Category: Biology
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Nitrate Reduction Test: Result, Principle, Procedure, and...
Continue ReadingNitrate Reduction Test Introduction
Biochemical tests are generally used to determine the ability of the microorganism to be stable in the enzymatic field. One such test is Nitrate reduction test, which helps us to differentiate between the species of the bacteria based on the ability or their inability to reduce nitrate into nitrite using an anaerobic respiration.
This anaerobic metabolism usually required an electron acceptor other than the atmospheric oxygen.
Many of the gram-negative bacteria use of the nitrate as the final electron acceptor.
Nitrate reduction test is one of vital test which helps us to determine the production of an enzyme known nitrate reductase, which helps us to reduce the nitrate.
Bacterial species is generally used to differentiate bacteria on the basis of their ability to reduce nitrate into nitrite or nitrogenous gases.
Nitrate Reduction Test Objective
• The main aim of the Nitrate Reduction Test is to determine the ability of an organism to reduce nitrate to nitrite.
• Nitrate Reduction Test also helps us to identify the different ways in which the nitrate is reduced in the bacteria.
Nitrate Reduction Test Principle
A heavy inoculum of the test organism is incubated into a broth, that contains nitrate. The organism which are all capable of producing a nitrate reductase enzyme has the capability to reduce nitrate to nitrite that is present in the broth which is further reduced in nitric oxide or nitrogen or nitrous oxide.
The nitrate reduction test is completely based on how the nitrate is reduced by the organism and its ability to form a red compound on it reacts with a sulfonic acid to form a complex known as nitrite-sulfanilic acid which then reacts with the alpha-napthylamine and produces a red precipitate known as Prontonisil which has an ability to soluble in water.
However, when the nitrate is alone present in the medium, there will be production of red color. If there is no formation of red color in the media in which the sulfamic acid and the alpha-naphthylamine is added and nitrite will not be present.
There are generally two explanations for the observation;
1. The nitrate should not be reduced, the strain is considered as nitrate-negative.
2. The nitrative can be reduced into nitrite that have been completely reduced into the nitric oxide or nitrous oxide or in the form of nitrogen which on reacting with nitrogen produces a nitrate-positive strain.
In case, when nitrate is not detected it is significant for the test that the organism is reduced into nitrate beyond the nitrite. This can be done my indirect means by adding a small amount of zinc powder into the culture.
Zinc powder helps in catalyzing the reduction of nitrate into nitrite. The development of red color can be seen on addition of the zinc which indicated that the nitrate has not been reduced by the particular organism as it does not have its capability to reduce nitrate.
In case if there is no change in color after adding zinc powder, it shows that the organisms reduced the nitrate present in the medium into nitrite or other compounds of nitrogen, and shows that it is a nitrate reducer.
Nitrate Reduction Test Reagents
Media:
Ingredients Gram per liter Peptone 5.0 Meat extract 3.0 Potassium nitrate 1.0 Nitrate Reduction Test Procedure
• Here the determination of nitrate reduction involves two steps of processes as follows
• Initially, the reduction of nitrate into nitrite is usually determined by addition of reagents of the nitrate A and B, if it is necessary, the reduction of nitrate beyond the nitrite is usually determined by adding the nitrogen reagent as zinc powder.
• First, the nitrate broths are inoculated in a bacterial suspension.
• The tubes are usually inoculated at a temperature of 30 to 37ºC for about 24 hours.
• After completing the process of incubation, the release of nitrogen gas is noted before addition of the reagents.
• Then about 6 to 8 drops of nitrogen reagents A and B are adding into the medium.
• After addition of the ingredients a medium is observed for any color change.
• In case, if there is no color change is noted, then zinc powder is added into the medium.
• Then the medium is being observed for at least 3 minutes for a change or formation of red color.
Nitrate Reduction Test Results
Positive Nitrate Reduction Test:
• In case of positive results, there will be a formation of a cherry red color while adding the reagents A and B.
• There will be no change in color or production of red color after adding the zinc powder.
Negative Nitrate Reduction Test:
• In case of negative result, there will a formation of red color only after adding the zinc powder into the medium containing the reagents
Nitrate Reduction Test Citations
- Nitrate reduction: new method for testing the antibiotic susceptibility of Haemophilus influenzae. Antimicrob Agents Chemother . 1978 May;13(5):791-5.
- Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system. J Microbiol Methods . 2010 May;81(2):208-10.
- The Use of the Nitrate Reduction Test in Characterizing Bacteria. J Bacteriol. 1919 May; 4(3): 267–290.
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Motility Test: Result, Principle, Procedure, and Reagents
Continue ReadingMotility Test
Generally, Motility is the ability of the organism to move itself by using a flagella or some other propellers which are unique for each of the organism and helps in its locomotion.
Where as bacteria confines a unique form of locomotion by using its specific fibrils which looks like a gliding sort of motion.
The motile bacteria move either by using a flagellum, a thread like locomotor which extends outward from the cell membranes.Â
Motility is too long to be recognized as it has it own important characteristic feature in the world of taxonomy.
Usually, the flagella are present primarily in all kind of basic bacilli’s, but few of them are flagellated cocci, which have their motility varying manner depending upon the different genera.
However, identification of motility is very important for categorizing the family of Enterobacteriaceae.
In microbiology, from early days, differentiation and classification of Enterobacteriaceae is identified by means of performing the kinds of motility test.
Motility Test Objective
• The main aim of the test is to detect the motility of the bacterium.
• To differentiate between the motile and a non-motile species of bacteria.
Motility Test Principle
Usually, the motility of the bacterium is demonstrated by using a semi solid medium of agar. As in the semi solid medium, motile bacteria swarm and it gives a diffused spread of growth which can be recognized in a naked eye.
The agar medium is usually used in the Sulphide Indole motility medium which is a combination of the differential medium where the test is performed using three different parameters such as reduction of Sulphur, Indole production, and motility.
This media has a very soft consistency which allows the bacterium to migrate along with and causing cloudiness in the medium, the inoculum is stabbed to the center of the semi solid medium deeply.
Bacterial motility is identified by a diffuse zone of growth which extends from the line of the inoculation. Whereas some organisms which grow along the entire medium shows very small areas of growth or there will be formation of nodules in a particular area.
