B.Sc. First Year (Semester II) Paper III
Basic Microbiology & Biomolecules
Unit I : Microbial Staining Techniques
Stain and Dye
* The term 'dye' is used to refer to a colouring agent that is used for general purposes, whereas the term 'stain' is used to refer to that dye which is used for biological purposes.
* A stain is any colouring organic compound that combined with another substance imparts a colour to that substance.
* Dye are used to colour non-biological materials whereas stains used for coloration of biological materials.
Chemical makeup of stain
* Benzene = organic compound
* Chromophore (Gk. Chroma = colour; phoros = to bear) = colour
* Auxochrome (Gk. auxein = to increase; chroma = colour) = ionization properties.
* Benzene + chromophore = chromogen
- Chromogen is a coloured compound only.
* Auxochrome with chromogen allows the dye to form salt compounds that adhere to cells.
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* These are anionic.
* Their chromogen has negative charge on ionization.
* They have affinity for the positive components of a cell.
* Acidic stains do not stain bacterial cells. They stain background with contrasting color.
* Examples : Eosin, rose bengal and acid fuschsin, India ink, Nigrosin, Picric acid.
Basic stain
* These are cationic.
* Their chromogen has a positive charge on ionization.
* They have affinity for the negatively charged molecules such as nucleic acids, many proteins, and the surfaces of bacterial and archaeal cells.
* Basic stains can stain bacterial cells as they possess negative charge.
* Examples : Methylene blue, basic fuchsin, crystal violet, safranin, malachite green.
Mordant
* Chemical which are required to bring about the staining reaction are called mordants.
* They helps to fix the color to the cell components.
* Basic mordant reacts with acidic stains and acidic mordant reacts with basic stains.
* Examples : Phenol in carbol - fuchsin (Ziehl - Neelsen technique) and iodine in the Gram stainin.
Leuco compound
* Chromophores are easily reduced by combining with hydrogen at the double bonds.
* Reduction of chromophore result is loss of color.
* These decolorized dyes are known as Leuco compound.
* They are used as an indicator of oxidation & reduction reactions.
* Examples - leucofuchsin used in periodic acid - schiff (PAS) stain.
Natural Stain
* These are very few in numbers.
* Examples :
1) Hematoxylin (Natural Black 1) , a naturally occurring flavonoid compound derived from the logwood tree, Haematoxylon campechianum.
2) Carmine (Carmine red) a natural red dye extracted from the dried females of the insect Dactylopius coccus var. Costa (cochineal).
Fluorochrome
* A fluorophore (or fluorochrome) is a flurorescent chemical compound that car re-emit light upon light excitation.
* Fluorochrome typically contain several combined aromatic groups or planar or cyclic molecules with several π bonds.
* They are notably used to stain tissues, cells or materials in a variety of analytical methods, i.e., fluorescent imaging and sperctroscopy.
* Examples : Fluorescein, Acridine orange, Acridine yellow.
Decolorizing agent
* They are used to remove dye from a stained microscopic specimen.
* Decolorizing agents can be as simple as water or ethanol.
* Example: Ethanol or an ethanol and acetone solution in Gram's staining; water in endospore staining
Theories of staining
* Physical theories :
1) Simple solubility e.g. Fat stains are effective because the stain is more soluble in fat than in 70% alcohol.
2) Absorption : This is a property by which a large body attracts to itself minute particles from a surrounding medium.
* Chemical theories :
It is generally true that acid stain basic element (cytoplasm) and basic dye stain acidophilic material (nucleus) however, this far from being complete truth, Indeed hematoxylin, which is an acid dye, does not stain the cytoplasm, but (in the presence of mordant) is one of the most widely used nuclear stains.
Development of staining methods
* A Weigert, a German scientist contemporary with Koch, had observed the use by Cohn and other, of various dyes to make clear the details of cell structures in histological preparations.
* The natural dyes carmine and hematoxylin were widely being used.
* Ehrlich improved methods, discovered by William Perkin a brilliant British chemist, of preparing very fine dyes from coal-tar distillates.
* In 1875, Weigert found that the dye methyl violet could be used to reveal bacteria in histological preparations.
* This method of making bacteria visible was adopted by Koch and soon came into wide use.
