Biomedical Engineering Reference
In-Depth Information
and sporulating cells as well as mature spores of Bacillus during endotoxin
production. Maskow et al. [ 30 ] monitored lipid storage in yeasts using this tech-
nique (scanning for the critical frequency). The operating scheme that keeps this
signal (the so-called permittivity) constant in continuous culture mode is known as
a permittistat [ 31 ].
Common to all these sensor types is that they need to be calibrated specifically
for the biological system under investigation against at least one reference gold
standard: both optical and electrical properties differ between biosystems.
3.1.3 In Situ Microscopy
One such gold standard, especially in animal cell cultivation, is the determination
of cell number concentration. A manual procedure involves a desktop microscope,
yet special microscopes have also been mounted and used in situ and fully auto-
matically; actually, the objective, illumination source (e.g., a LED), and probably
some mechanical actuators are inside the sterile barrier, and can be sterilized in
situ, whereas the rest, including the camera and computer, are of course outside.
The sample zone (in front of the objective) is on the order of 50 lm (and can
probably be adjusted to obtain ''optical dilution'' to adapt the method to the actual
cell density) for the observation of animal cells; the sample zone is usually open
and thus permits the acquisition of sequences of images (with exposure times of
1 ms or less). Microbubbles can be quite easily detected by image evaluation
software due to their size and almost perfectly spherical shape; because they
occupy part of the measured volume, this must be taken into account during
calculation of the cell number concentration.
Bluma et al. [ 32 ], Hoepfner et al. [ 33 ], Rudolph et al. [ 34 ], and Ulber et al. ([ 35 ]
including other optical sensors) have compiled comprehensive surveys of the in situ
instruments in use and also versions in bypass interfaced to the process via a flow
injection system. Recently, they reported the use of in situ microscopy also for the
monitoring of processes with immobilized enzymes and two liquid phases [ 36 ].
3.1.4 Online Flow Cytometry
Another technique (being more powerful than just counting; see Sect. 3.2.5 )is
counting cells with a flow cytometer (FCM). Such an instrument must be linked to
the bioprocess via an interface (see Sect. 3.2.4 ) that is at least able to dilute the
suspension (see also Sect. 3.3.1 ) and, probably, to stain the cells too. The first
group to employ such a setup used a degassing unit in bypass to the reactor and,
further, a sequential injection analysis (SIA)-type interface to first stain the cells,
wash them in a membrane-covered microchamber, and eventually feed them to the
flow cytometer [ 37 , 38 ]. The microchamber was also used to dilute the cells
appropriately in order to achieve a cell density of approximately 10 6 ml -1 (or less)
for the FCM measurement. Around 10 5 events were then evaluated at an event rate
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