Module 3

What is resolution?

Ability to distinguish two points that are close together (resolving power) or the distance between two objects at which the objects still can be seen seperate.

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 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.

What is simple staining?

a solution of a positively charged dye, such as methylene blue, crystal violet, safranin, or fuchsin, to bind to and stain the negatively charged membrane of the microorganism. This simple technique is often used to quickly observe the size, shape, and arrangement of cells.

What do confocal (laser scanning) microscopes do?

combine the usefulness of fluorescence microscopy with the ability to visualize cells in 3-D.

Why is staining often required?

due to the limitation of resolution on unstained cells because the flat and transparent regions of a cell may appear invisible under bright field conditions.

How do dark field microscopes differ from brightfield and phase contrast microscopes?

reflects light off of the specimen at an angle. This reflective approach does not permit the visualization of intracellular structures.

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

resolution and contrast

What is the difference between a brightfield microscope and a phase contrast microscope?

phase contrast microscopes are able to visualize certain structures that would otherwise be invisible. Provides detailed images live without staining.

gram negative bacteria appear?

pink/red

What are some contraindications of electron microscopes?

they are labor intensive, requires samples to be fixed (killed), and the process may alter the cell structure.

What is microscopy?

using light or electrons to magnify objects

What does a phase contrast microscope do?

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.

When was the first glimpse of the microscopic world seen?

1674 by cloth merchant and lens grinder Anonie VAn leeuwenhoek.

a 40x objective and a 10x ocular result in a total magnification of?

400x

what does giemsa stains help diagnose?

malaria as well as other blood parasites.

What is a nanometer?

(nm) measures cellular organelles and viruses

What is a micrometer?

(um) used to measure size of cells

electron microscopes use beams of _____________ (rather than light), which have significantly shorter _________________ than light, to increases its ______________ capacity to less than _______—that's 200x better!

1- electrons 2-wavelengths 3-resolution 4-1nm

UV light excites different __________________ at varying wavelengths, enabling scientists to use a wide array of ___________ during imaging. For instance, the ___________, _____________, and ______________ fluorescent proteins (GFP, YFP, and RFP, respectively) have become important tools in microscopy

1-fluorophores 2-colors 3 green, yellow, and red

_______________ controls the amount of light that passes through the sample and into the ______________ _______.

1-iris diaphragm 2-objective lens

The most common power of an occular lens is?

10x

Define the measurement micrometer.

A micrometer (µm) is defined as being one-millionth of a meter and is commonly designated at 10-6 meters.

Define the measurement nanometer.

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.

The simplest form of light or optical microscopes.

Brightfield microscopes

What is giemsa?

Combined with Wright's stain (stains blood cells), the resulting combinatorial stain can be applied to blood smears to determine the presence (or absence) of pathogenic bacteria—human (blood) cells appear purple, and bacterial cells appear as pink.

before the light reaches the sample, it first passes through a ________________ converging the light beams into a focused area on the sample

Condenser

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.

The differentiation in color in gram staining divides bacteria into what two categories?

Gram-positive or gram negative

Who was Hippocrates?

Greek physician deemed the father of medicine.

What did Thucydides realize as a survivor of the plague?

He could have contact with current sufferers of disease and not come down with the illness or symptoms again.

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.

What is the most common use of an acid fast stain?

Mycobacterium tuberculosis

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).

How was hygiene building seen in ancient civilizations?

Rome has sewage systems and built freshwater aqueducts which decreased waterborne illnesses.

The degree of magnification is directly proportional to?

The amount of light needed.

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.

One of his contemporaries was a historian named?

Thucydides

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 did Hippocrates observations make him question?

Whether there is a link between the environment and disease

What is differential staining?

a generalized term used for any staining technique that separates specimens into further subgroups. This process most often utilizes at least two dyes.

what is a wet mount?

a basic form of sample preparation for viewing live samples. A small liquid culture (usually just a drop) containing a microorganism of interest is prepared, added to a slide, and then covered with a glass coverslip. The coverslip is present to both protect the objective and the specimen while also holding the microorganism in place.

When Leeuwehhoek observed a droplet of water, what did he see?

algae, protozoa, and fungi!

