Botany

Botany Laboratory Experiments Stain Plant Specimens Stain and Mount



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Botany specimens from differing divisions based on their taxonomy respond to stains in a unique way.  What Bryophytes require might not be the same for Algae or Fungi, or even Pteridophytes.  


The most commons stains that are used are Aniline blue, Fast green, Safranin, Cotton blue, Methylene blue or  Crystal violet.   Media used for mounting too vary between Glycerine 10%, Glycerine jelly, Lactophenol, Erythrosine or Canada Balsam (or D.P.X. Mountant) depending whether they are for temporary or permanent preparations.


1.  Algae:  Temporary preparations


Single staining:  Aniline blue - 0.1% aqueous, Fast green - 0.5% aqueous, Safranin – 0.5% aqueous

Mounting media: Glycerine 10 % or glycerine jelly


2.  Fungi: temporary preparations


Single staining:  Cotton blue, Analine blue

Mounting media:  Lactophenol or glycerine 10%


3. Bryophytes:  Temporary preparations


Single staining:  Safranin and Fast green

Mounting media:  glycerine 10% or glycerine jelly


4.  Pteridophytes:  Temporary and permanent preparations


Double staining

Primary stains:  Safranin and Crystal violet

Secondary stains: Fast green and Erythrosine


Mounting media:  Glycerine 10% for temporary preparations and Canada Balsam or D.P.X.. Mountant for permanent preparations.


5.  Gymnosperms:  Temporary and permanent preparations


Double staining

Primary stains:  Safranin and Crystal violet

Secondary stains:  Fast green and Erythrosine


Mounting media:  Glycerine 10% for temporary preparations and Canada Balsam or D.P.X.. Mountant for permanent preparations.


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  Mixtures of some common stains


1   Crystal violet: 


• Crystal violet: 3 gms

• Distilled water: 80 ml

• Ethyl alcohol (95%):  20 ml, dissolved and mixed with 0.8 gms of ammonium oxalate. 


2  Methylene blue:


• Methylene blue: 0.3 gms

• (0.01%) distilled water - 100 ml

• Ethyl alcohol (95%):  30 ml, dissolved and mixed with potassium hydroxide


3 Safranin:


Safranin:  0.25 gms

Alcohol (95%):  10 ml

Distilled water: 100 ml


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Bacteria can be divided into gram negative and gram positive bacteria.  Gram’s stain is used for this purpose and help with microscopic studies of the same. 

Gram’s iodine solution:  iodine - 1 gm, potassium iodide, 2 gms and distilled water 300 ml.


* Steps to Gram’s staining:


• Stain specimen in crystal violet for one minute

• Wash briefly in water Immerse in Gram’s iodine solution for a minute

• Wash in water

• Blot dry

• Decolourize in 95% ethyl alcohol for 30 seconds; agitate

• Wash in water

• Counter-stain with safranin for 10 seconds

• Wash dry

• Mount with cover glass and examine 


After the above steps have been followed, it may be noted that some bacteria are colored violet from the violet stain while others will appear red, indicating the gram negative and gram positive bacteria.  

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