2. Tools In Microbiology

TOOLS OF MICROBIOLOGY

Microscope  

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instrument used to study MO Optical instrument used to observe tiny objects that cannot be seen by the unaided human eye. Simple Microscope - contains only one magnifying lens Anton van Leeuwenhoek Compound Microscope - contains more than one magnifying lens. Compound light microscope. Hans Jansen and son Zacharias. Photomicrographs - photographs taken through the lens system of compound microscopes.

PARTS OF A COMPOUND MICROSCOPE 

Magnifying Parts: -To enlarge objects of study - objectives: - Eyepiece or ocular objective: variable magnification - Scanner: 5x magnification, used to study larger organisms - Low Power Objective (LPO): 10x magnification - High Power Objective (HPO): 40x magnification - Oil Immersion Objective: 100x magnification

Illuminating Parts: - Parts that modify light and illuminate object of study - Abbe condenser: concentrates light - Mirror: reflects light or uses bulbs as main light source - Iris diaphragm: regulates the amount of light that hits the object of study  Mechanical Parts: - Supports the magnifying and illuminating parts - Used to focus the lenses - Draw tube, body tube, revolving nosepiece, dust shield, arm, stage, stage clips, coarse adjustment knob, base, inclination joint 

I. TYPES OF MICROSCOPE A. VISIBLE LIGHT MICROSCOPY 1. Bright- Field Microscope ---used to observe morphology of the organisms. ---does not resolve very small specimens (viruses) 2. Dark-field Microscope ---”Dark” background, light organisms ---used to detect Syphilis (Treponema pallidum)

VISIBLE LIGHT MICROSCOPY 3. Phase-Contrast Microscope ---Observe dense structures ---To facilitate detailed examination of the internal structures of living specimens. 4. Fluorescent Microscope ---Ultraviolet light ---used to show antibodies

B. ELECTRON MICROSCOPE 1. Transmission Electron Microscope ---Highest magnification (10,000100,000x) ---Cellular ultra structure and viruses ---2-D image 2. Scanning Electron Microscope ---Surface structure of cells and viruses ---3-D image ---magnification: (1000-10,000x)

Metric System – used to describe sizes of microorganisms 1. decimeter 10 2. centimeter 100 3. millimeter 1000 4. micrometer 1M – bacteria, protozoa 5. nanometer 1B - viruses

II. STAINING PROCEDURES OBJECTIVES: 1. Kill the organism 2. Preserve morphology 3. Anchor smear to slide A. Simple staining - Aqueous or alcohol solution of a single basic dye - Used to determine size, shape and morphological arrangement - Ex. Methylene blue

Simple Staining 

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Based on shape: - Cocci: round or spherical bacteria - Bacilli: rod-shaped or cigar-shaped bacteria - Spirals: coiled bacteria - Spirillum: flexible coiled bacteria - Spirochetes: rigid, coiled bacteria Based on Arrangement of cells: - Strepto: bacteria in chains - Staphylo: bacteria in clusters - Diplo: bacteria in pairs - Tetra: bacteria in 4s

B. Differential staining – use of 2 or more dyes that may differentiate one type of organism from one another.

1. Gram Stain - used to classify MO - Dr. Hans Christian Gram (1884) - Procedure: V I A S a. Crystal Violet (primary stain) b. Gram’s Iodine (Mordant) c. Alcohol (Decolorizer) d. Safranin (Counterstain)

Differential staining 2. Acid-Fast Stain (Ziehl-Nielsen) - Binds strongly to the bacteria that have a waxy material in their cell wall - Used to identify Mycobacterium, Nocardia - Procedure: C A M a. Carbolfuchsin (Primary stain) b. Acid-alcohol (Decolorizer) c. Methylene Blue (Counterstain)

Gram staining Color

Gram (+) Blue

Pink

Gram (-) Pink

Red

Peptidoglycan Thick Layer

Thin layer

Techoic acid in Present cell wall

Absent

Lipopolysacch Absent aride in cell wall

Present

C.Structural Stains - Observe capsules, spores, flagella 1. Negative stain - demonstrate the presence of capsules - capsule (unstained halo) around bacterial cells against dark background. 2. Endospore stain - Malachite green 3. Flagella stain - Carbolfuchsin