BIO4102

What does form mean (when describing colonies)?

The shape of the entire colony from above.

What does a punctiform colony shape look like?

What does a circular colony shape look like?

What does a filamentous colony shape look like?

What does a irregular colony shape look like?

What does a rhizoid colony shape look like?

What does a spindle colony shape look like?

What is the difference between a flat and a raised colony?

from the side, both are flat, but raised is thicker

What does elevation (with respect to colonies) mean?

This is the shape of the colony as it rises above the agar from a side view

What is the difference between a pulvinate and a convex colony?

Convex is like a half-oval and pulvinate is more like a half-circle

Describe what a umbonate colony looks like.

a boob

What does margin mean (with respect to colony formation)?

The shape of the exterior edge of a bacterial colony.

What does an undulate margin look like?

What does a lobate margin look like?

What does a erose margin look like?

What does a filamentous margin look like?

hairy

What does a curled margin look like?

waves

What method did we use to introduce new DNA into bacteria?

the Calcium Chloride/Heat Shock method

What do the calcium ions do in the Calcium Chloride/Heat Shock method?

the divalent cations of calcium (Ca2+) disrupt the plasma membrane integrity, neutralize the negative charges associated with the plasma membrane, and enable the DNA to interact with the bacterial cell more closely.

What does the "shock" do in the Calcium Chloride/Heat Shock method?

further destabilizes the membranes and enables some DNA to move across the membrane into the cytosol of the cell

What is the regulatory protein used in the Calcium Chloride/Heat Shock method? Tell me about it.

AraC. This is a repressor protein that can interact with a sequence of DNA around a promoter and inhibit RNA polymerase from transcribing the gene.

What will you see if the AraC protein is created and there is no arabinose?

GFP will not be transcribed (no glowing)

What will you see if the AraC protein is created and there is arabinose?

GFP will be transcribed (will glow)

What does ampR gene make?

B-lactamase inactivates the amicillin (bacteria becomes resistant)

What does the + and - on the tubes mean in the Calcium Chloride/Heat Shock Lab?

+ : has pGlo

- : has water

What all goes in the + and - tubes in the Calcium Chloride/Heat Shock Lab?

+ : CaCl2, E. coli, and pGlo

- : CaCl2, E. coli, and water

What do you expect to see in the + side of the before plate (no ampicillin)?

Regular growth

What do you expect to see in the - side of the before plate (no ampicillin)?

Regular growth

What do you expect to see in the + side of the after plate (no ampicillin)?

Glowing and regular growth

What do you expect to see in the - side of the after plate (no ampicillin)?

Regular growth

What do you expect to see in the + side of the before plate (with ampicillin)?

no growth

What do you expect to see in the - side of the before plate (with ampicillin)?

no growth

What do you expect to see in the + side of the after plate (with ampicillin)?

glowing growth

What do you expect to see in the - side of the after plate (with ampicillin)?

no growth

What are the two features of a properly prepared smear?

1) the microorganisms are transferred to the slide in a manner and amount that enables staining

2) that the microorganisms have been fixed to the slide.

What does heat-fixing a slide accomplish?

1. Adheres the bacteria to the slide surface

2. Kills most microorganisms without destroying structural features

3. Enables many stains to better penetrate or react with the microorganisms

What are two ways that you could ruin a stain before staining?

1. you heat-fixed the slide while it was still wet: the presence of liquid will result in microorganisms being “boiled” and destroying structural features needed for proper staining

2. you heat-fixed the slide for too long or at too hot of a temperature: using temperatures that are too high or using extended times can also destroy the smear

It is VERY important to make sure the smear is _____ before heat-fixing.

dry; the presence of liquid will result in microorganisms being “boiled” and destroying structural features needed for proper staining

What are the two categories you can sort stains into?

basic and acidic

Basic stains-

have a positive charge, which is attracted to the negative charges associated with many bacterial cell wall components. Most of the commonly used dyes, such as crystal violet and methylene blue, fall into this category.

Acidic stains-

have negative charges and are typically repelled by bacterial cell wall structures. These dyes provide contrast to the background (called negative staining) and include dyes such as nigrosin and India ink.

When a single dye is used, these procedures are referred to as ________ stains that provide contrast to observe cell shape and arrangements, but do not provide additional information about the microorganism.

simple

If a cell retains the primary stain it is referred to as "(positive/negative)" and if only the counter stain is retained it is referred to as "(positive/negative)."

positive, negative

This is best illustrated with the common Gram stain, where bacteria that retain the primary stain crystal violet are called Gram-positive, and those that only retain the counter stain are called Gram-negative. You will see this pattern with other differential stains used in this lab such as the Acid-fast stain and the Endospore stain.

What is the primary stain in the gram-staining protocol?

crystal violet

What does the crystal violet do in the gram-staining protocol?

