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4- Bacterial Stainكمنكمنكمنطكمطكنمing.pptx

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Types of stains ‫أنواع‬ ‫البقع‬ 1. Simple stain 2. Negative stain 3. Differential stains 4. Special stain 1. ‫وصمة‬ ‫عار‬ ‫بسيطة‬ 2. ‫وصمة‬ ‫عار‬ ‫سلبية‬ 3. ‫البقع‬ ‫التفاضلية‬ 4. ‫بقعة‬ ‫الخاصة‬
1. Simple stain ‫وصمة‬ ‫عار‬ ‫بسيطة‬ • It is used to show the basic morphology of bacteria. ‫يتم‬ ‫استخدامه‬ ‫إلظهار‬ ‫الشكل‬ ‫األساسي‬ ‫للبكتيريا‬ . • Staining can be performed with basic dyes such as methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial. ‫يمكن‬ ‫إجراء‬ ‫التلوين‬ ‫باستخدام‬ ‫األصباغ‬ ‫األساسية‬ ‫مثأل‬ ‫زرق‬ ‫الميثيلين‬ , ‫بشكل‬ ‫ايجابي‬ ‫متهم‬ ‫األصباغ‬ ‫التي‬ ‫تنجذب‬ ‫إلى‬ ‫سلبا‬ ‫المواد‬ ‫المشحونة‬ ‫من‬ ‫ميكروبية‬ .
Method: ‫طريقة‬ • Fix the dried smear ‫إصالح‬ ‫اللطاخة‬ ‫المجففة‬ • Cover the smear with Methylene blue stain for 1 minute. ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫الميثيلين‬ ‫األزرق‬ ‫وصمة‬ ‫عار‬ ‫لمدة‬ 1 ‫دقيقة‬ . • Wash off with clean water. ‫يغسل‬ ‫بالماء‬ ‫النظيف‬ • Wipe the back of the slide clean, and place it in a draining rack for the smear to air- dry. ‫امسح‬ ‫الجزء‬ ‫الخلفي‬ ‫من‬ ‫الشريحة‬ ‫ا‬ً‫ف‬‫نظي‬ ، ‫ثم‬ ‫ضعه‬ ‫في‬ ‫رف‬ ‫التصريف‬ ‫حتى‬ ‫تجف‬ ‫اللطاخة‬ ‫مجفف‬ ‫ه‬ ‫واء‬ . • Examine the smear microscopically, first with the 40 to see the distribution of material and then with the oil immersion objective to look for bacteria. ‫افحص‬ ‫اللطاخة‬ ‫مجهريا‬ ‫أوال‬ ‫باستخدام‬ 40 ‫لرؤية‬ ‫توزيع‬ ‫المواد‬ ‫ومن‬ ‫ثم‬ ‫مع‬ ‫الغمر‬ ‫النفط‬ ‫الهدف‬ ‫هو‬ ‫البحث‬ ‫عن‬ ‫البكتيريا‬ . • Results ‫نتائج‬ • Bacterial cells . . . . . . . . . . . . . . . . Blue ‫الخاليا‬ ‫البكتيرية‬ . . . . . . . . . . . . . . . . ‫أزرق‬
2. Negative stain ‫وصمة‬ ‫عار‬ ‫سلبية‬ • Negative stain is used to visualize capsules surrounding certain bacteria. ‫يتم‬ ‫استخدام‬ ‫وصمة‬ ‫عار‬ ‫سلبية‬ ‫ل‬ ‫تصور‬ ‫كبسوالت‬ ‫محيطة‬ ‫معينة‬ ‫بكتيريا‬ . • The negative stain has a negative charge and will not penetrate the cell because the negative charge on the surface of the bacterial cell wall. The capsules