Impact of temperature and time storage on themicrobial detection of oral samples byCheckerboard DNA–DNA hybridization method

Cassio do Nascimento a, Janine Navarro dos Santos a, Vinıcius Pedrazzi a,Murillo Sucena Pita a, Nadia Monesi c, Ricardo Faria Ribeiro a,Rubens Ferreira de Albuquerque Juniora,b,*a Faculty of Dentistry of Ribeirao Preto, Department of Dental Materials and Prosthodontics, Molecular Diagnosis

Laboratory, University of Sao Paulo, Av. Cafe s/n8, Monte Alegre, Ribeirao Preto, SP 14040-904, Brazilb Faculty of Dentistry, McGill University, Strathcona Anatomy & Dent, 3640, University Street, Montreal, QC, Canada

H3A 2B2c Faculty of Pharmaceutical Sciences of Ribeirao Preto, Department of Clinical Toxicological and Bromatologic Analysis,

University of Sao Paulo, Av. Cafe s/n8, Monte Alegre, Ribeirao Preto, SP 14040-903, Brazil

1–5

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 1

a r t i c l e i n f o

Article history:

Accepted 15 October 2013

Keywords:

DNA storage

Bacteria

Candida

Diagnosis

Clinical assessment

Checkerboard DNA–DNA

hybridization

a b s t r a c t

Purpose: Molecular diagnosis methods have been largely used in epidemiological or clinical

studies to detect and quantify microbial species that may colonize the oral cavity in healthy

or disease. The preservation of genetic material from samples remains the major challenge

to ensure the feasibility of these methodologies. Long-term storage may compromise the

final result. The aim of this study was to evaluate the effect of temperature and time storage

on the microbial detection of oral samples by Checkerboard DNA–DNA hybridization.

Methods: Saliva and supragingival biofilm were taken from 10 healthy subjects, aliquoted

(n = 364) and processed according to proposed protocols: immediate processing and pro-

cessed after 2 or 4 weeks, and 6 or 12 months of storage at 4 8C, �20 8C and �80 8C.

Results: Either total or individual microbial counts were recorded in lower values for

samples processed after 12 months of storage, irrespective of temperatures tested. Samples

stored up to 6 months at cold temperatures showed similar counts to those immediately

processed. The microbial incidence was also significantly reduced in samples stored during

12 months in all temperatures.

Conclusions: Temperature and time of oral samples storage have relevant impact in the

detection and quantification of bacterial and fungal species by Checkerboard DNA–DNA

hybridization method. Samples should be processed immediately after collection or up to 6

months if conserved at cold temperatures to avoid false-negative results.

# 2013 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: http://www.elsevier.com/locate/aob

1. Introduction

Molecular diagnosis techniques have been largely used to

detect and quantify microbial species colonizing the oral

* Corresponding author at. Faculdade de Odontologia de Ribeirao Preto,Preto - SP Brazil. Tel.: +55 16 3602 4095; fax: +55 16 3602 0547.

E-mail address: ralbuque@forp.usp.br (R.F. de Albuquerque Junior

0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.archoralbio.2013.10.007

cavity. Techniques using genetic materials are faster, more

sensitive and specific than conventional based culture

methods, which may fail to identify slow-growing, fastidious

or non-cultivable microorganisms.6,7 Molecular techniques

are not dependent on cell viability, favouring a rapid and

Universidade de Sao Paulo, Av. do Cafe s/n CEP: 14040-904 Ribeirao

).

d.

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 1 13

precise detection and identification of microorganisms that

present a phenotypically divergent behaviour. This represents

a relevant outcome in studies investigating oral anaerobic

infections in which cell death and DNA degradation may occur

during harvesting and transportation of samples.8,9 The use of

molecular techniques has revealed a large variation on the

microbiota from several regions of the human body.10–13 Some

of them have reported a multitude of non-cultivable bacterial

species that may be related to infectious diseases.

The Checkerboard DNA–DNA hybridization technique was

described by Socransky et al.14 This technique detect micro-

organisms by using whole-genomic DNA probes, which

permits, simultaneously, to identify and quantify up to 45

target bacterial species in up to 28 clinical samples. Thence-

forth, this technique has been applied in several areas of

dentistry to evaluate the microbial composition of oral biofilm

in health or disease.15–18 It has also been used to evaluate the

potential changes in the biofilm composition as result of

periodontal therapies19,20 and, recently, to identify and

quantify bacterial species colonizing implants and implant-

related sites.7,21 do Nascimento et al.22 have also reported the

use of this technique to detect Candida species harbouring the

oral cavity. This technique allows the direct detection of any

cultivable or non-cultivable bacterial or fungal pathogen.

The genetic material preservation is a primordial condition

in samples that will be submitted to molecular analysis. DNA

degradation and contamination may occur during storage

period, which may have a relevant impact in the final

detection of microorganisms.23 The oral cavity is colonized

by a large number of microorganisms, including pathogenic

and non-pathogenic species, such as bacterial and fungal

genera. Oral biofilm constitutes a complex microbiota com-

prehending these microorganisms enrolled in a matrix of

proteins, extracellular products and other salivary com-

pounds. Enzymes, toxins and other sub-products of cell lysis

may cause DNA fragmentation and degradation.24,25

Studies assessing the impact of storage conditions on the

genetic material from oral samples are too scarce in the applied

literature. Katsoulis et al.23 reported a significant reduction in the

bacterial counts detection of oral samples after 12 months of

storage at �20 8C. Nevertheless, this investigation was restricted

to evaluate only 2 different temperatures in a limited interval of

time without comparing between them. Considering the lacking

of information associated to the large number of studies currently

makinguseofgeneticmaterial fordiagnosisand,mostoftime,the

impossibility to process the samples immediately after harvest-

ing, we judge this theme of clinical relevance. Thus, the aim of this

study was to evaluate, by using Checkerboard DNA–DNA

hybridization technique, the effect of temperature and time

storageonthemicrobialdetectionoforalsamples.Thehypothesis

tested was that temperature and time of storage have influence in

the final detection and quantification of target species.

2. Materials and methods

2.1. Subject population

Ten healthy participants (5 men and 5 women; mean age 21.70

years) were selected among graduate students of Faculty of

Dentistry of Ribeirao Preto. Included participants should have

at least 24 teeth, no clinical signs of disease in the oral mucosa,

no dental caries and healthy gingiva. Excluded criteria were:

pregnant or lactating; periodontal treatment or antibiotics in

the previous 3 months; smoking; systemic disease, or

participants that required pre-medication for dental treat-

ment. The study was approved by the Faculty’s ethics

committee (Process 2010.1.1354.58.4) and all the participants

were informed on the nature of the investigation and gave

their written informed consent.

2.2. Samples preparation

Four millilitres of no-stimulated saliva were collected from each

participant and mixed into a Falcon tube. Additionally,

supragingival biofilm from first superior and inferior molars

of each subject was taken with sterile curettes and added to the

saliva tube to increase the microbial concentration. After

harvesting, the tube containing 40 mL of human saliva and

supragingival biofilm was vortexed during 3 min. Aliquots of

100 mL of the content were transferred to individual microtubes

totalling 364 samples of saliva. A volume of 50 mL of buffer TE

(10 mM Tris–HCl, 1 mM EDTA; pH 7.6) and 150 mL of 0.5 M NaOH

were individually added to the samples. The samples (n = 364)

were randomly divided in 17 groups (n = 28) according to the

proposed protocols for laboratorial processing:

(1) Samples processed immediately after harvesting (Con-

trol).

