Gram Stain

Principle

• Gram Staining ek differential staining technique hai jo Hans Christian Gram ne 1884 mein develop ki thi. Is technique ka principle yeh hai ki bacteria ki cell wall ki structure ke differences ki wajah se yeh bacteria ko do categories mein divide karta hai: Gram-positive aur Gram-negative. Gram-positive bacteria ke paas ek thick peptidoglycan layer hoti hai jo primary stain (crystal violet) ko retain kar leti hai, jabki Gram-negative bacteria ke paas thin peptidoglycan layer hoti hai aur ek outer lipid membrane hota hai, jo crystal violet ko retain nahi karne deta, isliye yeh counterstain (safranin) ko le lete hain.

• Gram-positive bacteria:

  • Thick peptidoglycan layer hoti hai.

  • Crystal violet stain ko retain karte hain aur purple/blue dikhte hain.

  • Example: Staphylococcus aureus, Streptococcus pneumoniae.

• Gram-negative bacteria:

  • Thin peptidoglycan layer aur outer membrane hoti hai.

  • Crystal violet ko lose karte hain aur safranin counterstain ko absorb karte hain, jo unhe pink/red dikhata hai.

  • Example: Escherichia coli, Salmonella typhi.

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Materials Required

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• Glass Slides (clean aur dry)

• Bacterial Culture (broth ya solid medium)

• Crystal Violet Stain (Primary Stain)

• Gram’s Iodine Solution (Mordant)

• Ethanol ya Acetone (Decolorizer)

• Safranin Stain (Counterstain)

• Distilled Water

• Dropper ya Pipette

• Bunsen Burner (heat fixation ke liye)

• Coverslips

• Microscope (oil immersion objective ke saath)

• Gloves aur Safety Goggles

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Procedure

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Step 1: Preparation of Smear:

• Ek clean glass slide pe smear banayein.

• Solid media ke liye: Ek isolated colony se bacterial culture ka loopful lekar slide pe place karein. Ek drop distilled water daalein aur sterile loop se bacteria ko evenly spread karein.

• Liquid culture ke liye: Bacterial broth culture ka ek drop slide pe daalein aur loop se spread karein.

• Smear ko air dry hone de, taki bacterial morphology distort na ho.

• Heat fixation: Slide ko Bunsen burner flame ke through 2-3 baar pass karein taki bacteria dead ho jayein aur slide pe adhere ho jayein.

Step 2: Staining with Crystal Violet:

• Smear ko crystal violet stain se flood karein aur 1 minute ke liye chhod dein. Crystal violet primary stain hai jo bacteria ke cell wall ko stain karta hai.

• Excess stain ko distilled water se rinse karein.

Step 3: Application of Gram’s Iodine (Mordant):

• Slide ko Gram’s iodine solution se flood karein aur 1 minute ke liye chhod dein. Iodine mordant ka kaam karta hai, jo crystal violet ke saath complex banata hai aur stain retention ko enhance karta hai.

• Excess iodine ko distilled water se rinse karein.

Step 4: Decolorization:

• Ethanol ya acetone (decolorizer) ko slide pe 10-20 seconds ke liye apply karein. Yeh step Gram-positive aur Gram-negative bacteria ko differentiate karne ke liye zaroori hai. Ethanol Gram-negative bacteria ke outer membrane ko dissolve karta hai aur crystal violet ko wash out kar deta hai, jabki Gram-positive bacteria crystal violet ko apni thick peptidoglycan layer ki wajah se retain karte hain.

• Jab runoff clear ho jaye, distilled water se rinse karein taki decolorization process stop ho jaye.

Step 5: Counterstaining with Safranin:

• Slide ko safranin stain se flood karein aur 30 seconds se 1 minute tak chhod dein. Safranin counterstain hai jo Gram-negative bacteria ko red/pink dikhata hai, jo crystal violet stain ko lose kar chuke hote hain.

• Excess safranin ko distilled water se rinse karein aur gently paper towel se blot karein taki slide dry ho jaye.

Step 6: Microscopic Examination:

• Smear pe immersion oil daalein.

• Slide ko 100x oil immersion objective ke under microscope pe examine karein.

• Pehle 10x objective se smear ke areas locate karein, phir 40x ya 100x oil immersion objective se bacteria ke detailed morphology ko observe karein.

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Observations

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• Gram-positive bacteria microscope mein purple/blue dikhenge kyunki unke paas thick peptidoglycan layer hoti hai jo crystal violet ko retain kar leti hai.

• Gram-negative bacteria microscope mein pink/red dikhenge kyunki unhone crystal violet ko lose kar diya hai aur safranin counterstain ko absorb kar liya hai.

Example:

• Staphylococcus aureus (Gram-positive): Purple cocci clusters.

• Escherichia coli (Gram-negative): Pink rods.

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Precautions

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• Ensure ki smear thin ho taki bacteria clump na ho aur identification mein difficulty na ho.

• Over-decolorization se bachain, kyunki isse Gram-positive bacteria bhi Gram-negative ki tarah dikh sakte hain.

• Hamesha fresh bacterial cultures use karein, kyunki purani cultures unreliable results de sakti hain.

• Staining chemicals ko carefully handle karein aur gloves, goggles, lab coat pehnein.

• Slide ko gently blot karein taki smear damage na ho.

• Staining chemicals ko proper disposal method se dispose karein.

References:

• Collee, J.G., Fraser, A.G., Marmion, B.P. (2006). Mackie & McCartney Practical Medical Microbiology (14th ed.). Elsevier.

• Horyn, J. et al. (2006). Gram Staining of Bacteria: Principles and Applications. Journal of Microbiology & Biology Education.

• Jorgensen, J.H., & Pfaller, M.A. (2015). Manual of Clinical Microbiology (11th ed.). American Society of Microbiology.
• Madigan, M.T., Martinko, J.M., & Bender, K.S. (2017). Brock Biology of Microorganisms (15th ed.). Pearson Education.