3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P85 OPTIMALIZATION OF METhOD FOR
QuANTIFICATION OF sTrePTOCOCCus
MuTANS TO DENTAL MATERIALS
ILOnA PRUDíKOVá, STAnISLAVA MATALOVá and
JOSEF JánČář
Faculty of Chemistry, Brno University of Technology
Purkyňova 118, 612 00 Brno, Czech Republic,
xcprudikova@fch.vutbr.cz
Introduction
The attachment of certain microorganisms to specific
surfaces in the human oral cavity and the resulting formation
of dental plaque on teeth and dental materials are primary
causes for oral diseases such as denture stomatitis, gingival
inflammation, and secondary caries, which may consequently
lead to unhealthy complications 1 . Secondary caries is the
most frequent reason for the replacement of restorative materials
including resin composites. That is reason, why antimicrobial
agents are incorporated and bacterial adhesion has
to be measured.
In the literature, several methods for quantification of
deposited oral microorganisms were described, but a lot of
them could be characterized as time, materials, instruments
and financially – consuming.
The amount of sorbed bacteria could be expressed using
radio-labelled microorganisms. Cells were radio-labelled using
[3H] – thymidine 2–7 , [3H] – uridine or [35S] – methionine.
The amount of adsorbed bacteria was measured in a scintillation
counter and was determined as radioactive counts per
minute (CPM) of the labelled bacteria after washing them
with buffer (KCl) to remove unbound bacteria.
Other tests for microorganisms quantification were carried
on examine dental materials. Discs from this dental materials
were inserted in petri dishes or tubes that contained
cell suspension and than they were incubated for 24 hours.
After incubation discs were removed and rinsed with distilled
water or PBS. Adherent bacteria were fixed with methanol or
glutaraldehyd and stained with acridine orange, crystal violet
or modified Gram stain. Quantitative analysis was performed
using a fluorescence microscope. The number of adherent
cells in several random fields was counted on each sample
and bacterial adhesion was expressed as percentage area coverage
8 .
These methods were used the most often, but except
them, other minor methods were tested. Capopreso at.al. 9 analysed
bacterial adhesion to dental alloys spectrophotometrically
in a microplate reader at 570 nm. The bacterial adhesion
of each specimen was quantified as the ratio between the optical
density at 570 nm and the surface area of the specimen.
Blunden 9 , Duskova 10,11 expressed the amount of deposited
microorganisms as percentage weight gain. Wu-Yuan 12 and
Wilbershausen 13 examined the attachment of oral bacteria
by SEM. For SEM, the samples were fixed in formaldehyd
or glutaraldehyd and dehydrated through a graded series of
aceton or ethanol. Boeckh 14 placed bacterial suspensions
s768
in conical cavities within the material and after incubation,
the suspensions were removed form the restoratives and the
numbers of viable bacteria were counted.
F. Ozer at al. used for testing antibacterial activity Tooth
cavity model. They prepared three cylindrical cavities in the
flat surface of human extracted tooth. Cell suspension and
brain heart infusion (BHI) broth were put in cavities and incubated
for 24 h at 37 ºC. The number of S. mutans recovered
was determined by the classical bacterial counting method
using 5% sheep blood agar 8 .
Experimental
The spectrophotometric Biuret method was used to
quantified amount of bacteria S. mutans adhering to the polymer
surface. In order to optimise Biuret method the composition
of liquid culture medium, the amount of bacteria suspension
used for the samples inoculation, the samples incubation
time and methods for the bacteria releasing from the samples
were tested.
B i u r e t M e t h o d
Under alkaline conditions substances containing two or
more peptide bonds form a purple complex with copper salts
in the reagent. Upon complexation, a violet color is observed.
The absorbance of the Cu 2+ protein complex is measured at
540 nm and compared to a standard curve. Bovine serum albumin
is used as a standard. This method was employed for
the quantification of the S. mutans in both culture medium
and on the discs.
Biuret agent was prepared by dissolving 1.5 g
CuSO 4 . 5H2 O + 6 g C 4 H 4 O 6 Kna . 4H 2 O in 500 ml H 2 O.
300 ml 10% naOH was added to the solution and distilled
water to make 1,000 ml.
When studying the amount of bacteria in the suspension
following procedure was used: 1 ml of the tested suspension
was mixed with 4 ml of Biuret agent and 1 ml distilled water.
Upon 10 seconds shaking period and 30 minute incubation,
absorbance was measured against a blank at 540 nm.
To evaluate the amount of bacteria adhered to the disc,
the bacteria had to be released from the disc. The samples
were placed into 2 ml distilled water and ultrasonic bath were
used. The time of ultrasonic bath was varied up to 8 minutes
(2, 4, 6 and 8 minutes) in order to find out conditions when
both the highest amount of bacteria is released and still no
damage of the disc occurs. Further the procedure is same as
in case of quantification of bacteria in suspension.
D e n t a l M a t e r i a l s a n d P r e p a r e o f
S a m p l e s
The discs were prepared from the commercially available
microhybrid composites (Adoro, Ivoclar Vivadent),
D3MA resin-based materials (Advanced Dental Materials)
and ceramic materials (Ivoclar Vivadent) as reference material.
The material was placed in a metal mold between a layer
of transparent foil and metal. It was cured in the Vectris cur-