Viral Transport Media (VTM) – Definition, Principle, Preparation, Application

What is Viral Transport Media (VTM)?

• Viral Transport Media (VTM) is a crucial component in the field of virology and diagnostic medicine. It refers to a specialized nutrient substance designed to facilitate the safe transportation and preservation of viral specimens from the collection site to the laboratory for further analysis, identification, and processing.
• Transporting viruses for laboratory testing poses unique challenges compared to other infectious agents. Viruses are highly labile and sensitive to environmental factors, making it essential to maintain their viability during transit. VTM plays a vital role in preserving the integrity and viability of viral specimens, ensuring that they remain suitable for analysis upon arrival at the laboratory.
• The primary objective of using VTM is to enhance the chances of successful virus isolation. The time interval between sample collection and inoculation into the culture greatly influences the probability of successful isolation. Therefore, it is crucial to minimize the time delay between collection and processing. VTM provides a stable environment for viruses, preventing their degradation and maintaining their viability until they can be examined in the laboratory.
• Another important aspect in maximizing the chances of successful virus isolation is to ensure that the collected specimen contains a sufficient number of virus particles. Early collection of samples during the patient’s illness helps to maximize the viral load present in the specimen. By using VTM, the viral particles are preserved and protected, increasing the likelihood of successful isolation and subsequent analysis.
• The use of suitable transport media significantly contributes to successful and reliable viral isolations. The composition of VTM typically includes a balanced mixture of nutrients, salts, buffers, and antibiotics that promote the survival and growth of viruses while inhibiting the growth of potential contaminants. These components help to maintain the viability of the viral specimen during transport and provide a suitable environment for their subsequent cultivation and identification in the laboratory.
• It is worth noting that the choice of laboratory plays a role in the success of viral isolation. Sending viral specimens to either a local laboratory or a reference laboratory can increase the chances of successful isolation. Reference laboratories often have specialized equipment, expertise, and resources to handle a wide range of viral pathogens, thus enhancing the likelihood of accurate diagnosis and identification.
• In summary, viral transport media (VTM) is a specialized nutrient substance used to transport and preserve viral specimens during their journey from the collection site to the laboratory. It helps maintain the viability of viruses, maximizes the viral load in the specimen, and increases the probability of successful virus isolation. The use of suitable transport media, along with timely sample collection and selection of an appropriate laboratory, contributes to reliable and accurate diagnostic outcomes in virology.

Definition of Viral Transport Media (VTM) 

Viral Transport Media (VTM) is a specialized nutrient substance used to safely transport and preserve viral specimens from the collection site to the laboratory for further analysis and processing.

Principle of Viral Transport Media (VTM)

The principle of Viral Transport Media (VTM) revolves around creating an optimal environment that protects the viability and integrity of viral agents during transportation and storage. The composition and design of VTM take into account the diverse characteristics of viral agents, including their composition, stability, structure, morphology, and size.

Enveloped viruses obtained from human samples are generally more susceptible to environmental conditions and require specific measures to maintain their viability. Non-enveloped dense viruses are comparatively more stable. To protect the viability of these viruses, different solutions are employed.

While some enveloped viruses, like herpes simplex viruses (HSV), can remain stable in non-frozen states under suitable conditions, most viruses require lower temperatures to maintain their infectivity. The stability and infectivity of viruses gradually decline over time, with the rate of decay being influenced by temperature. Cooling the viral specimens helps to enhance their stability.

Viral transport media are formulated to provide the appropriate conditions of temperature, pH, and nutrients to sustain the viability of viral cultures or specimens. The constituents of VTM are carefully selected to create an isotonic solution that maintains osmotic balance, contains proteins to protect the viral structure, incorporates antibiotics to control microbial contamination, and includes one or more buffers to regulate pH levels.

The primary objective of VTM is to sustain the viability of viral cultures or specimens during transportation and prevent the drying of the samples. This is particularly important for tests like the nucleic acid amplification test (NAAT), which require intact viral material for accurate and reliable results.

