Category: Biology
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Decarboxylase Test: Purpose, Indicator, and Results
Continue ReadingWhat is Decarboxylase Test?
Decarboxylase test is one of the biochemical tests. This test is commonly used to differentiate the members of the family Enterobacteriaceae on the basis of their ability to produce the enzyme decarboxylase.
However, the metabolism of amino acids differs depending on their characteristic as aerobic or anaerobic facultative anaerobes and as well as in gram-negative organisms.
Metabolism of amino acids occurs either by decarboxylation, deamination or hydrolysis depending on the metabolization of amino acid in an organism.
Decarboxylation often occurs in the presence of an enzyme deaminase and catalyzation occurs by breaking the bonds and it also binds to the rest of the amino acid in the amino group.
Three different decarboxylase enzymes are produced by the organisms, which catalyze the metabolism of amino acids, ornithine, arginine decarboxylase, decarboxylase, and lysine decarboxylase.
The production of these enzymes is taken as an important parameter for differentiating the bacteria present in the family of Enterobacteriaceae.
Apart from differentiating and identifying the Enterobacteriaceae, Ornithine decarboxylase teat is considered as one of the paramount importance especially in separating the members of the Klebsiella-Enterobacter Serratia group and also in identifying the species Proteus.
Decarboxylase Test Objective
 To detect the ability of an organism to produce an enzyme known as decarboxylase.
 To differentiate the members of the family Enterobacteriaceae on the basis of their ability to produce an enzyme decarboxylase.
Decarboxylase Test Principle
 The medias of Arginine, lysine and orthenine decarboxylase are used to detect the ability of an organism to decarboxylate or hydrolyze the amino acid and forms an amine which produces an alkaline ph.
 The basal medium is formed by using Moeller’s formula.
 The medium contains meat peptones and beef extracts which supplies nitrogenous nutrients to support the growth of bacteria.
 The media is composed of glucose which is a fermentable carbohydrate.
 The enzyme pyridoxal acts as a cofactor and enhances the decarboxylase activity. Bromocresol purple and cresol red is used as a pH indicator.
 Amino acids namely arginine, Lysine and ornithine are added once in the basal medium and it also helps in detecting the production of enzyme which decarboxylate or hydrolyze these substrates.
 After fermenting the glucose in the medium, acids are produced. These acids have the capability to lower the pH in the medium which results in change in color from purple to yellow.
 If the organism produces an enzyme decarboxylase, it results in decarboxylation or hydrolysis of amino acids, due to the response in the acid pH.
 Decarboxylation thus results in alkaline end products known as amines, which causes the medium to get back its original color, purple.
 In case if the organism does not ferment glucose, then the medium does not change into yellow, but in these cases too, the tests are performed by including a control without amino acids for comparison.
Decarboxylase Test Requirements
Media: Decarboxylase test medium Base is generally used for testing the amino acid decarboxylase activity. Where as the other medias like Motility-indole-ornithine medium and Lysine iron agar can also be used.
Reagents:
 Mineral oil
 Vaspar
 Liquid paraffin
 Petroleum jelly, which is maintained at 56ºC in the liquid form.
Supplies:
 Sterile sticks
 Inoculating loops
 Incubator
Decarboxylase Test Procedure
1. Preparation of Media
 Initially 9.02 grams of dehydrated powder is filled into a beaker containing 1000 milli liters of pure distilled or deionized water.
 The prepared solution is then heated till it boils, so that the medium dissolves completely.
 The media is initially divided into four equal parts. When one part is tube without adding any amino acid and the tube is labelled as control.
 Then the remaining three parts are dispensed into each of the tubes and L-lysine hydrochloride, L-arginine hydrochloride, and L-ornithine hydrochloride are separately added to bring a final concentration of about 0.5%.
 About 3 to 4 ml of the media is dispensed in a screw capped tubes and they are sterilized by autoclaving them at a 10 lbs. pressure in 115ºC for about 20 minutes.
 In order to avoid the false alkalinization on the surface of the medium, liquid paraffin is added to height of about 5mm before sterilizing.
2. Decarboxylase Test
 For Glucose-fermenting organisms
 A drop of 18-24hour brain heart infusion broth culture is add in each of the three decarboxylase broths.
 Here the control tube is not used for glucose-fermenting organisms.
 4mm layer of sterile mineral oil is added to each of the tubes and the tubes are incubated for 4 days at a temperature of 35 to 37ºC in an ambient air.
Glucose-Nonfermenting Organisms
 A suspension is prepared using brain heart infusion broth and it is prepared from an overnight culture on a sheep blood agar.
 Each of the four-decarboxylase broth is inoculated with four drops of the prepared suspension.
 Then a sterile oil is added to a height of about 4mm.
 The tubes are then incubated for 4 days at a temperature of about 35 to 37ºC in an ambient air.
 The tubes are then observed for color change for 24, 48, 72 and 96 hours.
Decarboxylase Test Results
 A positive test is found out by change in color of the medium from turbid purple to faded out yellow purple color.
 A negative test is detected by a bright yellow color or no change in color.
 The control tube must retain, its original color or sometimes it turns yellow. If turbidity or alkalinity of the purple color is seen in the control tube, then the test is considered as invalid one.
Decarboxylase Test Citations
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Methyl Red Test: Results, Procedure, and Principle
Continue ReadingMethyl Red Test
Methyl red test helps us to detect the production of sufficient acid during the process of fermentation of glucose and maintenance of conditions such as pH.
The pH of an old culture is sustained below the value of 4.5 which is shown by the change in color of the indicator which is added at the end of the period on incubation.
MR-VP broth was developed by Clark and Lubs which allowed both MR and VP tests to be performed in the same medium of inoculation by aliquoting the different portions of the tube.
What is Methyl Red Test?
Methyl red test is commonly known as MR test and it is used to detect the ability of the organism to produce and maintain the stable products of acids by fermenting the glucose.
Whereas methyl red test along with VP test is always performed together; as they are physiologically related to each other and they are performed in MRVP broth.
All of the members of the family Enterobacteriaceae has the capability to convert the substance of glucose into pyruvic acid by using Embden-Meyerh-of pathway, whereas the bacteria can metabolize the pyruvic acid further.
This test is one of the commonly performed biochemical test, which is used to detect the ability of the microorganism.
Methyl Red Test Principle
Generally, some bacteria have the ability to use glucose and convert it into a stable lactic acid, acetic acid or formic acid end product.
The bacteria first metabolize through mixed acid pathway to produce stable acid.