Staining
"Staining is an artificial coloration of a substance to facilitate examination if tissues, microorganisms, or other cells under under of microscope".
Objectives of staining
* To make microscope and semitransparent objects visible.
* To reveal their Morphology.
* To study various internal and external structures of organisms.
* To demonstrate purity of culture.
* To diagnose disease.
Simple Staining
* Simple staining implies the use of only a single stain, which is usually sufficient to reveal the morphological features of most microbial cells, including relative size, shape and characteristic arrangements for groups of cells.
* This method uses only single stain that does not differentiate between different types of organisms.
* Simple stains are used to stain whole cells or specific cellular component.
* Direct (Positive) staining : stain OBJECT
* Indirect (Negative) staining : stain BACKGROUND
a. Monochrome staining
Principle -
In Monochrome staining, the bacterial smear is stained with a single reagent, which produces a distinctive contrast between the organisms and its background.
Mechanism -
Basic stains with a positively charged chromogen are preferred because bacterial nucleic acid and certain cell wall components carry a negative charge hat strongly attracts and blinds to the cationic chromogen.
To purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells.
Stains -
The most commonly used basic stains are methylene blue, crystal violent, safranin and carbol fuchsin etc.
Procedure -
i) Preparation of a smear and heat fixing
* Using as sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to clean grease free microscope slide or transfer an isolated colony from a culture plate to a slide with a sterile water drop.
* Disperse the bacteria on the loop in the drop of water on the slide and make a thin and even smear on slide.
* Allow the smear to dry thoroughly .
* Hear-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells.
ii) Staining
* Cover the smear with methylene blue and allow the dye to remain in the smear for approximately one minute (staining time is not critical here: somewhere between 30 seconds to 2 minutes should give you an acceptable stain, the longer you leave the dye in it, the darker will be the stain).
* Using distilled water wash bottle, gentle wash off the excess methylene blue from the slide by directing a gentle stream of water over the surface of the slide.
* Wash off any stain that got on the bottom of slide as well.
* Wipe the back of the slide and blot the stained surface with a clean filter paper.
* Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective.
* Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply immersion oil directly to the smear and focus the smear under oil with the 100X objective.
i. Preparation of a smear and heat fixing
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ii) staining
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Observation -
* The bacterial cells usually stain uniformly and the colour of the cell depends on the types of dye used.
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b. Negative staining
* The main purpose of negative staining is to study the Morphological shape, size and arrangement of the bacteria cells that is difficult to stain e.g. Spirilla.
* It can also be used to stain cells that are too delicate to be heat-fixed.
* As heat fixation is not required in negative staining, the cells are not subjected to the distorting effect of chemicals and heat, due to which their natural size and shape can be seen.
* It is also used to prepare biological samples for electron microscopy.
* It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron- scattering power.
Principle -
Negative staining is based upon the principle that there is no reaction between the stain and the specimen. This is accomplished when the stain is used at a pH at which the interaction between stain and biological materials is negligible.
Mechanism -
* The acidic stain, with its negatively charged chromogen is used in negative staining.
* Since the surface of most bacterial cells is negatively charged, the cell surface repels the acidic stain.
* The glass of slide i.e. background will stain, but the bacterial cells will not.
* The bacteria will show up as clear spot against a dark background.
Stains -
Acidic stains such as 10% Nigrosin, 2% Congo red, India ink etc.
Procedure -
* Place a very small drop (more than a loop full, less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.
* Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.
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* Use another slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.
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* Push the slide away from the drop of suspended organisms to form a thin smear Air dry.
* Note: Do not heat fix the slide.
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* Focus a thin area under oil immersion and observe the unstained cells surrounded by the grey stain.
Observation -
Dark background with a light specimen.
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Differential Staining
* Differential staining is a staining process which uses more than one chemical stain.
* Using multiple stains can better differentiate between different microorganisms or structures/ cellular components of a single organisms.
* Two stains are used. One is called primary stain while other one is counterstain.
* Examples : Gram's Staining , Acid - fast staining
Gram's Staining
* The Gram staining is fundamental to the phenotypic characterization of bacteria.
* It is a method of staining used to distinguish and classify bacterial species into two large groups : gram - positive bacteria and gram - negative bacteria.
* The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.
* This gram stain technique continues to be a standard procedure in medical microbiology.