(2) Samples processed after 2 weeks of storage at room

temperature.

(3) Samples processed after 2 weeks of storage at 4 8C.

(4) Samples processed after 2 weeks of storage at �20 8C.

(5) Samples processed after 2 weeks of storage at �80 8C.

(6) Samples processed after 4 weeks of storage at room

temperature.

(7) Samples processed after 4 weeks of storage at 4 8C.

(8) Samples processed after 4 weeks of storage at �20 8C.

(9) Samples processed after 4 weeks of storage at �80 8C.

(10) Samples processed after 6 months of storage at room

temperature.

(11) Samples processed after 6 months of storage at 4 8C.

(12) Samples processed after 6 months of storage at �20 8C.

(13) Samples processed after 6 months of storage at �80 8C.

(14) Samples processed after 12 months of storage at room

temperature.

(15) Samples processed after 12 months of storage at 4 8C.

(16) Samples processed after 12 months of storage at �20 8C.

(17) Samples processed after 12 months of storage at �80 8C.

2.3. Microbiological assessment

The identification and quantification of each target species

were performed using the Checkerboard DNA–DNA hybrid-

ization technique proposed by Socransky et al.14 with a

modification according to do Nascimento et al.26 Thirty-eight

bacterial species including putative periodontal pathogens

(Aggregatibacter actinomycetemcomitans serotypes a and b,

Bacteroides fragilis, Capnocytophaga gingivalis, Campylobacter

Fig. 1 – Median (with maximum and minimum values),

upper and lower quartiles of total counts (T105 cells) of the

43 target species recovered from the proposed

experimental protocols. Different letters mean significant

differences sought by Friedman test followed by Dunn’s

post-test (E > B > C > A > D > F > G; p < 0.0001).

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 114

rectus, Escherichia coli, Eikenella corrodens, Enterococcus faecalis,

Fusobacterium nucleatum, Fusobacterium periodonticum, Klebsiella

pneumoniae, Lactobacillus casei, Mycoplasma salivarium, Neisseria

mucosa, Pseudomonas aeruginosa, Peptostreptococcus anaerobios,

Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella

intermedia, Prevotella melaninogenica, Parvimonas micra, Prevotella

nigrescens, Pseudomonas putida, Solobacterium moorei, Staphylo-

coccus aureus, Staphylococcus pasteuri, Streptococcus constellatus,

Streptococcus gordonii, Streptococcus mitis, Streptococcus mutans,

Streptococcus oralis, Streptococcus parasanguinis, Streptococcus

salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Trepo-

nema denticola, Tannerella forsythia and Veillonella parvula) were

investigated. Five Candida species frequently found harbour-

ing the oral microbiota of healthy and diseased subjects were

also evaluated (C. albicans, C. dubliniensis, C. glabrata, C. krusei

and C. tropicalis). Total and individual microbial counts

(number of cells colonizing surfaces) and incidence (%) of

each target species were evaluated for all the tested groups.

Whole genomic DNA probes from the 43 microbial species

were directly labelled with the thermostable alkaline phos-

phatase enzyme using the AlkPhos Direct Labelling and Detection

System (GE Healthcare, UK). Briefly, 100 ng of denatured DNA

were mixed with labelling buffer and alkaline phosphatase

enzyme. Formaldehyde was then added to covalently cross-

link the enzyme to the probe. The resulting alkaline

phosphatase-labelled probes were adjusted to a final concen-

tration of 1 ng/mL. The same set of labelled genomic probes

was used to evaluate all the samples in the proposed protocols.

Sensitivity and specificity tests were performed for each

labelled probe in order to optimize the amount of probe

needed to detect both 105 and 106 microbial cells of each

species with the lowest possible background.25

For microbiological assessment of the clinical samples,

microtubes containing samples were boiled for 5 min to

denature DNA. Then, the tubes were immediately cooled on

ice, and the samples were mixed with 800 mL of 5 M

ammonium acetate. The contents of each tube were individu-

ally applied to and concentrated on a nylon membrane

(Hybond N+, GE Healthcare, UK) and baked for 2 h at 80 8C.

As a standard reference, defined amounts of genomic DNA

corresponding to either 105 or 106 bacterial cells for each of the

target species evaluated were also applied to 2 standard lanes

in the same membrane set. The membranes were prehybri-

dized at 63 8C for 6 h in a hybridization solution (0.5 M NaCl;

0.4% w/v blocking reagent). After prehybridization, defined

amounts of labelled whole genomic probes from the target

species were individually applied to the membranes. The

hybridization process was performed overnight at 63 8C. After

washing, hybridization signals were detected by chemilumi-

nescence using CDP-Star reagent (GE Healthcare), and mem-

branes were exposed to ECL Hyperfilm-MP during 60 min (GE

Healthcare). Hyperfilm images were digitized and analyzed

with TotalLab Quant software (TotalLab Limited, UK).

2.4. Data analysis

The number of microbial cells recorded for each protocol could

be expressed in counts by comparing the intensity of

chemiluminescent hybridization signals found in samples

and standard control lanes containing 105 or 106 cells of each

target species. To compare the counts and incidences of each

target species in the different proposed protocols, data were

averaged across the different experimental conditions of

storage and samples processing. First, microorganisms were

analyzed as a pool of all 43 microbial species, and then

microorganisms were analyzed independently in order to

differentiate among the target species. Significant differences

between protocols were calculated using the Friedman test

with Dunn’s post-test. The differences in the incidence of

target species were detected by two-way ANOVA followed by

Bonferroni’s post-test. Differences were considered significant

when p < 0.05. GraphPad Prisma 5.02 statistical software

(GraphPad Software Inc., La Jolla, CA, USA) was used for data

analysis.

3. Results

3.1. Total microbial counts

Median (with maximum and minimum values), upper and

lower quartiles of total microbial counts (�105 cells) for each

protocol are summarized in Fig. 1. The total counts (median

and range: 25–75% �105 cells) of microorganisms recovered

from samples processed immediately after harvesting (Con-

trol: 2.78; range: 2.39–3.19) did not show significant differences

in relation to samples processed after 2 weeks of storage at

room temperature (2.86; range: 2.48–3.33), 4 8C (2.74; range:

2.39–3.14) or �80 8C (2.70; range: 2.33–3.15), and after 6 months

of storage at �80 8C (2.56; range: 1.63–4.09). Samples stored

during 2 weeks at �20 8C presented slightly higher cell counts

(3.28; range: 2.91–3.77; p < 0.05). Samples stored during 4

weeks at room temperature (3.42; range: 2.96–3.84), 4 8C (3.14;

range: 2.73–3.64) and �80 8C (4.74; range: 4.37–5.18) also

presented higher cell counts than control ( p < 0.001). Differ-

ently, samples processed after 4 weeks of storage at �20 8C

(1.60; range: 1.52–1.76), after 6 months stored at room

temperature (1.82; range: 1.34–2.25) and 12 months stored at

4 8C (1.27; range: 0.34–2.58), �20 8C (0.94; range: 0.03–2.58) or

�80 8C (1.65; range: 0.69–2.39) showed significant lower counts

when compared to control ( p < 0.01). The highest total cell

counts were recorded for samples processed after 4 weeks of

Fig. 2 – Median (with maximum and minimum values),

upper and lower quartiles of total microbial counts (T105

cells) evaluating period of storage. Different letters mean

significant differences sought by Friedman test followed

by Dunn’s post-test (C > B > A > D; p < 0.0001).

