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3 Microbiology: It is the science that deals with the study of micro-organisms (very small organisms) that are invisible to the naked eye It is the science that deals with the study of micro-organisms (very small organisms) that are invisible to the naked eye

4 Experiments and procedures involving handling of living pathogenic micro- organisms. Experiments and procedures involving handling of living pathogenic micro- organisms. Handling and examining cultures of microorganisms. Handling and examining cultures of microorganisms. → good aseptic technique → good aseptic technique (not contaminated/ not inadvertently dispersed) even if the microorganisms being studied are not considered to be pathogenic. even if the microorganisms being studied are not considered to be pathogenic. Any culture of any organism should be handled with respect for its potential pathogenicity.

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6 Sterilization: Sterilization: Killing or removal of all living micro- organisms (from a particular location or material). Killing or removal of all living micro- organisms (from a particular location or material). Sterile article: completely free of all living micro-organisms Disinfection: Disinfection: Destruction of vegetative conspiring micro-organisms. Destruction of vegetative conspiring micro-organisms..

7 Disinfectants: Disinfectants: Chemicals which cause disinfection Chemicals which cause disinfection Bacterial spores, mycobacteria, some viruses → considerable resistance Antiseptics: Antiseptics: Disinfectants which can be safely applied to skin & mucous membranes. Disinfectants which can be safely applied to skin & mucous membranes.

8 Contamination: Contamination: Introduction of undesirable m.o. Introduction of undesirable m.o. Asepsis: Asepsis: Processes designed to prevent m.o. from reaching a protected environment. Processes designed to prevent m.o. from reaching a protected environment. Aseptic technique: Practices used by microbiologists to exclude all organisms from contaminating media or contacting living tissues.

9 ☠ Lab coat & marker. ☠ No eating, drinking, or mouth pipetting. ☠ Benches disinfection. ☠ ☠ Inoculating loop sterilization. ☠ Aseptic technique. ☠ Discarded cultures & infectious materials. ☠ Broken or spilled living cultures. ☠ Broken or spilled living cultures. ☠ Microscope. ☠ Laboratory notes. ☠ At the end of each lab check: Gas tap is turned off. Water tap is closed properly. Microscope lamp is turned off. ☠ Finally wash your hands thoroughly.

10 Discard cultures and other infectious materials:  Petri dishes → Plastic bag → Autoclave.  Test tube cultures → wire basket → Autoclave.  Used pipettes → Plastic bag → Autoclave.  Used slides, covers, and pipettes → Jar containing a disinfectant.  Broken glass → swept in a dustpan → container for broken glass. NEVER place contaminated material in waste basket.

11 Broken or spilled living cultures:  Clothing → Autoclave plastic bag → Autoclave.  Flood the area with a disinfectant ( or paper towels are placed over the spills).  After min → wipe up & discard the waste in autoclavable dustpan→ Autoclave. 

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13 A Culture medium:- ❊ An artificial preparation which contains the essential elements and nutrients needed by the m.o to grow. (most bacteria &fungi) ❊ It may be: Liquid (broth) Liquid (broth) Solid (containing agar) Solid (containing agar) Semisolid (containing low conc of agar) Semisolid (containing low conc of agar) ❊ Strict intracellular organisms (e.g., some bacteria & all viruses)→ only cultures of living eukaryotic cells.

14 Inoculation: Inoculation: Culturing of sterile media with m.o [Inoculation loop]. Culturing of sterile media with m.o [Inoculation loop]. Incubation: Incubation: Placing the culture into the incubator at optimum temperature for growth. Placing the culture into the incubator at optimum temperature for growth.

15 Growth: Multiplication (↑number) to quantities sufficient to be seen by naked eye.. Multiplication (↑number) to quantities sufficient to be seen by naked eye.. Bacterial growth in the lab has 2 main forms: 1- Development of Colonies ( the macroscopic products of cell divisions of a single bacterium on solid media) 1- Development of Colonies ( the macroscopic products of cell divisions of a single bacterium on solid media) 2- Turbidity (macroscopic clumps) of a clear fluid medium. 2- Turbidity (macroscopic clumps) of a clear fluid medium.

16 Bacterial Growth

17 1- Macroscopical Examination (colony morphology): Characters of colonies.Characters of colonies. Hemolysis on blood agar.Hemolysis on blood agar. Pigment production.Pigment production. 2- Microscopical Examination: Examination of wet mount preparation.Examination of wet mount preparation. Examination of stained preparation.Examination of stained preparation.

