انتخاب محیط کشت مناسب برای نمونه های میکروب شناسی بالینی
انتخاب محیط کشت مناسب برای نمونه های میکروب شناسی بالینی
الف: دستگاه تنفسی بالایی- کشت حلق- در موارد عفونت استرپتوکوکی- بتا Strep Throat BA
(Throat.pharynx) کشت حلق در موارد عفونت گونوگوکی (Gonococcal pharyngitis) BA+TM medium
(Nasal) کشت بینی- کانال- بینی- تا نزدیک تیغه میانی BA+CA
(Sinus) کشت ترشحات آسپیره شده- از سینوسهای گونه و بینی یک BA برای شرایط بی هوازی و BA +CA
(middle ear) کشت ترشحات- از گوش میانی (مرتبط با پشت حلق) Otitis media-post pharynx BA+CA
(conjunctival) کشت ترشحات از چشم- در موارد التهاب ملتحمه و یا هر نوع ترشحات چرکی چشم BA+CA
ب- دستگاه تنفسی- پایینی- شامل نای، نایژک- تا بافت ریه
(Laryngeal) در موارد لارنیژیت- نمونه آسپیره شده از حنجره در موارد گروپ حلق- حنجره- هیچگاه سوآب به حلق نزنید چون سبب انسداد راه نای و خفگی می شود. BA+CA
(Bronchoalveolar) در موارد درگیری نای و نایژک ها (BAL)
BA+CA
در این موارد برای شناسایی احتمالی PCR TB بفرستند و یا حتی المقدور اسمیر AFB تهیه و رنگ شود.
در این مورد تقاضای رنگ آمیزی زیل نلسون (گرم) برای اسید فاست و یا با رنگ کینیون (سرد) رنگ آمیزی شود.
(Sputum) خلط: در ذات الریه های حاد باکتریال- نمونه خلطی قابل ارزش است که در رنگ آمیزی دارای تعداد قابل توجه 25-20 سلولهای التهابی نوتروفیلی و یا لنفوسیتی باشد- نمونه خلطی که فقط حاوی سلولهای پوششی (epithelial) است ارزش کشت و اسمیر ندارند.
(sputum) خلط: باید از عمق سینه بالا بیاید و در ظروف استریل دهان گشاد گرفته شود. نمونه خلطی که در لوله کشت گرفته می شود برای انجام کار باکتریولوژی بسیار خطرناک است.
(sputum) خلط- MacConkey یا EMB + CA + BA
Sputum در صورت تقاضای برای رنگ اسید فاست انجام دهید.
ولی کلاً موارد مشکوک به بیماری سل باید به بخش بهداشت انقلاب-- بلوار سیبویه انتقال یابد.
نمونه آبسه های ریوی (Lung-Abscess) که باید بوسیله سرنگ خالی از هوا کشیده شده باشد. BA+CA+EMB+Thio
لزوماً چند اسلاید رنگ آمیزی کنید.
نمونه آبسه های مغزی (Brain abscess):
BA´2+EMB+CA+Thio
چند اسلاید رنگ آمیزی کنید.
Aspirated Lung abscess: Thio BA´2+CA+EMB+
مایع پلور Pleural fluid BA+CA+Thio
در صورت تقاضا برای اسید فاست هم رنگ شود.
مایع پریکاردیال Pericardial fluid (مایع جنب پرده قلب)
BA+CA+Thio
ترشحات و چرک از مفاصل که یا بوسیله سرنگ کشیده شده باشد و یا ترشحات واض- از محل زخم با روش تمییز جمع آوری شده باشد. BA+CA+Thio+Smear
لزوماً برای کلیه چنین ترشحاتی رنگ آمیزی گرام انجام شود.
ترشحات زخم ها- جراحات- آبسه ها- جوش- کفیرگ و ... wound-discharge
BA+EMB(MaCconkey)+Thioglycolate+Smear
Deep abscess: از آبسه های عمیق تر- باید نمونه بوسیله سرنگ خالی از هوا کشیده شده باشد.BA+EMB+Thio در شرایط غیر هوازی نیز بگذارید. یک BA را در شرایط بی هوازی قرار دهید.