On the other hand, non-motile bacteria grow only in the soft agar tube and only in areas where they are inoculated.
Motility Test Reagents
Here SIM medium is generally used. It consists of the following ingredients.
Ingredients Gram per liter Pancreatic digest of casein 20.0Â Peptic digest of animal tissue 6.1 Fe (NH4)2(SO4)2.6H2O 0.2 Na2S2O3.5H2O 0.2 Motility Test Procedure
Initially, a straight needle is used to touch a young colony which has been kept for about 18-to-24-hour culture in a medium of agar.
Then the medium is stabbed once in the middle of the tube up to half an inch of the tube.
It should also be made sure that the needle should be removed from the tube as the same way it entered.
Then the medium is incubated at a temperature of 35 to 37ºC and it is observed frequently up to 7 days.
Within seven days, the diffused zone of growth which is flaring out from the line of inoculation can be seen.
Motility Test Results
Positive Motility Test​ Result: If the medium gives a positive result, then it gives a diffuse, hazy growth that spreads across the medium which is renders as slightly opaque one.
Negative Motility Test​ Result: In case of negative results, the growth is confined to the stab line, which has sharp defined margins and leaves the surrounding area of the medium opaque.
Motility Test Uses
• Motility Test​ is significantly used for differentiating the microorganisms on the basis of their motility performed in a laboratory setting
• Motility Test​ is also useful for assigning a taxonomic classification of the organisms.
• Motility Tests also plays an important role in characterizing the pathogens.
• Motility Test are usually employed for identifying the protocols which belongs to the family of Enterobacteriaceae.
• Motility Test​ is used for the specific species which helps to differentiate the gram-positive cocci, Enterococci, Faecalis, E. casseliflacus, Enterococcus faecium.
Motility Test Limitations
Some kind of organisms will not show the sufficient growth in the medium, which makes an accurate determination and the additional follow-up testing is needed.
It is also recommended that the biochemical, molecular, mass, spectrometry testing; are all performed in the colonies that are taken from the pure culture for complete identification of the species.
If the flagella of the bacteria are damaged due to heating, shaking or any other trauma it results in false-negative results. On the other hand, if the organisms are found to have a weak and unstable mode of motility, it leads to false-negative reactions.
While inoculating in a semi-solid media, it is important to note that the inoculating medium is removed along the straight line in the same way it is inserted. A fanning motion of removing a needle may result in false-positive results.
Motility Test Citations
- Evaluation of 15 motility media and a direct microscopic method for detection of motility in enterococci. J Clin Microbiol . 2002 Jul;40(7):2476-9.
- Use of Two Selective Media and a Broth Motility Test Can Aid in Identification or Exclusion of Bacillus anthracis. J Clin Microbiol. 2005 Sep; 43(9): 4336–4341.
- Bacterial Motility .J Bacteriol. 1934 Feb; 27(2): 165–174.
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MUG Test: Result, Principle, Procedure, and Reagents
Continue ReadingMUG Test
It has been reported that the enzyme beta-glucuronidase is generally present in many strains o the E. coli and also in other organisms like Salmonella, Shigella, Staphylococcus, Streptococcus along with E. coli which contains the enzyme Beta-glucuronidase.
Thus, the detection of this enzyme, Beta-glucuronidase is determined in the biochemical laboratories by using this method which helps us to identify and differentiate the certain organisms.
The substance known as MUG (4-methylumbellferyl-Beta-D-glucuronide is considered as a sensitive and a selective substrate for detecting the activity of Beta-glucuronidase.
Thus, MUG test is considered as a conjunction with the substrate like oxidase, indole and the fermentation of lactose, which is generally performed in an effective manner in order to identify the species of E. coli and the other such related organisms.
MUG is an acronym of 4-methylumberlliferyl Beta-D-glucuronide. Shortly, MUG test is used for rapid identification of the E. coli which has the capability to produce Verodoxin from the strain that do not produce it.
MUG Test Objective
The main aim of the test is to identify the activity of glucuronidase in the organisms by fluorescence, when it is observed under the UV light source having a long wave of about 365nm.
MUG Test Principle
E. coli and the other species of Enterobacteriaceae produces an enzyme known as Beta-D-glucuronidase, that hydrolyses the Beta-D-glucopyranoside-uronic derivatives into the aglycons and the d-glucuronic acid.
Here, the substrate 4-methylumbelliferyl-β-d-glucuronide which is impregnated into the disk and it is further hydrolyzed by the enzyme in order to yield methyl umbelliferyl moiety, that fluoresces blue under a long wavelength in the UV light.
Hence, if the test organism produces the enzyme known as glucuronidase which helps in breaking the substrate thus resulting in the formation of a fluorescence, which is noted as the positive test.
On the other hand, if there is no desired enzymatic activity, then the substrate will not be broken down into fluorescence on test, which is notes as a result for negative test.
MUG Test Reagents
Media: MUG disks are generally prepared by using the impregnating the MUG i.e., 4-mrthylumbelliferyl Beta-glucuronide onto a high-quality filter paper disk.Â
 MUG test
 2 to 3 isolated colonies of organisms
 Inoculating loops
 Petri dishes
 Test tubes
 Incubator
 Long wave UV light
MUG Test Procedure
Generally, two methods are used for performing the MUG test;
i. Direct Disk MUG Test
 Initially, the petri dish is made wet using a drop of water. It should not be saturated.
 With the help of a wooden applicator small portion of a colony is rubbed from an 18 to 24-hour culture.
 Then the culture is incubated in a closed container for about 35 to 37º C for about 2 hours.
 After incubating, the disk is observed under a ultra violet light, a long wave of about 360nm in a darkened room, fluorescence can be observed.
ii. Tube MUG Test
 Initially 0.25 ml of deionized water is taken in a clean glass tube.
 Then a heavy suspension is created using up to 3 to 4 colonies of the isolated test in the glass tube.
 With the help of the force, a MUG disk is placed in the tube and it is shaken strenuously to make sure that the adequate elution of the substrate in the surrounding medium.
 Further the medium is incubated aerobically for about one hour at a temperature of 35 to 37ºC.
