What it Takes To Start a Germ-Free Gnotobiotic Isolator Colony: Step #2

What it Takes To Start a Germ-Free Gnotobiotic Isolator Colony: Step #2

Step #2 Major Equipment Selection

The most obvious and arguably most critical piece of equipment in the colony is the isolator. The decision on isolator features is driven by the demands of the project at hand, including animal sourcing and the supplies involved in the research.

There are three basic types of isolators: rigid, flexible film and semi-rigid/ flexible front. As a manufacturer, I am frequently asked which style is best. My simple answer is: it’s mostly personal preference and previous experience. To expand on that, whatever can be done in one type can be done in the other two types. I only manufacture semi-rigid type isolators, but it would be dishonest to say only one style can work!

More important than the chamber type is the port or ports. Some of my clients are using a technique I’ll call small batch sterilization or packs. In this method, supplies like food & bedding are wrapped much like a surgical pack. They are covered in a double layer of surgical cloth, prepared and autoclaved. In this case a large rectangular port is particularly helpful.

A second method is the tried-and-true autoclave cylinders. In this case a 12” or 18” diameter round port will usually suffice. An alternate method is to use irradiated food and bedding. Irradiated packages work most efficiently in a rectangular port. However, that comes with a caveat — typical commercial irradiation is not sufficient for germ-free.

Perhaps the most compelling reason for a round port is the use of germ-free animal (mouse) shippers like those used by Taconic, which require a round port to transfer animals directly into the isolator. Alternatively, transfers can be made in pre-sterilized containers and a clean bench.

For years I was a proponent of rectangular ports. However, with the renewed focus on germ-free mice I’ve modified my view. Why be limited to one port? Why not have one rectangular and one round? A current trend and a majority of the isolators built by Park Bio for germ-free/ gnotobiotic use now have dual ports. Thus far this is unique to the semi-rigid design.

*There are several useful recently published text on this topic. I highly recommend “Gnotobiotics” by Trenton Schoeb and Kathryn Eaton published by Academic Press, 2017. There is an extremely useful chapter by Betty Theriault on starting a germ-free colony.  Another is “Gnotobiotic Mouse Technology” by Chriss Vowles, Natalie Anderson, Kathryn Eaton CRC Press, 2016. Trenton’s text gives extensive coverage of both semi-rigid and flexible film isolators. Chris’ focuses on flexible film, but there is also a wealth of other information in his excellent text!

Keep in mind although some techniques in the references are geared towards either semi-rigid or flexible film, nearly all can be adapted for use in whatever isolator you choose!

I have avoided mentioning IVC (individual ventilated cages). This is a viable solution for short term (less than 45 days) housing of gnotobiotic animals and/ or mono-associated animals. However, it is my opinion that they should never be considered for a foundation/ breeding colony of germ-free animals.

Directly related to the isolator themselves, and in fact a key part of what makes up a modern isolator, are the HEPA filters or in some cases a wrapped filter. The old test method of filtration has been without substantial change since the 1960’s. This method involves a filter body, consisting of a metal perforated core with filter media wrapped around the core, over the perforations, and held in place with clamps/tape.

The most common filter media was for years a fiberglass (pink) media that looks a bit like household insulation. In recent years, a substitute/ replacement for the fiberglass media has been a non-woven synthetic media often referred to as DW4. Wrap filters are not HEPA. They are probably 95-98% efficient at .3 microns. Most commonly wrap filters are validated by whether they are properly autoclaved or in place. Rarely are they validated by particle count.

The alternative to wrap filters are cartridge HEPA, which have around since the 1980’s and used in a variety of application both related to and unrelated to laboratory animals. The advantage with cartridge HEPA is they can (and should) be 100% tested to I. E.S.T. standards. They are true HEPA and not dependent on the technique of the person preparing the core wrapping. Generally the wrap filter is considered by most to be the least expensive. That school of thought can easily be proved wrong if you apply sound cost accounting principles. To break down the steps of both methods:

Wrapped Type

  1. Source and order the DW4 media
  2. Inventory the necessary tape, Mylar and media
  3. Cut the media to length, first though determine 3 wraps or 4
  4. Follow the ten-step set up procedure outline by * Chriss Vowles (see the reference in the text above).
  5. Autoclave
  6. Install in place on the sterilized isolator
  7. Validate

(at least 17 steps and hours of direct labor)

Cartridge Type

  1. Source and order
  2. Install on isolator
  3. Sterilize in place at the same time the isolator body/ chamber
  4. Validate

(Minimal labor)

This covers only the most expensive items needed. For a more complete list of required equipment, please see the textbooks I recommend earlier in this blog installment.

What it Takes To Start a Germ-Free Gnotobiotic Isolator Colony: Step #2

Positive Pressure Racks/ Germ-free/ Gnotobiotic Husbandry in IVC (individually ventilated cages)

The past decade has brought numerous improvements in IVC (individually ventilated cage positive pressure rack systems.) Studies sponsored by leading laboratory animal breeders and top notch research institutes around the world have conducted relatively long term studies and maintained germ-free/ axenic status. I happen to know two of the professionals who conducted the studies and can vouch that they are meticulous, dedicated and amongst the best I have ever met in the business. One is Gil Hecht at the Weizmann Institute in Israel, the other is Randi Lundberg formerly with Taconic.