The acid production varies from species to species depending on the specific enzymatic pathways which is seen in the bacteria.
This acid thus produced decreases the ph. value about 4.5 or in some cases even less than that, which is indicated by change in color from yellow to red when methyl red indicator is added.
During methyl red test, the bacteria which tests the growth of bacteria in a broth medium containing glucose.
When the bacteria have the ability to use the glucose by producing stable acid, the color changes from yellow to red on adding the methyl indicator in the broth culture.
Where as the mixed acid pathway gives 4 mol of acidic products which mainly involves lactic and acetic acid, 1 mol of neutral fermentation product, mostly ethanol is used here, 1 mol of carbon dioxide and 1 mol of H2 per mol of glucose is fermented.
When a large quantity of acids is producing, they cause a significant decrease in pH in the culture medium.
Methyl Red Test Reagents
MRVP Broth:
 Usually, the ph. in the MRVP broth is maintained at 6.9.
 1 litre of deionized water
 7.0 grams of Buffered peptone
 5 grams of Glucose
 5 grams of Dipotassium phosphate
Preparation of Methyl Red Solution
 Initially 0.1 gram of methyl red is dissolved in 300ml of 95% of ethyl alcohol.
 Then sufficient distilled water is added to make about 5oo ml of methyl red solution.
 Then the prepared solution is stored at a temperature of 4 to 8 ºC in a brown bottle.
 This solution which has been prepared can be stored up to one year.
Methyl Red Test Procedure
 Before inoculating the medium, it is allowed to equilibrate at a room temperature.
 Then the organisms are taken from the 18 to 24-hour pure culture, and it is lightly inoculated in the medium.
 Then the medium is incubated aerobically at a temperature of 37ºcelsius for about 24 hours.
 After incubating for 24 hours, 1 ml of aliquot broth is added into a clean test tube.
 Again, the broths are incubated for additional 24 hours.
 Two to three drops of methyl indicator are added to the aliquot.
 Then the change in color is observed immediately.
Methyl Red Test Results
 In positive reaction, the color changes to distinct red
 Some of the species like, Escherichia coli, Yersinia sps gives positive results in Methyl red test
 Whereas, in negative reaction, the color remains yellow and there will be no change in color
 Organisms such as Enterobacter aerogenes, Klebsiella pneumonia, etc. gives negative results in Methyl red test.
Methyl Red Test Uses
Basically, methyl test is paired up with Voges Proskauer test and it is used in differentiating the members of the family Enterobacteriaceae. But in recent times they are used to characterize the other groups of bacteria including those of Actinobacteria.
Methyl Red Test Limitation
 Usually, biochemical tests are recommended to perform on pure cultures for the complete identification.
 Methyl red test can be performed only if the medium is incubated for at least a minimum of 48 hours. If the tests are let to run very early them it results only in false-positive results.
 It is also important to take a note that only light inoculums are used in methyl red tests because when heavy inoculums are used the growth of bacteria will be inhibits and it results in invalid results
 Incubation can be followed for a maximum of five days, which is necessary for this test.
Methyl Red Test Citations
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Novobiocin Susceptibility Test: Purpose, Procedure, Risks, &...
Continue ReadingNovobiocin Susceptibility Test
Novobiocin Susceptibility test is usually done to differentiate the coagulase-negative Staphylococci and to identify and isolate as Staphylococcus saprophyticus, which is usually novobiocin resistant.
It is generally performed on urine isolates, especially the urine from women of the reproductive ages, as they contain coagulase negative.
Novobiocin is an aminocoumarin antibiotic, and it is produced from actinomycete Streptomyces nivens, and has its own antibacterial property.
Kloos and Schleifer in the year 1975, detected the simplified scheme for differentiating coagulase-negative Staphylococcus spp.
That includes novobiocin disk test. Staphylococcus saprophyticus, is one of the gram-positive coagulase negative species of Staphylococci.
It is an uropathogenic bacterium which acutes acute infection in the urinary tract.
This infection is uncomplicated, it is often found in young and middle-aged female patients.
Unlike other Coagulative Negative Staphylococcus, Staphylococcus saprophyticus is rarely resistant to the antibiotics and it is active against gram-positive organisms.
S. saprophyticus is one of the promising resistant of the antibiotic novobiocin.
Hence screening for coagulase-negative staphylococci from the urine cultures for novobiocin resistance is reliable presumptive for identifying saprophyticus.
Novobiocin Susceptibility Test Objective
Novobiocin susceptibility test helps to determine the susceptibility pattern of the bacterium with the antibiotic novobiocin.
Novobiocin Susceptibility Test Principle
As mentioned above, novobiocin is an antibiotic which interferes with the unpacking and repacking of DNA during replication of DNA and the bacterial cell cycle.
Novobiocin binds with the DNA gyrase and restricts the activity of adenosine triphosphate.
Susceptibility of the novobiocin is determined by placing a novobiocin antibiotic in a paper disk which is kept on a agar plate and then it is seeded with the organism under investigation.
As organism multiplies during the period of incubation and it produces lawn of confluency growth, cells are exposed to antibiotic diffusing into the agar from the paper disk.
If the bacterial species are susceptible to antibiotic novobiocin, then there will be a formation of transparent zone of inhibition around the area of the disk, which represents the area where the antibiotic concentration prevented the bacterial growth.
If there is no zone of inhibition then, it is said that the organism is resistance to the specific antibiotic used. Usually in the laboratories, Mueller-Hington agar plate is used.
The agar plate is seeded with the test organism to produce a confluent growth on the surface of the agar.
After seeding the test organism is completed, a novobiocin antibiotic disc is applied on the surface of the agar, and incubation is done and further the sensitivity of the organism to the antibiotic determined by performing a Kirby-Bauer method.
Novobiocin Susceptibility Test Plate
Novobiocin disks are prepared by impregnating 5µg of novobiocin into a high quality of about 6mm diameter filter paper disks or it can also be purchased, as it is also commercially available.
Novobiocin Susceptibility Test Procedure
 First the test isolated is kept in the pure culture for about 18 to 72 hours. Suspension is prepared of the test isolate in the tryptic soy broth which is equal to a Mc Farmland 0.5 standard or equivalent.
 Sterile swab is immersed in the suspension and it is rotated against the sides of the tube above the fluid level to remove the excess inoculum.
 Then the expressed swab is collected and inoculated into a blood agar or into the Muller Hinton plate by streaking the swab on the entire agar plate.
 This process is repeated in both the plates.
