATION
Results (slant/butt) Symbol Interpretation
Glucose fermentation only; Peptone
Red/yellow K/A
catabolized
Glucose and lactose and/or sucrose
Yellow/yellow A/A
fermentation
Red/red K/K No fermentation; Peptone catabolized
Red/no color change K/NC No fermentation; Peptone used aerobically
Glucose and lactose and/or sucrose
Yellow/yellow with bubbles A/A,G
fermentation; Gas produced
Red/yellow with bubbles K/A,G Glucose fermentation only; Gas produced
Red/yellow with bubbles and Glucose fermentation only; Gas produced;
K/A,G, H2S
black precipitate H2S produced
Red/yellow with black precipitate K/A, H2S Glucose fermentation only; H2S produced
Yellow/yellow with black Glucose and lactose and/or sucrose
A/A, H2S
precipitate fermentation; H2S produced
No change/no change NC/NC No fermentation
RESULT
Unknown bacterial culture can not ferment glucose and lactose
and/or sucrose.
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CATALASE TEST
INTRODUCTION
The catalase test identifies organisms which produce the catalase enzyme;
this enzyme converts hydrogen peroxide to water and oxygen gas. This enzyme
helps protect bacterial cells against hydrogen peroxide. Hydrogen peroxide is a
highly-reactive compound which damages cell components. It is sometimes
formed when the electron transport chain is used to produce energy.
PRINCIPLE
This test is performed to detect the presence of the enzyme catalase. Catalase
enzyme is found in most bacteria. It catalase present, it break the hydrogen
peroxide (H2O2) with the release of free Oxygen.
CATALASE
2H2O2 2H2O + O2
METHOD
9 One drop of 3%H2O2 is taken.
9 Touched a colony.
9 Observed the tube for bubble indicating a positive reaction.
INTERPRETATION
Bubbles positive
No bubbles negative
RESULT
` The unknown bacterial culture is catalase positive.
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OXIDASE TEST
INTRODUCTION
The oxidase test identifies organisms that produce the enzyme cytochrome
oxidase. Cytochrome oxidase participates in the electron transport chain by
transferring electrons from a donor molecule to oxygen.
PRINCIPLE
The oxidase test is a key test to differentiate between the families of
Pseudomonadaceae (ox+) and Enterobacteriaceae (ox-). The enzyme
cytochrome oxidase is involved with the reduction of oxygen at the end of the
electron transport chain. The colorless reagent used in the test will detect the
presence of the enzyme oxidase and, reacting with oxygen, turn blue or purple
within 15 seconds.
METHOD
9 A good amount of inoculum is taken from a plate culture and placed it
on a piece of filter paper.
9 One drop of the reagent is added. (If it is dark blue, it is old and should
not be used).
9 TIME the reaction: a positive reaction will occur within 15 seconds.
INTERPRETATION
Colorless Oxidase negative
Blue or purple color Oxidase positive
RESULT
The unknown bacteria are oxidase positive.
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UREASE TEST
INTRODUCTION
Some microorganisms have the ability to produce the enzyme urease. The
urease test is a useful diagnostic test for identifying bacteria.
PRINCIPLE
Hydrolysis of urea by the enzyme urease releases the end product ammonia,
the alkalinity of which causes the indicator phenol red (pH 6.8) to change from
yellow to red.
H2N
UREASE
C = O +H2O 2NH3 +CO2
H2N
METHODS
9 Christensen’s agar slant is inoculated with the bacterial culture.
9 Incubated at 37°C for 24 hours.
INTERPRETATION
Red colored slant Urease positive
Yellow colored slant Urease negative
RESULT
The unknown bacterial culture is urease negative.
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OBSERVED RESULTS & IDENTIFICATION CHARACTERISTICS
Name of
the Biochemical tests & Staining
sample
Catalase test
Oxidase test
Triple sugar
Morphology
Urease test
Citrate test
Indole test
Methyl red
iron test
staining
staining
& Gram
Motility
test
Unknown - - + - + + - + Bacillus
Gram -
ve
CONCLUSION
From the above staining and biochemical tests it is cconfirmed that the
unknown bacterial culture is Pseudomonas sp.
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Enumeration, Identification & Antibiotic Sensitivity Of
Microbes Associated With Urine
INTRODUCTION
A urine culture is a test to find and identify germs (usually bacteria) that may
be causing a urinary tract infection (UTI). Urine in the bladder normally is
sterile-it does not contain any bacteria or other organisms (such as
fungi).Clinical analysis of urine requires quantative determination of the number
of microorganisms/ml of urine sample. Bacterial count more than 105 /ml
indicate a urinary tract infection. Counts range from 0-104/ml are generally
common.