 After incubation, the tubes are taken out and they are detected for the presence of fluorescence by using a longwave of ultra violet for about 360nm in a darkened room.
MUG Test Results
Positive MUG Test Results: In case of positive result, there will be fluorescence, which resembles an electric blue.
Negative MUG Test Results: Whereas in case of negative results there will be absence of blue fluorescence.
MUG Test Uses
 This test is widely used for identifying the different genera which belongs to the family of Enterobacteriaceae in a presumptive manner.
 This test also helps us to characterize the Verotoxin producing E. coli. As the Verotoxin producing strains of E. coli does not have the capability to produce a MUG, and a negative test indicates the presence of a clinically important strain.
 This test also aids in detecting the Escherichia coli in the water and in food samples.
MUG Test Limitations
 In MUG test, the colonies that are isolated from the media containing dyes, are not suggested for test as it makes the interpretation difficult.
 This test is reliable only for oxidase-positive organisms as some of the oxidative negative organisms has its fluorescence naturally.
 It is also suggested that the tests such as biochemical, immunological, molecular or a mass spectrometry are generally performed in the colonies that is present in the pure culture for the complete identification.
 Where as some strains of the Shigella results as MUG positive. Serological testing is usually required to differentiate the species of Shigella and E. coli.
MUG Test Citations
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ONPG Test: Result, Principle, Procedure, and Reagents
Continue ReadingONPG Test Introduction
Biochemical tests helps us to determine the characteristics of the microbe in the medium. One such biochemical test is ONPG test. T
he ability of the bacteria to ferment the lactose is generally based on two enzymes named permease and the Beta-galactosidase.
Here permease allows the lactose to enter the cell wall of the bacteria easily then further it breaks down into glucose and the galactose molecules with the help of an enzyme Beta-galactosidase.
Glucose and the Galactose is further metabolized by the bacteria. On the other hand, some organisms lack the enzyme permease and it appears as a late or a non-lactose fermenter.
ONPG test is considered as one of the sensitive tests for fermenting the molecules like lactose. O-nitrophenyl-Beta-D-glucopyranoside is one of the artificial substrates which is incorporated into the medium used here which acts as the important substrate for the beta-galactosidase to ascertain the specific enzymatic activity and it also helps in aiding the identification and differentiation of the different organisms.
ONPG Test Objective
The main aim of the test is to determine the ability of the organism to produce an enzyme known as Beta-galactosidase.
ONPG Test Principle
O-nitrophenyl-beta-D-glucopyranoside, is an artificial substrate which is structurally similar to those of lactose, but this is exception to that of the glucose, substituted with an o-nitrophenyl group. Unlike substrate O-nitrophenyl-beta-D-glucopyranoside is capable of entering into the cell wall of the bacterium without the help of an enzyme permease.
While testing the organism into the broth medium, the organism is taken from a medium containing a high concentration of lactose and is inoculated into the ONPG broth.
If the organism contains the enzyme Beta-galactosidase, which helps in splitting the Beta-galactoside bond, which releases the O-nitrophenol an yellow-colored compound, which indicates the positive result.
Whereas in disk method, the organism that is being tested is taken from the medium containing a high concentration of the lactose. A dense suspension is prepared.
Further the ONPG disk is added to a 5.0 ml of the suspension. If the specific organisms’ posses the beta-galactosidase, the enzyme splits the Beta-galactosidase bond and results in the formation of a yellow color along with the change in suspension.
Organisms having a strong Beta-galactosidase activity helps in producing a positive reaction withing few minutes of inoculating the ONPG medium, where as the other organisms take up to 24 hours.
ONPG Test Reagents
ONPG Test Broth:
Ingredients Gram per liter Na2HPO4 9.46 Phenylalanine 4 ONPG 2 KH2PO4 0.907 ONPG Test Disk
ONPG differentiation disk is usually prepared by carefully impregnating the concentration of the ONPG into a 0.25 inch of diameter filter paper.
ONPG Test Procedure
ONPG test can be performed using two methods.
i. ONPG Test Disk Method
 Initially the ONPG disk is placed into a sterile tube and about 0.2ml of saline is added into it. Further the heavy inoculate is placed in the tube with a loopful of the test isolate.
 The inoculum is then incubated at a temperature of about 35 to 37ºC for about 4 hours.
 After incubation the disk is observed for any of the colour changes.
ii. ONPG Test Broth Method
 Initially the test medium is brought to a room temperature.
 Further the test medium is inoculated with the help of a heavy inoculum using a loop full of test isolate.
 Then the inoculum is incubated aerobically by loosening the caps and it is incubated at a temperature of about 35 to 37ºC.
 After incubation the development of yellow colour can be seen for positive results within one hour.
 If there is no change in colour in the medium after one hour of the incubation period, then the incubation is let to continue for next 24 hours.
ONPG Test Results
Positive ONPG Test: In case of positive test, the formation of yellow colour can be seen in the medium.
Negative ONPG Test: If the results are negative, then there will be no formation of the yellow colour.
ONPG Test Uses
 Generally, this test is used for differentiating the members of the Enterobacteriaceae form the other micro-organism depending on the activity of the enzyme Beta-D-galactosidase.
 This test also helps us to distinguish the lactose late fermenters from the non-lactose fermenters of the Enterobacteriaceae family.
ONPG Test Limitation
 Cultures which produce a natural yellow pigment cannot be tested using this media.
 For the complete identification of the biochemical, immunological and the molecular testing’s, the colonies that are taken from the pure culture is recommended.
 All the organisms that are tested is inoculated from the lactose that contains a medium.
ONPG Test Citations
- Common enterobacterial antigen and ONPG test in the taxonomy of the genus Yersinia. Contrib Microbiol Immunol . 1979;5:1-7.
- Evaluation of Four ONPG Tests for Enterobacteriaceae from Human, Animal and Selected Food Sources. J Food Prot . 1977 Oct;40(10):709-711.
- The ortho-nitrophenol (ONPG) test and acid from lactose in Gram-negative genera. J Clin Pathol . 1973 Nov;26(11):821-5.
