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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.

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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.

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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.

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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