Microorganisms commonly responsible for UTI are given below-
Gram positive bacteria- Staphylococcus aureus, Streptococcus sp. etc.
Gram negative bacteria - Pseudomonas sp., Klebsiella sp. Escherichia coli etc.
Fungi- Candida sp., Blastomyces darmatidis etc.
Protozoa- Entamoeba histolytica.
REQUIREMENTS
Media: ¾ Chistensen’s Urea agar
¾ Nutrient broth
¾ Nutrien agar Glass goods:
¾ MacConkey agar ¾ Test tubes
¾ Blood agar ¾ Petri dishes
¾ Muller Hingtone agar ¾ Conical flask
¾ Simmons citrate agar ¾ Beaker
¾ Triple sugar iron agar ¾ Pipettes
¾ MR-VP broth ¾ Hanging drop slide
¾ Tryptone water ¾ Slides and cover slips
Reagents:
¾ Gram staining reagents
¾ Oxidase reagents
¾ Catalase reagents
¾ Hydrogen peroxidase
Others:
¾ Staining tray
¾ Inoculating loop and niddle
¾ Cotton swabs
¾ Autoclave
¾ Incubator
¾ Microscope
¾ Wax marking pencil etc
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PROCERURE
Day-1
1. Mid stream urine is collected.
2. The appearance (clear and cloudy) and colour is observed.
3. Microscopically analyzed the urine sample for the presence of pus cells,
RBC, bacteria (both centrifuged and centrifuged, in case of
uncentrifuged urine, 50ul of urine is placed on a slide and covered with
22x22mm coverslip and examined.)
4. Inoculated the inoculum on the ready media.
5. Incubated the inoculated plates at 370C for 24-48 hrs.
Day-2
1. Colony characteristics are observed & counted for significant bacterial
growth.
2. Gram staining, motility staining procedure performed.
3. Presence of motility observed by Hanging drop method.
4. Several biochemical tests are performed (Simmons citrate agar
utilization tests, Triple sugar iron agar utilization tests, catalase test,
oxidase test, Methyl-Red test, Indole production test) to identify the
bacteria.
Day-3
1. Proper bacteria are isolated.
2. As significant bacterial growth observed, drug sensitivity test is
performed.
Medical Microbiology
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INDOLE PRODUCTION TEST
INTRODUCTION
The indole test is a biochemical test performed on bacterial species to
determine the ability of the organism to split indole from the amino acid
tryptophan. This division is performed by a chain of a number of different
intracellular enzymes, a system generally referred to as "tryptophanase."
PRINCIPLE
Indole is generated by reductive deamination from tryptophan via the
intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the
deamination reaction, during which the amine (NH2) group of the tryptophan
molecule is removed. Final products of the reaction are indole, pyruvic acid,
ammonia (NH3) and energy. The indole produced during the reaction is detected
by the addition of Kovac’s reagent (dimethylaminobenzaldehyde) which produces
a cherry-red reagent layer.
A positive result is shown by the presence of a red or red color in the surface
alcohol layer of the broth. A negative result appears yellow. A variable result can
also occur, showing an orange color as a result.
METHOD
9 Bacterial cultures is inoculated in tryptone broths and incubated at 37°C for
48 hours.
9 1 ml Kovac’s reagent is added and gently shaked and observed for a red
color ring in around the interface between the broth and the alcohol
reagent.
INTERPRETATION
Red- color Indole- Positive
Yellow color Indole - Negative
Orange color Indole - Variable
RESULT
SAMPLE RESULT
Sample 1 Orange colored layer
Sample 2 Yellow colored layer
So, sample1 is Indole-Variable & sample2 is Indole-Negative.
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METHYL-RED TEST
PRINCIPLE
The Methyl-Red test tests for the ability to perform mixed-acid fermentation.
MR-VP broth contains glucose, peptone, and a phosphate buffer. Organisms that
perform mixed-acid fermentation produce enough acid to overcome the buffering
capacity of the broth, so a decrease in pH results. Organisms that perform other
kinds of fermentation cannot overcome the buffering capacity of the broth.
After incubation, the pH indicator Methyl Red is added to the broth. Methyl
Red is red at pH below 4.4 (this would be a positive result) and yellow at pH
above 6.0. An orange color indicates an intermediate pH and would be
considered a negative result.
METHODS
9 Obtained two MR-VP broths in sterile test tubes.
9 Inoculated one broth using aseptic technique. The other broth
uninoculated (this will be a control).