- THE ONPG TEST IN DIAGNOSTIC BACTERIOLOGY. METHODOLOGICAL INVESTIGATIONS. Acta Pathol Microbiol Scand . 1964;60:376-86.
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Oxidation Fermentation Test: Result, Principle, Procedure, and...
Continue ReadingOxidation Fermentation Test Introduction
Oxidation Fermentation Test is also commonly known as Glucose test, is one of the biological techniques that is widely used to determine the metabolic way of the micro-organism in the field of microbiology, as micro-organisms are capable of metabolizing the carbohydrates like glucose.
Carbohydrates are one of the organic molecules which comprises of carbon, hydrogen and oxygen in the ratio of the 1:2:1.
Each organism uses carbohydrate in its own kind of way depending upon their enzyme synthesis in the body of the particular organism.
This test was developed initially by Hugh and Leif son in the year 1953, Where they developed an oxidative fermentation media for differentiating the oxidative bacteria and the fermentative bacteria.
Oxidative bacteria are the one which produces acids from the carbohydrates under aerobic conditions. Whereas the fermentative bacteria are the one which facilitates the acid production in both aerobic and anaerobic conditions.
However, the pattern of fermentation depends upon the characteristics of the specific species, genera or group of organisms for which the medium has been used for differentiation of microbes in the biochemical tests.
Oxidation Fermentation Test Objective
The main aim of the test is to detect the process of oxidation or fermentation of the carbohydrate by the bacteria.
Oxidation Fermentation Test Principle
It is considered that whether the organism is oxidative or fermentative, it can be detected by using Hugh and Leifson medium, which is most commonly referred to as OF (oxidation Fermentation) medium, which comprises of tryptone and the bromothymol blue as its indicator.
One of the sugar kinds namely glucose, xylose, mannitol, lactose, sucrose, xylose and maltose can be added in the medium, which plays an important role as the fermentable carbohydrates.
A specific organism is inoculated into two tubes, which are placed in each of the medium. After inoculated the tube is let to overlay with the melted paraffin or the mineral oil which produces an anaerobic environment in the medium.
On the other hand, the other tube is left open such that air can pass into the tube. Here the growth of micro-organisms takes place by using tryptophan that is present in the medium and results in the formation of dark blue color as a result of alkaline reaction.
In some cases, the organisms grow by utilizing the glucose that is present in the medium and produces acid, which turns the medium from bromothymol blue to yellow.
The oxidative utilization of the carbohydrate results in the acid production, by the formation of yellow color in the tube that is left open. Where as in fermentative utilization of the carbohydrate, results in acid production in both the tubes, both open and closed, which is indicated by formation of yellow color in the medium.
Hence the acid production in the tubes is considered as result of true fermentation, where the development of acid in the tubes that are open are a result of oxidative utilization of the carbohydrate that is present. However, Asaccharolytiv organisms will not produce acid in both in tubes.
Oxidation Fermentation Test Reagents
Hugh and Leifson’s Medium:
Ingredients Gram per liter Peptone 2.0 Sodium chloride 5.0 Dipotassium phosphate 0.30 Glucose 10.0 Bromothymol blue 0.03 Agar 3.0 Oxidation Fermentation Test Procedure
Initially a pure isolated colony id being obtained from an 18 to 24-hour culture. Then for each test organism, inoculate the tubes in a duplicate manner.
Then inoculate by medium by stabbing the agar from approximately four by one inch from the bottom of the tube.
Then a sterile mineral oil or a sterile melted paraffin or a sterile melted petrolatum is used in one of each duplicated tube, it is noted that the caps of the over tubes are tightened and the caps of the non-overlaid tubes are closed loosely.
Further the tubes are incubated aerobically at a temperature of about 35ºC for about 14 days.
Oxidation Fermentation Test Results
Positive Oxidation Fermentation Test: Positive carbohydrate utilization test is usually indicated by the development of a yellow color in the tube
Oxidative Results: In oxidative utilization, development of a yellow color can be seen in the open tube.
Fermentive Results: In fermentative utilization, yellow color can be seen in both open and closed tubes.
Negative Oxidation Fermentation Test: Negative carbohydrate test is identified by absence of the yellow color in the tube. The media in the tube remains greens or in some cases it turns blue.
Oxidation Fermentation Test Uses
Oxidative fermentation test greatly helps in identifying the gram-negative bacteria on the basis of their ability to oxidize or ferment a specific carbohydrate.
This test also helps us to determine whether the organism utilizes the substrates, carbohydrate for the production of acids and its byproducts.
Whereas the non-fermentative bacteria are routinely tested to find ability to synthesis acid from the six carbohydrates in the form of glucose, xylose, mannitol, lactose, sucrose and maltose.
Oxidation Fermentation Test Limitation
 It is usually recommended that the biochemical or the immunological tests including the molecular and the mass spectrometry testing should be performed by using only the pure culture for the complete identification.
 It should also be noted that the organisms that are grown slowly should not be used in the mediums, as it does not produce any results even after several days.
 Whereas some organism does not have the capability to grow in the oxidative fermentative medium, in such case, the other basal medium containing dextrose can be used, so that we can confirm the negative results.
 Use of some mineral oils may be acidic and it leads to erroneous results.
Oxidation Fermentation Test Citations
- Modified oxidation fermentation medium for detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis. J Clin Microbiol. 1983 Jul; 18(1): 56–62.
- Modified Medium for the Oxidation Fermentation Test in the Identification of Marine Bacteria. Appl Environ Microbiol. 1985 Jun; 49(6): 1541–1543.
- One-tube oxidation fermentation methods: limitations posed by atypical fermentative reactions. Appl Environ Microbiol. 1976 May; 31(5): 778–780.
- Modified medium for the oxidation fermentation test in the identification of marine bacteria. Appl Environ Microbiol . 1985 Jun;49(6):1541-3.
- One-tube oxidation fermentation test. Am J Clin Pathol . 1974 Mar;61(3):368-74.
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Starch Hydrolysis Test: Result, Principle, Procedure, and...
Continue ReadingStarch Hydrolysis Test Introduction
Biochemical tests are generally used to determine the ability of the microorganism to be stable in the enzymatic field.