This purple dye is a cationic dye that has a positive charge. The dye is attracted to the negative charges associated with both Gram-positive and Gram-negative cell walls.

However, most of the crystal violet on the Gram-negatives is associated with the outer membrane, while in the Gram-positive cells, it is complexed within the thicker peptidoglycan layer.

After the crystal violet has been added, what would you see in the microscope?

only of violet microbes

The next step in the gram-staining protocol uses a(n) ______ solution which functions as a Mordant.

iodine

What does the iodine/mordant do in the gram-staining protocol?

The Iodine complexes the Crystal Violet and supports its retention in Gram-positives in the subsequent step.

What is used as the decolorizer in the gram-staining protocol?

95% Ethanol

What does the decolorizing agent (ethanol) do?

The ethanol solution is able to solubilize the dye and outer membrane associated with Gram-negative cells. This results in the removal of the crystal violet dye associated with these cells. However, in Gram-positives the ethanol dehydrates the thick peptidoglycan layer (in essence collapsing the peptidoglycan matrix around the complexed crystal violet dye). This action, along with the amount of dye associated with the thicker peptidoglycan layer found in Gram-positives, helps to retain the crystal violet dye in these cells. It is important to note that excessive rinsing with ethanol will eventually remove the crystal violet dye even from Gram-positives.

What agent/step allows us to differentiate between the gram-positive and gram-negative cells in a gram-staining procedure?

the ethanol/decolorizing step

What allows ethanol to be able to differentiate between the gram-positive and gram-negative cells in a gram-staining procedure?

The difference in the amount of ethanol needed to decolorize Gram-negative and Gram-positive cells

After ethanol decolorization:

How would Gram-negative bacterium appear? How would Gram-positive bacterium appear?

Gram-negative would be clear

Gram-positives would be purple

If you looked in a microscope after the ethanol step, you would only see purple cells

What is used a counterstain in the gram-staining protocol?

safranin

What does safranin/counterstain do in the gram-staining protocol?

Enables the visualization of the Gram-negative cells. Dyes both of the cells.

What would you see if the counterstain was darker than the primary stain?

All of the cells would be the same color (the color of the darker counterstain)

At the conclusion of the procedure, the Gram-positive cells will appear ______, and the Gram-negative cells will appear ______.

purple (they are positive because they retained the primary stain), pink (from the Safranin counterstain)

What is the potential explanation for "Slide can be focused at 400X but not at 1000X"?

· The slide was placed with the sample facing down on the stage.

· You are focused on something on the bottom of the slide (dried dye etc.)

What is the potential explanation for "The slide is dark and grainy when observed at 1000X"?

Not enough light available for observation

What is the possible solution for "Slide can be focused at 400X but not at 1000X"?

· Turn the slide so the sample is facing up towards the objective.

· Remove the slide and clean the bottom of the slide.

What is the possible solution for "The slide is dark and grainy when observed at 1000X"?

· Open the aperture of the iris diaphragm

· Increase the intensity of the LED light source

What is the potential explanation for "Large purple geometric crystals or large purple "star bursts""?

The slide was too warm, when the crystal violet was applied causing the dye to precipitate onto the slide.

What is the potential explanation for "Nothing visible on slide"?

- Not enough bacteria placed on the slide (uncommon)

- Slide was not heat-fixed prior to staining protocol.

What is the potential explanation for "Gram-negative cells stain purple or a mix of purple and pink"?

- Smears were not decolorized enough to remove crystal violet from Gram- negative cells

- The smear was too thick and the ethanol was unable to decolorize the bacteria. This is not uncommonly observed in areas where the smear is thicker and you may want to observe areas of the slide with fewer bacteria.

What is the potential explanation for "Gram-positive cells stain light purple or pink"?

- Smears were heat-fixed too long.

- Insufficient iodine used.

- Cultures used are old.

- Too much water used in-between steps.

What are the parts of a compound light microscope?

Illuminator: the light source in the base of the microscope.

Abbe Condenser: a two lens system that collects and concentrates light from the illuminator and directs it to the iris diaphragm.

Iris Diaphragm: regulates the amount of light entering the lens system.

Mechanical Stage: a platform used to place the slide on which has a hole in the center to let light from the illuminator pass through. Often contains stage clips to hold the slide in place.

Body tube: Houses the lens system that magnifies the specimens

Oculars/Eye pieces: what you view through

Nose-piece: revolves and contains the objectives

Subculturing-

is transferring microorganisms from one media type to another. Various media types used in microbiology labs include agar slants, agar deeps, agar plates, and broths.

Innoculate-

Introduce cells to a medium

What are the advantages/disadvantages of an agar slant?

