Jan 15, 2025
Standardization of the Miniculture Technique for isolation of Leishmania spp.
- Laryssa Fabiano-Coelho1,
- Fernanda Nunes Santos1,
- Luciana de Freitas Campos Miranda1
- 1Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation (INI/Fiocruz)
Protocol Citation: Laryssa Fabiano-Coelho, Fernanda Nunes Santos, Luciana de Freitas Campos Miranda 2025. Standardization of the Miniculture Technique for isolation of Leishmania spp.. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldry5og5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 12, 2024
Last Modified: January 15, 2025
Protocol Integer ID: 111983
Keywords: Visceral Leishmaniasis, Canine Visceral Leishmaniasis, Tegumentary Leishmaniasis, Miniculture, Parasitological diagnosis , isolation of leishmania spp, standardization of the miniculture technique, microculture method, leishmania spp, handling microorganisms class ii, miniculture tube, tegumentary leishmaniasi, miniculture method, parasite isolation, miniculture technique, microorganisms class ii, microaerophilic environment, sterile material, microculture, culture method, biological sample, sterility during sample collection, microaerophilic environment with high co2 level, biological safety cabinet, use of tube, contamination, disadvantages of culture method, isolation of numerous biological agent, traditional culture method, possibility of contamination, miniculture, isolate, sterility, access to the isolate, bacteria, traditional method, fungi, biosafety standard
Abstract
Culture media are employed for the isolation of numerous biological agents causing diseases in humans and animals. The traditional culture method (TCM) is regarded as the gold standard in the diagnosis of Visceral and Tegumentary Leishmaniasis (VL / TL), as it is known that Leishmania spp. have a good growth on culture media used in TCM. The microculture and miniculture method consists in the use of tubes with a smaller volume, promoting a microaerophilic environment with high CO2 levels, which enhances the amastigote-to-promastigote differentiation. One of the disadvantages of culture methods is the possibility of contamination by bacteria and fungi, originating from the biological samples or the environment. It is important to ensure sterility during sample collection and processing. Therefore, the sample handling must be carried out with sterile material in a biological safety cabinet. This methodology requires trained personnel as well as sites adapted to biosafety standards for handling microorganisms class II. Although the microculture method is more sensitive than miniculture, the tubes used in this technique do not allow the parasite isolation and expansion. The miniculture tubes, like the traditional method, allows access to the isolate for post-culture analyses such as species characterization. Additionally, the miniculture showed better sensitivity compared to traditional methods and a shorter time to achieve positivity. Furthermore, miniculture is easy to implement, using the same equipment and materials as traditional culture but in smaller volumes, which can lead to cost reduction.
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Materials
Reagents:
- Antifungal 5' fluorocytosine (stock solution with 10,000 μg/mL)
- Bacteriological agar
- Calcium chloride
- Defibrinated and sterile rabbit blood
- Distilled water
- Hydrochloric acid (HCl)
- Penicillin (stock solution with 63,000 Units / mL)
- Schneider´s Insect Medium – S9895- SIGMA
- Sodium bicarbonate (NaHCO3)
- Sodium chloride (NaCl)
- Sodium hydroxide (NaOH)
- Sterile fetal bovine serum (FBS).
- Sterile human urine
- Sterile saline
- Streptomycin (stock solution with 50,000 µg /mL)
Materials:
- Beaker
- Bistoury
- Cell culture flasks
- Culture tubes
- Filter Paper
- Filter with a membrane porosity of 0.22 microns
- Microcentrifuge tubes
- Microscope slides and coverslips
- Pasteur Pipette
- Sterile Petri dish
- Sterile serological pipette
Equipments:
- Autoclave
- Biological safety cabinet
- Centrifuge
- Freezer
- Incubator (BOD)
- Inverted microscope
- Laminar flow cabinet
- Liquid nitrogen tank
- Magnetic stirrer
- Magnets
- Optical microscope
- pH meter
- Precision analytical balance
- Refrigerator
- Vacuum pump
Troubleshooting
Safety warnings
Every procedure must follow biosafety guidelines and good laboratory practices.
Ethics statement
For the development of this protocol, samples were provided by the Laboratory of Clinical Research in Dermatozoonoses (LapClinDermoZoo/FIOCRUZ). At the time of sample collection, the protocols were approved by the Ethics Committee on the Use of Animals (CEUA/FIOCRUZ - Licenses LW 19/20 and LW 19/21). Researchers intending to replicate this protocol must ensure prior approval from their respective ethics committee.
Before start
The tubes used for this procedure must contain a biphasic culture media composed of a solid phase of Novy and McNeal agar modified by Nicolle (NNN) and a liquid phase of Schneider's Insect Medium (S9895 – Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and antibiotics penicillin and streptomycin, according to the previously described protocol (https://dx.doi.org/10.17504/protocols.io.22tggen). The miniculture method, initially described by Boggild et al., 2008 for the diagnosis of cutaneous leishmaniasis in Peru, involved distinct approaches compared to our protocol. Unlike the original method, which used a single-phase medium and only aspirates, our adaptation employs a biphasic medium, biopsy fragments and aspirates, and evaluates two types of tubes for the miniculture method.