What is acid fast staining?

also known as the Ziehl-Neelsen stain, is a differential stain used to identify bacterial stains showing a high degree of resistance to decolorization.

What is negative staining?

an alternative to the positively charged simple staining dyes listed above. A negative stain can be thought of the inverse of a simple stain.

acidic dyes

anionic (-) chromophores that stain positively charged structures in acidic conditions (low ph). mostly used for negative staining

Rather than using halogen (brightfield) or UV (fluorescence) light, confocal microscopes use?

asers to focus on a single plane within an object and with a higher degree of accuracy

Unlike light or fluorescence microscopy where light is focused a single plane (2-D), confocal microscopy can?

capture images in either 2-D or 3-D.

basic dyes

cationic (+) chromophores that stain negatively charged structures in basic conditions (high ph). most common

chromophore

colored ion in dye

What is contrast?

difference of light absorbance between two objects and its background.

What is magnification and how does it occur?

it is the increased size of the object and results when radiation (light) bends as it passes through a lens.

light microscopy (brightfield, phase contrast, fluorescence, and confocal) cannot efficiently visualize viruses or even some subcellular compartments because?

its limited to a resolution of about 0.2 µM

What are the principles of microscopy?

magnification, resolution, contrast

What does phase contrast microscopy visualize that brightfield microscopy cant?

motility, such as swimming or gliding, without altering the cell morphology commonly brought about from treating the cells with a fixing agent.

For a microscope using two lenses (objective and ocular) the total magnification of a specimen is?

multiplicative

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

ocular lens

How are fluorescent proteins expressed in the cell?

nonspecifically illuminating the cell as a whole linked (coupled) to a normal cellular protein of interest whereby the fluorescent color is indicative of protein movement and localization or used as tags on molecules or antibodies used to designate the presence (fluorescence detected) or absence (no fluorescence) of a specific protein target.

A negative stain is only mildly invasive and may not kill the microorganism. As such, a negative stain is contraindicated for?

pathogenic samples.

What did evidence in literature and ancient text show?

people had the understanding that diseases could be contagious

gram positive bacteria appear?

purple

Stains are _______ that break down positively and negatively charged ions.

salts

What does heat fixation do?

samples are added to a glass slide and then passed through a flame until all liquid in the sample has been removed.

What do fluorescent microscopes do?

take advantage of fluorescent molecules called fluorophores to visualize cells on a dark background.

What is the objective lens?

the lens closest to the sample and yields the greatest amount of magnification.

What is perhaps the most important resource for studying biology at the microscopic level?

the microscope!

What is chemical fixation?

the use of paraformaldehyde, ethanol, or methanol. Although these processes fix the sample to the slide, it also kills the microorganism. As such, characteristics related to motility (movement) are not possible.

What is a electron microscope used for?

to visualize incredibly small specimens.

gram positive has a?

thick layer of peptidoglycan

gram negative has a?

thin layer of peptidoglycan

How can staining be used?

to examine tissues (muscle or connective), specific types of cells (blood, bacterial, etc.) or even organelles within an individual cell (nucleus, ER, etc.).

What are dark field microscopes used for?

to greatly increase the contrast between a specimen and background, resulting in a dark background with bright objects in it.

What are wet mounts used for?

to observe the motility and behavior of an organism

Unlike brightfield, the energy of the incoming light is in the form of the?

ultraviolet (UV) spectrum

What does a scanning electron microscope (SEM) do?

use a beam of electrons, but the image is obtained as the electrons reflect off (not through) the surface of the specimen.

What does the Scanning transmission electron holography microscopes (STEHM) do?

use an electron beam but coupled with a holography technique to study surfaces of proteins and subcellular structures. It has the capacity to magnify subatomic structures up to 20 million times larger than what can be viewed with the naked eye.

What do transmission electron microscopes (TEM) do?

use thin slices of a sample, heavily treated and coated in preservatives, and placed between the electron beam source and the detector.

who invented gram staining? How did it begin?

was first developed by Hans Christian Gram in 1884. It began with the observations that different types of bacteria react differently to various dyes.