Biowave II can be used to give an indication of bacterial cell density, by measuring the
optical density (OD) of a suspension of cells at 600nm (OD600). Usually, the reason for doing
this is so the researcher can know when cells are ready for harvesting.
It is important to note that for turbid samples such as cell cultures, the absorbance (OD)
measured is due to light scattering, and not due to molecular absorption. The reading is
affected by the optics of the system (distance between the cell holder and instrument exit slit,
geometry of this slit and the monochromator optics). Different spectrophotometer types
therefore give different readings for the same turbid sample.
1) Some users prefer that OD600 readings from Biowave II be comparable to those of other
instruments (for example, an older instrument around which laboratory procedures have been
written). To facilitate this, a correction factor is included in the Biowave II software. Results
obtained by comparing Biowave II to a “typical” general purpose spectrophotometer showed
OD600 from Biowave II to be approximately half that of the other instrument – suggesting that
a correction factor of 2.0 would be appropriate in this case. For this reason, the correction
factor 2.0 is the programmed default value in Biowave II. Of course, the user can change this
as required via the keypad: OD 600 – Parameters - Correction.
To determine the precise value for Correction, simply measure the same sample in Biowave
II and in the other spectrophotometer, taking care that the cells do not have time to
undergo significant further growth, or fall out of suspension between the two readings
(glycerol may be used for the latter). The value for Correction is found by dividing the
reading of the other instrument by the reading of Biowave II. This need be done only once.
2) For those users who want Biowave II to give an indication of actual cell density (in
cells/ml) rather than just an OD600 value, a second factor is used. This is to allow the
instrument to make the correct conversion from OD to cells/ml for the cell type being used.
To enter this value, go to OD 600 – Parameters – Factor. The highest number that can be
entered here is 9999 so, for the user’s convenience, a further number – either 1000 or
1000,000 – can be selected in the box called Multiplier. For E.coli, for example, an OD600
reading of 1.0 typically corresponds to around 8x108
cells/ml. In this case, the Factor entered
would be 800 and the Multiplier selected would be 1000,000.