JICDRO is a UGC approved journal (Journal no. 63927)

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Year : 2014  |  Volume : 6  |  Issue : 1  |  Page : 16-19

Recent advances in periodontal microbiology: An update on cultivation techniques

Department of Microbiology and Molecular Biology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India

Date of Web Publication18-Aug-2014

Correspondence Address:
Kishore G Bhat
Department of Microbiology and Molecular Biology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum - 590 010, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2231-0754.139087

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Microbial members of the subgingival plaque community play a major role in the initiation and progression of periodontal diseases. Majority of these bacteria are anaerobic in nature and several anaerobic systems have been used for their cultivation. Among them anaerobic jars are the most popular and are routinely used for the detection of periodontal pathogens from clinical samples. Despite best efforts, a significant portion of oral microbes have not yet been cultivated and several hypotheses have been put forth to explain this anomaly. This has led to renewed efforts to cultivate the oral bacteria so far identified only by their molecular signatures resulting in improvisation of existing culture techniques and devising novel methods of isolation. Several devices have been used on environmental samples successfully: One method called "minitrap" has been successfully adapted to oral cavity and has shown great promise in isolation of not yet cultivated oral bacterial species. These newer techniques are sure to shed more light on the role of microbes in the etiology of periodontal diseases.

Keywords: Anaerobes, culture, diffusion chamber, minitrap, not yet cultivated bacteria

How to cite this article:
Bhat KG. Recent advances in periodontal microbiology: An update on cultivation techniques . J Int Clin Dent Res Organ 2014;6:16-9

How to cite this URL:
Bhat KG. Recent advances in periodontal microbiology: An update on cultivation techniques . J Int Clin Dent Res Organ [serial online] 2014 [cited 2021 Jun 23];6:16-9. Available from: https://www.jicdro.org/text.asp?2014/6/1/16/139087

The human oral microbial flora is highly complex, comprising over 700 bacterial species among which nearly 40% remain uncultivated and are referred to as bacterial phylotypes. [1] This microbial community is predominantly anaerobic in nature and is responsible for the formation of a biofilm over the tooth surface in the form of subgingival and supragingival plaque. The members of the plaque community play a very important and decisive role in periodontal health and disease and have been grouped into several complexes. [2] Several bacteria especially those belonging to red and orange complexes are definitive pathogens involved in initiation and progression of periodontitis while some others are seen commonly in gingivitis and yet others are associated with periodontal health. [3] Many of these periodontal pathogens are also implicated in the etiology of several systemic diseases such as atherosclerosis, cardiovascular disorders, cerebro-vascular accidents and of late obesity. [4],[5]

Despite extensive research, our knowledge of the microbiota associated with various periodontal infections is far from complete. [6] This has naturally led to investigations into identifying oral bacteria other than the one which have already been studied, resulting in introduction of newer techniques and also improvements in the existing ones to study these microbes.

The techniques that are being used currently to study periodontal pathogens include microscopy, culture, immunological tests and molecular methods. [7] Among them culture holds the center stage and is considered to be a "gold standard" against which all the newer techniques are compared to establish their efficacy. Since majority of periodontal pathogens are anaerobic in nature, anaerobic culture techniques have been the major tool of oral microbiologists to study these microbes. There are several systems that can be used to create an anaerobic atmosphere for cultivation of oral microbes. These include biobags, use of pre-reduced anaerobically sterilized (PRAS) media, anaerobic chambers and anaerobic jars that are used with a combination of basal and selective media for the isolation of periodontal pathogens. [8] Each one of these systems has its own advantages and limitations. Among the aforementioned systems, anaerobic chambers and jars are most commonly used. However, anaerobic chamber is expensive to set up and maintain and are more useful for large scale studies of obligate (strict) anaerobes with a high sample load. On the other hand, jars with chemically generated (gaspak) anaerobic systems are most popular and are widely used in clinical microbiology laboratories for the study of periodontal pathogens in subgingival plaque samples [Figure 1]. They are inexpensive and easy to operate and maintain. Since majority of the periodontal pathogens are moderately obligate anaerobes, they survive exposure to atmospheric air for several hours. Hence the clinical samples can be processed on the working bench in the laboratory without any loss of viability before loading the plates in the anaerobic jar for incubation. [9] If used properly, anaerobic jars can match the anaerobic chambers in their efficacy to support the survival of periodontal pathogenic bacteria. [10] The only limitation is that the jar holds only a few plates at a time when compared to the chamber and one may have to use multiple jars to accommodate when the volume of samples is high.
Figure 1: Anaerobic jar for routine isolation of anaerobe in our lab with supplemented blood agar used for cultivation of black pigmented colonies