Starch hydrolysis test is one such test that helps us to identify the species of bacteria that has ability to hydrolyze starch which is present in the form of amylose and amylopectin, with the help of the enzyme a-amylase and the oligo-1,6-glucosidase.
Starch Hydrolysis Test is often performed to differentiate the species from the specific genera’s like Clostridium and the Bacillus. As the amylose and the amylopectin molecules are large, they cannot be passed through the cell wall of the bacterium easily.
As the bacteria use these enzymes as a source of carbon, which is very important for them, so these bacteria produce secretes a a-amylase and the oligo – 1, 6 glucosidases in the extracellular spaces, which helps to break the starch molecules into the smaller subunits of glucose which can enter directly into the glycolytic pathway.
Thus, during hydrolysis of the starch test, iodine reacts with the starch and results in the formation the brown color. And it also results in the clear zone around the growth of bacteria. Bacillus subtills is positive starch hydrolysis.
Starch Hydrolysis Test
Starch is one of the complex polysaccharides which can be seen, found abundantly in plants and it is commonly deposited in the form of large granules in the cytoplasm of the cell.
Starch consists of two major components namely amylose and amylopectin which are discussed above. In that amylose contain the units of D-glucose which is linked in a linear manner by the alpha – 1, 4 linkages, having two non-reducing ends and rhe reducing ends.
Amylopectin is one of the branched polysaccharides. These type of molecules contains a shorter glucose chain unit that are linked by an alpha-1, 4 and are joined together each other by an alpha-1, 6 linkages.
Here, the major component of the starch is hydrolyzed by an a- amylase, that is present in some kinds of bacteria as in fungi. This contains an ability to degrade the starch and it is used as a criterion for determining the amylase for producing a microbe.
Starch Hydrolysis Test Objective
 The main aim of the test is to determine the ability of an organism to hydrolyze the starch.
 This test also helps to differentiate the organisms based on their alpha-amylase enzyme activity.
Starch Hydrolysis Test Principle
Many kinds of bacteria produce an extracellular enzyme which are used to catalyze the chemical reactions that takes place outside the cell. At this time, nutrient sources like starch which are too large for the cell membrane to absorb, are breakdown into smaller molecules and they are transported into the cell through the process of diffusion.
However, in starch hydrolysis test, the test bacteria are grown on the agar plates which contains starch. Here, if the bacteria have its own ability to hydrolyze starch, in such way the rest of the medium that does not contains starch are kept non-hydrolyzed.
If there is no color change in the medium, when the hydrolysis process takes place iodine solution is being added here as an indicator to the agar plate after incubation. Whereas, on the other hand the non-hydrolyzed starch forms a dark blue color on adding iodine.
At the same time, a transparent clear zone can be seen, that is formed around the colonies which hydrolyses the starch, where the rest of the plate remains dark blue in color due to the reaction of iodine with starch.
Starch Hydrolysis Test Reagents
Media: The significant media used here for starch hydrolysis test is starch agar, which is one of the simple nutritive medium. Whereas the beef extract and the pancreatic digest of the gelatin provides the medium a rich source of nitrogen, vitamins, carbon, and amino acids. Agar acts as a solidifying agent, where as starch acts as the carbohydrate.
Composition of Media: Here about 5 grams of the peptic digest of the animal tissue is added per liter, sodium chloride of about 5 grams and yeast extract of 1.5-gram, Beef extract of about 1.5 gram, all each per liter are added along with the soluble 2 gram of agar in a pH of about 7.4.
Starch Hydrolysis Test Procedure
Initially, sterile technique is used to make a single line of streak in the inoculated organism that is present in the center of the labelled plate.
Then the inoculated plate is incubated at a temperature of about 37ºC for about 48 hours.
After incubation, the surface of the plates is flooded with the iodine solution using a dropper for about 30 seconds.
Then the excess iodine is poured off, then the clear zone is seen around the line of the bacterial growth.
Starch Hydrolysis Test Results
Positive Starch Hydrolysis Test: In positive results, a clear zone is formed around the area of the growth of the inoculated organisms after addition of iodine solution.
Negative Starch Hydrolysis Test: In case of negative result, a blue black or purple color of the medium is observed in the medium.
Starch Hydrolysis Test Citations
- Rate of starch hydrolysis in vitro as a predictor of metabolic responses to complex carbohydrate in vivo. Am J Clin Nutr . 1981 Oct;34(10):1991-3.Â
- STARCH HYDROLYSIS BY STREPTOCOCCUS EQUINUS. J Bacteriol. 1962 Feb; 83(2): 264–269.
- Limiting factors of starch hydrolysis. Eur J Clin Nutr . 1992 Oct;46 Suppl 2:S17-32.
- Biochemical characters and antibiotic susceptibility of Staphylococcus aureus isolates. Asian Pac J Trop Biomed. 2011 Jun; 1(3): 212–216.
- Improved Method for Detection of Starch Hydrolysis. Appl Environ Microbiol. 1982 Sep; 44(3): 747–750.
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Spot Indole Test: Result, Principle, Procedure, and...
Continue ReadingSpot Indole Test Introduction
Many of the biochemical tests are used to perform the characteristics of an organism and its reaction towards enzyme in a medium.
Spot indole test is used to determine the presence of an enzyme tryptophanase.
Tryptophanase breaks down the tryptophan to release the indole. So, this test is commonly known as indole test.
It is one of the biochemical tests, that is performed on the specific species of bacteria that helps in determining the ability of the organism for the conversion of tryptophan into indole.
This division is generally performed by the chain of the different intracellular enzymes. This system is commonly referred as Tryptophanase.
According to biochemical experiments indole is usually generated by the reductive deamination from the tryptophan through the intermediate compound known as Indole pyruvic acid.
Here, the Tryptophan catalyzes the reaction of deamination, when the amide group of the tryptophan molecule is removed. The final products of this process involve indole, pyruvic acid, energy, and ammonium. Here pyridoxal phosphate is required as a coenzyme.
Spot Indole Test
Spot Indole test is used to determine the presence of an enzyme tryptophanase, which helps in breaking the tryptophan to release indole which on reacting with cinnamaldehyde forms a blue-green compound.
However, the absence of an enzyme results in no change in color, which represents the indole negative.
