# Assessment of Seasonal and Diurnal Variations of Alkaline Phosphatase Activity in Pasteurized Milk
## 2 Materials and Methods
### Description of the Newly Developed Test \"AP Test\"
#### 2.3.2 \"AP Test\" Method of Use  
Place as many tubes in a metal or plastic stand as there are samples to be tested by the pasteurizer and number them. Using the pipette of variable volume 100-1000 \(\mu\)L, take a quantity of 400 \(\mu\)L of reagent R1 and place it in the tubes in ascending order, change the tip, adjust the quantity to be taken from 400 \(\mu\)L to 100 \(\mu\)L and add reagent R2 to each tube. Then change the variable-volume pipette and use the 10-100 \(\mu\)L pipette together with the corresponding tip. Take 10 \(\mu\)L of reagent R3 and place it in the tubes. Finally, take the 0.5 to 10 \(\mu\)L variable-volume pipette with a new tip and add 8.3 \(\mu\)L of the milk sample to be tested to each tube. Close the tube with either hydrophobic cotton or a plastic stopper and shake the contents of the tube gently for 2-3 s.  
Sampling Technique in the Research and Development Phase  
For the mast excretion of reproductive animals, initially, reproductive animals (sheep, goats, cows) were milked by hand [11; 12]. The milk was collected in sterile 150 mL containers and then thoroughly mixed to ensure homogeneity throughout the sample mass. After sampling, the samples were immediately placed in a portable refrigerator and transported to the laboratory of each dairy unit, where they were processed and analyzed on the same day. When this was not possible, they were kept at a temperature of 2-4 \({}^{\circ}\)C to be analyzed the following day. Samples of pasteurized milk (i.e., at time zero (0) after the end of the pasteurization process) were collected from the pasteurizer of each cheese unit directly and carefully using sterile containers. The use of sterile surgical gloves and personal surgical protection devices was mandatory in order to avoid contamination of the pasteurized milk by the user, since phosphatase is an abundant marker in the environment and contamination of the sample by exogenous phosphatase would affect our results. Receipt of raw milk as a positive control: From the samples collected mechanically (miking), one (1) sample was randomly selected to play the role of a positive control during the color imaging and comparison of the final samples. Raw milk as a percentage of phosphatase in pasteurized milk: From the samples collected mechanically (miking), one (1) sample was randomly selected to play the role of phosphatase source when added to pasteurized milk in specific proportions [13; 14; 15; 16]. Sample Handling and Homogenization Technique in the Research and Development Phase \begin{table}
\begin{tabular}{c c c} \hline \hline
**\% Raw Milk in Pasteurized Milk** & **Number of Samples** & \
\begin{tabular}{c} **“AP Test” Test Qualitative** \\ **Color Result** \\ \end{tabular} &
\begin{tabular}{c} **Time Interval for** \\ **Displaying the** \\ **Color Effect** \\ \end{tabular} \\ \hline
0.00\% or undetectable phosphatase & 10 & White color & 5 s \\
0.001\% or phosphatase 3.5 mV/L & 10 & Yellow-green color & 5 s \\
0.002\% or phosphatase 7 mu/L & 10 & Yellow-green color & 5 s \\
0.005\% or phosphatase 17.5 mV/L & 10 & Yellow-green color & 5 s \\
0.01\% or phosphatase 35 mV/L & 10 & Yellow-green color & 5 s \\
0.02\% or phosphatase 70 mV/L & 10 & Yellow-green color & 5 s \\
0.04\% or phosphatase 140 mV/L & 10 & Yellow-green color & 5 s \\
0.06\% or phosphatase 210 mV/L & 10 & Yellow-green color & 5 s \\
0.08\% or phosphatase 280 mV/L & 10 & Yellow-green color & 5 s \\
0.1\% or phosphatase 350 mV/L & 10 & Yellow-green color & 5 s \\ \hline \hline \end{tabular}  
* Preparation of Alkaline-Phosphatase-Contaminated New Samples  
\end{table} 
Table 1: Detection of LoD for “AP test” in pasteurized milk. Positive control = yellow-green color (presence of phosphatase in the sample). Negative control = white color (absence of phosphatase in the sample).