Chap 3 Observing Microorganisms Through A Microscope

Front 1

What is Resolution?

Back 1

-The ability of a lenses to distinguish fine detail and stucture.
-A microscope with the resolving power of 0.4nm can distinguish between two points great than or equal to 0.4nm
-Shorter wavelengths of light provide greater resolution

Front 2

Principles of Light Microscopy

Back 2

-Refractive index is the light bending ability of a medium
-Immersion oil is used to keep light from bending

Front 3

Brightfield(light)Microscopy

Back 3

-Dark objects are visible agaisnt a bright background.
-Light reflected off the specimen does not enter the objective lens.

Front 4

Darkfield Microscopy

Back 4

-Light objects are visible against a dark background
-Light reflected off the specimen
-Special condenser
-Useful in diagnonsis of SYPHILLIS( T. pallidum)
-Spirocette
-Used to examine microbes that are invisble in brightfeild microscopy, cannont be stained by standard methods, or are distorted by staining

Front 5

Phase Contrast Microscopy

Back 5

-Detailed examination of internal structures in living microbes
-NO STAINING REQUIRED
-Accentuates diffraction of the light that passes through a specimen

Front 6

Fluorescence Microscopy

Back 6

-Uses UV light
-Fluorescent substances absorb UV light and emit visible light
-Cells are stained with Fluorescent dyes
-Rapidly detect and identify microbes in tissues or clinical specimens
-Principle use is for immunoflurescence

Front 7

Fluorescent Micrscopy

Back 7

-Used to rapidly detect and identify fluorescent(yellow or green-colored)microbes:
Treponema pallidum-Syphillis

Mycobacterium tuberculosis- TB

Legionella pneumophilla- legionaires disease

Front 8

Electron Microscopy

Back 8

- Uses a beam of electrons
- Uses elctromagnetic lenses
-The shorter wave length of electrons give greater resolution
- less than 0.2 micrometer such as VIRUSES and RIBOSOMES

Front 9

Transmission Electron Microscopy

Back 9

-ultrathin sections of specimens
-Light passes through specimen, then an electromagnetic lens, to a screen or film
-Specimen may be stained ith heavy metal salts
-10,000-100,000x; resolution 2.5nm

Front 10

Scanning Electron Microscopy(SEC)

Back 10

-An electron gun produces a beam of electrons that scans the surface of the whole specimen
-Secondary electrons emmited from the specimen produce the image
-1000-10,000x; resolution 20nm

Front 11

Scanning-Probe Microscopy

Back 11

-Scanning tunneling microscoy uses a metal probe to scan the specimen
-Resolution 1/100 of an atom
-Detailed view of molecules inside cell(DNA)

Front 12

Atomic Force Microscopy

Back 12

-Uses a metal and diamond probe inserted into the specimen
-Produces 3-d images of bacterial surface layer protein or toxin
- Holes because the organism gives off gas Bacillus cases Gas gangrene

Front 13

Preparation of Specimens for Light Microscopy

Back 13

-A thin film of solution of microbes on a slide is a smear
-A smear is fixed by heat flame of the Bunsen burner or a slide warmer to attach and kill the microbes
- A smear is stained with a basic or acidic dye

Front 14

Unstained Specimens

Back 14

-Live or unstainded cells have little contrast
-Researchers make discoveries about cell behavior looking at live specimens

Front 15

Wet Mounts

Back 15

-A stain is rarely used
- Live organisms are seen under the high power objective
-Motility of an organism can be viewed on a wet mount
-Motile bacteris include Escherichia coli, Pseudomonas, and Proteus

Front 16

Simple Stains

Back 16

-Using a single basic dye
-Used to observe the shape and size of a cell
-cell morphology is shown

Front 17

Differential Stains-Gram Stains

Back 17

-Classifies bacteria into positive and negative
-Gram-postive tend to be killed by detergents and penicillin
-Gram-negative bacteria are more resistant to antibiotics

Front 18

Gram Stain Procedures

Back 18

1)Apply crystal Violet(primary stain colors all cells purple)2min rinse H20
2)Apply Iodine(mordant forms a complex with the crystal violet and holds the primary stainCV-1)1min rinse H20
3)Ethyl alcohol or Acetone(decolorizes and removes crystal violet-iodide complex from some bacteria)rinse H20 1min
4)Apply safranin9counterstain has contrasting color to the primary stain)rinse 30 sec

Front 19

Results of Gram Stain

Back 19

-Gram-positive bacteria resist decolorization and retain the crystal violet dye and stain purple
-gram-negative bacteria having much lipid in their cell wall are dcolorized and take up the safranin dye and stain pink

Front 20

Importance of Gram Stain

Back 20

1)1st step in id of bacteria
2)Useful in clinical diagnosis
3)Treatment

Front 21

Differential Stains: Acid Fast

Back 21

-Useful in diagnosis of tuberculosis caused by Myobacterium tuberculosis

Front 22

Steps in Acid Fast stain

Back 22

1) Ad Carbolfuchsin Dye(red)5min
2)Acid/Alcohol(decolorizer)20sec
3)Methylene Blue(counterstain)2min

Front 23

Acid Fast Stain result

Back 23

1)Cells that retain a carbolfuchsin stain in the presence of acid-alcohol are call acid fast such as myobacterium stain red
2)Non-acid fast cells lose the carbolfuchsin stain whne rinsed with acid-alcohol and take up the counter stain and appear blue

Front 24

What disease can be diagnosed using the acid fast stain?

Back 24

T.B. and Leprocy

Front 25

Special Stains

Back 25

-Staining the background instead of the cell
-Used to color or isolate a specific part of the microbe such as capsules,endospores or flagella

Front 26

Negative Staining

Back 26

-Is used to make capsules visible
-Capsules serve as virulence factor
-Capsule appears as a clear "halo"(Klebsiella pneumoniae, Streptococcus pneumoniae)

Front 27

Cryptococcus neoformans

Back 27

-a yeast
-has a thick capsule
-Capsule visible in India Ink

Front 28

What is the value of capsule to bacteria and yeast?

Back 28

Antiphagocytic

Front 29

Speacial Stains

Back 29

-Endospores are usually dormant, resting structures that allow bacteris to survive in harsh environments
-Endospores are usually produced by gram positive bacteria(Bacillus species and Clostridum species)

Front 30

SPecial Staining

Back 30

-Flagella are whiplike appendages that allow for bacterial movement
-Flagella Staining requires mordant to make th flagella wide enough to see
-number and arrangment of flagella can be observed