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General Microbiology Laboratory Manual Medical Technology Department Islamic University-Gaza Dr. Abdelraouf A. Elmanama (Ph.D. Microbiology) 2009 ‫‪General Microbiology Manual‬‬ ‫قال رسول ﷲ صلى ﷲ عليه وسلم‬ ‫َ‬ ‫ﺍجملﺬﻭﻡ ﻛﻤﺎ ﺗﻔﺮ ﻣﻦ ﺍﻷﺳﺪ( ) ﻻ ﻋﺪﻭﻯ ﻭﻻ ﻃﲑﺓ ﻭﻻ ﻫﺎﻣﺔ ﻭﻻ ﺻﻔﺮ‪ ،‬ﻭﻓﺮ ﻣﻦ‬ ‫صدق رسول ﷲ صلى ﷲ علي...

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General Microbiology Laboratory Manual Medical Technology Department Islamic University-Gaza Dr. Abdelraouf A. Elmanama (Ph.D. Microbiology)

2009

‫‪General Microbiology Manual‬‬

‫ق‬

‫س‬

‫ﷲ ص ﷲ ع يه وس م‬

‫َ‬ ‫ﺍجﺬﻭﻡ ﻛﻤﺎ ﺗﻔﺮ ﻣﻦ ﺍﻷﺳﺪ( ) ﻻ ﻋﺪﻭﻯ ﻭﻻ ﻃﲑﺓ ﻭﻻ ﻫﺎﻣﺔ ﻭﻻ ﺻﻔﺮ‪ ،‬ﻭﻓﺮ ﻣﻦ‬

‫ص‬

‫‪2‬‬

‫س‬

‫ﷲ ص ﷲ ع يه وس م‬

‫_______________________‬ ‫‪Abdelraouf A. Elmanama‬‬ ‫‪Ph. D Microbiology‬‬

‫‪General Microbiology Manual‬‬

‫وصف ل س‬ ‫سم ل ‪:‬أحي ء قيق ي أس سي ع ي‬ ‫قم ل ‪MEDI 3101:‬‬ ‫ل صل‪:‬اأو ‪2009‬‬ ‫) سع ع ي (‬ ‫ع لس ع إج لي ‪ :‬س ع واح مع‬ ‫وصف ل س ‪ :‬ي ن و ال س ال واضيع ال ع ق ب ي ي اس ا ال ي روس و وال ع مل مع أكثر الص غ اس ام ً في‬ ‫م را ال ي روبيول ي‪ ،‬وكي ي اس ا ال حوص ال يوكي ي ئي مع ال ر ل عض أنوا ال يري ال رضي وكي ي تعري ھ‬ ‫ا الحيوي و تصني ھ وتأثيراتھ ‪.‬‬ ‫وع ھ ومن ثم شر م صل ل‬ ‫س بق ‪ :‬أحي ء ع م ع ي‬ ‫م‬ ‫ل س ‪:‬‬ ‫م‬ ‫◌ً اسم‪:‬‬ ‫‪J121‬‬ ‫ل ب‪:‬‬ ‫‪2673‬‬ ‫لھ تف‪:‬‬ ‫مس ع ‪:‬‬ ‫م‬ ‫‪Microbiology Laboratory Manual prepared by Abdelraouf Elmanama‬‬ ‫‪:‬‬ ‫ل جع ل‬ ‫‪(Diagnostic Microbiology) author: BAILEY &SCOT’S‬‬ ‫م جع أخ ‪:‬‬ ‫‪ .‬اس ا ال ي روس و ب لش ل ال ي ال ي يح من خاله تعريف الشرائح ال يري ‪.‬‬ ‫أھ ف ل س ‪:‬‬ ‫‪ .‬كي ي الحصو ع مزا ب يري نقي وخ لي من ال و ‪.‬‬ ‫واس امھ في تعريف ال يري ‪.‬‬ ‫‪ .‬ص غ ال يري بأنوا الص غ ال‬ ‫بھ ف تعريف ال يري ‪.‬‬ ‫‪ .‬ت يق ال حوص ال يوكي ي ئي ال‬ ‫‪ .‬ر تش يص ال يري س ل ال را من ع ئ ‪ Enterobacteriaceae‬و ‪Pseudomonas‬‬ ‫‪ .‬ر تش يص ال يري موج ال را من ع ئ ‪ Staphylococcus‬و ‪Streptococcus‬‬ ‫‪ .‬اس ا ال حوص ال ي ي ئي وال ص ي في تش يص أنوا ال يري س بق ال كر‬ ‫وتصني ھ وكي ي آلي تأثيرھ ‪.‬‬ ‫ا الحيوي ا الحس سي أنوا ال يري ال‬ ‫‪ .