Examination of Tobacco Chewed saliva on different Fabrics using Starch-Iodine test: A Forensic Perspective.

 

Akanksha Singh^1,2, Moumita Sinha¹, Manju Sahu¹, Bharti Ahirwar³, Arjun Rao Isukapatla^1*

¹Department of Forensic Science, Guru Ghasidas Vishwavidyalaya, Bilaspur, India

²Department of Forensic Science, Gujarat Forensic Science University, Gujarat, India

³Department of Pharmacy, Guru Ghasidas Vishwavidyalaya, Bilaspur, India

 

ABSTRACT:

In the mouth of human and animals a watery substance produces called saliva which is thick, colorless opalescent fluid secreted by salivary glands. In humans saliva is composed of 99.5% of water, 0.5 % consists of electrolytes, WBCs, epithelial cells, enzymes, lysozymes, and mucus and antimicrobial agents. In the present study saliva stains and tobacco chewed saliva stains on different fabrics viz. silk, polyester, linen, jute, cotton, georgette, sarton were examined using starch Iodine test. The saliva samples with and without tobacco chewed samples taken in different clothes and examined with starch Iodine test. The starch Iodine test was found to give Positive results for saliva stains and tobacco chewed saliva stains on different fabrics, and it was also found that the stains on different fabrics gives positive results for saliva even after washing with water, but none of the fabrics gave positive results for saliva after washing with detergents. The ability of a fabric to retain stains of saliva after washing depends not only upon the chemistry and manufacturing of the fabrics but also upon the time of immersion of the fabric with water and detergent and also the method adapted to remove the stain from the fabrics. It is thus concluded that the enzyme present in saliva stick/adhere the fabrics which is generally not removed even after immersing in water and gives positive results where as the use of detergents on different fabrics removes the stains completely and fails to give result for saliva stains.

 

 

INTRODUCTION:

Saliva is a dilute watery fluid secreted by the salivary gland in which 69% constitutes of sub-mandibular glands, 26% parotid glands and 5% sublingual glands. The basic secretary units of salivary glands are called acini (clusters of cells). These clusters of cells secrete a liquid fluid that constitutes of water, electrolytes, enzymes and mucus. The small collected ducts within the salivary glands transfer to larger ducts, ultimately forming particular large ducts. Saliva plays a significant role in Forensic cases that can help investigator to solve criminal cases. It links a suspect to a crime and also helps in reconstructing the crime scene.

 

It plays conclusive evidence in sexual assault cases and other related offences. Saliva stains often remains unnoticed by the criminals due to its transparent color in nature or sometimes the criminals attempt to eliminate saliva or tobacco chewed saliva stained evidence at a crime scene by washing it.

 

The enzymes presents in saliva reduces biomass and helps mechanically cleansing of teeth, and also provides an optimal pH. (Humphrey et al. 2001). There are are at least four different variants (or versions) of alpha-amylase, two of them are observed in saliva, and the rest two are secreted in pancreas. Those versions of alpha amylase are almost identical at the enzymatic activity level (Whitehead and Kipps). Human pancreatic α-amylase (HPA), determined by the Amyl2 locus, which is produced by the pancreas and secreted into the duodenum through the pancreatic ducts. The HSA and HPA amino acid sequences are highly homologous in nature. Though, HSA is inactivated in the stomach by acids, while HPA is inactivated in the intestines lower portions and some activity of amylase remains in feces stains (Richard Li et al, 2008).  Amylase enzyme has an active site controlled in sub sites of these particular locations, each of which contains a glucose residue in it (Talamond, Noirot and de Kochko, et al, 2005). Types of proteins, enzymes, mucosa and their concentrations are varying with animal to animal (Young J.A and Schneyer, et al, 1981).

