Principles of Microscopy and Staining


acid-fast stain

staining technique that identifies acid-fast bacteria (those that are resistant to decolorization by acid alcohol because of their waxy or fatty cell walls) that are able to keep an acid-based stain inside their cells

coarse adjustment

technique used when the image appears very blurry, moving the specimen quickly into a range that must be further fine-tuned before a clear image is observed

compound light microscope

type of light microscope that uses two or three lenses and a beam of light to magnify the object being observed


lens that focuses the light so that it hits one point on the specimen being observed, such as microbes on a glass slide

confocal microscopy

technique that blocks out all light except for a small, direct beam of light—or a laser—that illuminates one small area of the specimen being observed

dark field microscopy

technique using a small disc, called a patch stop, that blocks direct light coming from the light source of the microscope

differential stain

technique that uses multiple, typically two, stains and can stain microbes in the same sample differently depending on certain properties such as the composition of the bacterial cell wall or the way the microorganism processes the chemicals used in the stain

electron microscopy

technique that uses a beam of electrons, instead of light, to illuminate a specimen

endospore stain

stain that results from use of the Schaeffer-Fulton stain and that distinguishes between endospores and bacteria

eyepiece lens

lens located just below the observer’s eye, which further magnifies the specimen

fine adjustment

technique used when the image is nearly in focus, allowing small adjustments to the position of the specimen in order to achieve sharp resolution of the image

Gram stain

method of staining used to differentiate types of bacteria based on cell wall structure


act of placing a small drop of water, oil, or glycerin, referred to as the immersion medium, on top of the glass covering the specimen

index of refraction

measure of how much faster light can travel in a material compared to in a vacuum

iris diaphragm

adjustable hole that allows some, but not all, of the light to travel to the objective lens

meter (m)

base unit of length measurement in the metric system

negative stain

dark stain used to cover the background of the slide while the microorganism(s) on the slide remains clear

numerical aperture

measure of the microscope's ability to gather light and resolve details of what is being observed when the object is a specific distance from the lens of the microscope

objective lens

first component of the microscope that magnifies the specimen

parfocal microscope

microscope containing objective lenses that keep the specimen being observed in focus even when the magnification is changed

phase-contrast microscopy

technique for observing live microbes, in which the specimens do not need to be fixed before observation


what happens to light at the boundary between two objects


the way in which the direction of light bends or changes as it passes from one medium to another


level of detail of an image

resolving power

ability of a microscope to differentiate between two points that are very close to each other

simple stain

stain that uses a single dye color and typically stains all microorganisms in a sample


slide preparation for which a microorganism (specimen) is spread in a thin layer onto a glass slide using a cotton swab and then left to dry

total magnification

technique that uses a beam of electrons, instead of light, to illuminate a specimen


distance between sequential peaks in a wave of light