‬تح ي ال‬ ‫‪ .‬كي ي معرف الع ال ق ير ل يري في العين اأس سي بواس ال ر ال‬ ‫‪ .‬تح ير اأوس الغ ائي ل يري‬ ‫الحصو ع ق من ال ع وم ي ع ال لب ع ب اي ال ريق من خا تع م ه ال ي مع ال ي روس و‬ ‫ل تج ل قع أ‬ ‫يحصل ع يه ل لب‪ :‬في تش يص ال يري ال ي ت ت ص غ ھ ومن ثم اس ا ال حوص ال يوكي ي ئي في تعريف ال يري ‪،‬‬ ‫ا الحيوي‬ ‫واخ ي ال‬ ‫ومن ثم عز ال يري ال رض وتش يصھ ب ل ر ال ع في ھ ا ال‬ ‫ا الحس سي ال ن س لھ‬ ‫لح س ‪ :‬ي م ت يب ال لب ع اس ا الح سو في تش يص ال يري ال رض ال عزول حسب ال حوص‬ ‫س‬ ‫ال ي ي ئي ال ي ھر معه في ال ع ل بإ خ ال ي ن ال وب ع نوعين من ال رامج ال ص‬ ‫ال س م ولي ً ل عريف ال يري‬ ‫ج ‪ 30‬ام ح ن ر نص ي‪.‬‬ ‫ت يع ل ج ‪:‬‬ ‫ج ‪ 10‬بحث ع ش ل بو بوينت مع الشر ‪.‬‬ ‫ج ‪ 10‬ح و و تق ير مع ي ونش ‪.‬‬ ‫ج ‪ 50‬ام ح ن ر نھ ئي‪.‬‬ ‫ي م ات ع م ع ام ح ل ص ي و ل ھ ئي احق ‪.‬‬ ‫ت يخ ام ح ن ‪:‬‬

‫‪3‬‬

‫_______________________‬ ‫‪Abdelraouf A. Elmanama‬‬ ‫‪Ph. D Microbiology‬‬

General Microbiology Manual

‫خطة طرح المساق‬ Course Titles 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Hours

Introduction

1.5

Introduction to the oil immersion compound microscope Simple stain, Gram stain Acid fast stain The spore stain, and negative stain Isolation of pure culture and sterile transfer Bacterial motility. Amylase Production add Gelatin Liquefaction Catalase Production Coagulase Test Oxidase Production Methyl Red & Voges-Peoskauer Tests (MR-VP) Tryptophan Hydrolysis (Indole Test) Citrate Utilization Test, Urease Test Nitrate Reduction Test Media preparation & Sterilization Single Media / Multiple Tests, Triple Sugar Iron Agar Selective and differential media Bacterial oxygen requirements Anaerobic bacteria The serial dilution method of bacterial enumeration and generation time Microbial control agents Gram positive coccus identification Pseudomonas identification Enterobacteriaceae identification Identification of unknown bacteria Microbes in the atmosphere Microbes in the soil

1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 3 1.5 1.5 1.5 1.5 1.5 1.5 1.5