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 1 15

storage at �80 8C (4.74; range: 4.37–5.18). Other, the lowest

value was recorded for samples stored during 12 months at

room temperature (0.00; range: 0.00–0.27).

Median (with maximum and minimum values), upper and

lower quartiles of total microbial counts (�105 cells) of tested

protocols comparing, individually, period and temperature of

storage are illustrated, respectively, in Figs. 2 and 3. Significant

differences were detected by Friedman test followed by

Dunn’s multiple comparisons post-test when factor period

of storage was analyzed ( p < 0.0001). Only samples stored

during 12 months presented significant reduced microbial

counts (0.89; range: 0.00–2.25; p < 0.001). When the factor

temperature was analyzed, samples processed immediately

after harvesting (2.78; range: 2.39–3.19) presented significant

higher total cell counts than storage at room temperature

(2.43; range: 1.12–3.23; p < 0.001) and �20 8C (2.43; range: 1.56–

3.38; p < 0.001). Samples stored at 4 8C (2.87; range: 2.26–3.51)

and �80 8C (2.81; range: 1.92–4.40) did not show differences

compared to control ( p > 0.05).

Fig. 3 – Median (with maximum and minimum values),

upper and lower quartiles of total microbial counts (T105

cells) evaluating temperature of storage. Different letters

mean significant differences sought by Friedman test

followed by Dunn’s post-test (A > C > B; p < 0.0001).

3.2. Individual microbial counts

The individual mean counts, standard deviations (�105 cells,

�SD) and respective p value for all the 43 target species

evaluated in the proposed protocols are displayed in Table 1.

All the species presented significant differences sought by

Friedman test followed by Dunn’s multiple comparisons post-

test ( p < 0.0001). Overall, most of the target species did not

show significant differences in the cell counts when compar-

ing samples processed after 2 weeks of storage in all the

proposed temperatures with samples processed immediately

after harvesting ( p > 0.05), except for S. gordonii, S. mitis, S.

moorei and S. sanguinis that presented higher cell counts after

storage at �20 8C ( p < 0.05). The samples processed after 4

weeks of storage at room temperature and 4 8C also presented

similar counts to those processed immediately after harvest-

ing ( p > 0.05). On the other hand, samples evaluated 4 weeks

after storage at �20 8C showed lower counts ( p < 0.01) and

samples stored at �80 8C showed higher counts ( p < 0.01). In

the samples assessed after 6 months of storage at room

temperature, 11 of 43 target species (about 25%) were found in

lower counts when compared to control samples ( p < 0.01).

The other samples of this period (4 8C, �20 8C or �80 8C)

presented similar or slightly higher counts for all species.

When samples processed after 12 months of storage were

compared to control, 98% of target species (except for P.

intermedia) were found in lower counts at room temperature,

55% at 4 8C, 69% at �20 8C and 40% at �80 8C ( p < 0.05).

3.3. Microbial incidence

When target species were evaluated as a pool of microorgan-

isms, without discriminating between species, the statistic

test two-way ANOVA have found significant differences

between tested protocols ( p < 0.0001; Fig. 4). Bonferroni’s

post-tests showed that only samples stored after 12 months at

room temperature presented lower incidence when compared

to control samples ( p < 0.05). Table 2 summarizes the

individual microbial incidence of each tested species in the

proposed protocols. All the protocols showed significant

differences sought by two-way ANOVA followed Bonferroni’s

post-tests ( p < 0.0001). C. glabrata, C. krusei, C. tropicalis, V.

parvula, T. denticola, T. forsythia, S. sanguinis, P. melaninogenica, P.

intermedia, P. gingivalis, E. coli, C. rectus, C. gingivalis, B. fragilis, A.

actinomycetemcomitans serotype a and A. actinomycetemcomitans

serotype b were the species that not presented significant

differences in the proposed protocols ( p > 0.05).

4. Discussion

Since the introduction of molecular diagnosis methods

detecting specific sequences of DNA or RNA that may or

may not be associated with disease, many methodologies

have been developed, improved and employed in diverse

studies to evaluate several species of microorganisms colo-

nizing various habitats of the human body.12,13 In dentistry,

these methods have been also extensively applied in epide-

miological27 and clinical studies.19 Depending on the objec-

tives, molecular assays may be qualitative – enabling only the

Table 1 – Mean, standard deviations (T105 cells, WSD) and respective p-value (p) for each target species individually assessed between proposed protocols.

Species Immediate 2 weeks 4 weeks

Control T.A. 4 8C �20 8C �80 8C T.A. 4 8C �20 8C �80 8C

Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p

C. tropicalis 3.23 0.64 A 3.71 0.94 A 2.97 0.62 A 3.62 0.65 A 2.86 0.60 A 3.61 0.75 A 3.55 0.56 A 1.50 0.44 B* 5.09 0.50 C**

C. krusei 3.07 0.58 A 3.47 0.99 A 2.73 0.42 A 3.32 0.48 A 2.72 0.60 A 3.32 0.62 A 3.31 0.51 A 1.51 0.55 B* 4.81 0.92 C**

C. glabrata 3.40 0.48 A 3.50 0.65 A 2.79 0.44 A 3.50 0.62 A 2.66 0.57 A 3.47 0.60 A 3.31 0.51 A 1.57 0.61 B* 5.20 0.82 C**

C. dubliniensis 3.00 0.52 A 3.21 0.85 A 2.74 0.53 A 3.53 0.90 A 2.56 0.52 A 3.20 0.73 A 3.25 0.79 A 1.57 0.17 B* 4.93 0.85 C***

C. albicans 3.15 0.54 A 3.13 0.63 A 2.66 0.49 A 3.17 0.40 A 2.95 0.59 A 3.34 0.53 A 3.12 0.52 A 1.54 0.10 B* 5.11 0.75 AV. parvula 3.51 0.46 A 3.24 0.77 A 2.72 0.54 A 3.45 0.49 A 2.80 0.64 A 3.59 0.65 A 3.14 0.69 A 1.57 0.12 B* 5.04 0.62 AT. denticola 3.06 0.47 A 3.24 0.62 A 2.36 0.36 A 3.16 0.41 A 2.83 0.47 A 3.54 0.63 A 3.18 0.66 A 1.52 0.10 B* 4.99 0.62 C**

T. forsythia 2.82 0.51 A 2.98 0.51 A 2.73 0.37 A 3.13 0.36 A 2.63 0.69 A 3.42 0.59 A 3.25 0.67 A 1.58 0.15 B** 4.93 0.55 C*

S. sobrinus 2.95 0.41 A 3.15 0.46 A 2.74 0.51 A 3.16 0.32 A 2.66 0.56 A 3.64 0.59 A 3.11 0.56 A 1.64 0.21 B** 4.95 0.50 C*

S. sanguinis 2.83 0.41 A 2.94 0.55 A 2.69 0.50 A 4.12 0.55 B*** 2.48 0.56 A 3.53 0.47 A 3.34 0.84 A 1.67 0.12 C** 4.86 0.62 B*

S. salivarius 2.94 0.49 A 3.27 0.55 A 3.02 0.43 A 3.35 0.33 A 2.66 0.45 A 3.53 0.71 A 3.27 0.84 A 1.65 0.16 A 4.93 0.64 B***