Agar slants are often used to maintain short-term stock cultures as they provide a large surface area relative to the space required to store them in the lab. These are also less prone to contamination due to the smaller opening compared to an agar plate.

What are the advantages/disadvantages of an agar deep?

These are typically inoculated by stabbing the media with a sterile needle. These are used for a variety of applications that include assessing motility of bacterial cultures.

What are the advantages/disadvantages of an agar plate?

providing a large surface on which to culture microorganisms. Like the agar slants, these are inoculated by streaking the surface with a sterile loop. The large surface area on these plates is often used to isolate colonies of bacteria.

What are the advantages/disadvantages of a broth tube?

can be inoculated by a sterile loop, needle, or pipette. Bacterial growth in liquid media can have distinct advantages, including the ease of scaling up cultures and the maintenance of bacterial arrangements that can be obscures when grown on solid media.

What is the closest total magnification you can have with a coverslip?

400X

What is the objective lens at 1000X total magnification?

100X

The _______ stain is a differential stain that distinguishes bacteria based on unique cell wall properties.

acid-fast

Acid-fast bacteria are taxonomically Gram-________, but often do not stain well when using Gram Stain techniques.

positive

Acid-fast bacteria have a waxy substance called ______ ____ in their cell walls, which comprises up to 60% of the cell wall components. This substance is primarily responsible for the unique characteristics that enable the acid-fast stain to differentiate these cells.

mycolic acid

In the acid-fast protocol, the primary stain in the acid-fast staining procedure is-

carbol fuchsin

In the acid-fast protocol, the carbol fuchsin (primary) stain contains ______ (organic solvent) that helps to solubilize the waxy cell wall and allow the stain to penetrate the cell.

phenol, but the phenol alone is not sufficient to allow much stain to penetrate the cell, and thus heat is required to further “loosen” the waxy mycolic acid.

In the acid-fast protocol, which cells will be stained by the carbol fuchsin (primary) stain? And what colors will the cells stained by this be?

all of them, reddish-pink

In the acid-fast protocol, what is the purpose of the water?

It cools the slide which locks the stain into the waxy acid-fast cells, and then cells are decolorized.

In the acid-fast protocol, the decolorizer used in the acid-fast staining procedure is _____ ______, which decolorizes all cells except acid-fast cells.

acid alcohol

After the decolorizer (acid alcohol) step in the acid-fast protocol,

What would the acid-fast and non-acid fast cells look like?

acid-fast: reddish-pink

non-acid-fast: clear

In the acid-fast protocol, the counterstain then used is _________ ______.

methylene blue

At the end of the staining procedure, acid-fast cells are __________ and all other cells are ______.

reddish-pink, blue

The endospore stain is a differential stain that stains-

bacterial endospores or spores.

In the endospore staining procedure, the primary stain in the endospore staining procedure is-

malachite green

Which two procedures require heat-fixing?

acid-fast staining and endospore staining

In the endospore staining procedure, once the cells have cooled, the cells are decolorized with _______, which selectively removes the malachite green from all vegetative cells but not from endospores.

water

In the endospore staining procedure, the counterstain then applied is _________.

safranin

At the end of the endospore staining procedure, the endospores are __________, and vegetative cells are _______.

dark green, pink

What is an anionic stain?

a negative stain

Why do we use negative stains?

Negative stains have the advantage of facilitating the observation of "fragile" bacteria that are sensitive to smear preparation techniques and/or cationic dyes. As described earlier, negative stains are anionic stains that, due to their negative charge, are not attracted to structures on the surface of bacterial cells. Thus, a negative stain will stain the "background" on a slide, and bacteria are visualized by the transmission of light through the bacteria.

The negative stain used in this lab is ________.

Nigrosin

If you found 48 microbes on the 10^-6 plate, how many microbes were in the 10^-4 tube?

4,800 microbes

What are the two questions that help us classify bacteria based on their interaction with oxygen?

1) does the bacteria USE oxygen in a metabolic process

2) is the bacteria able to DETOXIFY reactive oxygen species

Aerobes

require atmospheric O2 (~20%), and use O2 as the final electron acceptor in an electron transport system.

Microaerophiles

require O2 at concentrations below atmospheric levels, typically 2-10%. Microaerophiles have a limited ability to neutralize toxic oxygen, so excess O2 will kill the bacteria. However, microaerophiles do use O2 as final electron acceptor in the electron transport system.

Obligate Anaerobes

cannot survive in the presence of any oxygen. Obligate anaerobes lack the enzymes necessary to break down the toxic by-products of oxygen. In these bacteria, the final electron acceptor in the electron transport system is a molecule other than O2 (this is called anaerobic respiration).

Aerotolerant

organisms grow equally well in the presence or absence of oxygen. They do possess enzymes necessary to neutralize toxic oxygen by-products, but they don't use O2 as a final electron acceptor.