Characteristic Features of Viral transport media(VTM) and Composition

Characteristics of Viral Transport Media (VTM) encompass the key attributes necessary for effective transportation and preservation of viral specimens. An ideal VTM possesses several characteristics to fulfill these requirements:

1. Preservation of Virus Activity: A crucial characteristic of VTM is its ability to preserve the activity of the virus, even at room temperatures. This ensures that the virus remains viable during transportation, enabling accurate analysis and testing upon arrival at the laboratory.
2. Non-Toxic to Cell Cultures: VTM should be non-toxic to cell cultures. It should not interfere with the growth and behavior of the viral specimens in cell cultures, allowing for the detection of viral cytopathic effects without obstruction. This characteristic ensures that the VTM does not obscure or distort the appearance of viral cytopathic reactions.
3. Long Shelf Life: An ideal VTM has a long shelf life, whether stored in a frozen or non-frozen state. This longevity ensures that the VTM remains effective over an extended period, providing flexibility in transportation and storage options.
4. Applicability for Various Tests: VTM should be suitable for both culture isolations and direct tests, such as enzyme immunoassays or immunofluorescence. This characteristic allows for different testing methodologies to be employed on the viral specimens while maintaining their viability and integrity.

To achieve these characteristics, VTMs typically contain specific components:

• Protective Protein Component: VTMs often include a protein-based protective component that helps preserve the viral structure and activity during transportation. This protein component shields the virus from environmental factors that could potentially compromise its integrity.
• Antimicrobial Agents: VTM includes antimicrobial agents that help prevent the chances of contamination. These agents inhibit the growth of bacteria or fungi that may be present in the specimen, ensuring the integrity of the viral culture or specimen during transportation.
• Buffers: Buffers are essential components of VTM, as they help maintain the pH of the solution within the optimal range. This pH regulation is crucial for the viability of the viral specimens and supports their survival during transportation.
• Additional Ions and Minerals: Some VTMs may contain additional ions and minerals that aid in maintaining the viability of the viral specimens. These components provide necessary nutrients and support for the viral culture, enhancing its stability and preserving its activity.

Preparation of Viral Transport Media (VTM)

A variety of viral transportation media are available commercially that can be directly purchased and utilized under the conditions specified. One such medium is the COPAN Universal Transport Media and the Eagle Minimum Essential Medium (E-MEM). But, other media are able to be made locally in order to maintain the appropriate circumstances and the composition. The composition and the preparation of various viral transports differ according to whether the sample is taken from animals or human beings.

Preparation of Viral Transport Media (VTM) For specimens from humans

The preparation of Viral Transport Media (VTM) for specimens from humans involves specific steps and ingredients to ensure the preservation and viability of viral specimens during transportation. Here is a description of the preparation process:

For locally made VTMs for nasal and throat swabs from humans:

1. Add 10g veal infusion broth and 2g bovine albumin fraction V to sterile distilled water, bringing the total volume to 400 ml.
2. Add 0.8 ml of gentamicin sulfate solution (50 mg/ml) and 3.2 ml of amphotericin B (250 µg/ml) to the solution.
3. Sterilize the mixture by filtration, ensuring the medium is free from any contaminants.

For a larger volume of VTMs for humans, the following procedure can be followed:

1. Inactivate 500 ml of fetal bovine serum (FBS) by heating it in a water bath at 56.0°C +/- 1.0°C for 30 minutes. Alternatively, commercially inactivated FBS can be used if available.
2. Add 50 ml of amphotericin B and 50 ml of gentamicin to the inactivated FBS.
3. Filter sterilize the solution through a 0.20 to 0.45 µm filter unit (150 ml filter unit) to remove any potential contaminants.
4. Add 10 ml of the inactivated FBS to 500 ml of Hanks Balanced Salt Solution (HBSS).
5. Add 2 ml of the gentamicin/amphotericin B mixture to the HBSS with FBS solution.
6. Disperse the prepared solution into bottles and cap them securely. Mix the contents by gently inverting the bottles.
7. Label each bottle with the necessary information, including the date of production, additives used, and expiration date.
8. Aliquot 3 ml of the prepared medium into individual sterile conical screw-capped tubes, such as 16x100mm tubes. Ensure the lids are tightly closed after dispensing the medium. Label each container with the relevant information.
9. Store the VTMs at a temperature of 2-8°C to maintain their stability and viability.

By following these steps and utilizing the specified ingredients, VTMs for human specimens can be prepared effectively, providing the necessary conditions for preserving and transporting viral samples for further analysis and testing.