 After streaking the agar surface is allowed to dry for at least 15 to 20 minutes before applying it on the novobiocin disk.
 Using a sterile swab, lawn of growth is prepared on the entire surface of the plate in three directions and also along the entire edge of the plate.
 Then the forceps are dipped in an alcohol and flamed.
 Then the aseptically flames are used for applying the novobiocin disc to the surface of the inoculated plate.
 The discs are genteelly pressed down using a sterile forceps to ensure whether they adhere to the agar surface.
 The plates are aerobically incubated for about 18 to 24 hours at a temperature of about 35 to 37ºC.
 The diameter of the zone of inhibition is measure using a metric ruler or sliding capillaries.
Novobiocin Susceptibility Test Results
Positive Results: In case, if the results are positive, then the zone of inhibition will be greater than 16mm in diameter and it also indicates that the specific organism is sensitive to the antibiotic.
Negative Results: If the results are negative, then there will be a zone of inhibition which is less than or equal to 16mm, which indicates the novobiocin resistance.
Novobiocin Susceptibility Test Significance
 The CoNS is subdivided into two based on their novobiocin pattern of susceptibility. Here the CoNS group demonstrates the novobiocin susceptibility including epidermis, S. Captitits, S. haemolyticus, S. hominis, S. lugdenensis, S. warneri, S. saccharolyticus and other species.
 The novobiocin resistant group consists of species such as S. saprophyticus, S. xylosus, and kloosii.
 This test is basically used for differentiating Staphylococcus saprophyticus from other Coagulase negative staphylococci, especially in the clinical specimens.
Novobiocin Susceptibility Test Citations
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Lipase Test: Purpose, Procedure, Risks, and Results
Continue ReadingWhat is Lipid and Lipase Test?
Lipid is the common term in biology which is used to denote the type of fats present in our body.
Generally, fats are formed by linking the ester bonds between three molecules of the fatty acids along with one molecule of glycerol.
Where simple fats are known as triglycerides or triacylglycerols, as they are composed of glycerol with three long fatty acids chain.
The enzyme which breaks simple fats into smaller components of the fatty acids is known as Lipase.
As in human, some microorganisms also contains lipase and they are classified depending on their ability to synthesize and secrete lipase.
Different types of simple fats are used for determining the type of microorganism to be tested.
Lipase is one of the enzymes presents in our pancreas, and it is synthesized in pancreases and released in to our digestive tract, only when we swallow food materials.
Lipase thus helps in breaking down the fats present in our food materials.
Specific levels are maintained in producing and utilizing the lipase such as maintaining the normal digestion and the cellular functions.
But abnormally levels of the enzyme present in the blood indicates some of the health issues.
However, Serum lipase test helps in measuring the amount of enzyme lipase present in our body.
Lipase test helps in detecting the health issues such as acute pancreatitis, chronic pancreatitis, celiac disease or pancreatic cancer.
Thus, increase level of lipase is noted in our body when we have any of these issues.
Lipase Test Objective
 The aim is to determine the ability of the micro-organism to produces the lipase.
 This test also helps to determine the bacteria on the basis of the activity of lipase.
 Here the variety of lipid substrates, including corn oil, olive oil and the soyabean oil are used commonly to identify the differential characteristics, between the members of the family Enterobacteriaceae, Neisseria, staphylococcus and Clostridium.
 Several varieties of fungal species can also be detected by lipolytic ability.
Lipase Test Principle
Generally, fats are formed by the linkage of esters between three molecules of fatty acids along with one molecule of glycerol. Simple fats are also known as triglycerides or triglycerols.
Lipases breaks the simple fats into its smaller components as fatty acids and glycerol.
Many types of bacteria are classified based on their ability to produce lipases.
For determining and identifying the type of organism and the variety of simple fats used and are being tested.
However, the smallest and simplest test components of triglycerides is tributyrin, which is considered as a common constituent in the lipase testing media.
But tributyrin is too large to enter the cell, so lipase is released to break it into smaller components, which becomes easy for the cells to uptake.
After hydrolysis, the glycerol is converted into dihydroxyacetone phosphate, an intermediate form of glycolysis.
The fatty acids are catabolized by the process β-oxidation; which converts it into a variety of end products which can be used by the cell for the producing energy.
Tributyrin oil is one of the types of lipid known as triglyceride. Other lipase tests are used as a various source of fat involving soybean oil, corn oil, olive oil, peanut oil, egg yolk.
Tributyrin agar is a differential medium, which tests the ability of an organism to produces an exo-enzyme, known as lipase, that hydrolyses tributyrin oil.
Tributyrin oil is prepared in the form of an emulsion, so that the agar appears opaque. When the plate is inoculated with a lipase-positive organism, clear zones will appear around the growth as the evidence of lipase activity. If no clear zones appear, the organisms are lipase negative.
Lipase Test Media
Tributyrin agar: Peptic digest of the animal tissue at 5.0 gram/litre, Yeast Extract of 3.0 gram/litre, Agar 15.0 gram/litre, pH is maintained at 7.5.
Lipase Test Procedure
 First the tributyrin agar medium is inoculated in a single line streaking of the organism.
 Inoculated medium is incubated in a gas-Pak jar immediately after streaking anaerobically at a temperature of 35 to 37ºC for about 24 to 48 hours.
 Then the clear zone can be observed around the area of bacterial growth.
Lipase Test Results
 In case of positive result, a clear zone is observed around the area of bacterial growth.
 If the test is negative, then there will be not be any clear zone around the area of bacterial growth.
Lipase Test Importance
 The lipase test, is generally used for detecting the enumerate lipolytic bacteria, specifically in diary products which contains high amount of fats.
 It also helps in detecting the variety of other lipid substrates such as olive oil, corn oil, and soya bean oil and also helps in selecting the characteristics of other members such as Enterobacteriaceae, clostridium, staphylo coccus and Neisseria.
 Clinically the blood test for lipase helps in detecting most of the acute pancreatic diseases and other characteristics symptoms related to pancreas.
 Lipase test is often performed either amylase test which helps in detecting the pancreatic diseases.
Lipase Test Citations
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Optochin Susceptibility Test: Principle, Steps, and Importance
Continue ReadingOptochin Susceptibility Test
Optochin susceptibility test is usually done for identifying the species of Streptococcus pneumoniae.
Differentiating Streptococcus pneumoniae from the other species of Streptococci variants depends on demonstrating optochin susceptibility, bile solubility, reaction with a specific DNA probe, or detection of species-specific capsular polysaccharides.
