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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.
- 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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Triple Sugar Iron Agar Test: Principle, Objective,...
Continue ReadingTriple Sugar Iron Agar Test Introduction
Most of the biochemical tests are performed to determine the characteristics of the microorganisms and their reactions in certain mediums. Thus, it helps us to determine the nature of the organism inside the host body in different pathological conditions.
Triple sugar iron agar test is one such test, which is used the differentiate the different group of genera of the Enterobacteriaceae family, those of all gram-negative bacilli which can ferment the glucose thereby producing an acid. It helps us to distinguish them from the other species of gram-negative intestinal bacilli.
This test is generally performed in the biochemical laboratories, methods and procedures are discussed below.
Triple Sugar Iron Agar Test
This test is usually performed in a differential medium containing lactose, sucrose, and a small amount of glucose, along with the ferrous sulphate. This medium is used to differentiate the enteric based on the ability to reduce the sulfur and in fermenting carbohydrates.
Triple Sugar Iron Agar Test Objective
The main aim of the test is to whether the gram-negative bacilli have the capability to ferment the monosaccharides like glucose, lactose or sucrose which results in forming a hydrogen sulfide. To differentiate the members of the Enterobacteriaceae family from the other gram-negative rods.
Triple Sugar Iron Agar Test Principle
Triple sugar iron agar test is designed in such a way, to differentiate the different groups of the Enterobacteriaceae family, that are all gram-negative and are capable of fermenting glucose which initiates the production of acid and to differentiate them from the other species of gram-negative bacilli present in the intestine.
This differentiation is purely based on the fermentation of the monosaccharides like glucose, fructose and lactose which results in production of one of the acids, hydrogen sulfide.
This Triple Sugar Iron medium usually consists of about 10 parts of lactose, 10 parts of sucrose and 1 part of glucose and peptone. Here, phenol red and ferrous sulphate acts as an indicator which detects the presence of carbohydrates and it expresses it by the action of color change.
Usually here in the presence of carbohydrate in the medium, the colors change is from organ red to yellow, at the time of presence of acids. In case of oxidative decarboxylation of the peptone, the alkaline products in the medium built and results in increase in the ph. This change is indicated by change in color of the medium to orange red to deep red.
Whereas the sodium thiosulphate and the ferrous sulphate that are being present in the medium, detects the production of hydrogen sulphide which is indicted by formation of black color butt in the tube.
Initially glucose is utilized by a fermentative organism present in the medium, thus changes the entire medium into yellow when it becomes acidic within 8 to 12 hours. But this remains acidic even after 18 to 24 hours due to the presence of the organic acids and results in fermentation of glucose under the anaerobic conditions in the butt of the tube.
However, the slant reverts to the alkaline by change in color to red as of oxidation of the fermentation products under aerobic conditions on the slant. This change results in the formation of the carbon dioxide and water and oxidation of peptones in the medium to the alkaline amines. On addition to monosaccharides such as glucose, lactose and sucrose gets fermented.
Due to this fermentation products large amount of fermentation products are formed along the slant, thus neutralizing the alkaline amines and renders the slant acid, which is yellow in color because of 18-to-24-hour reaction. In case, if the slant and butt convert in alkaline form which shows that glucose is not yet fermented.
The organisms showing this reaction are usually called as non-fermenters and they derive their nutrition from the peptones that are present in the medium. And the presence or synthesis of carbon dioxide and hydrogen gas in the reaction is indicated by formation of bubbles or by observing the crack in the medium of the agar or in some cases it may also lead to separation of agar from the sides or from the bottom of the tube.
Whereas the production of hydrogen gas in the medium requires an acidic environment which leads to the blackening of the agar butt on reaction with ferric ammonium citrate in the tube.
Triple Sugar Iron Agar Test Reagents
Preparation of Triple Sugar Iron Agar:
Pancreatic Digest of Casein 15.0 grams Lactose 10.0 grams Sucrose 10.0 grams Sodium Chloride 5.0 grams Yeast Extract 3.0 grams Peptic Digest of Animal tissue 5.0 grams Beef extract 3.0 grams Dextrose 1.0 grams Ferric Ammonium Citrate 0.5 grams Sodium thiosulphate 0.3 grams Agar 12.0 grams Phenol red 0.024 grams Triple Sugar Iron Agar Test Procedure
Initially, a small amount of colony is collected by touching a well-isolated colony using a straight inoculating needle.