appear as rings around bacterial cells, and the background is dark. ‫وصمة‬ ‫عار‬ ‫سلبية‬ ‫لديها‬ ‫شحنة‬ ‫سالبة‬ ‫ولن‬ ‫تخترق‬ ‫الخلية‬ ‫ألن‬ ‫سلبي‬ ‫تهمة‬ ‫على‬ ‫سطح‬ ‫البكتيرية‬ ‫جدار‬ ‫ا‬ ‫لخلية‬ . ‫ال‬ ‫كبسوالت‬ ‫تظهر‬ ‫على‬ ‫شكل‬ ‫حلقات‬ ‫حول‬ ‫الخاليا‬ ‫البكتيرية‬ ، ‫و‬ ‫الخلفية‬ ‫مظلمة‬ . • Examples for negative stains: India ink, Nigrosin and Eosin. ‫أمثلة‬ ‫على‬ ‫البقع‬ ‫السلبية‬ : ‫حبر‬ ،‫هندي‬ ‫نيجروسينويوزين‬ . • Method: ‫طريقة‬ • Place a drop of India ink on a clean slide. ‫ضع‬ ‫قطرة‬ ‫من‬ ‫حبر‬ ‫هندي‬ ‫على‬ ‫شريحة‬ ‫نظيفة‬ . • Transfer colonies of the test organism and mix well with stain to a homogenous suspension. ‫انقل‬ ‫مستعمرات‬ ‫كائن‬ ‫االختبار‬ ‫واخلطها‬ ‫ًا‬‫د‬‫جي‬ ‫مع‬ ‫الصبغة‬ ‫إلى‬ ‫أ‬ ‫تعليق‬ ‫متجانس‬ . • Place the cover glass on the suspension. ‫ضع‬ ‫الغطاء‬ ‫الزجاجي‬ ‫على‬ ‫تعليق‬ • Examine under oil immersion lens (100x). ‫فحص‬ ‫تحت‬ ‫عدسة‬ ‫غمر‬ ‫الزيت‬ (100x).
Result: ‫نتيجة‬ The capsules appear as rings around bacterial cells, and the background is dark. ‫تظهر‬ ‫الكبسوالت‬ ‫على‬ ‫شكل‬ ‫خواتم‬ ‫حول‬ ‫الخاليا‬ ‫البكتيرية‬ ، ‫والخلفية‬ ‫مظلمة‬ . Note: ‫ملحوظة‬ - Bacterial cells are colorless or semi-transparent microorganisms - ‫الخاليا‬ ‫البكتيرية‬ ‫هي‬ ‫عديم‬ ‫اللون‬ ‫أو‬ ‫شبه‬ ‫شفافة‬ ‫الكائنات‬ ‫الدقيقة‬ - To stain the cell itself using a basic stain (crystal violet, safranin, methylene blue, and basic fuchsin) ‫لتلطيخ‬ ‫الخلية‬ ‫نفسها‬ ‫باستخدام‬ ‫وصمة‬ ‫عار‬ ‫األساسية‬ ( ‫البنفسجي‬ ‫البلوري‬ ، ‫والسافرانين‬ ، ‫وأزرق‬ ‫الميثيلين‬ ، ‫والفوشسين‬ ‫األساسي‬
3. Differential stain: ‫وصمة‬ ‫عار‬ ‫التفاضلية‬ A. Gram stain ‫غرام‬ ‫وصمة‬ ‫عار‬ B. Ziehl-Neelsen stain ‫زيل‬ ‫نيلسن‬ ‫وصمة‬ A. Gram-stain • The method is named after its inventor, the Danish scientist Hans Christian Gram (1850-1937). ‫تمت‬ ‫تسمية‬ ‫الطريقة‬ ‫على‬ ‫اسم‬ ‫مخترعها‬ ، ‫العالم‬ ‫الدنماركي‬ ‫هانز‬ ‫كريستيان‬ ‫جرام‬ (1850-1937). • The Gram stain is the most commonly used stain in the clinical microbiology laboratory. ‫صبغة‬ ‫جرام‬ ‫هي‬ ‫األكثر‬ ‫ا‬ً‫م‬‫استخدا‬ ‫وصمة‬ ‫عار‬ ‫في‬ ‫مختبر‬ ‫علم‬ ‫األحياء‬ ‫الدقيقة‬ ‫السرير‬ ‫ية‬ . • It places bacteria into one of two