Category: Biology
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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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Acetamide Utilization Test: Principle, Procedure, and Results
Continue ReadingAcetamide Utilization Test Introduction
Many of the biochemical tests are used to determine the ability of the micro-organism to utilize the chemicals and their reaction to certain enzymes. One such biochemical test is Acetamide utilization test.
This test is completely based on the acetamide agar, which is used to detect the ability of the organism to use acetamide by performing deamidation in the culture medium. This medium also contains carbon as its sole source along with inorganic ammonium salts.
What is Acetamide Utilization Test?
Acetamide utilization test is one of the biochemical tests which helps us to identify the aerobic organisms.
This test is based on the utility of acetamide by the organism during the process of deamidation.
This process occurs only during the presence of an enzyme acalamidase. This acetamide utilization test is performed to differentiate the group of fermentative organisms from the oxidative group of species.
This test is most commonly used for differentiating the Gram-negative, non-fermentative group of bacteria on the basis of their capability to utilize acetamide.
A medium containing acetamide as the sole source of carbon is used for utilizing the acetamide by the organism present in the growth medium.
Thus, the change in color in the growth medium of the medium, detects the growth of the organism present in the medium.
Acetamide Utilization Test Objective
The main aim of the test is to differentiate the bacteria based on their ability to use the acetamide as the sole source of carbon.
To identify the species of Aeruginosa from the other non-glucose-fermenting, Gram negative rods.
Acetamide Utilization Test Principle
As mentioned above, Acetamide utilization test is usually used to determine the ability of the specific organism to utilize acetamide as a source of carbon. This process usually takes place by deamidation.
The medium consists of acetamide. As a sole source of carbon along with the inorganic ammonium salts which serves as a sole of nitrogen.
The growth of organisms in the medium of acetamide agar indicates the positive Test for utilization of utilization.
During the metabolism of acetamide by the organism, the enzymatic action of acetamidase breaks the ammonium salts into ammonia. The ammonia thus released increases the alkalinity in the medium and results in change in the pH.
The change in the pH in the medium causes bromothymol blue indicator present in the medium to turn the color from green to blue, and it indicates the positive test.
When there is no release of ammonia in the medium, the color remains same and it indicates the negative result. In some cases, assimilation of the acetamide results in a formation of yellow color and it is mistaken for a positive result.
But the digestion of the acetamide by a deamination is limited to only some of the organisms. Hence this test is only performed for differentiating Pseudomonas aeruginosa from the other non-glucose fermenting, Gram-negative rods.
Acetamide Utilization Test Reagents
Media: Acetamide agar, it can be purchased commercially or it can also be prepared in laboratory in the following composition.
Ingredients Gram/liter Acetamide 3.0 Dextrose 0.2 Yeast extract 0.5 Potassium dihydrogen phosphate 1.0 Phenol red 0.03 Bacteriological agar 15.0 Sodium agar 5.0 Lab Supplies:
• Sterile inoculating loops or sticks
• Incubator
Acetamide Utilization Test Procedure
About 24.7 grams of the dehydrated powder or a lab prepared medium is added in a beaker containing about 1000 milli liters of distilled water.
The suspension is heated until it boils; so that the solution dissolves completely.
The dissolved medium is then dispersed into the tubes and it is sterilized in an autoclave at a temperature of about 121ºC for about 15 minutes.
After completing the autoclave process the tubes are taken out and they are cooled at a slanted position until it forms a butt at a depth of about 1.5 to 2.0 cm.
Acetamide Utilization Test
Here, a well isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating loop or a needle.
Then the acetamide agar inoculated tubes are stroked in the slant in the direction of back and froth using the sterile inoculating loops or sticks.
After streaking the caps of the tubes are closed loosely and adequate aeration is ensured.
Then the tubes are incubated at a temperature of 35 to 37ºC for 7 days.
After incubating they are examined daily for 4 days and at the 7th day before discarding in order to ensure the negative result.
Acetamide Utilization Test Result
Positive Result: In case of positive result, there will be change in change and a growth is detected in the medium
Negative Result: Here, negative result is indicated by no change in color and the slant remains green and there will be no change in growth
Controlled Organism
In case of controlling the quality for the acetamide utilization test, two different organisms are considered here as negative and positive controls.
