Microscopy

Observing Microorganisms Through a Microscope

Units of Measurement
Microorganisms are so small that metric prefixes may be unfamiliar
centi = 1/100 milli = 1/1000 micro = 1/1,000,000 nano = 1/1,000,000,000 or or or or 10-1 10-2 10-6 10-9

The Instruments

Compound Light Microscope Darkfield Microscopy Phase Contrast Microscopy Differential Interference Contrast Microscopy Fluorescence Microscopy Confocal Microscopy

Electron Microscopes Scanning Electron Microscopy Transmission Electron Microscopy Atomic Force Electron Microscopy

Compound Light Microscope

  

Uses visible light Has at least 2 sets of lenses Can achieve maximum 2000X magnification Resolution of objects as small as 0.2 Qm

Light Microscopy


light microscope
visible light passes through the specimen and then through glass lenses.  lenses refract light such that the image is magnified into the eye or a video screen.


Light Microscopes
 

Vary in magnification and resolving power. Magnification is the ratio of an objects image to its real size. Resolving power is a measure of image clarity.  It is the minimum distance two points can be separated and still viewed as two separate points.  Resolution is limited by the shortest wavelength of the source, in this case light.

Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Resolution of Light Microscopes



The minimum resolution of a light microscope is about 2 microns, the size of a small bacterium Light microscopes can magnify effectively to about 1,000 times the size of the actual specimen.  At higher magnifications, the image blurs. Fig. 7.1

Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Brightfield Illumination
 

Usual operations Specimens must be stained for viewing Best magnification and resolution with the oil immersion objective


Oil has same refractive index as glass

Darkfield Illumination
Light objects are visible against a dark background. Light reflected off the specimen enters the objective lens.
Figure 3.4a, b

Darkfield Microscopy


Useful for examining

Live organisms  Microorganisms which cannot be stained by standard methods  Treponema pallidum, the causative agent of syphilis


PhasePhase-Contrast Microscopy

Accentuates diffraction of the light that passes through a specimen.

Figure 3.4c

Differential Interference Contrast Microscopy



Accentuates diffraction of the light that passes through a specimen; uses two beams of light.

Figure 3.5

Fluorescence Microscopy


When illuminated with short P light some dyes emit light with longer P Enables viewing of cells located on an opaque surface such as a soil particle When illuminated with UV or halogen light source preparations glow

Bovine pulmonary artery endothelial cells. Photometrics, Ltd.

Fluorescence Microscopy
 

Uses UV light. Fluorescent substances absorb UV light and emit visible light. Cells may be stained with fluorescent dyes (fluorochromes).
Figure 3.6b

Fluorescent stain of cell

Confocal Microscopy


Uses fluorochromes and a laser light. The laser illuminates each plane in a specimen to produce a 3-D 3image.

Figure 3.7

Confocal Microscopy
3-D confocal microscopy of Salmonella-infected macrophage (green) with XY-slice showing bacteria (red) inside the cell

Exceptionally clear two-dimensional images Three-dimensional images obtained by computer construct

Electron Microscopy


  

Beam of electrons has shorter P so gives better resolution than visible light Electromagnetic lenses rather than glass Done in a vacuum Can resolve to 0.5nm and magnify up to 100,000 times. Specimen must be dry.dead

Transmission Electron Microscopy



 

Resolves objects as close as 2.5nm Magnification 10,000 to 100,000X UltraUltra-thin sections Specimens must be dehydrated Preparation of specimen may generate artifacts

(TEM)

Lambda Bacteriophage DNA (TEM x153,000)

Transmission Electron Microscopy (TEM)

  

Light passes through specimen, then an electromagnetic lens, to a screen or film. Ultrathin sections of specimens. Specimens may be stained with heavy metal salts.

Figure 3.8a

Scanning Electron Microscopy (SEM)

 

 

Resolves objects as close as 20 nm Magnification between 1,000 and 10,000X Whole specimens 3-dimensional view of specimen Specimen dehydrated

Slime Mold Fruiting Structure, Lamproderma sp. (SEM x290)

Scanning Electron Microscopy (SEM)



An electron gun produces a beam of electrons that scans the surface of a whole specimen. Secondary electrons emitted from the specimen produce the image.

Figure 3.8b

Electron micrographs

TEM

SEM

Scanning-Probe Microscopy ScanningScanning tunneling microscopy uses a metal probe to scan a specimen. Resolution 1/100 of an atom.
Figure 3.9a

ScanningScanning-Probe Microscopy


Atomic force microscopy uses a metal and diamond probe inserted into the specimen. Produces 3-D 3images.
Figure 3.9b

Scanning Tunneling Atomic Force

 

Thin metal probe scans specimen Resolving power much greater than electron microscopes no special specimen preparation detailed views of silicon chips & DNA molecule

 

Metal and diamond probe forced down along surface of specimen 3-dimensional image no special preparation of specimen is required views of detailed structure of biological molecules

Preparation of Microscopy Specimens



Microorganisms must be spread over the surface of a slide (smear) Microorganisms must be attached to the slide (fixed) Microorganisms must be colored (stained)

Making the Smear and Fixing It



Put a small amount of organism into a drop of water on a clean microscope slide & spread . When dry pass through the flame of a Bunsen burner or flood with methyl alcohol

Stains
 

Dyes Negative Stain



stains the background not the organism everything stained a single color distinguish among bacteria based on particular characteristics

Simple Stains


Differential Stains


Characteristics of Dyes



Basic Dyes
Chromophore is in the positive ion Used to stain most bacteria Used alone as simple stains in combination for differential stains




Acidic Dyes
Chromophore is in the negative ion Used in negative staining & for staining nuclear material

Gram Stain


The Gram Stain is the single most important test in microbiology. The principal utility of the Gram Stain rests on its speed and simplicity. Most bacteria may be divided in two groups by this procedure developed by the Danish physician Hans Christian Gram to differentiate pneumococci from Klebsiella pneumonia difference between Gram-positive and GramGramGramnegative bacteria is in the structure of the cell wall

Procedure

Results
G+ cocci G- rods

Websites with more samples of gram stained bacteria

GRAM STAINED IMAGES OF MEDICALLY IMPORTANT BACTERIA Loyola University Medical Center http://www.meddean.luc.edu/lumen/DeptWebs/microbio/med/gram/slides.htm GRAM STAIN TUTORIAL http://www.courses.ahc.umn.edu/pharmacy/5825/GSPage05.html

The primary stain in a Gram Stain is

1. 2. 3. 4.

safranin methylene blue crystal violet carbol fuchsin

Acid Fast Stain



Acid Fast Staining is used primarily for detection of organisms with a thick outer coat composed of true waxes, mycolic acids and phosphatides Mycobacteria are not decolorized and retain the stain, appearing pink under the light microscope (hence are 'fast', like colorcolor-fast clothes). Acid Fast Stain can also be used to identify several protozoa, such as Cryptosporidium and Isospora belli. These two coccidia have recently acquired greater clinical significance because of their widespread occurrence in immuno- compromised patients, immunosuch as those infected with HIV.

Special Stains


Spore Stain
Bacillus subtilis

Capsule Stain
Streptococcus pneumoniae

Flagella Stain
Pseudomonas aeruginosa