Page noDate

-

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
Glass goods: Test tubes Petri dishes Conical flask Beaker Pipettes Hanging drop slide Slides and cover slips Media: Nutrient broth Nutrien agar MacConkey agar Blood agar Muller Hingtone agar Simmons citrate agar Triple sugar iron agar MR-VP broth Tryptone water Chistensen’s Urea agar Reagents: Gram staining reagents. Oxidase reagents Catalase reagents Hydrogen peroxidase Others: Staining tray Inoculating loop and niddle Cotton swabs Autoclave Incubator Microscope 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. 4. 5. 6. Colony characteristics are observed. Presence of motility observed by Hanging drop method The gram, acid-fast and endospore staining are performed. The biochemical tests are performed.

Medical Microbiology

Page noDate-

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 + Kovac’s reagents

Indole + Pyruvic acid +NH3 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
Bacterial culture is inoculated in tryptone broth and incubated at 37°C for 48 hours. 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 Yellow color Orange color Indole- Positive Indole - Negative Indole - Variable

RESULT
The unknown bacterial culture is Indole negative.

Medical Microbiology

Page noDate-

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
Obtained two MR-VP broths in sterile test tubes. Inoculated one broth using aseptic technique. The other uninoculated (this will be a control). Incubated at 370emperature for two days. Broths obtained from the incubator. Added a dropful of Methyl Red to each broth. Observed the color (which should develop within a few minutes). broth

INTERPRETATION
Red color Yellow color Orange color MR- Positive MR- Negative MR- Variable

RESULT
The subjected bacterial culture is MR-negative.

Medical Microbiology

Page noDate-

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 Na2CO3

CO2 + 2Na+ + H2O

METHOD
A Simmon's Citrate agar slants is streaked with the organism and incubated at 37°C for 48 hours.

INTERPRETATION

Blue Green

Citrate positive Citrate negative

RESULT
The subjected unknown bacterial culture is Citrate positive.

Medical Microbiology

Page noDate-

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) Red/yellow Yellow/yellow Red/red Red/no color change Yellow/yellow with bubbles Red/yellow with bubbles Red/yellow with bubbles and black precipitate Red/yellow with black precipitate Yellow/yellow with black precipitate No change/no change Symbol K/A A/A K/K K/NC A/A,G K/A,G K/A,G, H2S K/A, H2S A/A, H2S NC/NC Interpretation Glucose fermentation only; Peptone catabolized Glucose and lactose and/or sucrose fermentation No fermentation; Peptone catabolized No fermentation; Peptone used aerobically Glucose and lactose and/or sucrose fermentation; Gas produced Glucose fermentation only; Gas produced Glucose fermentation only; Gas produced; H2S produced Glucose fermentation only; H2S produced Glucose and lactose and/or sucrose fermentation; H2S produced No fermentation

RESULT
Unknown bacterial culture can not ferment glucose and lactose and/or sucrose.

Medical Microbiology

Page noDate-

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
One drop of 3%H2O2 is taken. Touched a colony. Observed the tube for bubble indicating a positive reaction.

INTERPRETATION
Bubbles No bubbles positive negative

RESULT `
The unknown bacterial culture is catalase positive.

Medical Microbiology

Page noDate-

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
A good amount of inoculum is taken from a plate culture and placed it on a piece of filter paper. One drop of the reagent is added. (If it is dark blue, it is old and should not be used). TIME the reaction: a positive reaction will occur within 15 seconds.

INTERPRETATION
Colorless Blue or purple color Oxidase negative Oxidase positive

RESULT

The unknown bacteria are oxidase positive.

Medical Microbiology

Page noDate-

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 C = O +H2O H2N UREASE 2NH3 +CO2

METHODS
Christensen’s agar slant is inoculated with the bacterial culture. Incubated at 37°C for 24 hours.

INTERPRETATION
Red colored slant Yellow colored slant Urease positive Urease negative

RESULT
The unknown bacterial culture is urease negative.

Medical Microbiology

Page noDateOBSERVED RESULTS & IDENTIFICATION CHARACTERISTICS

Name of the sample Indole test

Biochemical tests & Staining Catalase test

Oxidase test

Triple sugar iron test

Unknown

-

-

+

-

+

+

-

+

Bacillus Gram ve

CONCLUSION
From the above staining and biochemical tests it is cconfirmed that the unknown bacterial culture is Pseudomonas sp.

