Molecular collections facility We have been identifying and classifying species for a long time but only relatively recently over the last 3 hundred years that we have become organized about it. With an estimate of 1.7 million species we have barely scratched the surface. Even a good conservative effort has us discovering and describing at least another 3 million though the final figure is expected to be much higher. And the vast majority of these described specimens sit in collections in museums and universities around the world. It is these collections that have been the bedrock for most morphological studies as well as being key for studies into climate change, invasive species and so on, and so are incredibly important repositories for our biological heritage. However we are no longer just thinking about the morphology of the specimens but rather their molecular makeup as well. Molecular sequencing is the relatively new tool which has often described as being the savior of taxonomic research but in reality is more likely to be one of the many weapons in our arsenal for species identifications. We can no longer think of it as being separate to our research or the impact it will have on specimen storage And as with morphology there are drawbacks, with one of the more obvious one being that of collecting for, extracting of, and long term storage of molecular material. Now the NHM is a physical collection that dates back a long time and has amassed a large number of specimens (I think the current guess is around 80 million). And these specimens are housed in many different ways depending on what they are, and also have been sourced, killed and preserved in a variety of fashions.
Within Entomology there were three main ways that the insects have been preserved either as a whole organism or part and this is pinned, slide or spirit. There are various other small collections such as SEM stubbs but this are incredibly small in terms of actual specimen numbers. The vast majority of the collection is pinned (25 out of 34 million). Some of our oldest collections date back to the late 17th Century.
Although not sounding so Plukenet was an English Botanist from the middle 1600’s. Plukenet published Phytographia (London, 1691–1692) in four parts in which he described and illustrated rare exotic plants. Within this he had squashed many insects. This page was probably done in 1690 and so represents some of the earliest preserved insects. These specimens are still of slight value for morphological identification and maybe soon for molecular as we develop even better techniques of recovering ancient DNA.
And there has been much over the last 300 years or so about how to collect and pin insects to enable them to be studied primarily for taxonomic studies based on their morphology. There are countless publications on the collecting and preservation of specimens from passive malaise traps to actively hunting some of the more challenging specimens. The long term storage and preservation has been discussed throughout this period as well, although only recently has there been any real empirical evidence looking at the various methods and how they affect long term storage.
It is a critical part of our jobs to ensure the long term safety of our specimens to avoid these sort of horrors..
Slides have been used as a method of preservation and observation for almost 200 years for microstructure of minerals and whole organisms and the microscopic parts of larger organisms. These have been affixed to glass, wood or plastic slides as liquid, solid or dry mounts and can be transparent for through light microscopy or opaque for reflected light. Many mounting media which were thought of as being perfect optically are now drying out, shrinking, crystallising and are even eating the specimens! As I am sure that you have seen yourselves but there is much heartache in my collection due to blackened mountants . There is an increasing requirement for the conservation of such collections and to understand why glues and mounting media are deteriorating so as to avoid future problems. The slides have many conservation issues but this is outside the remit of this talk so thankfully I will leave that curatorial hurdle to someone else!
Spirit material does however fall within the remit though as the preservation of specimens in different types of spirit and preservatives has a direct affect on the ability to recover DNA and other molecules from them. And within the entomology section there are over 7 million specimens preserved this way. The spirit collection has material contained in all manor of jars and a range of preservatives, as with most collections! You can see here the lovingly labeled formalin which is no longer used within the entomology collections. Instead the majority of our collection is preserved with IMS. Different preservatives have been used over the years and some of the material that was originally in Formalin has been transferred into IMS. We have very little in Ethanol.
Whilst researching the different methods of preservation that have been commonly used for spirit preservation I came across this method from the modern bible for life, wikipedia. I am not sure why this was the only method given but they help us in our curatorial knowledge by stating the best spirit preservation method for zoology specimens. I would not recommend 10% formalin as a long term preservation method!