 

The effects of pH, temperature, inhibitor concentration, substrate concentration and on the kinetics of amylase catalyze the reactions. Starch with a rich source of B polymorphic contents, like potato attacks slowly by amylase activity but can be digested faster (Slaughter et al, 2001). Saliva testing is an investigative technique that involves completely laboratory analysis of saliva to identify markers of immunologic, infectious, inflammatory, endocrine, and other different conditions. Saliva is a valuable biological material for examine steroid hormones such as proteins, cortisol, enzymes and antibodies, genetic material like RNA, and other different substances, including natural metabolites (Morgan CA 2000). Positive results for saliva identification were obtained when samples taken from plastic bottles, cigarette butts, buccal swabs, ceramic, mugs, plastic mugs, and soda cans (Sinelnikov et al. 2013). The specific activity of amylase enzyme (enzyme activity/protein concentrations) was determined for the identification of saliva stains. While examining the saliva stains during the first hour, amylase specific activity is found positive but during 1 to 28 days these results shows decreases in activity when the stains were kept at room temperature (Tsutsumi H).

 

In comparison to Semen, Saliva is harder to detect due to the lack of solid particles in the saliva sample. When dried saliva stains are found, they are colorless and difficult to detect (Vandenberg N 2006). On clothing saliva stains can be detected easily using alternate light sources such as by using the Poli light (Jackson D 2007). The alternate Polilight can be used to screen the and locate the possible saliva stains but is reliable in few cases only when compared to fluorescence screening, as only some saliva stains produce weak, indistinct fluorescence (Jean-Pascal Allery ). In majority of the cases where saliva stains is not diluted, it can be detected easily with the help of SALIgAE Spray (Silenieks et al. 2005). Human saliva samples taken in different types and colors of fabrics and was examined by Lumatec Superlight 400 emits light from 320 to 700 nm, which was able to detect stains both in darkness and in the presence of daylight (Anja Fiedler). However the preliminary test for detecting saliva identifies the α-amylase enzymatic activity, but not α -amylase molecule, these tests shows positive results (Myers et al, 2008).

 

MATERIALS AND METHODOLOGY:

The present study was conducted in Seven different and generally used fabrics (viz. Silk, Polyester, Linen, Cotton, Jute, Georgette, Sarton) in which saliva sample with tobacco content or tobacco chewed material by individuals were taken on different types of clothes and then these followed clothes were washed with different commercially available and commonly used detergents (viz. Surf excel, Ariel, Tide, Wheel, Nirma) to find out whether the detergent based washing clothes may gives the following positive results for the Starch Iodine tests.

 

Sample preparation:

Normal saliva (positive control) and tobacco chewed saliva samples were collected from different individuals and kept in a separate vials. Both the samples were pooled separately in different vials naming positive, negative and Tobacco chewed saliva samples, to prepare a standard stock solution and eliminate possible variations in amylase levels. Each fabrics pieces were cut into a size of 6x6cm. Stains were made from both the pooled saliva (i.e. normal saliva and tobacco chewed saliva). Each fabrics was stained separately with normal saliva and tobacco chewed saliva with 50μl of saliva each and left to dry at room temperature for 24 hrs.

 

Reagent Preparation:

Starch-Iodine Test:

Reagent I- 0.5% soluble starch solution i.e. 5mg of soluble starch in 1ml of water

Reagent II- Lugols Iodine solution

Examination of Stains on Different Fabrics Using Starch Iodine Test

After each fabrics get stained they were tested using starch-iodine test to examine the retaining capacity of each fabrics and to study the effects of saliva on different fabrics.

 

Methodology for conducting Starch-Iodine test:

Each fabric pieces are kept in a separate Petridis.

Group A - Contains all normal saliva (positive control) stained fabrics

Group B- Contains all tobacco chewed saliva stained fabrics

Group C- Contains all negative control (unstained fabrics)

 

Reagent I (0.5% soluble starch solution) is added to each Petridis of group A, B, C and is kept for incubation for 1hr. After incubation the Petridis are taken out from oven and reagent II (lugols iodine solution) was added and the color change was noted.

Washing of stains with water:

All stained fabrics (both normal saliva stain i.e positive sample and tobacco chewed saliva stain) were washed with water by immersing the stained fabrics for 10 min into a beaker filled with water at room temperature. A negative control was also washed in parallel. The washed fabrics are allowed to dry at room temperature and then tested with starch iodine test, and the colour change is successively noted.

