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Potassium Hydroxide Test: Result, Principle, Procedure, and...
Continue ReadingPotassium Hydroxide Test Introduction
Many of the biochemical tests are used to detect the ability of the micro-organisms to act against certain chemicals and enzymes.
Potassium hydroxide test is used to identify the gram-negative bacteria.
Potassium hydroxide is one of the inorganic compounds which has its chemical formula KOH, and it is most commonly referred to as caustic potash.
It can be used in many chemical and biochemical tests.
Potassium hydroxide is also known as Potassium hydroxide dissolves with the peptidoglycan which is present as a thin membrane in the cell walls of the gram-negative bacteria.
But this does not affect the gram-positive cell walls.
Disintegration of these gram-negative cell walls lyses the cell thus releasing the contents from the cell including those of DNA.
The DNA makes the solution viscous and makes the solution to stick to the plastics loop when touched.
However, Gram positive bacteria does not affect the Potassium hydroxide, as it has thicker peptidoglycan layer present in the cell wall.
Hence the cells will not be lysed, so that there will no release of DNA and there is no viscosity observed.
What is Potassium Hydroxide Test?
Potassium hydroxide test is also known as KOH string test.
Potassium hydroxide test mostly relies on the differential resistance of about 3% in the potassium hydroxide, between the gram positive and the gram-negative cells.
Here a small portion of a colony is mixed up with a small volume of 3% KOH.
If the cell lyses, the cellular DNA will be liberated making the mixture viscous or stringy.
The positive string test indicates a gram-negative organism.
Hence the potassium hydroxide can also be known as String test.
Potassium hydroxide test also helps in differentiating the Gram positive and the Gram-negative organisms and this test is also helpful in complementing the gram stain and the antibiotic disc test.
Potassium Hydroxide Test Objective
The main aim of the test is to differentiate between gram negative and the gram-positive organisms.
Potassium Hydroxide Test Principle
Similar to the Gram stain reaction, the potassium hydroxide test is basically used to identify the differences in the chemical composition of the bacterial cell wall.
During the presence of this Potassium hydroxide, Gram negative cells will be lysed, this makes the Potassium hydroxide solution to dissolve easily with the thin layers of the cells, known as peptidoglycan.
However, Disintegration of Gram-negative cell wall will lyse the cells making it to release all of it contents, including DNA.
As a result, a viscid chromosomal material is released along with the contents and it is caused by the suspension of the bacteria to become thick and stringy.
The viscous and the solution sticks to the loops.
On the other hand, Gram positive bacteria are not affected by the Potassium hydroxide as it has thicker peptidoglycan layer along the cell so there are no chances of cell lyses and no contents will be spilled out of the cell so no viscosity can be detected.
Potassium Hydroxide Test Procedure
About one drop of 3% of potassium hydroxide solution is placed in a clean microscopic slide.
Few colonies of a suspect organism are emulsifying along with the drop of potassium hydroxide placed in the slide and makes it as a dense suspension.
Then the mixture is continuously mixed for about 60 seconds and they are gently pulled using a loop such that to pull it against the suspension.
Then the medium is detected for any changes.
Potassium Hydroxide Test Result
For positive results, the organisms become thick, stringy and it forms a long strand within few seconds. This is seen only in the species of the Gram-negative bacteria.
In case of negative results, the organisms leave the suspension without any changes in the absence of the stringing. These results are mostly seen in Gram positive bacteria.
Potassium Hydroxide Test Uses
Potassium hydroxide tests are generally used in laboratories where the cultures are processed in large numbers, the above test is used is also used along with the gram stain in preliminary differentiation.
This test is also useful in reacting as a complement in the Gram stain and in antibiotic tests.
Potassium Hydroxide Test Limitation
Though, this test is useful, the test cannot be used to determine the results, as the negative tests does not prove any conclusive if an organism is Gram-positive.
In old cultures which are being cultures before 48 hours, has the chances of turning the negative results into positive after 30 seconds of mixing the bacteria along with the potassium hydroxide solution, thus gives unreliable results. This type of results are most common in species like Achromonacter genera such as Brucells melitenis, Pseudomonas paucimobilis, Moraxella species, etc.
False results mostly occur due to a heavy inoculum, where the solution appears in the form of gel, without a string form. In some cases, there is also chances of forming the inoculation with the mucoid colonies. On the other hand, False results occur when there is a very little or light inoculum or if there is too much of potassium hydroxide than normal.
Potassium Hydroxide Test Citations
- Evaluation of the KOH test and the antibiotic disk test in routine clinical anaerobic bacteriology. J Clin Microbiol . 1988 Oct;26(10):2144-6.
- The Sensitivity and Specificity of Potassium Hydroxide Smear and Fungal Culture Relative to Clinical Assessment in the Evaluation of Tinea Pedis: A Pooled Analysis. Dermatol Res Pract. 2010; 2010: 764843.
- The sensitivity and specificity of potassium hydroxide smear and fungal culture relative to clinical assessment in the evaluation of tinea pedis: a pooled analysis. Dermatol Res Pract . 2010;2010:764843.
- Successive potassium hydroxide testing for improved diagnosis of tinea pedis. Cutis . 2017 Aug;100(2):110-114.
- Use of Potassium Hydroxide (KOH) Test Reduces Antifungal Medication Prescription for Suspected Monilial Diaper Dermatitis in the Neonatal Intensive Care Unit: A Quality Improvement Project. Adv Neonatal Care . 2019 Dec;19(6):E3-E10.
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Nitrate Reduction Test: Result, Principle, Procedure, and...
Continue ReadingNitrate Reduction Test Introduction
Many of the biochemical test is usually used to detect the ability and to identify the species of bacteria b differentiating them based on the characteristics and their biochemical activities.
There are many factors such as protein, carbohydrate metabolism and the enzyme production.
These biochemical tests used in many of the clinical test to detect the pathogen and its nature of the disease-causing techniques.
Nitrite is one of the ions consisting molecule that is made up of nitrogen atom which is formed along with the oxygen atoms with the help of nitrogen bonds.
Nitrite is usually known as anion. Nitrite has the capability to inhibit the growth of bacteria hence it is used n many of the biochemical tests and also in clinical test and in food processing units.
In nitrate reduction test is based on the detection of the nitrite that is present in the medium after incubating it along with the organism.
Presence of nitrite in the medium reacts with the sultanic acid and results in the formation of the colourless complex.
This complex helps us to yield a red precipitate when the nitrate reagent is added to the medium.
What is Nitrate Reduction Test?
