.. تبسيط لشرح عملى الميكرو ..
دى تجيمعه لشرح عملى الميكرو
اللى فى ساحه حوار دكتور عبد العزيز
فكره
احد زملائنا
Summary of section one
MIC by broth dilution method
Equipments
1-two pipettes 5ml & 1ml
2- ten test tubes six are empty & four are filled (one filled with M.O& another with 4ml A.B(number it 1 using glass pencil) & two filled with n.broth
Summary of steps:- All the exp THREE steps ONLY
1-number the 6 empty test tubes 2,3,4,5,+ve and –ve using the glass pencil
then by using the 5ml pipette transfer 2ml broth to all these 6 empty tubes
2-Two fold serial dilution of the A.B using 5ml pipette
Transfer 2ml of A.B. from test tube A (numbered as 1) to test tube 2 & shake
well then transfer 2ml. from test tube 2 to test tube 3 & shake well then
transfer 2ml from test tube 3 to test tube 4
Repeat till reaching tube no. 5 & reject 2ml from test tube 5.... don't forget
3-By using 1ml pipette transfer 0.2 ml of M.O to test tubes 1,2,3,4,5 & +ve conrtol
i.e to all tubes except -ve control
Quiz on 1st section
1-MIC is the abbreviation of………& defined as……….0
2-A.B diluted by…….fold serial dilution in MIC determination by broth dilution methodology
3-MIC measured in the units of……….0
4-MIC = ………………in MIC determination by broth dilution method
5-The rule of +ve control in MIC determination by broth dilution is…….and it contain ……and……. And it shows…….after incubation while the –ve control rule is………and it contains…….only And it still………..after incubation
6-test tubes are incubated at…….۰c for……..hours in MIC determination by broth dilution method
لو سمحت يا دكتور ممكن حضرتك تشرح antibiotic senstivity
و ليه احنا بنستخدم saline في agar diffusion method
شكرا لحضرتك..........antibiotic sensitivity test
ال test ده بيتعمل علشان اشوف احسن antibiotic لعلاج infectious disease معين
الفكرة كلها انى بجيب antibiotic discs مختلفة و طبق اجار......بعمل culturing لل microorganism من العينة المفصولة من infectious disease على طبق الاجار...وبعد كده بحط ال antibiotic discs على سطح الاجار ده الى انا عملت culturing لل microorganism على سطحه
بدخل الطبق ال incubator وبعد 24 hours اشوف ايه هو ال antibiotic الى ادانى اعلى inhibition zone (تقاس بالمسطرة ) يبقى هو ده the most effecient antibiotic can be used in the treatement
لاحظ ان الشركات الى منزلة ال antibiotic discs بتكون منزلة معاه pamphlet وتقولك لو انت قست ال inhibition zone وطلعت مثلا:
اقل من 12 mm ده معناه ان microorganism resistant لل antiobiotic ده (اذن مينفعش استخدمه للعلاج)
اكتر من 12mm ده معنا ان microorganism sensitiveلل antiobiotic ده (اذن ممكن استخدمه للعلاج)
زى الجدول الى موجود فى شرح العملى
بالنسبة للسؤال التانى:
ال dilution بتاع ال antibiotic فى السكشن الاول MIC by broth diltuion method كنت بستخدم ال broth لعمل ال dilution لانها كانت هى ال medium المستخدم لنمو ال microorgainsm .... لو انت فاكر كنت بعد ما بخلص بحط فى الانابيب كلها 0.2 ml من microorganism ..... و بعد ما اعمل incubation for 24 hrs اشوف انهى انبوبه clear وانهى كانت turbid زى ما كنا منزلين فى demonstration
اما ال dilution بتاع ال antibiotic فى السكشن التانى MIC by agar diffusion method كنت بستخدم ال saline لعمل ال dilution لان الاجار هنا هو ال medium المستخدم لنمو ال microorgainsm .. فهنا مش محتاج broth فبحضر ال dilutions فى saline عادى.. وكنا بنحضر ال seeded agar (اجار فيه ال microorganism الى بقيس MIC of antibiotic against it) و نعمل ال cups..و احط drop من كل dilution فى كل cup... واجى تانى يوم بعد incubation period اشوف inhibition zone الى عملها كل antibiotic dilution نتيجة ال diffusion بتاعته فى الاجار....وبعد كده اقيس ال zoned ده واعمل الجدول وارسم ال curve ومن ال curve ممكن اطلع MIC
summary of section two
equipements
test tubes: 3 empty test tubes mark them with glass pencil 2, 3, 4 and 3 filled test tubes (one is saline test tube, one is antibiotic 4ml and the last one is wasserman of microorganism)0
