Contemporary Lab Skills - Tissue Culture and Centrifugation

Which of the following should be studied with a TISSUE culture?

Which of the following should be studied with a CELL culture?

Cells in a [blank_start]determined[blank_end] state remain the same after differentiating. [blank_start]Most[blank_end] cells are in this state. Cells in a [blank_start]plastic[blank_end] state are changing, usually before they differentiate. [blank_start]Stem[blank_end] cells are in this state.

List the Biosafety labs in order from MOST to LEAST restrictive. [blank_start]Level 4[blank_end] [blank_start]Level 3[blank_end] [blank_start]Level 2[blank_end] [blank_start]Level 1[blank_end]

Check all that apply to LEVEL 1 Biosafety labs.

Check all that apply to LEVEL 4 Biosafety labs.

As long as you wash your hands and arms up to the elbow and sanitize with >70% ethanol, you shouldn't have to worry about contaminating your culture due to personal hygiene.

Small pieces of jewelry are fine in a biosafety lab as long as they don't come into contact with your culture.

Which level would MOST LIKELY require a full-body containment suit?

Although Level 1 Biosafety Labs are generally safe, it is still recommended that you wear a lab coat, cap, gloves, and face mask in order to prevent contamination of your culture.

List the workspaces in order from LEAST to MOST protected/restricted. [blank_start]Laminar flow hood[blank_end] Air filtration system [blank_start]Containment hood[blank_end] Air filtration system Front shield Ultraviolet (uv) irradiation [blank_start]Glove box[blank_end] Closed system Interlock exchange [blank_start]Culture room[blank_end] Closed system Interlock exchange

The interior of an autoclave must have high pressure in order to stop water from expanding at high internal temperatures.

In order to sanitize using an autoclave, the object must be exposed to [blank_start]121[blank_end] degrees C at [blank_start]15[blank_end] psi for [blank_start]15[blank_end] minutes.

Which of the following are methods for FILTRATION sterilization?

Which of the following applies to Gamma irradiation?

Which of the following applies to Ultraviolet irradiation?

Culture media for cell/tissue growth MUST be well defined. Partially defined media will produce inconclusive results.

Liquid media is also known as

Which of the following are examples of liquid media?

Which of the following are examples of solid media?

Culture Containers [blank_start]Liquid Media[blank_end]: tubes flask bottles [blank_start]Solid Media[blank_end]: plates tubes magenta boxes

Which of these is an example of physical cell release?

Kelly attempted to cultivate individual bacterial colonies on a Petri plate with agar. However, when the culture had finished incubating, the colonies were in dense streaks throughout the plate with no colony isolation. Determine which step she most likely missed when streaking. Kelly: "I collected a sample from the suspension using a rod, and made a streak on a side of the agar, making sure to save room for my other streaks. I immediately took my rod and ran it through the first streak once and dragging it over the agar into my second streak. I did the same thing for my third and fourth streaks."

Which of the following are most effective at sanitizing metal instruments? (Choose 3)

Which of the following are valid incubation conditions?

Plants are usually incubated in __________ conditions.

Label the storage methods with their general conditions/uses [blank_start]Long-term storage[blank_end] -80 degrees C (freezer) -177 degrees C (liquid nitrogen) [blank_start]Glycerol stocks[blank_end] 50% glycerol Prevents cell death [blank_start]Freeze/thaw cycles[blank_end] Cell death Scrape surface

Disposal of Biohazardous waste requires some preparation before disposal. Incineration is performed [blank_start]off-site[blank_end] waste conditioning, while autoclaving is performed [blank_start]on-site[blank_end].

Which the following is the BEST describes a plant tissue culture? (Choose 3)

Match the medium/ingredient with its description [blank_start]Murashgee & Skoog (MS) Medium[blank_end]: -plant tissue culture [blank_start]Auxins[blank_end]: -Required for Somatic embryo induction, Callus culture, & plantlet regeneration -stimulates cell division/clumping, embryoid budding/clump formation, & root initiation -ABSOLUTE REQUIREMENT IN TISSUE CULTURE [blank_start]Cytokinins[blank_end]: -typically required for callus culture -sometimes promotes required for somatic embryogenesis -promotes plantlet regeneration -ABSOLUTE REQUIREMENT IN TISSUE CULTURE

[blank_start]Substantial[blank_end] amounts of reduced Nitrogen are required in tissue cultures. Organic nitrogen sources that function better at [blank_start]low[blank_end] concentrations include glutamine, glutamic acid, urea, and alanine.

[blank_start]Higher[blank_end] concentrations of auxins and [blank_start]lower[blank_end] concentrations of cytokinins yield roots. [blank_start]Lower[blank_end] concentrations of auxins and [blank_start]higher[blank_end] concentrations of cytokinins yield shoots. Other combinations yields [blank_start]callus[blank_end].

What is centrifugation typically used for?

Separation is based on:

Centrifugal force is expressed as...

Arrange the types of centrifuges according to their use: Bigger particles (Cells) [blank_start]Low-speed[blank_end] [blank_start]Micro-centrifuge[blank_end] [blank_start]HIgh-Speed[blank_end] [blank_start]Ultra-Centrifuge[blank_end] Smaller particles (DNA/RNA)

Which of the following centrifuges should be refrigerated? Why? (Choose 3)

The motion of particles during centrifugation is calculated using ___________.

Stoke's equation uses which of the following to calculate the motion of particles during centrifugation?