Control Incubation Results Pseudomonas aeruginosa It is incubated in an aerobic incubation of about 24 to 48 hours in a temperature of 33 to 37ºC It results in Acetamide positive with a growth and change in color to blue Escherichia coli In an aerobic incubation for about 24 to 48 hours in a temperature of about 33 to 37ºC It results in Acetamide negative, hence there will be no growth and there will also be no change in color Acetamide Utilization Test Uses
• Acetamide utilization test is usually used to detect the ability of the particular organism to utilize the acetamide as the only source of carbon.
• It is also used as qualitative test for differentiating Gram-negative bacteria into the fermentative and the oxidative group bacteria into fermentative and the oxidative group bacteria. Acetamide agar is used in the selective medium for isolating P. aeruginosa
• This medium has can also be used as a modification of Simmon Citrate agar to determine the ability of acetamide in acting a carbon source without the presence of peptone and other protein sources.
Acetamide Utilization Test Citations
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Bile Esculin Test: Principle, Procedure, and Results
Continue ReadingBile Esculin Test Introduction
Generally Biochemical tests are used to identify the species of bacteria by differentiating the species on the basis of their biochemical activities.
The factors such as protein and fat metabolisms and the enzyme production and ability of the organism to utilize the compounds in the mediums helps us in differentiating and identifying the specific species of the bacteria.
Bile Esculin test is one of the biochemical tests which is used widely used for differentiating the Enterococci from the group D viridians group of Streptococci.
What is Bile Esculin Test?
Bile esculin test is one of the biochemical tests which is performed in order to differentiate Enterococci and the group D streptococci from non-group-D viridians group Streptococci, based on their ability to hydrolyze esculin.
Many organisms have the capability to hydrolyze esculin, but only few species have the capability to hydrolase esculin in the presence of bile.
This property is used in the biochemical tests to differentiate the organisms.
The bile esculin test is usually performed in a medium of a selective agar known as bile esculin test that contains bile and esculin as their vital substances.
As agar consists of various types of bile salts, that inhibits the growth of other Growth-positive organisms and it also allows the selective isolation of the Enterococci and the Group D Streptococci.
Esculin is one of the derivatives of the glycoside-coumarin, which is also considered as the fluorescent compound as its hydrolysis, and it can be differentiated on the absence of fluorescence.
As years passed, bile esculin tests are modified accordingly and in recent times they are performed using bile esculin disks and they are performed more widely.
Bile-esculin disks are usually used for rapid differentiation of the Group D Streptococci and the non-Group D streptococci.
Bile Esculin Test Procedure
The main aim of the test is to identify the Enterococci and the Group D Streptococci on the basis of their ability to hydrolyze esculin in the presence of the bile.
To detect the difference between the members of the family Enterococci and Group D streptococci from other species of Non-Group D Streptococci.
Bile Esculin Test Principle
The basis of the esculin test is to hydrolyze the esculin that is hydrolyze at the presence of bile salts due to the enzymatic activity of enzyme esculinase.
Esculin is a glucoside containing glucose and hydroxycoumarin which are linked together by an ester bond with the help of an oxygen.
This test helps us to selects the organisms initially on the basis of their ability to grow in the medium containing about 4% of bile salts and they are detected on the basis of their ability to hydrolyze esculin.
Thus, the hydrolysis of esculin results in glucose and another compound known as esculetin.
On degrading esculin, Esculetin is produced which on hydrolysis reacts with the iron ions present in the medium and forms a compound known as phenolic iron complex, and results in the formation of dark brown or black color.
On the other hand, Esculin is a fluorescent compound, which on hydrolysis can be observed as a loss of fluorescence.
Once a bile is added into a medium, the micro-organisms get the capability to grow and it starts hydrolyzing the esculin.
The bile in turn inhibits the growth or production of the other Gram-positive organisms which in turn makes the medium more selective.
Availability of 4% of bile in the esculin medium inhibits most of the strains of the Streptococci excluding the Species S. bovis. Whereas it does not inhibit Enterococci or Listeria.
Bile Esculin Test Reagent
Media:
Bile esculin agar that slants with iron citrate is used.
Agar plate media is usually composed of Enterococcosel agar, or any other similar formulation.
Bile-esculin aide agar or a broth with iron citrate and azide is used. Azide inhibits most of the Gram-negative bacteria.
