Nursing Biochemistry Laboratory 1

Laboratory Manual

Laboratory Procedure Activity

Analysis of Saliva 12

INTRODUCTION

Saliva is a viscous fluid with a pH of around 6.8. It contains mucins, amylase, and other
proteins. Inorganic constituents include sodium, potassium, calcium, chloride, and
phosphate ions. Daily output of saliva is in the range of 1.0 – 2.0 liters.

Being a complex fluid, saliva influences oral health through specific and nonspecific
physical and chemical properties. The importance of saliva in everyday activities and the
medicinal properties it possesses are often taken for granted. However, when
disruptions in the quality or quantity of saliva do occur in an individual, it is likely that he
will experience detrimental effects on oral and systemic health. Often head and neck
radiotherapy has serious and detrimental side effects on the oral cavity including the
loss of salivary gland function and a persistent complaint of a dry mouth (xerostomia).
Thus, saliva has a myriad of beneficial functions that are essential to our well-being.
Although saliva has been extensively investigated as a medium, few laboratories have
studied saliva in the context of its role in maintaining oral and general health.1

Analyses of the properties of saliva using biochemical and physiological methodologies

can be traced back to at least over a century ago. It is obvious that in 1898, when
Chittenden and Mendel conducted their study on the influence of alcoholic drinks upon
digestion and secretion, the measurement of total organic constituents, salts, and
chlorine in saliva was already being performed routinely. In the late 19th century,
researchers had already learnt that saliva had digestive powers, mainly in the form of
amylolysis and proteolysis. Studies in the early 20th century had shown some evidence
of the dietary effect of saliva. Highly sensitive and high-throughput assays such as mass
spectrometry, microarray, and nano-scale sensors that can measure proteins and nucleic
acids with minimal sample requirement in a short period of time allowed scientists to
broaden the utility of saliva.2

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Bunsen burner 3 mL starch solution

iron ring 1 mL 0.1M phosphate buffer
iron stand 1 mL 0.1M sodium chloride, NaCl
funnel 3 mL 1N acetic acid, CH3COOH
spot plate 5 mL acetone, CH3COCH3
test tubes 1 mL 3M hydrochloric acid, HCl

1
Tiwari, Manjul. 2011, January-June. Science behind human saliva. National Center for Biotechnology
Information. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312700/.
2
Ibid.
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory 2
Laboratory Manual

test tube rack 5 mL 3M sodium hydroxide, NaOH

test tube holder 3 mL Benedict’s solution
beaker 1 mL 2.5M sodium hydroxide, NaOH
stirring rod 1 mL 0.05% copper(II) sulfate, CuSO4
graduated cylinder 1 mL 1% acetic acid, CH3COOH
droppers 5 mL 2% potassium oxalate, K2C2O4⋅H2O
watch glass 1 mL ammonium molybdate, (NH4)6Mo7O24
spot plate 1 mL nitric acid, HNO3
wire gauze 1 mL silver nitrate, AgNO3
filter paper 1 mL iodine solution
distilled water

PROCEDURE

Collection of Saliva

Rinse out the mouth to get rid of food particles. Take about 10 mL of distilled warm
water into mouth and move it about by the tongue for nearly a minute. Collect the fluid
in a clean beaker. Perform the tests with this diluted saliva.

1. Test for Amylase

To 2.5 mL of starch solution in a test tube, add one mL of 0.1M phosphate

buffer, pH 7.0, followed by 1 mL of 0.1M NaCl solution. Add 1 mL of salivary
fluid. Mix. Prepare a spot plate with each spot containing a drop of iodine
solution. At intervals of one minute, transfer one drop of the mixture to each
depression containing iodine in the spot plate. Blue color fades and disappears
with samples collected with increasing time intervals. Do this for 12 minutes.

Amylase hydrolyses -1,4 glycosidic bonds in starch at random. The break-down

products do not have the capacity to bind iodine.

2. Test for Calcium

To 2 mL of salivary fluid in a test tube, add 5 drops of 1% acetic acid and 5 mL

of 2% potassium oxalate solution. Observe a faint white precipitate form.
Calcium ions are precipitated as calcium oxalate under neutral or slightly acidic
conditions.

3. Test for Inorganic Phosphate

Mix 1mL of salivary fluid in a test tube, add 1 mL of ammonium molybdate

solution. Heat over direct flame and observe.

4. Test for Chloride

In a test tube, mix 1 mL of salivary fluid with 3-5 drops of nitric acid followed by
5 drops of silver nitrate solution. Observe.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory 3
Laboratory Manual

Isolation of Mucin from Saliva

Rinse the mouth with water and collect 15 mL of saliva in a 20 mL test tube. Add 3 mL
of 1N acetic acid drop wise, swirling the mixture after each addition. Observe the
formation of a thread-like precipitate. This is the mucin. Let it stand for at least 30
minutes. Decant, filter and wash the precipitate using two 2 mL portions of acetone.
Allow the precipitate to drain completely. Remove the filter paper from the funnel,
spread it out on a watch glass and allow the precipitate to dry. Use the precipitated
mucin to test for the possible carbohydrate and protein components of saliva.

5. Carbohydrate

Place about one-half of the isolated mucin in a test tube. Add 1 mL of 3M HCl
and immerse in a boiling water bath for 30 minutes. Cool and make faintly basic
with 3M NaOH. Do this by putting a “red” litmus paper in the test tube and add
NaOH drop by drop until the litmus paper turns “blue”. Add 3 mL of Benedict’s
solution. Heat again in boiling water bath until a visible change occurs.

6. Protein

Dissolve the remaining portion of mucin in 1 mL of 2.5M NaOH, then add 3 drops
of 0.05% CuSO4 solution. Mix. Observe the color change.

PROPER DISPOSAL: Dispose of solutions in the proper waste bottles (as

acid or basic wastes, and organic or inorganic wastes).

QUESTIONS
1. What is the purpose of each of the components of saliva?
2. How does each component help in the digestion of food?
3. Does digestion already happen in the mouth? If so, what type of digestion?
4. What impairment will a person experience without salivary glands?
5. Cite three salivary gland disorders and the corresponding medical treatment for
each.

REVIEW QUESTIONS (Answer this prior to performing the activity.)

1. When collecting saliva sample, why was there a need to rinse the mouth to get rid of
food particles?
2. Why does the blue color fade and disappear with samples collected with increasing
time intervals?
3. Does amylase hydrolyze other complex carbohydrates other than the -1,4 glycosidic
bonds in amylose?
4. What is the role of mucin in saliva?
5. Is it possible to determine the types of carbohydrate and protein found in mucin?

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory 4
Laboratory Manual

Group No.___ Date Performed :___________

Subject & Section:________ Instructor’s Initials :___________
Members:
1. _____________________
2. _____________________
3. _____________________

Data Sheet Activity

Analysis of Saliva 12

Data

Substances Test Reagents Observations/Results

Tested for
Amylase + 0.1M phosphate Intervals of 1 Minute (Color in 12 Spots)
buffer, 0.1M NaCl

Calcium + 1% CH3COOH,
2% K2C2O4⋅H2O

Inorganic Phosphate + (NH4)6Mo7O24,

heat

Chloride + HNO3, AgNO3

Carbohydrate + NaOH, Benedict’s

solution, heat

Proteins + NaOH, CuSO4

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.