S. pasteuri 2.94 0.43 A 2.87 0.66 A 2.93 0.49 A 3.36 0.38 A 2.87 0.47 A 3.62 0.56 A 2.93 0.08 A 1.62 0.80 B** 5.25 0.86 C*

S. parasanguinis 3.00 0.36 A 2.82 0.40 A 2.84 0.58 A 3.03 0.41 A 2.37 0.55 A 3.58 0.64 A 3.45 0.91 A 1.57 0.54 B** 4.66 0.53 C**

S. oralis 3.01 0.66 A 3.12 0.51 A 2.97 0.72 A 3.87 1.01 A 2.80 0.55 A 3.28 0.74 A 2.91 0.60 A 2.05 0.37 A 4.92 0.76 B***

S. mutans 2.83 0.48 A 3.95 0.51 A 3.29 0.55 A 4.93 0.50 A 3.79 0.70 A 3.52 0.43 A 3.08 0.73 A 5.17 0.71 B* 5.18 0.70 B*

S. moorei 2.35 0.38 A 3.17 0.48 A 2.68 0.36 A 3.44 0.44 B*** 2.77 0.46 A 3.74 0.53 B*** 3.71 0.85 B** 2.34 0.51 A 4.75 0.49 B*

S. mitis 2.76 0.52 A 3.68 0.66 A 3.08 0.54 A 4.51 0.46 B*** 3.24 0.45 A 3.71 0.42 A 3.61 1.16 A 2.53 0.48 A 4.75 0.45 B*

S. gordonii 2.96 0.67 A 3.01 0.40 A 3.06 0.55 A 4.34 0.66 B*** 2.61 0.39 A 3.52 0.60 A 3.36 0.57 A 2.14 0.49 A 4.77 0.42 B***

S. constellatus 2.70 0.42 A 3.01 0.50 A 2.75 0.52 A 3.43 0.39 A 2.93 0.51 A 3.47 0.53 A 3.28 1.11 A 1.82 0.20 A 4.69 0.48 B*

S. aureus 2.49 0.47 A 3.09 0.92 A 2.86 0.46 A 3.51 0.53 A 2.83 0.45 A 3.62 0.78 A 3.36 0.68 A 1.98 0.43 A 4.64 0.48 B*

P. putida 2.69 0.45 A 2.63 0.44 A 2.45 0.35 A 3.21 0.60 A 2.42 0.40 A 3.39 0.48 A 3.15 0.83 A 1.65 0.18 B*** 4.44 0.63 C**

P. nigrescens 2.49 0.49 A 2.42 0.49 A 2.40 0.42 A 3.27 0.53 A 2.31 0.39 A 3.26 0.45 A 3.25 1.04 A 1.70 0.18 A 4.48 0.88 B*

P. micra 2.61 0.67 A 2.69 0.43 A 2.73 0.49 A 3.04 0.58 A 2.46 0.64 A 3.11 0.56 A 3.10 1.03 A 1.63 0.84 B*** 4.32 0.65 C*

P. melaninogenica 4.51 0.84 A 3.97 0.71 A 4.45 0.58 A 5.08 0.67 A 4.02 0.49 A 4.11 0.63 A 3.90 0.72 A 2.04 0.30 B* 4.39 0.64 AP. intermedia 2.33 0.53 A 2.57 0.42 A 2.54 0.51 A 2.87 0.50 A 2.22 0.45 A 3.11 0.53 A 3.02 0.76 A 1.58 0.18 A 4.38 0.48 B*

P. gingivalis 2.31 0.50 A 2.59 0.49 A 2.35 0.48 A 2.91 0.52 A 2.51 0.93 A 3.26 0.47 B*** 2.96 0.73 A 1.58 0.29 A 4.32 0.42 B*

P. endodontalis 2.57 0.60 A 2.58 0.52 A 2.66 0.52 A 3.10 0.49 A 2.30 0.40 A 3.19 0.58 A 2.96 0.69 A 1.60 0.68 B*** 4.51 0.69 C*

P. anaerobios 2.80 1.13 A 2.80 0.46 A 2.52 0.48 A 3.23 0.58 A 2.35 0.54 A 3.01 0.57 A 2.75 0.47 A 1.93 0.46 A 4.41 0.64 B**

P. aeruginosa 2.43 0.56 A 2.60 0.51 A 2.52 0.45 A 2.78 0.39 A 2.49 0.50 A 3.22 0.62 A 2.83 0.60 A 1.72 0.42 A 4.43 0.46 B*

N. mucosa 2.24 0.34 A 2.38 0.56 A 2.50 0.49 A 2.94 0.53 A 2.48 0.56 A 3.29 0.56 B*** 3.06 0.75 A 1.64 0.23 A 4.40 0.55 B*

M. salivarium 2.96 0.73 A 2.75 0.48 A 2.74 0.51 A 3.38 0.48 A 3.01 0.50 A 3.42 0.78 A 3.23 0.80 A 1.68 0.80 B** 5.22 0.83 C**

L. casei 2.65 0.50 A 2.53 0.31 A 2.55 0.45 A 3.17 0.52 A 2.66 0.39 A 3.00 0.76 A 3.07 0.74 A 1.62 0.37 B*** 4.73 0.85 C*

K. pneumoniae 2.62 0.65 A 2.66 0.50 A 2.51 0.41 A 3.16 0.49 A 2.70 0.46 A 3.32 0.70 A 3.02 0.70 A 1.62 0.17 A 4.49 0.61 B*

F. periodonticum 2.64 0.81 A 2.68 0.75 A 2.46 0.50 A 3.10 0.60 A 2.50 0.54 A 3.22 0.75 A 3.11 0.50 A 1.54 0.65 B*** 4.65 0.56 C*

F. nucleatum 2.77 0.57 A 2.81 0.57 A 2.86 0.43 A 3.53 0.50 A 2.96 0.45 A 3.40 0.61 A 3.28 0.53 A 1.84 0.29 A 4.58 0.54 B*

E. faecalis 2.50 0.39 A 2.59 0.55 A 2.63 0.42 A 3.16 0.49 A 2.66 0.54 A 3.27 0.76 A 3.19 0.58 A 1.59 0.15 A 4.73 0.57 B*

E. corrodens 2.55 0.50 A 2.67 0.46 A 3.06 68.00 A 3.06 0.57 A 2.74 0.41 A 3.79 0.69 B** 3.29 0.70 A 1.83 0.26 A 4.95 0.62 B*

E. coli 2.62 0.48 A 2.52 0.50 A 2.83 0.56 A 3.17 0.49 A 2.63 0.42 A 3.48 0.61 A 3.30 0.56 A 1.65 0.28 A 4.91 0.49 B*

C. rectus 2.51 0.41 A 2.81 0.50 A 2.65 0.39 A 3.10 0.54 A 2.56 0.46 A 3.54 0.60 A 3.03 0.48 A 1.61 0.29 A 4.92 0.68 B*

C. gingivalis 2.78 0.67 A 2.65 0.58 A 2.77 0.44 A 3.09 0.47 A 2.73 0.54 A 2.98 0.80 A 3.07 0.65 A 1.59 0.29 B* 4.64 0.71 C*

B. fragilis 3.10 0.51 A 2.72 0.70 A 2.81 0.56 A 3.38 0.58 A 2.79 0.54 A 3.11 0.67 A 3.19 0.59 A 1.56 0.88 B* 4.76 0.62 C*