main groups: gram-positive (purple) or gram-negative (red). ‫ويصنف‬ ‫البكتيريا‬ ‫إلى‬ ‫واحدة‬ ‫من‬ ‫مجموعتين‬ ‫رئيسيتين‬ : ‫إيجابية‬ ‫الجرام‬ ( ‫أرجواني‬ ) ‫أو‬ ‫سلبية‬ ‫الجرام‬ ( ‫أحمر‬ ) • Some organisms are gram-variable or do not stain at all. ‫بعض‬ ‫الكائنات‬ ‫الحية‬ ‫متغير‬ ‫جرام‬ ‫أو‬ ‫ال‬ ‫وصمة‬ ‫عار‬ ‫على‬ ‫اإلطالق‬
Aim: ‫هدف‬ • The Gram staining technique is used to help identify pathogens in specimens and cultures by their Gram reaction (Gram positive or Gram negative) and morphology. ‫ستخدم‬ُ‫ت‬ ‫تقنية‬ ‫تلوين‬ ‫جرام‬ ‫للمساعدة‬ ‫في‬ ‫تحديد‬ ‫مسببات‬ ‫األمراض‬ ‫العينات‬ ‫و‬ ‫ال‬ ‫ثقافات‬ ‫بواستطهم‬ ‫رد‬ ‫فعل‬ ‫غرام‬ ( ‫غرام‬ ‫إيجابي‬ ‫أو‬ ‫سلبية‬ ‫الغرام‬ ) ‫و‬ ‫علم‬ ‫التشكل‬ ‫المورفولوجيا‬ . Required: ‫مطلوب‬ – Basic stain: Crystal violet stain - ‫أساسي‬ ‫وصمة‬ : ‫وصمة‬ ‫عار‬ ‫الكريستال‬ ‫البنفسجي‬ – Mordant: Lugol’s iodine ‫الذع‬ : ‫اليود‬ ‫لوغول‬ – Decolorizer: Acetone–alcohol ‫مزيل‬ ‫اللون‬ : ‫–األسيتون‬ ‫الكحول‬ – Counterstain: Safranin or Neutral red ‫وصمة‬ ‫عار‬ : ‫سفرانين‬ ‫أو‬ ‫أحمر‬ ‫محايد‬
Methods: • Fix the dried smear • Cover the fixed smear with crystal violet stain for one minute and wash off the stain with clean water. • Cover the smear with Iodine for one minute and wash off the stain with clean water. (Iodine fixes the crystal violet into the cell call of bacteria by acting as a chemical mordant) • Decolorize rapidly (few seconds) with ethanol, acetone or a mixture of both. Wash immediately with clean water. • Cover the smear with neutral red or safranin stain for 2 minutes. Wash off the stain with clean water. • Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry. • Examine the smear microscopically, first with the 40 objective to check the staining and then with the oil immersion objective to report the bacteria and cells Result • Gram positive bacteria . . . . . . . . . . . . . Dark purple • Gram negative bacteria . . . . . . . . . . . . . Pale to dark red
