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MicrobiologyMicrobiology is the science that deals is the science that deals with the study of too small organism with the study of too small organism

(micro(micro--organisms) that are invisible to the organisms) that are invisible to the naked eyenaked eye

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Laboratory Rules and

Safety

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Instructions and RulesInstructions and Rules☠ ☠ No eating or drinking.No eating or drinking.

☠ ☠ Lab coat & marker.Lab coat & marker.

☠ ☠ Aseptic technique.Aseptic technique.

☠ ☠ Benches must be Benches must be disinfected.disinfected.

☠ ☠ ☠ ☠ Avoid contamination.Avoid contamination.

(Discarded cultures & (Discarded cultures & infectious materials and infectious materials and broken or spilled living broken or spilled living cultures). cultures).

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☠ ☠ Microscope.Microscope.

☠ ☠ At the end of each lab At the end of each lab check:check:

Gas tap is turned off.Gas tap is turned off.

Water tap is closed Water tap is closed properly.properly.

Microscope lamp is Microscope lamp is turned off.turned off.

☠ ☠ Finally wash your Finally wash your hands thoroughly. hands thoroughly.

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Introduction to Microbiological Introduction to Microbiological Equipments and MaterialsEquipments and Materials

MediumMedium:-:-

❊❊ An artificial preparation contains the An artificial preparation contains the essential elements and nutrients needed essential elements and nutrients needed by the m.o to grow (media)by the m.o to grow (media)

Culture media Culture media

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❊ It may be: Liquid (broth) Solid (containing agar) Semisolid (containing low conc. of agar)

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Introduction to Microbiological Introduction to Microbiological Equipments and MaterialsEquipments and Materials

Inoculation:Inoculation: Culturing of sterile media with Culturing of sterile media with m.o [Inoculation loop].m.o [Inoculation loop].

Incubation:Incubation: Placing the culture into the Placing the culture into the incubator at optimum temperature for incubator at optimum temperature for growth.growth.

Sterile:Sterile: Free from any living microorganism Free from any living microorganism

Contamination:Contamination: Introduction of undesirable Introduction of undesirable m.o.m.o.

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Sterilization:Sterilization: Killing or removal of Killing or removal of all living all living micro-micro-

organisms (from a particular location organisms (from a particular location or material). or material).

Sterile article:Sterile article: completely free of all completely free of all living micro-organismsliving micro-organisms

Disinfection:Disinfection: Destruction of Destruction of vegetative conspiringvegetative conspiring

micro-organisms.micro-organisms.. .

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Disinfectants:Disinfectants: Chemicals which cause disinfectionChemicals which cause disinfectionBacterial sporesBacterial spores, , mycobacteriamycobacteria, , some some virusesviruses → → considerable resistanceconsiderable resistance

Antiseptics:Antiseptics: Disinfectants which can be safely applied to Disinfectants which can be safely applied to skin & mucous membranes. skin & mucous membranes.

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Contamination:Contamination: Introduction of undesirable m.o.Introduction of undesirable m.o.

Asepsis:Asepsis: Processes designed to prevent m.o. from Processes designed to prevent m.o. from reaching a protected environment.reaching a protected environment.

Aseptic techniqueAseptic technique:: Practices used by Practices used by microbiologists to exclude all organisms from microbiologists to exclude all organisms from contaminating media or contacting living contaminating media or contacting living tissues.tissues.

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Discard cultures and other infectious Discard cultures and other infectious materials:materials:

Petri dishesPetri dishes→ Plastic bag → Autoclave.→ Plastic bag → Autoclave.Test tube culturesTest tube cultures → wire basket → → wire basket →

Autoclave.Autoclave.Used pipettesUsed pipettes → Plastic bag → Autoclave. → Plastic bag → Autoclave.Used slides, covers, and pipettes Used slides, covers, and pipettes → Jar → Jar

containing a disinfectant.containing a disinfectant.

Broken glassBroken glass → swept in a dustpan → → swept in a dustpan → container for broken glass.container for broken glass.

NEVER place contaminated material in waste NEVER place contaminated material in waste basket.basket.

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Broken or spilled living cultures:Broken or spilled living cultures:

Clothing Clothing → Autoclave plastic bag → Autoclave.→ Autoclave plastic bag → Autoclave.

Flood the area with Flood the area with a disinfectanta disinfectant ( or paper towels are placed over the spills). ( or paper towels are placed over the spills).

After After 20- 30min20- 30min→ wipe up & discard the waste in → wipe up & discard the waste in autoclavable dustpanautoclavable dustpan→ Autoclave→ Autoclave..

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Microscopical Examination:Microscopical Examination:Characters of cells.Characters of cells.

