Micro lab quiz pt 2

What type of media is BHI (Brain heart infusion)

Enriched non-selective media

What type of organisms is BHI used to grow?

Yeasts, molds, bacteria, and many pathogens

What are the colony morphological characteristics that can be viewed with the naked eye, a hand lens, or a colony counter?

Colony shape, margin (edge), surface, elevation, textuse, and optical properties

What is a margin?

The appearance, shape, or edge of the microorganism

What are some examples of margins?

entire (smooth, no irregularities), undulate (wavy) lobae (lobed)

What is a whole colony?

a visible cluster of microorganisms that have grown on or within a media.

A single colony on a plate is typically composed of…

a visible mass of cells that originated from one cell undergoing many rounds of replication.

Colony morphology

can vary from one microbial species to the next.

Colony morphology is typically analyzed for five basic categories. These include shape, margin, elevation, pigment production, and

texture

Smooth, rhizoid, lobate, and filamentous would be terms used to describe the ___________ of a colony.

Margin

What is the function of the nosepiece

Holds the objective lenses and allows for easy switching between them

What is the function of the objective lenses

Magnifies specimens for detailed observation

What is the function of the stage clips

Holds the slide in place on the stage so it doesnt move

What is the function of the stage

Where the specimen slide is placed

What is the function of the condenser

To focus light onto the specimen

What is the function of the coarse adjustment knob?

Larger knob on the side of microscope, used to move the stage up and down

What is the function of the fine adjustment knob

small knob on the side of microscope to fine-tune the focus of your specimen

What is numerical aperture?

The measure of the lens ability to “capture” light coming from the specimen and use it to make the image

What is resolving power?

A measurement of how far apart two points must be for the microscope to view them as being separate

How do we determine total magnification?

magnification by the objective lens X magnification by the ocular lens = Total magnification

What is the role of wavelength of light used in bright field microscopy?

It determines the maximum resolving power (resolution) and defines the limits of what limit of detail can be seen

What does a shorter wavelength mean

Higher resolution and you can see details better

If the total magnification of a microscope can be increased by simply adding more lenses, why are bright-field light microscopes not outfitted to magnify things greater than 1300×?

Because the resolution limit for a light microscope is about 0.2μm, magnifications above this will not produce clear images.

Fluorescent microscopy differs from other types of light microscopy in that it requires

the use of an ultraviolet light source.

Why is it important to center an object you are analyzing in the field of view of the microscope before switching to a higher power lens?

As magnification increases, field diameter decreases and objects at the periphery will no longer be in the visible field.

Most microscopes are parfocal. This means that the distance from the nosepiece opening to the focal plane of each lens has been standardized. What does this mean to you with regard to how you use the microscope?

Once the specimen is in focus under a low-power lens, the microscope can be switched to a higher-power lens without moving the stage and the image will be in or nearly in focus no matter what lens is used.

Spheres

Cocci (singular coccus)

Rods

Bacilli (singular bacillus)

The shape is…

the morphology of bacteria

Spirals

spirilla (singular spirillum)

Slightly curved rods

Vibrios

Short rods

coccobacilli

Flexible spirals

Spirochetes

Single cells

Cocci, bacilli, spirilla, spirochaetes, and vibrios

Pair of cells

Diplo

Chains of cells

Strepto

four cells fused together

Tetrads

Cube

Sarcina

Irregular cluster

staphylo

How does a basic dye work?

The charged portion of a colored molecule allows it to act as a dye through ionic or covalent bonds between the colored molecules and the cell.

What is a basic stain

When the charged portion of the colored molecule becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion

The term coccus describes a bacterial shape that is _______________.

Spherical

During the simple stain procedure, why is it necessary to heat-fix the cells?

To adhere them to the slide.

Chromogens that function as basic stains in the simple staining procedure color the bacterial cell by

adhering to the negatively charged cell.

When preparing a bacterial smear for staining, why is it important to air dry the slides before heat-fixing?

To prevent boiling during the heat-fix that would damage the cells.

Chromogens that function as acidic stains in the negative staining procedure make the bacterial cell visible by

adhering to the glass of the slide.

Negative staining is preferable to simple staining when

determining the accurate size is crucial.

Negative staining allows for more accurate size determination because

No heat-fix is involved

Why must the stain be spread very thin in the negative staining procedure?

To allow individual cells to be visualized clearly.

What negative stain dye did we use in class to visualize bacteria (the one with no heat fixing)

Nigrosin

Are the cells stained or unstained in a negative stain?

The cells remain unstained in a negative stain

Explain the steps of the gram stain procedure

1.) Put a drop of crystal violet stain on your slide

2.) Sterilize your loop, cool it, then get some desired bacteria on your loop

3.) Mix the bacteria witch the crystal violet on your slide

4.) Wait one minute, heat fix, and rinse the slide using distilled water (while holding the slide with a slide holder)

5.) Keep rinsing until the water coming off of the slide is clear

6.) Add gram Iodine stain, let it dry for one minute

7.) Do the rinsing step again, same rules

8.) decolorize the slide with a gram decolorizer and use water until the runoff is clear

9.) Immediately counterstain the smear with safranin for one minute

10.) Rinse slide again the same way

11.) Gently blot dry in the blotting paper

12.) When dry observe under the microscope. You will need to use oil immersion when you get to 100x

Does gram negative bacteria turn purple at any point?

Yes. When the crystal violet stain is used it dyes both gram positive and gram negative cells purple. Then the decolorization step makes the gram negative cells transparent again (gram positive stays purple). Finally, when Safranin is added the gram negative cells turn pink/red

Does gram positive bacteria change color?

No it stays purple after the crystal violet stain is added

What color is gram positive bacteria

purple

What color is gram negative bacteria

pink/red

What are the structural differences between gram positive and gram negative cells

Gram neg. cell walls have a higher lipid content and a thinner layer peptidoglycan layer than gram pos. cell walls

What is the most likely thing to go wrong in the gram stain procedure?

The decolorization step. It is posible to over-decoloize by leaving the decolorizer on for too long and get reddish gram positive cells. It is also possible to under-decolorize and produce purple gram negative cells. Which are both wrong due to poor technique

Upon completion of the Gram stain, Gram-positive cells will appear

Purple

In the Gram-stain procedure, the primary stain crystal violet functions to

stain all bacterial cell walls

In the Gram-stain procedure, Gram-negative, but not Gram-positive, cell wall structures are decolorized because

the alcohol removes the lipids of the Gram-negative wall, making the wall more porous.

Differential stains function to

highlight differences between cells or parts of a cell