Select all that apply to sedimentation constants

1 Svedberg (S) = ______ seconds (s)

Which of the following will calculate centrifugal force?

label the abbreviations with their meaning in following equations for Centrifugal force. RCF = (w^2)r/g RCF = 11.18r/(rpm/1000)^@2 RCF = [blank_start]relative centrifugal force[blank_end] w = [blank_start]angular velocity[blank_end] g = [blank_start]gravitational force[blank_end] r = [blank_start]radial distance[blank_end] of particle from axis of rotation rpm = [blank_start]revolutions per minute[blank_end]

When centrifuging, equivalent masses MUST lay opposite each other in the rotor. This is more critical at lower g-forces because the lower speed allows for greater imbalance.

Why must centrifuges be balanced before use?

This is a [blank_start]fixed-angle[blank_end] rotor. (Type of Separation - Efficiency) Pelleting - [blank_start]Excellent[blank_end] Rate zonal - [blank_start]Poor[blank_end] Isopycnic - [blank_start]Good[blank_end]

This is a [blank_start]swinging bucket[blank_end] rotor. (Type of separation - Efficiency) Pelleting - [blank_start]Inefficient[blank_end] Rate zonal - [blank_start]Good[blank_end] Isopycnic - [blank_start]Adequate[blank_end]

This is a [blank_start]vertical[blank_end] rotor. (Type of Separation - Efficiency) Pelleting - [blank_start]Poor[blank_end] Rate zonal - [blank_start]Good[blank_end] Isopycnic - [blank_start]Excellent[blank_end]

This is a [blank_start]continuous flow[blank_end] rotors. (Type of separation - Efficiency) Pelleting - [blank_start]Poor[blank_end] Rate zonal - [blank_start]Excellent[blank_end] Isopycnic - [blank_start]Adequate[blank_end]

Centrifugal rotors are rated for [blank_start]maximum operating speed[blank_end]. The rating is established by [blank_start]the manufacturer[blank_end] based on [blank_start]construction materials[blank_end] and [blank_start]design[blank_end].

De-rating is the reduction in maximum operating [blank_start]speed[blank_end] after a period of use. The de-rating schedule is established by [blank_start]the manufacturer[blank_end]. It prolongs the useful life of the rotor, albeit at a lower maximum operating [blank_start]speed[blank_end].

The picture depicts [blank_start]pelleting[blank_end], which happens when using a [blank_start]fixed angle[blank_end] rotor to centrifuge until the particles sediment to the bottom of the tube.

[blank_start]Differential pelleting[blank_end] happens after repeating centrifugation at higher RCFs. The pellets sediment to the bottom based on size and mass ([blank_start]Nuclei[blank_end] > [blank_start]Chloroplast[blank_end] > [blank_start]Mitochondria[blank_end])

This is [blank_start]rate zonal centrifugation[blank_end], which separates particles based on mass and shape. The sample is stabilized by a [blank_start]sucrose[blank_end] gradient so that sedimentations happen in layers (sedimentation constants proportional to [blank_start]molecular weight[blank_end]). A [blank_start]swinging bucket[blank_end] rotor is used for analytical purposes and small-scale preps, while a [blank_start]vertical[blank_end] rotor is used for larger particles.

This is [blank_start]isopycnic centrifugation[blank_end], or equilibrium density gradient centrifugation. In this kind of centrifugation, migration stops when particle [blank_start]density[blank_end] equals medium [blank_start]density[blank_end]. For this kind of centrifugation, a [blank_start]vertical[blank_end] rotor is fast and produces high resolution. A [blank_start]fixed angle[blank_end] rotor produces a high resolution, and a [blank_start]swinging bucket[blank_end] rotor has a high capacity.

Label as it applies to the pictured centrifugation ([blank_start]isopycnic[blank_end]): Typical gradients: [blank_start]CsCl[blank_end] (most common) [blank_start]Percoll[blank_end] (common) [blank_start]Ca2SO4[blank_end] (occasionally) Typical starting density = [blank_start]1.55[blank_end] CsCl/mL Density or buoyant density (p) in CsCl Protens = [blank_start]1.3[blank_end] dsDNA = [blank_start]1.6-1.7[blank_end] RNA = [blank_start]1.75-1.8[blank_end] p is used to separate and characterize [blank_start]DNA[blank_end] p = 1.660 + 0.098*(G + C) where (G + C) = [blank_start]mole[blank_end] fraction

In plants, G + C (the mole fraction) may be too similar to separate and characterize DNA using isopycnic centrifugation. In this case, one would have to isolate the organelles to get the DNA.

Label with the corresponding type of gradient

Which of the following are continuous density gradients?

Which type of gradient is the most common?

Which of the following would likely show a step gradient after centrifugation?

The picture depicts a [blank_start]gradient maker[blank_end]: a [blank_start]two[blank_end]-chambered apparatus that controls the mixing of two solutions to form a gradient.

Which of the following are self-forming gradients?

The method used to separate the supernatant from pellet with minimal cross-contamination depends on ____________.

Which of these are methods of removing a supernatant?

Which of these are resuspension methods?

Which of these are methods of recovering bands?

One way of concentrating samples is through [blank_start]precipitation[blank_end], where one centrifuges to pellet and collects the precipitate. Then one resuspends the pellet in the buffer to the desired concentration. [blank_start]Nucleic Acids[blank_end] - Ethanol - Isopropanol [blank_start]Proteins[blank_end] - Acetone - Ammonium sulfate - Isopropanol - Trichloroacetic acid

One way of concentrating samples is [blank_start]spin columns[blank_end], where one centrifuges a tube with 2 chambers with a [blank_start]molecular weight[blank_end] cut off membrane separating the compartments. Before centrifugation, the upper chamber contains a [blank_start]dilute[blank_end] sample. The lower chamber collects the [blank_start]filtrate[blank_end] during centrifugation, while the lip surrounding the membrane collects the [blank_start]concentrated sample[blank_end] in the upper chamber.

To recover samples from small-volume solutions, pulse-spin the tube to accumulate liquid at the ____________ of the tube.