Biomedical Engineering Reference
In-Depth Information
colony may not be formed from a single cell. This method (plate counts) is more suitable for
bacteria and yeasts and much less suitable for molds. A large number of colonies must be
counted to yield a statistically reliable number. Growth media have to be selected carefully,
since some media support growth better than others. The viable count may vary, depending
on the composition of the growth medium. From a single cell, it may require 25 generations
to form an easily observable colony. Unless the correct medium and culture conditions are
chosen, some cells that are metabolically active may not form colonies.
In an alternative method, an agar-gel medium is placed in a small ring mounted on
a microscope slide, and cells are spread on this miniature culture dish. After an incubation
period of a few doubling times, the slide is examined with a microscope to count cells.
This method has many of the same limitations as plate counts, but it is more rapid, and cells
capable of only limited reproduction will be counted.
Automatic cell counters are based on the relatively high electrical resistance of cells.
Commercial particle counters employ two electrodes and an electrolyte solution. One electrode
is placed in a tube containing an orifice. Avacuum is applied to the inner tube, which causes
an electrolyte solution containing the cells to be sucked through the orifice. An electrical
potential is applied across the electrodes. The voltage applied across the orifice is of the
same magnitude as that required for the dielectric breakdown of microbial membranes.
Therefore, the voltage must be carefully adjusted. As cells pass through the orifice, the elec-
trical resistance increases and causes pulses in electrical voltage. The number of pulses is
a measure of the number of particles; particle concentration is known, since the counter is
activated for a predetermined sample volume. The height of the pulse is a measure of cell
size. Probes with various orifice sizes are used for different cell sizes. This method is suitable
for discrete cells in a particulate-free medium and cannot be used for mycelial organisms.
In an alternative automatic method, the cells are measured via image recognition. When
dilute cell culture is passed through an orifice, images are taken continuously (with a fast
image capturing camera) and subsequently digitally analyzed on a computer. Special tech-
nique such as polarized light may be required to illuminate the cells. This method can
measure both cell number and cell sizes.
Alternatively, the number of particles in solution can be determined from the measure-
ment of scattered light intensity with the aid of a phototube (nephelometry). Light passes
through the culture sample, and a phototube measures the light scattered by cells in the
sample. The intensity of the scattered light is proportional to cell concentration. This method
gives best results for dilute cell and particle suspensions.
11.1.2. Cell Mass Concentration
Cell mass holds direct perceivable link to the substrate consumption and product forma-
tion. There are two kinds of cell mass quantification methods: direct methods and indirect
methods.
11.1.2.1. Direct Methods
Determination of cellular dry weight is the most commonly used direct method for deter-
mining cell mass concentration and is applicable only for cells grown in solids-free medium.
If noncellular solids, such as molasses solids, cellulose, xylan, or corn steep liquor, are
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