THE BUDAPEST TREATY:
CODE OF PRACTICE FOR IDAs

OBLIGATIONS OF THE IDA

PRACTICALITIES

Content

Test methods and criteria for viability testing

• for fungi and yeasts
• for bacteria
• for plasmid bearing (genetically modified) mo
• for isolated plasmid DNA
• for bacteriophages
• for plant cell cultures
• for plant viruses
• for plant seeds
• for animal cell cultures
• for animal viruses

Contamination of deposited cultures

Responsibility for authenticity and purity of the deposited cultures

Requests for information about a deposited culture or the related deposit documents

End of the period of storage

TEST METHODS AND CRITERIA FOR VIABILITY TESTING

The IDA is obliged to test the viability of each deposited mo. Hence the depositor must provide the information necessary to perform the test.

In general the testing of the purity of the deposited cultures is performed simultaneously with the viability test.

Although the identity of the mo is not checked extensively, procedures for purity control commonly bring, to a varying degree, attention to the taxonomic positioning of the mo. If the IDA notices discrepancies between the identity or the properties of the organism and the description given by the depositor, it is recommended that the IDA notifies the depositor of this fact. The depositor can then check the authenticity of the deposited culture.
If during the course of discussions about the identity of the organism the IDA is required to take further action (e.g. carry out the identification of the mo), the depositor may be charged for this additional service.

For fungi and yeasts

To test the viability of fungi and yeasts IDAs inoculate the organism onto the recommended media and incubate under the recommended conditions. Viability is proven by observation of growth of the organism (i.e. visible increase of cell material).

Purity is verified macroscopically and microscopically.

For bacteria

To test the viability of bacteria IDAs inoculate the organism onto/into the recommended media and incubate under the recommended conditions.
Colony formation or increase in cell number (in case of liquid cultures) must be observed.

Starting from an active culture, the minimal criteria for confirming viability range from 10 - 12 colonies from the original biomass to 1 colony provided that this single colony can be successfully subcultured. The colonies obtained should look "normal" and should be of the type expected for the particular bacterium being deposited.

In cases where only a few colonies are obtained from frozen or freeze-dried preparations and where the IDA does not propagate the material, one of the following options can be taken:

1. request replacement samples from the depositor, repeat the viability test and issue BP/9 if the new samples are cultured successfully. In this case, the date of receipt of the replacement samples is deemed the date of deposit.
2. issue a viability statement (BP/9) but immediately request new samples under rule 6.2. of the BT.

Since option 2 can not guarantee the viability of the replacement samples, option 1 is to be preferred.

Examination of purity is usually done macroscopically (colony morphology) and microscopically (cellular morphology). If both cell and colony morphology appear to be the type expected for the bacterium being deposited, then these observations are considered to be sufficient for issuing BP/4 and BP/9.

For plasmid bearing (genetically modified) mo

To test the viability of plasmid bearing (genetically modified) mo, the organism is inoculated on an appropriate selective medium. Viability is proven by growth of the organism on this selective medium.

To check the purity, the organism is streaked on the appropriate medium with and without selective pressure. In addition, microscopic analysis is recommended.

For isolated plasmid DNA

The presence of plasmid DNA is proven electrophoretically in an agarose gel. At the same time the approximate amount and the size of the DNA can be estimated.
The viability of the plasmid is proven by the transformation of a suitable host with the plasmid and the subsequent inoculation of this host/plasmid combination onto a selective medium.

• In special cases the restriction pattern of the plasmid DNA can also be determined.
• Other examinations might be necessary in order to comply with other regulations.
• In case it is not available in the public collection the depositor should also supply a suitable host strain.
  
  

For bacteriophages

The viability of phages is tested by applying the spot-test or by plating bacteria and phages together in a top layer.
The number of plaque-forming units (pfu) per ml of lysate is determined by the serial dilution method or by the spot titre method. A minimum of 10⁷ pfu/ml is required to have a sufficiently safe quantity to store the lysate for the purposes of the BT.

Purity of the phage lysate can be tested by streaking the lysate on an uninoculated agar plate.

For plant cell cultures

When plant cell cultures are deposited in the form of a callus, viability is proven by growth on an appropriate medium. A definite increase of cell mass must be observed.
For samples in the form of a suspension culture, growth in an appropriate medium must result in a definite increase in cell density. Frozen samples are first thawed and transferred to an appropriate medium. The growth of the cells is observed until the cell mass has at least doubled.

The viability test will be deemed negative, if the cell number has not increased considerably (i.e. doubled) after a period of at least two months.

Depositors should be encouraged to give the normal growth characteristics of the culture. This information will assist the IDA make the appropriate determination as to viability.

The purity of the cell culture is checked by microscopic examination. If contamination with microorganisms is suspected, further tests are to be conducted.

• To test the viability of plant cells several laboratory tests are available (FDA, TTC, reduction). Nevertheless, because a positive result obtained from one of these tests does not guarantee that a cell culture will regrow after cryopreservation, the result of the viability test must be based on the observation of obviously growing cultures.
• Since plant cell cultures may be mixed populations of genetically different cells (ploidy changes, chromosome changes and transposon activation occur in cell cultures), tests must be conducted to determine whether cryopreservation changes the specific characteristics of a cell culture.

