PlPath(505)DETECTION AND DIAGNOSIS OF PLANT

DISEASES

Presented byAnkitaH-2016-70-MMSc (plant pathology)1st year

bull Submitted to- Dr IM Sharma Dr Monica Sharma

STAINING IN PATHOGEN DIAGNOSIS

Staining ndash auxiliary technique increases the visibility generate extra information regarding cell Stains are used tobull Define and examine bulk tissues for highlighting (for example- sieve tubes of phloem xylem vessels)bullCell populations (classifying different bacterial cells for instance)bullOrganelles within individual cells

Stains- chemical substances used to stain cells are organic compound containing a benzene ring a chromophore and an auxochrome group

bullTypes- 1 Acidic- Anionic Used to stain basic component of cell like

cytoplasmic component Eg Picric acid acid fuchsin eosin etc

2 Basic ndash Cationic Used to stain acidic component of cell like

nucleic acid Eg Methylene blue crystal violet safranin etc

3 Neutral ndash Having no charge Made by mixing aqueous solution of certain

acidic and basic dyes

Staining techniquesDirect staining - The organism is stained and background is left unstainedNegative staining - The background is stained and the organism is left unaltered

Stains are classified asbull Simple stainbull Differential stainbull Structural or special stains

Simple StainingbullThe staining process involves immersing the sample (before or after fixation and mounting) in dye solution followed by rinsing and observation bullSimple staining is one step method using only one dyebull Basic dyes are used in direct stain and acidic dye is used in negative stain bullUsed to study the morphology better to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
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• Slide 3
• Slide 4
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• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

bull Submitted to- Dr IM Sharma Dr Monica Sharma

STAINING IN PATHOGEN DIAGNOSIS

Staining ndash auxiliary technique increases the visibility generate extra information regarding cell Stains are used tobull Define and examine bulk tissues for highlighting (for example- sieve tubes of phloem xylem vessels)bullCell populations (classifying different bacterial cells for instance)bullOrganelles within individual cells

Stains- chemical substances used to stain cells are organic compound containing a benzene ring a chromophore and an auxochrome group

bullTypes- 1 Acidic- Anionic Used to stain basic component of cell like

cytoplasmic component Eg Picric acid acid fuchsin eosin etc

2 Basic ndash Cationic Used to stain acidic component of cell like

nucleic acid Eg Methylene blue crystal violet safranin etc

3 Neutral ndash Having no charge Made by mixing aqueous solution of certain

acidic and basic dyes

Staining techniquesDirect staining - The organism is stained and background is left unstainedNegative staining - The background is stained and the organism is left unaltered

Stains are classified asbull Simple stainbull Differential stainbull Structural or special stains

Simple StainingbullThe staining process involves immersing the sample (before or after fixation and mounting) in dye solution followed by rinsing and observation bullSimple staining is one step method using only one dyebull Basic dyes are used in direct stain and acidic dye is used in negative stain bullUsed to study the morphology better to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Staining ndash auxiliary technique increases the visibility generate extra information regarding cell Stains are used tobull Define and examine bulk tissues for highlighting (for example- sieve tubes of phloem xylem vessels)bullCell populations (classifying different bacterial cells for instance)bullOrganelles within individual cells

Stains- chemical substances used to stain cells are organic compound containing a benzene ring a chromophore and an auxochrome group

bullTypes- 1 Acidic- Anionic Used to stain basic component of cell like

cytoplasmic component Eg Picric acid acid fuchsin eosin etc

2 Basic ndash Cationic Used to stain acidic component of cell like

nucleic acid Eg Methylene blue crystal violet safranin etc

3 Neutral ndash Having no charge Made by mixing aqueous solution of certain

acidic and basic dyes

Staining techniquesDirect staining - The organism is stained and background is left unstainedNegative staining - The background is stained and the organism is left unaltered

Stains are classified asbull Simple stainbull Differential stainbull Structural or special stains

