# Optical Properties of Buffers and Cell Culture Media for Optofluidic and Sensing Applications
## 3 Results and Discussion  
Transmission spectra for all the considered liquids are presented in Figure 3. In general, transmission of all the liquids was limited by the transmission spectrum of water, which is the main ingredient of all of them. The IR part of the spectra (\(\lambda\) > 700 nm) followed typical water transmission characteristics with two absorption peaks at 950 and 1200 nm and almost no transmission at 1400 nm. Transmission spectra of TBE and PBS were similar to that of water in the visible range as well. Simple salts (TRIS, boric acid, EDTA, NaCl, phosphate buffer, and KCl) added to water did not change its absorption in the visible spectral range. However, compared with buffers, the transmission of CCM was significantly different. EXPRESS 5 showed a significantly reduced transition below 600 nm, which is related to the absorption of nutrients (e.g., glucose, sodium pyruvate). The DMEM transmission characteristics had, in turn, a very broad and deep absorption peak with a maximum at 560 nm. Figure 2: Scheme of the setup used for measuring scattering properties. This peak was caused by a high concentration of phenol red dye--phenolsulfonphthalein absorption in pH 8.2 (\(\lambda_{\text{max}}=\) 570 nm). To verify the influence of long-term handling on buffers and CCM, we also measured their transmission after 3 and 8 months (Figure 4). During 8-month-long storage, all the media were kept in the dark and at a temperature of 4 \({}^{\circ}\)C. For the TBE buffer (Figure 4c), we did not observe any change in transmission. This buffer was stable during the 8-month-long storage. High stability is related to the composition of TBE, which contains TRIS, boric acid, and EDTA, offering slightly alkaline pH conditions. Moreover, the presence of EDTA is very important due to its chelator activity, particularly for magnesium ions (Mg\({}^{2+}\)). These ions act as cofactors and are required for the activation of many enzymes; therefore, the absence of Mg\({}^{2+}\) may block bacteria growth. In the case of PBS, we observed a gradual reduction of the transmission peak centered at 1100 nm (Figure 4d). The visible part of the spectrum remained unchanged after the first 3 months, and later between the third and eighth months, we noticed a reduction in transmission from 10% to 25% at \(\lambda=\) 600 nm and \(\lambda=\) 400 nm, respectively. This effect was possibly induced by the crystallization of salts in the buffer. However, in comparison with the CCM, the process was less intense. For CCM, we observed a well-visible reduction of transmission properties for both of the liquids. Their transmission properties remained comparable for the first 3 months, but between the third and eighth months, an intensive decrease of transmission was observed. For EXPRESS 5, this effect was related to strong light scattering caused by medium degradation and/or by bacterial or fungi infection. CCM are usually used for no longer that 1 month from the date of first use and it is recommended by the manufacturer to use them within 2-4 weeks. Moreover, CCM should be used in a clean-room environment and a chamber with laminar flow to reduce the likelihood of infection. Every use of the medium increases the probability of bacterial or fungi infection. The observed effect may have been caused by nutrient degradation due to the half-life of the molecules or the fact that the media were measured at normal atmosphere (with a low CO\({}_{2}\) concentration, \(\sim\)0.4%). In cell cultures, the CO\({}_{2}\) concentration in the incubator is at a much higher level (usually 5% or 10%), and the CO\({}_{2}\) dissolved in the medium is converted into H\({}^{+}\) and HCO\({}_{3}\)\({}^{-}\) ions, which helps to stabilize the pH. CCM that are not kept in an atmosphere with a high CO\({}_{2}\) concentration show limited pH buffering properties and are more likely to degrade.