_______________________ Abdelraouf A. Elmanama Ph. D Microbiology

4

General Microbiology Manual

Table of Contents Exercise Introduction Microbiology Laboratory safety rules Glossary of terms Exercise 1: Introduction to the oil immersion compound microscope Exercise 2: Staining technique Exercise 2.1: Simple Stains Exercise 2.2: A. The Gram stain Exercise 2.2: B. The acid-fast stain Exercise 2.3: A. The spore stain Exercise 2.3: B. Negative Stain (CAPSULE) Exercise 3: Aseptic technique Exercise 3: A. sterile technique Exercise 3: B. sterile transfers Exercise 3: C. Isolation of pure cultures Exercise 4: Bacterial Motility Exercise 5: Catalase Test Exercise 6: Coagulase Test Exercise 7 : Amylase production Exercise 8 : Gelatin Liquefaction Exercise 9 : bacterial metabolism and carbohydrate fermentation Exercise 10: Oxidase Production Exercise 11: Methyl Red and Voges-Peoskauer Tests Exercise 12: Tryptophan hydrolysis ( Indole Production ) Exercise 13: Citrate Utilization Exercise 14: Urease Test Exercise 15: Nitrate production Test Exercise 16: Media Preparation & Sterilization Exercise 17: Single Media \ Multiple media Exercise 12: Selective and differential media Exercise 19: Bacterial oxygen requirements Exercise 20: Anaerobic bacteria Exercise 21: The serial dilution method of bacterial enumeration Exercise 22: Bacterial generation time Exercice 23: Microbial control agents Exercice 24: A. Gram positive coccus identification Exercice 24: B.Pseudomonas identification Exercice 24: C. Enterobacteriaceae identification Exercise 24: D. Identification of unknown bacteria Exercise 24: E. Microbes in the atmosphere Exercise 24: F. Microbes in the soil Selected website Appendices _______________________ Abdelraouf A. Elmanama Ph. D Microbiology

Page 7 7 9 11 16 17 20 25 28 32 36 36 39 41 45 53 55 59 60 63 65 67 70 71 73 76 80 88 92 96 99 102 106 109 112 115 117 119 123 124 126 127 5

‫‪General Microbiology Manual‬‬

‫م ل ل ي وبي ل جي)‪(J121‬‬

‫ح‬ ‫ح !!!!!!!! أنت ت ل في بي خ بي ل جي ل ع‬ ‫ل ل ع يك إت إ ش‬ ‫ل ح ص ل ي ومغ‬ ‫ق ل ل ء بإج ء ل حص ل ق‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬

‫يجب ل‬

‫ل‬

‫لخ‬ ‫لسام ‪.‬‬

‫لول ءب‬

‫ب ل لي‪:‬‬

‫إل ل‬ ‫آو ش‬ ‫أو ج ب‬ ‫ي ع م ب ت أكل و ل‬ ‫يجب غسل لي ين ب ل ء و لص ب ق ل ل ء ب ج ل حص‪.‬‬ ‫ل ق ي من أ عي م ض أث ء ل مل م ھ ‪.‬‬ ‫ل‬ ‫يجب ت ء لق‬ ‫يجب ت ء ل ف أبيض ل يف‪.‬‬ ‫ل‬ ‫خ ل ل ف م ع تغ ي أك‬ ‫ل ل وضع غ ء ل أ‬ ‫ع أخ‬ ‫عي م ض ‪.‬‬ ‫ل‪.‬‬

‫أث ء إج ء ل حص ل ق‬

‫يجب ل‬

‫ف أك‬

‫ان ھ ء من إج ء ل حص ل ق‬

‫ع‬

‫ماح‬

‫قسم ل ح ليل ل ي – لج م‬

‫يجب ل‬

‫ض يجب‬

‫سي ‪.‬‬

‫ل ق ل ل قت ل ح أو ل وج م ه و أ‬

‫ش ل م ع حسن ھ م م‬ ‫إسامي ‪-‬غ‬

‫أن ال لب‪ ....................................... /‬ق قرأ م و‬ ‫ال وقيع‪..............................:‬‬ ‫‪6‬‬