This leads me to repeat the question that so many people involved in biome and gut flora research have asked, ‘is it necessary to use isolators for long term studies and/ or long term husbandry of stock animals, can we use IVC instead?’ The short answer is yes. An odd comment for the manufacturer of isolators to make! The no answer comes with a few key caveats; does the institute have prior germ-free experience, do we have the requisite and adequate staff (with both aptitude and training) , have we identified the level of risk are our researchers willing to take.

Not every institute has the resources of Weizmann or of a large commercial breeder like Taconic. Researchers are faced with minimal support from their university and are therefore dependent on grants. The end result is underfunded, under staffed germ-free facilities and under trained technicians. That is where the benefit of isolators shine. Although they still require trained staff they are less technique dependent and able to run well in lean funding situations! Many facilities start with just 1 or 2 isolators. A total commitment of less than 50 cages and under twenty thousand dollars in equipment cost. Even at that level there is redundancy so research can continue even if 1 isolator was contaminated.

The one constant even in the face of recent IVC improvements is they are inherently technique dependent. The other is that despite the success that both Weizmann and Taconic have had in Rack systems (IVC) they still maintain large isolator facilities. The reason, one size does not fit all. For some the rack systems are the answer for others traditional isolators work best. A third factor is that isolators are still the highest level of biosecurity available. That is across all manufacturers’ regards of isolator brand. Any isolator is a higher level security than any positive pressure rack system (IVC).

Some manufacturers of isolator offer products that have changed very little since the 1980’s. I supposed you could glean they are tried and true, ‘why fix what isn’t broken’. I deviate from that norm in that customer feedback should drive design and constantly seek to find the better ways to approach the same task. At Park Bioservices we were faced with clients wanting to interface with the Taconic germ-free shipper and or autoclave cylinders. We had long contended that our rectangular ports were easier to use than the old traditional round ports. However round was better for ease of connecting with shippers and cylinder. Our great improvement (at least I thought so) were port adapters. They clamped onto rectangular ports and allowed the germ-free shipper to attach. Problem solved? The answer from clients was a resounding NO! We had to rethink our strategy. This led us to offering isolators with 2 ports, one round the other rectangular, one isolator with the advantage of both ports. This was the optimal solution and by all reports from customers a winner.

Let me end here by saying that of the alternatives available today isolators are the most flexible (no pun intended). They can handle from 4 cages to a 100 plus cages and be configured as needed by the facility/ institution.

Aticles referenced with permission of the authors.
The Randi Lundberg article.
The Gil Hecht Article

What it Takes To Start a Germ-Free Gnotobiotic Isolator Colony: Step #2

What it Takes to Start a Germ-Free Gnotobiotic Isolator Colony: Step #1

Step #1: Time and That One Key Person to Manage it Time. To put it in one word, starting a germ-free gnotobiotic isolator colony, takes time — lots and lots of time. From planning, to mustering the resources together, designing, and obtaining funding, it is time that is the principal component of the process. I’ll deviate from my overly simplistic description to elaborate a bit. It is fundamental and absolutely necessary to commit to having full time staffing for the creation of any germ-free isolator. It cannot be managed by part-time staff or graduate students. To be successful requires hiring one key person who will run the operations. As the primary investigator, finding that person should almost immediately follow your initial impetus to start such a project. Not only will this person be insurmountably helpful once the project begins, but he/she should also be there to plan what equipment is needed, hire staff (if more than a tiny facility is planned), make sure enough space and resources are allocated, and to layout the work flow pattern. This key person is obsessive in the pursuit of perfection. To the beginner, the uninitiated, this person appears to be wasting time on the smallest of details. To a big picture person, the meticulousness is painful to watch, but, in this industry, it’s the small oversights that lead to failure. Maintenance and routine observation of minutia that may escape a researcher being pulled in 20 different directions should be the purview of this key person. They will see the pinhole leak, the autoclave cycle that failed, the leaky cap, or the faulty door gasket. Make no mistake: this sucks a person’s time like the vortex of a black hole, but it will make or break your success. For the investigator who is busy with the high-level science, writing, teaching, and attending meetings, this will all go unseen. It doesn’t take a village to start a germ-free isolator colony, but it does take at least one fastidious, detail oriented, and impossibly well-organized individual. This person might be difficult because they have a tendency not to delegate tasks and do everything themselves. This type of person isn’t easily satisfied. If they have staff working under them, they watch them as if under a microscope. From a manufacturers point of view, I know this type well. They send hand schematics of what they want, they follow up again and again, and they ask so many questions it’s impossible to keep track without a score card. They demand top quality and quick resolutions to their issues. I get messages all hours of the day and night, leaving me wondering when they sleep. They drive me crazy with their relentless pursuit of perfection. In short, they take nothing for granted. Undoubtedly, they make it difficult, but much less difficult than trying to resolve an issue once an error has shipped. If you’re planning a lab and you don’t have the person I described, start the search process now! Step #2: to be continued…