(Urine) نمونه های ادرار- نمونه گرفته شده با سوزن (سوپراپیوبیک) BA+EMBکه این نمونه ادرار را با لوپ 0.001cc (یک لاندا) کشت دهید. در کودکان و نوزادان لزوماً از لوپ cc 0.001 یا یک لاندا ادرار برای کشت استفاده کنید.
نمونه ادرار تمیز گرفته شده صبحگاهی (وسط ادرار) BA+EMB
نمونه ادرار راندوم (ادراری را که چند ساعت در مثانه باشد) BA+EMB در بزرگسالان از لوپ 0.01cc – 10 لاندا استفاده شود و بصورت شطرنجی کشت دهید تا شمارش کلنی آسان باشد.
Stool
نمونه کشت مدفوع- در کودکان و خردسالان زیر 2 سال
XLD+ Selenite broth +EMB
نمونه کشت مدفوع- در مواردی که به شیگلا و یا سالمونلا مشکوک باشد: XLD+SS+EMB+ Selenite broth
نمونه کشت مدفوع در مواردی که به انترو هموراژیک ای کلی (EHEC) Enterohemorrhagic E Coli مشکوک باشد.
(SMAC agar-Sorbitol MacConkey agar)+GN broth
نمونه کشت مدفوع برای موارد مشکوک به وبا
TCBS + Peptone water
Tetrathionate citrate Bile Salt Sucrose agar
نمونه کشت مدفوع برای مورد آلودگی و مسمومیت غذایی با استافیلوکوک Baird-Parker agar
نمونه کشت مدفوع برای موارد آلودگی با کمپیلو باکتر ژژونیmedium (اسکیرو) Skirrow می باشد. برای این محیط باید از خون تازه اسب ساخته شود و در شرایط کم هوازی قرار گیرد.)میکروآئروفیلیک(
محیط کشت:
محیط کشت غنی آگارخوندار BA=Blood agar
این محیطی با خصوصیات مناسب برای رشد اکثر باکتریهای بیماری زا است. بیشتر باکتریهای ایجاد کننده عفونت در انسان و حیوان می تواند بر آن رشد کند.
بعلاوه باکتریهای همولیز کننده خون را از باکتریهای غیر همولیز کننده جدا می کند محیط نباید هیچگونه آلودگی داشته باشد- سطح آن صاف و رطوبت سطح محیط کم باشد محیط نباید خشک و چروکیده باشد. در صورتیکه حتی یک کلنی در کناره های پلیت هم بود سعی کنید استفاده نکنید.
محیط شکلات آکار- همان محیط BA است. CA= Chocolate agar که خون در دمای 80 درجه لیز شده و رنگ شکلاتی بخود گرفته است. این محیط حاوی فاکتورهای رشد مانند NAD (نیکوتین اسید آدنین دی نوکلئوتید) است یا همان فاکتور V این محیط مغذی برای رشد باکتریهای مانند هموفیلوس آنفولانزا- موراکسلا کاتارالیس، نایسر یا مننژیتیدیس و سایر باکتریهای نیازمند به مواد غنی است. این محیط بهتر است در ظرف شمع (Candle Jar) قرار گیرد. یا در شرایطی که CO2 محیط 10% بیشتر از هوای معمولی باشد.
EMB) methylene blue agar ( Eosin
این محیط برای رشد باکتریهای گرام منفی راد انتروباکتریال ها است. برخی که لاکتوز را سریعاً و قوی تخمیر می کنند روی محیط- جلای فلزی ایجاد می کنند. (metallic sheen)
غیر تخمیریها مانند پسودوموناس و برخی آسینتوباکتر و برخی باکترهای دیگر بر آن رشد می کنند و کلنی های بنفش رنگ ایجاد می کنند.
XLD=xylose lysine dextrose
این محیط برای رشد بهتر شیگلاو سالمونلاها طراحی شده و برای کشت مدفوع نسبت به SS agar ترجیح دارد.
محیط اسکیرو:
این محیط ها بیشتر و بهتر برای رشد دادن کمپیلوباکترها و هلیکوباکترپیلوری طراحی شده اند. که برای هر کدام باید یک تقویت کننده آنتی بیوتیکی خاص اضافه شود.