Aa serotype b 2.91 0.51 A 2.98 0.69 A 3.28 0.64 A 3.39 0.59 A 3.39 0.54 A 3.39 0.56 A 3.62 0.61 A 1.71 0.12 A 5.05 0.67 B*

Aa serotype a 3.28 0.66 A 3.19 0.68 A 3.54 0.60 A 3.60 0.70 A 3.83 0.63 A 3.98 0.81 A 3.74 0.61 A 1.77 0.25 B* 4.93 0.48 C*

a r

c h

i v

e s

o

f o

r a

l b

i o

l o

g y

5

9 (

2 0

1 4

) 1

2 –

2 1

16

Table 1 (Continued )

Species 6 months 12 months

T.A. 4 8C �20 8C �80 8C T.A. 4 8C �20 8C �80 8C

Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p Mean �SD p

C. tropicalis 1.84 0.85 B* 2.84 0.84 A 2.78 0.73 A 2.63 0.91 A 1.38 1.45 B* 2.19 1.98 A 1.49 1.44 B** 2.23 0.66 B*** B < A < CC. krusei 2.09 0.76 A 3.80 1.05 A 3.52 1.12 A 3.86 1A78 A 0.18 0.27 B* 1.79 1.66 A 1.42 1.73 A 2.43 0.89 A B < A < CC. glabrata 2.18 0.42 B* 3.81 0.99 A 3.00 0.62 A 3.67 1.33 A 0.14 0.29 B* 1.64 1.80 B* 1.27 1.86 B* 2.16 0.89 B* B < A < CC. dubliniensis 1.91 0.58 B** 2.90 0.58 A 2.23 0.56 B*** 2.14 1.04 A 2.02 1.15 B*** 1.12 1.29 B* 1.68 2.04 B** 2.05 0.53 B** B < A < CC. albicans 1.94 0.51 B* 2.80 0.62 A 2.37 0.53 A 6.34 1.71 A 0.07 0.22 B* 0.90 0.92 B* 1.59 1.84 B** 1.86 0.61 B* B < AV. parvula 1.77 0.58 B* 3.89 1.05 A 3.28 1.08 A 3.80 1.35 A 0.08 0.32 B* 1.64 0.69 B* 2.20 1.62 B*** 1.86 1.32 B** B < AT. denticola 1.88 0.52 B** 2.87 0.70 A 2.69 0.82 A 3.80 1.35 A 0.99 0.88 B* 1.79 0.81 B* 1.87 1.84 B* 1.95 0.63 B** B < A < CT. forsythia 1.65 0.65 B*** 3.05 0.74 A 3.94 0.68 C*** 2.46 0.63 A 0.10 0.26 B* 0.69 0.54 B* 0.85 1.05 B* 1.23 1.08 B** B < A < CS. sobrinus 1.96 0.94 A 3.94 0.68 A 3.78 1.73 A 2.98 1.29 A 0.06 0.25 B* 1.27 0.55 B* 0.83 0.81 B* 1.97 0.47 A B < A < CS. sanguinis 1.69 0.61 C*** 3.32 0.73 A 2.84 0.88 A 2.65 1.35 A 0.05 0.17 C* 1.92 0.60 A 1.08 0.90 C* 1.77 1.39 A C < A < BS. salivarius 1.91 0.87 A 6.97 1.63 B* 7.77 2.22 B*** 6.39 2.52 B*** 0.06 0.22 C* 0.67 0.56 C* 0.50 0.73 C* 1.65 0.65 A C < A < BS. pasteuri 1.86 0.54 A 3.52 0.90 A 3.14 0.97 A 2.52 1.40 A 0.05 0.22 B* 1.29 0.62 B** 0.65 0.84 B* 1.87 1.84 A B < A < CS. parasanguinis 2.05 0.73 A 5.04 0.68 C* 3.88 1.27 A 3.54 1.39 A 0.04 0.15 B* 3.19 0.89 A 1.78 1.30 A 0.85 1.05 B* B < A < CS. oralis 1.70 0.44 C*** 4.12 0.76 A 3.83 1.66 A 2.68 1.72 A 0.00 0.02 C* 1.82 0.70 A 1.57 0.93 C* 1.88 0.52 C** C < A < BS. mutans 1.53 0.60 A 4.20 0.83 B*** 4.32 0.95 B*** 5.48 0.98 B*** 0.02 0.13 C** 1.98 1.11 A 1.68 0.84 A 1.94 0.19 A C < A < BS. moorei 1.52 0.54 A 3.81 0.66 B* 3.34 1.47 A 4.79 2.25 B*** 0.00 0.02 C* 0.87 0.48 A 0.81 0.75 A 1.59 1.84 A C < A < BS. mitis 1.43 0.71 A 5.52 1.32 B* 5.06 1.91 B*** 4.12 1.71 A 0.02 0.12 C* 2.23 0.66 A 2.29 0.78 A 2.20 1.62 A C < A < BS. gordonii 1.48 0.75 A 5.14 1.54 B*** 4.61 2.31 A 4.35 1.91 A 0.03 0.15 C* 1.99 0.98 A 0.47 0.62 C* 1.20 1.53 A C < A < BS. constellatus 1.44 0.57 A 2.91 0.88 A 2.71 0.77 A 1.73 1.12 A 0.24 0.94 C* 0.47 0.62 C* 0.21 0.51 C* 1.22 1.62 C*** C < A < BS. aureus 1.55 0.68 A 5.14 2.22 B* 4.35 1.37 B*** 3.59 1.77 A 0.00 0.00 C* 1.22 1.13 A 0.72 0.85 C*** 1.01 1.73 A C < A < BP. putida 1.57 0.61 A 3.04 0.80 A 2.73 0.84 A 1.57 1.12 A 0.00 0.00 B* 0.37 0.70 B* 0.24 0.36 B* 0.63 0.70 B* B < A < CP. nigrescens 1.73 0.52 A 3.87 0.85 B*** 3.07 1.45 A 3.03 1.37 A 0.00 0.01 C* 0.47 0.67 C* 0.38 0.45 C* 0.83 0.81 C** C < A < BP. micra 1.56 0.55 A 3.02 0.89 A 2.52 0.66 A 1.64 1.02 A 0.04 0.14 B* 0.76 0.96 B* 1.99 1.13 B* 1.07 1.67 B* B < AP. melaninogenica 1.76 0.91 B* 4.88 0.89 A 4.45 0.66 A 5.13 1.45 A 1.61 1.26 B* 3.60 0.85 A 6.04 1.82 A 1.57 1.60 B* B < AP. intermedia 1.50 0.46 A 3.54 0.84 B** 3.33 1.37 A 3.80 0.98 B* 3.32 1.65 A 4.24 1.03 B* 4.44 2.08 B* 1.08 0.90 A A < BP. gingivalis 1.42 0.69 A 3.02 0.49 A 3.48 0.85 B*** 2.65 0.60 A 2.33 0.53 C* 1.40 1.11 A 1.75 1.89 A 2.26 1.87 A C < A < BP. endodontalis 1.55 0.54 A 2.91 0.67 A 2.95 0.94 A 2.20 1.04 A 0.01 0.04 B* 0.21 0.46 B* 0.58 1.09 B* 2.20 1.62 A B < A < CP. anaerobios 1.93 1.05 A 3.96 0.68 B*** 3.24 1.17 A 3.65 1.70 A 0.01 0.02 C* 0.15 0.39 C* 0.53 1.00 C* 1.38 1.45 A C < A < BP. aeruginosa 1.83 0.71 A 3.19 0.68 A 2.99 0.98 A 1.86 0.99 A 0.02 0.12 C* 0.24 0.48 C* 0.56 1.34 C** 0.86 1.47 A C < A < BN. mucosa 1.96 0.70 A 3.21 0.79 A 2.71 0.76 A 1.34 0.64 A 0.00 0.05 C* 0.22 0.43 C* 0.85 1.48 C*** 1.23 1.04 A C < A < BM. salivarium 1.86 0.61 A 2.93 0.77 A 2.97 0.57 A 1.47 0.73 B* 0.07 0.22 B* 0.89 0.59 B* 0.75 0.95 B* 2.23 1.09 A B < A < CL. casei 1.97 0.77 A 3.21 0.51 A 2.86 0.80 A 1.77 0.66 B*** 0.04 0.23 B* 0.72 0.47 B* 0.47 0.71 B* 3.32 1.65 A B < A < CK. pneumoniae 2.00 0.74 A 4.10 0.76 B** 3.61 0.88 A 1.94 0.97 A 0.00 0.00 C* 0.90 0.59 C* 0.63 0.74 C* 2.23 0.66 A C < A < BF. periodonticum 1.97 0.47 A 3.12 0.72 A 3.02 0.85 A 2.04 1.04 A 0.00 0.00 B* 0.65 0.55 B* 0.43 0.80 B* 1.73 0.88 A B < A < CF. nucleatum 2.05 0.53 A 3.28 0.61 A 3.74 0.86 A 2.55 1.46 A 0.01 0.06 C* 1.09 0.88 C** 1.22 1.62 C*** 1.80 0.68 A C < A < BE. faecalis 1.63 0.71 A 2.67 0.74 A 2.85 0.47 A 1.62 1.04 A 0.03 0.16 C* 1.59 1.04 A 1.57 1.60 A 2.05 0.53 A A < B < CE. corrodens 1.96 0.66 A 3.48 0.79 A 2.64 0.53 A 2.25 1.38 A 0.11 0.38 C* 2.06 1.45 A 1.55 1.63 A 1.86 0.61 A C < A < BE. coli 1.95 0.76 A 3.27 0.69 A 3.59 0.77 A 2.88 1.53 A 0.15 0.33 C* 1.84 1.35 A 1.93 2.00 A 1.12 1.03 C*** C < A < BC. rectus 1.52 0.85 A 2.52 0.47 A 3.27 0.97 A 2.30 1.79 A 4.45 1.61 B* 3.37 1.53 A 4.61 1.84 B* 1.95 0.63 A A < BC. gingivalis 2.27 0.62 A 3.47 0.35 A 3.23 0.78 A 2.82 0.17 A 0.99 0.85 B* 2.55 1.26 A 2.95 2.11 A 1.71 0.94 B** B < A < CB. fragilis 2.23 0.66 A 3.42 0.54 A 3.20 0.84 A 3.74 2.37 A 4.79 1.13 C** 3.88 1.20 A 4.97 2.70 A 1.97 0.47 B** B < A < CAa serotype b 1.95 0.63 A 3.48 0.45 A 3.15 0.76 A 3.88 2.17 A 4.19 1.12 B*** 5.86 0.84 B* 7.08 2.06 B* 1.24 0.95 A A < BAa serotype a 1.92 0.61 B** 3.89 0.72 A 3.29 0.66 A 3.89 2.14 A 1.57 1.38 B*** 3.59 1.59 A 3.41 1.85 A 1.65 0.65 B** B < A < C

