Lab 1 Flashcards
Koehler Illumination
compound microscope
has two or more magnifying lenses, the ocular and the objective lenses.
binocular microscope
has two eyepieces.
brightfield microscopy
have a single light source that directs light through an iris diaphragm and a condenser before reaching the specimen.
Light passing through the specimen is collected by the objective lens before passing through a series of mirrors to the ocular lens.
As a result of this single light path, the image that you view through the eyepiece is laterally reversed, upside down and 2-dimensional.
As light passes through the specimen, regions of the specimen that alter the path of the light create contrast, allowing us to view the specimen.
A microscope has three lens systems:
1) the condenser lens which focuses the light from the light source through the specimen,
2) the objective lens which magnifies the image of the specimen and
3) the ocular lens which inverts the image to make it suitable for viewing, and further magnifies the image.
Condenser aperture diaphragm.
• This diaphragm controls the amount of light entering the lens system.
• This feature is useful for viewing unstained biological specimens that are translucent.
• Reducing the amount of light improves contrast, making the specimen “stand out” against the background.
• Your microscopes have an iris (condenser) diaphragm with a lever (just below the stage) that opens and closes the diaphragm to let in varying amounts of light.
• Use the condenser diaphragm to reduce the amount of light and increase the contrast of the image.
ocular micrometer
A precise measurement of microscopic objects can be made using an ocular micrometer.
Objective lens
10X
Ocular micrometer calibration value
100 μm/eyepiece unit
Objective lens
40X
Ocular micrometer calibration value
25 μm/ eyepiece unit
Objective lens
100X
Ocular micrometer calibration value
10 μm/eyepiece unit
To calculate a specimen’s size using an ocular micrometer
use the micrometer to measure the specimen in eyepiece units (e.p.u), and then multiply the e.p.u. by the calibration for the appropriate objective lens
• There are no units assigned to the grid in eyepiece (they are referred to as eyepiece units or e.p.u.)
- This is because the grid system is mounted onto the ocular lens, and the perceived distances between the grid bars changes when the objective lenses are changed. Therefore, the micrometer must be calibrated for each objective lens.
Drawing the cell and adding a scale bar to the drawing
A scale bar functions to let the person observing your drawing or image know how big the actual size of the cell was that you drew or photographed.
- By looking at the scale bar included with the drawing, the person looking at the image can figure out what the actual size of the cell is and have an idea of the magnification of the image (how many times larger is your image/drawing than the actual cell).
The line (scale bar) you draw must represent the same ratio of the cell image relative to the cell’s actual/real size.
- Determine the actual size of this cell μm using the ocular micrometer.
For this example, the 40X objective was used to take a image. If the cell measured 2 e.p.u. using the ocular micrometer, then to figure out the actual size of the cell:
2 e.p.u. X 25 μm/ e.p.u. = 50 μm.
The cell is 50 μm in real life. - Determine the size of the image/ drawing.
Use a ruler to measure the length of the image/drawing. Say the cell is 5 cm in diameter (across). - Now you can use a simple ratio to determine how long to draw a scale bar (X) that accurately represents 10 μm:
actual/real size of cell (50 μm) = 10 μm. ……………………………………………..scale bar
———————————……..—————
Size of drawing (5cm)……………… X
X = 1 cm
By solving for X, you know how long of a line to draw that represents 10 μm. Draw a line that is 1 cm long under the image and label it 10 μm. This line is drawn to the same scale as the image is.
Knowing the actual/real size of the cell, you can double check your work by seeing if the scale bar on the image will determine the actual/real size of the cell accurately.
Micropipettes
are instruments used to measure very small volumes of liquid [less than 1 ml].
different sized micropipettes that measure liquids in the ranges of 10 - 100 μL (yellow pipette) and 100 - 1000 μL. (blue pipette).
Absorption spectrum
A graph showing the amount of light absorption for a specific molecule for many wavelengths is called an absorption spectrum.
- absorption spectrum is used to determine which wavelength to set on the spectrophotometer to measure the molecule of interest.
Standard curve
A standard curve is a graph that shows the relationship between the concentration of a substance and its absorbance.
- prepared by measuring the absorbance of known concentrations of the substance you are trying to measure.
- Because a standard curve is the result of known concentrations of a substance plotted against the absorbance of those concentrations, the slope of the line graph represents the relationship between concentration and absorbance for that substance.
- relationship between concentration (x) and absorbance (y) is provided by m = slope.
- absorbance increases in direct proportion to the concentration of the compound.
Dilutions
The linear relationship between absorbance and concentration is only true at lower sample concentrations. If you measure an absorbance value of an unknown sample with a high concentration (the measured absorbance value is greater than the highest absorbance value of the standard curve), you can no longer assume the relationship is linear.
The sample must be diluted, and the dilution must be accounted for when determining concentration of the undiluted sample.
- often expressed as a fraction, or a “one in X” ( ex: 1/5) in which you are reducing the concentration of your solution to a fraction of the original. What this means is that your sample is one part (or unit volume) of your sample in a total of X parts (or unit volumes).
scientific or research hypotheses.
These hypotheses specify the independent and dependent variables under investigation and the potential relationship between them.
• independent variable in an experiment is the part the researcher is changing/manipulating.
• dependent variable is what is being measured in response to the changes/manipulations.