Most of the clinical microbiology laboratories depends on the optochin susceptibility test.
Initially Optochin susceptibility was first described for differentiating pneumococci, from other α-hemolytic streptococci in the year 1915, but this test is being virtually unused in the laboratories in the mid-1950s.
Optochin is a chemical named as ethylhydrocupreine hydrochloride which is completely soluble in water.
Optochin is used to identify Streptococcus pneumoniae, and alpha hemolytic Streptococcus which is mostly susceptible to the chemical, Optochin.
Where as the species of alpha-hemolytic Streptococcal species are resistant to optochin.
This test helps in determining whether bacterium is either sensitive or resistant to the chemical.
This test is also widely used in the form of filter paper discs or impregnated with ethylhydrocupreine hydrochloride, and they are applied directly on the inoculated plates before incubating.
Optochin test is one of the tests which can be performed in shorter time than the other tests like bile solubility test.
Features of Optochin Susceptibility Test
Streptococcus pneumonia is most commonly found in the respiratory tract of the humans, as other pathogens like Streptococci, and it has a hemolytic pattern which helps in distinguishing it from the other alpha-Streptococci and the Lacto cocci.
Optochin is a chemical that is used in the presumptive identification of alpha-hemolytic Streptococcus pneumoniae, which is sensitive to optochin.
Where as the other species like alpha-hemolytic Streptococcus species are resistant to optochin.
Optochin is completely soluble in water.
This test is widely used in the form of filter paper disks.
Bowen and Jeffries in the year 1955 impregnated the disks with the reagent ethylhydrocupreine hydrochloride that are applied directly to inoculated plates before incubation to demonstrate the susceptibility of pneumococcus for identification.
Optochin Susceptibility Test Principle
Streptococcus pneumoniae is generally found in the respiratory tract and it also has its hemolytic pattern which is identical to other alpha-hemolytic Lacto bacilli and streptococci.
Streptococcus pneumoniae is sensitive to the chemical optochin, which is not able to form colonies in its presence and as and its change in surface tension and causes the cell membrane of Streptococcus pneumonia to lyse.
Optochin is a chemical which is quinine derivative and it has a capacity to soluble in water where all the alpha-hemolytic streptococcal species are resistant to this chemical.
Optochin susceptibility test is usually employing in identifying the streptococcus which is sensitive to the chemical optochin.
For determining the susceptibility of the organism, Streptococcus pneumonia, filter paper disks are impregnated with the chemical optochin and it is used in a disk diffusion test.
This test is usually performed on blood agar, which is a zone for creating inhibition of the lysis of the cell membranes in the Streptococcus pneumonia cells surrounding the disk which helps in determining the positive test.
This test is very easy to perform and it also economic and has a sensitivity of more than 95%.
Optochin Susceptibility Test Requirements
• 5% of sheep blood agar.
• Optochin disks- each disk should be impregnated with 5µg of optochin
• Incubator
• Sterile forceps
• Sterile inoculating loops
Optochin Susceptibility Test Procedure
 With a help of an inoculating loop, 2 to 3 streaks are drawn on a well isolated colonies of a pure culture medium, which is tested on 5% of blood agar.
 Optochin disks are placed on the inoculated surface of agar with the help of sterile forceps.
 Ensure whether the disks are adhered firmly to the surface of the agar by using a sterile forceps or loops.
 Then the plates are placed in an incubator at temperature of about 35 to 37ºC.
 Observe the growth on the surface of the blood agar plates and finally the inhibition of the diameter zone is measured including the diameter of the disk plate.
Optochin Susceptibility Test Results
Positive Results:
 If the zone of inhibition is detected as 14mm or greater than that and a diameter of 6mm disk, it results in the positive condition.
 If the zone of inhibition is formed lesser than 14mm the strain is identified as pneumococcus only if it is soluble in bile.
Negative Results:
 If there is no detection of any formation of zones, the test is negative and it indicates the absence of Streptococcus pneumoniae.
Optochin Susceptibility Test Limitations
 Usually, Streptococcus pneumoniae are isolated and they must be incubated in a carbon-dioxide enriched environment, as some isolates will grow poorly or it does not grow at all.
 Optochin susceptibility is one of the presumptive tests which is recommended as one of the important biochemical tests which is used to perform the complete identification process.
 Any zone of inhibition which is less than 14mm is not enough for performing the test of pneumococci, as the strain is important for identifying the pneumococcus with confirmation by a positive solubility test of bile or serology is performed.
Optochin Susceptibility Test Citations
- Assessment of the Optochin Susceptibility Test to Differentiate Streptococcus pneumoniae from other Viridans Group Streptococci. Clin Lab . 2021 Mar 1;67(3).
- Identification of Streptococcus pneumoniae: Development of a Standardized Protocol for Optochin Susceptibility Testing Using Total Lab Automation. Biomed Res Int . 2017;2017:4174168.
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Deoxyribonuclease Test (DNase Test): Principle, Steps, and...
Continue ReadingDeoxyribonuclease Test
Deoxy ribonucleic acid is one of the large molecule sized polymer, which is being composed of multiple nucleotides in the form of monomer and it is large in size and it is not able to enter cell membrane of the bacteria directly.
Thus, microorganisms like bacteria produces an enzyme known as Deoxyribonuclease, which helps in breaking down the DNA into smaller monomers, so that the cell membrane can easily absorb or intake it.
The nucleotides thus taken are used for making nucleic acid in the bacteria, and it also acts as a source of nitrogen, carbon and phosphorus.
Some microorganism even produces extracellular DNase which breaks down the larger mass of DNA into smaller monomers, so that it is easy for the taking up the vital nutrients that are necessary for the organism through cell membrane by means of protein transports that are present on the membrane of the cell.
But the degradation of DNA is a virulent factor, which causes serious infections in the hosts cell and it results in various drastically symptoms.
The ability to produce DNase can be used in differentiating a different pathogenic organism. Thus, Deoxyribonuclease is an enzyme which helps in catalyzing the hydrolytic cleavage of phosphodiester bonds, which forms the back bone of the DNA, which results in degradation of the DNA.
Deoxyribonuclease is also considered as one of the types of nuclease, a generic form for enzymes that are capable of hydrolyzing phosphodiester bonds.
DNA ribonuclease test is often called as DNA hydrolysis test or DNase test, which helps in determining the ability of the organism to hydrolyze DNA and to utilize it as a source of energy and carbon for the growth.
What is Deoxyribonuclease Test (DNase Test)?