Anaerobic metabolism usually requires an electron acceptor other than the atmospheric oxygen.
Many of the species of the Gram-negative bacteria uses the nitrate as the final acceptor of electron.
Nitrate reduction test is a test which determines the production of the enzyme known as nitrate reductase, that results in the reduction of nitrate.
Bacterial species are differentiated according to their ability to reduce nitrite or nitrogenous gases.
Nitrate Reduction Test Objective
The main aim of the test is to determine the ability of an organism to reduce the nitrate or nitrite.
To identify the different ways of reduction of bacteria by the nitrate.
Nitrate Reduction Test Principle
To perform this test, a heavy inoculum of the test organism is collected and it is incubated in the broth containing nitrate.
The organisms which have the capability to produces the enzyme, nitrate reductase reduces the presence of nitrate in the broth, thus it forms its reduced form known as nitrite.
Nitrite further reduces to nitric oxide, nitrous oxide or nitrogen.
This test is purely based on the detection of nitrite and its ability to form a red colored compound on reacting with sulfamic acid and forms a complex known as nitrite sulfamic acid.
Sulfanilic acid further reacts with the alpha-naphthylamine which results in the formation of the red precipitate known as Prontosil, this prontosil is completely soluble in water and it is called as azo dye.
It is also noted that only when nitrate is present in the medium, formation of red color can be observed.
If there is no formation of red color then it can be proceeded by adding sulfanilic acid and Alpha-naphthylamine , when nitrite is absent in the medium.
Usually, this observation is explained in two ways, Nitrate does not have reduces, if the strain is noted as nitrate-negative.
The nitrate reduces into nitrite, which is further reduced into nitrous oxide or nitrogen, which on this reduced form does not reacts with nitrite, then the strain is said to be nitrate-positive.
However, if nitrate is not detected, it is important to test if the organism has the capability to reduce nitrate beyond nitrite.
This is usually done by adding a small amount of zinc powder catalyzes in order to reduce the nitrate into nitrite.
On adding zinc, the formation of red color can be observed, which indicates that nitrate was not reduced to nitrite.
In other case, where is no change in color even addition of zinc powder, then it indicates that the organism is reduced into any of the nitrogen compounds.
Nitrate Reduction Test Reagents
Nitrate Broth:
Peptone – 5 grams per litre
Meat extract – 3 grams per litre
Potassium nitrate – 1 gram per litre
Nitrate Reduction Test Procedure
Nitrate reduction is usually determined in two step process. Initially the reduction of nitrate to nitrite is determined by adding the nitrogen reagents into the medium.
If necessary, the reduction of nitrate beyond nitrite beyond is determined by adding nitrate reagent C, which is in the form the zinc dust.
Initially the nitrate broths are inoculated into the nitrate broth into the bacterial suspension.
Incubate the tubes at the optimum temperature of about 30 to 37ºC for 24 hours.
After incubating the formation of nitrogen gas is noted before adding the reagents.
To this medium, about 6 to 8 drops of the nitrate reagent A and the same amount the nitrate reagent B are added, once it is added, color changes can be observed in a minute or some times even less than that.
On the other hand, if there is no color change, then zinc powder is added and formation of any changes can be observed within 3 minutes of adding.
Nitrate Reduction Test Result
Positive Test:
In positive tests, the development of red color can be detected after addition of the reagents A and B.
And in Zinc powder addition, the medium should not develop the red color and it results in positive results.
Negative Test:
Development of a red color when zinc powder is added.
Nitrate Reduction Test Citations
- Simple disk technique for detection of nitrate reduction by anaerobic bacteria. J Clin Microbiol . 1977 Mar;5(3):315-9.
- 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.
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Kligler Iron Agar Test: Principle, Procedure, and...
Continue ReadingKligler Iron Agar Test
Many of the biochemical tests are performed to detect the ability of the micro-organism to utilize the enzyme in the culture medium. Kligler iron test is also one of the biochemical tests, which employs a medium for the identification of the species of Enterobacteriaceae, based on the fermentation of the double sugar and the production of hydrogen sulphide.
Kligler Iron Agar Test was first determined by Kligler in the year 1918 to describe the medium for detecting the production of hydrogen sulphide and it also helps us to differentiate the species of Salmonella.
This was first modified by Bailey and Lacey by substituting the phenol red indicator instead of Andrade indicator in the medium.
The medium used in the test is commonly known as KIA. It is also recommended that determination of the Hydrogen sulphide depends on the production of the Enteric gram-negative bacilli and also in detecting the production of H2S by using some strains of Pseudomonas.
What is Kligler Iron Agar Test?
Kligler iron agar test is used for detecting the fermentation of carbohydrate in the medium.
Here the reaction of the KIA helps us to include or exclude a particular bacterial species to isolate it from the family of Enterobacteriaceae.
If the organisms cannot ferment the carbohydrate or glucose present in the medium, then alkaline Slant in alkaline butt reaction can be observed in the Klinger Iron Agar.
This reaction is sufficient for excluding and isolating the species belonging to the Enterobacteriaceae family.
Klinger Iron Agar is also helpful in identifying the species named Salmonella shigella along with the other members of the Enterobacteriaceae family.
Kligler Iron Agar Test Objective
The main aim of the test is to differentiate the organisms by demonstrating the medium with the hydrogen sulphide production and to determine the fermentation of dextrose and lactose.
Kligler Iron Agar Test Principle
The medium of Klinger iron consists of lactose and the glucose in addition to peptone, HM peptone B and the yeast extract, which helps us to enable the difference between the species of the enteric bacilli.
Generally, in this test, phenol red indicator is used as a pH indicator, which brings about a change in colour in the medium when the acid response is detected during the fermentation process of the sugars.
Fermentation of the dextrose results in the production of acid, which in turn changes the colour of the medium from red to yellow.
If there is only minimum amount of sugar present in the medium, the indicator remains red and there will not be any change in the colour.
On the other hand, when lactose is fermented, there will be large amount of acid production in the medium and it also avoids reoxidation and results in the yellow colour of the whole medium.
The combination of ferrous sulphate and the sodium thiosulphate enables us to detect the production of hydrogen Sulphide and it is evidenced by a black colour or through the formation of butt.
In some cases, it can also be determined by ring formation near or top of the butt.
The production of butts and yellow slants in the lactose fermentation is due to production of high amount of acid in the medium which induces the change in pH under the aerobic conditions.
Whereas, the tubes which does not show any change in colour indicates that there is no presence of glucose or lactose in the medium.