As you can see here…it was be absolutely useless for molecular work. There has been a lot of work studying the different types of preservative for spirit detailed in many papers, theses and workshops, and so I will not go into them here. Suffice to say there is much agreement in the fact that no one agrees - one size does not fit all. The table here is cribbed from a thesis looking into spirit preservation and shows some of the different modes of fixation and the subsequent preservation and the affect on both DNA recovery and morphology. For molecular studies ethanol at a strength of 80% plus has been found to be the most effective for long term recovery of DNA. However as we know this has a desiccating affect on the specimens leaving them delicate and brittle. It also results in the shrinkage of specimens. IMS as used by collections world wide (ours is dominated by IMS – it’s cheap!) also has had some degree of success of DNA extraction. This is heavily dependent upon the ratio of IMS with water, with the latter having a very destructive effect on DNA.
Specimens and their DNA are all affected by the same agents of deterioration to greater of lesser degrees. Light, humidity and temperature are just a few of the factors that can result in the destruction of all or part of the specimens. But it wasn’t just the collection of whole specimens in different preservations that was building up. Previously most of the pinned material that was being sent out to researchers around the world for molecular analyses was a grey area. These were sometimes returned minus a leg or whatever, or sometimes nothing at all except the DNA aliquot. Sometimes we were not even getting the DNA. And Why? Because we had no idea as to what we doing with this DNA –up to a couple of years ago this was being stored in fridges and freezers in a higgledy-piggledy fashion far and wide across the museum.
Nowadays all of our pinned material and all of the spirit material are housed in collection facilities that offer temperature and humidity controlled environments. These are in place to ensure that the problems of the past that affected both the morphology and the molecular structure of the specimens theoretically no longer happened. Moisture, heat, contamination are all being monitored with all of the information being electronically housed on our main database system which is now able to start to be able to be used in a predictive rather than a reactive fashion. The pinned collections are in cabinets that protect them from light and pests and the environmental conditions within and around these cabinets are permanently monitored.
The spirit collections, the pickles, live in their own spirit building, much to the amusement of the general public, and these are being permanently flushed so there is no build up of fumes. A large majority of the spirit collections has recently been surveyed and topping up has occurred. From these surveys we have determined that the majority of our material would not be suitable at all for molecular work using present methods as some of the specimens were being stored in IMS that had almost completely evaporated (some jars had dropped from 80% to 5%). These inventories have helped us decide as collections managers whether we should send material out for molecular research. Had we had done this survey the year before the 100 ticks used for sequencing would have been retained and thus saving the researcher time and money. This has impacted upon recent collection development projects, one of which was the establishment of a long term malaise study with specimens being preserved directly into 100% ethanol. These have been running in the wildlife garden at the Museum over the past year to provide fresh UK material for molecular studies. We are confident that within the UK that we have described over 95% of our fauna but we know very little about the molecular makeup of many of these species.
So that was good – we have moved or in the process of moving the entomology collections into high quality storage facilities but we also needed to sort out the present and future molecular collections to ensure that they too would provide viable long term material. But it is with our molecular collection facilities and operations that we have recently undergone a vast consolidation process and upgrade. After a long period of investigation across the museum (it is a museum after- all and time is often of little meaning) of hidden and relic collections a designated Molecular collections facilities at the NHM opened for Business in August 2012. Armed with the knowledge of how large the existing collections were and upon consultation determined the subsequent required capacity the development of a world class bio-repository which can store up to 2 million samples of high quality genetic resources such as tissues or cell extracts with streamlined operations to deposit large quantities in one time. All of the Molecular samples and specimens that enter this facility are curated in future proofed storage systems. We hope.
For medium term storage there are three main ways that we have set up to preserve material; Cold storage +4oC Frozen Storage -20oC Ultracold storage -80oC All of these are set up to a monitoring system and there is an alarm system to contact both security and the lab managers at any time of day or night! As well as on the main power systems these are also all connected to back up generators. Each bank of ultracold freezers (4 rows of four) is on different circuits to enable specimens to be moved into other freezers incase one of the circuit’s malfunctions.