Indole test is one such biochemical tests, which is basically performed on the species of bacteria to determine the ability of the organism in conversion of the tryptophan into indole.
This division is usually performed by a numerous chain of reaction that contains variety of intracellular enzymes, which are generally referred to as tryptophanase.
Indole is generated here by a reducing deamination from tryptophan through the intermediate molecule known as indole pyruvic acid.
Here Tryptophanase catalyzes the deamination reaction during the process of removing an amine group from the tryptophan. Whereas the final products of the reactions are indole, pyruvic acid, ammonium, energy.
Spot Indole Test Objective
The main aim of the test is to determine the ability of the organism to produce the indole by the action of an enzyme tryptophan.
Spot Indole Test Principle
Principle of the spot indole test helps in mediating the enzyme intracellular enzyme produces the indole by hydrolytic activity against the amino acid Tryptophan.
Bacteria produces the enzyme tryphtophase plays a role in degrading this amino acid tryptophan and converts it into pyruvic acid, along with ammonia and indole.
Indole is usually detected by its ability of combine with some aldehydes and its results information of a colored substance.
Whereas in positive indole test, bacteria forms a blue-green compound which is formed by the reaction of the indole with the cinnamaldehyde which is visualized easily.
This reaction is usually happening during the process of condensation, which is formed by the acid, that helps in splitting of the protein.
Whereas the absence of the enzyme results in no change in color and implies the negative reaction of the indole.
Spot Indole Test Procedure
• Initially a piece of filter paper is saturated using 1% of p-dimethylaminocinnamaldehyde reagent.
• With the help of a wooden stick or using a bacteriologic loop a small portion of the bacterial colony is removed from the small portion of the colony of bacteria present in the agar and the sample is rubbed on the surface of the filter paper.
• Then the change in color in the medium is observed within one to three minutes.
Spot Indole Test Results
Positive Spot Indole Test: If the result is positive it results in development of a blue color within 3 minutes.
Negative Spot Indole Test: If the result is negative, there will be no change in color. In some cases, there will be a development of pale pink color.
Spot Indole Test Limitation
 Colonies are only tested if the cultured media doesn’t contain any glucose as glucose inhibits production of indole.
 It should also be noted that the bacterial inoculum should not be selected from the Macconkey agar and the EMB agar, as the color of the lactose fermenting colonies present in the medium has a maximum of chances to interfere with the test interpretation along with the indicators used in the media, which all results in false positive results.
 On the other hand, Some strains like proteus, vulgaris, Providencia and Aeromonas species gives the false negative reaction during spot indole test.
 As the adjacent colonies are likely to take up the indole in the diffused form, positive tests are only considered as a valid one, if only the pure cultures are cultured.
 While undergoing these tests, Kovacs indole reagent is generally used as a substitute for the spot test reagent, which is usually used as a substitute for the reagent in the spot test. Anyhow this reagent that is being used in the spot test is detected as a less sensitive one for detecting the role of indole in an Indole spot reagent.
Spot Indole Test Citations
- An evaluation of the practicality of the spot-indole test for the identification of Escherichia coli in the clinical microbiology laboratory. Am J Clin Pathol . 1982 Nov;78(5):755-8.
- The spot indole test for identification of swarming Proteus. Am J Clin Pathol . 1985 Jan;83(1):87-90.
- Spot indole test: evaluation of four reagents. J Clin Microbiol . 1982 Apr;15(4):589-92.
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PYR Test: Result, Principle, Procedure, and Reagents
Continue ReadingPYR Test
PYR test is generally used for detecting the activity of pyrrolidonyl aryl amidase in the species of Streptococcus pyogenes which belongs to the group A family of Streptococcus and other species like Enterococcus spp, coagulase negative staphylococci and some species of Enterobacteriaceae.
This is also known as PYR (L-Pyrroliodonyl Beta-naphthylamide) that serves as a substrate foe determining the pyrroliodonyl peptidase.
Facklam, Thacker, Fox and Eriquez is reported that 98 percentage of the group A streptococci and the 96 % of the group D Enterococci hydrolyse PYR. Although the species of Aerococcus are rarely isolated in the clinical laboratory. These organisms are also expected to hydrolyse the PYR.
However, Facklam et.al reported that 98 percent of the group A streptococci and 96 percentage of the Enterococci hydrolyse the PYR. Although Aerococcus species are found very rarely isolated from the clinical laboratory and these organisms are also expected to hydrolyse PYR.
Facklam et. el later reported that there are about 98 percent of the group B Streptococci, 100 percent of the non-group A, B, D Streptococci 100 % of group D non-enterococci and the 83% of the viridians streptococci yield negative PYR test results.
PYR Test Principle
PYR is one of the rapid methods for presumptively identifying the bacteria based on their pyrrolidonyl arylamidase enzyme.
The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl Beta-naphthylamine.
This Beta-naphthylamine is usually detected during the presence of the N, N-methylamino cinnamaldehyde reagent which results in the production of the bright red precipitate.
After the process of hydrolysis of the substrate with the help of peptidase, which results in the formation of a b-naphthylamide and results in the formation of a red colour upon the addition of 0.01% of the cinnamaldehyde reagent.
On rubbing a visible inoculum of the micro-organisms in a small area in the disk, that is impregnated using a substrate by hydrolysing happens within 2 minutes, during when the cinnamaldehyde reagent is added. However, the reaction is usually detected by change in colour.
PYR Test Procedure
Generally, there are about two methods used for performing the PYR test.
1. Broth PYR Test Method: Initially the PYR broth is inoculated for about 18 to 24 hours with a 3 to 5 colonies. Then the inoculated broth is incubated along with the tube aerobically at a temperature of 35 to 37ºC for about four hours, After completing the process of incubation about 2 to 3 drops of PYR reagent is added and the tube is noted for change in colour. Usually for positive test the development of the red colour within 1 to 2 minutes.
