Wet and gram staining

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رنگ آميزي ساده، گرم و wet mount گروه ميكروب شناسي دكتر مهاجري پاييز 1387

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هدف رنگ آميزي: ايجاد كنتراست بين باكتري و محيط اطراف جهت مشاهده بهتر Even with the microscope, bacteria are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. The most common method to increase contrast is to stain part or all of the microbe

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Types of dyes (stain) Stains are chemicals containing chromophores, (groups that impart color). Their specificity is determined by their chemical structure and charge they carry. Accordingly there are 3 types of dyes: Basic dye (cationic dye) is a stain that has positively charged chromophore. Examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue. Acid dye (anionic dye) has a negatively charged chromophores.Examples of acid dyes are Nigrosine and sodium eosinate. Neutral dye: has both a negatively and positively charged chromophores (net charge is neutral) Example: eosin methylene blue

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Staining There are several staining methods that are used routinely with bacteria. These methods may be classified as: Simple stain: use only one dye, it will react with different types of bacteria in an identical fashion. e.g.: methylene blue Differential stain: use 2 or more dyes, that react differentially with different types bacteria giving varying results depending on the organism being treated. e.g.: Gram’s staining Structural stain: Special stain to examine an internal or external structure of bacterial cell: capsular stain, flagellar stain

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Heat fixation accomplishes three things: kills the organisms causes the organisms to adhere to the slide alters the organisms so that they more readily accept stains (dyes)

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If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed. If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps. If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains

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رنگ آميزي گرم (Gram staining) Hans Christian Gram 1884 1) تهيه اسمير 2) فيكس كردن - متانول 95% : مزيت آن حفظ شكل RBC، عيب آن سميت - شعله مستقيم 3) رنگ كريستال ويوله يك دقيقه - رنگ قليايي براي ديواره باكتري

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4) شستشو لام با آب روان 5) يديدوره (Gram’s Iodine) يك دقيقه - لوگل بعنوان دندانه (mordant) تركيب با كريستال ويوله و ايجاد كمپلكس بزرگ Crystal- Iodine 6) رنگبري با الكل- استن (نسبت مساوي از الكل 95% و استن) مهمترين مرحله، بسيار حساس، ضخامت لايه PG خارج كردن كريستال ويوله از CW باكتريهاي گرم منفي

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7) شستشو با آب روان 8) سافرانين 30 ثانيه - بعنوان رنگ زمينه (counterstain) - رنگ كردن سلولهاي رنگ نشده 9) شستشو با آب روان باكتريهاي گرم مثبت < < بنفش باكتريهاي گرم منفي < < قرمز ** ممكن است باكتريها در حين رنگ آميزي از بين نرفته باشند

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در موارد زير باكتريهاي گرم مثبت ممكن است به رنگ باكتريهاي گرم منفي (قرمز) در آيند - رنگ آميزي باكتريهاي بسيار جوان يا پير - باكتريهايي كه قبلاً تحت اثر آنتي بيوتيك ها قرار داشته اند - حضور آنزيم هاي اتولايزين - طولاني بودن مرحله رنگبري با الكل- استن (*) RBC و WBC به رنگ قرمز در مي آيند

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روشهاي ديگر تفكيك G+ve از G-ve 1- LANA (L- آلانين-4- نيترو آنيليد)، در مجاورت باكتريهاي گرم منفي زرد مي شود. 2- KoH 3% در مجاورت باكتريهاي گرم منفي string ايجاد مي كند، علت: KoH 3% باعث بيرون ريختن محتويات هسته باكتري و افزايش ويسكوزيته مي شود. 3- G+ve ها حساس به ديسك ونكومايسين (5μg) و G-ve ها حساس به ديسك كليستين و پلي ميكيسن

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استثناء: لاكتوباسيل ها G+ve اند ولي ونكومايسن مقاومند موراكسلا و آسينه توباكتر G-ve اند ولي به ونكومايسين حساسند

Simple stain procedure : 

Simple stain procedure Prepare a heat fixed bacterial smear Leave to cool Using a dropper, cover the film with crystal violet (methylene blue or safranin) Leave for 30 sec in case of using crystal violet,2- 3 min for safranin or methylene blue Wash gently, dry between 2 filter papers Add oil and examine using oil immersion lens

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Simple Stain Technique use basic dyes such as crystal violet or methylene blue positively charged dyes are attracted to negatively charged materials in the cytoplasm

Negative stain procedure : 

Negative stain procedure Prepare an air dried bacterial smear Do not heat fix !!! Add one drop of nigrosine on the side of the slide Holding a second slide at a 45 degree angle, allow the drop to spread along the angled slide. Allow the dye to thoroughly air dry. Do not wash  Apply immersion oil to the smear and observe under light microscope.

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Negative Stain Technique use dyes such as nigrosin or Congo red acidic negatively charged dyes repelled by negatively charged cytoplasm leave cells clear and unstained

Microscopical examination : 

Microscopical examination Staphylococcus aureus Simple stain with crystal violet Escherichia coli Simple stain with Safranin

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Staphylococcus aureus Negative stain

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Principle of staining technique: 1- Crystal violet: all cells are stained violet. 2- Iodine acts as a mordant (fixes the dye) all cells remain violet. 3-Alcohol acetone decolorizes gram negative cells only: Gram-positive remains violet , gram-negative becomes colorless. 4-Counter stain with Safranin: Gram-positive remains violet while gram negative becomes red.

Benefit of Gram Staining : 

Benefit of Gram Staining To get a gram-reaction and define/identify cellular morphology and arrangement

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Wet mount A drop of the microorganism is placed directly on the slide and observed Hanging drop A drop of culture is placed on a coverslip (glass slide cover) and inverted over a hollowed-out slide and observed

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http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/shoeb/Gram.html http://www.youtube.com/watch?v=OQ6C-gj_UHM p_mohajeri@hotmail.com

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