# Biology 2290F/G Study Guide - Final Guide: Hemocytometer, Diffraction Grating, Radiant Flux

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10 Nov 2012
School
Department
Professor
Biology 2290
Dean Unit
Spectronic 20 Optical System and Spectrophotometry
- Radiant energy is focused onto a diffraction grating which splits the
light into its component wavelengths
- Different wavelengths can be selected by changing the position of the grating, allowing only a narrow range of
wavelength to pass through the slit and reach the sample
- Any light not absorbed (transmitted) by the sample reaches a phototube that converts it to electrical current
- This current is measured by a galvanometer (amp meter)
- Output information is both transmittance and absorbance, but the machine only measures transmittance
- It compares measured transmittance to that of a reference blank to determine absorbance
- Absorbance is the amount of light absorbed by a sample
- Transmittance is the fraction of light that passes through the sample
- Lambert’s Law: for a thin layer of ample, the same proportion of light is absorbed regardless of the radiant power of
the incident light
o Imagine a sample divided into many thin layers, the first layer absorbs half the light (and transmits half), the
second layer will also absorb half the light it receives, etc…
o Layers of infinite thinness would exponentially decrease light quantity
o Absorbance is a logarithmic function of transmittance
o   
 
(Io = radiant power of incident light)
- Beer’s Law: the absorbance of a solution is proportional to the number of absorbing particles (or concentration of
the absorbing material) in solution
o A = E x l x c
A = absorbance (no units)
E = absorption coefficient (litres/mole/cm) probability that a given type of photon will be
absorbed by a given type of molecule, varies with wavelength and solvent used
C = concentration of solution (mol/L)
L = path length through the spectrophotometer tube (usually 1cm)
- The blank used in experiments should contain all of the substances in the sample except for the material whose
absorbance you wish to measure
- Spectrophotometers are important in experiments where observation is not always possible, allows you to follow
the course of a biological process
- To make a standard curve of absorbance and concentration you make a number of tubes with different
concentrations of your material of interest and record the absorptions
o Graph this, the slope of the line will be your E value for that material
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- A at a chosen time gives the change in concentration of the solution, which can be used with a standard curve to
find the average rate of reaction
o Slope of the standard curve = E
o 
  


You can then use the volume used in the experiment to get moles/minute
Microscopy
- Resolution (d) is the smallest distance by which two objects must be separated in order to allow them to be imaged
as two separate objects.
- Abbe’s equation gives the theoretical limit of resolution in an optical system
o   
 (0.612 = constant, n sin = numerical aperture of lens, = wavelength of light)
o N = refractive index of material medium, = aperture angle
o To attain maximal resolution want to increase n (use immersion lens and immersion oil) and make as close
to 90 as possible, decrease to about 400nm (shortest to which eye is sensitive)
Counting Cells
- There are two ways to count cells: haemocytometer and spectrometry
- Using the haemocytometer you place the cells on the plate and count the outer four squares and the middle square
o Cells on the bottom or right borders count, top and left borders do not
o Do this for both grids
- Find the average number of cells per square
- Each square counted has a volume of 0.004mm3
- Multiply the average cell count by 250 (= 1/0.004) and then by 1000 (1000mm3 in 1cm3 = 1mL) to get cells/mL
- To use the spectrometer you first need to use the haemocytometer to count cells and then make a standard curve of
A400 vs. # of cells/mL
- The slope of this graph can then be used to determine subsequent readings
o #cells/mL = A400 / slope
Photon Fluence Rate
- The number of photons in the 380-700nm range incident per unit time on a unit surface
- Direct relationship between number of molecules that are photochemically altered and number of photons within
380-700nm range that are absorbed, regardless of photon energy, useful to know how many photons hit surface and
not how much energy they deliver
- Measured with a quantum sensor, units: mols of photons per second per square metre)
- 1 mol of photons contains 6.02 x 1017 photons
pH Calculations
- [H+][OH-] = 1 x 10-14
- pH = -log[H+] and [H+] = 10-pH
- pOH = -log[OH-] and [OH-] = 10-pOH
- pH + pOH = 14
-  

Buffer Solutions
- HA A- + H+ weak acid and it’s conjugate base in solution creates this equilibrium
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## Document Summary

Radiant energy is focused onto a diffraction grating which splits the light into its component wavelengths. Different wavelengths can be selected by changing the position of the grating, allowing only a narrow range of wavelength to pass through the slit and reach the sample. This current is measured by a galvanometer (amp meter) It compares measured transmittance to that of a reference blank to determine absorbance. Any light not absorbed (transmitted) by the sample reaches a phototube that converts it to electrical current. Output information is both transmittance and absorbance, but the machine only measures transmittance. Absorbance is the amount of light absorbed by a sample. Transmittance is the fraction of light that passes through the sample. Lambert"s law: for a thin layer of ample, the same proportion of light is absorbed regardless of the radiant power of the incident light.