‫رق‬ُ‫ط‬ : • ‫إصالح‬ ‫اللطاخة‬ ‫المجففة‬ • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫الثابتة‬ ‫بـ‬ ‫الكريستال‬ ‫البنفسجي‬ ‫وصمة‬ ‫عار‬ ‫ل‬ ‫دقيقة‬ ‫واحدة‬ ‫واغسل‬ ‫البقعة‬ ‫بها‬ ‫ماء‬ ‫نظيف‬ . • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫اليود‬ ‫لمدة‬ ‫دقيقة‬ ‫واحدة‬ ‫واغسل‬ ‫البقعة‬ ‫بالماء‬ ‫النظيف‬ . ( ‫يعمل‬ ‫اليود‬ ‫على‬ ‫تثبيت‬ ‫اللون‬ ‫البنفسجي‬ ‫البلوري‬ ‫في‬ ‫نداء‬ ‫الخلية‬ ‫للبكتيريا‬ ‫عن‬ ‫طريق‬ ‫العمل‬ ‫كمادة‬ ‫كيميائية‬ ) • ‫إزالة‬ ‫اللون‬ ‫بسرعة‬ ( ‫بضع‬ ‫ثوان‬ ) ‫مع‬ ‫اإليثانول‬ ‫أو‬ ‫األسيتون‬ ‫أو‬ ‫خليط‬ ‫منهما‬ . ‫يغسل‬ ‫على‬ ‫الفور‬ ‫بالماء‬ ‫النظيف‬ . • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫أحمر‬ ‫محايد‬ ‫أو‬ ‫سفرانين‬ ‫وصمة‬ ‫لمدة‬ ‫دقيقتين‬ . ‫اغسل‬ ‫البقعة‬ ‫بالماء‬ ‫النظيف‬ . • ‫امسح‬ ‫الجزء‬ ‫الخلفي‬ ‫من‬ ‫الشريحة‬ ‫ا‬ً‫ف‬‫نظي‬ ، ‫ثم‬ ‫ضعيها‬ ‫في‬ ‫رف‬ ‫تجفيف‬ ‫حتى‬ ‫تجف‬ ‫الل‬ ‫طاخة‬ ‫في‬ ‫الهواء‬ . • ‫افحص‬ ‫اللطاخة‬ ‫مجهريا‬ ‫أوال‬ ‫باستخدام‬ 40 ‫هدف‬ ‫للتحقق‬ ‫من‬ ‫تلطيخ‬ ‫ثم‬ ‫مع‬ ‫هدف‬ ‫غمر‬ ‫النفط‬ ‫لإلبالغ‬ ‫عن‬ ‫البكتيريا‬ ‫والخاليا‬ ‫نتيجة‬ • ‫البكتيريا‬ ‫إيجابية‬ ‫الجرام‬ . . . . . . . . . . . . . ‫األرجواني‬ ‫الداكن‬ • ‫البكتيريا‬ ‫سالبة‬ ‫الجرام‬ . . . . . . . . . . . . . ‫شاحب‬ ‫إلى‬ ‫اللون‬ ‫األحمر‬ ‫الداكن‬
Factors that affect on Gram-reaction: ‫العوامل‬ ‫التي‬ ‫تؤثر‬ ‫على‬ ‫تفاعل‬ ‫الجرام‬ : • Old colonies (culture) • Over decolorization. • Too old iodine • Cell wall damage due to antibiotic or excessive heat fixation. • Thick smear • ‫المستعمرات‬ ‫القديمة‬ ( ‫الثقافة‬ ) • ‫زيادة‬ ‫إزالة‬ ‫اللون‬ . • ‫اليود‬ ‫قديم‬ ‫ًا‬‫د‬‫ج‬ • ‫تلف‬ ‫جدار‬ ‫الخلية‬ ‫بسبب‬ ‫مضاد‬ ‫حيوي‬ ‫أو‬ ‫غ‬َ‫ل‬‫ا‬َ‫ب‬ُ‫م‬ ‫فيه‬ ‫تثبيت‬ ‫الحرارة‬ . • ‫مسحة‬ ‫سميكة‬
B. Ziehl-Neelsen Stain (Acid-fast stain) • it is used to identify acid-fast organisms, mainly Mycobacteria, which can not be stain by Gram-stain. • The carbol fuchsin stain binds to the mycolic acid in the mycobacterial cell wall. • After staining, an acid decolorizing solution is added. This removes the red dye from the background cells, except mycobacteria which retain (hold) the dye and are therefore referred to as acid fast bacilli. • Next, the smear is counter-stained with malachite green or methylene blue which stains the background material, providing a contrast color against which the red AFB can be seen.
