Albert’s stain

Allied Guru

Principle

  • Albert’s stain ka principle is based on Corynebacterium species ke granules ko stain karne ki ability.
  • Yeh granules polyphosphate ke accumulation ka result hote hain.
  • Albert’s staining method special dyes ka use karti hai jo in granules ko bind karte hain, unhe blue-black rang de dete hain, jabki puri bacterial cell ko greenish hue milti hai.
  • Albert’s staining mein ek combination of dyes use hoti hai jo bacterial cells ko aur unke granules ko selectively stain karti hai.
  • Corynebacterium species ke metachromatic granules microscope par blue-black dikhte hain, jo unhe baaki bacteria se easily distinguish karne mein madad karte hain.

Requirements

Albert’s staining ko perform karne ke liye aapko yeh materials chahiye honge:

  • Microscope: Ek compound light microscope, preferably oil immersion lens (100x), taaki aap granules ko clearly dekh sakein.

  • Glass slides: Jisme bacterial smear prepare kiya jaa sake.

  • Inoculating loop: Bacterial sample ko slide par transfer karne ke liye.

  • Heat source: Smear ko slide par fix karne ke liye.

  • Staining rack: Slides ko staining ke dauran rakhne ke liye.

  • Distilled water: Slides ko rinse karne ke liye.

  • Staining containers: Staining reagents ko store karne ke liye.

Reagents

  1. Albert’s Stain Solution: Yeh ek mixture hota hai do main components ka:

    • Alberts’ Solution A: Isme methyl violet aur iodine hoti hai, jo bacteria ko stain karti hai aur granules ko bind karti hai.

    • Alberts’ Solution B: Isme potassium iodide hota hai, jo iodine staining mein madad karta hai aur granules ka color enhance karta hai.

  2. Acid-alcohol solution: Yeh decolorizing agent hota hai, jo non-granular areas se excess stain ko remove karta hai.

Sample

  • Bacterial Culture: Albert’s stain ka use zyada tar Corynebacterium diphtheriae ko detect karne ke liye kiya jaata hai. Sample kuch bhi ho sakta hai, jaise:

    • Throat swabs: Diphtheria ke suspected cases ke liye.

    • Tissue biopsies: Corynebacterium species ke infections ko diagnose karne ke liye.

    • Sputum aur other respiratory samples: Jahan patient ko respiratory symptoms ho jo diphtheria ke ho sakte hain.

  • Smear Preparation: Bacteria ka thoda sa sample slide par rakha jaata hai, aur usko ek thin, even layer mein spread kiya jaata hai. Phir smear ko air-dry karne ke baad heat-fix kar liya jaata hai taaki bacteria slide par chipak jaayein.

Procedure

  1. Prepare the smear:

    • Bacterial sample ka ek chhota drop clean glass slide par rakhein.

    • Inoculating loop ka use karke sample ko evenly spread kar lein.

    • Smear ko air-dry hone dein aur phir slide ko flame ke upar 2-3 baar pass karke heat-fix kar lein.

  2. Stain with Albert’s Solution A:

    • Albert’s Solution A (methyl violet aur iodine mixture) ko smear par kuch drops dal kar smear ko completely cover kar lein.

    • Stain ko 5-10 minutes ke liye room temperature par chhod dein.

  3. Decolorize with acid-alcohol:

    • Acid-alcohol solution se slide ko gently rinse karein, excess stain ko remove karte hue, lekin granules ko intact rakhte hue.

    • Distilled water se rinse kar ke decolorization process ko stop kar dein.

  4. Apply Albert’s Solution B:

    • Albert’s Solution B (potassium iodide solution) ko smear par apply kar ke 5 minutes tak chhod dein.

    • Yeh step granules ko zyada intense color deta hai aur unki visibility improve karta hai.

  5. Final rinse and drying:

    • Distilled water se slide ko rinse karein aur extra stain ko remove kar dein.

    • Slide ko air-dry hone dein.

  6. Microscopic examination:

    • Slide ko compound light microscope se examine karein, oil immersion lens ka use karte hue.

    • Granules dark blue-black dikhai denge, jabki bacterial cell ka baaki hissa greenish dikhai dega.

Results

  • Positive Results:

    • Bacteria mein characteristic metachromatic granules dark blue-black dikhai denge.

    • Bacterial cell ka baaki hissa greenish hoga.

    • Corynebacterium diphtheriae aur dusre Corynebacterium species ke granules easily visible honge.

  • Negative Results:

    • Agar bacteria mein metachromatic granules nahi hain, toh granules ka dark staining nahi hoga.

    • Cells greenish dikhai denge, bina kisi dark inclusions ke.

Applications

  1. Diphtheria Diagnosis: Albert’s staining ka primary use Corynebacterium diphtheriae ko identify karne ke liye hota hai. Metachromatic granules ka presence diphtheria ko confirm karne mein madad karta hai.

  2. Identification of Corynebacterium Species: Albert’s staining dusre Corynebacterium species ko bhi identify karne mein madad karta hai, jo opportunistic infections cause karte hain.

  3. Bacterial Research: Yeh method Corynebacterium species ka study karne aur unke morphology aur granule formation ko samajhne mein use hota hai.

  4. Educational Purpose: Albert’s stain microbiology labs mein students ko bacterial granules dikhane ke liye use hota hai, jo unke understanding ko enhance karta hai.

  5. Confirmation of Infection in Clinical Specimens: Yeh effective method hai diphtheria aur dusre Corynebacterium infections ko confirm karne ke liye throat swabs aur clinical samples mein.