Biosci 221 Lab 3 Quiz

staining, stock bacteria, and population counts

What is an acidic stain and how is it helpful for viewing the size and morphology of the bacteria?

What is an acidic stain and how is it helpful for viewing the size and morphology of the bacteria?

Acidic stains are when we use acidic agents, such as Nigrosin, to stain bacteria and view them in their original shape and size due to the lack of heat fixation. The negative charge of the stain repels the negative charge of the bacteria’s cell, making it appear clear.

What is a basic stain and how is it useful in staining?

A basic stain is when we use a positively-charged dye to stain the negatively-charged bacteria and view them easily under the brightfield lens.

What are the average sizes of the bacteria you are working with?

0.5um-15um

How do you measure bacteria?

Using the ocular micrometer

How is oil immersion used?

A drop of immersion oil is used ONLY on the 100x objective lens to view microorganisms more clearly since the oil increases the resolving power.

How do we view differential stains?

By always using brightfield on a 100X objective lens with oil immersion.

What is a differential stain?

A differential stain is a staining process where we use several staining agents to differentiate bacteria by their shape or cell wall structures.

When do we use a differential stain and what does that tell us about the organisms that are positive or negative?

We might use a differential stain in a mixed sample of organisms to differentiate cell types. Bacterial cells with gram-positive cell walls are stained purple with the dye crystal violet, while bacterial cells with gram-negative cell walls are counterstained pink with safranin dye.

Which differential staining methods do we use?

Simple staining

Negative staining

Gram staining

Endospore staining

Acid-fast staining

What are the advantages of negative staining?

A negative stain is very good for observing cell morphology, arrangement, and size because the slide is NOT heat-fixed.

What is the purpose of heat fixation?

To fix the bacteria onto the slide so it doesn’t wash away with the staining agents and DI water.

What are the main problems when we make a smear preparation that is too thick or uneven?

We won’t be able to view bacteria clearly. A thick/uneven smear cannot be properly decolorized (so no clear shapes) and might wash off the slide despite fixation.

What are arrangements? (diplo-, strepto-, tetrads, sarcinae, staphylococci, palisade)

Arrangements are how bacteria are shaped and arranged in groups.

• Diplo- means they occur as a pair

• Strepto- means they are in a chain

• Tetrads are in groups of four

• Sarcinae are in groups of eight (3D like)

• Palisades are when bacilli are side-by-side (like a picket fence)

• Staphylococci are when cocci are in grape-like clusters.

Can there be various arrangements for the same bacterium?

Yes, always

What conditions affect how you see arrangements?

how thick your slide is and how well it has been spread around

What are the stock bacteria we have that may form tetrads?

Micrococcus luteus

Sporosarcina ureae

Kocuria rosea

Staphylococcus saprophyticus

What are the stock bacteria that may form sarcinae?

Sarcina aurantiaca

Sporosarcina ureae

Kocuria rosea

What are the stock bacteria that may form staphylococcal arrangements?

Staphylococcus epidermis

Staphylococcus saprophyticus

What are the stock bacteria that may form palisade arrangements?

Corynebacterium pseudodiphtheriticum

What are the stock bacteria that may form diplococci?

Sporosarcina ureae

Lactococcus lactis

Kocuria rosea

Moraxella catarrhalis

What are the stock bacteria that may form streptobacilli?

Bacillus cereus

Bacillus subtilis

Klebsiella aerogenes

Why is a counterstain always lighter than a primary stain?

Primary stain is usually darker and attaches to the cell walls first. Darker colors usually obscure lighter colors.

How do you use the following objective lens when we are observing bacteria: 10X, 20X, 40X, and 100X?

Find and focus on bacteria using the coarse and fine focus on the 10X lens. Continue to focus on the 20X and 40X lenses, then use immersion oil to fully focus on the 100X lens.

Name 2 things that may go wrong when you are making a negative stain

1. Putting too much dye onto the slide results in it not being able to thin out when smearing

2. Smearing it incorrectly that there is no difference between the thick and thin side

What is the dye used in negative staining?

