This study evaluated salivary pH levels and the presence of red complex bacteria in healthy individuals, those with gingivitis, and those with chronic periodontitis. 60 subjects were divided into the three groups based on a clinical examination. Salivary pH was measured using a digital pH meter and red complex bacteria were assessed using the BANA test on subgingival plaque samples. Results found significantly lower salivary pH levels in subjects that tested positive for red complex bacteria by the BANA test. The study concluded that there is a positive correlation between BANA test results and pH, with more periodontal destruction associated with lower pH and positive BANA results.

1. EVALUATION OF RED COMPLEX ORGANISMS AND
SALIVARY pH IN HEALTH, GINGIVITIS AND CHRONIC
PERIODONTITIS –
A CLINICO-MICROBIOLOGICAL STUDY

2. Presented by: Dr Harshavardhan Patwal
Guided by: Dr. Nandini Manjunath
Prof & H.O.D
Dept of Periodontics
A. J. Institute of Dental Sciences, Mangalore

3. INTRODUCTION
• Periodontal diseases are multifactorial infections elicited by a
complex of bacterial species that interact with host tissues and cells
causing the release of broad array of inflammatory mediators which
lead to distruction of periodontal structures.
• The ecological factors provided by the environment of the oral
cavity are directly proportional to the species richness and species
biodiversity of the microorganisms that reside on the teeth.

4. INTRODUCTION
• The main ecological factors are pH, saliva, temperature and redox
reactions .
• The majority of the organisms prefer neutral pH levels (pH7).
Saliva acts as a buffer maintaining the pH in the oral cavity
between 6.75-7.25.

5. OBJECTIVES OF THE STUDY
:
1.To estimate the level of salivary ph in healthy, gingivitis,
chronic periodontitis patients.
2. To estimate the “Red Complex” organisms in healthy,
gingivitis, chronic periodontitis .

6. MATERIALS AND METHODS
• Study sample consist of 60 adult subjects (both male and female)
of 35-60 years of age.
• A brief case history was recorded and an informed consent was
taken for all the patients.
• Clinical examination was recorded and study subjects were
divided into 3 groups ( healthy, gingivitis, chronic periodontitis)
based on the objectives.

7. INCLUSION CRITERIA
• Patients with healthy periodontium was considered as a control
group.
• Subjects with 30% periodontal pockets with probing depth of
equal to or more than 5mm in each quadrant .
• Patients who have not received any antimicrobial therapy for the
last 6 months.

8. EXCLUSION CRITERIA:
• Inability to provide information or cooperate to dental examination
• Inability to accept periodontal treatment
• Patients diagnosed with diabetes mellitis, cardio-vascular or kidney
diseases or any nerve condition for which prophylactic antibiotic
treatment before the dental examination is necessary.
• Smokers and individuals who consume alcohol.
• Pregnant and lactating women , women taking oral contraceptives .
• Malignancy.

9. CLINICAL PARAMETERS:
• Complete periodontal examination was done using the
parameters such as plaque index (sillness and loe) and gingival
index (loe and sillness) probing pocket depth and clinical
attachment level.
• Red complex organisms are assessed using BANA test.
• Salivary ph was measured using a Digital pH meter (systronics
MK-6).

10. COLLECTION OF SALIVA
:
• Subjects were instructed not to eat or rinse within 60 minutes
prior to sample collection. Whole saliva was collected simply by
drooling into a sterilized vial with the forward tilted head or by
allowing the saliva to accumulate in the mouth and then
expectorate into a vial.
• The resulting saliva was stored in at -20°C until the
determinations are performed.

11. Unstimulated saliva is collected by tilting the head of the
patient downward and collecting it in the cup .

12. WORKING OF pH METER
 PRINCIPLE
• Measurement of voltage difference between 2 electrodes placed
in a solution .

13. PARTS OF pH METER
1. Calomal electrode :external reference electrode whose
electrical stimulation depends on test solution .
2. Glass electrode
3. Electronic meter

14. PARTS OF ELECTRONIC METER
1. Calibration key : to
enter the calibration
of known solution.
2. Confirmation key
To confirm the
calibraton value
3. Arrow keys :
manually select pH
of buffer .
4. MR – recall stored
value
5. Memory key : to
store in memory .

15. REFERENCE ELECTRODE WITH
GLASS ELECTRODE IS CALLED
COMBINATION ELECTRODE

16. CALIBRATION OF PH METER
:
• Step 1 – wash electrode with distilled water .
• Step 2- calibrate pH meter with pH 7.0 buffer
• Step 3 – when ready blinks on the screen press confirmation
key
• Step 4 – again rinse the electrode with distilled water
• Step 5 – place the electrode in pH of 4.0
• Step 6 – press calibration key
• Step 7 – check the pH of test solution .

17. CALIBRATING
SOLUTIONS

19. DETERMINATION OF “RED
COMPLEX” ORGANISMS :
• BANA test is a enzymatic chair side test. It is a modern chair
side para-clinical method designed to detect the presence of one
or more anaerobic bacteria ,commonly associated with
periodontal diseases.
• This test is very sensitive detecting small quantities of
pathogens, no meaningful diffrences could be found between
DNA probes. Immunological reagents and BANA test.

20. PRINCIPLE OF BANA TEST :
• Peptides of the 3 bacterial “Red Complex” species
(T.denticola,P.gingivalis,B.forsythus) can hydrolyse the peptide
analog N-benzoyl DL-Arginine- napthalamide. One of the
hydrolytic products of this reaction is B-naphthylamide, which
reacts with a reagent, which is imbedded in the upper strip of
the test, producing a permanent blue color .
• Blood and saliva do not interfere with the test .