Preparation of Viral Transport Media (VTM) For specimens from Animals

When preparing Viral Transport Media (VTM) for specimens from animals, two types of VTMs are commonly used, each serving specific purposes:

1. Transport Medium 199: This medium is widely employed for the collection and transport of clinical specimens from animals of various species. Transport Medium 199 provides a suitable environment for preserving the viability of viral specimens during transportation. It is compatible with both egg inoculation and tissue culture inoculation methods.
2. PBS-Glycerol Transport Medium: The PBS-Glycerol transport medium is a glycerol-based solution that offers longer-term stability for specimens when immediate cooling is not feasible. It provides a protective environment for the viral specimens, ensuring their viability during transportation. However, this medium is not suitable for tissue culture inoculation. Instead, it is typically used for preserving specimens intended for egg inoculation.

When preparing VTMs for animal specimens, it is crucial to select the appropriate medium based on the intended purpose, cooling capabilities, and inoculation methods. The choice between Transport Medium 199 and PBS-Glycerol transport medium depends on factors such as the nature of the specimen, intended testing methods, and the availability of immediate cooling options.

Transport medium 199 Preparation

To prepare Transport Medium 199, the following steps can be followed:

1. Start with a tissue culture medium 199 containing 0.5% bovine serum albumin (BSA). This serves as the base medium for the preparation.
2. To 1 liter of the tissue culture medium 199 with BSA, add the following components:

• Benzylpenicillin: Add 2 x 10^6 IU/liter.
• Streptomycin: Add 200 mg/liter.
• Polymyxin B: Add 2 x 10^6 IU/liter.
• Gentamicin: Add 250 mg/liter.
• Nystatin: Add 0.5 x 10^6 IU/liter.
• Ofloxacin hydrochloride: Add 60 mg/liter.
• Sulfamethoxazole: Add 0.2 g/liter.

3. After adding the above components, thoroughly mix the solution to ensure proper distribution of the additives.
4. Sterilize the prepared medium by filtration. This filtration step is crucial to remove any potential contaminants and ensure the medium’s sterility.
5. Once sterilized, distribute the Transport Medium 199 into 1.0-2.0 ml volumes in screw-capped tubes. The volume can be adjusted based on the specific requirements and intended use.

By following these steps, the Transport Medium 199 can be prepared for use in preserving and transporting viral specimens. The components added to the base medium help protect the viability of the viral specimens during transportation, while the sterilization step ensures the medium’s sterility.

PBS-Glycerol transport medium Preparation

To prepare PBS-Glycerol transport medium, you can follow these steps:

1. Prepare a phosphate-buffered saline (PBS) solution by adding the following amounts of chemicals: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4. Add distilled water to the mixture to make 1 liter.
2. Autoclave the prepared PBS solution to ensure its sterility.
3. Mix the sterile PBS solution with sterile glycerol in a 1:1 ratio to make 1 liter of PBS-Glycerol transport medium.
4. To the 1 liter of prepared PBS-Glycerol solution, add the following components:

• Benzylpenicillin: Add 2 x 10^6 IU/liter.
• Streptomycin: Add 200 mg/liter.
• Polymyxin B: Add 2 x 10^6 IU/liter.
• Gentamicin: Add 250 mg/liter.
• Nystatin: Add 0.5 x 10^6 IU/liter.
• Ofloxacin hydrochloride: Add 60 mg/liter.
• Sulfamethoxazole: Add 0.2 g/liter.

5. Thoroughly mix the solution to ensure proper distribution of the additives.
6. Dispense 1.0-2.0 ml of the prepared transport medium into sterile plastic screw-cap vials. The volume can be adjusted based on the specific requirements and intended use.
7. Store the vials containing the PBS-Glycerol transport medium. If you plan to use them within a short period (1-2 days), they can be stored at room temperature. If you need longer-term storage, store the vials at -20°C to maintain the stability of the medium.

By following these steps, you can prepare PBS-Glycerol transport medium for preserving and transporting viral specimens, providing longer-term stability when immediate cooling is not possible.

