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A simple device for sampling apical sub-gingival plaque
Journal o f Microbiological Methods 4 (1986) 293- 297 Elsevier
293
JMM 00145
A simple device for sampling apical sub-gingival plaque L.S. Tipler Department o f Dental Sciences, University o f Liverpool, PO Box 147, Liverpool L69 3BX (England) (Received 20 August 1985) (Revised version received 10 December 1985) (Accepted 16 December 1985)
Summary The construction of a device useful for sampling the anaerobic apical region of periodontal pockets is described. It is re-useable, easily manipulated and can be flushed with oxygen-free gas during sampling or used as a free structure.
Key words:
Sampling device - Strict anaerobes - Subgingival plaque
Introduction There have been many studies implicating bacteria in the etiology of periodontal disease [1-4]. In advanced periodontal lesions where there is detachment of the periodontal membrane, alveolar bone loss and pocket formation, a niche favouring anaerobic bacteria is produced. Since these bacteria are at the site of tissue destruction, efforts have been made to isolate and study them in detail. This involves sampling from the apical region of pockets and can be achieved using an oxygen-free gas flushed syringe developed by Newman et al. [5]. This article describes the construction of a similar device which is smaller and therefore more manoeuverable.
Construction of sampling device See Figs. 1 and 2 for the component parts mentioned below. 'Butterfly-21' infusion set needles (Abbott Laboratories Ltd., Kent) formed the foundation of the device. One wing was removed to reduce its overall width and the needle was cut and polished to produce a blunt needle 5 mm in length. (a) Non-flushed modification: the clear plastic tubing was cut approximately 2 cm from the 'Butterfly' sleeving as in Fig. 1. (b) Flushed modification (not illustrated): a small hole was made in the side of
0167-7012/86/$3.50 © 1986 Elsevier Science Publishers BN. (Biomedical Division)
294
SAMPLING
DEVICE
ASSEMBLED a
d,, e,,, OPEN
..... \
g
Fig. 1. Illustration of the 'assembled' and 'open' device. (a) Alumiuium foil cap. (b) 'Butterfly' sleeving. (c) Clear plastic tubing. (d) Calcium alginate covered broach tip at maximum protrusion of 5 mm. (e) Blunt needle 5 mm in length. (f) Shoulders of broach prevent further travel. (g) Reinforced aluminium foil extension of cap. the clear plastic tubing approximately 2 cm from the 'Butterfly' sleeving. A suitable gas sterilising filter fits into the adaptor at the end of this tubing. The barbs of a medium Nerve Broach (Pulpadent, Ash Dental Supplies, Manchester) were lightly covered with calcium alginate fibres (Clagitex, Medical Alginates Ltd., Middlesex). The broach was then either inserted down the plastic tubing (non-flushed) or through the hole in the side of the tubing (flushed) and was then pushed along to the opening of the blunt needle. N.B. Shoulder position of broaches vary. Since these shoulders act as a brake against the needle end inside the broach (see Fig. 2), a size allowing 5 mm protrusion of the covered broach tip through the needle end was used. This, together with the 5 mm needle length, allows a pocket depth of up to 10 mm to be sampled. A small aluminium foil cap (Fig. 3) was fitted over the needle end of each device to protect the broach from contamination with more coronal sub-gingival bacteria during sampling. One side of the cap was extended and reinforced to aid its recovery after sampling. The assembled devices were packaged individually in sealed gas permeable plastic tubing and were sterilised using ethylene oxide. The efficiency of the sterilisation procedure was assessed using indicator spores of Bacillus stearothermophilus (Oxoid Ltd.) which were not viable after exposure to ethylene oxide alongside the packaged devices. The device cannot be autoclaved. Use of device
Sampling is achieved by introducing the assembled device (Fig. 1) into the
295
a
\
gl
4,,
Ira...
[ d Fig. 2. A schematic representation of the sampling device. Key as for Fig. 1.
periodontal pocket through the more coronal plaque, withdrawing it slightly and redirecting it laterally over and away from the cap. The broach is then gently pushed out o f the needle into the anaerobic apical region of the pocket and a sample taken. Figure 4 shows the relative size and position o f the device during sampling. After reprotecting the sample from contamination by more coronal plaque by withdrawing it back into the needle, the whole device is removed from the pocket. The broach tip
"-~Ist
fold
3rd fold 2nd fold
Reinforced Foil extension (15mm) t
t B
Fig. 3. Aluminium foil cap construction and dimensions (mm).