21. DIRECTIONS OF USE :
• Anaerobic microorganisms associated with periodontal disease
are found in the subgingival plaque. To obtain specimens for
testing, sites should be cleared of supragingival plaque.
• A Gracey curette is used to obtain subgingival plaque specimens ,
which are placed on the lower matrix. Before taking another
specimen, wipe the curette on a clean piece of cotton or other
suitable wipe to prevent carry-over of plaque.
• The upper matrix is moistened with saline solution and the test is
folded so as the two matrices are coming in contact. It is
incubated for 5 minutes at 55 Celsius degrees temperature. If
BANA positive species are present when the test is opened, a
permanent blue coloration on the upper matrix is found . The
higher the concentration of bacterial species, the darker blue
coloration is present on the test. According to the result, the test
can be positive, weak positive, or negative .

22. BANA STRIP:

23. COLLECTION OF SUBGINGIVAL
PLAQUE :

24. BANA KIT

25. INCUBATING STRIP FOR 5
MINUTES UNTIL THE BEEP
SOUND .

26. COMPARASION OF TEST
RESULTS

27. STATISTICAL ANALYSIS
• Statistical analysis was carried out using chi square tests and
fishers exact test .

28. COMPARISON WITH pH AND BANA
Crosstab
BANA Total
NEGATIVE WEAKLY
POSITIVE
POSITIV
E
pH 6.26-7.25 Count 2 3 0 5
% within
BANA
66.7% 42.9% 0.0% 33.3%
6-6.25 Count 1 4 0 5
% within
BANA
33.3% 57.1% 0.0% 33.3%
5.5-5.9 Count 0 0 5 5
% within
BANA
0.0% 0.0% 100.0% 33.3%
Total Count 3 7 5 15
% within
BANA
100.0% 100.0% 100.0% 100.0%

29. HERE IT IS NOTED THAT SIGNIFICANTLY LOWER PH WHEN
BANA IS POSITIVE. ALL 5 CASES WHERE BANA WAS
POSITIVE THE PH WAS <5.9 P VALUE 0.001. THIS IS
SIGNIFICANT AT THE 5 % ERROR MARGIN.
Chi-Square Tests
Value P VALUE
Fisher's Exact Test 13.114 .001
N of Valid Cases 15

31. CONCLUSION :
• There is a positive correlation, both clinically and statistically,
between the BANA test results and the pH seeing the current
stage of periodontal destruction. The BANA test results are not
correlated with the degree of oral hygiene evaluated against the
plaque index, so the quality and not quantity of bacterial plaque
influence the test results.
• Among these possibilities, the microbial-enzymatic BANA test is a
quick, chair-side test with a very good sensibility, giving the
clinician details about the microbial composition of the subgingival
plaque and consequently about the clinical evolution of the
periodontal disease. BANA test is also offering therapeutic
orientation regarding the need for antimicrobial therapy.

32. REFERENCE :
1.Stanley C.holt & Jeffrey L.ebersole Porphyromonas gingivalis,Treponama
denticola, and Taneralla forsythia:the “red complex”,a prototype polybacterial
pathogenic consortium in periodontitis . perioontology 2000 2005,(38),72-122.
2.Marsh PD, Are dental diseases examples of ecologica; catastropies.
Microbiology2003 143(3) 279-294.
3.Marsh P.D , Devine DA How is the development of dental biofilms influenced
by the host. J.Clin.Periodontol 2011 ,38(11) 28-35.
4.Gracia .F. Hicks, M.J Maintaining the integrity of the Enamel surface the role
of dental biofilm, saliva and preventative agents in the enamel
demineralization and remineralization . J.A.D.A 2008, 139(2) 255-345..
5.Arnaud alves bezerra junior,Debora pollos,Jose roberto cortelli,clintia helena
coury saraceni,Celso Silva Queiroz.Evaluation of organic and inorganic
compounds in saliva of patients with chronic periodontal disease.
Rev.odonocienc2010.;25(3):234-238.
6.Suncica Travan,Fei li,Nisha J D’silva,Elizabeth H Slate and KeithL.kirkwood
Diffrential expression of mitogen activating protein kinase in periodontitis. J
Clin Periodontol 2013;40:757-764.

33. REFERENCES :
7.Mrinal K Bhattacharjee,Claiborne B. Childs, and Emdad Ali . Sensitivity of the
Periodontal Pathogen Aggregatibacter Actinomycetemcomitans at Mildly Acidic pH. J
Periodontol June 2011;(.82) .No.6.917-925.
8.Holt SC, Bramanti TE . Factors in virulence expression and their role in periodontal
disease pathogens. Crit Rev Oral Biol Med 1991:(2):177-281.
9.Kortsik C, ElmerA, Tamm I. Pleural effusion due to Histoplasma capsulatum and
idiopathic CD14 lymphocytopenia. Respiration 2003(70):118-122.
10.Fine DH, Furgang D, Gold Man D, saliva from subjects harbouring
actinomycetemcomitans kills streptococcus mutans in vitro . J periodontol
2007;(78):518-526.
11. Bretz W, Loesche W. Charecteristics of Trypsin like activity in subgingval plaque
samples, J. Dent. Res 1987;( 66):1668-1672.
12.Loesche,W.J , Bretz, W.A., Kerschensteiner, D.,Stoll, J.,Socransky, et al.Development
of diagnostic test for anaerobic periodontal infections based on plaque hydrolosis of
benzoyl-DL-arginine-napthylamide. J clin microbiology 1990(28),1551-1559.
13.Cristina Gabriela Puscasu, Anca Silvia Dumitriu, HoriaTraian Dumitriu. The
significance of BANA test in diagnosis of certain forms of periodontal disease.
OHDMBSC- 5-(.3)-september,2006.