However, the gas production is detected by formation of the individual bibles or by splitting of the agar, due to the formation of the cracks in the butt of the medium.
Kligler Iron Agar Test Reagents
Ingredients Gram/Litre Peptone 15.0 HM peptone 3.0 Yeast extract 3.0 Protease peptone 5.0 Lactose 10.0 Dextrose 1.0 Ferrous sulphate 0.2 Sodium chloride 5.0 Sodium thiosulphate 0.3 Phenol red 0.02 Agar 15.0 Kligler Iron Agar Test Procedure
Initially, a well isolated colony from a solid culture medium is picked from the centre of the medium using an inoculating needle.
Here the medium used for identification of the colonies includes MacConkey Agar, Bismuth Sulphide Agar, or Deoxycholate Citrate Agar are used as the plating medias.
Further the stab is taken from the centre of the medium into the deep tube which has about 3 to 5mm of depth.
Then the inoculating medium is withdrawn and it is streaked against the surface of the slant.
The caps of the tubes are closed loosely before incubating so that there will be an aeration in the medium.
Then the tubes are incubated aerobically at a temperature of about 35ºC for about 18 to 48hours.
After incubation the tubes are observed for acid production which can be detected by change in colour or formation of butts.
Kligler Iron Agar Test Result
The results are interpreted by Carbohydrate fermentation, Kligler Iron Agar colour reactions and by Hydrogen sulphide production.
Carbohyderate Fermentation
Slant Reaction:
Positive reaction- Yellow colour (acid)
Negative reaction- Red colour (alkaline)
Butt reaction:
Positive result _ Yellow (acid)
Negative result – Red (alkaline)
Kligler Iron Agar Color Reactions
Formation of red slant or yellow butt – Presence of dextrose and absence of lactose.
Formation of yellow slant or yellow butt – Presence of dextrose and lactose
Formation of red slant or red butt – Absence of dextrose and presence of lactose.
Production of Hydrogen Sulfide
Positive Test: Here the positive result is determined but formation of black colour throughout the medium and a black ring at the juncture of the slant and butt. In some cases, it is also determined by formation of black precipitate in the butt.
Negative Test: Here the negative test is indicated when there is no formation of black colour.
Gas Production
Positive Test: Formation of bubbles in the medium along with cracking or displacement of the medium. In some cases, it also leads to separation of the medium.
Negative Test: There will no bubbles and displacement or separation of the medium does not take place.
Kligler Iron Agar Test Citations
- The phenylalanine test on Kligler’s iron agar for the identification of Proteus. Nature . 1959 May 30;183(4674):1537-8.
- Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens. J Clin Microbiol. 1994 Jun; 32(6): 1583–1585.
- Biochemical differentiation of the Enterobacteriaceae with the aid of lysine-iron-agar. Appl Microbiol . 1966 Mar;14(2):212-7.
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Salt Tolerance Test: Principle, Procedure, and Result
Continue ReadingSalt Tolerance Test Introduction
Many of the biochemical test are performed in order to detect the ability of the organism to grow in a particular medium and to detect the utilization of the enzymes by them.
Once such biochemical test is salt tolerance test, which helps us to detect the ability of the bacteria to grow in the presence of the variable amount of sodium chloride and which is used to characterize the variety of bacteria.
Salt Tolerance Test also accounts the organism’s ability to tolerate the various osmotic concentrations. E. faecalis, E. zymogenes, E. liquifaciens, and E. durans, which are present among the species of Enterococcus, and are considered as salt tolerant.
What is Salt Tolerance Test?
Salt tolerance test is usually used to identify the group of enterococcal group D Streptococcus based on the ability to tolerate the salt contents.
Thus, the capability of the bacteria to grown in the variable amount of Sodium chloride containing medium, helps us to distinguish the variety of bacteria’s, including those of Viridians Streptococci.
This test is used particularly for identifying the Species of Enterococcal Group D organisms, as it has its specialized ability to grow in a medium containing of 6.5% of the Sodium chloride.
This test is often performed with Bile Esculin tests in most of the laboratories, to distinguish the species of Enterococcus from the Group D species of Streptococci and also from Streptococcus bovis and Streptococcus lactis.
Enterococci is considered as one of the Significant causes for endocarditis, which has a high degree of mortality rate.
Salt Tolerance Test and Broth
In Salt tolerance test, Brain Heart infusion broth is used most commonly, it is supplemented with 6.5% of Sodium chloride and the Bromocresol purple as the indicator to denote the pH of the medium.
The use of this indicator helps us to read the results easier.
The broth also consists of dextrose.
Where the fermentation of this dextrose results in acid production, which leads to the change in pH and the color of the medium changes from purple to yellow.
Salt Tolerance Test Objective
The main aim of the test is to determine the ability of the organism to grow in high concentrations of the salt.
It is also used to differentiate the variety of Enterococcus species from the Non- enterococcus species.
Salt Tolerance Test Principle
Generally, Salt has its own characteristic to act as a selective agent and it also has the capability to interfere with the membrane permeability and the osmotic equilibrium.
The salt tolerance in the medium is calculated as a selective and the differential one according to the capability of the organisms to produce heavy growth in the broth and on the solid agar medium within 48 hours.
The salt tolerance medium was first formulated by Hajna.
Where as the high salt concentration inhibits a range of the bacteria which allows the salt-tolerant organisms like enterococci to grow in the medium, the quadric formaulation includes the carbohydrates, that can be fermented, dextrose, color indicator, bromocresol purple.
Organisms which have the capability to grow in the slaine medium, utilize the sugar and it releases the acid as its by product during their metabolism in the medium.
Which results in the decrease in the pH, the indicator bromocresol changes its purple color into yellow color.
Enterococci are usually resistant to high concentration of the salts and it often shows growth in the medium which contains the species like Enterococcus faecalis, Enterococcus zymogenes, Enterococcus liquefactions and Enterococcus durans which are known as salt tolerant species of the Enterococcus.
Salt Tolerance Test Reagents
The medium is composed of Sodium chloride of about 6.5% which is often used in brain heart infusion broth, commonly called as BHI and it can be used instead of individual components along with the sodium chloride and the indicator.
Components Required:
Herat digest of about 10 grams
10 grams of Enzymatic digest of animal tissue
Sodium chloride
Bromocresol purple indicator (it is added Per 1000 ml)
Salt Tolerance Test Procedure
Initially, one or two colonies are inoculated from 18 to 24-hour culture into the 6.5 of the sodium chloride broth without an indicator or 6.5 % of Nacl broth with indicator.
