cell bio 2 lab final exam review flashcards

Lag phase

stopping/slowing of cell growth as it prepares to divide

     Exponential phase

cells divide quickly with short intervals in between

Log phase or logarithmic growth

when the exponential phase is plotted in log form and it makes a straight line

Doubling time

how long it takes population to double, found on a log phase graph

The use of the slope of the log form graph

It can be used to compare among different graphs to figure out the optimal growth conditions

stationary phase:

no increases in cell numbers are noted because all the resources are almost used up, visually the slope increase slows down

Death phase

the cells in solution start to die off because there are no more resources left

Cell growth curve is plotted in…

time on x axis and organisms/mL on y axis

process that we did for cell growth kinetics lab

1.       Prepare liquid cultures of whatever organism (we compared yeast and E.coli) and monitor their growth.

2.       Change variables as desired (we changed incubation temperatures)

3.       Measure at different time intervals (we did 0, 1, 2, 4, and 17)

4.       Then we can measure optical density, do dilution plating, and direct cell counts with hemacytometer (which we only did for yeast)

Optical density equation (done through spectrophotometry)

cells per ml/ OD unit (values over 1 are irrelevant)

doubling time equation

time/(3.3 log(end cell density/beginning cell density)

hemocytometer counts equation

(cell count x10^4)/ numbers of squares counted

Spectrophotometric readings and real life example

·       Cells have a certain wavelength they emit so we can measure the overall wavelength of a sample to find out roughly how many cells are in there

·       We can analyze at 600 nm to find absorbance of cells that they emit at that wavelength

Dilution plating:

·       We have to dilute the solution (we did 10^-2, 10^-4, 10^-6, 10^-7 of bacteria and 10^-2, 10^-4, and 10^05 of yeast) in order for it to properly grow on the agar plates

·       Total dilution was 1 million fold (so the first 2 are 0.1 mL bacteria to 9.9 mL solution and last 2 are 1 mL bacteria to 9 ml solution)

Hemocytometer counts

·       We did this technique on yeast. Usually this technique is done if the cells are big enough to be looked at under a regular microscope.

·       The plates for this technique have a certain volume. It is 3 mm square and each little square is 0.25 mm long. We count the number of cells in each group of 16 little squares and multiply it by 9 to get the number of cells in the sample total

Differential centrifugation

This technique separates parts of the cell depending on weight, size, or density

Rate zonal centrifugation (sedimentation velocity or zone configuration)

the parts are separated by their sedimentation coefficient which depends on mass and shape. Larger particles are easier to pellet because they are heavy, but the less heavy parts are still in solution

Isotonic centrifugation (density gradient centrifugation)

separate parts based on buoyant density, and happens because of the steep gradient

rcf equation

1.12 x radius x (rpm/1000)² 

rpm formula

sqrt(rcf/1.12 times radius) times 1000

Marker enzyme

enzyme activity that is specific to a certain organelle

Acid hydrolases

a class of degradative enzymes that are most “happy” in acidic environments. An example of this is the fluid inside of the lysosome

Acid phosphatase

the hydrolytic enzyme inside of lysosomes, and its function is to break phosphor-ester bonds

why do we utilize acid phosphotase?

Because the enzyme is non-specific and will bind to product containing the phosphor-ester bond, we can make it bind to a synthetic product that makes a yellow product so we can measure it with the Spectro Vis at 405 nm.

Biuret assay (sensitivity greater than 1 mg/mL):

uses an amine end terminus, is only good for food because it has a high protein concentration

Bradford Assay (sensitivity greater than 10 micrograms/ml) :

in this technique, we used proteins of known concentrations to measure protein concentrations/purity depending on what it emits at 405 nm

Lowry procedure (sensitivity greater than 1 microgram/mL)

another protein assay method [unfortunately not given any info on this one]

Fluorescamine precure

sensitivity of less than 1 microgram/mL. This method is very expensive because of how accurate and specific it is.

Why do we use protein assays?

With these methods, we can directly measure the effects of temperature and pH on how the enzyme works or how much protein is in the sample

beer lambert equation

concentration= absorbance value/ (extinction coefficient x path length [usually 1])

Polyacrylamide gel electrophoresis (PAGE)

Using this technique, proteins can be identified depending on how far it goes through the gel, as the negative charge on them (whether it be natural or artificial) causes the migration

  Why is denaturing gel needed…

the technique requires boiling and SDS is in the buffer which causes denaturation. SDS has a net negative charge so it migrates to anode (smaller go higher up than bigger)

Native gel- s (technique)

This technique requires no boiling, no sds, so no denaturing. Separation is based on native charge. This is used to purify protein

Types of actins which we looked at in creeping cells

G actin (actin monomers) and F actin (what makes up microfilaments)

Immunofluorescence

light is absorbed at one wavelength and released at another (blue comes out at green, UV comes out as blue, Green comes out at red)

Gel Casting:

We used discontinuous gel electrophoresis which has 2 matrixes (stacking and separating)

2 specific examples of gel staining agents/technique:

Coomassie blue or fluorescent staining

Antigen means… (definition)

antibody forming

monoclonal antibody

this type binds directly to the substrate

polyclonal antibody

this type binds to the antibody itself

real life example of affinity labelling

we used phalloidin which has a high affinity for filamentous actin so it binds to it there

Chemiluminescence definition

emits light but does not release heat because of it

Karyotype

analysis of chromosomes stuck in metaphase, shown visually in an array or spread

what karyotypes show

shows deletions, insertions, translocations, inversions, monosomy, triploidy, and trisomy, or if there is too many or too few chromosomes

How did we stop the chromosomes when they are in metaphase?

By adding an anti cancer drug that blocks microtubule polymerization, so stops the mitotic spindle to form.

HELA cells

collected malignant cancer cells that keep dividing and are sold commercially

Human chromosomes are

metacentric, submetacentric, or acrocentric

Mouse chromosomes are…

telocentric

Steps we took to make chromosome spreads:

1.       Stop at metaphase

2.       Swell them with KCl

3.       Fix them in methanol/acetic acid

4. yeet them onto the plate to break the cells open to expose chromosomes

Polymorphic chromosomes definition

diversity between chromosomes

Variable number of tandem repeats (VNTR)

repeated copies of the same sequence that are next to each other on chromosome

Steps of PCR reaction:

1.       Denaturation at 94 degrees Celsius

2.       Primer bonding at 65 degrees celcius

3.       Extension at 72 degrees Celsius