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Despite the best efforts made by oral microbiologists, a sizable portion of the microbiota of the subgingival plaque remains uncultivated and their presence in the oral cavity has been detected by using molecular techniques such as polymerase chain reaction (PCR) as well as molecular cloning and sequencing. [11] When these microbes are growing as members of plaque community, their physiological and nutritional needs are met due to interspecies cooperation and they thrive in their natural environment. Successful cultivation of these microbes depends on our ability to recreate these natural conditions in the laboratory. Unfortunately, the growth requirements of many oral anaerobic bacteria are not yet known leading to our inability to grow them in vitro.[12]

It was realized more than 100 years ago that all the bacteria from the oral cavity visualized under the microscope cannot be grown in culture media in the laboratory, leading to what is called as "the great plate count" anomaly. Many theories subsequently have been formulated to explain this phenomenon. These include;

  1. Lack of essential nutrients or growth factors in the artificial culture medium,
  2. Overfeeding conditions where in too much of nutrients are incorporated into the medium,
  3. Toxicity of the culture medium itself, leading to inhibition of bacterial growth,
  4. Production of substances (metabolic end products, toxins) inhibitory to certain bacteria by other species present in the immediate vicinity,
  5. Metabolic dependence on other species present in the community for growth,
  6. Disruption of inter-bacterial communication systems induced by bacterial disruption during sample processing and
  7. Bacteria could be present in a viable but dormant state. [13],[14],[15]

It is important to remember that a given species has not yet been cultivated does not imply that it will remain uncultivated forever. The best example is that of oral treponemes; only a decade ago the number of Treponema species identified was around five. Today 10 different species have been identified and named and efforts are being made to identify several other spirochetes. [16] Experience with these bacteria has shown us that no single method or culture medium is suitable for isolation of diverse microbial community of oral cavity.

Currently, there is a growing trend to develop specific and innovative approaches and culture media that allow cultivation of previously uncultivated bacteria many of which may have pathogenic potential and are of clinical importance. [17] The proponents of molecular techniques may argue that when one has access to sophisticated molecular tools such as PCR, hybridization and mass spectroscopy, is there any need to depend on culture methods? But it is of utmost importance to remember that mere identification of genome sequences on their own cannot provide any quantitative data about metabolism, physiology or pathogenicity of oral microbes. For this purpose, microorganisms have to be made available in the laboratory. Several advances in isolation and cultivation techniques have been made over the last few years that could help us to broaden our knowledge about oral organisms in great detail. [18] Hence, microbial culture remains a principal tool for the study of microbes and the lack of access to cultures of many of them prompted a more recent resurgence of cultivation efforts, especially with oral microbes. Some of these methods involve a substantial departure from conventional techniques, some include new tools to mimic the natural milieu and increased length of incubation and lowered the concentration of nutrients. [19]

One such method considers the possibility of cultivating microbial species inside diffusion chambers incubated in the natural environment of these species. [20] The device used here essentially consists of a metal disk with central orifice. The inner and outer parts of the orifice are sealed with 0.02 μm pore size polycarbonate membrane creating a sealed space [Figure 2]. This space is filled with microbes and the device is then introduced into the natural environment of the bacteria to be studied. It is expected that diffusion through the membrane will automatically provide natural conditions for these bacteria, allowing their cultivation with no prior knowledge of their growth requirements.
Figure 2: Model of diffusion chamber used for bacterial isolation. (Adapted from ref. 20)