Simple StainingbullThe staining process involves immersing the sample (before or after fixation and mounting) in dye solution followed by rinsing and observation bullSimple staining is one step method using only one dyebull Basic dyes are used in direct stain and acidic dye is used in negative stain bullUsed to study the morphology better to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

bullTypes- 1 Acidic- Anionic Used to stain basic component of cell like

cytoplasmic component Eg Picric acid acid fuchsin eosin etc

2 Basic ndash Cationic Used to stain acidic component of cell like

nucleic acid Eg Methylene blue crystal violet safranin etc

3 Neutral ndash Having no charge Made by mixing aqueous solution of certain

acidic and basic dyes

Staining techniquesDirect staining - The organism is stained and background is left unstainedNegative staining - The background is stained and the organism is left unaltered

Stains are classified asbull Simple stainbull Differential stainbull Structural or special stains

Simple StainingbullThe staining process involves immersing the sample (before or after fixation and mounting) in dye solution followed by rinsing and observation bullSimple staining is one step method using only one dyebull Basic dyes are used in direct stain and acidic dye is used in negative stain bullUsed to study the morphology better to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Staining techniquesDirect staining - The organism is stained and background is left unstainedNegative staining - The background is stained and the organism is left unaltered

Stains are classified asbull Simple stainbull Differential stainbull Structural or special stains

Simple StainingbullThe staining process involves immersing the sample (before or after fixation and mounting) in dye solution followed by rinsing and observation bullSimple staining is one step method using only one dyebull Basic dyes are used in direct stain and acidic dye is used in negative stain bullUsed to study the morphology better to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Simple StainingbullThe staining process involves immersing the sample (before or after fixation and mounting) in dye solution followed by rinsing and observation bullSimple staining is one step method using only one dyebull Basic dyes are used in direct stain and acidic dye is used in negative stain bullUsed to study the morphology better to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Commonly used simple stains are1048698 Methylene blue1048698 Dilute carbol fuchsin1048698 Polychrome methylene blue

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Differential stainingbullDifferential stains use two or more stains and allow the cells to be categorizedinto various groups or typesbull It usually provides more information about the characteristics of the cell wall (thickness)bullTwo step methodbullIt includes- 1)Gram staining2)Acid fast staining

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

GRAM STAINING

Gram staining PrinciplesbullGram staining is used to determine gram status to classify bacteria broadlybull It is based on the composition of their cell wallbull Gram staining uses crystal violet to stain cell walls iodine as a mordant and acid fuchsin or safranin counterstain to mark all bacteriabullGram-positive bacteria stain dark blue or violet bullTheir cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria

Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
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Gram Staining Technique1 Crystal violet acts as the primary stain2 Gramrsquos iodine acts as a mordant (Helps to fix the primary dye to the cell wall)3 Decolorizer(acetone or ethanol) is used next to remove the primary stain (crystal violet) from Gram Negative bacteria4 Finally a counter stain (Safranin) is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
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• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Gram Reaction-bullGram-positive bacteria are those that are stained dark blue or violet by Gram staining bullWhere Gram-negative bacteria which cannot retain the crystal violet stain instead taking up the counter stain (safranin or fuchsine) and appearing red or pink bullGram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall bullGrampositive cell walls typically lack the outer membrane found in Gram-negative bacteriabullGram-negative bacteria are those bacteria that do not retain crystal violet dyein the Gram-staining protocol

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
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• Slide 20
• Slide 21

ACID-FAST REACTION

bullThe ZiehlndashNeelsen stain also known as the acid-fast stain widely used differential staining procedurebullIn this type some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms bullZiehl- Neelsen Procedure1 Make a smear Air Dry Heat Fix2 Flood smear with Carbol Fuchsin stain(Carbol Fuchsin is a lipid soluble phenolic compound which is able to penetrate the cell wall)3 Cover flooded smear with filter paper4 Steam for 10 minutes Add more Carbol Fuchsin stain as needed5 Cool slide6 Rinse with distilled water

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
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• Slide 18
• Slide 19
• Slide 20
• Slide 21

SPECIAL STAINS (kind of differential stain)bull Stain for endosporesbull Stain for capsulesbull Stain for flagellabullStain for bacterial nucleus