Triple Sugar Iron Agar is first inoculated by stabbing the center of the medium to the end of the tube. Further the surface of the agar slant is streaked.
The tube of the cap is closed loosely, and it is inoculated in an ambient air chamber for about 18 to 24 hours at a temperature of 35 to 37ºC.
After incubation the tube is observed and opted if there is any color change in the butt and slant or any cracks or blackening of the medium.
Triple Sugar Iron Agar Test Results
Result (Slant/butt) Interpretation Red /Yellow color Fermentation of glucose alone and catabolisation of peptone. Yellow/Yellow Fermentation of monosaccharides such as Glucose, sucrose, lactose. Red/Red No fermentation, Peptone is catabolized under aerobic and anaerobic conditions. Yellow/ Yellow bubbles Glucose and lactose or sucrose fermentation, gas produced Red/Yellow with bubbles Fermentation of glucose alone, Gas produced. Red/Yellow with bubbles and formation of black precipitate. Fermentation of glucose alone, Hydrogen sulphide gas is produced. Yellow/Yellow with bubbles and black precipitate Glucose, lactose, sucrose fermentation, Production of hydrogen sulphide gas Red/Yellow with black precipitate Glucose fermentation alone, production of hydrogen sulphide gas Yellow/Yellow with black precipitate Fermentation of glucose, lactose or sucrose, production of hydrogen sulphide gas. Triple Sugar Iron Agar Test Citations
- Effects of storage temperature, light and time on stability of triple sugar iron agar and its productivity for Escherichia coli and Salmonella typhimurium. Folia Microbiol (Praha) . 1983;28(6):446-51.
- Multidrug-resistant Escherichia coli and Salmonella spp. isolated from pigeons. Vet World . 2020 Oct;13(10):2156-2165.
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Drawbacks of Rutherford Model: Experiments and Conclusion
Continue ReadingWhat is Rutherford Model?
Greek philosopher Democritus was concerned to know that how a piece of matter can be cut into so many smaller pieces. He also believed that it could reach a point where it wouldn’t be able to be cut further into smaller pieces. These smallest pieces of matter are named as Atomos. It was an incident in about 450 B.C. However, this idea was not further discussed for more than 2000 years.
Then, British Chemist named John Dalton restored the idea of atom in 1800 and provided suggestion for atoms. After the discovery of atom, many atomic models were projected by scientists to define the atomic structure and discovery of subatomic particles such as electron, proton, and neutron.
Then, around 1911, a great physicist, Rutherford discovered the nucleus and proposed Rutherford’s Atomic Model. Rutherford upturned Thomson’s model in 1911 with his the most famous experiment which came to known as gold foil experiment in which he discussed that the atom has a tiny and heavy nucleus which is present in the centre of an atom.
Rutherford proposed an experiment to use the alpha particles which are emitted by a radioactive element. He projected that if Thomson model of atom was correct, then the beam of alpha particles would go straight through the gold foil. It was then noted that most of the beams went straight through the foil, but a few of them were deflected. Rutherford thus presented his physical model for subatomic structure of particles, as a description for the amazing experimental results.
He stated that the atom comprises of a central charge, this is at present termed as atomic nucleus, though Rutherford himself did not use the term nucleus in his research paper and this nucleus is surrounded by a cloud of circling electrons.
Features of Rutherford Model
The main points of Atomic Nuclear Model proposed by Rutherford is given below;
• Most of the atom has empty spaces within it as most of the alpha particles go straight through gold foil.
• Most of the mass that was present inside the atom was focused in a small area in the Centre, which is named as the nucleus.
• The electrons rotate around the nucleus in their fixed paths termed as orbits.
• The size of the nucleus is very small as compared to the size of an atom. According to calculation of Rutherford. He stated that the size of the nucleus is 105 times smaller than an atom.
• There is also a very strong electrostatic force of attraction present between the positive and negative charges of an atom which makes the atom stable.
Drawbacks of Rutherford Model
The model of the atom projected by Rutherford is still known as the classical model and was very much acknowledged at that time, though, later, it was proved that there were certain facets that this model was not able to explain.