Different letters, in the horizontal, mean significant differences between proposed protocols sought by Friedman test followed by Bonferroni’s post-test.

Aa: Aggregatibacter actinomycetemcomitans.* p < 0.001.** p < 0.01.*** p < 0.05.

a r

c h

i v

e s

o

f o

r a

l b

i o

l o

g y

5

9 (

2 0

1 4

) 1

2 –

2 1

1

7

Table 2 – Mean percentages (%) of individual incidence of target species in the proposed protocols.

Immediate 2 weeks 4 weeks 6 months 12 monthsa

Control RT 4 8C �20 8C �80 8C RT 4 8C �20 8C �80 8C RT 4 8C �20 8C �80 8C RT 4 8C �20 8C �80 8C p value

C. tropicalis 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 85.71 89.28 75.00 100.00 >0.05C. krusei 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 46.42 82.14 53.57 100.00 >0.05C. glabrata 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 25.00 92.85 64.28 96.42 >0.05C. dubliniensis* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 14.28 71.42 64.28 100.00 <0.05C. albicans* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 14.28 75.00 71.42 100.00 <0.05V. parvula 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 21.42 100.00 92.85 82.14 >0.05T. denticola 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 100.00 85.71 100.00 >0.05T. forsythia 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 21.42 85.71 67.85 60.71 >0.05S. sobrinus*** 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 428.00 100.00 85.71 100.00 <0.001S. sanguinis 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 48.00 100.00 89.28 78.57 >0.05S. salivarius* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 10.71 96.42 50.00 100.00 <0.05S. pasteuri* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 7.14 100.00 75.00 85.71 <0.05S. parasanguinis* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 14.28 100.00 100.00 67.85 <0.05S. oralis* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 7.14 100.00 89.28 100.00 <0.05S. mutans* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 100.00 100.00 100.00 7.14 96.42 96.42 78.57 <0.05S. moorei* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 7.14 96.42 85.71 71.42 <0.05S. mitis* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 100.00 100.00 100.00 7.14 100.00 96.42 92.85 <0.05S. gordonii* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 10.71 100.00 89.28 67.85 <0.05S. constellatus* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 10.71 64.28 25.00 78.57 <0.05S. aureus** 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 0.00 89.28 78.57 57.14 <0.01P. putida** 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 0.00 53.57 46.42 89.28 <0.01P. nigrescens* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.57 64.28 64.28 85.71 <0.05P. micra* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 10.71 85.71 96.42 64.28 <0.05P. melaninogenica 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 100.00 100.00 100.00 85.71 100.00 100.00 92.85 > 0.05P. intermedia 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 100.00 100.00 82.14 >0.05P. gingivalis 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 25.00 92.85 82.14 82.14 >0.05P. endodontalis* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 7.14 28.57 39.28 92.85 <0.05P. anaerobios* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.57 25.00 42.85 85.71 <0.05P. aeruginosa* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.57 35.71 42.85 64.28 <0.05N. mucosa* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.57 42.85 50.00 82.14 <0.05M. salivarium* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 10.71 92.85 67.85 100.00 <0.05L. casei* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.57 92.85 50.00 96.42 <0.05K. pneumoniae** 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 0.00 96.42 75.00 100.00 <0.01F. periodonticum** 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 0.00 82.14 46.42 82.14 <0.01F. nucleatum* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 3.57 96.42 78.57 96.42 <0.05E. faecalis* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 96.42 100.00 100.00 100.00 7.14 100.00 92.85 100.00 <0.05E. corrodens* 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 10.71 96.42 78.57 100.00 <0.05E. coli 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 35.71 82.14 100.00 64.28 >0.05C. rectus 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 89.28 100.00 100.00 100.00 100.00 100.00 100.00 100.00 >0.05C. gingivalis 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 85.71 100.00 96.42 82.14 > 0.05B. fragilis 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 >0.05Aa serotype b 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 71.42 >0.05Aa serotype a 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 89.28 100.00 100.00 100.00 >0.05

RT: room temperature. Aa: Aggregatibacter actinomycetemcomitans.a Differences between protocols sought by two-way ANOVA followed by Bonferroni’s post-test ( p < 0.0001).* p < 0.05.** p < 0.01.*** p < 0.001.

a r

c h

i v

e s

o

f o

r a

l b

i o

l o

g y

5

9 (

2 0

1 4

) 1

2 –

2 1

18

Fig. 4 – Mean of total incidence (%) of microbial species

found in the tested protocols (*significant differences

detected by two-way ANOVA followed by Bonferroni’s

post-test; p < 0.05).

