Carbohydrate detection using Qualitative tests.
Carbohydrates are class of heterogeneous compounds.They were wrongly believed to be hydrates of carbon.Carbohydrates are polyhydroxy ketones or derivative there of or polyhydroxy aldehyde. Classification of carbohydrate is based on their complexity a)Monosaccharides or simple sugars. b)Polysaccharides (starch,dextrin,cellulose) or Heteropolysaccharides (mucopolysaccharides).
Many of the reactions which carbohydrates undergo are common to all the monosaccharides . Differences in structure of sugar frequently affects the rate of the reaction,sometimes the ability to react at all. Carbohydrates possesses hydroxyl group and carbonyl group-hence reactions of the carbohydrates are similar to those of alcohols,aldehydes and ketones.
NAME OF THE METHOD: Mollisch Test, Benedict’s Test
REAGENTS USED : Mollisch reagent(5% solution in naphthol in alcohol) and Benedict’s reagent.
MISCELLANEOUS : 1% solution of the sugars to be analused, clean test tubes,pipettes, Boiling water bath for Benedict’s Test, sulphuric acid.
1) In 5ml of 1% sugar solution in a test tube add 2 drops of Mollisch reagent.
2) Mix it thoroughly.
3) Incubate the test and allow 3ml of conc. Sulphuric acid to flow from the sides of the tubes,thus forming a layer of acid beneath the sugar.
OBSERVATION: A reddish violet zone appears at the junction between two liquids.Hence Purple Or reddish Violet colour indicates the presence of Carbohydrates.
The reaction is due to formation of furfural derivative by the action of acid on sugars,thus the test is not highly specific test,for Carbohydrates as it is positive for furfural and furfural yielding derivatives. Concentration of Carbohydrates may give a red instead of violet colour.
1) To 5ml of Benedict’s reagent add 8 drops of the sugar solution.
2) Mix vigorously for 2 minutes or place the test tube in boiling water bath for 3 minutes.
3) allow the fluid to cool.
OBSERVATION:Depending upon the concentration of the reducing sugar different coloured precipitates are obtained.
a) Brick red b) Yellow c)Green d)Blue
The huge bulk of the precipitate formed makes the test very precipitable.The test is highly specific.
Capsule Staining is performed to observe bacterial capsule using MANEVAL’S METHOD.
Capsule is an envelope or slime layer surrounding the cell wall of certain bacteria which gives protection to the cell wall.The capsule;s composition,as well as its thickness,varies with individual bacterial species. Polysaccharides,polypeptides, and glycoproteins have all been found in capsules.Often,a pathogenic bacterium with a thick capsule will be more virulentthan a strain little or no capsule since the capsule protects the bacterium against the pathogenic activity of the host’s pathogenic cells.
Congo red (Maneval’s A) which is an acidic dye is used as negative stain, staining the background leaving the organism unstained. Maneval;s B consists of phenol,acetic acid,FeCl3 and acid fuchsin.The cells are stained by the basic dye-acid fuchsin in the presence of mordants like acetic acid and FeCl3 with a chemical intensifier phenol.The capsular boundary and the background can be stained with acidic dye like congo red which under acidic condition (glacial acetic acid in Maneval’s B) turns blue in colour, making the background blue.The capsule remains unstained.
The Protocol followed is as follows:
1) Place a drop of Congo reg on a clean grease free slide.
2) Mix it with a drop of suspension of the culture.
3) Spread it gently in the form of a thin film.
4)Allow it to dry slightly, and then still moist in the centre of the smear, add Maneval’s solution B and allow it to react for a minute.
5) Discard the excess stain and dry in the air.
6) Observe under Oil immersion lens.
Using Capsule staining,the colorless capsule is detected under oil immersion lens on Blue background with Pink coloured bacilli.
Through the staining procedure the presence of capsule was confirmed.Capsule acts as virulent factor possessed by the cell that protects bacteria from from phagocytosis , rendering the strain more pathogenic.
Cell wall staining is done to demonstrate the cell wall of the bacteria by CHANCE’S METHOD.
The cell wall of bacteria is a rigid structure which protects the cell wall against severe physical conditions and gives a definite shape to the cell.The gram positive bacterial cell wall consists of 20-80 nm thick peptidoglycan or murein layer with considerable amounts of teichoic acids and teichuronic acids as well as polysaccharides.The gram negative bacterial cell wall has a 1-5 nm peptidoglycan layer surrounded by a 7-8 nm thick outer membrane containing lipoproteins and lipopolysaccharides.