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A more advanced method is the isolation chip that works on the same principle mentioned above, but consists of hundreds of miniature diffusion chambers each loaded with an average of one cell per chamber. This combines microbial growth and isolation into a single step and overcomes the deficiencies of diffusion chamber. [20]

The above mentioned methods have been successfully used for isolation of marine and soil bacteria. Sizova and colleagues have made certain changes in the design of the diffusion chamber so that it could be adapted to oral cavity. This technique advocates the use of "minitrap". [21] This is a modified diffusion chamber containing sterile agar and separated from the outside environment by membranes. It basically consists of three surgical steel plates each with 72 holes of 400 μm in diameter. This device is inserted into a window that is precut in the palatal appliance and is affixed with superglue [Figure 3]. It is incubated in the subject's mouth for 48 hours and during this period, the organisms get trapped in the agar and grow with constant diffusion of nutrient materials from the surroundings. The appliance is then removed and placed in an anaerobic glove box. The minitrap is separated from the appliance and disseminated using the grown material to test under anaerobic conditions for further growth. The investigators could grow 30 new species by this technique, ten of which were known earlier by their 16S rDNA sequences and the other twenty being completely new to human oral cavity and are likely to be novel genera.
Figure 3: Minitrap device used in the oral cavity for enrichment and isolation of oral bacteria. (Adapted from ref. 21)

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The most notable outcome of all these novel approaches is the domestication of the newly discovered species by repeated subcultures in liquid media that would pave the way for further studies on their physiology and metabolic nature. [22],[23] Currently, different investigators are making efforts to improvise the existing devices so that they can be adapted to the oral cavity easily and without causing discomfort to the subjects during the study period. Finally, we should remember that most of the periodontal pathogens that have been identified so far are moderate anaerobes that have the ability to survive in the presence of traces of oxygen. What if the not yet cultivated oral bacterial species are strict anaerobes? Then probably the maintenance of strict anaerobic environment during the sample processing and isolation procedures may well help us in cultivating and identifying several more oral bacteria that may have a role in periodontal diseases. [24]

To conclude, a combination of novel and traditional culture techniques offers great promise in isolation and subsequent domestication of oral bacteria. These efforts will go a long way in closing the gap between microorganisms that could be cultivated in culture and those present in the oral cavity but as yet not cultivated. The additional knowledge gained by using these newer isolation methods would hopefully fill in the lacunae about the microbial role in periodontal infections. It sure looks like the periodontal microbiology is in for an exciting time!