‫ب‬ . ‫زيل‬ ‫نيلسن‬ ‫الصبغة‬ ( ‫الصبغة‬ ‫المقاومة‬ ‫لألحماض‬ ) • ‫يتم‬ ‫استخدامه‬ ‫لتحديد‬ ‫الكائنات‬ ‫سريعة‬ ‫الحموضة‬ ، ‫خاصة‬ ‫المتفطرات‬ , ّ ‫أي‬ ‫ال‬ ‫يمكن‬ ‫صبغها‬ ‫بصبغة‬ ‫جرام‬ . • ‫ال‬ ‫كاربول‬ ‫فوكسين‬ ‫وصمة‬ ‫عار‬ ‫تربط‬ ‫فطريحامض‬ ‫في‬ ‫ال‬ ‫جد‬ ‫ار‬ ‫الخلية‬ ‫الفطرية‬ . • ‫بعد‬ ‫تلطيخ‬ ، ‫أ‬ ‫إزالة‬ ‫اللون‬ ‫الحمضي‬ ‫يتم‬ ‫إضافة‬ ‫الحل‬ . ‫هذا‬ ‫يزيل‬ ‫صبغة‬ ‫حمراء‬ ‫من‬ ‫خاليا‬ ‫الخلفية‬ , ‫يستثني‬ ‫المتفطرات‬ ‫التي‬ ‫تحتف‬ ‫ظ‬ ‫بها‬ ( ‫يمسك‬ ) ‫ال‬ ‫صبغ‬ ‫ولذلك‬ ‫يشار‬ ‫إليها‬ ‫باسم‬ ‫العصيات‬ ‫الحمضية‬ ‫السريعة‬ . • ‫التالي‬ ‫هو‬ ‫اللطاخة‬ ‫مكافحة‬ ‫ملطخة‬ ‫مع‬ ‫المسرطنة‬ ‫أو‬ ‫أزرق‬ ‫الميثيل‬ ‫ين‬ ‫الذي‬ ‫يصبغ‬ ‫مادة‬ ‫الخلفية‬ ، ‫مما‬ ‫يوفر‬ ‫ا‬ً‫ن‬‫لو‬ ‫ا‬ً‫ن‬‫متباي‬ ‫يمك‬ ‫ن‬ ‫أن‬ ‫يكون‬ ‫عليه‬ ‫اللون‬ AFB ‫األحمر‬ ‫تستطيع‬ ‫رؤيتها‬ .
Mycobacterium tuberculosis bacteria. Illustration of the Ziehl-Neelsen stain result. The reagents used are carbol fuchsin, acid alcohol, and the malachite green counterstain Acid-fast bacilli stain red and the background is green. ‫السل‬ ‫الفطري‬ ‫بكتيريا‬ . ‫رسم‬ ‫توضيحي‬ ‫للزيل‬ - ‫نيلسن‬ ‫نتيجة‬ ‫وصمة‬ ‫عار‬ . ‫الكواشف‬ ‫المستخدمة‬ ‫هي‬ ‫كاربولفوكسين‬ ‫والكحول‬ ‫الحمضي‬ ، ‫و‬ ‫المسرطنة‬ ‫مضاد‬ ‫العصيات‬ ‫المقاومة‬ ‫لألحماض‬ ‫تصبغ‬ ‫باللون‬ ‫األحمر‬ ‫و‬ ‫ال‬ ‫خلفية‬ ‫يكون‬ ‫أخضر‬ .
Required: – Carbol fuchsin stain – Acid alcohol, 3% v/v (M. tuberculosis), 1% (M. Leprae) – Malachite green Method: • Heat-fix the dried smear • Cover the smear with carbol fuchsin stain • Heat the stain until the vapor just begins to rise. Allow the heated stain to remain on the slide for 5 minutes. Wash off the stain • Cover the smear with 3% v/v acid alcohol for 5 minutes. Wash with water. • Cover the smear with a malachite green stain for 2 minutes. • After drying the smear, examine it under an oil immersion lens.