Peptone-yeast esculin broth, but it is used mostly in the anaerobic atmosphere.
Esculin agar is used without bile or aside but it usually contains iron citrate.
Lab Supplies:
Long wave-UV light
1% of ferric ammonium citrate.
Bile Esculin Test Procedure
About 64.5 grams of the dehydrated powder is added in a beaker containing 1000 milli liters of the distilled or deionized water.
The prepared solution is heated till it boils to dissolve the solution completely.
The dissolved medium is then dispensed into the tubes.
Further the tubes are sterilized in an autoclave at a temperature of about 121ºC for about 15 minutes.
After the completion of the autoclave processing the tubes are taken out and they are cooled in a slanted position at a temperature of about 40 to 45ºC.
The same position is maintained until the butts are formed at a depth of about 1.5 to 2.0 cm.
Esculate Hydrolysis
Esculate hydrolysis is detected either by using tube test or by disk test. Disk test is considered as one of the rapid tests.
I. Bile Esculin Tube Test
An isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating needle.
The bile inoculating tubes are then inoculated by streaking the surface of the slant by using light inoculum from the agar plate and the tubes are inoculated with 10µl loopful of 0.5 McFarland standard suspension using a sterilized water.
Then the cap of the test tubes is left loosened in order to ensure aeration.
Then the tubes are left to incubate aerobically at a temperature of about 35 to 37ºC for about 24 hours and for a maximum of 7 days until the color changes are observed.
For esculin broth without iron citrate, the tubes are observed daily for the decent of fluorescence.
In the absence of the fluorescence 2 to 3 drops of 1.0% ferric ammonium citrate is added to the esculin tube and the color change is observed.
I. Bile Esculin Disk Test
In disk test, esculin test is moistened with the one drop of distilled water.
And it is ensures, that it is not saturated. By using a sterile loop, two to three isolated colonies are picked from 18 to 24-hour culture.
Then the disk is observed for the development of a dark brown or black color for about 10 minutes in a room temperature.
Bile Esculin Test Result: Tube Test
A positive result in a tube test is determined in the medium containing ferric ammonium citrate is usually detected by blackening of the medium.
A negative result in the tube test is detected by absence of change in colors. On the other hand, the medium will fluorescence under the UV light.
For esculin broth without iron citrate, a positive test is usually observed by blackening of the medium on addition of ferric reagent or by the loss of fluorescence in the medium.
Whereas here, the negative tests occurs when the bile esculin medium losses its ability to let grow the organisms on the medium containing bile.
Bile Esculin Test Result: Disk Test
In disk test, the positive result is determined by development of dark color or black color
Where as the negative disk test is determined by absence of color.
Bile Esculin Test Citations
- Esculin hydrolysis by Enterobacteriaceae. J Clin Microbiol . 1977 Aug;6(2):111-6.
- The use of bile – esculin agar for the taxonomic classification of the family Enterobacteriaceae. Antonie Van Leeuwenhoek . 1977;43(1):31-5.
- Presumptive identification of group D streptococci: the bile-esculin test. Appl Microbiol . 1970 Aug;20(2):245-50.
- Bile-esculin test for presumptive identification of enterococci and streptococci: effects of bile concentration, inoculation technique, and incubation time. J Clin Microbiol . 1998 Apr;36(4):1135-6.
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Bacitracin Susceptibility Test: Principle, Procedure, and Results
Continue ReadingBacitracin Susceptibility 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 Bacitracin susceptibility test. Bacitracin susceptibility test is one of the antimicrobial tests which are used for identifying a group A streptococcus from the other Beta-hemolytic species of Streptococci.
What is Bacitracin Susceptibility Test?
Bacitracin susceptibility test is considered as an antimicrobial disk tests used foe identifying the Group A Streptococci from the other species of Beta-hemolytic Streptococci.
Antimicrobial susceptibility tests are usually performed in order to differentiate a particular species from another specific genus.
Here, antimicrobial tests are used in order to determine the susceptibility of a bacterial species to a specific antibiotic.
Bacitracin
Bacitracin is one of the bactericidal drugs which is used to treat the superficial skin infections. But these are rarely injected for systemic uses.
Bacitracin is obtained from the Bacillus subtilis which is one of the polypeptide antibiotics.