Identification of BacteriaIdentification of Bacteria

Macroscopical Macroscopical Examination:Examination:

Characters of colonies.Characters of colonies.

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Biochemical Tests.Biochemical Tests.

Identification of BacteriaIdentification of Bacteria

Additional Tests:Additional Tests:• such as seriological testssuch as seriological tests

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Colony vs. CellColony vs. Cell

Colonies Cells

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Colony vs. CellColony vs. Cell

Colonies Cells

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The MicroscopeThe Microscope

The microscopeThe microscope is the most important tool used is the most important tool used for examination and identification of for examination and identification of microorganisms. microorganisms.

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The MicroscopeThe Microscope

Uses of microscope:Uses of microscope:1.1. Identification of microbial groups. Identification of microbial groups.

(Bacterial, Fungi, Protozoa)(Bacterial, Fungi, Protozoa)

2.2. Morphological studies of m.oMorphological studies of m.o

(size, shape, arrangement…..)(size, shape, arrangement…..)

3. Physiological studies 3. Physiological studies (motility ….)(motility ….)

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The MicroscopeThe Microscope

Types of microscopes:Types of microscopes:

1.1. Optical microscope:Optical microscope: Use light beams and lensesUse light beams and lenses The most common one used in the labThe most common one used in the lab

2. 2. Electronic microscope:Electronic microscope: Use electron beams and magnetic fieldsUse electron beams and magnetic fields Used for examination of viruses and sections Used for examination of viruses and sections

of bacteriaof bacteria

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Optical microscope vs. Optical microscope vs. Electronic microscopeElectronic microscope

Electronic microscopeElectronic microscopeOptical microscopeOptical microscope

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Optical microscope vs. Optical microscope vs. Electronic microscopeElectronic microscope

Electronic microscopeElectronic microscopeOptical microscopeOptical microscope

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Optical microscope vs. Optical microscope vs. Electronic microscopeElectronic microscope

Electronic microscopeElectronic microscopeOptical microscopeOptical microscope

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The MicroscopeThe Microscope

Optical microscope:Optical microscope:There are two typesThere are two types a-a- Simple microscope: single system Simple microscope: single system

of lensesof lensesb-b- Compound microscope: has two Compound microscope: has two

lens system, the ocular lens and the lens system, the ocular lens and the objective lensobjective lens

The two lenses system give greater The two lenses system give greater magnification magnification

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Components of the compound Components of the compound microscopemicroscope

Ocular lens

Objective lens

Lamp box

Iris diaphram

Stage

Coarse adjustment

Fine adjustmentSlide movement knob

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Theoretical principles of Theoretical principles of microscopymicroscopy

Magnification:Magnification:

It means enlargement of the linear It means enlargement of the linear diameter of an object.diameter of an object.

It is the function of two lens system It is the function of two lens system [the ocular and the objective lens][the ocular and the objective lens]

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Theoretical principles of Theoretical principles of microscopymicroscopy

Total magnification =Total magnification =

mag. power of ocular lens X mag. power of mag. power of ocular lens X mag. power of objective lens objective lens used used

N.B N.B (oil immersion lens is used)(oil immersion lens is used)

Objective lens mag. PowerObjective lens mag. Power

1.1. Scanning lens 4×Scanning lens 4×

2.2. Low power objective lens 10×Low power objective lens 10×

3.3. High power objective lens 40×High power objective lens 40×

4.4. Oil immersion lens 100× Oil immersion lens 100×

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Theoretical principles of Theoretical principles of microscopymicroscopy

The magnification power of ocular lens The magnification power of ocular lens (eye piece) is (eye piece) is 10×10×

The total magnification will be The total magnification will be 40, 100, 40, 100, 400, 1000 times.400, 1000 times.

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Theoretical principles of Theoretical principles of microscopymicroscopy

Working distance:Working distance:

It is the distance between the objective It is the distance between the objective lens and the slide.lens and the slide.

General rule, as the magnification of General rule, as the magnification of the lens increase the working distance the lens increase the working distance

decrease.decrease.

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Theoretical principles of Theoretical principles of microscopymicroscopy

Resolution:Resolution: It is the ability of a lens to reveal two It is the ability of a lens to reveal two

closely adjacent points as separate and closely adjacent points as separate and distance.distance.The resolving power of the oil immersion lens depends on depends on the addition of special oil (Ceder wood oil) between the specimen slide and the objective lens.

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Resolution:Resolution:

Higher Resolution; note the “Sharpness” of the Image.

Lower Resolution; it is less sharp.

Whi

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Oil

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The Mineral Oil has the same Index of Refraction as glass (so light does not bend).

Air has a different Index of Refraction from glass (so light bends).

Air has a different Index of Refraction from water (so light bends).

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oil

Objective lens

Lost lightSaved light

specimen

Light source

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