two pipettes (5ml and 1ml)0
melted agar flask and two petri dishes
glass dropper (pastuer pipette)0
loop
steps
three steps
1- preparation of antibiotic dilutions by two fold serial dilution method
as (MIC broth) transfer to three empty test tubes (2, 3 and 4) 2ml saline using 5ml pipette then transfer 2ml from the antibiotic test tube to test tube 2, shake well then take 2ml from it and transfer to test tube 3 and so on till reaching test tube 4
2-preparation of seeded agar
when the melted agar cool to about 50-55 transfer to it 0.1ml microorgainsm using 1ml pipette, shake the flask well clockwise and anticlockwise, then pour agar to the two plates continously
3-preparation of cups and application of each A.B dilution to the corresponding cup
هنرسم الطبق ونحدد ال center بتاعته زى ما اتفقنا ونقيس 2cm او 2.5cm من جدار الطبق علشان نحدد اماكن cups و نستخدم ال wasserman tube علشان نعمل ال cups ونشيلها باستخدام loop
بعد كده باستخدام dropper نعمل application ل two drps من كل A.B dilution مع مراعاة ان
1 يبقى ادامه 2 و 3 ادامها 4
وابدا ب4 ثم 3 ثم 2 ثم 1
Quiz on 2nd section
1- MIC defined as.......while MBC defined as.............0
2-what's meant by inhibition zone
3-inhibition zone measured in the units of.................0
4-M.O added to agar when its temperature becomes.....................0
5-what's meant by seeded agar
6-during application of the A.B in MIC determination by agar diffusion methodology to corresponding cups using Pastuer pipette we start with the highest conc to the lowest conc (true or false)0
7-How to calculate MIC in MIC determination by agar diffusion methodology
8-A.B diluted by ............. during MIC determination by agar diffusion methodology
9- the result of antibiotic combinations may be either ........... or ........ or ............0
10- what's meant by culture sensitvity testing
لو سمحت ياdr....ايه الbrowns standard opacity tubeده ...يعنى هو ال salineولا ايه ...وايه الفرق بينه وبين الbrowns opacity tubesوليه بنستخدم ال tubes دى .....معلش يا drاصلى قرات السكشن ومش فاهمه الحاجات دى ..فارجو التوضيح .....شكرا
brown opacity test tubes دول عبارة عن انابيب فيها baso4 راسب ابيض....الهدف من استخدامهم هو تظبيط عدد ال bacteria فى ال vaccine product الاخير الى تلاته فى عشرة اس تمانية cell/ml
فعندنا اتنين test tubes
brown opacity test tube II دى فيها baso4 راسب ابيض ال opacity بتاعته بتكافؤ ال opacity بتاعت bacterial suspension عدد ال cellsالى فيه ستة فى عشرة اس تمانية cell/ml
brown opacity test tube I دى فيها baso4 راسب ابيض ال opacity بتاعته بتكافؤ ال opacity بتاعت bacterial suspension عدد ال cellsالى فيه تلاته فى عشرة اس تمانية cell/ml
بعد ما مبنعمل ال bacterial susp وانقله للانبوبة الفاضية بقارن ال opacity بتاعته ب opacity بتاعت brown opacity test tube II ده...لو زى بعض يبقى انت اتاكدت ان عدد الcells فعلا ستة فى عشرة اس تمانية cell/ml
كنا بعد كده بنحط ال test tube الى فيها ال bacterial susp فى ال water bath درجه حرارته 60 لمده ساعة بعد كده باخد عشرة ملى من ال bacterial suspension الى عدد ال bacteria الى فيه ستة فى عشرة اس تمانية cell/ml واحطهم فى vaccine bottle واحط عليهم عشرة ملى phenol
لما بنحط عشرة ملى phenol على العشرة ملى bacterial suspension الى عدد ال bacteria الى فيه ستة فى عشرة اس تمانية cell/ml مش كده عدد cells هيقل للنصف ويبقى تلاته فى عشرة اس تمانية cell/ml وهو ده العدد الى انا عاوزه فعلا فى ال product الاخير....اتاكد ازاى....انى اقارن ال opacity بتاعت الproduct الاخير ب opacity بتاعت brown opacity test tube I
كنت بقول هل ينفع احضر heat killed bacterial vaccine من ال bacillusاحنا كنا سمعنا يا د. ان حضرتك اديت سؤال بيجى ف الشفوى لسكشن حضرتك ..ياريت من فضلك لما حضرتك تدخل تانى تقولنا عليه .......وشكراااااا
