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Microbiology Lab Bio 231 اﻷﺣﯾﺎء اﻟدﻗﯾﻘﺔ اﻟﺟزء اﻟﻌﻣﻟﻲ

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Microbiology Lab Bio 231 اﻷﺣﯾﺎء اﻟدﻗﯾﻘﺔ اﻟﺟزء اﻟﻌﻣﻟﻲ المملكة العربية السعودية Kingdom of Saudi Arabia Ministry of Higher Education وزارة التعليم العالي KING ABDULAZIZ جامعة الملك عبد العزيزUNIVERSITY Supervised فرع كلية العلوم للبنات Faculty of Sciences قسم اﻷحياء Dept. of Biological Sciences Microbiology Lab Bio 231 اﻷحياء الدقيقة الجزء العملي إعداد: إشراف: أ.اﻻء النهاري أ.د. رقية قشقري أ.أهله جمال د.هدى الشيخ أ.أروى زهيري د.منال قطان Weeks # Date The Subjects Introduction to Microbiology 1 11-2-2013 28-3-1434 Health and Safety in microbiology laboratories Controlling of Microbial 18-2-2013 2 Growth 8-4-1434 Microbial Culture Media Bacterial isolation and purification 25-2-2013 3 techniques 15-4-1434 Bacterial Morphology 4-3-2013 4 Staining and Description 22-4-1434 Simple Stain 5 11-3-2013 29-4-1434 Gram Stain The quantitative measurement of 6 18-3-2013 6-5-1434 bacterial growth 7 Holiday 8 1-4-2013 Introduction to Mycology 20-5-1434 8-4-2013 9 Introduction to phycology 27-5-1434 15-4-2013 10 Introduction to Protozoa 5-6-1434 Introduction to virology and Immunology 11 22-4-2013 12-4-1434 29-4-2013 12 Revision 19-4-1434 1 Lecture 1 INTRODUCTION TO MICROBIOLOGY Microbiology is the study of living organisms of microscopic size, which include bacteria, fungi, algae and protozoa and the infectious agents at the borderline of life that are called viruses. It is concerned with their form, structure, reproduction, physiology ,metabolism and classification. It includes the study of their distribution in nature, their relationship to each other and other living organisms, their effects on human beings and on other animals and plants, their abilities to make physical and chemical changes in our environment and their reactions to physical and chemical agents. 2 Health and Safety in microbiology laboratories اﻷمن والسﻻمة في مختبرات اﻻحياء الدقيقة Dont Do • Wear lab coat. • Do not play in the laboratory. • Wear gloves. • Do not use any laboratory. • Tie long hair neatly at the back of equipment until you are told to do the neck so. • Wear shoes that enclose your entire • Do not perform any laboratory feet. procedure until you are told to do • Wash your hands with disinfectant so. soap when you arrive at the lab and • Do not bring food or drinks into the again before you leave. laboratory. • Disinfect work areas before and • do not take cultures out of the after use laboratory. • Label everything clearly. • Never pipette by mouth. • Inoculating loops and needles should be flame sterilized in a Bunsen burner before you lay them down. • Sterilize equipment and materials. • Dispose of all solid waste material in a biohazard bag and container. 3 Laboratory Safety Signs 4 Instruments and tools used in microbiology laboratories اﻻجهزة واﻻدوات المستخدمة في معامل اﻻحياء الدقيقة Compound Microscope .1 أول من صنع المجاهر هو العالم الهولندي Antonyvan Leuwenhoek فان لوفينهوك عام )1632( والذي اخترع المجهر البسيط والمكون من عدسة واحدة. المجهر المركب من اهم المجاهر المستخدمة في الفحص اﻻولي في معمل اﻻحياء الدقيقة. السبب في تسمية هذه المجاهر بالمجاهر المركبة ﻻحتوائها على عدستي تكبير أحداهما موجودة في العدسة العينية Ocular Lenses والثانية موجودة في العدسة الشيئية .Objective Lenses 5 اجزاء المجهر الضوئي المركب Parts and Uses of Compound Microscope Part Functions eyepiece lens Ocular A series of lenses that magnify 10 times. Body tube Raised and lowered in focusing Base supports entire microscope Arm supports upper half of microscope Objective Lenses Usually four magnifications: 4x lower power, 10x low power, 40x high power and 100x oil immersion. Revolving Nose Piece can be rotated to change from one objective to another Stage Support the mechanical stage and microscopic slides. Iris diaphragm Control the amount of light striking the object Condenser Condenses the light waves Coarse adjustment moves body tube up and down rapidly for propose of approximate focusing Fine adjustment moves body tube up and very slowly for propose of definitive focusing 6 Parts of Compound Microscope 7 Safety Cabinet .2 غرف عزل الميكروبات المجهزة بنظام دفع الهواء واﻻشعة الفوق بنفسجية لتحد من عملية التلوث او اﻻصابة خﻻل عزل الميكروبات الممرضه. Autoclave .3 جهاز للتعقيم ويتم فيه التسخين بواسطة الضغط. Hot air oven .4 اﻷفران التي تسمح بتسخين الهواء إلى درجات حرارة مختلفة وعادة ما يستخدم هذا الجهاز في تعقيم اﻷدوات التي ﻻ تتأثر بالحرارة العالية مثل الزجاجيات بأنواعها. Incubator .5 جهاز يستخدم لتحضين الميكروبات في درجات حرارة مناسبة , حيث يحتوي على جدار عازل يوفر منطقة معقمة ومعزولة لتنمية الميكروبات مع القدرة على التحكم في درجة الحرارة, غالبا ما يحتوي على باب خارجي بداخله باب اخر زجاجي يسمح بمراقبة نمو الميكروبات داخل البيئات بدون تلوثها. Water