18 3-Biochemical Tests: (The ability to attack various substances e.g., carbohydrate breakdown; (The ability to attack various substances e.g., carbohydrate breakdown; or to produce particular metabolic products e.g., enzymes. 4-Additional Tests: such as seriological tests such as seriological tests

19 Colony vs. Cell Colonies morphology (Macroscopical examination) Cells (Microcopical examination)

20 Colony vs. Cell Colonies morphology (Macroscopical examination) Cells (Microcopical examination)

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22 The microscope It is the most important tool used for examination and identification of microorganisms. It is the most important tool used for examination and identification of microorganisms. It produces magnified images of micro- organisms.

The microscope Types of microscopes Types of microscopes 1- Light microscope: 1- Light microscope:  Use light beams and lenses  The most common one used in the lab  Max. magnification is about times. 2- Electron microscope:  Use electron beams and magnetic fields  Magnification power is times or more.  Used for examination of viruses and sections of bacteria

24 The light microscope

25 The electron microscopy

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27 Light microscope vs. Electronic microscope Electron microscope light microscope

28 Light microscope vs. Electronic microscope Electron microscope Light microscope

29 Light microscope vs. Electronic microscope Electron microscope Light microscope

30 Electron microscope

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32 The Light Microscope Types: a- Simple microscope: single system of lenses a- Simple microscope: single system of lenses b- Compound microscope: has two lens system, the ocular lens and the objective lens The two lenses system give greater magnification The two lenses system give greater magnification

33 Components of the compound microscope Ocular lens Objective lens Lamp box Iris diaphram Stage Coarse adjustment Fine adjustmentSlide movement knob

34 Theoretical principles of the light microscope MagnificationResolution

35 Theoretical principles of the light microscope Magnification: It means enlargement of the linear diameter of an object. It is the function of two lens system [the ocular and the objective lens].

36 Total magnification = mag. power of ocular lens mag. power of ocular lens × mag. power of objective lens used × mag. power of objective lens used The magnification power of ocular lens (eye piece) is 10× The magnification power of objective lens Objective lens mag. Power Objective lens mag. Power 1. Scanning lens 4× 2. Low power objective lens 10× 3. High power objective lens 40× 4. Oil immersion lens 100×

37 The total magnification will be: 40 times → when using the scanning lens 40 times → when using the scanning lens 100 times → when using the low power lens 100 times → when using the low power lens 400 times → when using the high power lens 400 times → when using the high power lens 1000 times → when using the oil immersion lens 1000 times → when using the oil immersion lens

38 Working distance: It is the distance between the objective lens and the slide. It is the distance between the objective lens and the slide. General rule: As the magnification of the lens increase the working distance decrease. As the magnification of the lens increase the working distance decrease.

39 Resolution: It is the ability of a lens to reveal two closely adjacent points as separate and distinct points.

40 Resolution

41 Resolving power = 0.61× wavelength of light used 0.61× wavelength of light used numerical aperture. numerical aperture. Numerical aperture = n sin θ n = refractive index of the medium through which light passes before entering the objective lens. n = refractive index of the medium through which light passes before entering the objective lens. θ = an angle equal to half the angle of the maximum cone of light that may enter the objective. θ = an angle equal to half the angle of the maximum cone of light that may enter the objective.

42 The max. resolution power of the compound light microscope with the oil immersion lens is 0.2 µm The max. resolution power of the compound light microscope with the oil immersion lens is 0.2 µm (blue filter, the condenser at the highest position, oil immersion lens). ( µm → for the electron microscope) ( µm → for the electron microscope)

43 oil Objective lens Lost light Saved light specimen Light source The resolving power of the oil immersion lens depends on the addition of special oil (Ceder wood oil) between the specimen slide and the objective lens.

44 Uses of the light microscope 1. Identification of microbial groups. (Bacterial, Fungi, Protozoa) 2. Morphological studies of m.o (size, shape, arrangement…..) (size, shape, arrangement…..) 3. Physiological studies (motility and reproduction….) (motility and reproduction….)

45 Care of the microscope Transport: Use both hands. Uncluttered workstation. Avoid Electrical cord dangling → foot entanglement. Dust cover. Lens care: Cleaning lens tissue moistened with Xylene,  Occasional wiping off the top surface of the condenser with lens tissue.

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48 Examination of living bacteria for motility (Hanging – drop technique) For non-pathogenic bacteria : Direct observation of a drop from a liquid containing bacteria → an excellent method of studying motility Hanging – drop technique e.g. Hanging – drop technique In stained slide preparation the cells are heat- killed prior to staining→ the motility in not observable.

49 True motility:- it is the active directional movement of the organism from place to place. (several times the long dimension of the organism (several times the long dimension of the organism & In different directions) & In different directions) Brownian movement:- is a vibratory nondirectional movement of the cells due to their bombardment by water molecules in the suspension

50 Materials:  Culture of Proteus vulgaris  Slide, coverslip.  Plasticine.

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