**3. Research methods**

This study investigated the algal status such as viability using FCM after treatment of algae with the test condition. For this study, the author used *Parachlorella kessleri* (C-531 strain) (new nomenclature for *Chlorella kessleri*) as the model organism representing algae. The alga was obtained from the Institute of Applied Microbiology (IAM) culture collection at The University of Tokyo. Before experiments, algae were grown on CA agar plates at pH 7.2 [19] under an LD cycle (12 h light/12 h dark) at approximately 1100 lux of natural white fluorescent light and 23 ± 2°C. The algae were scratched with an inoculating needle and were suspended in fresh CA liquid medium at pH 7.2.

*Chlorella* (initial density of 1.0 × 104 cells/ml adjusted using hemocytometry) grown in CA medium as a control condition for 1 week under an LD cycle and algae treated with metal eluate for 1 week as a test condition were prepared, respectively, as described in earlier reports [1, 12, 14]. Moreover, algae treated with heat for 5 min at 100°C were prepared. Here, the test conditions were reference standards subjected, respectively, to metallic eluate from steelmaking by-products and heat stress. A detailed description of the metal eluate reveals that the metal eluate was made from stainless steel slag (**Table 1**) subjected to a leaching test based on JIS K0058-1: 2005 (method for chemicals in slags Part 1: Leaching test) [12, 14, 20–23]. **Table 1** presents compositions of stainless steel slag particles used for this study [12, 14, 20–22]. In brief, slag used for this study mainly contains SiO<sup>2</sup> , CaO, Al<sup>2</sup> O3 , MgO, MnO, and Cr<sup>2</sup> O3 [12, 14]. Here, all Fe and Cr compounds are described, respectively, as FeO or Cr<sup>2</sup> O3 because it is generally difficult to distinguish FeO and Cr<sup>2</sup> O3 formed from Fe and Cr in a suspended metal solution at the occasion of elemental analysis after alkali fusion of stainless steel slag [1, 12, 14].

After elution from slag at pH 6 adjusted with HCl, the solution was filtrated with a 0.45 μm pore filter to eliminate slag particles. Then the solution was used for bioassay with *Chlorella* as a test solution including trace metals. **Table 2**, which shows components of the metal eluate used for this study, includes environmental quality standards for soil pollution, marine pollution, and water pollution, along with other standards for eluent and drinking water for reference. In this study, CA medium containing eluates was first made from 25 vol% of the concentrated CA medium, which had four times that amounts of respective chemicals for making CA medium, and 75 vol% of mixture of arbitrary amounts of eluate, a definite number of algae (1.0 × 104 cells/ml), and ultrapure water. Therefore, nutrient amounts of CA medium containing eluates were the same as those of CA medium alone, but the concentrations of chemicals derived from eluate differed from those of CA medium without eluate as described in reports of earlier studies [1, 12, 14].

To characterize each algal sample using FCM, this study used a cell analyzer (Muse™; Merck Millipore Corp., Hayward, CA) with a green laser operating at 532 nm as an excitation light source, a photodiode for detection of FSS, and two fluorescence filters of a 680/30 nm band pass (BP) filter suitable for chlorophyll fluorescence (red fluorescence) and a 576/28 nm BP filter suitable for chlorophyll degradation (yellow fluorescence) (**Figure 4C**) [1, 11, 12, 14].

This study was undertaken to evaluate the correlativity between algal properties and the test condition. To evaluate the correlativity among multiple properties of algae and each stress factor, PCA of multivariate analysis was used for this study using software for multivariate analysis (Institute of Statistical Analyses, Inc.). A dimensional reduction technique, PCA, reduces multi-dimensional information to arbitrary one-dimensional information, which is a

**3. Research methods**

referred and partly modified from the literature [1, 13, 15, 18].

86 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

CA liquid medium at pH 7.2.

This study investigated the algal status such as viability using FCM after treatment of algae with the test condition. For this study, the author used *Parachlorella kessleri* (C-531 strain) (new nomenclature for *Chlorella kessleri*) as the model organism representing algae. The alga was obtained from the Institute of Applied Microbiology (IAM) culture collection at The University of Tokyo. Before experiments, algae were grown on CA agar plates at pH 7.2 [19] under an LD cycle (12 h light/12 h dark) at approximately 1100 lux of natural white fluorescent light and 23 ± 2°C. The algae were scratched with an inoculating needle and were suspended in fresh

**Figure 4.** Fluorescence characteristics of algae and microphotographs of *Chlorella*-like alga experimentally isolated from ciliate *Paramecium bursaria*. A bright field image of *Chlorella*-like alga (A) and the corresponding fluorescence image derived from chlorophyll (B) are shown. Panel C presents fluorescence characteristics of *Chlorella*-like alga obtained using fluorescence spectroscopy. Emission spectra of algae are shown with (dotted line, heated algae) and without heat treatment (solid line, control algae). Yellow (dotted arrow) and pink (solid arrow) areas, respectively, represent detection ranges of yellow and red fluorescence channels for FCM used for this study (see *Research methods*). Panels A-D were


**Table 1.** Chemical compositions of steel slag used for this study (mass%) referred from the literature [1, 12–14].


dataset from a new axis produced by PCA [15]. According to results of the correlation matrix analysis for the data, the author calculated the contribution rate of each component, the factor loading of each parameter, and the score plot of each component. Here, each factor loading (PC1-3) generally indicates correlation factors between each parameter and each component (**Figure 5**). The statistical results obtained using PCA were interpreted to evaluate the algal

Efficient Interpretation of Multiparametric Data Using Principal Component Analysis as…

http://dx.doi.org/10.5772/intechopen.71460

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After treatment of algae with and without eluate, the algae were quantified using hemocytometry. Here, CA medium containing several concentrations (0–70 vol%) of the metallic eluate was used for the experiment using hemocytometry. The algal proliferation ratio (average ± standard error) was expressed as a proportion of the number of algae treated

**Figure 5.** Outline from FCM analysis to PCA analysis of multivariate analysis. All data from FCM were extracted and subjected to PCA. Only one or two parameters are used to produce a one-dimensional (1D) histogram and 2D scatter or contour plots in conventional FCM analysis. These graphs, however, ignore some parameters of data (left below in **Figure 5**). To incorporate all information of the data, PCA transforms multi-dimensional data into a new smaller set of

status between control and test conditions.

variables (right below in **Figure 5**).

with eluate to that of control without eluate [1, 12, 14].

