3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures
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Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology<br />
P85 OPTIMALIZATION OF METhOD FOR<br />
QuANTIFICATION OF sTrePTOCOCCus<br />
MuTANS TO DENTAL MATERIALS<br />
ILOnA PRUDíKOVá, STAnISLAVA MATALOVá and<br />
JOSEF JánČář<br />
Faculty of Chemistry, Brno University of Technology<br />
Purkyňova 118, 612 00 Brno, Czech Republic,<br />
xcprudikova@fch.vutbr.cz<br />
Introduction<br />
The attachment of certain microorganisms to specific<br />
surfaces in the human oral cavity and the resulting formation<br />
of dental plaque on teeth and dental materials are primary<br />
causes for oral diseases such as denture stomatitis, gingival<br />
inflammation, and secondary caries, which may consequently<br />
lead to unhealthy complications 1 . Secondary caries is the<br />
most frequent reason for the replacement of restorative materials<br />
including resin composites. That is reason, why antimicrobial<br />
agents are incorporated and bacterial adhesion has<br />
to be measured.<br />
In the literature, several methods for quantification of<br />
deposited oral microorganisms were described, but a lot of<br />
them could be characterized as time, materials, instruments<br />
and financially – consuming.<br />
The amount of sorbed bacteria could be expressed using<br />
radio-labelled microorganisms. Cells were radio-labelled using<br />
[3H] – thymidine 2–7 , [3H] – uridine or [35S] – methionine.<br />
The amount of adsorbed bacteria was measured in a scintillation<br />
counter and was determined as radioactive counts per<br />
minute (CPM) of the labelled bacteria after washing them<br />
with buffer (KCl) to remove unbound bacteria.<br />
Other tests for microorganisms quantification were carried<br />
on examine dental materials. Discs from this dental materials<br />
were inserted in petri dishes or tubes that contained<br />
cell suspension and than they were incubated for 24 hours.<br />
After incubation discs were removed and rinsed with distilled<br />
water or PBS. Adherent bacteria were fixed with methanol or<br />
glutaraldehyd and stained with acridine orange, crystal violet<br />
or modified Gram stain. Quantitative analysis was performed<br />
using a fluorescence microscope. The number of adherent<br />
cells in several random fields was counted on each sample<br />
and bacterial adhesion was expressed as percentage area coverage<br />
8 .<br />
These methods were used the most often, but except<br />
them, other minor methods were tested. Capopreso at.al. 9 analysed<br />
bacterial adhesion to dental alloys spectrophotometrically<br />
in a microplate reader at 570 nm. The bacterial adhesion<br />
of each specimen was quantified as the ratio between the optical<br />
density at 570 nm and the surface area of the specimen.<br />
Blunden 9 , Duskova 10,11 expressed the amount of deposited<br />
microorganisms as percentage weight gain. Wu-Yuan 12 and<br />
Wilbershausen 13 examined the attachment of oral bacteria<br />
by SEM. For SEM, the samples were fixed in formaldehyd<br />
or glutaraldehyd and dehydrated through a graded series of<br />
aceton or ethanol. Boeckh 14 placed bacterial suspensions<br />
s768<br />
in conical cavities within the material and after incubation,<br />
the suspensions were removed form the restoratives and the<br />
numbers of viable bacteria were counted.<br />
F. Ozer at al. used for testing antibacterial activity Tooth<br />
cavity model. They prepared three cylindrical cavities in the<br />
flat surface of human extracted tooth. Cell suspension and<br />
brain heart infusion (BHI) broth were put in cavities and incubated<br />
for 24 h at 37 ºC. The number of S. mutans recovered<br />
was determined by the classical bacterial counting method<br />
using 5% sheep blood agar 8 .<br />
Experimental<br />
The spectrophotometric Biuret method was used to<br />
quantified amount of bacteria S. mutans adhering to the polymer<br />
surface. In order to optimise Biuret method the composition<br />
of liquid culture medium, the amount of bacteria suspension<br />
used for the samples inoculation, the samples incubation<br />
time and methods for the bacteria releasing from the samples<br />
were tested.<br />
B i u r e t M e t h o d<br />
Under alkaline conditions substances containing two or<br />
more peptide bonds form a purple complex with copper salts<br />
in the reagent. Upon complexation, a violet color is observed.<br />
The absorbance of the Cu 2+ protein complex is measured at<br />
540 nm and compared to a standard curve. Bovine serum albumin<br />
is used as a standard. This method was employed for<br />
the quantification of the S. mutans in both culture medium<br />
and on the discs.<br />
Biuret agent was prepared by dissolving 1.5 g<br />
CuSO 4 . 5H2 O + 6 g C 4 H 4 O 6 Kna . 4H 2 O in 500 ml H 2 O.<br />
300 ml 10% naOH was added to the solution and distilled<br />
water to make 1,000 ml.<br />
When studying the amount of bacteria in the suspension<br />
following procedure was used: 1 ml of the tested suspension<br />
was mixed with 4 ml of Biuret agent and 1 ml distilled water.<br />
Upon 10 seconds shaking period and 30 minute incubation,<br />
absorbance was measured against a blank at 540 nm.<br />
To evaluate the amount of bacteria adhered to the disc,<br />
the bacteria had to be released from the disc. The samples<br />
were placed into 2 ml distilled water and ultrasonic bath were<br />
used. The time of ultrasonic bath was varied up to 8 minutes<br />
(2, 4, 6 and 8 minutes) in order to find out conditions when<br />
both the highest amount of bacteria is released and still no<br />
damage of the disc occurs. Further the procedure is same as<br />
in case of quantification of bacteria in suspension.<br />
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<br />
S a m p l e s<br />
The discs were prepared from the commercially available<br />
microhybrid composites (Adoro, Ivoclar Vivadent),<br />
D3MA resin-based materials (Advanced Dental Materials)<br />
and ceramic materials (Ivoclar Vivadent) as reference material.<br />
The material was placed in a metal mold between a layer<br />
of transparent foil and metal. It was cured in the Vectris cur-