module 3

define the measurements micrometer and nanometer

A micrometer (µm) is defined as being one-millionth of a meter and is commonly designated at 10-6 meters. A nanometer (nm) equals 10-9 m or one-billionth of a meter.

The acid-fast stain is most often used to identify what specific microorganism?

Acid-fast stains are used to identify bacterial stains showing a high degree of resistance to decolorization. Mycobacterium tuberculosis is the most common use for an acid-fast stain.

What is the primary difference between TEM and SEM?

During transmission electron microscopy the electron passes through the sample whereas during scanning electron microscopy the electron is reflected off the sample creating a three dimensional 'shell' model of the specimen.

What is one limitation of fixing your sample?

Fixation requires you to irreversibly kill your sample. Thus, determining the motility (cell movement) of a sample is impossible. Fixation also runs the risk of distorting the specimen shape and arrangement.

Gram staining is based on what basic principle?

Gram staining, developed by Hans Christian Gram in 1884, began with the basic observation that different types of bacteria react differently to various dyes. Some bacteria readily take up a specific dye while others do not.

What is a key determinant in a bacteria being Gram-positive?

Gram-positive bacteria have a thick peptidoglycan layer. The Gram stain exploits this characteristic by using the dye combinations of Crystal violet and Iodine. Crystal violet is retained by the thick peptidoglycan cell wall and forms a stable complex with iodine (upon its addition) effectively trapping the dyes in the cell. The resulting mixture is a purple coloration of the cell.

What is the purpose of heat fixing a sample?

Heat fixing ensures the samples tightly adhere to the glass slide prior to staining (and washing) procedures.

As light passes through a microscope, what is the last piece that light passes before reaching your eyes?

Once light passes through the sample and the objective lens it is directed through the ocular lens, or eyepiece, directly into your eye.

Phase-contrast microscopy provided what benefits to imaging?

Phase contrast microscope can provide detailed images of live cells without staining. By using specialized condensers and objectives, a phase contrast microscope amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable.

What are the two critical factors that influence your ability to see an object?

Resolution and contrast. Resolution refers to the distance between two objects at which the objects still can be seen as separate. Poor or low resolution means two (or more) objects may appear as one. The contrast is the difference in light absorbance between two objects. Poor contrast gives a high background and makes the visualization of multiple objects difficult. For instance, trying to identify 2 dark colored objects at night (low light = low contrast) versus the same 2 objects in the middle of a sunny afternoon (bright light against 2 dark objects = high contrast).

Unlike brightfield microscope, fluorescence microscopes illuminate samples through what spectrum?

The energy of the incoming light is in the form of the ultraviolet (UV) spectrum.

If you wish to increase the amount of light going into a microscope, what part would you adjust?

The iris diaphragm controls the amount of light that passes through the sample and into the objective lens. Thus, as you open the iris more light is permitted to pass through to illuminate the sample.

How is the total magnification of an object calculated?

Total magnification is calculated by multiplying the power of the objective and the power of the eyepiece. For instance, a 40x objective with a 10x eyepiece would make an object appear (40 x 10) 400 times larger (400x).

What is the distinguishing feature of dark field microscopy?

Unlike bright field or phase contrast microscopy where light passes directly through the sample, dark field microscopy reflects light off of the specimen at an angle. The resulting image is an exceptionally dark background and a vibrant specimen.

What is the primary purpose of a wet mount?

Wet mounts are most often performed to visualize live cells as well as the motility and behavior of an organism.