Capsule stainingbullThe purpose of the capsule stain is to reveal the presence of the bacterial capsule bullCapsule may appear as clear halo when a fresh sample is stained by Grams or Leishman stainbullGenerally we are using Negative stains like - India ink or Nigrosin

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
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• Slide 21

Endospore StainingbullBacterial endospores are metabolically inactive highly resistant structures produced by some bacteria as a defensive strategy against unfavorable environmental conditions bullPrimary stain - is malachite green which stainsboth vegetative cells and endospores and heat is applied to help the primary stain penetrate the endospore bullDecolorized with water which removes the malachite green from the vegetative cell but not the endosporebull Safranin ndash counterstain for any cells which have been decolorizedbull At the end of the staining process vegetative cells will be pink and endospores will be dark green

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
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• Slide 11
• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Flagella stainbullThey are very fragilebull Here staining is preeceded by using of some precipitating agent like tannic acid or iron chloridebullLiefsonrsquos stain Carbol fuchsin or Fontanarsquos solution is used to demonstrate the flagella

Nucleus staining of bacteriabullHere nuclear material is present in a region called nucleoid devoid of nuclear membranebullSince cytoplasm has a strong affinity for most stains and it may interfere with observation of nuclear materialbullIt should be hydrolysed first with HCl bullLater stained with Giemsa stainbullNuclear bodies will appear purple coloured

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
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• Slide 12
• Slide 13
• Slide 14
• Slide 15
• Slide 16
• Slide 17
• Slide 18
• Slide 19
• Slide 20
• Slide 21

Lactophenol cotton blue staining in fungibullItrsquos a rapid and routine examination of all types of fungibullIt stains the fungal cytoplasm and provides a light blue background against which walls of hyphae can be seen as non-stained regionbullIt contains four components-a)Phenol serves as a fungicideb)Lactic acid act as a clearing agentc)Cotton blue stains cytoplasmd)Glycerine gives a semipermanent preparation

Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
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Nuclear staining in fungibull Fungi are eukaryotic bullGot organized nuclei bounded by nuclear membrane with characteristics pores a nucleolus and chromatin strandsbullFungal nuclei are oftenly stained with Hematoxylin Giemsa Feulgen or Acetocarmine

bullExample of some fluorescent dye used in microscopic study of fungi bullAcridine orange- nucleus- orange colourbullAlexafluor ndash cell wall(chitin)- green colourbullCalcofluor white- cell wall(chitin and cellulose)- green colourbullDAPI- nucleic acid- blue colourbullHoechest 33258- dsDNA- green colour

Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
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Staining in ActinomycetesbullGram positive prokaryotes characterized by formation of branched filamentous bodybullAlso known as mold-like bacteriabullGiemsa stain Crystal violet Methyl violet Hematoxylin Methylene blue and Carbol fuchsin are some stains used for thembullKnown to show acid fast reactionbullFor differentiating the substrate and aerial mycelium the culture grown on cellophane is stained in Sudan IV staindipped in 70 ethyl alcoholwashed in water before mountingbullStain is retained by aerial hyphae because of the lipid content of outer wall and substrate hyphae appear colourless

Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
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Staining in vesicular arbuscular mycorrhizal fungi(VAM)bullMycorrhiza is an assosciation of a fungus with roots of plantsbullStandard mounting media for determining VAM fungi are water lactoglycerine or lactophenolbullStaining is done in Cotton blue or Melzerrsquos reagentbullCan be seen directly by observing the washed VAM infected roots under compound microscopesbullBy squashing roots with lactofuchsin as a mounting media can help to visualise vesicles or arbuscles

Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
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Staining in PhytoplasmabullThey are different from other bacteria by the absence of cell wall bullHighly pleomorphic and sensitive to osmotic lysis so known to present in phloem cellsbullMost reliable method for demonstrating the presence of phytoplasma or MLOrsquos is electron microscopybullUnder lowndashpower light microscopy we can go for using Dienesrsquo stainbullComposition of Dienesrsquo stain-Methylene blue Azure II Maltose Sodium carbonate and Distilled waterbullCells with infection appears blue

• PlPath(505)DETECTION AND DIAGNOSIS OF PLANT DISEASES
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