Then the tube is further incubated at temperature of about 35 to 37ºC in an ambient air for about 48 hours.
Then the incubated medium is examined for the presence of turbidity or growth or formation any colonies without an indicator. In some cases, there will also be a color change in the media with an indicator, here the color changes from purple to yellow.
Salt Tolerance Test Result
Positive Test: Here the positive result is indicated by visible turbidity or growth in the broth, and a color change from purple to yellow with the use of indicator.
Negative Test: Here the negative test is indicated by no turbidity or absence of color change, and the color remains the same.
Salt Tolerance Test Uses
This test is often used to differentiate the species of enterococci from the non-enterococci species.
It is also used to differentiate the species of non-beta hemolytic strains of the catalase from the negative gram-positive cocci based on their ability to grow in a medium containing 6.5% of sodium chloride broth.
Where as the Aerococcus species such as the A. viridians and Aerococcus Urinae has also has the capability to grow in the medium containing 6.5% of sodium chloride, hence this salt tolerance broth has the capability to differentiate the different species of Aerococcus from the other similar organisms such as Stomatococcus and Helcoccus possessing the same characteristics.
Salt Tolerance Test Limitation
It is often insisted that the biochemical immunological, molecular or mass spectrometry test is performed on the colonies from pure culture for complete identification of the test.
Some strains of pseudococcus, Leuconostoc and Beta-hemolytic streptococcus species grows in the Salt tolerance Broth.
Infusion broth having 6.5% Sodium chloride produces slow reactions in the medium which results in making the interpretations difficult.
Usually, a light inoculum must be used while inoculating a broth. • If too heavy inoculum is used it produces turbidity and results in a false positive result.
Salt Tolerance Test Citations
- THE SALT-TOLERANCE TEST. Triangle . 1964 Oct;7:268-72.
- FURTHER STUDIES WITH THE SALT TOLERANCE TEST IN NORMAL INDIVIDUALS AND IN PATIENTS WITH ADRENAL CORTICAL HYPERFUNCTION. J Clin Invest . 1949 Sep;28(5 Pt 2):1091-3.
- Taxonomic study of a salt tolerant Streptomyces sp. strain C-2012 and the effect of salt and ectoine on lon expression level. Microbiol Res . Feb-Mar 2014;169(2-3):232-8.
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Biuret Test for Protein: Principle, Procedure, and...
Continue ReadingBiuret Test Introduction
Many of a biochemical test are used to detect the ability of the microorganism to utilize the enzymes and the proteins in the medium. Biuret test is one such test, which is used to detect the proteins in a compound.
What is Biuret Test?
Protein are one of the complex molecules which are formed by millions of amino acids.
Amino acids are known as amphoteric electrolytes as they have carboxyl and the amino groups and it acts like an acid and base, it has one positive charge and one negative charge and these ions are considered as electrically neutral and they do not migrate in the electrical field.
The two amino acids are linked together with the help of a peptide bond which results in the formation of a dipeptide and this process is often called as condensation reaction.
However, the amino acids are linked together with the help of three peptide bonds and they are known as tripeptide and as well as the chain elongates, it is also called as polypeptide.
Biuret is one of the compounds which is formed by heating urea at 180ºC, which results in condensation with two molecules of urea.
The peptide bond in the Biuret usually gives a positive result in the tests.
Biuret test is considered as one of the generally performed tests for the compounds such as proteins, which have their two or more peptide bonds.
Biuret Test Objective
The main aim of the test is to detect the protein in the given sample or a solution.
To detect the presence of the peptide pond.
Biuret Test Principle
As mentioned above, biuret test is one of the biochemical tests, which is used to detect the presence of a peptide bond in the compounds or substances given.
This test is purely based on the structure of peptide which consists of about two peptide linkages and results in producing a violet or purple color when it is treated along with the copper sulfate.
During the presence of an alkaline solution, the blue colored copper II ion, forms a complex with the peptide bonds.
These peptide bonds does not share their pair of electrons with the nitrogen and oxygen that is present in the water.
The colored coordination complexes is usually formed between the ion carbonyl oxygen and the amide nitrogen (=NH) of the peptide bond.
On formation, of this complex the solution changes its color blue to purple. When the purple color changes deeper, in the number of peptide copper complexes.
This compound containing at least two H2N-C, H2N-CH2-, H2N-CS- or similar groups they are joined together either directly or with the help of a carbon or a nitrogen atom.
One copper ion is appropriately linked to about 6 peptide bonds present in the molecule of protein which reacts and also the number of a protein molecules present in reaction system.
The Biuret reagent is a solution composed of Sodium hydroxide or potassium hydroxide along with hydrated copper II sulfate, and potassium sodium tartrate.
Sodium hydroxide and potassium hydroxide thus provides the alkaline medium and the potassium sodium tartrate is added to chelate and to stabilize the cupric ions in the solution or in order to maintain their solubility in alkaline solution.
Biuret Test Reagents
5% of egg white (albumin)
Biuret reagent
Water bath
Pipettes
Dry test tubes
Biuret Reagents:
Copper sulfate
Sodium hydroxide
Sodium potassium tartarate (Rochelle Salt)
Preparation of Biuret Reagents
This reagent is prepared by adding sodium hydroxide and copper sulfate solution, making it alkaline.
To prepare 1000 ml of a Biuret reagent, about 1.5 gram of a pentavalent copper sulphate and about 6 gram of sodium potassium tartarate and dissolve in a 500ml of distilled water.
Sodium potassium acts as a chelating agent and it also helps stabilize the copper ion.
Further 375 ml of a two molar sodium hydroxide and it is mixed both the solution in volumetric flask and make it final volume to 1000 ml by adding water.
Biuret Test Procedure
About 1ml of test solutions are taken in a dry test tubes and in another tube take 1 ml distilled water is added.
To this about 1ml of biuret reagent to all the test tubes and it is mixed well.
Then the test tube is observed for the developed for blue colors.
Then there will be change in color to the purple.
Hence, it is concluded that there is a presence of peptides and proteins and it resulted in positive tests.
It is also to be noted that in a biuret test of protein, Histidine is the only amino acid to give a positive result.
Biuret Test Result
Observation Interpretation If there is no color change and the solution remains blue Absence of proteins or peptides Here the test is detected as negative The solution changes into deep purple It determines the presence of proteins. Here the test is detected as positive. Biuret Test Uses
This test is generally used to detect the amount of protein present in the urine.
Biuret reaction with the protein is applied to determine quantitative analysis of the total protein by using the spectrophotometric analysis.