 

Washing of tobacco chewed saliva stained fabrics with different detergent:

For washing 1g of detergent was mixed in 500ml of water, and fabrics were washed for 10 min. All the fabrics were washed by hand in a beaker at room temperature. During washing, the fabrics were well agitated by hand in order to remove the maximum amount of tobacco chewed saliva stain following the conventional method routinely practiced. One type of fabric was washed with all five brands of detergents. A negative and positive control is also washed in parallel. After washing, the fabrics were left to dry at room temperature, and then subjected to starch-iodine test for the detection of saliva and the results in the form of colour change is noted.

 

Starch-iodine test on pre starched cotton cloth:

A pre starched cotton cloth is stained with positive and negative saliva sample and is subjected to starch-iodine test, then the results are noted respectively.

 

RESULTS AND DISCUSSION:

The data from the below tables shows that the presence of α-amylase activity in Starch-iodine test is indicated by brownish color, while a dark blue starch iodine complex is observed in all negative controls. The presence of brownish color indicates that the starch is being hydrolyzed by the action of α-amylase present in saliva and is no longer available for complexing. It was observed in the present study that the fabric retains saliva even after washing with water which was indicated by the presence of brownish color, but after washing with different detergents saliva stains were completely removed which was indicated by dark blue color after adding Lugol's iodine solution. Thus, none of the fabrics retain saliva after washing with detergents. It was also found in the study that the pre-starched cotton cloth gave negative result even for the positive saliva sample, this may be due to the presence of starch already in the cotton cloth which may not be hydrolyzed by the given positive saliva sample.

Table-4.1: Examination of saliva on different fabrics

SI. No.	Fabric Type	Colour observed after addition of Lugol's Iodine solution		Alpha-amylase activity
		Normal saliva	Tobacco chewed saliva	Normal saliva	Tobacco chewed saliva
1.	Silk	Brownish Coloured observed	Brownish Coloured observed	Present (Fig.4.2)	Present (Fig.4.3)
2.	Polyester	Brownish Coloured observed	Brownish Coloured observed	Present (Fig.4.2.1)	Present (Fig.4.2.3)
3.	Jute	Brownish Coloured observed	Brownish Coloured observed	Present	Present
4.	Sarton	Brownish Coloured observed	Brownish Coloured observed	Present	Present
5.	Georgette	Brownish Coloured observed	Brownish Coloured observed	Present	Present
6.	Linen	Brownish Coloured observed	Brownish Coloured observed	Present	Present

 

Table-4.2: Examination of saliva on different fabrics after washing with water.

Sl. No.	Fabric Type	Colour observed after addition of Lugol's Iodine solution		Alpha-amylase activity
		Normal saliva	Tobacco chewed saliva	Normal saliva	Tobacco chewed saliva
1.	Silk	Brownish Coloured observed	Brownish Coloured observed	Present	Present
2.	Polyester	Brownish Coloured observed	Brownish Coloured observed	Present	Present
3.	Jute	Brownish Coloured observed	Brownish Coloured observed	Present (Fig.4.3.2)	Present (Fig.4.3.3)
4.	Sarton	Brownish Coloured observed	Brownish Coloured observed	Present (Fig.4.4.1)	Present (Fig.4.4.3)
5.	Georgette	Brownish Coloured observed	Brownish Coloured observed	Present	Present
6.	Linen	Brownish Coloured observed	Brownish Coloured observed	Present	Present

 

Table-4.3: Examination of saliva on different fabrics after washing with different commercially available detergents.

 

Table-4.4: Examination of saliva on pre starched cotton cloth using starch-iodine test.

 

 

Fig.4.1.1 (Polyester) normal saliva Brownish color observed

 

Fig.4.1.2 (Polyester) negative control dark blue color observed

 

Fig.4.1.3 (Polyester) tobacco chewed saliva. Brownish color observed

 

Fig: 4.1 Examination of Saliva on Silk fabric

 

 

Fig.4.2.1 (Polyester) normal saliva Brownish color observed

 

Fig.4.2.2 (Polyester) negative control dark blue color observed

 

Fig.4.2.3 (Polyester) tobacco chewed saliva. Brownish color observed

 

 

Fig 4.2: Examination of saliva on polyester fabric

 

 

Fig.4.3.1 (Jute) negative control Dark blue color observed

 

Fig.4.3.2 (Jute) positive control Brownish color observed

 

Fig.4.3.3 (Jute) Tobacco chewed saliva Brownish color observed

 

 

Fig 4.3: Examination of saliva on Jute Fabric after washing with water.