Instruction for use of Viral transport medium tube with swab

When using a Viral Transport Medium (VTM) tube with a swab, the following instructions can be followed:

1. Prior to use, ensure that the sample tube and swab are sterilized either by the medical institution or the patient.
2. Take note of the relevant sample information on the tube, such as patient details and sample collection time.
3. Inject a suitable amount of VTM solution into the sample tube, ensuring that it is enough to preserve the sample.
4. Depending on the specific sampling requirements, take the sample swab in the appropriate location. For example:
   • Nasal swab: Gently massage the swab’s head through the nostrils or nasal passages, rotating it three times before removing it. Repeat for the other nostril.
   • Pharyngeal swabs: Wipe the bilateral tonsils and the posterior wall of the pharyngeal cavity with swabs.
   • Gargle: Gargle with 10mL normal saline, swirling it in the mouth and throat before spitting it out into the sample tube.
   • Nasal lotion: Inject 50mL normal saline into the nostril, instruct the patient to close the pharyngeal apex, and then release the saline into the sample tube.
   • Nasopharyngeal aspirates: Use a collection device attached to a negative pressure pump to extract tracheal and bronchial secretions from the nasopharynx.
   • Autopsy specimens: Take tissue samples during autopsy, separating samples if necessary.
   • Samples for mycoplasma, chlamydia, and Ureaplasma: For males, use sterile cotton swabs to rotate around the urethra.
5. Place the sample swab into the sample tube, making sure it is immersed in the VTM solution.
6. Tighten the cover of the tube securely to prevent any leakage.
7. Label the sample tube with relevant information, including patient details, sample type, and collection date.
8. If possible, deliver the collected clinical specimens to the laboratory within 48 hours. If not, store the samples at a temperature of -70°C or lower until they can be transported.

It is important to follow the standard sampling methods for each specific sample type to ensure accurate and reliable results. Proper collection, storage, and transportation of the specimens are crucial for successful analysis in the laboratory.

Precautions/cautions

When handling and using Viral Transport Media (VTM), it is important to take certain precautions and exercise caution. Here are some key precautions to consider:

1. Prompt Handling and Storage: After the sample is taken, examine the disposable sampler as soon as possible and immediately store it at a low temperature of 2-8°C. Samples should be stored at this low temperature for up to 2-8 hours and not exceed 48 hours. For long-term storage, maintain temperatures lower than 20°C, preferably at -70°C or even lower, such as -196°C.
2. Prohibition for Bacteria Sampling: VTM is not intended for sampling bacteria. The preservative liquid in the VTM contains antibiotics that inhibit bacterial growth. Therefore, it should not be used for bacterial sampling purposes.
3. Avoid Direct Contact: When collecting samples from patients, it is important to avoid direct contact with the patients themselves. This helps prevent potential transmission of infectious agents and ensures safety during the sampling process.
4. Proper Sampling Technique: Follow the prescribed method of sampling accurately to ensure precise location and even collection of the sample. Adhering to proper sampling techniques is essential to maintain the quality of the sample and ensure reliable test results.
5. Expiration Date and Packaging: Do not use the VTM product beyond its expiration date or if the packaging is damaged. Using expired products or those with compromised packaging may affect the integrity and reliability of the sample and the test results.

By adhering to these precautions, you can ensure the proper handling, storage, and use of Viral Transport Media (VTM) and minimize potential risks during the sampling process.

Viral transport media uses (VTM)

Viral Transport Media (VTM) has various uses in the field of specimen collection and transportation. Here are some key uses of VTM:

1. Collection and Transport of Viral Specimens: VTMs are primarily used for the collection and transport of specimens containing viruses. They provide an appropriate environment to preserve the viability and integrity of viral particles during transportation from the collection site to the laboratory. This ensures accurate and reliable testing and analysis of viral specimens.
2. Preservation of Other Organisms: In addition to viruses, VTMs can also be used to maintain the viability of other organisms such as mycoplasma, chlamydiae, and ureaplasma. The composition and characteristics of VTMs are designed to create a suitable environment that supports the survival and integrity of various microorganisms during transportation.
3. Maximizing Viral Particle Yield: VTMs play a crucial role in maximizing the number of viral particles in a sample. By providing optimal conditions for viral preservation, VTMs help prevent viral particle loss and maintain their infectivity. This is particularly important for diagnostic and research purposes where a higher viral particle yield enhances the sensitivity and accuracy of tests.
4. Transportation of Swabs and Sample Materials: Liquid transport media, which include VTMs, are primarily used for transporting swabs or materials that have been collected using swabs. The liquid medium allows for the release and suspension of sample materials into the medium, ensuring their preservation during transportation. This is especially useful in situations where refrigeration facilities are not readily available.
5. Facilitating Sample Collection in Resource-Limited Areas: VTMs are particularly valuable in areas where refrigeration facilities may be limited or unavailable. By providing a suitable medium for sample collection and transportation, VTMs enable the safe and effective transport of specimens even in resource-limited settings. This ensures that samples can reach the laboratory for analysis without compromising their viability.