Medical Microbiology

Morphology & Gram staining

Urease test

Citrate test

Methyl red test

Motility staining

Page noDate-

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 belowGram 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: Nutrient broth Nutrien agar MacConkey agar Blood agar Muller Hingtone agar Simmons citrate agar Triple sugar iron agar MR-VP broth Tryptone water Reagents: Gram staining reagents Oxidase reagents Catalase reagents Hydrogen peroxidase Others: Staining tray Inoculating loop and niddle Cotton swabs Autoclave Incubator Microscope Wax marking pencil etc

Chistensen’s Urea agar Glass goods: Test tubes Petri dishes Conical flask Beaker Pipettes Hanging drop slide Slides and cover slips

Medical Microbiology

Page noDatePROCERURE 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

Page noDate-

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
Bacterial cultures is inoculated in tryptone broths and incubated at 37°C for 48 hours. 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 Yellow color Orange color Indole- Positive Indole - Negative Indole - Variable

RESULT
SAMPLE Sample 1 Sample 2 RESULT Orange colored layer Yellow colored layer

So, sample1 is Indole-Variable & sample2 is Indole-Negative.

Medical Microbiology

Page noDate-

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
Obtained two MR-VP broths in sterile test tubes. Inoculated one broth using aseptic technique. The other uninoculated (this will be a control). Incubated at 370emperature for two days. Broths obtained from the incubator. Added a dropful of Methyl Red to each broth. Observed the color (which should develop within a few minutes). broth

INTERPRETATION
Red color Yellow color Orange color MR- Positive MR- Negative MR- Variable

RESULT
SAMPLE Sample 1 Sample 2 RESULT Orange color Yellow color

So, sample1 is MR-Variable & sample2 is MR-Negative.

Medical Microbiology

Page noDate-

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
A Simmon's Citrate agar slants is streaked with the organism and incubated at 37°C for 48 hours.

INTERPRETATION
Blue Green Citrate positive Citrate negative

RESULT
Sample 1 Sample 2 Control Blue colored slant Blue colored slant Green colored slant

So, both Sample 1& Sample 2 are citrate positive.

Medical Microbiology

Page noDate-

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 Yellow/yellow Red/red Red/no color change Yellow/yellow with bubbles Red/yellow with bubbles Red/yellow with bubbles and black precipitate Red/yellow with black precipitate Yellow/yellow with black precipitate No change/no change

K/A A/A K/K K/NC A/A,G K/A,G K/A,G, H2S K/A, H2S A/A, H2S NC/NC

Glucose fermentation only; Peptone catabolized Glucose and lactose and/or sucrose fermentation No fermentation; Peptone catabolized No fermentation; Peptone used aerobically Glucose and lactose and/or sucrose fermentation; Gas produced Glucose fermentation only; Gas produced Glucose fermentation only; Gas produced; H2S produced Glucose fermentation only; H2S produced Glucose and lactose and/or sucrose fermentation; H2S produced No fermentation

RESULT
SAMPLE Sample 1 Sample 2 RESULTS (SLANT/BUTT) Yellow/yellow Red/red

So, sample1 can ferment glucose and lactose and/or sucrose & sample2 cannot ferment glucose and lactose and/or sucrose.

Medical Microbiology

Page noDate-

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
One drop of 3%H2O2 is taken. Touched a colony. Observed the tube for bubble indicating a positive reaction.

INTERPRETATION
Bubbles No bubbles positive negative

RESULT
SAMPLE Sample 1 Sample 2 RESULT Positive positive

So, sample1 is catalase positive & sample2 also catalase positive.

Medical Microbiology

Page noDate-

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
A good amount of inoculum is taken from a plate culture and placed it on a piece of filter paper. One drop of the reagent is added. (If it is dark blue, it is old and should not be used). TIME the reaction: a positive reaction will occur within 15 seconds.

INTERPRETATION
Colorless Blue or purple color Oxidase negative Oxidase positive

RESULT SAMPLE Sample 1 Sample 2 RESULT Colorless Blue or purple color

So, sample1 is oxidase negative & sample2 is oxidase positive.

Medical Microbiology

Page noDate-

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 C = O +H2O H2N

UREASE
2NH3 +CO2

METHODS
Christensen’s agar slant is inoculated with the bacterial culture. Incubated at 37°C for 24 hours.

INTERPRETATION
Red colored slant Yellow colored slant Urease positive Urease negative

RESULT
SAMPLE Sample 1 Sample 2 RESULT Red colored slant Yellow colored slant

So, sample1 is Urease positive & sample2 is Urease negative.

Medical Microbiology

Page noDate-

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 Sample1. Sample2.

Sensitive antibiotics Amikacin, Ciprofloxacin Ciprofloxacin, Gentamycin

Resistant antibiotics Ceftazidin, Vancomycin Penicillin, Ticareillin

Medical Microbiology

Page noDate-

OBSERVATION
The number of bacterial colony - >105/ml

Name of the sample Methyl red test

Biochemical tests & Staining Triple sugar iron test

Motility staining

Sample 1 Sample 2

variable

+/-

+ +

+ -

+ +

+

+ -

+

Bacillus Gram -ve 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

Morphology & Gram staining

Catalase test

Oxidase test

Urease test

Citrate test

Indole test