Microbiology Lab Exercise 4

Microscopy

What is the most common method used to directly detect organisms in clinical specimens and characterize bacteria grown in culture?

Light Microscopy

Where visible light is passed through the sample and then through a series of lenses that reflect the light in such a way as to magnify or enlarge the organisms present. aka bright field microscopy

Two

You should use __________ hands to carry the microscope (one to hold the arm and the other to hold the base)

No! Always pick up the microscope to move it

If the microscope needs to be moved can you slide it across the lab bench?

Lens paper

Use _______ to clean the glass surface of the ocular and objective lens.

True

True or false: when using a slide with oil you should go from low power directly to the oil immersion lens?

High

When using a slide with oil you should not go past _________ power to avoid getting oil on the lens.

Lowest

When finished using your microscope remove the slide from the stage and place the _________ power in observation position.

Lowest

When finished with the microscope the scanning objective lens should be in the observation position, the slide holder should be in the center of the stage and the stage should be in the _________ position.

True

True or false: it is better to use one eye when looking through a binocular microscope because it will help you see a more focused image.

Eyepieces

These can be adjusted to the width of your eyes and contain lenses that you look through as well as a black pointer. Magnification is printed on the side but for most microscopes is 10x

10x

The magnification of most ocular lenses is _______

Nose-piece

The structure that holds the objective lenses located under the ocular lenses and above the stage. It can be rotated to place the objective lens needed in the observation position.

Objective Lens

The lenses found above the stage and contained within cylinders screwed into the nosepiece. There are usually 4 lenses (4x, 10x, 40x, and 100x)

Scanning Objective

The objective lens with 4x magnification and red band

Low-Power Objective

The objective lens with 10x magnification and yellow band

High-power objective (or high-dry)

The objective lens with 40x magnification and blue band

Oil Immersion

The objective lens with 100x magnification and white band

Stage

The flat surface that holds the slide or material to be examined.

Stage Clips

Structure used to hold the slide in place.

Condenser lens

The structure that collects and concentrates the light for enhanced viewing. The condenser should be in the uppermost position for viewing of bacteria and other microorganisms.

Iris Diaphragm

A structure within the condenser that helps to collect and concentrate light for enhanced viewing. This should be fully open when using high-power objective lenses to admit the maximum amount of light.

Light Source

This is built into the microscope and utilizes a 6V, 20W tungsten halogen bulb.

On/off switch

The button located on the base of the microscope that will turn the microscope light on or off.

Arm

Curved structure connecting the eyepiece/head to the base.

Base

The bottom stand that supports the microscope.

Coarse focus adjustment knob

Large knob used for rapid movement of the objective lens toward or away from the specimen for general focus. Produces large up and down movement of the stage.

Fine focus adjustment knob

Smaller knobs inside the coarse knob used for detailed, precise focusing. Used to bring the object into final focus on higher powered lenses.

Stage movement control knobs

Knobs that move the slide to adjust the viewing position of the specimen.

Brightness control knob

Turned forward or backward to change the degree of light intensity. If it isn't turned all the way up the colors don't look right at 1000x magnification.

Coarse

The __________ focus adjustment knob should be used on low power objectives only

Fine

The ________ focus adjustment knob should be used on higher power lenses to achieve final focus.

Rheostat

What is another term for the brightness control knob?

Magnification

The enlargement of the image by the ocular and objective lenses.

Multiply

To find the total magnification of the image _________ the magnification of the ocular lens by the magnification of the objective lens.

Resolution

Which criterion for optimum observation is the most important?

Resolution

The ability to distinguish between two closely placed objects and see them as two distinct objects.

Wavelength

Resolution is based on the physical characteristics of the lens system and the _______ of light

Working Distance

The distance between the bottom of the objective lens and the surface of the slide under observation.

Closer

The higher the power of the objective lens the ________ it will be to the slide or the shorter the working distance.

Parfocal

Meaning that when the low power objective lens is focused very sharply the rest of the lens system will be in focus or only need very small adjustments using the fine focus knob.

Coarse Focus Knob

Never use the _____________________ when the oil immersion lens is in position.

Columination

Collecting light into a bundle (done by the condenser)

Refraction

The bending of light

Immersion Oil

This substance is used as it has the same refractive index as the glass of the slide, so it helps collect the slide to the lens, minimizing the light loss and improving resolution.

Contrast

The principle of light microscopy defined by the ability of objects to stand out from their background.

Stains

Chemicals or dyes used to enhance contrast.

Positive stain

A stain that colors the microorganism to make it stand out against a white background.

Negative Stain

A stain that colors the background but not the microorganisms to form a silhouette of the microorganism against a dark background.

Resolving Power

(0.61 * wavelength of light)/ Numerical aperture of objective lens

liquid

If the bacteria are growing in a ______ medium you can transfer 10-12 loopfuls directly to the slide.

Petri dish

If the bacteria are growing on a _________ add a drop of water to the slide and add a small amount of bacteria from the colony to the slide and stir to produce a smooth mixture.

Fix

After adding the bacteria you have to _______ the bacteria to the surface of the glass slide which is usually done with heat

Heat

To fix bacteria to the slide you have to use ______ from a Bunsen burner or heating block.

stain

Once the bacteria has been fixed, add your ______ and let it sit for 20-30 seconds. Then rinse the slide with gently flowing stream of water.

Stains

These are helpful because they can distinguish between bacterial cells and nonliving material, it can also determine the shape, relative size, and cell arrangement of the bacteria under observation. It can also detect the presence or absence of endospores.

Differential Staining

Staining that uses multiple dyes to distinguish between different types of cells or structures based on their distinct chemical or physical properties.

Safranin

What was the second stain used by Christian Gram?

Purple

What color are gram-positive bacteria?

Pink

What color are gram-negative bacteria?

Crystal Violet

What was the primary stain used by Christian Gram?

Crystal Violet, Gram’s Iodine, Acetone/Alcohol Mix, Safranin

What are the chemicals added to the sample in a Gram Stain?

Counterstain

A different color stain added after the primary stain has been removed from certain bacteria.