BIO LAB 9

Purpose of a chi-square test

Determine whether an observed distribution of experimental results was significantly different from an expected distribution (chance or not)

Purpose of a t-test

Compare the means/averages of two groups (ex. control and “treated” group) to determine whether the means of the two groups are significantly different from each other

Null hypothesis

States that the means of the two samples are equal and not statistically different; degree of difference is due to chance

Null hypothesis is accepted when

t value is less than/equal to table value for that d.f.

Null hypothesis is rejected when

t value is greater than table value for d.f.; means two mean values are statistically significant, so treatment does have a significant effect

Variable

What the mean measures; can be height, weight, blood pressure, etc

Treatment

The group opposite to the control group; received a variable that sets them apart, such as a medication

Sampling

Selection of test individuals; usually selected to try and represent a whole population

Accuracy

Refers to true value, with all sources of error and bias eliminatedP

Precision

Reproducibility of a measurement

Turbidimetry

Using a colorimeter to measure the amount of light transmitted through suspensions of cells

Light in turbidimetry

Will be reflected and scattered (instead of absorbed); this is because the cells are so LARGE

Difference between dissolved and suspended particles

Dissolved particles will never settle out of solution; suspended particles would settle, in time, to the bottom of the container

Different example of settling process

Centrifuge with the milk proteins; separated casein from supernatant fluid

Cells being observed in turbidimetry here

Yeast cells

Bulleted steps for turbidimetry procedure

• Turn on SpectroVis Plus, set wavelength to 500nm

• Weigh ut 0.5g of dry yeast

• Transfer 25mL of GYE into Erlenmeyer flask

• Swirl flask to suspend cells

GYE

Yeast growth solution; provides water and nutrients to dry yeast cells from packet to bring them out of dormant state

What happens when you mix dry yeast and GYE

Dry yeast cells will resume active metabolism and begin to divide mitotically to produce more yeast cells

Most common yeast in baking/brewing

Saccharomyces cerevisiae

Normal yeast cell dimensions

3 μm to 10 μm (larger than bacterial, smaller than most eukaryotic cells)

Bud

A daughter cell produced in mitosis; instead of splitting, original cell produces this enlarging “bud” attached to its side, which ultimately detaches

(receives a full set of chromosomes)

Why yeast suspension occurs “milky”

Because it has an incredibly large number of suspended cells and reflects light of all wavelengths (“white” light)

1:100 dilution of original suspension yielded

An average cell count of 31.3 cells per 0.00625 cubic mm

Concentration of cells in 1:100 dilution

5 × 10⁶ cells/mL; calculated by 31.3/0.00625 mm

Concentration of undiluted suspension

5 × 10⁸ cells/mL

Caution for pipetting in this experiement

Be sure to swirl it to ensure uniform distribution of cells throughout, and not them settling to the bottom (since they are suspended and not dissolved)

General procedure for assay setup

• Set up 6 large test tubes

• Pipette individual amounts of GYE into each

• Then pipette 1mL of flask’s suspension into tube 1, swirl to mix

• Measure 1mL of this tube and put into tube 4

• Then transfer 2mL from tube 1 into tube 3

• Then transfer 3mL from tube 1 into tube 2

• Then transfer 6mL from tube 4 to tube 5

• Then transfer 3mL from tube 4 to tube 6

• Set colorimeter wavelength to 500nm, and select appropriate graph

• Calibrate machine using blank

• Collect %T for each sample, and record in table

• Calculate concentration (# of cells per mL), and then using this value calculate log of concentration

Composition of the “blank”

3mL GYE without yeast

Dilution values for each tube

• Tube 1: 1/10 (1mL solvent, 9mL solute)

• Tube 4: 1/100 (1/10 × 1/10)

• Tube 3: 2/100 (2/10 × 1/10)

• Tube 2: 3/100 (3/10 × 1/10)

• Tube 5: 6/1000 (1/100 × 6/10)

• Tube 6: 3/1000 (1/100 × 3/10)

Vernier Spectral Analysis setting

% Transmittance vs. Concentration (Beer’s Law)

How to calculate concentration of cells per mL

5 × 10⁸ cells/mL times (dilution value)How

How to calculate log of cell concentration

Base 10 log

3 techniques used for separation of biological materials

• Filtration

• Centrifugation

• Use of selectively-permeable membranes

Solid vs mobile phase

How different types of molecules can separate, by binding onto these “phases”

• Solid phase: ex. filter paper

• Mobile phase: dissolving, usually through chromatographic solvent/solvent system

Origin

Point of application

Solvent front

Leading edge of the advancing liquid

Properties of molecules in chromatography determine…

Whether binding or dissolving predominates (shown in solubility through moving solvent)

Solid phase in paper chromatography

Cellulose fibers

Solid phase in thin layer chromatography

Silica gel or other material

Affinity

Tendency of molecules to bind or to dissolve

One major benefit of chromatography

Being able to separate molecules from complex mixtures

Two main materials in this procedure

• Dyes (3 different ones)

• Filter paper circles (4)

Why dyes are being used

Are visible to the naked eye and do not need chemical treatment to make them visible

Solvent the dyes are dissolved in

Methyl alcohol

Setup “dye” procedure for chromatography:

• Get 4 filter paper circles, 11cm in diamater

• Mark a tiny dot in center for origin, then draw 5mm diameter circle around the dot

• With pencil GENTLY mark the edge of each paper with A, B, C, D

• Use a microcapillary for each dye to drop a dot on origin spot, then blow on the spot to evaporate the alcohol

• Apply a second tiny drop on top of the dry first one, blow to dry again, and repeat 4-6 times (until all microcapillary’s liquid has been applied)

• IMPORTANT: keep dye spot as small as possible

• Repeat these steps for B, C, D

Dyes A, B, C, D

• A: alone

• B: alone

• C: alone

• D: all 3 combined

“Wetting” procedure for chromatography

• Fill 4 glass Petri dish halves with 1/3 full DI water

• Place paper “A” horizontally on top of one of dishes, so the “wick” hangs down into the water

• Then place empty dish half upside down on top of paper circle, so that paper is sandwiched between them and will not fall

• Note start time, and do the same with 3 other papers

• Let the chromatographic solvent move across papers for 15-20 minutes and watch as solvent passes dye spot

Why 3 dyes spread apart different

Have different properties and therefore differ in solubility in water and in their affinity for cellulose fibers

How to mark for Rf value

• When moving edge of solvent is about ½ inch from dish edge, remove papers from fishes and mark location of the solvent front on each paper

• After papers dry, mark center of each dye band that moved away

• Measure distance from origin to that center point

In such procedures, molecules that move with the solvent…

Tend to spread out and form a band, or zone, moving away from the origin

Similarity in molecules and bands

The more similar two types of molecules are in their properties, the more likely their bands will overlap

Resolution

Degree of separation; we are striving for highest degree of resolution, meaning no overlap of bands at all

Rf value calculation + range

Distance the band moved / distance the solvent moved

• Values are between 0 (no movement from origin) and 1 (moved to solvent front)

Elution

Separation & purification of the dye; do this by cutting out the band of each dye and soak in a solvent to redissolve dye off the paper