Nigrosin

What are the stains and the procedures of Gram staining?

1. Begin with a heat-fixed emulsion

2. Cover the smear with crystal violet stain for 1 minute

3. Grasp the slide with a slider holder and gently rinse the slide with DI water

4. Cover the smear with iodine stain for 1 minute

5. Grasp the slide with a slider holder and gently rinse the slide with DI water

6. Decolorize with acetone/alcohol by allowing it to trickle down the slide until the runoff is clear. Gently rinse the slide with DI water immediately

7. Counterstain with safranin stain for 2 minutes. Rinse with DI water

8. Gently blot dry in a tablet of bibulous paper.

Name 4 things that may go wrong when you are Gram staining

1. If the smear is too thick, proper decolorizing will not be possible.

2. If the smear is overheated during heat fixing, the cell walls will rupture.

3. Older cultures contain more ruptured and dead cells; they may stain gram negative even if the bacteria are gram positive.

4. Excess water left on the slide will dilute reagents

What could have gone wrong if your Gram-positive rod looks like a Gram-negative rod?

When cells die off (too old), they don’t pick up the stain as well

What could have gone wrong if your Gram-negative cocci looks like a Gram-positive cocci?

Slide too thick, difficulty in decolorizing, age of the culture, etc.

What bacteria are endospore positive?

Sporosarcina ureae

Bacillus cereus

Bacillus subtilis

What is an endospore?

A structure for the preservation of the DNA while conditions are poor

What does the heat do in the endospore staining process?

The heating process makes the spore coat more permeable to the stain

When are endospores formed?

When stressed

If you don't find endospores does that mean it's negative?

Not necessarily; they may not be stressed or perhaps a procedure mistake

Do you have to do an endospore stain to see endospores?

No, an endospore can be seen in simple and Gram stains, as a clear or empty space in a vegetative cell.

What are the stains used in endospore staining?

Malachite Green

Safranin

What is the endospore staining procedure?

1. Begin with a heat-fixed emulsion

2. Cover the smear with a strip of bibulous paper. Apply malachite green stain. Steam for 5-10 mins. Keep the paper moist with the stain the entire time. Make sure you refill the beaker of water.

3. Grasp the slide with a slide holder. Remove the paper and dispose of it properly. Gently rinse the slide with DI water

4. Counterstain with safranin stain for 1 min. Rinse with DI water.

5. Gently blot dry in a tablet of bibulous paper.

What is the primary and secondary stain in endospore staining? Is there a mordant?

Primary = Malachite Green

Secondary = Safranin

The heating process acts as the mordant in this stain procedure

Are there important endospore-forming bacteria?

Clostridium difficile causes the disease C-diff and because of its ability to produce endospores it is not readily killed by disinfectants, and can easily be transmitted in healthcare facilities

What is acid-fast staining?

In the Acid-Fast stain, a very concentrated stain solution of kinyoun carbolfuchsin (hot pink) is used in order to penetrate and stain the cell walls

Why is AF staining important?

This stain is a valuable diagnostic tool for finding tuberculosis and nocardial diseases in patient specimens

What AF orgs do we have?

One; Mycobacterium smegmatis

What is the AF procedure?

1. Begin with a heat-fixed emulsion

2. Apply kinyoun carbolfuchsin stain for 5-10 mins

3. Grasp the slide with a slide holder. Gently rinse the slide with DI water.

4. Continue holding the slide with a slide holder. Decolorize with acid-alcohol until the runoff is clear. Gently rinse the slide with DI water

5. Counterstain with methylene blue stain for 1 min. Rinse with DI water

6. Gently blot dry in a tablet of bibulous paper

What is the most important step in AF?

The decolorization step

What do a positive and negative AF look like?

The bacteria will be purple if positive (i.e. Mycobacterium smegmatis) and blue if negative

What can go wrong with the AF procedure?

the organism doesn't retain the stain or it fails to stain properly

Know how to do dilution

1. Individual dilution

2. Total dilution

3. How much plating: 1.0 ml (x1) or 0.1 ml (x 10^-1)

4. (30-300) x DF