Bright-Field Microscope (specimens are visualized b/c of differences in contrast aka density between specimen and surrounding)

Limit of resolution is about 0.2 micrometers

-Bright field

-Phase contrast

-Dark field

-Fluorescence

1) Add culture to slide, allow to air-dry

2) Heat Fix, flood slide with stain

3) Put onto microscope, place drop on slide

gram-positive bacteria appear purple 

gram-negative bacteria appear red

*VERY IMPORTANT B/C IT WILL HELP TO QUICKLY INFORM DECISIONS ABOUT INFECTION THERAPY*

1) FLOOD THE HEAT-FIXED SMEAR WITH CRYSTAL VIOLET FOR 1 MIN

2)ADD IODINE SOLUTION FOR 1 MIN

3)DECOLORIZE WITH ALCOHOL FOR 20 SECONDS

4)COUNTERSTAIN WITH SAFRANIN FOR 1-2 MINS

Bright-Field Microscopy 

Dark-Field Microscopy 

*light only reaches the specimen only from the sides 

*the only light reaching the lens is the one that is scattered by the specimen

*this makes the specimen appear light on a dark background

Phase-Contrast Microscopy 

*phase ring in the objective lens amplifies differences in the refractive index of cell and surroundings

*improves contrast of a sample without the use of a stain

*allows for the visulization of live samples (wet mounts)

*resulting image are dark cells on a light background

Fluorescent Microscopy 

*used for microbes that fluoresce

*either they fluoresce naturally or stained with DAPI Dye

Confoccal Scanning Laser Microscopy

*uses computerized microscope

*generates a 3d image with layers

*commonly used for biofilm studies

Atomic Force Microscopy

*a tiny stylus measures weak repulsive forces between it and the specimen 

-uses electrons instead of photons to image cells and structures 

-2 types of electron microscopes

*TEM*

*SEM*