Bacitracin often interferes with the peptidoglycan and help in producing the bacteria and also in inhibiting their growth.
Bacitracin blocks the bactoprenol from transporting the sugars such as NAM and NAG sugars across the cell membranes, which further inhibits the production of peptidoglycan.
The growth of Group A Beta-hemolytic Streptococcus is inhibited by the Bacitracin. Where as the other species of beta-hemolytic streptococci are not inhibiting.
This helps us to differentiate the two different group of organisms. However, Bacitracin disks are mainly used against the Streptococcus pyrogens which helps in inhibiting the growth of an organism.
Bacitracin results in forming the zone of inhibition of about 12mm against the S. pyrogens, hence it helps us to determine the positive control of the organism while testing.
Bacitracin Susceptibility Test Objective
The main aim of the Bacitracin susceptibility test is to differentiate the Group A Beta-hemolytic Streptococci form the other species of Beta-hemolytic Streptococci.
To detect the pattern of antibiotic susceptibility in various organisms against Bacitracin.
Bacitracin Susceptibility Test Principle
Generally, the growth of Group A Beta-hemolytic streptococci on the blood agar is inhibited by using 0.04 units of Bacitracin disks.
Whereas the other similar species like Micrococci and Streptococci are also inhibited but this 0.04-unit disc, whereas the other Coagulate-negative Staphylococci are resistant to this.
Bacitracin susceptibility test discs are simply a filter paper disc, which are impregnated with 0.04 units of Bacitracin.
These impregnated discs are then placed on an agar, which allows the antimicrobial to diffuse along with the medium thus inhibiting the growth of the organisms.
This test result is evaluated after incubating based on the zone of inhibition that are formed around the discs.
If the growth is seen at the edges of the disk, the it is deemed as resistant, in other case if the zone is present in a circular zone around the stick, then it represents the inhibition and susceptibility of the organisms.
Usually, Bacitracin disks are time saving experiments along with minimum labor and materials, if they are used in the form of screening test before serological grouping.
It is also said that Group A Streptococci are more sensitive to Bacitracin than the Beta-Hemolytic strains of the other groups.
Hence it is advisable to perform Bacitracin susceptibility test through antimicrobial disks for a rapid diagnosis, especially for Group A Streptococci.
Micro Organisms Tested
This test is generally used for Penicillin-susceptible test or for stick colonies of the Gram-positive cocci, which are found in groups and catalyzes the positive and negative coagulates.
Here lemon-Yellow colored colonies are not detected as they are assumed as Micrococcus.
Bacitracin Susceptibility Test Reagents
Media Used:
Blood agar or
Muller Hinton Agar
Supplies Used:
Bacitracin 0.04-unit discs
Sterile forceps
Swab
Inoculation broth.
Bacitracin Susceptibility Test Procedure
Usually, two different kinds of methods are used for Bacitracin Susceptibility test based on the kind of culture media they are used.
The test can be performed either by using pure culture of an organism or directly via clinical samples. The methods of Bacitracin susceptibility tests are listed below.
1. Hebert’s Method Using Blood Agar Plates
Initially 0.1 McFarland suspension of the organism is performed using a over night culture of the organism.
Different sections of blood agar plate are inoculated which results in forming a lawn culture.
Here each section is inoculated in one specific direction and the area of inoculation should be at a separation of 10mm between each of the discs that were placed.
These are then left to dry for about 10 minutes and after drying they are placed on the agar using sterile forceps.
Then the disc is tapped using the sterile stick and adherence is ensured.
The plates are then incubated at a temperature of about 35 to 37ºC for about 24 hours.
After a period of incubation, the zone of incubation is observed thus results are measured.
Further the results are confirmed using serological testing.
2. Muller-Hinton Agar Method
This method is also used to observe the susceptibility of the fast-growing organisms. Here 0.5 Mc Far land suspension of the organisms is prepared using an over night culture of the organism.
The MHA plates disks are inoculated using a suspension with sterile swabs to form a bacterial lawn on the agar.
Then the medium is allowed to dry. After drying the antibiotic disks are placed on the agar placed using the sterile forceps and the discs are placed by maintaining a distance of about 10mm.
Then further the discs are tapped using sterile sticks.
Then the inverted plates are incubated at a temperature of about 35 to 37ºC for about 24 hours.
After incubation the zone of inhibition is observed and measured.