Biuret Test Citations
- The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission. J Hosp Infect . 2006 Nov;64(3):288-92.
- Comparison of tests for failure of passive transfer in neonatal calf serum using total protein refractometry and the biuret method. Prev Vet Med . 2021 Apr;189:105290.
- Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens. J Am Vet Med Assoc . 2016 Apr 1;248(7):789-94.
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Pyruvate Broth Test: Principle, Procedure, and Result
Continue ReadingPyruvate Broth Test Introduction
Many of the biochemical tests are used to detect the ability of the organism to utilize the organic and chemical substances in the medium, one such biochemical test is Pyruvate broth test.
Biochemical tests help us to identify the species of bacteria on the basis on their ability to differentiate the biochemical activities of the various species of bacteria.
As bacterial physiology differs from one species of the organism to the another. Thus, ability of the bacteria to form the organic compounds which helps to metabolize the certain compounds of the carbohydrates and the other related compounds that is widely used for identifying the microorganism.
One such test is pyruvate test, which tests the ability of the specific species to the substrate pyruvate.
What is Pyruvate Broth Test?
Pyruvate is considered as the conjugate base of pyruvic acid, which is used as a key in intermediating the biological processes.
Pyruvate is generally producing as the end product of the process of glycolysis which acts as a connective link between various biochemical processes including gluconeogenesis, fermentation, cellular respiration, fatty acid synthesis etc.
However, by accompanying these processes, pyruvate plays a key role in providing energy to the living cells through Krebs cycle, which is one of the metabolic pathways in all living cells.
In Krebs cycle, Pyruvate is decarboxylated into acetyl coA which is then converted into acetyl CoA.
Pyruvate broth is usually used to determine the ability of the microorganism to utilize the pyruvate.
The presence of this test substrate pyruvate helps us to differentiate the characteristics of an organism which metabolize the pyruvate and other which does not have the capability to metabolize.
This characteristic is useful in differentiating the organisms such as Enterococcus faecalis from Enterococcus faecium.
Here pyruvate broth is made up of pancreatic digest that includes casein and the yeast extracts which provides the bacteria healthy supplements like amino acids, vitamins and other necessary growth factors, generally phosphate is added here in the broth to maintain the stable pH needed for the culture medium.
Due to the pyruvate breakdown it results in the change in pH to the acidic form in the culture.
Pyruvate is one of the active substrates where, the organisms can utilize it and breakdown the substrate thus generating the host of metabolic acids, and it is detected by the pH indicator known as Bromothymol blue.
At the time of acid generation, the culture medium changes its color from a blue green to yellow.
Pyruvate Broth Test Objective
The main aim of the test is to determine the ability of an organism to utilize the pyruvate to synthesis the acidic products.
Pyruvate Broth Test Principle
To perform this test, pyruvate broth test is added to the culture broth, which helps us to determine the capability of the organism to utilize pyruvate, thus results in formation of the metabolic acids.
Acid produces as a result of metabolic waste when pyruvate broth is being inoculated with bacteria so that they are capable of metabolizing the pyruvate.
The acid production decreases the pH of the medium which results in change in color from.
Bromothymol blue is used as an indicator as it one of the acid base indicators which suits the medium.
It generally has a greenish blue at an alkaline pH and during acid production, it changes its color to yellow indicating the fermentation in the pyruvate.
Thus, yellow color is one of the indicatives of the positive reaction of the fermentation.
Pyruvate Broth Test Reagents
Pancreatic digest of casein of about 10 gram per Liter
Sodium pyruvate – 10 grams
Yeast extract of about 5 grams
Potassium hydrogen phosphate- 5 grams
Sodium chloride – 5 grams
Indicator- Bromothymol blue- 40 grams/liter
Pyruvate Broth Test Procedure
Before performing the procedure, initially the medium is allowed to reach the temperature.
Then the direct inoculum is prepared overnight so that it can be inoculated into the broth.
Initially, the pH of a medium was set at 7.3 and then the pyruvate broth is inoculated with an organism cultured for about 18 to 24 hours overnight from the Todd-Hewitt broth culture for blood agar.
The tube is then incubated in an ambient air for 24 to 48 hours at a temperature of about 35ºC.
The culture should be observed continuously for a color change. Usually, the tubes are observed maximum for about 5 days.
Pyruvate Broth Test Result
A change in color of the broth from greenish-blue to yellow, indicates a positive control for pyruvate broth test.
Whereas, if there is no color change then it results in a negative result.
Quality Control
In pyruvate broth test, positive control is usually determined for Enterococcus faecalis, and the negative control is determined for two species namely, Enterococcus faecium and Staphylococcus bovis.
Pyruvate Broth Test Uses
Pyruvate broth test is usually used in differentiating the species of Enterococcus faecalis from the Enterococcus faecium.
This test can also be used as a part of identification of the other organisms which have the capability to utilize pyruvate.
Pyruvate Broth Storage and Shelf Life
Usually, pyruvate broth is stored in an upper right position at a temperature of about 4 to 8ºC. It is also important that the broth should not be placed in a direct sunlight.
By following these conditions, the broth has a shelf life of about 26 weeks, calculated from the date of manufacture.
Pyruvate Broth Test Citations
- Susceptibility of anaerobic bacteria to sulfamethoxazole/trimethoprim and routine susceptibility testing. Antimicrob Agents Chemother . 1978 Sep;14(3):384-90.
- Identification of Enterococcus spp. with a Biochemical Key. Appl Environ Microbiol. 1999 Oct; 65(10): 4425–4430.
- Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance. J Appl Bacteriol . 1983 Feb;54(1):115-25.
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Leucine Aminopeptidase Test: Principle, Procedure, and Result
Continue ReadingLeucine Aminopeptidase Test Introduction
Many of the biochemical test is usually used to detect the characteristics of the micro-organism especially to detect their activity when exposed or left in a medium of certain enzymes.
Mostly the biochemical tests are followed for bacteria which acts as a pathogen in the hosts organism.
Leucine Amino peptidase test is one among the biochemical test, which is commonly referred to as LAP test.
This test is followed by using the serum of the host. This test is usually used to detect the presence of an enzyme leucine amino peptidase for analyzing the preliminary characteristic of a catalase negative, a gram-positive coccus.
This is especially for a non-hemolytic coccus. Leucine Aminopeptidase is one of the cytosolic metallo-exopeptidase which helps in catalyzing the removal of the amino acids from the peptide, that is generated during the process of hemoglobin degradation.
What is Leucine Aminopeptidase Test?
