With a Spectrophotometer

The concentration of cells in a culture can also be determined with a spectrophotometer

by measuring the amount of 600-nm light scattered by the culture. The level of absorbance
(A) at 600 nm will depend on the distance between the cuvette and the detector and will
vary among spectrophotometers, often by a factor of 2. It is thus wise to calibrate each
instrument by recording the OD600 (sometimes expressed as A600) of a culture that contains
a known number of cells determined by some other method, such as observation on a
count slide or titering for viable colonies (UNIT 1.3).
If the culture is visibly turbid, also measure a 10-fold dilution of it. For a culture grown
in rich medium, a good rule of thumb is that each 0.1 OD unit is roughly equivalent to 1
× 108 cells/ml.
Calculate the number of cells/ml from whichever suspension (the undiluted or the diluted)
has an OD600 <1.

Contributed by Karen Elbing

Clark & Elbing LLP
Boston, Massachusetts

Roger Brent
The Molecular Sciences Institute
Berkeley, California

Growth in Liquid
Media

1.2.2
Supplement 59 Current Protocols in Molecular Biology
 About TURBIDITY...

A final widely used method for the determination of cell number is a

turbidometric measurement or light scattering. This technique depends on
the fact that as the number of cells in a solution increases, the solution
becomes increasingly turbid (cloudy). The solution looks turbid because light
passing through it is scattered by the microorganisms present and the
turbidity is proportional to the number of microorganisms in the solution. The
turbidity of a culture can be measured using a photometer or a
spectrophotometer. The difference between these instruments is the type of
light they pass through the sample. Photometers, such as the Klett-
Summerson device, use a red, green or blue filter providing a broad spectrum
of light. Spectrophotometers use prisms or diffraction gratings supplying a
narrow band of wavelengths to the sample. Both instruments measure the
amount of transmitted light, the light that makes it from the light source
through the sample to the detector.

A spectrophotometer or photometer quantifies the amount of turbidity of a

culture. The amount of light scattered from a solution is proportional to cell
number. The instruments measures light that is not scattered by the sample.

When measuring light scattering it is important to consider the wavelength of

light used a bacterial culture. Microorganisms may contain numerous
macromolecules that will absorb light, including DNA (254 nm), proteins (280
nm), cytochromes (400-500 nm), and possible cell pigments. When
measuring bacteria by light scattering it is best to pick a wavelength where
absorption is at a minimum and for most bacterial cultures wavelengths
around 600 nm are a good choice. However, the exact wavelength chosen is
species specific.

The amount of light transmitted through a sample is inversely proportional to

cell number and can be expressed in the following equation.

The transmittance equation:

The ratio of light hitting the sample (Io) to light passing through a sample (I) is
the transmittance.

Where T is the light transmitted, Io is the light entering the sample and I is the
light passing through to the detector.
Due to the nature of light scattering, transmittance decreases geometrically as
the cell numbers increase. It is more intuitive to think of the units increasing as
growth increases and for most bacterial analysis, transmittance is converted
into absorbance using the following equation.

Absorbance:

The absorbance of a sample it the negative log base 10 of the transmittance.

Absorbance increases in a linear fashion as the cell number increases. When

measuring growth of a culture the term optical density (OD) is normally
used to more correctly represent the light scattering that is occurring;
under optimal conditions, little light is actually absorbed by the culture so
the term absorbance is misleading. For most unicellular organisms
changes in OD are proportional to changes in cell number (within certain
limits) and therefore can be used as a method to follow cell growth. If a
precise cell number for a given OD is desired, a standard curve can be
generated, where viable plate count or cell mass is plotted as a function of
OD. It also wise to develop a standard curve to verify that the OD is actually
an accurate portrayal of cell growth. After the standard curve is made, it is
then possible to simply measure the OD of the culture and read the cell
number from the curve.

The turbidity of a culture is dependent upon the shape and internal light-
absorbing components of the microorganism and therefore turbidity
readings are species-specific and cannot be compared between different
microbes or even between different strains of the same species. As above,
there are microbes that change cell size or shape at different stages of
growth, which introduces some inaccuracy to this method of cell counting.
Also both living and dead cells scatter light and are therefore counted.
However, the method is very rapid and simple to perform and provides
reliable results when used with care, so it is an extremely common method of
real time analysis of prokaryotic populations. In fact it is one of the methods
we will use for measuring cell number in the experiment on bacterial growth.
Turbidometric measurements also do not destroy the sample.

NOTE:

Using an absorbance spectrophotometer to monitor light scattered by non-

absorbing suspended cells is common practice in life science laboratories.
Such applications, more than any other, accentuate the differences
amongst the optical systems of the numerous spectrophotometer
designs.