طبعا مش هينفع علشان ال bacillus لما بتتعرض لدرجة حرارة 60 او لما بتتعرض لاى drastic conditions مش بتموت ...بتكون spore وتفضل عايشة وبالتالى لم اشيلها من درجة الحرارة ده ال spores هتتحول تانى ل vegetative cells و بالتالى لما اجى احقنها مش هتعمل immunization ده هتعمل infection
summary of 3rd section
instrument
1-two slant agar of bacteria from which the vaccine will be prepared
2-two sterile distelled water tubes
3-empty test tube
4-brown opacity test tubes I and II
5-loop
6-pipette 5ml
7-phenol 1% test tube
8-vaccine bottle
steps : all the steps are three steps
1-preparation of bacterial suspension
using 5ml pipette transfer 6.5ml water to each slant agar then put the pipette on the rack and its tip beside the flame as it will be used at the end of the experiment
then sterile the loop by red heating then scratch carfully the microorganism from the agar surface ....then roll each slant between hands for one min then pour the contents of each tube to the empty test tube
after this step the opacity of bacterial suspension should be the same as opacity of brown opacity test tube II
2- heat killing
write ur name using glass pencil on the tube and put it in water bath its temp 60 for about 1 hr
3- transfer 10ml of bacterial susp to the vaccinebottle using the 5ml pipette and add to which 10ml phenol 1%
after this step the opacity of bacterial suspension should be the same as opacity of brown opacity test tube I
sterility test summary
transfere one ml from the sample to the fluid thioglycolate medium and 1ml to the fluid sabauroud's medium
quiz on 3rd section
1- brown opacity test tube prpared by adding........on.........0
2-brown opacity test tube II conc is .........the conc of brown opacity test tube I
3-vaccine is defined as...........0
4-heat killed bacterial vaccine may be prepared from spore forming bacteria as bacillus (true or false)0
5-stock vaccine is....... while autogenous vaccine is....0
6-storage temp of the vaccine is..........0
7-heat killing occur at........C
8-thioglycolate medium used to detect..........while sabauroud's medium used to detect.......0
9- and ........... and .............. and ..............are are examples on aerobic bacteria, anaerobic bacteria and fungi respectively
summary of section four
equipements
test tubes: 2 empty test tubes mark them with glass pencil S2, T2 and 4 filled test tubes (one is saline test tube, one is standard antibiotic 4ml S1, one is the test antibiotic 4ml T1 and the last one is wasserman of microorganism)0
three pipettes (two 5ml and one 1ml)0
melted agar flask and two petri dishes
two glass dropper (pastuer pipette)0
loop
steps
three steps
1- preparation of antibiotic dilutions by two fold serial dilution method
transfer to two empty test tubes (S2 and T2) 2ml saline using 5ml pipette then transfer 2ml from the antibiotic test tube S1 to test tube S2 shake well
then using the other 5ml pipette take 2ml from test tube T1 to test tube T2 and shake well
2-preparation of seeded agar
when the melted agar cool to about 50-55 transfer to it 0.1ml microorgainsm using 1ml pipette, shake the flask well clockwise and anticlockwise, then pour agar to the two plates continously
3-preparation of cups and application of each A.B dilution to the corresponding cup
هنرسم الطبق ونحدد ال center بتاعته زى ما اتفقنا ونقيس 2cm او 2.5cm من جدار الطبق علشان نحدد اماكن cups و نستخدم ال wasserman tube علشان نعمل ال cups ونشيلها باستخدام loop
بعد كده باستخدام dropper نعمل application ل two drps من كل A.B dilution مع مراعاة ان
S1 يبقى ادامه S2 و T1 ادامها T2
لاحظ ان كل A.B هيتحط ب dropper من 2 droppers الى نازلين
مع مراعاة ان ابدا ب s2 ثم s1 بال dropper الاول
و t2 ثم t1 بال dropper التانى
.gif "Fop (18)")