Leucine Aminopeptidase Test is usually used o detect the activity of the catalase-negative gram-positive cocci.
This test specially differentiates the species Aerococcus and the Leuconoctoc from the other species like Streptococcus, Enterococcus, Lactococcus and Pediococcus.
Generally, it is one the rapid test followed for detecting the enzyme Leucine aminopeptidase.
Here, Leucine-Beta-naphthylamide impregnated disk acts as a substrate for detecting leucine aminopeptidase.
After hydrolyzing activity of the substrate by enzyme, it results the Beta-naphthylamine to produces a red color while adding the cinnamaldehyde reagent.
Leucine Aminopeptidase Test, is usually performed with the other jests in the form of conjugation experiments, for identifying the species of Streptococci and the other catalase negative gram-positive cocci.
Leucine Aminopeptidase Test Objective
The main aim of the test is to perform a preliminary characterization of the catalase negative, gram positive cocci.
Leucine Aminopeptidase Test Principle
Leucine aminopeptidase test is one of the rapid tests, used for detecting the presence of an enzyme leucine amino peptidase.
Here, the disks are impregnated with Leucine-Beta Naphthylamide, that is hydrolyzed by the enzyme known as leucine aminopeptidase, which is produced by the LAP positive organism.
This enzymatic activity thus results in the release of a substrate known as Beta-naphthylamine, Beta-naphthylamine, combines with the p-dimethylaminocinnamaldehyde reagent, at the time of adding it to the culture and results in the formation of highly visible red Schiff base.
Leucine Aminopeptidase Test Procedure
Initially, a Leucine Aminopeptidase disk is placed in a sterile petri disk in an aseptic condition and it is allowed to warm as of the room temperature.
Then it is slightly dampening the Leucine Aminopeptidase disk with the reagent grade water or with a small quantity of the sterile distilled water.
Then a small amount of several colonies of an 18-to-24-hour culture is applied on a small area of the Leucine Aminopeptidase disk using a wooden applicator stick.
Then the prepared medium is incubated at a room temperature for about 5 minutes.
After incubation the medium is taken out from the incubator and one drop of cinnamaldehyde reagent is added and the changes are noted within a minute.
Leucine Aminopeptidase Test Result
Positive Test: In case of positive result, development of red or pink color in the medium can be observed. For positive control presence of Enterococcus faecalis is determined.
Negative Test: In case of negative result, there will be no change in color. In few cases, a slight yellow color can be noted. For negative control Aero coccus viridians can be determined.
Leucine Aminopeptidase Test Uses
LAP test is usually used in combination with the other tests like PYR and some biochemical tests. This helps us to differentiate between the catalase, gram positive cocci.
Generally, the organisms like Streptococcus pneumoniae and Streptococcus pyogenes, Pedi coccus, Lactococcus and Enterococcus species are all considered and gives a positive result for LAP test.
Where as the other species like Beta-hemolytic Streptococci, Aero coccus and Leuconostoc species are all considered as Negative control, and thus helps us in identifying the specific species.
Aminopeptidase test have been used in performing a Gram analysis of the bacteria that is isolated from the Scleroderma citrinum mycorrhizae, the mycorrhizae and the bulk soil.
Leucine Aminopeptidase Test
The test organism is confirmed as Gram-positive coccus and catalase negative before initiating the Leucine aminopeptidase test.
Adequate inoculum is ensured, or else there will be many chances for getting negative results.
Streptococci is usually tested prior to 48 hours of incubation or it is sub cultures before testing.
Clinical Uses Leucine Aminopeptidase Test
This test is performed in a clinical laboratory, using a blood sample. For collecting a blood, a specific person should have a fasting for about 8 hours.
Leucine Aminopeptidase is one of the types of protein and it is also known as enzyme which is normally found in the cells of the liver, blood and bile, placenta and urine.
This test is often suggested by the diagnostics when there is abnormal functioning of the liver or any damage is detected in the liver or any tumor in the liver.
Leucine Aminopeptidase Test Citations
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Sulphur Reduction Test: Principle, Procedure, and Result
Continue ReadingSulphur Reduction Test Introduction
Many of the biochemical tests are performed in order to distinguish the different species of bacteria, based on their enzymatic activity. One such biochemical test is Sulphur reduction test.
These biochemical tests play an important role in identifying the microbes. These tests are performed based on the fermentation activity of the certain substances such as carbohydrates.
In recent times, Microbial tests performed biochemically shortens the time to identify the certain microbes. It can also be performed economically with less cost and it also has the accuracy in its results.
What is Sulphur Reduction Test?
Sulphur reduction test is usually performed in the Sulphide Indole Motility medium, which is a conjugation of different medium which tests the three variety of parameters such as Sulphur Reduction, indole production and the motility.
As the name says, this test is commonly used to test a microbe for the ability to synthesize the gas hydrogen sulfide.
Sulphur Reduction Test Objective
The aim of the test is to detect the ability of the organism to reduce Sulphur.
To differentiate the species, of gram-negative enteric bacilli based on their production of sulphide.
Sulphur Reduction Test Principle
Here, the organism that produces enzyme thiosulfate reductase, which reduces the Sulphur into hydrogen sulfide gas.
This occurs when strain degrades the amino acid cysteine while the degradation process of protein or during the process of protein degradation or during the process of anaerobic respiration, which shuttles the electrons to Sulphur instead of oxygen.
In Sulphur indole motility tubes, the medium contains casein and the animal proteins as the source of amino acid, sodium thiosulphate, as a source of sulfur and the ferrous ammonium sulphate, which acts as the hydrogen sulphide indicator.
Cysteine is a Sulphur containing amino acids that is present in the SIM medium.
The enzyme cysteine desulfurizes and thiosulfate reductase catalysis the reactions of hydrolysis and it produces hydrogen sulphide.
Thus, the produced hydrogen sulphide gas, combines with the ferrous ammonium sulfates and forms an insoluble, black color ferrous sulfide precipice.
This black color acts as an indicator during the presence of a hydrogen sulphide.
In the laboratory, a fresh medium of the cultured organisms is inoculated in the center of the medium in a single stab with the help of a straight needle.
After the process of incubation, the tube is further observed for the production of hydrogen sulphide gas, which is observed by the blackening of the medium.
SIM medium is generally used for this Sulphur reagent test, it consists of nutrients, iron and sodium thiosulphate.
One of the vital nutrient present in the medium is peptone, which is made up of amino acids like those of tryptophan.
However, if the organism has the capability to reduce Sulphur into hydrogen sulfide, the reduced hydrogen sulphide will combine with the iron and results in the formation of the ferric sulphide, which is shown as a black precipitate.