Thayer-martin (Martin lewis) medium
این محیط همان شکلات آکار است که به آن مقادیر کم کلیستین (C) و انکوماسین ((V و نووبیوسین (N) اضافه شده- زمانی که از محیط های آلوده برای تشخیص میکروب سوزاک بخواهید جدا کنید بهتر است از این محیط استفاده کنید- نوع دیگر این محیط کوتریمکسازول زدن هم اضافه می کنند برای نمونه های رکتوم در موارد مشکوک به Proctitis گونوکوگی
در موارد عفونت های قارچی کاندیدایی، کرپیتوکوکی
Candida albicans and Cryptococcus neoformans
اگرچه این دو قارچ روی محیط های مانند مولر هیتون و آگار ساده و یا BA آگار هم رشد می کنند- ولی می توان از محیط Mycocel medium و یا سابرو دکستروز استفاده کنید.
در مواردی که کنار بلاد آگار BA´2 دو نوشته شده منظور این است که یک محیط بلاد آگار در شرایط بی هوازی قرار گیرد.
امروزه در آزمایشگاه های معمولی و یا تخصصی برای کشت سل (مایکوباکتریوم توبرکلوزیس کمپلکس) کار انجام نمی شود و کلیه نمونه ها باید به مراکز بهداشتی (در شیراز خیابان سیبویه) مرکز بهداشت ارجاع داده شود.
در صورتیکه برای تشخیص نمونه ای- روشهای ملکولی (PCR) یا Multiplex PCR خواستند به مرکز تحقیقات میکروب شناسی بالینی ارجاع داده شود.
برای کشت خون در مورد باکتریمی و سپتی سمی در بیشتر مراکز همان محیط کشت خون متداول همراه با سدیم پلی انتول سولفانات SPS+Tryptic soy broth+ استفاده می شود. در صورتیکه محیط BACTEC خواستند به مرکز نمازی و یا شهید فقیهی ارجاع داده شود.
SPS+TSB Routine Blood Culture medium
برای کودکان و نوزادان از محیط که 5 یا 10 سی سی حجم دارد استفاده کنید چون برای این سنین فقط 5/1 – 5/0 سی سی خون کافی است. برای افراد بزرگسال از محیط 50 سی سی استفاده کنید و 5 سی سی خون اضافه نمائید.
تقسیم کشت خون BACTEC یک سیستم اتوماسیون کشت خون است. که فقط در حال حاضر در بعضی مراکز مانند نمازی، شهید فقیهی و بیمارستان پیوند و امیر وجود دارد. این سیستم همیشه نیازمند به شیشه های (Bottle) های مخصوص است که در آن مراکز گاهاً یافت می شود.
پی نوشت:
آگار خوندار BA=Blood agar
محیط خوندار شکلاتی رنگ CA=Chocolate agar
محیط اِی اِم بی EMB=Eosin methylene Blue agar
ایکس اِل دی XLD=xylene Lysine dextrose
SS=Salmonella-Shigella agar
TCBS=Thiosulfate citrate bile salt sucrose agar
تی سی بی اس- مخصوص کشت باکتری مولد (وبا)
TSB+SPS = Tryptic Soy broth+sodium polyenthol- sulfonate
محیط مایع برای کشت خون
Blood-Culture medium
TM=Thyer-martin (Martin Lewis) medium
SD= Sabouraud dextrose agar
محیط سابرو- برای کشت بیشتر قارچها- مخمرها و قارچهای (مولد)
Thio=Thioglycolate medium
محیط- برای رشد میکروبهای بی هوازی اختیاری و بی هوازی ها باید توجه کرد که رشد بی هوازیها در پایین ترین قسمت لوله محیط کشت صورت می پذیرد.
Selenite broth: SF broth
برای تقویت رشد سالمونلا و برخی شیگلاها. البته باید حداکثر بعد از 6 ساعت که مدفوع در آن قرار گرفت روی محیط جامد (XLD) و ... پاساژ داده شود.
محیط آگار- حاوی مواد مغذی برای: Skirrow-medium
رشد کمپیلو باکترها و هلیکوباکترها: البته برای هر کدام از این دو باکتری باید Supplement های آنتی بیوتیک خاص اضافه شود- به کتب مرجع مراجعه نمایند. محیط Skirrow بعد از قرار دادن نمونه بر آن باید در شرائط (کم هوازی) microaerophilic قرار گیرد.
Dr.baratOboudi
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