Chapter 3 Flashcards by Daphmy Francois

Direction and magnitude of bending Is determined by the refractive indexes of the two media forming the interfaces

Bending of light

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Measure of how greatly a substance slows the velocity of light

Measure of the light bending ability of a medium

Refractive index

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Ability of a lens to separate or distinguish small objects that are close together

Resolving power

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Wavelength of light/ 2x nunerical aperture

Resolving power

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Function of the diameter of the objective lens to its focal length

Numerical aperture

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Bright field microscope
Dark field microscope
Phase contrast microscope
Fluorescence microscope

Light microscope

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Produces dark image against brighter background

Has several objective lenses

Bright Field microscope

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Used to study living microorganisms
Contains opaque disc that will block light from directly entering the objective lens. Only light reflected from specimen enters the objective lens

Darkfield microscopy

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No staining/Study living microbes
Image appear dark against light background. Has special objectives and a condenser that make cellular component visible which differ only slightly in their refractive indexes

Phase contrast microscopy

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Ability of substances to absorb short wavelength of light (UV) and give of longer wavelength light (visible)

Fluorescence

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Fluorescence dyes

An be chemically combined with an antibody, complex used in diagnosis to check the presence of an antigen(microbe)

Fluorochromes

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Uses ultra violet or near ultraviolet wavelength light source
Organisms with fluorchrome appear luminescent against dark background

Fluorescence microscope

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Transmission electron microscope (TEM)

Scanning electron microscope (SEM)

Electron microscope

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Examine objects smaller than 0.2 um like viruses and internal cellular structures
Electrons used
Images are black & white
Electromagnetic lenses instead of glass lenses

Electron microscopy

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Electrons scatter when they pass thirty ultra thin sections of a specimen
Transmitted electrons are used to produce image
Objects 10,000-100,000
Internal structures
Resolution 2.5nm

Transmission electron microscope

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Uses electrons reflected from surface of a specimen to create 3D image
No sectioning
Objects magnified 1,000-10,000x
External images

Scanning electron microscope.

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Bacterial smear is a fries preparation of bacterial cells on a glass slide
Require only small amount of the microbial culture

Smear

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Done by passing the air dried smear several times over the flame or using blow dryer
Smear is fixed
Coagulates bacterial protein so bacteria stick to the slide surface

Heat fixation

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Done to study the microbial properties and to group the microbes in specific groups for diagnosis

Staining

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Coloring microbes with dye that creates contrast between bacteria and background and emphasis microbial structures

Staining

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Organic compound contains benzene ring plus a chromosphere and auxochrome group

Stain

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Salts composed of a positive and negative ion, one of which is colored and is non as the chromophore

Stains

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Contains positive ions 
Crystal violet 
Methylene blue 
Malachite green 
Safranin

Basic dye

Contains negative ions
Eosin
Acid fuchsin
Nigrosin

Acidic dye

Used to observe the overall shape size and capsules | No heat fixation required

Negative staining

Simple Differential Special

Kinds of staining

Aqueous or alcohol solution of single basic dye Creates contrast between Bactria and background Highlights entire microorganism to study cell shape, size, arrangement Heat fixed

Simple staining

``` Methylene blue Crystal violent Carbolfuchsin Safranin Mordant** (sometimes) ```

Simple staining

Requires the use of at least three chemical reagents that are applied sequentially to a heat fixed smear 1. Primary stain 2. Decolorizing agent 3. Counterstain

Differential staining

Imparts it's color to all cell

Primary stain

Bases on chemical composition of cellular components.

Decolorizing agent

Contrasting color to that of the primary stain

Counterstain

Differential staining procedure developed by Hans Christian Gram in 1884

Gram staining

Primary stain crystal violet (basic purple dye) applied to heat stain

Gram staining step 1

After washing primary stain iodine (mordant) is applied that will make crystal violent complex Bacteria at this point appear dark violet or purple

Gram staining step 2

Alcohol, alcohol acetone solution applied as decoloring agent and removes purple color from Gram negative bacteria ** Gram Positive will retain purple color

Gram Staining step 3

Alcohol rinsed and the slide is then stained with Counterstain Safranin(basic red dye)

Gram Staining step 4

Have thicker layer if Peptidoglycan in cell wall. CV-1 complex is not washed by alcohol wash. They retain the CV-1 complex and remain purple

Gram positive bacteria

Have thinner layer of Peptidoglycan and also have layer of lipopolysachharide as part of their cell wall. Alcohol disrupts layer and CV-1 complex is washed through ***will remain colorless unless counter stain Safranin applied, become pink

Gram negative

Penicillins Cephalosporins *cannot penetrate lipopolysachharide layer of gram negative**

Beta Lactams

Uses to distinguish Myobacterium species and some species of Nocardia Bind strongly to waxy material of the cell wall

Acid fast staining

Carbol fushion(red dye) applied to fixed smear

Acid fast staining step 1

After cooling & washing smear washed with alcohol decolorizer * Non acid fast lose stain * *Acid fast retain dye, more soluble in lipid cell wall than in acid alcohol

Acid fast staining step 2

Counter stain methylene blue is used to stain non acid fast bacteria blue

Acid fast staining step 3

Schaeffer-Fulton stain 1. Heat fixed smear 2. Primary stain Malachite green applied, heated (stain wall) 3. Washed to remove excess 4. Counter stain Safranin applied to stain other parts pink

Endospore special stains

Determines microbes virulence Gelatinous covering that is soluble in water. India Ink or Nigrosin stains background dark Safranin used to stain cells Capsules look like Halos around stained cells

Capsule staining

Mordant used to build diameter When stained with Carbolfuchsin becomes visible under light microscope. Number and location used in diagnosis

Flagella Staining

Scanning

10x

Low power

40-45x

High power

90-100x

Oil immersion

Ocular lens

10x

Ocular lens x objective lens

Total magnification

Any kind of microscope that uses visible light to observe specimen Are compound microscope

Light microscope

Microscope remains in focus even objectives are changed

Parfocal

Acid fast color

Red

Non acid fast color

Blue