Deoxyribonuclease test is one of the biochemical tests which is being performed to differentiate the organisms on the basis of their ability to produce the enzyme Deoxyribonuclease.
This test is used presumptively to differentiate one of the species of bacteria known as Staphylococcus aureus, which also plays an important role in producing deoxyribonuclease, the enzyme from the other species of staphylococci.
As Staphylococcus aureus produces heat stable enzyme, a thermo nuclease.
To detect this enzyme, initially the organisms are destroyed using heat and then the free DNase reacts in the medium.
This test also gives positive results for Vibrio, Helicobacter, Moraxella, Aeromonas, Serratia.
Deoxyribonuclease Test Principle
This test is used to determine the ability of a particular organism to produce the enzyme DNase.
DNase are the extracellular endonucleases, which cleaves the DNA and there by releases free nucleotides and the phosphates.
To detect these enzymes, DNase agar is used to detect the hydrolysis of the DNA, without the use of any indicators.
When without using any of the indicator, DNA agar helps us to observe the hydrolysis of DNA by clearing the agar after adding HCL.
This acid helps in precipitating the unhydrolyzed DNA and makes the medium opaque.
Hence, DNase producing the colonies produces, colonies which hydrolyses the DNA and results in clear zone around the growth of bacteria.
When DNase is left to react with methyl green it produces, mint green color by combining with DNA.
On hydrolyzing DNA, complex is released and the methyl green changes into a colorless compound at changes its structure when a pH of about 7.5.
Hence it results in the formation of clear halo like appearance around the areas where the organisms producing DNase are grown.
If toluidine blue O is added to the DNase agar.
It results in the formation of complex in the DNA and changes its structure on hydrolyzing the DNA resulting in bright pink color.
Deoxyribonuclease Test Materials
Media: DNase agar or DNase agar with methyl indicator is generally used.
Reagents: Hydrolytic acid is used only when DNase agar is used without using an indicator.
Supplies used: Inoculating loop, Bunsen burner.
Deoxyribonuclease Test Procedure
Initially the Surface of the agar plates are dried before using, Each plate is divided into sections by drawing the line below the glass plate.
Then agar medium is inoculated by either of the following methods.
Spot Inoculation Method
o Touch the colony of the organism under test using a loop and inoculate it on the small area of the test agar plate, in the center of the marked area in any one of the sections, to form a thick plaque of growth about 5-10mm in diameter after incubating.
o The incubation is done at a temperature of about 37ºC for 18 to 24 hours.
o Band or Streak line inoculation.
o Here heavy inoculum is used and a line of about 3 to 4 com long is drawn from rim to the center of the DNase agar plate.
o Plate is then incubated at a temperature of 37ºC for about 18 to 24 hours.
DNase Agar Test Without Indicator
o The plate is being flood with 1N of hydrochloric acid.
o After flooding, the plate is left to stand for few minutes until the reagent is being absorbed into the plate.
o Excess hydrochloric acid is decanted and the plate is further examined in a dark background.
Deoxyribonuclease Test Result
1. Positive Deoxyribonuclease Test: When DNA is hydrolyzed, methyl green is released which turns the medium colorless around the test organism.
2. Negative Deoxyribonuclease Test: In case of negative result, there will be no degradation of DNA, and the medium remain green without any change or absence of color.
Organisms Tested for Deoxyribonuclease Test
Positive organisms in DNase test:
o Serratia Marcescens
o Staphylococcus aureus
o M. Catarrhalis
o Campylobacter jejuni
DNase test negative organisms:
o Staphylococcus epidermidis
o Neisseria gonorrhoeae
Deoxyribonuclease Test Citations
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Gelatin Hydrolysis Test: Definition, Principle, Procedure, and...
Continue ReadingGelatin Hydrolysis Test
Generally, gelatin is used to define an animal protein or a collagen which is a component of a vertebrate in a connective tissue. Gelatin is used as a solidifying agent in a food for a long day ago, Organisms that produces proteolytic enzyme, specifically gelatinases help in hydrolysing the gelatin into a polypeptide and individual amino acids.
During this process, gelatin losses its structure and converts in to liquid form. Robert Koch used gelatin in his culture in the form of a nutrient gelatin which is one of the oldest solid culture media.
Gelatin usually dissolves in a water at 50 degree Celsius and it exists as the liquid at a temperature of above 25 degree Celsius and it further solidifies and forms a gel like substance when it is cooled below 25ºC.
What is Gelatin Hydrolysis Test?
Gelatin hydrolysis test is also called as Gelatin liquefication test, as this test involves the process of liquefication of gelatin in the presence of an enzyme gelatinase.
Gelatinase is considered as one of the most important enzymes in various pathogenic organisms as it is produced extracellularly, and hydrolysis gelatin which is derived from the connective tissues of the vertebrates in the form of collagen.
This enzyme also works as a virulent factor which dissolves the connective tissues of the host and aids in producing invasive infections.
Gelatin is a protein that hydrolyses in the presence of an enzyme gelatinase by breaking down the complex structure into monomeric amino acids.
This test is being followed from early days in the form of presumptive test to identify the pathogenic organisms like Serratia, Pseudomonas, Flavobacterium and Clostridium.
Gelatin Hydrolysis Test Principle
Gelatin is a type of protein derived from the animal tissues, collagen and it forms a solid structure at low temperature. This protein is metabolised or degraded by a group of enzymes known as gelatinase.
Gelatinases are the proteolytic enzymes which liquifies the gelatin into polypeptides and individual amino acids.
The degradation of the gelatin into polypeptides, is followed by covering the polypeptides into amino acids.
Gelatinase is very important in bacteria as gelatin is comparatively a large polymer and thus it cannot be transported into the cell membranes, as gelatinase breaks this compound into the smaller peptides it can easily be transported into the cell and it is utilised by the bacteria.
In hydrolysis test, media containing gelatin is used and its hydrolysis is detected either by liquification of the media or by flooding the media with mercuric chloride, as mercuric chloride helps in precipitating the gelatin and make the hydrolysed area to look clear.
This test is commonly used to determine the ability of an organism to produce the extracellular proteolytic enzymes, gelatinases, which hydrolyses the gelatin, a component of the vertebrate connective tissue.
This reaction usually occurs in a series of two steps, in first reaction process, Gelatinases hydrolyses gelatin into polypeptides and further the polypeptides are converted into amino acids.
The amino acids are taken up by the cells and they are used for their own metabolic purposes. Generally, the presence of gelatinases can be detected using a nutrient gelatin medium.
