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ORIGINAL ARTICLE
Year : 2015  |  Volume : 4  |  Issue : 4  |  Page : 218-221

The simplest technique for cultivation and maintenance of Balantidium coli


1 Department of Microbiology, Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly, Uttar Pradesh, India
2 Department of Microbiology, M.M. Institute of Medical Sciences and Research, Ambala, Haryana, India

Date of Web Publication20-Oct-2015

Correspondence Address:
Rajesh Bareja
Department of Microbiology, Shri Ram Murti Smarak Institute of Medical Sciences, Bhojipura, Bareilly - 243 202, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.167650

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  Abstract 

Introduction: Balantidium coli (B. coli) is considered as the largest protozoon and the only parasitic ciliate known to infect humans. Objective: This study describes the cultivation and maintenance of B. coli isolated from a stool specimen of patient during the routine examination. Materials and Methods: B. coli was identified microscopically and then successfully cultivated and maintained in home-produced culture media. Four media, Water (W), water with fecal matter (WF), WF and blood (WFB), and water with serum (WS) were used to cultivate and maintain the parasite. Inoculated culture media were observed daily and then weekly. Results: WF and WFB exhibited good growth of the parasite as well as maintenance up to 5 months. W and WS showed good maintenance of the parasite up to 7 months and no growth, respectively. Conclusion: The present study depicts the simple and cost-effective techniques that help us in cultivation and maintenance of B. coli for a long time without doing subcultures again and again.

Keywords: Balantidium coli, cultivation and maintenance, water with fecal matter, water with fecal matter and blood


How to cite this article:
Bareja R, Pottathil S, Grover PS, Singh H. The simplest technique for cultivation and maintenance of Balantidium coli. Int J Health Allied Sci 2015;4:218-21

How to cite this URL:
Bareja R, Pottathil S, Grover PS, Singh H. The simplest technique for cultivation and maintenance of Balantidium coli. Int J Health Allied Sci [serial online] 2015 [cited 2019 Sep 21];4:218-21. Available from: http://www.ijhas.in/text.asp?2015/4/4/218/167650


  Introduction Top


Balantidiosis is a zoonotic disease and is acquired by humans via the fecal-oral route from the normal host, the pig. Humans may remain asymptomatic, or may develop dysentery similar to that caused by Entamoeba histolytica.[1] Death is an infrequent consequence of balantidiosis but in developing countries like India, with undernourished and over parasitized populations, it can make the difference between a healthy life and chronic debilitation.[1]Balantidium coli (B. coli) can become an opportunistic parasite in immunosuppressed hosts living in urban environments, where pigs are not a factor in infection. As opportunistic organisms, the trophozoites of B. coli tend to become invasive and penetrate the linings of the mucosa and sub-mucosa of the damaged intestine and within the lymphoid tissue of affected hosts, from which they travel throughout the rest of the body.[2],[3]

Microscopy is still the first choice for the identification of these ciliates in stool samples. Establishment of all these organisms in culture is far from a routine procedure and is usually less sensitive than microscopy as a detection mechanism. In contrast to bacteria, these organisms are difficult, expensive, and labor-intensive to maintain in the diagnostic laboratory. Balantidium is an often-neglected pathogen. Research on Balantidium has been sparse. In the present study, we devised in vitro techniques for cultivation and maintenance of B. coli that are easy and cost-effective.


  Materials and Methods Top


Isolation of Balantidium coli

A freshly collected diarrheic stool sample from the patient was subjected to routine examination. Fresh wet mounts were carried out to detect trophozoites of B. coli using a light microscope viewed at low power (×100) and high power (×400) magnifications. Sample preparation and identification were done following the protocol of Garcia.[4] Because of their large size and spiraling motility, balantidia can readily be recognized in wet mount slide preparations, even at a low magnification. The Institutional Ethical Committee approved this study. The study received a waiver of informed consent because the sample used was collected for use in approved routine test.