Principle :
"The Gram staining procedure differentiates organisms of the domain Bacteria according or cell wall structure. Gram- positive cells have a thick peptidoglycan layer and stain blue to purple. Gram - negative cells have a thin peptidoglycan layer and stain red to pink".
Steps :
Mechanism :
Gram - positive cell wall :
* Gram - Positive bacteria have a thick mesh - like cell wall which is made up of peptidoglycan (50 - 90% of cell wall), which stains purple.
* Peptidoglycan is mainly a polysaccharide composed of two subunits called N- acetyl glucosamine and N- acetyl muramic acid.
* As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of peptids by means of a transpeptidase enzyme, resulting in the shape and rigidity of cell wall.
* The thick peptidoglycan layer of Gram - positive organisms allows these organisms to retain the crystal violet - iodine complex and stains the cells as purple.
Gram - Negative Cell wall :
* Gram - negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall).
* Therefore, they lose the crystal violet - iodine complex during decolorization with the alcohol, rinse, but retain the counter stain Safranin.
* As a result, gram negative cells appear reddish or pink.
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| Gram positive VS Gram negetive cell wall |
Procedure :
* Take a clean, grease free slide.
* Prepare the smear of suspension on the clean slide with a loopful of sample.
* Air dry and heat fix.
* Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
* Flood the gram's iodine for 1 minute and wish with water.
* Then, wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
* Add Safranin for about 1 minute and wash with water.
* Air dry, Blot dry and observe under microscope.
Stain Reaction :
The four basic steps of the Gram Stain are :
1) Application of the primary stain crystal violet (CV) to a heat fixed smear of bacterial culture.
* CV dissociates in aqueous solutions into CV+ and Cl- ions.
* These two ions then penetrate through the cell wall and cell membrane of both Gram - positive and Gram - negative cells.
* The CV+ ions later interacts with negatively charged bacterial components and stains the bacterial cells purple.
2) Addition of Gram's Iodine
* This step is known as "fixing the dye".
* Iodine (I - or - I3 -) acts as a mordant and as a trapping agent.
* A mordant is a substance that increases the affinity of the cell wall for stain by binding to the primary stain, thus forming an insoluble complex which gets trapped in cell wall.
* In the Gram stain reaction, the crystal violet and iodine form an insoluble (CV - I) which serves to turn the smear a dark purple color.
* At this stage, all cells will turn purple.
3) Decolorizations with 95% ethyl alcohol
* This step is known as "Solvent treatment".
* Alcohol/ acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall.
* The CV - I complex is then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless.
* On the other hand, alcohol has a dehydrating effect on the cell walls of gram positive bacteria which causes the pores of the cell wall to shrink.
* The CV - I complex gets tightly bound into the multi-layered Gram positive cell wall thus staining the cells purple.
4) Counterstain with safranin
* The decolorized Gram negative cells can be rendered visible with a suitable counterstain, which is usually positively charged safranin.
* Safranin stains them red.
* Red colour which adheres to the Gram positive bacteria is masked by the purple of the crystal violet.
* Basic fuschin is sometimes used instead of Safranin in rare situations.
Observation :
Summary
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| Gram's staining Summary |
Acid-Fast staining
* Acid fast stains are used to differentiate acid fast organisms such Mycobacteria.
* Acid fast bacteria have a high content of mycolic acids in their cell walls.
* Acid- fast bacteria are also known as acid-fast bacilli of simply AFB.
* Stained bacteria that are not decolorized by acid alcohol are called acid fast bacteria.
* They are a group of bacteria sharing the characteristic of acid fastness.
* Acid fastness is a physical property that gives a bacterium the ability to resist decolorization by acids during staining procedures.
* This means that once the bacterium is stained, it cannot be decolorized by acids routinely used in the process.
* Bacteria displaying acid fastness include:
1) Genus Mycobacteria - M. leprae, M. tuberculosis, M. smegmatis etc.
2) Genus Nocardia - N. brasiliensis, N. cyriacigeorgica, N. farcinica etc.
