Comparative study of five culture media for in vitro culture of Trichomonas gallinae from birds in Assam
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2022-11-01 |
| Journal | Journal of Entomology and Zoology Studies |
| Authors | M Saikia, K Bhattacharjee, Sarmah PC, Deka DK, RANJEET NEOG |
| Institutions | Assam Agricultural University |
| Analysis | Full AI Review Included |
Executive Summary
Section titled âExecutive SummaryâThis study compared the in vitro efficacy of five different culture media for optimizing the growth and viability of the flagellate protozoan, Trichomonas gallinae, obtained from domestic birds.
- Core Achievement: Medium 199 demonstrated superior performance, yielding the highest parasite concentration (16,000 cells/ml) and maintaining high motility (+++) for the longest duration (up to 7 days).
- Optimization Target: The research successfully identified the optimal culture medium for reliable harvesting and subculture of T. gallinae under standardized in vitro conditions.
- Initial Conditions: All media were inoculated with a standardized initial concentration of 1 x 103 cells/ml of trophozoites.
- Incubation Parameters: Cultures were maintained at a controlled temperature of 37 °C in an aerobic BOD incubator for seven days.
- Comparative Results: Modified Diamond media and RPMI 1640 supported growth but viability dropped significantly after 4 days. Nutrient broth showed the poorest performance, reaching the death stage by 72 hours.
- Method Validation: Culture methods were confirmed to be superior to wet mount preparation or staining for detecting the flagellate, establishing the optimized culture protocol as the gold standard for diagnosis.
Technical Specifications
Section titled âTechnical Specificationsâ| Parameter | Value | Unit | Context |
|---|---|---|---|
| Initial Inoculation Density | 1 x 103 | cells/ml | Standardized across all five media |
| Incubation Temperature | 37 | °C | Aerobic condition (BOD incubator) |
| Culture Monitoring Period | 7 | days | Total study duration for growth assessment |
| Highest Concentration (Medium 199) | 16,000 | cells/ml | Measured at 48-72 hours post-inoculation |
| Highest Concentration (Modified Diamond) | 4,400 | cells/ml | Measured at 48-72 hours post-inoculation |
| Highest Concentration (MEM) | 3,800 | cells/ml | Measured at 48-72 hours post-inoculation |
| Modified Diamond Media pH | 6.5 | N/A | Adjusted using hydrochloric acid |
| Sterilization Pressure (Modified Diamond) | 15 | lb/in2 | Autoclaved at 121 °C |
| Fetal Calf Serum Inactivation Temp | 56 | °C | Maintained for 30 minutes |
| Penicillin G Concentration | 100,000 | IU/100ml | Added to all media as antibiotic mixture |
| Streptomycin Sulfate Concentration | 0.1 | gm/100ml | Added to all media as antibiotic mixture |
Key Methodologies
Section titled âKey MethodologiesâThe study utilized standardized laboratory procedures for media preparation, culture initiation, and quantitative assessment of parasite growth and motility.
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Modified Diamond Media Preparation:
- Ingredients: Trypticase (20.0 g), yeast extract (10.0 g), maltose (5.0 g), L-cysteine hydrochloride (1.0 g), and ascorbic acid (0.2 g) dissolved in 1,000 ml distilled water.
- Sterilization: Autoclaved at 121 °C under 15 lb/in2 pressure for 15 minutes.
- Supplementation: 10% fresh inactivated fetal calf serum (inactivated at 56 °C for 30 minutes) and a standardized antibiotic mixture were added.
- pH Control: Final pH adjusted to 6.5 using hydrochloric acid.
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Commercial Media Utilization: Four commercially available media (Nutrient broth, Medium 199, MEM, and RPMI 1640) were purchased and supplemented with the same antibiotic mixture used for the Modified Diamond media.
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Sample Collection and Inoculation:
- Oral swabs were collected from the oropharynx and crop of visually inspected birds (pigeon, duck, chicken, quail) using a sterilized cotton swab.
- Swabs were inoculated into tubes containing the respective culture media.
- Initial inoculation density was standardized to 1 x 103 cells/ml.
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Incubation and Monitoring:
- Cultures were incubated at 37 °C in an aerobic BOD incubator.
- Growth and motility were assessed every 24 hours for 7 days (168 hours).
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Quantitative Assessment (Cell Counting):
- Trophozoite concentration was determined using a Neubauer chamber under 400X magnification.
- Viability Staining: Trypan blue (0.3% solution) was used to differentiate live (unstained) from dead (blue) parasites, facilitating accurate live cell counting, especially at high concentrations.
- Motility Scoring: Motility was qualitatively scored (+++ = High, ++ = Moderate, + = Less, +/- = Weakly motile, - = No motility/dead).
Commercial Applications
Section titled âCommercial ApplicationsâThe optimized culture protocol provides a robust, standardized method critical for bio-engineering and diagnostic applications in veterinary and poultry industries.
- Veterinary Diagnostics: Provides a highly sensitive and reliable gold standard protocol (Medium 199) for the detection and confirmation of T. gallinae infection in domestic and game birds, crucial for disease control programs.
- Bioprocess Engineering: The established growth kinetics and optimal media formulation (Medium 199) are essential for scaling up parasite production for research purposes, including antigen preparation, drug efficacy testing, and potential vaccine development.
- Laboratory Automation and Quality Control: The standardized parameters (37 °C, 1x103 cells/ml inoculation, 7-day viability) enable the development of automated high-throughput screening systems for anti-protozoal compounds.
- Poultry and Game Bird Industry Management: Reliable culture techniques mitigate the significant health and economic impact of trichomoniasis by allowing rapid and accurate identification of infected flocks.
- Media Formulation Science: The comparative data highlights the specific nutritional requirements met by Medium 199 (a complex eukaryotic cell maintenance medium) that are superior to simpler broths (Nutrient broth) or other basal media (MEM, RPMI 1640) for this specific flagellate.
View Original Abstract
A study was carried out in order to compare the in vitro efficacy of five different culture media against the flagellate protozoa, Trichomonas gallinae. For in vitro culture, oral swabs of T. gallinae were collected randomly from oropharynx and crop after visual inspection of birds as pigeon, duck, chicken and quail with a sterilized cotton swab and cultivated in five media namely modified Diamond media, Medium199, Minimum Essential Medium (MEM), RPMI 1640 and Nutrient broth. The initial inoculation of trophozoites into each media was 1X103cell/ml. Among all the five media, Medium 199 showed the highest growth rate and motility of the organisms till 7 days after initial inoculation. All media were found suitable for harvesting and sub culture of T. gallinae under in vitro condition.