Micro chap 3/exam 1

1 milimeter (mm) is the size of a lower case "o"

1 mm= 1000 Mm(micrometer)

1Mm= 1000 nm (nanometers

Light travels in waves.

• Red:780-622 nm
• Orange: 621-597 nm
• Yellow:596-577 nm
• Green: 576-492 nm
• Blue: 491-455 nm
• Violet:454-390 nm

Visible light waves are between 780-400nm

Magnification: degree of which the scope enlarges the specimen's image

Resolution: a measure of clarity of the image

You can magnify something as big as you want, but if you do not have resolving power then it will be blurry and pointless

The resolution limit is ~1/2 wavelength in nm

The resolution limit for a light microscope with the eye is 200 nm the smallest visible wavelength is violet at 400nm

other things that can limit resolution: quality and cleanliness of the lens

In order for a specimen to be visible under a microscope, it must distort the light waves passing though it

immersion oil is used in order to bend the light waves directly into the objective lens and it get a better picture of the MO. Otherwise, the light waves would miss the objective lens.  The oil acts as an extension of the glass lens directly onto the slide

Ocular lens is 10x magnification. Multiply this by whatever the objective lens is:

• 4x times 10x=40 total mag
• 10x times 10x = 100 total mag
• 40x times 10x =400 total mag
• 100 times 10x =1000 total mag

10 Mm-100Mm

1Mm-10Mm

30nm-200nm

1nm-10nm

• 
• Most commonly used is the brightfield microscope.  It is best for stained cells, as live cells are transparent.  It's application is that its used to view stained cells

• 
• Only the light that travels through the MO is seen.  That means that the MO does not to be stained and it can be viewed live.  It's application is to check motility and to look for cells in a sample

Flourencent dye molecules (flourochromes) are added to the MO and a UV lamp is used (light waves too short for human eye to see).  The flourochromes make the light waves longer so that we can see them, typically a green color.

• how does it work?  Its a key and lock system for antibodies that attach to the MO.  Specific flourochromes are added to specific antibodies that attach to the MO and create an immunofluoresence
• *a very good way to detect specific MO in a sample

• Transmission Electron microscope
• shoots electron beams of a much shorter wavelength.  This allows us to view much smaller MO.  There is a vacuum in the TEM because e- beams are scattered by air and dust

-application is to view high resolution pics of SLICES of cell interiors (harsh prep and cells are dead)

The microscope actually scans the exterior of the cell and therefore the application us ti see high resolution pics of the cells surface (also dead)

because the resolution limit is 1/2 the wavelength, shorter wavelengths let us view smaller objects

• magnification: 1x
• resolution: 100Mm (size of the smallest visible object)

• magnification: 1000x
• resolution: .2Mn (200nm)

• Magnification: 1000-10,000 x
• Resolution: 10nm

• Magnification: 10,000-100,000x
• Resolution: 2.5 nm

• Use the aseptic technique to put the smear on the slide
• Air dry and then heat fix

Simple stains are one step.  The bacteria is usually (-) charged and the dye is (+) charged such as crystal violet, safranin, and methalyne blue

Used to simply view bacteria, not to ID them necessarily

Negative stains are a (-) bacteria and a (-) stain.  The bacteria are not distorted as much.

They use several steps and different stains such as a counterstain in order to stain the MO for viewing

Primary stain will color the MO you are looking for.  The decolorizer is normally some kind of acid which removes the stain from some MO (such as gram -).  The counterstain stains those MO which were decolorized in order to provide a method of ID as well as contrast

H.C Gram (Danish) in 1884 developed the staining technique we call the Gram stain

• 1. Apply crystal violet to heat fixed slide for 1 minute
• 2. Rise off Cv with water
• 3. Apply iodine as a mordant for 1 minute
• 4. Decolorize with 95% ETOH
• 5. Counterstain with safranin

Gram + will be dark purple from the cv, while the gram - will be pink from the safranin.  The gram + have thick walls which hold in the cv, while the thin walls of the gram - are easily decolorized

• 1.  Apply Carbohlfucshin to the heat-fixed smear
• 2. gently heat this slide over steam for 5 min
• 3. Decolorize with 95%ETOH/3%HCL
• 4. Counter stain with methalyne blue

Mycobacterium have thick waxy exteriors that make them hard to dye, but once they are, they will stay pink from the carbohlfucshin.  Non-acid-fast will appear blue from the counterstain.

• 1. Apply malachite green to heat fixed smear
• 2. Heat slide over steam for 5 min
• 3. Rinse heavily with water
• 4. Counterstain with safranin

Also very difficult to stain, but one they are, they will hold onto the malachite green.  Dead and vegetative cells will be pink from the counterstain.

Endospores are dormant and durable forms of the cells.

Bacillus and Clostridium are two genera that form spores

To ID the MO.

Gram stains are done first to help ID. There are no known acid fast or endosore that are gram -.