   References Top

1.Dewhirst FE, Chen T, Izara J, Paster BJ, Tanner AC, Yu WH, et al. The human oral microbiome. J Bacteriol 2010;192:5002-17.  Back to cited text no. 1
2.Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134-44.  Back to cited text no. 2
3.Feng Z, Weinberg A. Role of bacteria in health and disease of periodontal tissues. Periodontol 2000 2006;40:50-76.  Back to cited text no. 3
4.Paquette DW, Brodala N, Nichols TC. Cardiovascular disease, inflammation and periodontal infection. Periodontol 2000 2007;44:113-26.  Back to cited text no. 4
5.Ritchie CS. Obesity and periodontal disease. Periodontol 2000 2007;44:154-63.  Back to cited text no. 5
6.Artimage GC. Comparison of the microbiological features of chronic and aggressive periodontitis. Periodontol 2000 2010;53:70-88.  Back to cited text no. 6
7.He XS, Shi WY. Oral microbiology: Past, present and future. Int J Oral Sci 2009;1:47-58.  Back to cited text no. 7
8.Van Horn KG, Warren K, Baccaglini EJ. Evaluation of the AnaeroPack system for the growth of anaerobic bacteria. J Clin Microbiol 1997;35:2170-3.  Back to cited text no. 8
9.Bartlett JG, Sullivan-Sigler N, Louie TJ, Gorbach SL. Anaerobes survive in clinical specimens despite delayed processing. J Clin Microbiol 1976;3:133-6.  Back to cited text no. 9
10.Doan N, Contreras A, Flynn J, Morrison J, Slots J. Proficiencies of three anaerobic culture systems for recovering periodontal pathogenic bacteria. J Clin Microbiol 1999;37:171-4.  Back to cited text no. 10
11.Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 2005;43:5721-32.  Back to cited text no. 11
12.Lewis K. Persister cells, dormancy and infectious diseases. Nat Rev Microbiol 2007;5:48-56.  Back to cited text no. 12
13.Rodríguez-Valera F. Approaches to prokaryotic biodiversity: A population genetics prospective. Environ Microbiol 2002;4:628-33.  Back to cited text no. 13
14.Wade W. Uncultivable bacteria - the uncharacterized organisms that cause oral infections. J R Soc Med 2002;95:81-3.  Back to cited text no. 14
15.Wade W. Non-culturable bacteria in complex commensal populations. Adv Appl Microbiol 2004;54:93-106.  Back to cited text no. 15
16.Ellen RP, Galimanas VB. Spirochetes at the forefront of periodontal infections. Periodontol 2000 2005;38:13-32.  Back to cited text no. 16
17.Green BD, Keller M. Capturing the uncultivated majority. Curr Opin Biotechnol 2006;17:236-40.  Back to cited text no. 17
18.Zengler K. Central role of the cell in microbial ecology. Microbiol Mol Biol Rev 2009;73:712-29.  Back to cited text no. 18
19.Connon SA, Giovannoni SJ. High-throughput methods for culturing microorganisms in very low nutrient media yield diverse new marine isolates. Appl Environ Microbiol 2002;68:3878-85.  Back to cited text no. 19
20.Epstein SS, Sizova M, Hazen A. New approaches to cultivation of human microbiota. In: Fredricks DN, editor. The Human Microbiota: How Microbial Communities Affect Health and Disease, 1 st ed. USA: Wiley Blackwell; 2013.  Back to cited text no. 20
21.Sizova MV, Hohmann T, Hazen A, Paster BJ, Halem SR, Murphy CM, et al. New approaches for isolation of previously uncultivated oral bacteria. Appl Environ Microbiol 2012;78:194-203.  Back to cited text no. 21
22.Vartoukian SR, Palmer RM, Wade WG. Strategies for culture of 'unculturable bacteria'. FEMS Microbiol Lett 2010;309:1-7.  Back to cited text no. 22
23.Bollmann A, Lewis K, Epstein SS. Incubation of environmental samples in a diffusion chamber increases the diversity of increased isolates. Appl Environ Microbiol 2007;73:6386-90.  Back to cited text no. 23
24.Tanner AC, Mathney JM, Kent RL, Chlamers NI, Hughs CV, Loo CY, et al. Cultivable anaerobic microbiota of severe early childhood caries. J Clin Microbiol 2011;49:1464-74.  Back to cited text no. 24

   Authors Top

About the Author Dr. Kishore Bhat who holds a post-graduate degree in molecular Microbiology currently heads the Department of Microbiology at Maratha Mandal's NGH Institute of Dental Sciences & Research centre, Belgaum. It is a unique department that is specialized in Oral Microbiology and has facilities for processing oral microbes with various levels of identification that include anaerobic culture, immunological tests and molecular technique. This department is recognized as centre for excellence and a nodal reference centre for oral microbiology by RGUHS Bangalore. Dr. Bhat is a recognized Ph.D guide at RGUHS and has 62 publications to his credit. He has two ongoing grants one from Vision Group of Sciences & Technology Karnataka for Infrastructure development for Oral Microbiology and second one from RGUHS, Bangalore to Periodontal pathogens by Elisa. Currently he is working on 1. Isolation and characterization of Oral spirochetes from periodontally healthy and diseased individuals 2. Evaluation of loop mediated isothermal amplification to detect P. gingavalis in patients with chronic Periodontitis. He is also honorary consultant microbiologist at KLE's Dr. Prabhakar Kore Basic Sciences Research Centre, Belgaum.


  [Figure 1], [Figure 2], [Figure 3]

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