Use of the spectrophotometer

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Use of the spectrophotometer
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Transmittance

• As bacterial number increases, the culture
  becomes more dense and turbid
• Less light can pass through
• The transmittance decreases

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We will measure transmittance with a
spectrophotometer
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Turn the machine on and let it warm up for 15
minutes

• The on-off switch is in the back of the machine
• Press it to 1 to turn the machine on

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The machine should flash BLA (blank) when it is
turned on
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Then, it should read 0 T
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Press the mode button to make sure you are
measuring T (transmittance) and not A
(absorbance)
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This machine is NOT set correctly it is reading
A

• Press the mode button to change it to T

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This is now correct the machine is reading T
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Set the wavelength to 686
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You will put your media or culture into the
cuvettes and then into the appropriate slot in
the machine
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Close the compartment after you put your cuvette
in
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Place control media (with no bacteria) into the
machine

• Set the machine to 100 transmittance
• Notice that the media is clear

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Place your sample into the machine and read the
transmittance

• It should read less than 100T
• Notice that it is cloudier than the control media
  and there will be less transmittance

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Readings

• As the culture becomes less dense (with increased
  dilutions), the readings should increase