‫ل اب س ل ج ب ل‬

‫ب ل لي‪:‬‬

‫تأك من إع ج يع أ و ل س م إل م نھ ل صص ‪.‬‬ ‫تأك من إغا ك ف أجھ ل ي تم س مھ ‪.‬‬ ‫ع ن ف م ق ل ل ل صص بك‪.‬‬ ‫ح‬ ‫‪.‬‬ ‫قم ب ع ل ف أوا ثم لق‬ ‫به ل ج ب مامس لق‬ ‫في لحقي ل‬ ‫يجب وضع ل ف ل س‬ ‫ل صص به‪.‬‬ ‫إع ل ق ل‬

‫‪ /‬ي ع ل ج ع في ل‬

‫بأ‬

‫ب ل لي‪:‬‬

‫من ل قت‪.‬‬ ‫لا م في م ق ل ل لاس‬ ‫‪ ‬تح ي أ و وك ف ل‬ ‫ع ع لح ‪.‬‬ ‫‪ ‬يجب ع ل ج في ل ل إا ل و وبح و ن و ح‬ ‫ع ل ص من أ و لح ل س م في لح وي ل صص ل لك‪.‬‬ ‫‪ ‬ح‬ ‫ل‬ ‫‪ ‬في ح ت ضك أ ح ع ضي أو نس أ من ل ح ليل ل ي ي ئي أو ل ي‬ ‫ل ف ع ل ل ل قي ب إج ء لا م ل ح ع سام ك‪.‬‬ ‫إبا ل‬

‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬

‫س‬

‫ل‬

‫فع‬

‫ل‬

‫ل‪.‬‬

‫من إ ش ا وع يه أتعھ ب ال زا‬ ‫_______________________‬ ‫‪Abdelraouf A. Elmanama‬‬ ‫‪Ph. D Microbiology‬‬

General Microbiology Manual

Introduction Welcome to the microbiology laboratory. The goal of the laboratory is to expose students to the wide variety of lives in the microbial world. Although the study of microbiology includes bacteria, viruses, algae and protozoa, this lab will concentrate primarily on the bacteria. Microbiological techniques are important in preparing the students for the much harder task of identifying the pathogenic microorganisms in a clinical and environmental specimen. In this manual, I started each experiment with a brief theoretical introduction revealing the theoretical basis on which the experiment is based on, so that there will be a strong conjunction between the practical and theoretical sessions. Included in this manual also, the safety precautions which are essential for every one in the field of microbiology. Bacteria belong to the kingdom Monera. This kingdom contains more biological diversity than all other kingdoms combined. Most people tend to associate bacteria with disease, but less than ten percent of all bacteria cause disease. Many bacteria cannot even live at the temperatures found in and on the human body. In this lab, most of the bacteria with which we will be working are non-pathogenic (do not cause disease). However, some of the bacteria are opportunistic; that is, they can cause disease in an ill or injured person. Therefore, treat all bacteria as if they are pathogenic (cause disease).

Laboratory Safety Rules These rules are for the safety of the students, instructors and support staff. Please read and follow them. Failure to follow safety rules may result in removal from the class. 1. Wear a lab coat in lab. We will be working with a variety of materials that can cause permanent stains on some fabrics. Also, a lab coat can help protect from accidental contamination by microorganisms. 2. No eating or drinking during lab. Many pathogens spread by ingested food and drink. In addition, food can carry microorganisms that might contaminate laboratory cultures. 3. Keep long or fluffy hair tied up and out of the way. Hair can contaminate and be contaminated by microbial cultures. 4. Always wear shoes in lab. 5. Thoroughly wash your hands with soap and water before and after lab. Thorough and frequent hand washing easily and effectively controls the spread of many pathogens. 6. Clean the lab bench with disinfectant before and after lab. This helps to prevent contamination of cultures, books, clothing, etc.