bath .6 الحمام المائي يستخدم للتسخين الغير مباشر باستخدام الماء, يستخدم عادة في اذابة البيئات واجراء التفاعﻻت المختلفة. Centrifuge .7 جهاز الطرد المركزي يستخدم لفصل العينات المختلفة اعتمادا على سرعة الدوران واختﻻف تراكيز وكثافة المواد, وتختلف انواعه وأشكاله وأحجامه. Balance .8 و يستخدم لقياس كتلة المواد المختلفة وله أشكال مختلفة فمنها ما يقيس لرقمين عشريين وهو اﻷكثر شيوعا ومنها ما يصل لقراءة خمس أرقام وأكثر. Inoculation loop or Inoculation needle .9 ادوات التلقيح تختلف انواعها وأشكالها حسب استخداماتها. بعضها لﻻستخدام المتكرر ويمكن تعقيمها وبعضها اﻻخر يستخدم مره واحدة فقط ويتم التخلص منها. Petri-dishes .01 طبق بتري عبارة عن وعاء مسطح دائري الشكل وشفاف مع غطاء، يصنع اما من الزجاج او البﻻستيك، لها اغراض مختلفة ولكن اهمها تستخدم كأوعية للبيئات التي تستخدم لتنمية الميكروبات. Glass Ware .11 مختلفة اﻻشكال ومتعددة اﻻستخدامات Some Glassware Used in Microbiology Lab 8 Pipettes .12 الماصات متعددة اﻻشكال واﻻستخدامات واﻻحجام, منها الزجاجية او البﻻستيكية, متكررة اﻻستخدام او التي تستخدم لمرة واحدة فقط. Slides and cover slips .11 الشرائح واغطيتها الزجاجية تستخدم لفحص العينات بواسطة المجهر الضوئي Cork borers .11 الثاقب الفليني يستخدم غالبا لقطع دسك دائري من اﻻنسجة والبيئات المختلفة Bunsen flame .11 اللهب 9 Lecture 2 Controlling of Microbial Growth Methods of destroy, remove or inhibit the growth of microorganisms. Definitions Sterilization: The process to destroy all viable microbes (bacteria, viruses, fungi) including endospores. Sterilant: Material or method used to remove or kill all microbes. Disinfection: The process to destroy vegetative pathogens, not endospores and resistant organisms or bacterium spores. Disinfection Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. 10 Disinfectant: A substance or method used to kill microbes on surfaces. Antiseptic agents: Use of chemical agents on skin or living tissues to inhibit or eliminate microbes. Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them. Bactericide: An agent that kills bacteria (Most do not kill Endospores). Microbial Control Methods Physical Chemical Mechanical Methods Methods Methods 11 Physical methods 1. Heat Sterilization: Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents There are 2 procedures depending on the tolerance of the material used: a) Dry heat Dry oven: Time and Temperatures: 170⁰C for 1 hour or 160⁰C for 2 hours Note: Exposure time begins only after the sterilizer has reached the target temperature. Do not overload the sterilizer. Uses: To sterilize Forceps, Scissors, Scalpels, Swabs. Disadvantages: Plastic and rubber items cannot be dry-heat sterilized because temperatures used (160–170 C) are too high for these materials. 12 Flaming Holding Inoculation loop, Forceps and spatulas in a Bunsen flame till they are red hot. b) Moist heat: • Temp below 100oC: Pasteurization • Temperature at 100oC: Boiling. • Steam under pressure: Autoclave. Uses: • Serum or body fluids. • Liquids and Drinks • Media Autoclave: Autoclave works on the principle of Steam under pressure; it was invented by Charles Chamberland in 1879. Time and Temperatures: 121oC/10-40min Disadvantages: -Non stainless steel metal items corrode. -May damage plastic and rubber items. 13 2. Radiation: Two types of radiations are used NON –IONISING Electromagnetic rays with longer wavelength, Used in rapid mass sterilization of Syringes and catheters such as UV. IONISING High penetrative power rays, Used to Sterilize plastics Syringes, catheters, grease fabrics metal foils. Such as X- rays, gamma rays. Note: No appreciable increase in the temperature – COLD STERILISATION Chemical Methods The chemical compounds used can be: 1. Liquid Sterilization 2. Gas Sterilization Disadvantages: Generally, chemical sterilization procedures have the disadvantages of presenting health hazards to users (e.g. poisonous, flammable). 1. Liquid Sterilization Alcohol: Act as surfactants dissolving membrane coagulating proteins of vegetative bacterial cells and fungi, Such as 70% aqueous solution of ethyl alcohol and isopropyl alcohol. Uses – Disinfection of clinical thermometer. – Disinfection of the skin and Venupuncture. 14 Phenol: Phenol and phenolic compounds (e.g. lysol) lyse the cell membrane and denature proteins at 1-2% (aqueous solution). Uses: – Various combinations are used in the control of pyogenic cocci in surgical & neonatal units in hospitals. – Aqueous solutions are used in treatment of wounds. ALDEHYDES: Formaldehyde is bactericidal, sporocidal & has a lethal effect on viruses. Glutaraldehyde is effective against Tubercle bacilli, fungi and viruses Uses: To treat corrugated rubber anesthetic tubes, Face masks, Plastic endotracheal tubes, Metal instruments and polythene tubing. 2. Gas Sterilization: Primary gas used in hospitals and clinics, such as Ethylene
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