Limitations of Viral Transport Media (VTM)

While Viral Transport Media (VTM) is widely used and beneficial for preserving and transporting viral specimens, it does have certain limitations that should be taken into account. Here are some limitations of VTM:

1. Contaminant Growth: During the long period of transport, there is a possibility of some growth of contaminants in the VTM. These contaminants may arise from the environment or even from the specimen itself. This can potentially interfere with the accurate detection and isolation of the target virus.
2. Time Sensitivity: It is crucial to inoculate the specimen onto the appropriate culture medium as soon as possible after transportation. The longer the time lapse between sample collection and inoculation onto the culture medium, the higher the risk of reduced viability and potential loss of the virus. Therefore, minimizing the time interval is essential for optimal results.
3. Variable Effectiveness: VTMs may not be equally effective for all viruses. Some viruses may be more labile than others, meaning they are more susceptible to environmental changes and may have reduced viability even when using VTMs. Therefore, it is important to consider the specific characteristics of the virus being tested and select the appropriate transport medium accordingly.
4. pH Changes: Certain antibiotics and chemicals used in VTMs can alter the pH of the solution. This pH change can potentially impact the viability of the organism being transported. Maintaining the appropriate pH balance is crucial for the survival and integrity of the viral specimen, and any pH alteration should be considered when using VTMs.

Examples of Viral transport media

There are several examples of Viral Transport Media (VTM) available in the market. Here are a few examples:

1. MDS Viral Transport Medium: MDS Viral Transport Medium is a specially formulated medium designed for the collection and transportation of viruses. It is specifically intended to preserve the viability and virulence of the virus sample. MDS Viral Transport Medium contains Hanks’ Balanced Salt Solution, which serves as a base medium. It includes a protective protein component to prevent microbial contamination and buffers to regulate the pH of the solution.
2. Universal Transport Medium: The Universal Transport Medium is an FDA-cleared collection and transport media suitable for preserving clinical specimens containing viruses. It is specifically designed for viral molecular diagnostic testing. The transport medium is typically packaged in a plastic tube with a screw cap. It is capable of keeping organisms alive for up to 48 hours at room temperature or when refrigerated, facilitating reliable viral testing.
3. Remel Viral Transport Media: Remel is a well-known brand in the field of microbiology. They offer viral transport media that are trusted by clinical, industrial, and research laboratories. Remel’s viral transport media are designed to provide optimal conditions for the collection, preservation, and transportation of viral specimens.

These are just a few examples of commercially available viral transport media. Each VTM product may have its specific composition and features, but they are all aimed at ensuring the viability and integrity of viral specimens during transportation, enabling accurate testing and analysis in laboratories.

Media	Components
Swab-tube combination Culturette Virocult	Swab-tube combination containing modified Stuart medium, antibiotics Collection swab, plastic transtube, small sponge saturated with phosphate buffer, D-glucose, lactalbumin hydrolysate, chloramphenicol, and cycloheximide
Cell culture medium MEM	MEM with amino acids, fetal bovine serum, buffers, antibiotics
Broth-based media Nutrient broth Tryptic soy broth Tryptose phosphate broth	Beef extract, peptone Tryptone, soytone, dextrose, sodium chloride, dipotassium phosphate Tryptose phosphate broth, 0.5% BSA or 0.5% gelatin, antibiotics, phenol red (optional)
Veal infusion brot	Veal infusion, proteose peptone, sodium chloride
Bentonite containing media Bentonite transport	HB597 tissue culture powder, Tris buffer, EDTA, bentonite, antibiotics, with or without rabbit serum-coated bentonite
Richards Viral Transport	Phosphate buffer, organic buffers, sucrose, amino acids, bovine serum, phenol red, antibiotics
Leibovitz-SPG	Leibovitz #15, sucrose, phosphate buffer, BSA, antibiotics
Carr-Scarborough Viral/ chlamydial transport	Phosphate-buffered sucrose, L-15 medium, glutamic acid, bovine albumin, gentamicin

FAQ

References

• https://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf
• Johnson, F. B. (1990). Transport of viral specimens. Clinical Microbiology Reviews, 3(2), 120–131. doi:10.1128/cmr.3.2.120
• https://bioactiva.com/pub/media/sebwite/productdownloads//v/i/viral-transport-medium.pdf
• https://www.pppmag.com/article/2638