‫مطلوب‬ : - ‫كاربول‬ ‫فوكسين‬ ‫وصمة‬ - ‫الكحول‬ ‫الحمضي‬ ، 3% ‫حجم‬ / ‫حجم‬ ( ‫م‬ . ‫السل‬ ), 1% ( ‫م‬ . ‫الجذام‬ ) - ‫المسرطنة‬ ‫طريقة‬ : • ‫قم‬ ‫بإصالح‬ ‫اللطاخة‬ ‫المجففة‬ ‫بالحرارة‬ • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫كاربول‬ ‫فوكسين‬ ‫وصمة‬ • ‫تسخين‬ ‫وصمة‬ ‫عار‬ ‫حتى‬ ‫البخار‬ ‫يبدأ‬ ‫للتو‬ ‫في‬ ‫االرتفاع‬ . ‫اسمح‬ ‫للبقعة‬ ‫الساخنة‬ ‫بالبقاء‬ ‫على‬ ‫الشريحة‬ ‫لمدة‬ 5 ‫دقائق‬ . ‫اغسل‬ ‫البقعة‬ • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫بنسبة‬ 3% ‫حجم‬ / ‫حجم‬ ‫الكحول‬ ‫الحمضي‬ ‫ل‬ 5 ‫دقايق‬ . ‫يغسل‬ ‫بالماء‬ . • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫بـ‬ ‫أ‬ ‫المسرطنة‬ ‫وصمة‬ ‫عار‬ ‫لمدة‬ 2 ‫دقيقة‬ . • ‫بعد‬ ‫تجفيف‬ ‫اللطاخة‬ ، ‫قم‬ ‫بفحصها‬ ‫تحت‬ ‫عدسة‬ ‫مغمورة‬ ‫بالزي‬ ‫ت‬ .
Results ‫نتائج‬ • Acid facid bacilli (AFB).. . . . . . . . . . Red, straight or slightly curved rods ‫حامض‬ ‫فاسدعصيات‬ ( ‫أف‬ ‫ب‬ ).. . . . . . . . . . ‫أحمر‬ ، ‫ستريت‬ ‫ز‬ ‫ر‬ ‫أو‬ ‫قضبان‬ ‫منحنية‬ ّ ً‫ال‬‫قلي‬ • Cells and Background. . . . . . . . . .. . Green ‫الخاليا‬ ‫والخلفية‬ . . . . . . . . . .. . ‫أخضر‬
4. Special stain a. Albert stain: • Its used to stain the volutin or metachromatic, granules of Corynebacterium diphtheria. Required – Toluidine blue staining granules with a black color and malachite green staining bacterial cells with a Green color – Albert’s iodine fixative (mordant ) Method • Fix the dried smear using alcohol • Cover the smear with the toluidine blue-malachite green stain for 3–5 minutes. Wash off the stain. • Cover the smear with Albert’s iodine for 1 minute. Wash off with water. • after drying, examine microscopically (oil lens).