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 1 19

identification of microorganisms (such as PCR followed by

pyrosequensing), semi-quantitative (such as Checkerboard

DNA–DNA hybridization) or quantitative (such as real time

PCR), which enable both detection and quantification of

microbial species. Each one has its own feasibility and

characteristics. Considering the relevance of the preservation

of the genetic material in all of these methodologies and the

lack of information on DNA stability after storage, we have

investigated, in this controlled in vivo study, the impact of

temperature and time storage in the detection of microorgan-

isms from clinical oral samples by using Checkerboard DNA–

DNA hybridization method.

The hypothesis stated concerning that temperature and

time of storage have influence in the final detection and

quantification of target species was proved by findings of this

investigation. After testing all the proposed protocols using

the samples processed immediately after harvesting as

standard of comparison, overall, we could find that samples

stored up to 6 months either at room temperature, 4 8C or

frozen at �20 8C and �80 8C presented similar total microbial

counts when compared to control (immediate processing).

Contrary, all the long-term stored samples (12 months)

showed a striking reduction in the total microbial counts,

mainly those stored at room temperature. The impact of factor

‘time’ or ‘temperature’ in reducing the total microbial counts

was proved when isolated in the statistical analysis. Regarding

the individual microbial counts, most of species (about 91%)

did not show significant differences when comparing samples

processed after 2 weeks of storage in all the proposed

temperatures with control. Similar to our findings for total

microbial counts, 98% of target species assessed after 12

months of storage were found in reduced counts. The

microbial incidence was also significantly reduced in samples

stored during 12 months in all temperatures.

Our findings suggest that the period of time and tempera-

ture in which samples remain stored have an important

impact on the final results of hybridization signals detected by

Checkerboard DNA–DNA hybridization. Similar data were

described by our group in a preliminary study investigating the

impact of time on oral samples stored at �20 8C.28 Similar to

findings observed in the present investigation, in that study,

samples stored during 12 or 24 months showed significant

reduced bacterial counts when compared to samples pro-

cessed immediately after harvesting. In the present study,

cold temperatures did not show to influence samples stored

up to 2 weeks. In contrast, they have proved to be relevant in

preserving the genetic material from samples stored during 4

weeks and 6 months. According to our findings, Ivanova and

Kuzmina29 have reported that temperature is a crucial factor

to obtain high quality DNA in PCR reactions after long-term

samples storage. These authors also observed best results in

long-term storage under cold temperatures. Typically, cold

temperatures are required to long-term success storage.30,31

Although our results and several other similar studies have

reported that cold storage may prevent contamination and

favour DNA stability in long-term, we have to consider it with

caution since many studies have related significant DNA loss

due to repeated freeze-thawing, evaporation and denaturation

of frozen samples.32–34 Additional factors that may compro-

mise DNA integrity must be consider, including limited DNA

quantity, presence of nucleases, other chemical agents and, in

some cases, unfavourable transport conditions.35

A rationale for the higher counts reported in some

protocols, when compared to control group, may be related

to the differences in the DNA amount present in each aliquot

prepared for the assessment, even we had tried to standardize

all the aliquots vortexing the tube containing saliva and

supragingival biofilm collected from the participants before

starting to aliquot samples. These data did not invalidate our

findings since values observed were similar and comparable to

control standards.

We can find only few studies proposing alternatives in

addition to different temperatures to avoid DNA damage and

degradation during storage, including different media solu-

tions and dry-down approaches. Some authors have shown

that DNA samples may remain stable for long periods of time

(up to 1 year), but some of them have experienced some degree

of sample instability.36,37 However, differently from Checker-

board DNA–DNA hybridization analysis, these studies have

evaluated samples with genetic material extracted before

storage. The findings observed in this investigation may be of

clinical significance to help studies of oral samples requiring

optimized genetic material storage. In addition, data may be

useful in methodologies in which DNA/RNA extraction is not

needed prior to analysis and, especially, in cases where

immediate transport under controlled conditions or immedi-

ate laboratorial processing is not possible. Additional testing

on a wider range of samples stored prior to DNA extraction is

recommended to establish a sound basis for storage protocols

of oral samples.

The results must be interpreted according to the method-

ology applied and cannot be generalized. The microbial counts

found in the tested groups may be the consequence of various

effects. The limit of detection in this methodology is 104

microbial cells. It means that counts in the 1–1000 range could

have been present in the patients but could not be detected by

the method. Calibration of the probe concentration to increase

sensitivity could have prevented these failures to occur. In

addition, an increase of cross-reactions may arise when the

probe concentration is increased. Other molecular methods

more specific, such as real-time PCR, may be applied to

overcome these limitations.

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 120

Within the limitations of this study, we can conclude that

temperature and time of oral samples storage have relevant

impact in the detection and quantification of bacterial and

fungal species by Checkerboard DNA–DNA hybridization

method. Long-term stored samples (12 months) presented

a significant reduction in the total and individual microbial

counts. Cold temperature showed to be effective in preserv-

ing DNA of samples stored during 6 months. According to the

protocols tested in this investigation, samples should be

processed immediately after collection or up to 6 months if

conserved at cold temperatures to avoid false-negative

results.

Funding

This study was supported by grants from Fundacao de Amparo

a Pesquisa do Estado de Sao Paulo (FAPESP). Processes n8 2010/

17807-6 and 2011/10008-3.

Competing interests

The authors declare that they have no conflict of interest.

Ethical approval

The study was approved by the local ethics committee (Ethical

Committee of the Faculty of Dentistry of Ribeirao Preto) and all

the experiments were undertaken with the understanding and

written consent of each subject according to the ethical

principles (Process No.: 2010.1.1354.58.4).

r e f e r e n c e s

1. Haffajee AD, Roberts C, Murray L, Veiga N, Martin L, TelesRP, et al. Effect of herbal, essential oil, and chlorhexidinemouthrinses on the composition of the subgingivalmicrobiota and clinical periodontal parameters. J Clin Dent2009;20(7):211–7.

2. Goncalves LS, Souto R, Colombo AP. Detection of Helicobacterpylori, Enterococcus faecalis, and Pseudomonas aeruginosa in thesubgingival biofilm of HIV-infected subjects undergoingHAART with periodontitis. Eur J Clin Microbiol Infect Dis2009;28(11):1335–42.

3. Miani PK, do Nascimento C, Sato S, Filho AV, da Fonseca MJ,Pedrazzi V. In vivo evaluation of a metronidazole-containinggel for the adjuvant treatment of chronic periodontitis:preliminary results. Eur J Clin Microbiol Infect Dis 2012;31(July(7)):1611–8.

4. Cortelli JR, Fernandes CB, Costa FO, Cortelli SC, Kajiya M,Howell SC, et al. Detection of periodontal pathogens innewborns and children with mixed dentition. Eur J ClinMicrobiol Infect Dis 2012;31(6):1041–50.