2. Disk PYR Test Method (Rapid): Disk method is one of the rapid methods used for performing the PYR test. In the process, the wet PYR test disc on the strip using distilled water for about 10µl. Instead of distilled water, deionized water can also be used. It should be noted that the disk should not be flooded while wetting. After wetting about 5 to 10 colonies of the test strain is taken from an 18 to 24-hour culture and it is placed on the surface of the disc using an inoculating loop and it is smeared lightly on it. Then the disc is incubated for about 1 to 2 minutes in a room temperature. After incubating the disc is taken out and a drop of N, N-dimethylaminocinnamaldehyde is added. After addition formation of red colour is observed within 2 minutes.
PYR Test Results
Positive PYR Test Results: If the result is positive bright pink or cherry red colour is observed within 2 minutes. For positive control, the species belonging to Group A Streptococci, Group D Enterococci, Coagulase negative Staphylococcus species such as hemolyticus, S. lugdunesis, Corynebacterium hemolyticum. Citrobacter, Aerococcus, S. schleiferi.
Negative PYR Test Results: : In case if the test is detected as negative result, then there will be no colour change. Sometimes there will be a blue colour in the medium. If the medium is appeared to be pale pink, then the reaction is considered as negative. Mostly the species belonging to Group B Streptococci, Streptococcus mitis, S. bovis, S. equis, S. milleri are tested as negative.
PYR Test Quality Control
Positive Control: Enterococcus faecalis, Streptococci’s pyogenes
Negative Control: Streptococcus agalactiae.
PYR Test Limitation
PYR test is used as a presumptive separation of the group A Streptococci and the Group D Enterococci from the other species of streptococci species.
Additional testing is done using a pure culture, and it is recommended for complete identification of the characteristics and nature of the species.
If the disk or filter paper is too moist it results in false, negative tests.
False-negative results happen if the selective medium or the biochemical agars are used for providing the inoculum.
Escherichia coli and indole positive proteus obtained from the medulla containing a high tryptophan content yields a blue-green development. This results in negative reactions.
On the other hand, some less commonly encountered species of lactococci and aerococci results in PYRase positive. In case, if the results are read after 20 seconds of performing, then it results in Non-specific colour reactions.
PYR Test Citations
- Presumptive identification of streptococci by pyrrolidonyl-beta-naphthylamide (PYR) test. Zhonghua Yi Xue Za Zhi (Taipei) . 1997 Apr;59(4):259-64.
- Value of the L-pyrrolidonyl-beta-naphthylamide hydrolysis test for identification of select gram-positive cocci. Diagn Microbiol Infect Dis . 1986 Jan;4(1):43-7.
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Beta Lactamase Test: Result, Principle, Procedure, and...
Continue ReadingBeta Lactamase Test Introduction
Biochemical tests are generally used to determine the ability of the microorganism to be stable in the enzymatic field. Beta Lactamase Test also helps us to determine the characteristics of the microbe in the medium.
However, these tests are usually preferred for identifying the different species of bacteria based on their ability to differentiate the biochemical and enzymatic activities of the chosen species in the culture. Basically, Bacterial physiology varies from one type of organism to the other.
This ability of the bacteria to form an organic compound by metabolizing the certain forms of carbohydrates and compounds related to it are performed by biochemical tests.
Beta lactamase test is one such biochemical test which is used for differentiating the bacteria based on the characteristic, that can produce an enzyme known as beta-lactamases which are mediated by the genes that are being present on plasmids and chromosomes.
Beta Lactamase Test
As mentions above, different bacteria produce different and an important class of enzymes known as beta-lactamases, that are being mediated by the genes that are present of the plasmids or in the chromosomes.
Generally, these enzymes confer a resistance against several penicillin antibiotics by cleaving action of the beta-lactam ring of the penicillin and cephalosporin antibiotics, which results in inactivating the drugs.
These are then capable of inactivating the penicillinase-labile penicillin. Such as amoxicillin, ampicillin, Penicillin, Mezlocillin and carbenicillin.
Beta-lactamases thus plays an important role in resisting the bacteria against the agents of Beta-lactam and ring of the penicillin’s and the cephalosporin antibiotics, which results in inactivating the drugs, now a day’s various assays are available to detect the presence of Beta-lactamases such as iodometric method, acidimetric method by using the chromogenic substrates.
Beta Lactamase Test Objective
The main aim of the test is to detect the ability of the enzyme beta-lactamase, that confers the penicillin resistance to the various bacterial organisms.
Beta Lactamase Test Principle
The most useful tests in the clinical laboratories for the detecting the beta-lactamase is chromogenic cephalosporinase test.
This test disk is usually consisting of a chromogenic cephalosporin that is being used as a substrate.
Organism that contains Beta-lactamases when applied to the disk for exerting the effect by opening their Beta-lactam ring present in the substrate.
This process thus results in formation of a coloured product that is conspicuous, and it also allowed the detection.
On hydrolysing the bacterial inoculum, it produces a deep pink colour. If there is no formation of any of the colours, then it indicates the absence of a Beta-lactamase.
Beta Lactamase Test Disk
Cefinase disk is considered as one of the best examples for the commercially available chromogenic tests. This disk helps in incorporating the nitrocefin as the basic substrate.
It also exhibits a rapid colour change from yellow to red due to the presence of amide bond in the beta lactam ring which hydrolyses the ring with the helps of the enzyme, Beta-lactamase.
When the bacterium produces this enzymes, Beta-lactamase in the significant quantity the coloured disc turns red in the specific areas where the isolated area is being smeared.
Beta Lactamase Test Procedure
Initially the disk is dispensed from the cartridge into an empty petri dish using a single disk dispenser. In some cases, instead of petri dish, microscopic slides can also be used.
Then the disc is moistened with one drop of the sterile distilled water.
Then with the help of a sterile loop or an applicator stick several well-isolated colonies are removed and the surface of the disks are smeared. Then the change in colour of the disk can be observed.
Beta Lactamase Test Results
Positive Results: If the result is positive then there will be a change in colour from yellow to pink-red colour on the area particularly where culture is spread.
Negative Results: If the results are negative there will be no colour change in the medium.
Beta Lactamase Test Uses
 Beta Lactamase Test helps us to detect the whether the certain species are resistance to drugs such as penicillin or ampicillin. Few examples are that
 N. gonorrhoea is resistance towards penicillin.
 H. influenzae is resistance to ampicillin.
 Staphylococcal is resistance to penicillin.