296
Fig. 4. Illustration of the relative size and position of the device during sampling. is then pushed out of the needle while in a steam of sterile oxygen-free nitrogen gas (B.O.C.) and snipped off into pre-reduced sterile transport fluid. The cap is recovered by gently pulling the reinforced extended side which remains outside the pocket throughout the procedure. The device has been used to obtain isolates from periodontal lesions anatomically similar to those sampled by Newman et al. [5]. Many of these isolates were obligate anaerobes [6], some of which have been included in subsequent studies to determine the role of anaerobic bacteria in the etiology of chronic periodontal disease in humans [7]. The device is re-usable and therefore economical as well as being small and easy to manage. By altering broach and needle lengths a variety of tools for sampling bacteria from enclosed, and therefore possibly anaerobic sites can be made.
297
Acknowledgements I w o u l d like t o t h a n k Dr. T . H . M e l v i l l e a n d Mr. K . S . L a s t f o r t h e i r s u p e r v i s i o n a n d a d v i c e d u r i n g t h e d e v e l o p m e n t o f t h e device, A l s o Mr. J. A b b o t t f o r his m e c h a n i c a l e x p e r t i s e a n d Mr. J. S. B a i l i e a n d Mr. J. H . H i b b a r d f o r t h e i l l u s t r a t i o n s .
References 1 Listgarten, M.A. and Hellden, L. (1978) Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. J. Clin. Periodontol. 5, 115- 132. 2 Slots, J. (1979) Subgingival microflora and periodontal disease. J. Clin. Periodontol. 6, 351 -382. 3 Van Palenstein Helderman, W.H. (1981) Microbial etiology of periodontal disease. J. Clin. Periodontol. 8, 261 - 280. 4 Moore, W. E. C., Holdeman, L. V., Smibert, R.M., et al. (1982) Bacteriology of severe periodontitis in young adult humans. Infect. lmmun. 38, 1137-1148. 5 Newman, M.G., Socransky, S.S., Savitt, E.D., Propas, D.A. and Crawford, A. (1976) Studies of the microbiology of periodontitis. J. Periodontol. 47, 373- 379. 6 Tipler, L.S. (1984) Studies of bacterial populations associated with the onset and progression of chronic periodontal disease in humans. Ph.D. Thesis, University of Liverpool, England. 7 Tipler, L.S. and Embery, G. (1985) Glycosaminoglycan-depolymerizing enzymes produced by anaerobic bacteria isolated from the human mouth. Archs. Oral Biol. 30, 391-396.
293
JMM 00145
A simple device for sampling apical sub-gingival plaque L.S. Tipler Department o f Dental Sciences, University o f Liverpool, PO Box 147, Liverpool L69 3BX (England) (Received 20 August 1985) (Revised version received 10 December 1985) (Accepted 16 December 1985)
Summary The construction of a device useful for sampling the anaerobic apical region of periodontal pockets is described. It is re-useable, easily manipulated and can be flushed with oxygen-free gas during sampling or used as a free structure.
Key words:
Sampling device - Strict anaerobes - Subgingival plaque
Introduction There have been many studies implicating bacteria in the etiology of periodontal disease [1-4]. In advanced periodontal lesions where there is detachment of the periodontal membrane, alveolar bone loss and pocket formation, a niche favouring anaerobic bacteria is produced. Since these bacteria are at the site of tissue destruction, efforts have been made to isolate and study them in detail. This involves sampling from the apical region of pockets and can be achieved using an oxygen-free gas flushed syringe developed by Newman et al. [5]. This article describes the construction of a similar device which is smaller and therefore more manoeuverable.