For plant viruses

In order to revitalize the virus in desiccated infected leaves the leaves are to be ground with a few drops of inoculation buffer until a green paste is achieved. This paste is to be diluted with inoculation buffer to yield 2-3 ml of final inoculum. The inoculum is to be rubbed onto the leaves of the appropriate propagation host(s) which had been dusted with a sterile abrasive, such as Celite or Carborundum. After a few minutes the inoculated leaves are to be rinsed with tap water.

For plant seeds

Germination is the sole criterion for the determination of seed viability.

Germination can be considered to occur at the initial imbibition stage or at the emergence and development of a seedling. For the purposes of a patent deposit, however, the first sign of radical emergence constitutes germination and hence viability.

For plant seeds, viability should not be confused with storage ability or longevity of the deposit. Although in principle a single seed germinating in a batch of 400 can render the batch viable, it is preferred that 85% of the seeds germinate.
If less than 85% of the seeds germinate, the depositor must be notified that it is unlikely that his deposit will survive the 30 years storage period and that a new deposit will be required at a later stage. This eventuality can be covered under rule 6.2.

• If required by the guidelines specified by the International Board for Plant Genetic Resources (IBPGR) or the International Seed Testing Association (ISTA), all seeds tested should be germinated under dormancy breaking procedures.
• Alternative methods for viability testing, e.g. tetrazolium topography, should be used only in exceptional circumstances and then only by suitably trained personnel.
  

For animal cell cultures

For animal cell cultures a minimum number of 4 x10⁶ viable cells per ampoule (or 2 x 10⁶ cells for adherent cultures) is required to confirm viability. For an acceptable deposit a good recovery and growth of the cells must be observed. Growth can be measured by counting the cells in a counting chamber. The number of viable cells must increase within one and a half weeks.

The purity of the culture is to be rigorously verified against the presence of bacteria (mycoplasma, etc.) and fungi.

For animal viruses

Viability tests are performed in vivo on eggs and on primary cells.
Many different assays can be used to test the viability of animal viruses, e.g. RT assays.
A minimum number of infective particles corresponding at least to 100 times the minimum detectible level is required.

Viruses are checked for purity against the presence of bacteria (mycoplasma, methylotrophic bacteria, etc.) and fungi.

Responsibility for authenticity and purity of the deposited cultures

In practice the IDA and the depositor share the responsibility for the purity of the deposited culture.
The depositor must ensure that a pure culture is transmitted to the IDA (if more than one component must be present, the culture is to be recognized as a 'mixed' culture).

The IDA must check the purity of the culture before accepting it and must notify the depositor if any contaminants are found. Also, the IDA must take all the necessary measures to ensure that the culture remains uncontaminated.

The final responsibility for the authenticity of the culture lies with the depositor. The IDA is not obliged to check the identity or the performance (e.g. product expression) of the culture. Most IDAs are technically not in a position to perform this task. Nevertheless, it is important that the IDA takes such measures as are needed to enable the depositor to fulfil his responsibilities.

If it is the case that the IDA must propagate the material in order to have sufficient material to preserve the mo, the IDA must send a sample of the propagated material to the depositor and request him to verify the authenticity of the culture. As before, the depositor should be made aware that if he does not provide a written confirmation or rejection of the authenticity within a certain time limit (e.g. three months), the propagated material is to be regarded as identical to the original deposited culture (see also 'Contamination of deposited cultures'). In case the authenticity of the culture has been rejected, the IDA must ask the depositor to transmit a new sample of the culture. According to rule 6.2 the depositor has to add, among other things, a written statement alleging that the mo which is the subject of the new deposit is the same as that which was the subject of the previous deposit.

When a sample of a culture for which the IDA did not receive a confirmation or rejection of authenticity is to be furnished to a third party, the IDA should provide a statement that "the culture has not been checked by the depositor".

Request for information about a deposited culture or the related deposit documents

According to rule 9.2. the IDA should not disclose information that a particular mo has been deposited with it under the BT.

Information about a deposit is given only to the person who, after request, is entitled to obtain a sample of the deposited mo according to rule 11 of the BT.
According to rules 11.4. and 7.6., this person is also entitled to receive the following information:

• the accession number given to the deposit
• a copy of the receipt (international form BP/4)
• an indication of any properties of the mo which are or may be dangerous to health or to the environment
• upon request, an indication of the conditions which the IDA employs for the cultivation and storage of the mo
• upon request, the most recent scientific description and/or proposed taxonomic designation of the deposited mo.

This information is to enable the recipient to handle and analyse the microorganism correctly.

Further information (e.g. concerning the relations between the IDA and the depositor, the delivery of samples or any other kind of file inspection) should not be made available to third parties.

All requests for information from parties other than those mentioned in rule 11, should be accompanied by the written permission of the depositor.
The IDA should ensure that the necessary criteria are fulfilled before releasing any information. The IDA should also ensure that any request for information, even from the depositor, is given in a written statement.

End of the period of storage

The IDA must store the deposited mo for a period of at least 30 years after the date of deposit (or for a period of at least 5 year after the most recent request for the furnishing of a sample of the deposited organism was received by the IDA).

It is advisable that IDAs make suitable arrangements about what to do with the deposited material when this period is over. Such arrangements should be specified in a contract between the IDA and the depositor. Possible arrangements are:

• destruction of the material
• return the material to the depositor
• make the material available to the public

In the case of absence of such arrangements, civil law is applicable.

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