And for the long term storage there are cryo-storage in liquid nitrogen which stores the material at approximately -196oC. As with the medium term storage there is full back up generator power, temperature monitoring and alarm systems and air conditioning ensuring a secure biorepository. Cryo-facilities have so far been shown to be the best method for long term tissue storage and they also look cool. The added advantage of these facilities that if they were to break down or loose power that unlike the freezers they would not heat up as rapidly and would keep the samples chilled for a much greater period.
As well as these storage facilities there are ambient storage facilities such as the super-dry cabinets. These control the humidity and oxygen levels that can be independently controlled (the latter by purging with nitrogen gas) to optimize the storage for dried specimens, silica gel and FTA cards.
These FTA ‘Whatman’ cards are a relatively new method of DNA preservation that the museum is experimenting with. They work in a very simple fashion and enable me to now go on fieldwork not only armed with machetes but now hammers. What more could a girl want. A part of, or the whole organism (just the legs in the case of my mosquitoes), is placed on the card within the circles shown here, and once full the lips are folded over and you smash the specimens! These in the field can then be preserved in bags of silica and this provides very good quality DNA. These can then either be carried back or sent in the post for storage until DNA analyses are required. This is often very helpful as there are very strict regulations concerning alcohol on planes. They are a proprietarily system though and we do not know what the makeup of the chemicals that are on the cards that breakdown the material and embed the DNA into the tissue. As with all systems that rely on us and an external company there are obvious concerns.
These FTA Cards are one of the many ways that the Collection facilities staff are working alongside researchers and curators to discuss logistics for collection expeditions to help users decide on the most appropriate field processing and procedures for collecting molecular samples in the field. To this end there are compatible consumables such as tubes, ethanol resistant cryomarkers, as well as storage buffers (RNAlater, Analar ethanol etc) as well as the use of dry shippers. These dry shippers, once primed can transport samples at -150oC and have data loggers for temperature monitoring during collection and transportation, thus reducing any possible genetic degradation from the sampling event till they are deposited in the biobank. Previously I have used dry ice to take samples back from abroad which is fine if you are in France but removing samples from Vietnam to the UK is not possible. Dry shippers themselves have their own logistical issues – we were not allowed to put them in the hold of certain airlines and so had to gain special permission.
And finally all of these archival objects that are in cryo-vials have a 2d barcode matrix on the bottom of them to enable them to be added to the main database of the museums collections. We use Ke-Emu for all of our collections management from specimen level identifications, location within building, accession and permit information and our loans system. Since the ratification of the CBD in 93’ there have been increased legislation when it comes to the transfer of genetic material which is only going to become more regulated.
These facilities then will enable us to have long term storage of the DNA and other tissues as well as being able to track them. This is especially important as nowadays there are many restrictions placed upon the sharing of genetic resources and often one of the requirements for permits is the ability to track all subsequent use of the genetic material. So now we have the majority of the entomological material, be it specimens or components of, in stable facilities. We are able to comply with permits and international legislation of genetic material as well as linking the constituent parts of the specimen back to the main part. As ancient DNA techniques become more successful and more of the specimens are sequenced these long term storage facilities will become major repositories in their own right. However, the most simplest change that we can add to our specimens to facilitate future work is to have the names and percentage values of any chemical used to preserve and fix specimens as well as the method of how we say in the UK ‘bumping them off’
Mc alister ecn2013
Molecular storage facilities
and their role in modern
methods in taxonomy
Dr Erica McAlister
Department of Life
Traditional methods for Entomological
• Morphological taxonomy
– Pinned collection
– Slide collection
– Spirit collection
• Zoology specimen
• Wet specimens
• "Wet" specimen collections are stored in 70%
ethanol (EtOH) with various additives after fixing
with 10% formalin. Modern specimens are stored
in screw-top glass jars with polypropylene lids if
they are small or stored in stainless steel tanks if
they are large. Specimens are also stored in 10%
Different spirit preservations and impact on DNA
Adapted from Carter 2003
Fair to good
Problems of the past