This is the reason for blackening of the medium and it indicates the reduction of the Sulphur in the medium and gives a positive result.
Sulphur Reduction Test Reagent
SIM Agar: For preparation of a Sulphide, indole moetilty agar, the pancreatic digest of casein is taken into about 20-gram, peptic digest of an animal tissue about 6 grams, agar-3.5 grams.
Sulphur Reduction Test Procedure
Initially an 18-to-24-hour culture is collected using a straight needle. Here the stab is taken in a single depth of about half an inch from the middle of the tube.
Then it is incubated at a temperature of about 35 to 37ºC and it is observed regularly for a maximum of 7 days. Blackening of the medium is observed, it is noted on the line of inoculation.
Sulphur Reduction Test
Positive Results: It results in darkening of the medium, where black precipitate is seen, or in some cases, it can also be seen in blackening of the line in the inoculation which indicates the presence of certain bacteria that produces hydrogen sulphide gas.
Negative Result: Negative result is often indicated by lack of black precipitate in the medium.
Sulphur Reduction Test Uses
This test is often used to differentiate the Sulphur reducing members of the family Salmonella shigella and the Genera of protists from the species such as Negative Moranella morganii and providencia rettgeri.
This test can also be used for producing the hydrogen sulfide which can be used for diagnosing and identifying the enteric bacteria.
Sulphur Reduction Test Limitation
Although, there are many advantages of this test, there are some limitations such that the reaction given by the SIM medium is not enough to speciate the organism, hence there is a need of additional biochemical and serological tests.
It should also be noted that inoculum is taken from the solid medium.
As taking an inoculum from a liquid or a broth suspension delays the initiation of the growth process, which also leads to the errors in the results.
Sulphur Reduction Test Citations
- Biochemical Differentiation and Comparison of Desulfovibrio Species and Other Phenotypically Similar Genera. J Clin Microbiol. 2005 Aug; 43(8): 4041–4045.
- Indirect sulfur reduction via polysulfide contributes to serious odor problem in a sewer receiving nitrate dosage. Water Res . 2016 Sep 1;100:421-428.
- Realizing high-rate sulfur reduction under sulfate-rich conditions in a biological sulfide production system to treat metal-laden wastewater deficient in organic matter. Water Res . 2018 Mar 15;131:239-245.
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Coagulase Test: Result, Principle, Procedure, and Uses
Continue ReadingCoagulase Test Introduction
Many types of biochemical and antimicrobial tests are being performed in a laboratory in order to detect the specific characteristic of a micro-organism especially a pathogen and its reactions to the certain enzymes.
One such biochemical test is Coagulase test. Coagulase test is generally used to differentiate the bacterium, Staphylococcus aureus, positive from the coagulase negative Staphylococci.
Staphylococcus aureus synthesis an enzyme named Coagulase which helps in converting the soluble from of fibrinogen present in the plasma into the insoluble form.
What is Coagulase Test?
Coagulase test is one the biochemical test which is used to differentiate Staphylococcus aureus from other species of Streptococci such as S. epidermidi and S. saprophyticus on the basis on their ability to synthesis the enzyme, coagulase.
The coagulase test is very important to differentiate the species of the gene Staphylococci, and it is divided into two types namely Positive Staphylococci and the Coagulase Negative Staphylococci.
The enzyme, coagulase acts as a essential factor in many of the organisms and it interacts with the factor fibrinogen that is present on the surface of the host cell.
Organisms which contain coagulase usually contains a protective barrier around themselves, that increases their pathogenicity and their resistance against the hosts immune system.
Coagulase is categorized into two ways; free coagulase and as bound coagulase, each of which is determined by different methods.
The bound coagulase is known as clumping factor and it is usually detected by using a slide test.
The free coagulase is usually detected by a test tube which results in the formation of a clot.
Coagulase Test Objective
• The aim of the main of the test is to detect free coagulase and bound coagulase produces by different organisms.
• To differ coagulase positive coagulase Staphylococci from the coagulase negative staphylococci.
• To identify and differentiate the species of Staphylococcus aureus from other Staphylococcal species.
Coagulase Test Principle
Coagulase is one of the enzymatic proteins, which is thermostable and it is a thrombin-like substance, that contains fibrinogen into fibrin and results in the clot or clumping.
In Staphylococcus aureus, two different forms of coagulase are found. Free coagulase and the bound coagulase.
I. Bound Coagulase Test
The clumping factor, known as bound coagulase is detected rapidly by slide test, however, this test requires several colonies and it lacks sensitivity.
Staphylococcus aureus, produces a substance in its cell wall protein, which binds to a Fc moiety of a human immunoglobin G and it behaves like a coagulase-reacting factor.
When the latex or the erythrocyte particles are coated with Immunoglobin G and the human fibrinogen, a staphylococcus agglutinates, either of the clumping factor that is present in the cell wall of bacteria.
Thus, the presence of the clotting factor is demonstrated by the ability of the organism to act upon the factor fibrinogen, which is present in the plasma to form a clot during the process of slide assay.
However, the test for clumping factor is rapid and it also requires several colonies, and the factor may not present in all the organisms of S. aureus.
II. Free Coagulase Test
This is one of the other types of coagulase which is released by the organism.
Free coagulase is different from the bound coagulase in the mechanisms of clotting as free coagulase requires the activation of a plasma-coagulase reacting factor known as CRF.
It is known as the modified or derived form of Pro-thrombin, to form a coagulase complex, CRF.
The plasma added to the tube is preferably rapid plasma that acts as a binding factor.
The complex formed then reacts with the fibrinogen to form fibrin clot inside a test tube.
Coagulase Test Reagent
Supplies:
Loops or sterile sticks
Glass or plastic tubes
Glass slides
Reagents:
Frozen Plasma with EDTA
Here, most preferably rabbit plasma is used and human plasma is not commonly used for this test, as it is less sensitive and it causes potential infections with the human pathogenic viruses
5% of calcium chloride
Coagulase Test Procedure
Usually, coagulase test is experimented using two methods such as Tube test and Slide test.
I. Coagulase Slide Test
For slide test, about 10µl of physiological saline or deionized water is added to a slide.
Then using an inoculating loop several colonies from a fresh culture are collected and are emulsified into the water, in order to get a smooth milk-colored suspension.
Further, a drop of rabbit plasma is placed on the slide and clumping can be observed immediately.
Mostly clumping does not exceeds more than 10 seconds.