1 Not detected.

2 Reportable detection limit.

3 These data from a previous study reported by Takahashi et al. [17].

4 Standard value is not applied to coastal waters.

5 Standard value is applied to coastal waters.

6 The Cd value has changed from 0.1 to 0.03 mg/L since December 2014.

7 Habitable river or lake for aquatic life.

8 Habitable coastal water for aquatic life

9 Habitable coastal water that requires conservation in particular for nidus and nursery ground.

10Total concentrations of both calcium and magnesium are limited for water hardness.

<sup>11</sup>Habitable lake for aquatic life.

<sup>12</sup>Total N contents derived from nitrite nitrogen.

<sup>13</sup>Total N contents derived from both nitrite nitrogen and nitrate nitrogen.

**Table 2.** Environmental quality standards regarding pollutions and others for effluent and drinking water, and concentrations of elements of eluate (mg/L) referred from the literature [1, 12–14].

dataset from a new axis produced by PCA [15]. According to results of the correlation matrix analysis for the data, the author calculated the contribution rate of each component, the factor loading of each parameter, and the score plot of each component. Here, each factor loading (PC1-3) generally indicates correlation factors between each parameter and each component (**Figure 5**). The statistical results obtained using PCA were interpreted to evaluate the algal status between control and test conditions.

**Origin of slag Eluate of stainless** 

Total As ND<sup>1</sup>

Chromium (VI)

Total Al ND

Total Si 1.8

Standard value is not applied to coastal waters.

<sup>12</sup>Total N contents derived from nitrite nitrogen.

Standard value is applied to coastal waters.

Habitable river or lake for aquatic life.

Habitable coastal water for aquatic life

<sup>11</sup>Habitable lake for aquatic life.

These data from a previous study reported by Takahashi et al. [17].

The Cd value has changed from 0.1 to 0.03 mg/L since December 2014.

<sup>13</sup>Total N contents derived from both nitrite nitrogen and nitrate nitrogen.

concentrations of elements of eluate (mg/L) referred from the literature [1, 12–14].

**Regulated substances**

**Substances out of regulation**

1

2

3

4

5

6

7

8

9

Not detected.

Reportable detection limit.

**steel slag**

88 Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

(RDL<sup>2</sup>

Total Be ND (RDL: 0.0005) 2.5

Total V ND (RDL: 0.001) 1.5

Total Zn 0.099 2 0.03<sup>7</sup>

Total N 0.4 0.1–1<sup>11</sup>

Total P ND (RDL: 0.1) 0.005–1<sup>11</sup>

Habitable coastal water that requires conservation in particular for nidus and nursery ground. 10Total concentrations of both calcium and magnesium are limited for water hardness.

**Table 2.** Environmental quality standards regarding pollutions and others for effluent and drinking water, and

F− ND (RDL: 0.1) 0.8 15 0.84 84

Total Ca 9.3 30010 Total Fe ND 10 0.3 Total Mg 0.9 30010 Total Mn 0.028 10 0.05

**Environmental quality standards**

**Marine pollution**

ND (RDL: 0.005) 0.05 0.5 0.05 0.5 0.05

: 0.001) 0.01 0.1 0.01 0.1 0.01

**Water pollutant**

> , 0.028 ,

0.01<sup>9</sup>

0.2–18

0.02–0.098

16

**Effluent standard**

, 230<sup>5</sup> 1

2 1

, 15<sup>5</sup> 0.8

100 0.04<sup>12</sup>, 10<sup>13</sup>

**Drinking water standard**

**Soil pollution**

Total B 0.16 1 1<sup>4</sup> 104

Total Cd ND (RDL: 0.0001) 0.01 0.1 0.01 0.03<sup>6</sup> 0.003

Total Cu 0.003 0.001 3 3 1 Total Pb ND (RDL: 0.0005) 0.01 0.1 0.01 0.1 0.01 Hg ND (RDL: 0.0001) 0.0005 0.005 0.0005 0.005 0.0005 Total Ni 0.001 0.001 1.2 0.02 Total Se 0.012 0.01 0.1 0.01 0.1 0.01

After treatment of algae with and without eluate, the algae were quantified using hemocytometry. Here, CA medium containing several concentrations (0–70 vol%) of the metallic eluate was used for the experiment using hemocytometry. The algal proliferation ratio (average ± standard error) was expressed as a proportion of the number of algae treated with eluate to that of control without eluate [1, 12, 14].

**Figure 5.** Outline from FCM analysis to PCA analysis of multivariate analysis. All data from FCM were extracted and subjected to PCA. Only one or two parameters are used to produce a one-dimensional (1D) histogram and 2D scatter or contour plots in conventional FCM analysis. These graphs, however, ignore some parameters of data (left below in **Figure 5**). To incorporate all information of the data, PCA transforms multi-dimensional data into a new smaller set of variables (right below in **Figure 5**).