 

 

Fig.4.4.1 (Sarton) positive control Brownish color observed

 

Fig.4.4.2 (Sarton) negative control Dark blue color observed

 

Fig.4.4.3 (Sarton) Tobacco chewed saliva Brownish color observed

 

 

Fig 4.4: Examination of saliva on Sarton Fabric after washing with water.

 

 

Fig.4.5.1 (Georgette) positive control Dark blue color observed.

 

Fig.4.5.2 (Georgette) negative control. Dark blue color observed

 

Fig.4.5.3 (Georgette) Tobacco chewed Saliva Dark blue color observed

 

 

Fig 4.5: Examination of saliva on Georgette Fabric after washing with detergents.

 

 

Fig.4.6.1 (Linen) positive control Dark blue color observed.

 

Fig.4.6.2 (Linen ) negative control Dark blue color observed

 

Fig.4.6.3 (Linen) Tobacco chewed saliva Dark blue color observed

 

 

Fig 4.6: Examination of saliva on Linen Fabric after washing with detergents.

 

 

Fig.4.7.1 (pre starched cotton) Positive Control Dark blue color observed

 

Fig.4.7.1 (pre starched cotton) Negative Control Dark blue color observed

 

Fig.4.7.3 (pre starched cotton) Tobacco chewed saliva Dark blue color observed

 

 

Fig 4.7: Examination of saliva on pre starched cotton Fabric.

 

A study was conducted to detect the presence of tobacco chewed saliva on seven different white fabrics after washing with water and five commercially available detergents. After applying the tobacco chewed saliva to the fabrics, the diameter of the stains were noted. Every fabric obtained a different diameter depending on its ability to absorb the stain. Fabric that absorbed more stain showed a larger diameter whereas, fabric that absorbed less tobacco chewed saliva stain showed a smaller diameter. The fabrics with greater absorption, viz. polyester, georgette retained more stain and can therefore be more useful for detection of the stains. Also it can be subsequently help in generating the DNA profile. The absorption of stain by the fabrics depends upon the chemistry, the weave of the fabric, water wicking and chemical modification. After washing both with water and detergents the visibility of the stains were noted, water washed stains were partially visible by naked eyes on some of the fabrics which was confirmed by the presence of brownish color developed after adding lugol's iodine solution, but the stains washed with detergents were completely removed. Also it was found that stains become less visible and less retained by fabrics when the time of washing is increased.

 

CONCLUSION:

Saliva is one of the important body fluid in criminal investigations, which helps in the digestion of starch and lubrication of food. The presence of α-amylase in saliva is detected by principle of Starch Amylase activity which confirms the presence or absence of Saliva. Starch is a complex compilation of simple sugars (or polysaccharides) which breaks down into simpler substances, and forms two sugar maltose (a disaccharides), the enzyme required to break down starch is salivary amylase. Many information can be gathered in crime scene investigations through saliva examination, the epithelial cells of the buccal cavity found in saliva can be used for DNA profiling too. Saliva is generally found in sexual assault cases, on gags (cloth used to tie the mouth) in kidnapping cases or other cases, which can link a suspect to the crime scene. From the above study it is concluded that saliva is retained in fabrics even after washing with water due to the fact that the enzyme present in saliva sticks the fabrics which is generally not removed by washing with water but detergents completely removes the adhered enzyme of saliva. If the criminal attempts to eliminate saliva stained fabrics from the crime scene it can be still detected by using starch-iodine test. Rather after washing with detergents which are highly impossible to detect these types of saliva stains from the different types of fabrics. A pre-starch starched cotton cloth gives false negative result of saliva, thus saliva cannot be detected easily.

 

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Accepted on 20.08.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4285-4290.