* There are two types of acid fast staining : hot method (e.g. Ziehl-Neelsen method) and cold method (e.g. Kinyoun method)
Principle : (some for both hot cold acid fast staining method )
" The cell wall of acid fast bacteria contains a lipid called Mycolic acid which makes them resistant to simple aqueous stains. Thus, a lipid- soluble stain (primary stain) is required to stain them which is retained even after decolorization step. whereas primary stain is lost from the non acid fast bacteria upon decolorization and cells get stained with counterstain".
Mechanism : (same for both hot and cold acid fast staining method)
* The cell wall of Mycobacterium sps typically contain waxy substance (Mycolic acid) that makes it impermeable to staining by aqueous staining solutions.
* These bacteria can not be stained by simple or even by Gram staining.
* They can however be stained by lipid-soluble stain and one stained can not be readity decolourized by weak mineral acid.
* Hence, these bacteria are called acid fast bacilli and the staining method is called acid fast staining.
Reagents: (same for both hot and cold acid fast staining method)
* Primary stain : 0.3% Carbol-fusion,(Dissolve 50g phenol in 100ml ethanol (95%) or methanol (95%). Dissolve 3g Basic fushsin in the mixture and add distilled water to bring the volume to 1L)
*Decolorization Solution : Add 30ml hydrochloric acid to 1L of 95% denatured alcohol. Cool and mix well before use. Alternate decoloring reagent (without alcohol): slowly add 250ml sulfuric acid (at least 95%) to 750ml distilled water. Cool and mix well before using.
Counterstain: 0.3% methylene blue (Dissolve 3.0g methylene blue in 1L distilled water).
Ziehl - Neelsen method
* Acid fast staining technique was developed in 1882 by Paul Ehrlich.
* It is named for two German dactors who modified the stain : the bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen in 1890.
* Z.N. staining or Ziehl - Neelsen staining is a bacterilogical stain used to identify acid-fast organisms mainly mycobacteria.
* Ziehl - Neelsen is a not method of acid fast staining.
* The primary stain used in acid-fast staining is carbolfuchsin.
* It is lipid - soluble and contains phenol, which helps the stain penetrate the cell wall.
* This is further assisted by the addition of heat.
* The smear is then rinsed with a very strong decolorizer, which removes the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms.
* The decolorized non-acid-fast cells then take up the counterstain.
Procedure:
* Prepare and fix the specimen smear prior to staining.
* Heat-fix the dried smear.
* Flood the smear with carbol fuchsin stain.
* Heat the stain to evaporate; replenish stain as needed. Also, prevent stain from boiling. Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
*Wash off the stain with clean distilled water. Heated slides must be cooled prior to washing.
* Decolorize the smear with acid alcohol for 5 minutes or until the smear is sufficiently decolorized.
* Wash well with clean water.
* Flood the smear with methylene blue stain for 1-2 minutes.
* Wash off the stain with clean water.
* Wipe the back of the slide clean and place it in a draining rack for the smear to air dry.
* Examine the smear microscopically, using the 100X oil.
Observation:
*Acid fast : Bright red colored
*Non-acid fast: Blue color
* The kinyoun method or kinyoun stain cold method) developed by joseph J. Kinyoun, is a peocedure used to stain acid-fast species of the bacteria.
* It is a variation of a method developed by Robert Koch in 1882.
* It does not require heating.
* In the Ziehl-Neelsen stain, heat acts as physical mordant while phenol (carbol of carbol fuschin) acts as the chemical mordant.
* Since the kinyoun stain is a cold method (no heat applied), the concentration of carbol fuschin used is increased.
Structural staining
* Structural staining is used observe certain structures on bacteria.
* This is important because certain structures on bacteria can be antigenic or acts as an endotoxin.
* Structural stains are more complex than simples ones and use more than one stain to differentiate cellular components.
* They are used to examine structural differences between bacterial groups or to provide contrast to different structures within the same organisms.
* Examples of structural staining methods are cell wall staining, PHB staining, endospore staining etc.
cell wall staining
* Bacterial cell wall is present beneath the capsule and surrounding the cytoplasm.
* It is a rigid structure that gives definite shape to the bacterial.
* It is made made up of peptidoglycan.
* And because of its thickness and chemical nature, it is resistant to the staining.
* Thus, it is normally difficult to separately stain the cell wall of the cells.
* There are various staining techniques used to stain cell wall like chance's method, Ringer's method and Dyar's method.
