1. Making A Standard Curve

Spectrophotometry is a common lab technique that uses _____ to measure the ________ of a component in a solution

light
concentration

Absorption or scattering?
How much light a sample absorbs

Absorption

Absorption or scattering?
Loss of light due to suspended substances

Scattering

Absorption or scattering?
Represents concentration of component

Absorption

Absorption or scattering?
Represents turbidity of the solution

Scattering

Measure of absorbance and scattering through a sample

Optical density

How is optical density measured?

Ratio of incident light and light transmitted

Optical density = ____ + ____

absorption
scattering

Beer-Lambert Law:
Amount of light absorbed by a solution is ______ _______ to the concentration of the absorbing substance and the path length through the solution

Directly proportional  

Light that strikes a sample

Incident light

Light that passes through sample and exits on other side

Transmitted light

What type of equipment can be used to measure optical density (2)?

Spectrophotometer
Plate reader

How are OD measures used to determine cell concentration?

Compare OD measure of the unknown sample to the OD values of known concentrations from a standard curve

Why is 600nm used for bacterial cultures?

Bacterial cells cause more scattering at 600nm

More absorbance at 600nm = (more/less) bacteria present

more

A graph that shows the known relationship between a measured variable and known quantity, allowing determination of unknown values by comparison

Standard curve

What is a standard curve used to calculate?

The concentration of a sample

What are ‘standards’ and how are they made?

Set of known values made from solutions prepared in a dilution serires

How are datapoints on a standard curve plotted?
x-axis =
y-axis =

x-axis = concentration
y-axis = OD

What does the line on a standard curve represent?

The best-fit relationship between OD and concentration across the known standards

Two strategies to quantify bacterial cultures are

Plate counts
Optical density

Plate counts or optical density?
Direct representation of only viable cells
More accurate but more laborious
Requires incubation time
More prone to human error

Plate counts

Plate counts or optical density?
Measures both live and dead cells
Less accurate but less laborious
No incubation time
Less prone to human error

Optical density

Laboratory technique that is used to dilute a sample or solution by a specific factor

Serial dilution

What are two uses of serial dilutions?

Reduce a sample to a countable number of cells/colonies
Standard curve generation

2-fold or 10-fold dilution set up?
50% of previous dilution + 50% dilutant for a given tube

2-fold

2-fold or 10-fold dilution set up?
10% of previous dilution + 90% dilutant for a given tube

10-fold

How are bacterial cells harvested from a parent culture?

Centrifuging overnight culture to form a cell pellet

How are bacterial cells washed from a parent culture?

Removing supernatant from cell pellet, then washed by resuspending (e.g. in PBS)

Mixing solution with tip of pipette by drawing liquid and re-dispensing throughout the solution

Pipette mixing

What is the purpose of a vortex?

Resuspend pellet/mix solution thoroughly

Pellet or supernatant?
The solid collection of cells in bottom of centrifuge

Pellet

Pellet or supernatant?
Liquid on top of pellet

Supernatant