Beta Lactamase Test Limitation
 Beta Lactamase Test is limited for using it directly for detecting the Beta-lactamase production in the organisms that produces the enzyme those activities are known already. This also includes the Beta-lactams that are more commonly used for the therapeutic eradication of the organisms.
 Beta Lactamase Test is not supposed to replaced entirely by the convectional susceptibility because the other factors cannot be participated here for influencing the other results of the tests.
 Whereas the detection of the activity of the Beta-lactamase in the species of Staphylococci takes a longer period than others, up to one hour.
 Only one disk can be used at a time to detect the bacterial strain for determining the presence or absence of a Beta-lactamase.
Beta Lactamase Test Citations
- An update on β-lactamase inhibitor discovery and development. Drug Resist Updat . 2018 Jan;36:13-29.
- An Improved Extended-Spectrum-β-Lactamase Detection Test Utilizing Aztreonam plus Clavulanate. J Clin Microbiol . 2017 Dec 26;56(1):e01309-17.
- β-Lactamase inhibitors: an update. Mini Rev Med Chem . 2013 Nov;13(13):1846-61.
- Three decades of beta-lactamase inhibitors. Clin Microbiol Rev . 2010 Jan;23(1):160-201.
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Casein Hydrolysis Test: Result, Principle, Procedure, and...
Continue ReadingCasein Hydrolysis Test Introduction
Biochemical tests are generally used to determine the ability of the microorganism to be stable in the enzymatic field. This test also helps us to determine the characteristics of the microbe in the medium.
However, these tests are usually preferred for identifying the different species of bacteria based on their ability to differentiate the biochemical and enzymatic activities of the chosen species in the culture.
Basically, Bacterial physiology varies from one type of organism to the other This ability of the bacteria to form an organic compound by metabolizing the certain forms of carbohydrates and compounds related to it are performed by biochemical tests.
Casein hydrolysis test also helps us to determine the ability of the micro-organism to degrade the protein casein.
Casein is one of the major milk proteins and it is one of the macro molecules which is one of the subunits linked together by the peptide bonds.
The basic principle used in this test is that medium will be opaque due to the colloidal suspension of casein.
Hydrolysis of this reaction causes the milk agar to clear the area that is present around the growth area, as the casein protein is converted into its soluble and a transparent form.
Casein Hydrolysis Test
Casein, the major milk protein, one of the macro molecules comprising of amino acid, which makes a major source of about 85% of protein that is being an important part in forming the white color of the milk.
Casein is generally considered as too large which cannot enter the cell membrane.
To assimilate with the cell membrane, protein should undergo a step-by-step process of degrading them into peptones, and other smaller compounds such as polypeptides, dipeptides and into to other components such amino acids, which make up the building block.
These proteases play an important role in cleaving the peptide bond by introducing a water molecule into it.
This reaction further liberates the amino acids that are low in their molecular weight and are transported via the cell membrane which helps them in synthesizing the structural and functional cellular proteins.
Casein Hydrolysis Test Objective
The main aim of the Casein Hydrolysis Test is to determine the ability of the specific organism to degrade the casein proteins. To differentiate the organism based on their ability to produce an exo enzyme namely proteinase.
Casein Hydrolysis Test Principle
Some micro-organisms have its ability to degrade the protein, casein by producing the proteolytic enzymes namely known as proteinase. This is also known as caseinase. Usually for demonstrating an activity a lab milk aga is being used for the experiments.
Here the medium is generally composed of the nutrient agar being supplemented with the milk containing the protein substrate known as casein.
Like the other substances as protein, mil protein, casein is also a colloidal suspension which provides the medium its color and opacity as it reflects light rays rather than the transmitting them.
Following the inoculation and incubation of agar in the plate culture the organism will exhibit a zone of proteolysis, that is usually demonstrated by the clear area that is being surrounded by a growth of bacteria.
This loss of opacity is due to the result of hydrolytic reaction yielding soluble, non-colloidal amino acids which represents the positive reaction.
Absence of a protease activity in the medium that is surrounding the growth of organisms and remains opaque, in case of negative reaction.
Casein Hydrolysis Test Reagents
Media:
Ingredients Gram per Liter SM powder 28.0 Tryptone 5.0 Yeast extract 2.50 Dextrose 1.0 Agar 15.0 Casein Hydrolysis Test Procedure
Initially the organisms are inoculated on the plate either in a form of straight line or in a zig zag manner.
The plate is incubated at a temperature of about 25 to 37ºC.
Then the milk agar plate cultures for examined for the presence or absence of a clear area or for the formation of zone of proteolysis, which surrounds the growth of the bacteria, which is used as the test organisms.
Casein Hydrolysis Test Results
Positive Casein Hydrolysis Test: In case of positive test, a clear zone is observed around the growth of the colonies. In some cases, it may also be present beneath the growth of the colonies.
Negative Casein Hydrolysis Test: Whereas, in case of the negative test, no clearing is observed neither around nor beneath the inoculum.
Uses of Casein Hydrolysis Test
Casein Hydrolysis Test helps us to identify the bacteria that is grown in the milk.
It also helps us in differentiating the Enterobacteriaceae, Bacillaceae and the other families.
Casein Hydrolysis Test can also be used for differentiating the aerobic actinomycetes based on the proteolysis of casein.
Casein hydrolysis test is used for identifying the organism which helps in hydrolyzing the casein. Some such organisms include Streptomyces, Pseudomonas and Actinomadura.
Casein Hydrolysis Test Limitation
Generally, it is suggested that the immunological, biochemical, molecular or the mass spectrometry tests are performed on the colonies from the pure culture for complete identification of the appropriate species.
Casein Hydrolysis Test Citations
- Hydrolysis of casein: a differential aid for the indentification of Serratia marcescens. J Clin Pathol. 1972 Dec; 25(12): 1083–1085.
- Use of casein, tyrosine, and hypoxanthine in the identification of nonfermentative gram-negative bacilli. Med Microbiol Immunol . 1979 May 15;167(2):71-5.
- Characterization of casein hydrolysates derived from enzymatic hydrolysis. Chem Cent J. 2013; 7: 62.
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