Microscopy Compound Microscope The Compound Microscope is the

  • Slides: 16
Download presentation
Microscopy

Microscopy

Compound Microscope • The Compound Microscope is the type of microscope we use in

Compound Microscope • The Compound Microscope is the type of microscope we use in the lab. It uses visible light as it’s light source for illumination. • The average compound microscope has resolving power of. 2 m (1 m =. 000001 m) • The resolving power is the ability of the microscope to distinguish between 2 pts, or to show detail. • Staining is used to make the specimen contrast sharply with the medium so that it is easier to see.

Fluorescence Microscopy • This particular microscope uses fluorochromes to attach to organisms and allow

Fluorescence Microscopy • This particular microscope uses fluorochromes to attach to organisms and allow them to fluoresce. Fluorochromes are small molecules that only fluoresce once they have attached to their target. • Immunofluorescence is a particular type of marker used in fluorescence microscopy. The fluorochromes are first attached to a specific antibody (Ab). The antibody is usually specific for a particular type of cell, virus, or other type of specimen. • This type of microscopy is useful for detecting viruses and other org. within cells, tissue, or other clinic specimen that normally we wouldn’t be able to see or detect otherwise.

 • For example, let’s say that we suspect someone has Hepatitis is caused

• For example, let’s say that we suspect someone has Hepatitis is caused by a virus. Viruses are so small that they can’t be seen under a compound microscope. In addition, they also spend much of their replication time within host cells and you can’t see into a cell with a compound microscope. – So we take a sample of blood from the patient. – Then we get some antibodies that are specific for the hepatitis virus. – Next we put the fluorochrome on the antibody. –.

– Then we put the fluorochrome, antibody combination together on the specimen. – If

– Then we put the fluorochrome, antibody combination together on the specimen. – If the virus is present, then the antibody will bind to the virus or virus infected cell. Upon binding, it will “turn on the switch” for the fluorochrome. The fluorochrome will now fluoresce indicating the specimen is positive for that virus. – If the virus is not present, then the antibody won’t be able to bind and the fluorochrome won’t fluoresce.

Electron Microscopes • Electron Microscopes are used to examine objects smaller than. 2 m.

Electron Microscopes • Electron Microscopes are used to examine objects smaller than. 2 m. There are 2 types that are used. • SEM: Scanning Electron Microscope – This type of electron microscope creates and image of the surface structures of the organism. – It bounces a beam of electrons (e-s) off the organism to create the image. As the electrons come into contact with the specimen the e-s bounce off in different directions. A computer then records the direction it went and extrapolates the shape of the surface of the specimen by the direction that each e-s bounces off.

 • TEM: Transmission Electron Microscopy – The Transmission Electron Microscipe uses ultra thin

• TEM: Transmission Electron Microscopy – The Transmission Electron Microscipe uses ultra thin slice of the specimen to create images of inside cellular structures. – In this case the image is created by sending the e-s through the specimen and recording the trajectory of the e-s as they exit out the other side. • These are both great ways for us to learn more about a particular organism. Unfortunately a live specimen can never be viewed this way because the preparations are toxic to the cells.

Biological Stains Used in the Lab • To prepare specimens to be viewed under

Biological Stains Used in the Lab • To prepare specimens to be viewed under the compound microscope there are two types of stains that are used. – 1. Basic Stain: The basic stain is composed of a positively (+) charged ion that attaches to the negatively (–) charged cell membrane. • Crystal violet and safranin are examples of basic stains. – 2. Acidic Stain: An acidic stain is composed of a negatively (-) charged ion that is repelled by the negatively (–) charged membrane. • Eosin and Acid fuschin are examples of acidic stains.

 • There are two different ways that we can use basic and acidic

• There are two different ways that we can use basic and acidic stains to better view and identify microbes. – 1. Simple Stain: A simple stain is when we use one stain, basic or acidic and color the organism to view under the microscope. It helps us to determine the shape of the organism. – 2. Differential Stain: A differential stain is when we combine 2 or more stains to gather more information. • For example, a gram stain uses 2 stains. One stain indicates the organism is G+ and the other indicates the organism is G-. • Other types of differential stains are endospore stains, capsule stains, acid-fast stains, and flagella stains.

The Basic Gram Stain Procedure • Let’s say that you have a culture that

The Basic Gram Stain Procedure • Let’s say that you have a culture that has two different kinds of organisms growing in it. One organism is a cocci and the other is a rod. You want to determine what the gram reaction is for each. • The first step is to heat fix the specimen. (You smear the specimen on the slide and wave it through the bunsen burner to slightly bake it onto the slide. You don’t want it to wash off during the staining procedure. !) • The second step is to apply the Primary stain. The primary stain is always the first stain applied in a differential staining procedure. In this case the primary stain is crystal violet. It is a basic stain so it attaches to all the cells present because they all have a (-) charged cell membrane.

 • The next step is to apply iodine to the specimen. The iodine

• The next step is to apply iodine to the specimen. The iodine acts as a sealant to tightly bind the primary stain to the G+ cell wall. This is called a mordant. • Then the specimen is washed with alcohol. The alcohol dissolves lipids. Remember that the Gcell wall is made up of an outer membrane that contains lipids. The alcohol wash removes the cell wall from the G– organism. • Next the secondary stain is applied. In a differential stain, the secondary stain is the second type of stain used in the process. In this case, safranin is the secondary stain that is used. It is a basic stain too. It attaches only to the unstained cells, ie the G- organisms on the slide.

 • The final result is that the G+ cells are stained purple and

• The final result is that the G+ cells are stained purple and the G- cells are stained magenta or pink. • The gram stain works simply because there is a difference in the cell walls between G+ and G- organisms. • See “Insight 4. 2” on page 98 of your textbook for an excellent explanation of the gram stain procedure.

Endospore stain • An endospore is a special structure made by some bacteria to

Endospore stain • An endospore is a special structure made by some bacteria to store genetic material for a new cell. • Because endospores are so resistant to many environmental pressures they can’t be stained with ordinary dyes because don’t penetrate the wall. • The Primary stain for an endospore stain is malichite green. Heat is used with this stain to force it into endospore. • The Secondary stain is safranin. Safranin stains the cell wall of the microbe giving a contrast between the endospore and the organism.

Capsule Stain • Remember that a capsule often indicates virulence. • A Negative stain

Capsule Stain • Remember that a capsule often indicates virulence. • A Negative stain (India Ink) is used to show the outline of the capsule. It will not bind to capsule but it will stain around the capsule. • The the Secondary stain, methyl-blue is applied to stain the organism inside the capsule. • The clearing around the stained orgaism is the capsule.

Acid-fast Stain • Some microbes have cell walls that are constructed a little differently

Acid-fast Stain • Some microbes have cell walls that are constructed a little differently that how we have discussed. The organism, Mycobacterium has a waxy substance in its cell wall called mycholic acid. The mycholic acid makes it difficult to stain this organism using a basic dye. So a different type of stain is used for it. • The acid-fast stain is used to distinguish mycobacterium from other types of rods. • Carbol fuschin is the primary stain for this technique. Like the endospore stain, heat is used to force the stain on the organism. • A Secondary stain is added after to bind to any organism that did not bind to the primary stain.

Flagella Stain • A flagella stain is a very tedious and delicate staining procedure.

Flagella Stain • A flagella stain is a very tedious and delicate staining procedure. • It uses a mordant and carbol fuschin to build up the diameter of the flagella until they become visible. • This type of staining procedure is valuable because the arrangement of the flagella can be used as a diagnostic aid. • No homework for this chapter.