Bacitracin Susceptibility Test Results
Zone of Inhibition Media Result Zone of inhibition is 6mm or less than that Blood agar or Muller Hinton Agar Resistant Zone of inhibition is greater than 10mm Blood agar or Muller Hinton Agar Susceptible Zone of inhibition is between 6mm to 10mm Blood agar or Muller Hinton Agar It indicates probable susceptibility so the tests should be repeated Bacitracin Susceptibility Test Citations
- The bacitracin sensitivity test for identifying beta-haemolytic streptococci of Lancefield group A. J Postgrad Med . 1981 Apr;27(2):86-9.
- Variations in bacitracin susceptibility observed in Staphylococcus and Micrococcus species. J Clin Microbiol . 1986 May;23(5):963-4.
- The bacA gene, which determines bacitracin susceptibility in Streptococcus pneumoniae and Staphylococcus aureus, is also required for virulence. Microbiology (Reading) . 2000 Jul;146 ( Pt 7):1547-1553.
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Acetate Utilization Test: Principle, Procedure, and Results
Continue ReadingAcetate Utilization Test Introduction
In the modern world, apart from life style changes, we come across many medical terms as a part of our daily activities, such as Lab test, diagnosis, vaccine, chemicals and so on, which became most common terms as a part of other things we hear.
Many tests are performed in a laboratory and in biochemical labs to detect the nature of the pathogen or a disease. Acetate utilization test is one such test which is used to determine whether the organism has the ability to use acetate as a sole source of carbon.
What is Acetate Utilization Test?
The acetate utilization test is generally used to test the ability of the organism to utilize the acetate as the single source of carbon.
This test is also performed as a qualitative test for the differentiation of the Gram-negative bacteria into fermentative and the oxidative group of bacteria.
This test is also being used to differentiate the species of Shigella from E. coli and the non-fermentative negative bacteria.
Acetate agar which contains sodium acetate as the sole source of nitrogen is used for inoculating the organism. Growth is indicated for the positive test of acetate utilization test.
When the bacteria metabolize acetate, the ammonium salts are broken down into ammonia, which increases the pH medium of the culture, this increase in pH turns the bromothymol blue indicator in the medium changes from green to blue.
Acetate Utilization Test Principle
In acetate utilization test, Acetate agar is employed as a test organism. This acetate agar has the ability to utilize acetate.
The culture medium is composed of Sodium acetate which acts as a sole carbon source and the inorganic ammonium salts as the source of the nitrogen. Where the growth of organisms suggests the positive results for the utilization of acetate.
During the metabolism process of acetate, by the bacteria, the ammonium salts are broken into ammonium, that elevates alkalinity.
The shift in the pH changes bromothymol blue indicator in the medium from green to blue. This medium is generally used for differentiating the Shigella spp from Escherichia coli.
As Shigella spp doesn’t have the capability to metabolize the acetate, However, approximately of about 94% of Escherichia coli plays a major role in utilizing the acetate.
Acetate Utilization Test Reagents
Media: The culture media is composed of Sodium acetate agar.
Ingredients Gram/liter Sodium Chloride 5.0 Magnesium sulfate 0.1 Ammonium phosphate-monobasic 1.0 Potassium phosphate-dibasic 1.0 Sodium acetate 2.0 Agar 20.0 Bromothymol blue 0.08 Sterilized sticks and inoculating loops
Sterile pipette
Incubator
Sterile saline.
Acetate Utilization Test Procedure
Procedure for Acetate utilization test involves two steps;
1. Preparation of media
2. Utilization test
1. Preparation of Media
For the preparation of media, 69.1 grams of the dehydrated powder is added in beaker along with 1000 milliliters of the deionized or the distilled water.
Instead of dehydrated powder lab-prepared media can also be used.
The prepared suspension is then heated till boiling; so that the medium is dissolved completely.
Then the dissolved medium is dispensed into tubes and they are sterilized in an autoclave at 121ºC for about 15 minutes.
After completing the process of autoclaving the tubes are taken out and cooled at a slanted position to a temperature of about 40 to 45º.
The position is then maintained in order to obtain butts of depth 1.5 to 2.0 cm.
2. Utilization Test
The isolated colony is taken from an 18 to 24-hour culture with the help of a sterile inoculating needle.