Cultivation ofBalantidum coli

Following culture media were used for cultivation and maintenance of B. coli:

  • Water (W): Water was collected in a sterile bottle from a water cooler to that a water purifier was already installed. Culture and microscopic examination were done to rule out any bacterial and parasitic contamination. 10 mL of water was used for culture of B. coli
  • Water + Fecal mater (WF): 2–3 g of fecal mater, that is, negative for any parasite is mixed in 10 mL of water in another sterile bottle
  • Water + Fecal mater + Blood (WFB): In another sterile bottle, 0.5 mL of fresh blood was mixed with water and fecal mater
  • Water + Serum (WS): 0.5 mL of serum was mixed with 10 mL of the water.


Inoculum

A measured inoculum (0.05 mL) of that diarrheic specimen, which was positive for B. coli trophozoites, is passed to all four culture bottles containing a fresh medium. Duplicate cultures were also set up. All bottles were incubated at room temperature (25°C) and observed after 24 h, 48 h, 72 h, and then weekly regularly [Table 1]. After incubation if no trophozoites are seen further 48-h incubation is warranted, and this is followed by reexamination. If there are still no trophozoites seen, the culture is discarded as negative. There was no replenishment of the culture media used during the observed period. However, various subcultures were done using 0.05 ml of inoculum from previously positive culture and inoculated in fresh medium to observe the unceasing growth of B. coli.
Table 1: Growth and maintenance of B. coli in various culture media

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  Results Top


Numerous motile trophozoites were observed under low power and then high power after 24 h of inoculation [Figure 1] and [Figure 2]. General structures such as cilia, vestibulum, macronucleus, and contractile vacuoles are clearly seen. After 1-month slightly, decrease in the count was observed as shown in [Table 1]. In subcultures, numerous motile trophozoites were observed again. B. coli survived for 7 months, 4 months, but a decrease in the count and no growth in culture medium W, WF, WFB, and WS, respectively. Growth was observed in subcultures except WS. The cultures grew and maintained well at room temperature up to several months.
Figure 1: Numerous trophozoites of Balantidium coli under low power magnification (×100)

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Figure 2: Trophozoites of Balantidium coli under high power magnification (×400)

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  Discussion Top


There were a number of early attempts at maintenance or cultivation of B. coliin vitro. The first report of cultivation of the ciliate B. coli is that of Barret and Yarbrough, using the simple saline-serum medium used for Blastocystis hominis.[5] Biphasic media in tubes were often used with an agar, inspissated egg yolk, or serum butt overlaid with nutrient medium. Bacteria present in the sample can overgrow in an enriched medium, requiring the addition of antibiotics (e.g., penicillin-streptomycin) to suppress bacterial proliferation. Zaman maintained monoxenic cultures of B. coli with Escherichia coli, using antibiotics to control bacterial overgrowth.[6] Diamond's trypticase (casein digest), yeast extract, serum, and porcine gastric mucin (TYSGM) medium for Entamoeba and other enteric parasites also supports the xenic growth of balantidia.[7] The medium contains TYSGM. The starch powder is added to tubes at the time of inoculation of the medium. A defined medium with cysteine HCl and i-inositol was used for short-term physiologic experiments with the organisms, but little in the way of results appeared in the literature.[8]

Established cultures of all parasites are handled in essentially the same way. Xenic cultures of E. histolytica and Dientamoeba fragilis are routinely passaged at 48–72 h intervals. Xenic isolates of B. hominis and B. coli can usually be kept on this schedule also.[7] The preparation and cultivation of these xenic cultures are complex, costly, and difficult to handle. Studies showed the results of these media were also compromising. Cultures have been established in (LE (Lock-egg slant medium) modification of Boeck and Drbohlav's medium), and Robinson's media, but as mentioned before it is difficult to keep many isolates for long.[9],[10] Whether this is a deficiency of the media or is dependent on the bacterial flora composition or on some other factor is not known.[7] Positive cultures containing B. coli survived for 14 days with subcultures done every 24 h. Cultures of the isolate observed at 48 h without subcultures resulted to an increased bacterial density and the protozoa gradually decreased in number.[11]