Chance's Method
Principle :
"Bacterial cell is stained pink with new fuschin and then only cytoplasm is decolorized with congored solution while cell wall remains pink"
Mechanism :
* As we know cell wall and cytoplasm are acidic in nature where cell wall is more acidic than cytoplasm.
* So when we apply the first stain that is 0.5% new fuchsine which is the basic stain it stains the cytoplasm as well as the cell wall.
* But here we want to stain the cell wall so the smear is treated with the second stain that is 0.5% congo red solution.
* As we know congo red is a selective decolorizing agent it selectively decolorizes the less acidic portion that is cytoplasm.
* Hence cell wall remains stained by 0.5% new fuchsine stain.
Requirements:
* Clean grease free slide.
* Nichrome wire loop.
* Bacterial suspension.
* New fuchsine solution.
(Basic fuschin - 0.5g; Distilled water - 100ml)
* 0.5% congored solution.
Procedure:
* Take a clean, grease free slide.
* Prepare the smear of the suspension on the clean slide with a loopful of sample.
* Air dry and do not heat fix.
* New fuchsine is poured on smear and kept for about 3-4 minutes and rinse with water.
* Prepare a thin film of congored on dried smear with the help of another slide.
* Air dry, Blot dry and observe under microscope.
Observation:
Ringer et al. method
* Here mordant is used.
* In this method, tannic acid acts as a mordant that accelerates the reaction between cell and stain.
* Congored acts as decolorizing agent that decolorizes cytoplasm.
* And crystal violet (basic stain) stains the cell wall.
Principle :
" Bacterial cell wall is made positively charged by treatment with cationic surface agents e.g Tannic acid, so that it can take up anionic stain. But cytoplasm retains negative charge and thus, can be stained with contrasting basic stain".
Requirements:
* Clean grease free slide.
* Nichrome wire loop.
* Bacterial suspension.
* 4% tannic acid.
* 0.5% crystal violet solution.
* 0.5% congored solution.
Procedure :
* Take a clean, grease free slide.
* Prepare the smear of suspension on the clean slide with loopful of sample.
* Air dry and do not heat fix.
* Tannic acid solution is poured on smear and allow it to react for 30 minutes.
* Pour crystal violet and keep it for 1-2 minutes.
* Gently wash the slide with water.
* Prepare a thin film of congored on smear and allow it to react for 3 minutes.
* Air dry, blot dry and observe under microscope.
Observation :
PHB staining
* Bacterial cell inclusion bodies are also known as granules.
* These granules function as energy storage and these are also involved in reducing osmotic pressure.
* Some of these granules are polyphosphate granules, sulphur granules, glycogen granules and PHB granules etc.
* Poly (3-hydroxybutyrate) (PHB) granules are important storage compounds of carbon and energy in many prokaryotes.
* These granules serve as lipid reserve materials in bacteria.
* PHB granules are produced by microorganisms e.g. Bacillus megaterium apparently in respnse to conditions of physiological stress; mainly conditions in which nutrients are limited.
* PHB granules allow survival of the cells in the absence of suitable carbon sources.
* The PHB polymer is primarily a product of carbon assimilation (from glucose or starch) and is empolyed as a form of energy storage molecules to be metabolized when other common energy sources are not available.
* These granules are visible under a light microscope by staining with sudan black.
Principle :
" Sudan Black B is a slightly basic dye that combines with the acidic groups in the pilid compounds, hence staining the PHB granules".
Mechanism :
* In Burdon's method of PHB staining, Sudan black B stain is used.
* It is a fat soluble stain which stains lipid layer by getting dissolved in it.
* Safranin is the counter stain which stains the cytoplasm of bacterial cells.
Requirements :
* Clean grease free slide.
* Nichrome sire loop.
* Bacterial suspension.
* Sudan black B stain.
* Xylene
* 0.5% safranin solution.
Procedure :
* Take a clean, grease free slide.
* Prepare a thin smear of suspension on the clean slide with a loopful of sample.
* Air dry and heat fix.
* Sudan black B stain is pored on smear.
* Remove excess stain and dry it.
* Rinse the smear with xylene and blot dry.
* Flood the dried smear with 0.5% safranin.
* Allow to react for 5-10 minutes.
* Air dry, Blot dry and observe under Microscope.
Observation :