9 Incubated at 370emperature for two days.
9 Broths obtained from the incubator.
9 Added a dropful of Methyl Red to each broth.
9 Observed the color (which should develop within a few minutes).
INTERPRETATION
Red color MR- Positive
Yellow color MR- Negative
Orange color MR- Variable
RESULT
SAMPLE RESULT
Sample 1 Orange color
Sample 2 Yellow color
So, sample1 is MR-Variable & sample2 is MR-Negative.
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SIMMON’S CITRATE AGAR TEST
INTRODUCTION
Simmons citrate agar tests the ability of organisms to utilize citrate as a
carbon source. Simmons citrate agar contains sodium citrate as the sole source
of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen,
other nutrients, and the pH indicator bromthymol blue. This test is part of the
IMViC tests and is helpful in differentiating the Enterobacteriaceae.
PRINCIPLE
Organisms which can utilize citrate as their sole carbon source use the
enzyme citrase or citrate-permease to transport the citrate into the cell. These
organisms also convert the ammonium dihydrogen phosphate to ammonia and
ammonium hydroxide, which creates an alkaline environment in the medium. At
pH 7.5 or above, bromthymol blue turns royal blue. At a neutral pH, bromthymol
blue is green, as evidenced by the uninoculated media.
If the medium turns blue, the organism is citrate positive. If there is no
color change, the organism is citrate negative. Some citrate negative organisms
may grow weakly on the surface of the slant, but they will not produce a color
change.
METHOD
9 A Simmon's Citrate agar slants is streaked with the organism and
incubated at 37°C for 48 hours.
INTERPRETATION
Blue Citrate positive
Green Citrate negative
RESULT
Sample 1 Blue colored slant
Sample 2 Blue colored slant
Control Green colored slant
So, both Sample 1& Sample 2 are citrate positive.
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TRIPLE SUGAR IRON TEST
INTRODUCTION
Triple sugar iron agar (TSI) is a differential medium that contains lactose,
sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH
indicator phenol red. It is used to differentiate enterics based on the ability to
reduce sulfur and ferment carbohydrates.
PRINCIPLE
As with the phenol red fermentation broths, if an organism can ferment any
of the three sugars present in the medium, the medium will turn yellow. If an
organism can only ferment dextrose, the small amount of dextrose in the
medium is used by the organism within the first ten hours of incubation. After
that time, the reaction that produced acid reverts in the aerobic areas of the
slant, and the medium in those areas turns red, indicating alkaline conditions.
The anaerobic areas of the slant, such as the butt, will not revert to an alkaline
state, and they will remain yellow.
METHOD
TSI medium is inoculated with an inoculating needle by stabbing the butt and
streaking the slant and incubated at 37°C for 24 hours.
INTERPRETATION
Results (slant/butt) Symbol Interpretation
Red/yellow K/A Glucose fermentation only; Peptone catabolized
Yellow/yellow A/A Glucose and lactose and/or sucrose fermentation
Red/red K/K No fermentation; Peptone catabolized
Red/no color change K/NC No fermentation; Peptone used aerobically
Yellow/yellow with bubbles A/A,G Glucose and lactose and/or sucrose fermentation;
Gas produced
Red/yellow with bubbles K/A,G Glucose fermentation only; Gas produced
Red/yellow with bubbles and K/A,G, Glucose fermentation only; Gas produced; H2S
black precipitate H2S produced
Red/yellow with black precipitate K/A, H2S Glucose fermentation only; H2S produced
Yellow/yellow with black A/A, H2S Glucose and lactose and/or sucrose fermentation;
precipitate H2S produced
No change/no change NC/NC No fermentation
RESULT
SAMPLE RESULTS (SLANT/BUTT)
Sample 1 Yellow/yellow
Sample 2 Red/red
So, sample1 can ferment glucose and lactose and/or sucrose &
sample2 cannot ferment glucose and lactose and/or sucrose.
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CATALASE TEST
INTRODUCTION
The catalase test identifies organisms which produce the catalase enzyme;
this enzyme converts hydrogen peroxide to water and oxygen gas. This enzyme
helps protect bacterial cells against hydrogen peroxide. Hydrogen peroxide is a
highly-reactive compound which damages cell components. It is sometimes
formed when the electron transport chain is used to produce energy.
PRINCIPLE
This test is performed to detect the presence of the enzyme catalase. Catalase
enzyme is found in most bacteria. It catalase present, it break the hydrogen
peroxide (H2O2) with the release of free Oxygen.