To overcome all these difficulties, in this study, some new culture media are devised. These media are not only cost-effective but also easy to handle and yielding good results. There is no need to passage again and again for maintenance of this parasite. The culture mediums "WF" and "WFB" showed better cultivation and maintenance of the parasite. They can maintain the parasite up to 5 and 4 months, respectively. The culture medium "W" also showed some cultivation property in 1st week. However, the maintenance of B. coli in this medium was up to 7 months. No the culture medium "WS" neither showed cultivation property or maintenance. One of the frustrations faced by some authors was the lot-to-lot variability in the medium components. An item from the same supplier with the same catalog number may support abundant growth 1-time and not the next.[7] The media was used in this study do not require any specification and dependability on manufacturer. We can prepare it in our laboratory easily, whenever required.

Staining such as hematoxylin-eosin could not stain the internal structure of parasite, but the stain concentrates progressively in the cytoplasm, obscuring all internal detail [Figure 3].
Figure 3: H and E, stained preparation of Balantidium coli trophozoites

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Trophic ciliates did not survive longer than 24–48 h in cultures maintained at temperatures over 40°C.[12] We kept the culture positive for B. coli, at 42°C in water bath for 24 h to observe the survival of this parasite but could not be discovered it. The cultures grew well at room temperature.


  Conclusion Top


The development of these new techniques will encourage propagation and longer survival of B. coli in culture. These mediums "WF" and "WFB" support the growth of the organisms and routine maintenance of parasite that was difficult or time-consuming. We hope that the difficulties associated with cultivation and maintenance of these organisms will not prevent people from choosing to work with them.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Schuster FL, Ramirez-Avila L. Current world status of Balantidium coli. Clin Microbiol Rev 2008;21:626-38.  Back to cited text no. 1
    
2.
Cho HS, Shin SS, Park NY. Balantidiasis in the gastric lymph nodes of Barbary sheep (Ammotragus lervia): An incidental finding. J Vet Sci 2006;7:207-9.  Back to cited text no. 2
    
3.
Headley SA, Kummala E, Sukura A. Balantidium coli-infection in a Finnish horse. Vet Parasitol 2008 25;158:129-32.  Back to cited text no. 3
    
4.
Garcia LS. Diagnostic Medical Parasitology. Washington, DC: ASM Press; 2007.  Back to cited text no. 4
    
5.
Barret HP, Yarbrough N. A method for the cultivation of Balantidium coli. Am J Trop Med 1921;1:161-5.  Back to cited text no. 5
    
6.
Zaman V. Balantidium coli. In: Kreier JP, editor. Parasitic Protozoa. Vol. 2. New York: Academic Press; 1978. p. 633-53.  Back to cited text no. 6
    
7.
Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 2002;15:329-41.  Back to cited text no. 7
    
8.
Klaas J 2nd. Two new gastric mucin cultivation media and a chemically defined maintenance medium for Balantidium coli. J Parasitol 1974;60:907-10.  Back to cited text no. 8
    
9.
Von Brand T, Rees CR, Jacobs L, Reardon LV. Studies on reducing substances and gas formation in cultures of Endamoeba histolytica and a single species of symbiotic bacterium. Am J Hyg 1943;37:310-9.  Back to cited text no. 9
    
10.
Robinson GL. The laboratory diagnosis of human parasitic amoebae. Trans R Soc Trop Med Hyg 1968;62:285-94.  Back to cited text no. 10
    
11.
Nilles-Bije ML, Rivera WL. Ultrastructural and molecular characterization of Balantidium coli isolated in the Philippines. Parasitol Res 2010;106:387-94.  Back to cited text no. 11
    
12.
Cox FE. The cultivation of Balantidium coli throughout its viable temperature range. Ann Trop Med Parasitol 1961;55:305-8.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1]



 

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