_______________________ Abdelraouf A. Elmanama Ph. D Microbiology

7

General Microbiology Manual 7. Keep the lab bench free of unnecessary materials. Don't use the lab bench as a storage area for coats, books, etc. 8. Do not take cultures from the lab area. 9. Dispose of all contaminated materials in autoclave bags. When in doubt, ask the instructor. 10. Immediately report all accidents and spills to the instructor. Cover spills with disinfectant-soaked paper towels for at least 15 minutes before disposing of them. 11. Read all assigned materials before the lab session. Experiments will go smoother and have greater chances of success when you know what you will be doing ahead of time. 12. Treat all microbial cultures as if they are pathogens. Better safe than sorry. 13. When in doubt, ask the instructor. The only stupid questions are those that are intended as such.

NOTES: 1. Personal belongings are not to be stored in the laboratory. 2. You will be assigned to a group consisting of four students and you will work together in a semester long project. 3. Please read the safety instructions posted in the lab.

_______________________ Abdelraouf A. Elmanama Ph. D Microbiology

8

General Microbiology Manual

Glossary of terms Aerobic: Requires oxygen (opposite of anaerobic). Agar: Powder added to media for solidification. Air-dry: Drying of slide suspension in air before heat fixing and staining. Analog: Similar structure, but not identical. Antibody: Specific, protective protein produced by the immune system in response to an antigen. Antigen: Foreign, non self immunogenic material that elicits an immune response. Atrichous: Without flagella, nonmotile. Autoclave: Moist heat method of sterilization using pressure. Axial filament: A structure for motility used by the Spirochment bacteria. BHI: Brain heart infusion, a really good enrichment medium. Broth: media without agar. Brownian movement: Vibrations of an object seen in a microscope, not true motility. Candle jar: Candle burns in a closed container producing a carbon dioxide incubator, containing 2-10%O2 and around 10% CO2. CFU: Colony- forming unites CAN: Columbia naladixic acid media, selective (for Gram positive) and differential medium. Coliforms: Gram- rods which ferment lactose, non spore forming. Colony: A visible mass of bacteria growing on solidified medium, a clone. Differential stain: Uses 2 or more dyes which allow differentiation between different bacteria groups or structures. Counter stain: The 2nd dye added to a smear, taken in after the wall is decolorized, e.g. safrinin, methylene blue. Declorizer: The reagent used to remove the primary dye from the cell wall in a differential stain e.g. acid alcohol, acetone- alcohol.. _______________________ Abdelraouf A. Elmanama Ph. D Microbiology

9

General Microbiology Manual Primary dye: The 1st dye used in a differential stain, e.g. malachite green, crystal violet. EMB: Eosin methylene blue medium, selective (for Gram negative) and differential medium. Exoenzyme: Enzyme excreted away from the cell. Facultative anaerobe: Uses oxygen when present but can either ferment or an aerobically respire without it. Fastidious: Hard-to- grow bacteria, requiring grow factors or particular nutrients. Microaerophilic: Likes a reduced oxygen concentration. Obligate aerobe: Requires oxygen to grow. Fecal coliforms: Gram- rod which ferment lactose, non spore forming, GI flora in animals, in feces. Genus: Category of organisms with like features and closely related, divided into species. Heat- fix: Use of flame to 1. Coagulate proteins of suspension, causing adherence to slide. 2. Kill the microbes. IMVIC: Acronym= indole, methyl red, Voges- proskauer, citrate. MIC: Minimal inhibitory concentration of antibiotic that inhibits a bacterium. NA\NB: Nutrient agar or nutrient broth. Pathogenic: disease- causing. PCA: Plate count agar medium general all- purpose enrichment. Phenotype: Expression of gene as a trait. Plate count agar: Variation of nutrient agar, for optimizing counts of bacteria in sample. Streak plate: Procedure where pre-made agar plates have a sample of bacterium placed on tope of the agar and spread via a glass rod. Zone of inhibition: Area of no bacterial growth around a chemical on a disc indicates sensitivity.

_______________________ Abdelraouf A. Elmanama Ph. D Microbiology

10

General Microbiology Manual

Exercise 1: Introduction to the oil immersion compound microscope. Introduction Many students are probably familiar with the compound microscope from using it in previous biology classes. Figure 1 represents a typical compound microscope. A basic microscope consists of two lenses and the associated hardware to make viewing of specimens easier. The uppermost lens, called the ocular, is the part through which a person looks. The lower lens is the objective. Usually, several objective lenses are mounted on a turret, allowing rapid changing of objective lenses. The body tube holds the ocular and objective lenses in place. Most microbiological specimens are mounted on glass slides and placed on the stage.