When an organism produces gelatinase, the enzymes liquifies the growth medium by liquifying the gelatin that is present in the medium.
Gelatin Hydrolysis Test and Microorganism
Gelatin hydrolysis test is generally used to detect the micro-organisms Gram negative rods require gelatine for the identification of specific fluorescent pseudomonas. Such as Pseudomonas putida (negative) from pseudomonas fluorescens (positive). Whereas the gram-positive rods are needed for identifying the pathogens in species level.
Gelatin Hydrolysis Test Materials
Nutrient Gelatin media is generally used for the purpose of demonstrating the hydrolysis of gelatin by adding mercuric chloride or by using liquification of gelatin.
Ingredients Gram/litre Tryptose 20.0 Gelatin 10.0 Magnesium sulphate 0.01 Beef extract 3.0 Agar 15.0 Reagents:Â
• Mercuric chloride
Supplies:
• Inoculating needle
• Incubator
• Pipettes
Gelatin Hydrolysis Test Procedure
1. Preparation of the media
First, the media is prepared. 128 grams of dehydrated medium mixed with 1000 millilitres of warm distilled water in a beaker.
The prepared solution is heated with agitation to bring about the boiling point and the media is let to dissolve completely.
Then the prepared medium is dispensed into series of test tubes and it is autoclaved at 15lbs pressure for about 15 minutes. If agar plate method is used, the medium is autoclaved in the beaker.
The tubes are cooled after autoclaving to about 49 to 50 degree Celsius by keeping it in an upright position.
2. Gelatin Hydrolysis
Gelatin hydrolysis can be identified by nutrient gelatin slab method or by agar plate flooding method by using mercuric chloride.
a. Stab Method of Gelatin Hydrolysis Test
Gelatin medium in a tube is inoculated with 4 to 5 drops of a 24-hour broth medium.The inoculated tubes are then incubated at a temperature of about 37 ºC in the air for 24 to 48 hours, If the organisms have the capability to grow at 25ºC then the incubation should be done at 25ºC. After the first incubation, the tubes are placed at 4ºC for one 24 hours.
b. Plate Method of Gelatin Hydrolysis Test
In plate method, heavy inoculum of a culture is taken using an inoculating loop and it is inoculating using nutrient gelatin medium. And the plates are kept in an incubator, by setting a temperature of 37ºC for about 24 to 48 hours.
Gelatin Hydrolysis Test Result
1. Tube Method: In positive result, either partial or total liquefication of the gelatin can be observed in the tubes. In negative result, complete solidification of gelatin is seen at a temperature of 4ºC.
2. Plate Method: In positive result, clear zone around the colonies is noted after adding mercuric chloride. If there is no clear zone after adding mercuric chloride, it indicates the negative result.
Gelatin Hydrolysis Test Uses
 Gelatin hydrolysis test is usually used to test the capability of an organism to produce gelatinase.
 This test also helps in identification of Serratia, Pseudomonas, Flavobacterium and clostridium.
 Gelatin hydrolysis test also helps in distinguishing the gelatinase positive staphylococcus aureus from the gelatinase negative non-pathogenic S. epidermidis.
 Using this test; genera of bacteria such as Serratia and proteus are differentiated from other members of Enterobacteriaceae family
Gelatin Hydrolysis Test Citations
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Hippurate Hydrolysis Test: Definition, Principle, Procedure, and...
Continue ReadingHippurate Hydrolysis Test
Hippurate hydrolysis test is one of the biochemical tests, which is used to differentiate the micro-organisms on the basis of their ability to hydrolyze Hippurate into benzoic acid and glycine by the action of the enzyme hippuricase, present in the bacteria.
Hippuricase is one of the constitutive enzymes which helps in hydrolyzing the Hippurate and helps in producing amino acid, glycine.
Glycine can be detected by the oxidizing the Ninhydrin reagent, that results in the production of a deep purple color.
Hippurate hydrolysis test is used in the identification of Gardnerella vaginalis, campylobacter jejuni, Listeria monocytogenes and group B streptococci; to detect the ability of the organism to hydrolyze Hippurate.
What is Hippurate Hydrolysis Test?
In olden days this test was performed using a ferric chloride indicator to identify the benzoic acid, but those traditional methods would take a longer time to resolve.
But now a days with the modern techniques, this test has been modified and it is used as a rapid test by detecting the glycine by adding ninhydrin as an indicator.
This test can also be used to distinguish the Group B streptococci from other groups A, C, F and G which cannot hydrolyze sodium Hippurate.
Other group of viridians like Group D can be also detected by using Hippurate sodium hydrolyze.
This test is considered as one of the class of test which differentiates the bovine-β-hemolytic Group B streptococci from human β-hemolytic Group B species of streptococcus.
Hippurate Hydrolysis Test Objective
Hippurate hydrolysis test is generally used to detect the production of the enzyme hippuricase; for identifying the presumptive of different microorganisms. Its main aim is to differentiate bacteria based on their ability to produce hydrolyzed Hippurate.
Hippurate Hydrolysis Test Principle
Hippurate test is based on the ability of the organism to hydrolyze sodium Hippurate into glycine and benzoic acid with the action of an enzyme hippuricase.
This test is primarily used in identifying the campylobacter jejuni, Gardnerella vaginalis, Listeria monocytogenes and streptococci’s agalactiae.
This ability of the few bacterial species to hydrolyze the Hippurate was classically tested using a ferric chloride indicator which helps in detecting the benzoic acid.
However, now a days a 2-hour rapid method is opposed to 48-hour tractional method, for detecting the Hippurate hydrolysis.
The rapid is done by using ninhydrin as an indicator, which on reaction with protein or amino acids detects glycine.
As glycine is deaminated by oxidizing action of ninhydrin, which results in the reduction of ninhydrin and the substance resembles in purple color.
The test medium used here should contain only Hippurate as a source of protein as ninhydrin acts with the free amino acids that is present.
Thus, rapid Hippurate hydrolysis test helps in detecting the by- product of the benzoic acid which is seen as a sensitive and classical method.
Hippurate → Glycine + Benzoic acid
Glycine + Ninhydrin → Purple colored complex
The two important reagents used in this test are Hippurate solution and Ninhydrin.
1. Hippurate Solution
Hippurate reagent can be found commercially in the form of dehydrated tubes or in the form of disks or tablets. This can also be prepared in the laboratory in the form of 1% of Hippurate solution, for preparing in the laboratory, one gram of sodium Hippurate is added into 100 ml of distilled water.