II. Coagulase Tube Test
For tube test, Initially the plasma is diluted with the physiological saline by adding, 0.2ml of plasma in 1.8 ml of saline.
Then about 5 ml of diluted plasma is then added into a test-tube along with 5 drops of test organism culture.
Then the test tube is mixed thoroughly and it is incubating at a temperature of about 37ºC for about one hour.
Then the tube is finally observed for formation of a clot.
In case, if there is no formation of any clumps, then the is tube is examined for 30 minutes for a regular interval of 6 hours.
Coagulase Test Result
Slide Test:
o Usually, a positive test is demonstrated by the agglutination of the bacterial cells after addition of plasma.
o Whereas, the negative test is demonstrating when is no formation of clumps.
Test Tube:
o In tube test, few criteria are followed for determining the positive and negative results.
Positive Results:
o In positive result, Complete clot is formed or there will be any degrees of clot formation within 24 hours.
o If there is no clot formation, after adding 1 to 2 drops of calcium chloride, after 24 hours.
Negative Results:
o In case of negative results, there will be no formation of clumps or clots.
o If there is no clot observed after 24 hours in a temperature of 35ºC, then 2 to 3 drops of calcium chloride is added and it results in formation of a clot.
Coagulase Test Citations
- Interpretation of the tube coagulase test for identification of Staphylococcus aureus. Appl Microbiol . 1975 Apr;29(4):502-5.
- Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob . 2010 Aug 13;9:23.
- Evaluation of direct tube coagulase test in diagnosing staphylococcal bacteremia. J Clin Diagn Res . 2014 May;8(5):DC19-21.
- Microtube coagulase test for detection of coagulase-positive staphylococci. J Clin Microbiol . 1982 May;15(5):848-51.
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Carbohydrate Fermentation Test: Principle, Procedure, and Uses
Continue ReadingCarbohydrate Fermentation Test Introduction
Many of the biochemical test is used to detect the nature of the bacteria to detect their ability of utilizing the enzymes in their medium.
Carbohydrate fermentation test is one of the biochemical tests, which is used to detect the ability of a bacterium to use carbohydrate.
Carbohydrate Fermentation Test helps in testing the presence of an acid or a gas produced during the fermentation of a carbohydrate.
What is Carbohydrate Fermentation Test?
Carbohydrates are one of the organic molecules that comprises of carbon, Hydrogen and Oxygen in the respected ratio of CH2O.
Carbohydrates are classified into three types based on their structure and the number of carbon atoms it is present.
The types include monosaccharides and polysaccharides. The energy is released from the carbohydrates during the process of the catabolism, which results in the breakdown of these complex organic molecules.
Whereas this power for breaking carbohydrate comes from the micro-organisms like bacteria, yeast and fungi. However, these utilizations of the carbohydrate and its process of breakdown is different depending upon the enzyme system of the respective organism.
The pattern of fermentation is the characteristic species of certain organisms.
Due to these reasons, the property of the fermentative reaction is used extensively for differentiating the micro-organisms.
Carbohydrate Fermentation Test Objective
The main aim of the test is to detect the ability of the micro-organisms to ferment the carbohydrate and to produce the products of organic acids.
To determine the capability of the micro-organisms to synthesis gaseous and the fermentative products.
Carbohydrate Fermentation Test Principle
Carbohydrate fermentation is one the process in where the micro-organism produces energy in the form of Adenosine Triphosphate, ATP. Which is one the vital source of energy.
When glucose enters a cell, it catabolizes aerobically, where the oxygen molecule serves as a final electron acceptor.
In case of anaerobic respiration, the inorganic irons serve as the electron acceptors.
The metabolic end product formed after fermenting the carbohydrate results in organic acid and gaseous products.
Fermentative dehydration of the carbohydrates by the micro-organisms in the presence of oxygen is carried out in the fermentation tubes, which is known as Durham tube, which helps in detecting the production of gas.
Generally, a fermentation process is composed of basal medium which contains specific carbohydrates along with the pH indicator.
During fermentation of a carbohydrates, organic products are obtained. During these processes of metabolism there will a change in color of the medium.
This color change occurs only when a sufficient amount of acid is produced, as the organism utilize the peptones produced by the alkaline by products.
Thus, the degradation of the peptones in the culture results in production of the alkaline end products, which results in change in color of the culture from yellow to pink.
Carbohydrate Fermentation Test Procedure
I. Preparation of carbohydrate fermentation broth:
Initially, trypticase is weighed and dissolved along with the Sodium chloride and phenol red in about of 100 ml of distilled water and it is transferred into a conical flask.
About 1 % of the desired carbohydrate is then added into all the flasks.
Further Durham tubes is inserted into all the tubes in an inverted position.
Tubes should be filled fully.
Then it is sterilized in an autoclave at a temperature of about 115ºC for 15 minutes.
Here it should be noted that phenol red carbohydrate fermentation broth is should not be overheated, as it results in breaking the molecules and it forms compounds with a characteristic flavor and color.
This process is usually known a caramelizing of the sugar.
Then the sugar is transferred into the screw capped tubes or in the fermentation tubes and it is labelled properly.
II. Inoculation of bacterial culture into the fermentation tubes:
About one drop of inoculation is collected from the 18 to 24-hour culture broth and it is inoculated into a tube in an aseptic condition.
Then the tubes are inoculated at a temperature of about 37ºC for about 18 to 24 hours and they are examined for the production of acid and gas.
Carbohydrate Fermentation Test Result
Observation Result Interpretation Change in color of the medium from pink to yellow color It results in acid production Organism ferments the given carbohydrate and it produces organic acids which reduces the pH of the medium and turns the medium into the acidic medium The change in color of the medium to yellow color and there will be a production of gas in the Durham tube It results in gas and acid production Here, the organism ferments carbohydrates and the organic acids and it also releases gas and it is determined by air bubbles present in the medium in an inverted Durham tube. There will be no change in color and the medium remains same No fermentation takes place Here the organism does not utilize the available carbohydrate and it grows by means of using the other sources present in the culture medium. Carbohydrate Fermentation Test Citations
- Rapid carbohydrate fermentation test for confirmation of the pathogenic Neisseria using a Ba(OH)2 indicator. J Clin Microbiol . 1977 Jan;5(1):15-9.
- A cost-effective carbohydrate fermentation test for yeast using microtitre plate. Indian J Med Microbiol . Apr-Jun 2015;33(2):293-5.
- A simple carbohydrate fermentation test for identification of the pathogenic Neisseria. J Clin Microbiol . 1975 Jul;2(1):72-3.
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