A turbid suspension of saline is prepared by using 18-to-24-hour culture from a noninhibitor plate of culture.
The acetamide agar tube is inoculating by streaking the surface of a slant with the light inoculum which is picked from the culture.
The slant is then streaked back and froth with the loop or using an inoculating stick.
The cap or the test tubes are loosened to ensure whether the inoculum is getting sufficient aeration.
The tubes are then incubated aerobically at the temperature of about 35 to 37ºC for about seven days.
As incubation at 35 to 37ºC is not sufficient for thee Enterobacteriaceae, incubation at 30ºC is followed for seven days for the non-fermentation of Gram-negative rods.
The test tubes are examined regularly for at least 7 days before discarding the samples.
Acetate Utilization Test Result
For a positive result, the growth is represented as a change of color green to intense blue along the slant. Where as for negative result, the growth is absent and there will be no color change and the slant remain green as same.
Bacterial Control
Mostly two different organisms are taken for the positive and negative controls as the form of quality control for the acetate utilization test.
Control Incubation Results Shigella flexneri Aerobic incubation is followed for 24 to 48 hours at temperature of about 33 to 37ºC Acetate negative, where there is no growth and no change in color and the medium remains green Escherichia coli Aerobic incubation is followed for 24 to 48 hours at temperature of about 33 to 37ºC Acetate positive where the growth is shown and change of color to intense blue from green Acetate Utilization Test Uses
• This test is usually used to test the ability of the organism to utilize acetate as the source of carbon.
• This test is also used in the form of qualitative test for differentiation of Gram-negative bacteria for fermentation and oxidative group of bacteria.
• Acetate agar can also be used as a selective media for the isolation of Escherichia coli.
Acetate Utilization Test Citations
- Acetate tolerance and the kinetics of acetate utilization in diabetic and nondiabetic subjects. Am J Clin Nutr . 1990 Jan;51(1):112-8.
- Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies. PLoS One. 2014; 9(10): e109255.
- Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase. J Bacteriol . 1995 Oct;177(20):5826-33.
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Mitosis: Definition, Stages, Types, Diagram, and Facts
Continue ReadingMitosis: Introduction
Cell cycle is the sequential process taking place to regulate the growth of organism; cell divides to produce a genetic replica and enters the stage of cell growth.
Cell growth involves the synthesis of organic material and integrates information across its counter parts for synchronous development of the whole body.
The cell synthesis phase lasts till a cell reaches its maturity; on initiation the cell again divides to produce new cell and the process continues.
Cell cycle is a sequential development of cell between two cell divisions. The cycle is genetically controlled and are programmed in every cell and are specific for each region.
Varied species has variable time length of cell cycle decided by physiological and influences pertaining to their niche.
Two Phases of cell cycle are: Interphase and Mitotic Phase.
Interphase involves G1, synthesis and G2 phase; chromosomal replication and development is regulated by the phase; determines the quality and quantity of chromosome entering the daughter cells and a balance is maintained by the phase.
Karyokinesis and Cytokinesis division, segregation of chromosome and cell takes place during Mitotic Phase.
The notable feature of cell cycle is eukaryotic organisms though diverse and distinct have a common type of cell division over the Kingdom of Eukaryotes is a scientific wonder and research have emphasized that timing of a cell entering the cell cycle is essential in cell cycle regulation.
Phases of Cell Cycle
The cell cycle is common for all eukaryotic organisms; travelling through 2 major phases based on the cell division: Interphase and Mitotic Phase.
Interphase consists of 3 phases Gap 1 phase, Synthesis Phase and Gap 2 Phase.
Similarly, mitosis has four phases Prophase, metaphase, anaphase, and telophase.
The development of cells through these phases are influenced and facilitated by heterodimeric protein kinases – Cyclin and Cyclin Dependent Kinases.
Mitosis Phase
The changes in above phases are minimal or not clearly visible in microscopes whereas the changes in M Phase are easily detectable.
The phase has 4 parts in which the division takes place systematically and continuously.
The cell stages are easily visible in plant parts as the specialized dividing region – Meristem is prevalent in roots and shoots are continuously dividing providing a mechanical support and functional integrity to the plants.
The 4 phases are: Prophase, Metaphase, Anaphase and Telophase. Each phase has a distinctive change to be identified and Eukaryotic cells replicates in the same order in most of the organisms.