5. Finoti LS, Anovazzi G, Pigossi SC, Corbi SC, Teixeira SR,Braido GV, et al. Periodontopathogens levels and clinicalresponse to periodontal therapy in individuals with theinterleukin-4 haplotype associated with susceptibility tochronic periodontitis. Eur J Clin Microbiol Infect Dis 2013.http://dx.doi.org/10.1007/s10096-013-1903-z. [in press].

6. Domaracki BE, Evans A, Preston KE, Fraimow H, Venezia RA.Increased oxacillin activity associated with glycopeptides incoagulase-negative staphylococci. Eur J Clin Microbiol InfectDis 1998;17(3):143–50.

7. Barbosa RE, do Nascimento C, Issa JP, Watanabe E, Ito IY,Albuquerque Jr RF. Bacterial culture and DNA Checkerboardfor the detection of internal contamination in dentalimplants. J Prosthodont 2009;18(5):376–81.

8. Whelen AC, Persing DH. The role of nucleic acidamplification and detection in the clinical microbiologylaboratory. Annu Rev Microbiol 1996;50:349–73.

9. Pitt TL, Saunders NA. Molecular bacteriology: a diagnostictool for the millennium. J Clin Pathol 2000;53(1):71–5.

10. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L,Sargent M, et al. Diversity of the human intestinal microbialflora. Science 2005;308(5728):1635–8.

11. Dethlefsen L, McFall-Ngai M, Relman DA. An ecological andevolutionary perspective on human–microbe mutualismand disease. Nature 2007;449(7164):811–8.

12. Grice EA, Kong HH, Renaud G, Young AC, Bouffard GG,Blakesley RW, et al. A diversity profile of the human skinmicrobiota. Genome Res 2008;18(7):1043–50.

13. Vitali B, Biagi E, Brigidi P. Protocol for the use of PCR-denaturing gradient gel electrophoresis and quantitativePCR to determine vaginal microflora constitution andpathogens in bacterial vaginosis. Methods Mol Biol2012;903:177–93.

14. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE,Levin AE. ‘‘Checkerboard’’ DNA–DNA hybridization.Biotechniques 1994;17(4):788–92.

15. Aberg CH, Sjodin B, Lakio L, Pussinen PJ, Johansson A,Claesson R. Presence of Aggregatibacteractinomycetemcomitans in young individuals: a 16-yearclinical and microbiological follow-up study. J ClinPeriodontol 2009;36(10):815–22.

16. Mannaa A, Carlen A, Dahlen G, Lingstrom P. Intra-familialcomparison of supragingival dental plaque microflora usingthe checkerboard DNA-DNA hybridisation technique. ArchOral Biol 2012;57(12):1644–50.

17. Kim K, Heimisdottir K, Gebauer U, Persson GR. Clinical andmicrobiological findings at sites treated with orthodonticfixed appliances in adolescents. Am J Orthod DentofacialOrthop 2010;137(2):223–8.

18. do Nascimento C, Miani PK, Pedrazzi V, Goncalves RB,Ribeiro RF, Faria AC, et al. Leakage of saliva through theimplant–abutment interface: in vitro evaluation of threedifferent implant connections under unloaded and loadedconditions. Int J Oral Maxillofac Implants 2012;27(3):551–60.

19. Krohn-Dale I, Bøe OE, Enersen M, Leknes KN. Er:YAG laser inthe treatment of periodontal sites with recurring chronicinflammation: a 12-month randomized, controlled clinicaltrial. J Clin Periodontol 2012;39(8):745–52.

20. Endo MS, Martinho FC, Zaia AA, Ferraz CC, Almeida JF,Gomes BP. Quantification of cultivable bacteria andendotoxin in post-treatment apical periodontitis before andafter chemo-mechanical preparation. Eur J Clin MicrobiolInfect Dis 2012;31(10):2575–83.

21. do Nascimento C, Monesi N, Ito IY, Issa JP, Albuquerque JrRF. Bacterial diversity of periodontal and implant-relatedsites detected by the DNA Checkerboard method. Eur J ClinMicrobiol Infect Dis 2011;30(12):1607–13.

22. do Nascimento C, de Albuquerque Jr RF, Issa JP, Ito IY,Lovato da Silva CH, de Freitas Oliveira Paranhos H, et al. Useof the DNA Checkerboard hybridization method fordetection and quantitation of Candida species in oralmicrobiota. Can J Microbiol 2009;55(5):622–6.

23. Katsoulis J, Lang NP, Persson GR. Proportional distribution ofthe red complex and its individual pathogens after sample

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 1 2 – 2 1 21

storage using the checkerboard DNA–DNA hybridizationtechnique. J Clin Periodontol 2005;32(6):628–33.

24. Kruszewska H, Misicka A, Chmielowiec U. Biodegradation ofDNA and nucleotides to nucleosides and free bases. Farmaco2004;59(1):13–20.

25. Socransky SS, Haffajee AD, Smith C, Martin L, Haffajee JA,Uzel NG, et al. Use of checkerboard DNA–DNA hybridizationto study complex microbial ecosystems. Oral MicrobiolImmunol 2004;19(6):352–62.

26. do Nascimento C, de Albuquerque Jr RF, Monesi N,Candido-Silva JA. Alternative method for direct DNAprobe labeling and detection using the checkerboardhybridization format. J Clin Microbiol 2010;48(8):3039–40.

27. Vettore MV, Leao AT, Leal Mdo C, Feres M, deFigueiredo LC, Sheiham A. Periodontal bacterial load: aproposed new epidemiological method for periodontaldisease assessment. J Contemp Dent Pract2010;11(1):E049–56.

28. do Nascimento C, Muller K, Sato S, Albuquerque Jr RF. Effectof sample storage time on detection of hybridization signalsin Checkerboard DNA–DNA hybridization. Can J Microbiol2012;58(4):502–6.

29. Ivanova NV, Kuzmina ML. Protocols for dry DNA storage andshipment at room temperature. Mol Ecol Resour2013;13(5):890–8.

30. Smith LM, Burgoyne LA. Collecting, archiving andprocessing DNA from wildlife samples using FTA databasingpaper. BMC Ecol 2004;4:4.

31. Molina Md. Armstrong TK, Zhang Y, Patel MM, Lentz YK,Anchordoquy TJ. The stability of lyophilized lipid/DNAcomplexes during prolonged storage. J Pharm Sci2004;93(9):2259–73.

32. Shikama K. Effect of freezing and thawing on the stability ofdouble helix of DNA. Nature 1965;207(996):529–30.

33. Davis DL, O’Brien EP, Bentzley CM. Analysis of thedegradation of oligonucleotide strands during the freezing/thawing processes using MALDI-MS. Anal Chem2000;72(20):5092–6.

34. Smith S, Morin PA. Optimal storage conditions for highlydilute DNA samples: a role for trehalose as a preservingagent. J Forensic Sci 2005;50(5):1101–8.

35. Bonnet J, Colotte M, Coudy D, Couallier V, Portier J, Morin B,et al. Chain and conformation stability of solid-state DNA:implications for room temperature storage. Nucleic Acids Res2010;38(5):1531–46.

36. Lee SB, Crouse CA, Kline MC. Optimizing storage andhandling of DNA extracts. Forensic Sci Rev 2010;22:131–44.

37. DNA Bank Network, Long Term DNA Storage WorkshopProceedings, http://www.dnabank-network.org/publications/Workshop Long-term DNA Storage Summaryand Abstracts.pdf [accessed 15.06.13].