2. Ninhydrin
Ninhydrin can also to purchased commercially. But mostly it is prepared in the laboratories while performing the diagnosis, for laboratory preparation, of the ninhydrin, 50ml of 1-butanol is added into a dark colored glass bottle. Then 3.5 grams of ninhydrin is added to the bottle and it is mixed well.
Hippurate Hydrolysis Test Materials
 Sterilised wooden stick and inoculating loops
 Incubator
 Test tubes
 Distilled water
Hippurate Hydrolysis Test Procedure
1. Preparation of Hippurate Solution
If dehydrated Hippurate is used, 0.2ml of distilled water is added at a pH of 6.8 to 7.2 is added with the test reagent. Then the two drops of distilled water are added to an empty tube for disk or tablets If Hippurate solution is prepared in the laboratory, 0.4ml of the reagent is added to the tube for each test.
2. Hippurate Hydrolysis Test
A heavy suspension is prepared in a tube from an 18 to 24-hour culture. The colony is then picked up carefully where the agar which contains protein should not be taken. The tube is then incubated for 30 minutes, at 35ºC to 37ºC.
Then the tube is observed for every for 10 minutes intervals till the deep blue colour; The change in colour appears usually appears within 15 minutes, after the addition of ninhydrin solution.
Hippurate Hydrolysis Test Result
 A positive Hippurate hydrolysis test results in the appearance deep blue which almost looks like a crystal violet within 30 minutes.
 A negative Hippurate hydrolysis test, results in the appearance of a faint blue colour or there will be no colour change.
Control Organism in Hippurate Hydrolysis Test
 Streptococcus agalactiae: it indicates Hippurate positive
 Streptococcus pyogenes: It indicates Hippurate negative
Hippurate Hydrolysis Test Citations
- Rapid, colorimetric test for the determination of hippurate hydrolysis by group B Streptococcus. J Clin Microbiol . 1976 Jan;3(1):49-50.Â
- A rapid hippurate hydrolysis test for the presumptive identification of group B streptococci. Pathology . 1983 Jul;15(3):251-2.
- Evaluation of the rapid hippurate hydrolysis test with enterococcal group D streptococci. J Clin Microbiol . 1977 Mar;5(3):290-2.
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Esculin Hydrolysis Test: Principle, Procedure, Purpose, and...
Continue ReadingEsculin Hydrolysis Test
Biochemical tests are considered as the most important method to detect the micro-organisms and other pathogens. One such biochemical test is Esculin hydrolysis test.
It is one of the differential tests, which helps in differentiating the bacteria depending on the ability of an organism to hydrolyse esculin. This test is generally based on the hydrolysis of the esculin, a glucoside into a glucose and esculetin by a micro-organism which has constitutive β-glucosidase or esculinase enzyme.
What is Esculin Hydrolysis Test?
Esculin hydrolysis test is used to differentiate the type of bacterial species like the gram-positive and gram-negative bacteria, which also includes along with it a a broad spectrum of aerobes, anaerobes and facultative anaerobes.
This test is generally used as a taxonomic tool in identifying the variety of microbes, including Enterobacteriaceae family, streptococcus genera and Listeria, non-fermentative gram negative-bacilli and anaerobes.
Esculin hydrolysis test thus helps in differentiating the bacteria based on their ability to hydrolyse esculin.
This test is usually done selectively by adding bile in the medium, and it is commonly called as bile esculin test.
Hydrolysis of esculin by bacteria can be done by detecting the growth in the supporting media such as Vaugh-Levine, bile-esculin, or Pfizer selective enterococcus media or by non-growth supporting media like Patho Tech or Rapid spot tests.
This test fully depends upon the hydrolysis of esculin into glucose and esculetin with the help of microorganism which constitutes β-glucosidase or an enzyme esculin’s.
Objective of Esculin Hydrolysis Test
• This test helps in detecting the ability of an organism to hydrolyse esculin by producing the enzyme esculinase.
• This test also helps in differentiating the members of the family Enterobacteriaceae.
Esculin Hydrolysis Test Principle
The basis of esculin test is to hydrolyse the esculin during the presence of bile salts, by the enzyme esculinase.
Esculin is a glucosidase consisting of glucose and the hydroxycoumarin which is linked together by an ester bond through oxygen.
The bile esculin test selects an organism on the basis of their ability to grow in a medium containing 4% of salts which is followed by selection on the basis of their ability to hydrolysis of the esculin, which results in the formation of the compound called esculetin.
After the degradation of esculetin it reacts with the iron ions in the medium to form phenolic iron complex which results in a dark brown or black colour.
In the other sense, esculin is a fluorescent compound and on hydrolysis can be observed by loss of fluorescence.
When bile is added to the medium, micro-organisms grow in order to hydrolyse esculin. Bile inhibits the growth of other gram-positive organisms and makes the medium selective.
Esculin Hydrolysis Test Materials
Media: Esculin agar is used for detecting the hydrolysis of esculin. This medium is a differential medium and they can be made selective by adding bile.
Composition of Esculin agar is listed below;
Ingredients Gram/Liter Casein enzymic hydrolysate 13.0 Yeast extract 5.0 Beef heart infusion in a solid state 2.0 Sodium chloride 5.0 Ferric chloride 0.5 Agar 15.0 Reagents: In esculin spot test, 0.02% of esculin solution is prepared using distilled water.
Supplies:Â
• Long wave UV light up to 360nm
• Sterilised sticks, needles, or inoculating loops
• Pasteur pipettes
• Boiling heat block
Esculin Hydrolysis Test Procedure
1. Preparation of the media
About 41.5 grams of dehydrated powder is added in 1000 millilitres of distilled or deionised water and it is mixed thoroughly, after mixing the solution is heated till it reaches the boiling point and the medium to let to dissolve completely.
The solution is then dispensed into a tube having screw caps and it is sterilised in the autoclave at 121 degree Celsius for about 15 minutes.
Then the tubes are taken out from the autoclave and they are cooled by placing them in a slanted position up to a temperature of about 40 to 45º.
This position is maintained at the same angle to achieve the butts of about 1.5 to 2,0 cm depth.
2. Esculin Hydrolysis Test
Esculin hydrolysis test is usually observed in two methods namely tube test or esculin spot test. The spot test is often known as rapid test.
I. Esculin Hydrolysis Tube Test
In tube test, first the light inoculum is taken from the 18 to 24-hour culture using a sterile inoculating needle from the centre of the well-isolated colony.