I. Prophase
The prophase is marked by chromosomal condensation and disintegration of cellular components and assembly of cytoskeletons for cell division.
RNA synthesis is inhibited.
The nuclear envelope disintegrates.
The chromatin condensation during G2 phase completes forming Chromatids.
Chromatids are held together by centromere.
The formation of cytoskeleton is initiated along with microtubules.
Kinetochores are formed.
II. Metaphase
Nuclear membrane is eliminated completely chromosomes are completely condensed.
The cytoskeleton – spindle fibers develop and attach to the kinetochores.
This phase also signifies with equatorial alignment of chromosome.
The phase has a checkpoint where it makes sure all the chromatids are well connected to the kinetochores.
Feeble connection leads to unequal distribution of chromosomes in the daughter cells forming defective cell when not eliminated becomes lethal to the organism.
III. Anaphase
Chromosomal split forms daughter chromatids; travels to the opposite poles.
The chromosomes are V – Shaped as they are dragged to the opposite sites by the shortening of the spindle fibers.
So that daughter cells have an equal number of chromosomes to function regular cellular activities.
IV. Telophase
Microtubules disappear and chromosomes decondense to chromatin mass.
Nuclear envelope starts to form.
The disintegrated organelles form again with 2 nuclei and nucleoli.
This reappearance of the cell organelles and equal distribution of the cellular materials ensures each cell to function independently in the cell cycle.
These phases mark the Karyokinesis were the nucleus and other cell parts are newly formed.
V. Cytokinesis
Cytokinesis is the formation of daughter cells after mitosis; indicated by furrow which starts to differentiate two daughter cells grows gradually forming a cell plate while the organelles formed gets segregated.
Cell plate represents the lamella between 2 cell walls.
Mitosis Regulation
The cell cycle is completely regulated by Cyclins and Cyclin dependent protein kinases; belongs to serine – threonine sub classes; Phosphorylates and dephosphorylates the activating components to proceed through the cell cycle depending on the external cues.
The commitment to enter cell division is irreversible when a cell traverse from G1 phase to synthesis Phase is controlled by CYC – CDK complexes as all other phase to enter Mitotic phase.
Mitotic Phase requirements are dealt by the previous phases were DNA replication and chromosomal condensation along with mitotic transcriptional factors and other proteins are synthesized to ensure the cell division which is also supported by the phytohormones.
The main CYC – CDK complex in mitotic cycle is the CYCD-CDKA whose expression is essential for daughter cells to develop well depending on the nutrient provided.
The transition from G2 to M phase is mediated by CYCB – CDKB from G2 phase ensures a proper cell division; expressed from S phase by minimal accumulation gradually increases in quantity to transit from G2 phase to Mitotic phase.
Mitotic CYC – CDK complexes are synthesized during S phase but remains phosphorylated which induces inactivation of the compound.
Following DNA replication phosphatase cdc 25 is activated; dephosphorylation of threonine and tyrosine removes the inhibition from Wee 1 kinase promotes expression of Mitotic CDK – CYC inducing mitosis and forms 2 daughter cells.
Endoreduplication
In endoreduplication the cells undergo continuous division without the formation of cell wall (i.e.) CYTOKINESIS increasing the chromosomal number of the plant.
This is referred as ploidy and in plants; increases the yield and stability of the plant; is under debate.
Endoreduplication are predominant in endosperms. The protein expression producing the mitotic phase is inhibited by regulatory mechanism for the expression of endoreduplication.
The main feature in endoreduplication is that it initiates after a mitosis takes place.
Several factors are involved in the ubiquitination of the cyclin to ensure the cellular restriction to a particular function.
Mitosis Citations
- Chromatid cohesion during mitosis: lessons from meiosis. J Cell Sci . 1999 Aug;112 ( Pt 16):2607-13.
- Mitosis: towards a molecular understanding of chromosome behavior. Curr Opin Cell Biol . 1991 Feb;3(1):59-66.
- Mitosis in filamentous fungi: how we got where we are. Fungal Genet Biol . 1999 Jun;27(1):1-25.
- Abnormal mitosis in reactive astrocytes. Acta Neuropathol Commun . 2020 Apr 15;8(1):47.
- Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints. Chromosome Res . 2011 Apr;19(3):291-306.
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