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 Methods for Collecting Saliva
MAHVASH NAVAZESH
Department of Dental Medicine and Public Health
University of Southern California
School of Dentistry
Los Angeles, California 90089-0641

INTRODUCTION

Human saliva is produced by three paired major glands (parotid, submandibular,

and sublingual) and numerous minor glands. The parotid glands are located under
and ventral to the ears. Parotid saliva enters the oral cavity via Stensen’s ducts, which
are located on the buccal mucosa near the maxillary second molars. The sub-
mandibular glands are located at the midpoint of the mandible. The sublingual glands
are located under the tongue in the floor of the mouth. The submandibular saliva and
sublingual saliva enter the oral cavity via Wharton’s ducts, which are located in the
floor of the mouth. Minor salivary glands are scattered throughout the mouth and are
located on the buccal mucosa, soft palate, and inner surfaces of the lips.
Accurate measures of salivary flow rate and composition are essential for many
clinical, experimental, and diagnostic protocols. Saliva can be collected under un-
stimulated (resting) or stimulated conditions. Salivary flow can be stimulated by a
variety of agents: gustatory and masticatory stimuli have been used most frequently
to increase salivary flow rate. The most commonly used stimulants are paraffin wax,
rubber bands, gum base, and citric acid. Pharmacologic’ and electrical* stimulants
have been used as therapeutic aids in the management of patients with salivary gland
hypofunction. In contrast, unstimulated saliva is defined as the saliva collected with
no apparent source of stimulation. It can be collected as whole saliva or from
individual glands. Whole saliva represents secretions from the major and minor
glands. Individual gland secretions refer to saliva from parotid, submandibular, and
sublingual glands. Individual gland secretions are superior to whole saliva for many
compositional analyses because whole saliva contains nonsalivary elements such as
desquamated epithelial cells, food debris, bacteria, gingival crevicular fluid, and
leukocytes. However, for assessment of overall salivary gland dysfunction, whole
saliva is superior and clinically more r e l e ~ a n t . ~
Salivary flow rates vary significantly among individuals and in the same in-
dividual under different condition^.^ Therefore, it is crucial that the method of saliva
collection be standardized.
Unstimulated salivary flow rate is affected by many factors, the most important
of which is potentially the degree of hydration. It has been reportedS that an 8%
reduction in body water content can cause a 100% reduction in the salivary flow rate.
Hyperhydration, exposure to light, olfactory stimuli, and body positioning can also
influence the flow rate. Shannon6 compared flow rates in a group of subjects under
different body positions. He reported higher flow rate values in the standing position
and lower values in the lying position as compared with the flow rate in the sitting
72
NAVAZESH: COLLECTION 73

position. Thus, it is best to collect saliva while the subject is sitting upright with the
head slightly tilted forward and the eyes open. Subjects should refrain from smoking,
eating, or drinking for 1-2 hours prior to the test session. It must be remembered that
salivary flow rate and composition are affected by seasonal and diurnal factors.
Parotid salivary flow rate has been shown to reach its peak value during the winter
season,' and salivary flow rate shows a circadian rhythm with the peak value in the
afternoon.8 Therefore, the time of day for saliva collection should be standardized
and the time of year should be considered as a potential factor influencing flow rate
in long-term salivary studies.
A subject's medical status and medications may also affect salivary flow and
composition."" When stimulated saliva is collected, additional factors such as dura-
tion of stimulation,'* nature of the stimulant,13and salivary gland size14 affect the
flow rate and composition. If compositional analyses are to be done, saliva should
be collected into chilled tubes, kept on ice, and frozen until analysis.

METHODS FOR COLLECTING WHOLE SALIVA

There are different methods available for collecting whole saliva. Regardless of
the method used, subjects should be instructed to rinse the mouth thoroughly with
deionized water prior to the collection trial and to void the mouth of saliva. The
subject should be seated comfortably with eyes open, head tilted slightly forward,
and (for unstimulated saliva collection) instructed to rest for 5 minutes and to
minimize orofacial movements. Five minutes is an adequate collection period. The
following are the four most common methods for collecting whole saliva:
(1) Draining method: Saliva is allowed to drip off the lower lip into a preweighed
or graduated test tube fitted with a funnel and the subject expectorates into
the test tube at the end of the collection period. The Proflow Sialometer'"
(Proflow, Incorporated, Amityville, New York) is a graduated collection
vessel attached to a funnel and is commercially available (FIGURE 1).
(2) Spitting method: Saliva is allowed to accumulate in the floor of the mouth
and the subject spits it out into the preweighed or graduated test tube every
60 seconds.
(3) Suction method: Saliva is continuously aspirated from the floor of the mouth
into a test tube by a saliva ejector or an aspirator.
(4) Swab (absorbent) method: Saliva is collected (absorbed) by a preweighed
swab, cotton roll, or gauze sponge placed in the mouth at the orifices of the
major glands and is removed for reweighing at the end of the collection
period.
In a comparative study15of these methods, it was found that the suction and swab
methods introduced some degree of stimulation and variability and thus are not
recommended for unstimulated whole saliva collection. The swab method was found
to be the least reliable. Draining and spitting provide similar types of information
about unstimulated whole saliva and are both reproducible and reliable. The spitting
method is also recommended for stimulated whole saliva collection. Saliva could be
stimulated by applying 0.1-0.2 mol/L citric acid to the tongue at a fixed interval (e.g.,
74 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Sialometer'": a graduated tube used for collecting whole saliva.