Therefore, the certain drawbacks of Rutherford’s model of an atom is given below, and it was felt that some findings still need to be done in the field of atomic structure. Drawbacks of Rutherford model of atom:
1. According to Maxwell, electrons should release electromagnetic radiations because an accelerated charged particle has the tendency to release electromagnetic radiation. Rutherford also stated that the electrons revolve around the nucleus in their fixed paths named as orbits. But due to the presence kinetic motion of the electrons, the radiations further cause the shrinking of the electrons. Hence, electron should fall in nucleus of an atom in less than 10 seconds because electrons will keep on losing energy. Consequently, Rutherford’s model was unable to explain Maxwell’s theory.
2. If electrons would fall in nucleus this should make the atom very unstable, but it is shown that atoms are very stable particles.
3. Rutherford further explained the revolving of electrons around the nucleus of atoms in fixed paths or orbits, though, he was unable to explain how the electrons were placed inside the atom, thus results in making the model an incomplete model of the atom.
4. The Rutherford model of atom also helped to concentrate a great deal of charge on atom and mass of atom to a very small space, but was unable to feature any structure to the remaining electrons and atomic mass of atom. It did give reference to the atomic model of Hantaro Nagaoka, in which the electrons are commonly arranged in more than one rings, with the definite symbolic structure as of the stable rings of Saturn.
Rutherford Model Citations
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Molality: Definition, Calculation, Equation, and Formula
Continue ReadingWhat is Molality?
A final way to express the concentration of a given solution is by calculating its molality. The molality (m) of a solution is defined as the moles of solute divided by the kilograms of solvent in which solute is dissolved. For instance; A solution that comprises of 1.0 mol of NaCl dissolved into 1.0 kg of water is referred to as a “one-molal” solution of sodium chloride. The symbol for molality is represented in a lower-case m.
Molality Formula
Molality(m)=Number of moles of solute divided by Mass of solvent in kgs.
SI unit of molality = mol kg-1.
Molality (m) = moles of solute/kilograms of solvent = mol/kg
Molality Characteristic
1. Molality is denoted as a property of a solution.
2. It is an intensive property thus it will not differ from sample to sample for a given solution.
3. The number of moles of solute and the mass of solvent are generally not affected by pressure and temperature of system. Henceforth, molality unlike molarity, is not dependent on temperature and pressure.
4. Molality is referred to a non-volumetric concentration such as mole fraction and mass fraction.
Molality vs Molarity
Molarity (M) is commonly defined as the number of moles of solute per liter of solution.
Molarity = moles of solute/liters of solution
Molality (m) is commonly defined as the number of moles of solute per kilogram of solvent.
Molality = moles of solute/kilograms of solvent
Molarity is referred to measurement of the moles in the total volume of the solution, whereas molality is a measurement of the moles in connection to the mass of the solvent.
Molality varies from molarity only in its the denominator. While molarity is expressed in the liters of solution, molality is expressed in the kilograms of solvent.
Concentrations expressed in molality are commonly used to study properties of solutions which are related to vapor pressure and temperature changes. Molality is often used because its value generally does not change when temperature of system is change thus it is an independent quantity. On the other hand, the volume of a solution, is somewhat dependent upon temperature.
Molality and molarity are related in value for dilute aqueous solutions because of the fact that the density of those solutions is fairly close to 1.0 g/mL. This further means that 1.0 L of solution has approximately a mass of 1.0 kg. As the solution becomes more and more concentrated, its density will not even be close to 1.0 g/ml and the molality value will differ from molarity.
How to Calculate Molality? Example 1
What is the molality when 0.830 mol is dissolved in 1.60 L of solvent?
Molality = 0.830 mol / 1.60 kg
The answer is 0.518 m.
How to Calculate Molality? Example 2
Calculate the molality of a solution prepared from 30.22 grams of NaCl in 5.00 kg of water.
Given;
Solute = 30.22 gm of NaCl
Solvent = 5.00 Kg of water
Molar mass of solute that is NaCl = 58.44 gm/mol
number of moles of solute = 30.22 / 58.44
= 0.51 mol
Molality or m = number of moles of solute (n)/ weight of solvent in kg
Thus,
m = 0.51 / 5.00 = 0.102 moles/kg
Molality Citations
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