CATALASE
2H2O2 2H2O + O2
METHOD
9 One drop of 3%H2O2 is taken.
9 Touched a colony.
9 Observed the tube for bubble indicating a positive reaction.
INTERPRETATION
Bubbles positive
No bubbles negative
RESULT
SAMPLE RESULT
Sample 1 Positive
Sample 2 positive
So, sample1 is catalase positive & sample2 also catalase positive.
Medical Microbiology
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OXIDASE TEST
INTRODUCTION
The oxidase test identifies organisms that produce the enzyme cytochrome
oxidase. Cytochrome oxidase participates in the electron transport chain by
transferring electrons from a donor molecule to oxygen.
PRINCIPLE
The oxidase test is a key test to differentiate between the families of
Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -). The enzyme
cytochrome oxidase is involved with the reduction of oxygen at the end of the
electron transport chain. The colorless reagent used in the test will detect the
presence of the enzyme oxidase and, reacting with oxygen, turn blue or purple
within 15 seconds.
METHOD
9 A good amount of inoculum is taken from a plate culture and placed it on
a piece of filter paper.
9 One drop of the reagent is added. (If it is dark blue, it is old and should
not be used).
9 TIME the reaction: a positive reaction will occur within 15 seconds.
INTERPRETATION
Colorless Oxidase negative
Blue or purple color Oxidase positive
RESULT
SAMPLE RESULT
Sample 1 Colorless
Sample 2 Blue or purple color
So, sample1 is oxidase negative & sample2 is oxidase positive.
Medical Microbiology
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UREASE TEST
INTRODUCTION
Some microorganisms have the ability to produce the enzyme urease. The
urease test is a useful diagnostic test for identifying bacteria.
PRINCIPLE
Hydrolysis of urea by the enzyme urease releases the end product ammonia,
the alkalinity of which causes the indicator phenol red (pH 6.8) to change from
yellow to red.
H2N
UREASE
C = O +H2O 2NH3 +CO2
H2N
METHODS
9 Christensen’s agar slant is inoculated with the bacterial culture.
9 Incubated at 37°C for 24 hours.
INTERPRETATION
Red colored slant Urease positive
Yellow colored slant Urease negative
RESULT
SAMPLE RESULT
Sample 1 Red colored slant
Sample 2 Yellow colored slant
So, sample1 is Urease positive & sample2 is Urease negative.
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KIRBY-BAUER DISK-DIFFUSION METHOD
INTRODUCTION
The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing
in a clinical laboratory where a large number of isolates are tested for
susceptibility to numerous antibiotics.
PRINCIPLE
An agar plate is uniformly inoculated with the test organism and a paper disk
impregnated with a fixed concentration of an antibiotic is placed on the agar
surface. Growth of the organism and diffusion of the antibiotic commence
simultaneously resulting in a circular zone of inhibition in which the amount of
antibiotic exceeds inhibitory concentrations. The diameter of the inhibition zone
is a function of the amount of drug in the disk and susceptibility of the
microorganism.
METHOD
1. Made a suspension at an appropriate turbidity of the bacterial culture to be
tested.
2. Placed a sterile cotton swab in the bacterial suspension and remove the
excess fluid by pressing and rotating the cotton against the inside of the tube
above the fluid level. The swab is streaked in at least three directions over
the surface of the Mueller-Hinton agar to obtain uniform growth. A final
sweep is made around the rim of the agar. Be sure to streak for confluency.
3. Allowed the plates to dry for five minutes.
4. Using sterile forceps placed antibiotic disks.
5. Incubatde the plates within 15 minutes after applying the disks. The plates
are incubated soon after placing the disks
6. Following overnight incubation, measured the diameter of the zone of
growth inhibition around each disk to the nearest whole mm. Examined the
plates carefully for well-developed colonies within the zone of inhibition.
7. Using a standard table of antibiotic susceptibilities, determine if the strain
is resistant, intermediate, or susceptible to the antibiotics tested.
RESULT
Sample Sensitive antibiotics Resistant antibiotics
Sample1. Amikacin, Ciprofloxacin Ceftazidin, Vancomycin
Sample2. Ciprofloxacin, Gentamycin Penicillin, Ticareillin
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OBSERVATION
The number of bacterial colony - >105/ml
Name of
the Biochemical tests & Staining
sample
Triple sugar iron
Motility staining
Methyl red test
Gram staining
Morphology &
Catalase test
Oxidase test
Urease test
Citrate test
Indole test
test
Sample 1 variable +/- + + + - + - Bacillus
Gram -ve
Sample 2 - - + - + + - + Bacillus
Gram -ve
CONCLUSION
From the observed result it is concluded that isolated sample-1 is Klebsiella
sp. & sample-2 is Pseudomonas sp.
Medical Microbiology