Construction of sampling device See Figs. 1 and 2 for the component parts mentioned below. 'Butterfly-21' infusion set needles (Abbott Laboratories Ltd., Kent) formed the foundation of the device. One wing was removed to reduce its overall width and the needle was cut and polished to produce a blunt needle 5 mm in length. (a) Non-flushed modification: the clear plastic tubing was cut approximately 2 cm from the 'Butterfly' sleeving as in Fig. 1. (b) Flushed modification (not illustrated): a small hole was made in the side of
0167-7012/86/$3.50 © 1986 Elsevier Science Publishers BN. (Biomedical Division)
294
SAMPLING
DEVICE
ASSEMBLED a
d,, e,,, OPEN
..... \
g
Fig. 1. Illustration of the 'assembled' and 'open' device. (a) Alumiuium foil cap. (b) 'Butterfly' sleeving. (c) Clear plastic tubing. (d) Calcium alginate covered broach tip at maximum protrusion of 5 mm. (e) Blunt needle 5 mm in length. (f) Shoulders of broach prevent further travel. (g) Reinforced aluminium foil extension of cap. the clear plastic tubing approximately 2 cm from the 'Butterfly' sleeving. A suitable gas sterilising filter fits into the adaptor at the end of this tubing. The barbs of a medium Nerve Broach (Pulpadent, Ash Dental Supplies, Manchester) were lightly covered with calcium alginate fibres (Clagitex, Medical Alginates Ltd., Middlesex). The broach was then either inserted down the plastic tubing (non-flushed) or through the hole in the side of the tubing (flushed) and was then pushed along to the opening of the blunt needle. N.B. Shoulder position of broaches vary. Since these shoulders act as a brake against the needle end inside the broach (see Fig. 2), a size allowing 5 mm protrusion of the covered broach tip through the needle end was used. This, together with the 5 mm needle length, allows a pocket depth of up to 10 mm to be sampled. A small aluminium foil cap (Fig. 3) was fitted over the needle end of each device to protect the broach from contamination with more coronal sub-gingival bacteria during sampling. One side of the cap was extended and reinforced to aid its recovery after sampling. The assembled devices were packaged individually in sealed gas permeable plastic tubing and were sterilised using ethylene oxide. The efficiency of the sterilisation procedure was assessed using indicator spores of Bacillus stearothermophilus (Oxoid Ltd.) which were not viable after exposure to ethylene oxide alongside the packaged devices. The device cannot be autoclaved. Use of device
Sampling is achieved by introducing the assembled device (Fig. 1) into the
295
a
\
gl
4,,
Ira...
[ d Fig. 2. A schematic representation of the sampling device. Key as for Fig. 1.
periodontal pocket through the more coronal plaque, withdrawing it slightly and redirecting it laterally over and away from the cap. The broach is then gently pushed out o f the needle into the anaerobic apical region of the pocket and a sample taken. Figure 4 shows the relative size and position o f the device during sampling. After reprotecting the sample from contamination by more coronal plaque by withdrawing it back into the needle, the whole device is removed from the pocket. The broach tip
"-~Ist
fold
3rd fold 2nd fold
Reinforced Foil extension (15mm) t
t B
Fig. 3. Aluminium foil cap construction and dimensions (mm).
296
Fig. 4. Illustration of the relative size and position of the device during sampling. is then pushed out of the needle while in a steam of sterile oxygen-free nitrogen gas (B.O.C.) and snipped off into pre-reduced sterile transport fluid. The cap is recovered by gently pulling the reinforced extended side which remains outside the pocket throughout the procedure. The device has been used to obtain isolates from periodontal lesions anatomically similar to those sampled by Newman et al. [5]. Many of these isolates were obligate anaerobes [6], some of which have been included in subsequent studies to determine the role of anaerobic bacteria in the etiology of chronic periodontal disease in humans [7]. The device is re-usable and therefore economical as well as being small and easy to manage. By altering broach and needle lengths a variety of tools for sampling bacteria from enclosed, and therefore possibly anaerobic sites can be made.
297
Acknowledgements I w o u l d like t o t h a n k Dr. T . H . M e l v i l l e a n d Mr. K . S . L a s t f o r t h e i r s u p e r v i s i o n a n d a d v i c e d u r i n g t h e d e v e l o p m e n t o f t h e device, A l s o Mr. J. A b b o t t f o r his m e c h a n i c a l e x p e r t i s e a n d Mr. J. S. B a i l i e a n d Mr. J. H . H i b b a r d f o r t h e i l l u s t r a t i o n s .
References 1 Listgarten, M.A. and Hellden, L. (1978) Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. J. Clin. Periodontol. 5, 115- 132. 2 Slots, J. (1979) Subgingival microflora and periodontal disease. J. Clin. Periodontol. 6, 351 -382. 3 Van Palenstein Helderman, W.H. (1981) Microbial etiology of periodontal disease. J. Clin. Periodontol. 8, 261 - 280. 4 Moore, W. E. C., Holdeman, L. V., Smibert, R.M., et al. (1982) Bacteriology of severe periodontitis in young adult humans. Infect. lmmun. 38, 1137-1148. 5 Newman, M.G., Socransky, S.S., Savitt, E.D., Propas, D.A. and Crawford, A. (1976) Studies of the microbiology of periodontitis. J. Periodontol. 47, 373- 379. 6 Tipler, L.S. (1984) Studies of bacterial populations associated with the onset and progression of chronic periodontal disease in humans. Ph.D. Thesis, University of Liverpool, England. 7 Tipler, L.S. and Embery, G. (1985) Glycosaminoglycan-depolymerizing enzymes produced by anaerobic bacteria isolated from the human mouth. Archs. Oral Biol. 30, 391-396.