In the miniculture technique, two tubes with different volumes can be used:
- Miniculture 1: Vacuum blood collection tube (13 x 75 mm), made of clear PET plastic, without additives, sterile, with total capacity of 4 mL. In this study the technique was standardized with the VACUPLAST‱ tube (Code: GD040SADT)
- Miniculture 2: Polypropylene microtube with an external screw cap and sealing ring, sterile and autoclavable, with total capacity of 2 mL. In this study the technique was standardized with the Nalgene‱ cryogenic vials (Code: 5012-0020)
Step 1 - Preparation of Tubes
In the biological safety cabinet, the tubes should be opened and positioned to facilitate the distribution of the solid phase in a bevel shape.
After this, the tubes should be exposed to ultraviolet light (UV) for at least 30 minutes.
Following the aseptic step, the tubes should be prepared as described below:
- Miniculture 1:
- NNN = 1 mL
- Schneider = 1.5 mL
- Miniculture 2:
- NNN = 0.5 mL
- Schneider = 1.0 mL
Step 2 - Collection of Clinical Samples
Veterinarians perform biopsies of the skin and spleen and aspirates of the bone marrow and lymph nodes in dogs with two positive serological tests for canine visceral leishmaniasis, following euthanasia.
Lymph nodes and bone marrow aspirates should be seeded on the same day and maintained for a maximum of 6 hours under refrigeration at 4-8°C.
In the biological safety cabinet, 0.2 mL of each aspirate should be seeded in duplicate in the culture tubes.
- If it is not possible to collect the necessary volume, the collected aspirate volumes should be equally distributed in the culture tubes.
Tissue fragments are kept in microcentrifuge tubes containing sterile saline solution with penicillin, streptomycin and 5' fluorocytosine, as previously described in protocol (https://dx.doi.org/10.17504/protocols.io.22tggen).
In the biological safety cabinet, these fragments are washed in another tube containing the same saline solution, gently agitated.
Subsequently, the tissue fragment is transferred to a third tube containing the same solution and stored at 4-8°C overnight.
After this period, in the biological safety cabinet, the fragments should be divided into two similar sizes on a sterile Petri dish and seeded separately into the culture tubes containing biphasic culture medium.
The tubes should be labeled with the encoded patient data and incubated in a Biochemical Oxygen Demand incubator (BOD) at 26-28°C.
For diagnosis, a small aliquot of the liquid phase from the culture tubes is collected using a sterile pipette.
This aliquot is placed on a microscope slide, and the examination is performed under an optical microscope with magnification lenses of 100x and 400x.
Microscopic observations should be performed at the following intervals after sample seeding:
- 1st reading: 3 to 5 days
- 2nd reading: 7 days
- 3rd reading: 14 days
- 4th reading: 21 days
- 5th reading: 28 days
The culture is considered positive when one or more Leishmania promastigotes is detected in any of the readings performed.
Step 3 - Maintenance of Parasites Isolated
After parasite isolation in culture, perform the first passage between 1 and 7 days, according to the intensity of parasite growth.
Inoculate the entire volume of samples to be expanded into a traditional culture tube containing biphasic NNN medium and Schneider's Insect Medium (S9895 – Sigma-Aldrich) supplemented with 10% FBS and antibiotics penicillin and streptomycin.
Maintain the tubes in a BOD incubator at a temperature of 26-28°C and monitor parasite growth by observation of an aliquot under the optic microscope.
After 5-7 days from the initial subculture, based on parasite growth, transfer the entire volume of traditional culture tubes to cell culture flasks (75 cm²) containing 2 mL of NNN and 3-5 mL of Schneider's Insect Medium (S9895 – Sigma-Aldrich) supplemented with 10% FBS and antibiotics penicillin and streptomycin with an additional supplement of sterile human urine (5%), which was obtained from a healthy male child. The urine should be sterilized on the same day of collection by a vacuum filtration system with 0.22 µm pore size membrane.
Monitor parasite growth using an inverted light microscope.
Add culture medium to the flasks at intervals of 2-3 days according to the parasite growth until it reaches the final volume of 10 mL.
Then, the total culture volume can be centrifuged and the parasite pellet obtained can be cryopreserved in liquid nitrogen for an indefinite period.
Protocol references
BOGGILD, A. K. et al. Optimization of microculture and evaluation of miniculture for the isolation of Leishmania parasites from cutaneous lesions in Peru. The American Journal of Tropical Medicine and Hygiene, v. 79, n. 6, p. 847–852, dez. 2008.
MIRANDA, L. F. et al. Parasitological diagnosis of American Tegumentary Leishmaniasis - Isolation of Leishmania in Culture.Protocols.io, 20 maio 2019.
Acknowledgements
CAPES, Oswaldo Cruz Foundation, Brazilian Ministry of health.