4. ‫وصمة‬ ‫عار‬ ‫خاصة‬ ‫أ‬ . ‫ألبرت‬ ‫وصمة‬ ‫عار‬ : • ‫يستخدم‬ ‫لصبغ‬ ‫فولوتين‬ ‫أو‬ ‫متبدل‬ ‫اللون‬ , ‫حبيبات‬ ‫ل‬ ‫الوتدية‬ ‫الخناقية‬ . ‫مطلوب‬ – ‫حبيبات‬ ‫تلوين‬ ‫باللون‬ ‫األزرق‬ ‫طولويدين‬ ‫مع‬ ‫أ‬ ‫لون‬ ‫أسود‬ ‫و‬ ‫تلطي‬ ‫خ‬ ‫الملكيت‬ ‫األخضر‬ ‫الخاليا‬ ‫البكتيرية‬ ‫مع‬ ‫اللون‬ ‫االخضر‬ - ‫مثبت‬ ‫اليود‬ ‫ألبرت‬ ( ‫الذع‬ ) ‫طريقة‬ • ‫إصالح‬ ‫اللطاخة‬ ‫المجففة‬ ‫باستخدام‬ ‫الكحول‬ • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫بال‬ ‫التولويدين‬ ‫األزرق‬ ‫الملكيت‬ ‫األخضر‬ ‫وصمة‬ ‫عار‬ ‫لمدة‬ 3-5 ‫دقائق‬ . ‫اغسل‬ ‫البقعة‬ . • ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫اليود‬ ‫ألبرت‬ ‫لمدة‬ 1 ‫دقيقة‬ . ‫يغسل‬ ‫بالماء‬ . • ‫بعد‬ ‫التجفيف‬ ، ‫فحص‬ ‫مجهريا‬ ( ‫عدسة‬ ‫زيتية‬ ).
Results ‫نتائج‬ Bacteria cells . . . . . . . . . . . . . . . . . . . . . . Pale green ‫خاليا‬ ‫البكتيريا‬ . . . . . . . . . . . . . . . . . . . . . . ‫أخضر‬ ‫باهت‬ Metachromatic granules . . . . . . . . . . . . . Black ‫حبيبات‬ ‫متبدلة‬ ‫اللون‬ . . . . . . . . . . . . . ‫أسود‬
b. Endospore stain: It is used to identify bacterial spore (shape and its location in bacteria). Required: o Malachite green o Safranin Method:  Heat-fix the dried smear  Cover the smear with Malachite green  Heat stain until steaming arise and leave the heated to remain for 5 mins  Wash with water  Cover the smear with safranin for 2 mins and wash off the stain.  After smear drying, examine microscopically (oil lens).
‫ب‬ . ‫وصمة‬ ‫عار‬ ‫اإلندوسبور‬ : ‫يتم‬ ‫استخدامه‬ ‫لتحديد‬ ‫الجراثيم‬ ‫البكتيرية‬ ( ‫شكلها‬ ‫وموقعها‬ ‫في‬ ‫البكتيريا‬ ). ‫مطلوب‬ : o ‫المسرطنة‬ o ‫سفرانين‬ ‫طريقة‬ :  ‫قم‬ ‫بإصالح‬ ‫اللطاخة‬ ‫المجففة‬ ‫بالحرارة‬  ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫المسرطنة‬  ‫وصمة‬ ‫عار‬ ‫الحرارة‬ ‫حتى‬ ‫تبخير‬ ‫تنشأ‬ ‫واتركيها‬ ‫ساخنة‬ ‫لترتاح‬ ‫لمدة‬ 5 ‫دقيقة‬  ‫يغسل‬ ‫بالماء‬  ‫قم‬ ‫بتغطية‬ ‫اللطاخة‬ ‫ب‬ ‫سفرانين‬ ‫ل‬ 2 ‫دقيقةواغسل‬ ‫وصمة‬ ‫عار‬ .  ‫بعد‬ ‫تجفيف‬ ‫اللطاخة‬ ، ‫قم‬ ‫بفحصها‬ ‫مجهريا‬ ( ‫العدسة‬ ‫الزيتية‬ ).
Results: ‫نتائج‬ Spore .............................. Green color ‫بوغ‬ .............................. ‫اللون‬ ‫األخضر‬ Vegetative cell.................. Red color ‫الخلية‬ ‫الخضرية‬ .................. ‫لون‬ ‫أحمر‬ Thank You

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Editor's Notes

  1. Capsules of bacteria are non-ionic and therefore stain will not adhere to their surfaces. The best method to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, Congo red) and to stain the cell itself using a basic stain (e.g., crystal violet, safranin, methylene blue and basic fuchsin).
  2. مرسخ لوني Mordant
  3. ethanol, acetone or a mixture of both
  4. الإيثانول أو الأسيتون أو خليط منهما