lecture 2

Question 1

FLUORESCENT MICROSCOPY

Answer 1

-humans see 390-700 nm
-Uv light is 10-400 nm range so invisible to us
-flourescent uses UV light excite a fluorescent substance
-the fluorescent dye is attached to the sample of interest on a slide
-Tagged to a primary antibody to a specific region in a specimen–like a protein

Question 2

Auramine or Auramine-Rhodamine
Stain:

Answer 2

FLUORESCENT STAIN FOR ACID FASTNESS
- dye binds directly to the mycolic acid in the cell wall
Decolorizer: Flood with acid-alcohol
Quencher: Potassium permanganate reduces non specific fluorescence
Result =yellow or orange fluorescence against a greenish background on a fluorescent microscope

Question 3

Kinyoun & Ziehl-Neelsen
NON FLUOROCHROME STAINS FOR ACID FAST BACILLI

Answer 3

basically the same reagents
use different methods to treat cell wall for penetration of Carbolfuchsin

-Primary stain is Carbolfuchsin (a phenolic compound) –penetrates the cell wall

-Kinyoun Acid Fast stain technique: Higher concentration of phenol increases uptake of Carbolfuchsin
-Decolorizer that is 3% acid alcohol

–Ziehl-Neelsen (is a modification of Kinyoun)-Uses heat to increase uptake of Carbolfuchsin

Question 4

carbol Fuchsin

Answer 4

lipid soluble, phenolic compound, which is able to penetrate the cell wall

Question 5

Ziehl – Neelsen Procedure

Answer 5

-Make a smear. Air dry. Heat fix.
-Flood Carbol Fuchsin
-Cover with filter paper
-STEAM for 10 minutes.
-Cool slide
-Rinse with DI water
- Flood slide with acid alcohol. 3% HCl and 95% ethanol. Or, 20% H2SO4

The waxy cell wall prevents the stain from being removed by acid alcohol (decolourizer) once it has penetrated the cell wall. The acid alcohol will remove the stain from the other cells.

BLUEISH BACKGROUND WITH PINK CELLS

Question 6

Acridine Orange
FLUOROCHROME STAIN

Answer 6

-Binds to nucleic acid of cells
-Fluorescent orange colour
-presence of organisms that lack a cell wall e.g. Mycoplasma sp.

Question 7

Calcofluor White
FLUOROCHROME STAIN

Answer 7

Binds to cellulose and chitin (found in the cell walls of fungi)
Screening of clinical specimens (e.g. respiratory specimens) for fungal elements

Question 8

DARK FIELD MICROSCOPY

Answer 8

-makes unstained, transparent specimens clearly visible by using light to enhance contrast.
-An opaque disc placed under condenser lens
-prevents central light from touching the sample only the oblique light interacts with specimen
-Organism appears “bright” on a black background
x10 or x40 objectives only – no oil

Question 9

DARK FIELD MICROSCOPY detection

Answer 9

-NO staining involved
-uses ANNUALR DIAPHRAGM
Detects live organisms -motility
Detects difficult to stain organisms
Detects organisms like Spirochetes
Detects shapes of cell structures

Question 10

The wave is made up of

Answer 10

-crest (peak) and a trough (dip)
-repeating pattern of crests & troughs

length of one crest & trough (1 wave) is a wave cycle
a wavelength is 2 cycles

phase of a wave is measured in degrees, where 360 is one wavelength
-If you add two different light waves together, depending on their phases the intensity of light can be affected.
synchronized - light
async- darker

Question 11

PRINCIPLE OF PHASE CONTRAST MICROSCOPY

Answer 11

• converts phase shifts into changes in the intensity of the light

-A change in light must be ½ λ to be noticed by the human eye***

The phase changes that are visible to us are caused when light passes thru the specimen and shift ¼ λ & then a phase plate in the objective that shifts the light another ¼ λ = ½ λ

Question 12

Refractive index (RI)

Answer 12

change in speed when light passes from one medium to another- causes a phase shift

Question 13

PHASE CONTRAST MICROSCOPE

Answer 13

-2 different plates an annular diaphragm in the condenser and a phase plate in each objective
-must be superimposed on each other
-Size of aperture varies with each annular diaphragm and the size of the phase plate varies with each objective

1 dia for 10x, 2 dia for 40 and 3 dia for 100x

Question 14

NEGATIVE (BRIGHT) PHASE CONTRAST

Answer 14

-phase plate set on denser through so it goes around the u letter
-2 light waves one that hits the specimen with phase shifted 1/4 wavelength and the other that did not hit the specimen with phase 1/4 shifted
-therefore in this phase there is direct and obstructed light that reinforce each other= constructive interference producing a bright image on a dark background

Question 15

POSITIVE PHASE CONTRAST

Answer 15

-on a lesser through so the light goes through the actual U lettering

-The direct light that did not hit the specimen is not phase shifted at all
-This means both light waves are out of phase = destructive interference
-Direct light allowed thru but light that was phase shifted is lower in intensity
-Produces a dark image on a bright background

Question 16

USES OF PHASE CONTRAST
advantages

Answer 16

-allows observations of transparent specimen
-Tissue, parasites, fungus, semen

-can see cells in a natural state
-Can observe a living organism
-Ideal for thin specimens
Can see structures that are otherwise invisible such as organelles

Question 17

USES OF PHASE CONTRAST
disadvantages

Answer 17

-Not ideal for thick organisms - may appear distorted
-Phase artifacts like shade-off & halo effect

Question 18

the principle of fluorescent microscopy

Answer 18

-UV light provided tungsten or mercury vapor
-UV light goes through an excitation filter letting light in if its a specific wavelength to excite the fluorophore
-if the light is at a longer wavelength than the excitation light (need a low level that we cant see) its called the stokes law
-the longer wavelength lets us see
-fluorescence decreases as total magnification increases

Question 19

flourescence and affect on acid fast organisms

Answer 19

• some bacteria has a high lipid and wax content - mycolic acid
  -this makes them resistant to decolorization even with acid alcohol - acid fast orgs
  -no gram staining bacteria and parasites (beaded appearance)

Question 20

annualr diaphragm

Answer 20

central cone of light is let through a big disk with little disk

Question 21

phase plate

Answer 21

found in the objectives
a u shaped groove cut out
thucker through negative phase plate
thinner trough - positive plate
-its like an etched U in a square so positive goes in the lettering and negative goes around the letter

direct light hits the trough
obstructed light does not hit the trough