15-60 seconds). Gustatory stimulants will interfere with some salivary analyses such
as buffering capacity and will not elicit a constant flow rate because of the saliva
diluting effect. Mechanical (masticatory) stimulation will not interfere with saliva
composition; however, it is difficult to maintain a constant force of stimulation
(mastication) throughout the collection period. Standard-size gum base or paraffin
wax (1.5 g, mp = 42 "C) can be used as a stimulant. The frequency of stimulation
can be controlled by a metronome at about 70 chewslmin.
To familiarize the subject with the method, it is wise to run a (l-2)-min trial
collection prior to the actual 5-min collection period. For stimulated saliva, the first
2-min collected sample should be discarded.
NAVAZESH: COLLECTION 75

METHODS FOR COLLECTING SALIVA FROM

INDIVIDUAL GLANDS

Parotid saliva can be collected with a Lashley cupI6or a modified Carlson-

Crittenden device.”J* The collector consists of a plastic or metal cup with an inner
and outer chamber (FIGURE 2). The inner chamber is attached to plastic tubing that
carries saliva to the collection vessel. The outer chamber is attached to a rubber bulb
or suction-inducing device via plastic tubing and the cup is placed over Stensen’s
duct. This method is simple and reliable. The collection cup is available commer-
cially from Stone Machine Company (Colton, California).
Secretions from the submandibular and sublingual glands often enter the oral
cavity via a common duct, making it difficult to collect secretions from each gland
separately. Tapered polyethylene tubing (approximately 0.5-1.5 mm) may be used
for cannulation of Wharton’s duct and for collection of submandibular saliva. How-
ever, extreme caution must be used when placing the cannula because the wall of the
duct is thin and may rupture.
Custom-made collection devices have been introduced by various investiga-
tors.’’+-21These devices, referred to as “segregators”, usually contain a central cham-
ber for the collection of submandibular saliva and one or two lateral chambers for
the collection of sublingual saliva. Polyethylene tubing connects the chambers to
graduated receiving vessels. Fabrication of these collection devices is time-consum-
ing because a mold of the floor of the subject’s mouth has to be made and the device
has to be fabricated and adjusted on an individual basis. This is inconvenient for
individuals with inflammatory or ulcerative soft-tissue disorders affecting the floor
of the mouth. A simple method for collection of mixed submandibular and sublin-
gual saliva was introduced by Fox et a1.22in 1985. Stensen’s ducts are blocked,

FIGURE 2. A Carlson-Crittenden collector used for collecting parotid saliva.

76 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 3. A micropipette used for collecting submandibular and sublingual saliva. (Cour-
tesy of Jonathan Ship.)

Wharton’s ducts are isolated, and saliva is collected from the floor of the mouth with
a micropipette by use of gentle suction (FIGURE 3).
Minor gland secretions can be collected by pipette or absorbent filter paper2*zs
from the inner surface of lips, palate, or buccal mucosa. Secretions may also be
collected with an absorbent paper strip26and the quantity of saliva may be deter-
mined with a PeriotronB (Proflow, Incorporated), which measures small volumes of
fluids. For collection of saliva from minor salivary glands, the mucosa is dried first
and, after a 2 m i n interval, saliva samples may be obtained by touching the develop-
ing beads of saliva with the absorbent paper. The quantity of saliva may then be
determined utilizing a PeriotronB.
In recent years, human salivary gland function and dysfunction have received
increasing clinical and research attention. However, it is difficult to obtain conclusive
information based on comparisons of available data because the collection methods
are not consistent. This report has focused on the most common methods available
for collecting whole and individual gland saliva. It is hoped that more clinicians and
investigators will follow standardized methodology for collection of saliva to en-
hance the value of saliva as a diagnostic tool.

REFERENCES
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patients with Sjogren’s syndrome. J. Dent. Res. 67( 10): 1334-1337.
NAVAZESH: COLLECTION 77

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