Preliminary tests for Saliva
Preliminary tests for the detection of saliva typically involve a combination of visual examination and chemical analysis. Visual examination may include looking for the presence of saliva stains with the help of alternating light sources such as UV light. Chemical analysis includes the detection of enzyme activity (such as amylase) present in saliva. These tests, however, are not exclusive to salivary amylase and might give positive results from other sources containing amylase. Therefore, preliminary tests must be followed by a confirmatory test to confirm the findings.
1. Visual examination
Saliva stains are generally difficult to visualize by the naked eye, especially on fabrics or surfaces with complex patterns. Unlike some bodily fluids that may leave more noticeable stains, saliva stains are often colorless, making them challenging to identify without specialized lighting techniques or equipment. When exposed to UV light, saliva may exhibit a faint blue or whitish fluorescence, resembling the fluorescence observed in semen stains. However, the fluorescence intensity is generally less pronounced than seminal stains.
2. Lugol’s Starch-iodide test
Lugol’s Iodine solution contains iodine molecules that fit tightly between the interconnected sugar molecules of starch, causing the iodine to exhibit a distinctive blue-black color. The color changes observed in Lugol's Iodine solution when starch interacts with iodine are primarily due to the concentration and arrangement of iodine molecules in the solution.
When starch is broken down by the action of amylase in the presence of saliva, the iodine-starch complex is disrupted. This leads to a change in the concentration and arrangement of iodine molecules, causing the solution to exhibit a yellow-brown color. The color change signifies the breakdown of the starch and the release of iodine molecules from the complex.
Figure: Principle of Lugol’s Starch-Iodine test for the detection of Saliva
✏️ Preparation of Reagents:
a) Lugol’s Iodine solution: Dissolve 1g of iodine and 2 g of potassium iodide (KI) in 200 ml of water.
b) Starch solution: Dissolve 5g soluble starch in 10 ml of water.
✏️ Procedure:
Obtain a sample of the suspected saliva stain. This can be done by swabbing the area or by cutting out a small piece of the material on which the stain is found.
Add 2 drops of Lugol’s iodine solution to the sample. Iodine reacts with the amylase enzymes present in saliva to produce a blue-black color.
Add 2-3 drops of starch solution to the sample. The starch solution is added to enhance the visibility of the color change.
The presence of a blue-black color change indicates the presence of saliva.
Figure: Procedure for Lugol’s Starch-Iodine test for the detection of Saliva
3. Phadebas® Forensic Tube test
Phadebas® tube test is performed using the commercially available Phadebas® tablets that contain a blue dye cross-linked to starch. When starch is hydrolyzed and digested, the dye is released, giving a blue color.
✏️ Procedure
Take three test tubes and label them ‘Test’, ‘Positive control’, and ‘Negative Control’ or ‘A’, ‘B’, and ‘C’ respectively.
Add a small piece of the test sample with the suspected stain to test tube ‘A’. Add a small piece with a known saliva stain as a positive control in test tube ‘B’. Add a small piece with water stain as a negative control to test tube ‘C’.
Add 1 ml of distilled water and ¼ piece of Phadebas® tablet to each tube.
Vortex the tubes to ensure thorough mixing.
Incubate the tubes at 37°C for 30 minutes
Observation: A dark blue color of equal or greater intensity than the positive control is regarded as a positive result for the test sample. A blue color that is less intense than the positive control but darker than the negative control is considered inconclusive. No blue color is considered as a negative result.
4. Phadebas® Forensic Press test
Phadebas® press test is performed using the commercially available Phadebas® paper that contains a blue dye cross-linked to starch. When starch is hydrolyzed and digested, the dye is released, giving a blue color.
Place the item to be tested on a flat surface and dampen the item with water using a spray bottle.
Place a piece of Phadebas® paper over the area to be tested, with the blue reagent side in contact with the item, and trace a rough outline of the area being tested.
Spray water liberally onto the Phadebas® paper. The paper must not dry out during the test period but it also should not be too wet.
Cover the paper with a clean glass board on and place a weight on top of the glass. Use a 4 kg or heavier weight, to ensure good contact between the item and the paper.
Observe the test for maximum 40 minutes and record the time of positive reaction.
Observation: Positive areas appear as pale blue zones on the non-reagent side of the Phadebas® paper.