Then the esculin agar tubes are inoculating by streaking a slant on the surface using the light inoculum which has been picked up from the culture.
Then the caps of the tube are checked for adequate aeration. Then further the tubes are incubated in the air at 35 to 37ºC for about 24 hours and any change is colour is observed up to 7 days of incubating.
In case, if esculin broths are used the tubes are observed daily and checked whether there is any loss in fluorescence of UV light. If there is loss of fluorescence, 1% of ferric ammonium id added in drops into the tube and colour changes are observed.
II. Esculin Hydrolysis Spot Test
In rapid spot test, the prepared 0.2% of esculin solution is autoclaved and it is placed on the filter paper; then the filter paper is placed on a standard microscopic slide and they are positioned on the supporting rods.
Esculin solution is pipetted over the filter paper and over saturation must be avoided. Then the inoculum is taken from a 24-hour colony and streaked in middle of the filter paper.
Then the prepared filter paper along with a slide is placed in an inoculum at 37ºC for about 10 to 15 minutes.
Klebsiella generally gives a positive result within 15 minutes, but it should be holded for 30 minutes to check the proper result.
Hand-held wood lamp is used in the subdued light to observe the loss of fluorescence.
Esculin Hydrolysis Test Result
Esculin Hydrolysis TubeTest: Blackening of the medium detects the positive result and lack of colour change detects the negative result.
Esculin Hydrolysis Spot Test: Loss of fluorescence and black colour under a UV light demonstrates a positive result and bright fluorescence indicates the negative result.
Esculin Hydrolysis Test Citations
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Indole Test: Principle, Procedure, Purpose, and Results
Continue ReadingWhat is Indole Test?
Indole test is one of the biochemical tests which is conducted on the bacterial species to detect the ability of pathogen to produce indole from tryptophan in the presence of a group of enzymes known as tryptophanase.
Generally, it is a qualitative test, which is performed as part of the IMViC test and is used to differentiate the members of the Enterobacteriaceae family.
It is very important in identifying the different species of bacteria like Escherichia coli, proteus, Morganella, etc.
This plays a central role in characterizing the coliforms and it is denoted by various differences of the indole test and also in combination with other biochemical tests.
It is used as a traditional method to distinguish Indole negative Enterobacter and Indole Positive E. coli and Klebsiella.
Variation of the test is known by Ehrlchs reagent that is used when the test has been performed in non-fermenters and anaerobes.
Indole Test Purpose
Indole test is used to detect the ability of the organism to produce enzyme tryptophanase.
Indole Test Principle
As mentioned before, Indole test is one of the biochemical tests which differentiates the coliform from other members of the Enterobacteriaceae; by detecting the ability of the pathogen to produce enzyme known as tryptophanase.
Tryptophanase hydrolyses the amino acid known as tryptophan into indole, ammonia and pyruvic acid.
It is usually known as intracellular enzyme or endoenzyme. Pyruvic acid thus produced can be used by the organism in the Krebs’s cycle or it enters into the glycolysis pathways and it is used to synthesize other necessary compounds for the cell.
The culture medium used for indole test involves Sulphide, Indole, motility (SIM) or nutrient peptone, either of the given media provides necessary amino acid a tryptophan which acts as a substrate the reaction.
Thus, the organism is able to produce indole as a main product and ammonia as a byproduct.
Kovac’s reagent is used as the reagent for this test which reacts as a side product in catabolizing the tryptophan and this results the indole to form a Rosindole dye which is cherry red in color.
Hence the formation of cherry red color denotes the positive indole test and it is not in the sense that E. coli is positive to indole and Klebsiella is negative to it.
Indole Test Materials
• Filter paper
• Watch glass
• Test tubes
• incubator
• Peptone broth-enriched with amino acids and tryptophan
• Bacterial samples of Escherichia coli and Klebsiella species
• Kovac’s reagent.
Indole Test Procedure
Indole test can be performed in two ways such Rapid Spot test and tube test.
1. Rapid Spot Indole Test
 To perform rapid spot test, a piece of filter paper is taken and is it moistened using the reagent.
 An isolated colony from a cultured medium is taken using an inoculated loop and it is rubbed against the moistened filtered paper.
 Then the filter paper is observed for color change
Rapid Spot Indole Test Interpretation
 If the appearance of blue color is observed in the filter paper within 20 minutes, then it indicates the positive result of indole test.
 If there is absence of any colors in the filter paper then it denotes the negative result for indole test.
2. Tube Indole Test
 Either peptone broth or medium is prepared using Sulphide, Indole and motility in the test tubes and the test tubes are autoclaved at 15lbs/inch pressure for about 15 minutes.
 By using the sterile wire, broth is inoculated with the samples that were provided and the test tubes are labelled with the specific name of the organism.
 Further the test tubes are incubated at 37ºC for 24 to 48 hours.
 After the proper incubation, test tubes are taken from the incubator and are added with four to eight drops of Kovac’s reagent in each of the test tubes, in such a way that reagent touches the walls of the test tubes.
 After adding the reagents, the test tubes are placed in between the palms and they are shaken to mix the reagent such that it mixes well with the culture.
 The test tube is left undisturbed till the solution stands and development of cherry red color is observed at the surface of the media.
Tube Indole Test Interpretation
 Indole test is diagnosed as positive if it appears in the cherry red color; it also indicates the presence of E. coli.
 Indole test shows negative result, if there is no formation of red color, and indicates the presence of Klebsiella.
Uses of Indole Test
• Indole test is generally used to test the ability of an organism to utilize tryptophan and to produce indole.
• This test is used to differentiate the members of the Enterobacteriaceae family as a part of IMViC test.
• This test also helps in differentiating the Proteus mirabilis from other species of Proteus.
• This test also differentiates the indole positive E. coli and the Indole negative Enterobacter and klebsiella.
• Along with this, this test also helps in differentiating K. pneumoniae, which is indole positive from K. oxyotoca, an indole positive and also in differentiating Citrobacter freundii, an indole Negative from the Citrobacter koseri, an indole positive species.
Indole Test Citations
- Indole-spot test in bacteriology. Tech Bull Regist Med Technol . 1963 Mar;33:47-50.
- Product in indole detection by Ehrlich’s reagent. Anal Biochem . 2015 Sep 1;484:21-3.
- Technique of indole test. Acta Pathol Microbiol Scand . 1959;46:349-60.
- Spot indole test: evaluation of four reagents. J Clin Microbiol . 1982 Apr;15(4):589-92.
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