Figure (1): A typical compound microscope. Individual microscopes may vary somewhat from this illustration. _______________________ Abdelraouf A. Elmanama Ph. D Microbiology

11

General Microbiology Manual Usually, clips or clamps hold the slide firmly to the stage. A light source and a condenser lens are located beneath the stage. The condenser focuses the light through a hole in the stage. The condenser usually includes an iris that varies the amount of light passing through the specimen. After passing through the specimen, the light goes through the objective and ocular lenses, and then into the eye of the observer. As light passes through various substances (glass, air, specimens, etc.), it bends. This bending of light is called refraction. The refractive index of a substance is a measurement of the extent that the substance bends light. Excessive refraction can cause distortion of the image. At magnifications of less than 500 x, the distortion is minimal. But at higher magnifications, the distortion becomes so great that image details are lost. An oil immersion lens helps to remedy this problem by eliminating the air gap between the specimen and the objective lens. A drop of special immersion oil is placed on the microscope slide, and the oil immersion objective lens is maneuvered so that it is touching the oil. Immersion oil has the same refractive index as glass so that the light passes through the slide, specimen, oil and objective lens as if they were a single piece of glass.

Figure (2): Changes in image composition coincide with changes in depth of focus. Depth of focus is inversely proportional to magnification and aperture diameter.

In this lab, you will become familiar with the use of the microscope (particularly oil immersion microscopy) and will compare the relative size and shape of various microorganisms. Most bacteria range in size between 0.5-2.0 micrometers (μm) There are three common shapes of bacteria: the coccus, the bacillus, and the spiral. Figure 3 represents a typical shape of bacteria.

_______________________ Abdelraouf A. Elmanama Ph. D Microbiology

12

General Microbiology Manual

Figure (3): represents a typical shape of bacteria.

Some Concepts to Consider Resolution: Resolution is the ability to distinguish between two points; The closer the two points, the higher the resolution. Magnification: Relative enlargement of the specimen, the total magnification of the image is calculated by multiplying the magnification of the ocular by the magnification of the objective. Depth of focus: thickness of a specimen that can be seen in focus at one time; as magnification increase the depth of focus decrease. Field of vision: the surface area of view; the area decrease as magnification increase. Numerical aperture (N.A.): the amount of light reaching the specimen; As N.A. increase the resolution increase.

_______________________ Abdelraouf A. Elmanama Ph. D Microbiology

13

General Microbiology Manual

Materials Each student/team: Microscope. Immersion oil. Lab supplies: Prepared stained slides of bacteria. Selected other prepared slides.

Procedure 1. Obtain a prepared slide of mixed bacteria. Mount the slide onto the stage of the microscope. 2. Start with the lowest power objective in place. Using the course adjustment knob, move the objective lens to its lowest point. Look through the ocular and focus upward with the coarse adjustment until an image comes into view. Use the fine adjustment to obtain maximum clarity. From this point on, do not use the coarse adjustment; doing so can result in damage to the lens, slide or both. Adjust the iris to allow enough light for maximum visibility and contrast. Usually, this will be about half the maximum iris opening. Too much light can wash out the details of the image. 3. Move the slide to a point of interest. Move the next objective lens into place and adjust the fine focusing knob, and adjust the iris as necessary. Repeat this step with the highest power, non-oil lens. 4. Note that as the power of the objective lens increases, the distance between the objective and the specimen (working distance) decreases. Also, as magnification increases, the field of view (visible area) and depth of field/focus (visible thickness) decrease. Moving the fine adjustment up and down allows viewing of other areas along the depth of thickness of the specimen (Figure 8). 5. To use the oil-immersion lens, move the turret halfway between the high-power air (non-oil) lens and the oil lens. Place a drop of immersion oil directly on the slide. Mov...