The cell wall has low affinity for stains and is not stained in most of the usual staining procedures.Basic dye like New fuchsin have positive charge on the dye portion of molecule and react with negatively charged bacterial cell surface.Both the cell wall & the cytoplasm are stained by the New fuchsin. Congo red acts as decolorizer and decolorizes the cytoplasm.Congo red being an acidic dye reacts with the basic dye NEW fuchsin on the surface of the cell, thus the cell wall is stained red and the cytoplasm light pink to colorless.
1) The smear is to be prepared. (DO NOT HEAT FIX)
2) It is then treated with 0.5% New fuchsin for 1 minute.
3) The stain is discarded and the slide is washed thoroughly.
4) It is then treated with 0.5% Congo red for 5 minutes.
5) The slide is then washed, dried and observe under Oil immersion lens.
To demonstrate the cell wall of bacteria
To demonstrate the cell wall of bacteria
The cell wall of Bacillus subtilis was stained red in color which appeared rod shaped with colorless cytoplasm.The presence of cell wall was confirmed using Chance’s method by using Bacillus subtilis.
The negative stain is particularly useful for determining cell size and arrangement.Overall bacterial morphology can be determined with the use of harsh staining or heat fixing techniques that change the shape of cells.This may be required when the bacterium does not stain well (eg.,some of spirochetes) or to confirm observations made on shape and size of bacteria observed in either a wet-mount or hanging drop slide.Negative staining is also good for viewing capsules.
Anionic dyes (acidic)have a negatively charged chromophore. At neutral pH there is repulsion between the negative charge on the bacterial cell wall and thus stain cannot penetrate the cells.This is called Negative or Relief staining.This indirect,background staining is achieved by mixing bacteria with an acidic stain such as nigrosin,India ink,or eosin,and then spreading out the mixture on a slide to form a film.Hence only the background gets stained.After staining,cells would be seen as clear and bright bodies against a dark background.
The Protocol followed for Negative Staining is as follows:
1) With a glass marker mark the name of the culture in the far left corner of the slide and mark a square/circular area in the corner on the under surface of the slide.
2) Invert the slide and place it on the blotting paper on the bench.
3) Sterilize with wire loop,cool it and take a loopful of the bacterial culture on the slide.
4) Mix a small drop of Nigrosin with the culture on the slide and spread into an even smear.Alternatively it may be drawn out in the form of a film using another slide
5) Allow the smear to just dry enough so that no cracks appear.
6) Do not heat fix the smear.
7) Place a drop of oil on the smear and observe under oil immersion lens.Ensure that the field is completely flooded with light and the iris diaphragm is open for observing the bacteria.
Hence,colorless oval and rod shaped cells are observed in cluster against a dark background.
WHAT IS STAINING?
Staining is a technique used in microbiology (microscopy) to enhance contrast in microscopic images.In medicine and biology stains are frequently used to highlight structure of biological tissues.It is basically used to study the morphology of cell or other eg. lamellar structure of semi crystalline polymer.
GRAM STAINING METHOD:
Gram staining differentiates bacterial species into two groups-Gram Positive & Gram negative.
Hans Christian Gram invented the method.
Cell wall of Gram-positive bacteria is made up of peptidoglycan that is about 50-90% of the cell envelope and stained purple by primary stain crystal violet.Gram-negative bacteria have a thinner layer about 10% of cell envelope and hence do not retain the primary stain,thereby getting stained pink/red by the counter stain.
Crystal violet dissociates in aques solution into CV+ and chlorine ions that penetrate through the cell wall and cell membrane of both gram positive and negative cells.The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple with primary stain.The iodine ion from the mordant interacts with the CV+ and forms the CVI complex within the membrane
When a decolorizer such as alcohol or acetone is added it interacts with the lipids of the cell membrane.Due to multilayered nature of the peptidoglycan of gram positive cells the CVI complex gets trapped and cannot be removed by the addition of the decolorizer. Hence Gram-Positive bacteria retain the primary stain and get stained purple. Alcohol dissolves the outer Lipid membrane of Gram-Negative bacteria, thus leaving the peptidoglycan layer exposed and increases the Porosity of the cell wall. Hence the CVI complex is removed on decolorization rendering the Gram-Negative bacteria colorless. A counter stain, which is usually positively charged (safranin or basic fuchsin), stains the Gram Negative cells pink or red differentiating them from the purple Gram-Positive cells.
Thus, Characterizing of bacteria is done by this method.