[EricHugo]

Fixation instructions

I have pasted a bit of text on fixation below from Histotechnique for information purposes. The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues or soon after death to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated.

Formalin is used for all routine tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly.

There are two major groups of fixatives for the work required in this project, classified according to mechanism of action:

* Aldehydes
* Alcohols

Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin, although for coral tissues, 10% formalin in seawater will work. There are a number of available fixatives that are better, including a modified Helley’s solution, paraformaledhyde recipes and a brand called Z-fix, that are especially good for coral tissue. If anyone has access to these chemicals, let me know via email because this would be the best for most of the histology assays I will use. I can provide detailed instructions if this is something you can do.

Glutaraldehyde fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde. I do not expect to have to do EM at this point, although I might in the future. However, I can use tissue from affected live corals and really don’t want anyone unskilled to use glutaraldehyde. It is pretty nasty stuff.

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are not sectioned, there is no problem from induced brittleness. Because coral tissue is only two cell layers thick, alcohol works. It is difficult to work with the tissue later on, and some assays cannot be performed if alcohol is used as a fixative, but I will be able to get some results if they are used. They are the most easily available for most persons. Ideally, ethanol should be used by purchasing small bottles of pure grain alcohol from a liquor store and diluting it to 70% with freshly made artificial seawater. I would use about 1/4 of a teaspoon of sodium bicarbonate (baking soda) in the mix, as well, to act as a buffer, if the fixative volume is around 250-500ml (8-16 ounces).

The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Because corals have skeletons, this must be included since the fixative will soak into porous skeleton and be unavailable to penetrate coral tissue.

Sources for materials

90% rubbing (isopropyl) alcohol for surface sterilization is available at most major drugstores.

70% rubbing (isopropyl) alcohol for fixation and less effective surface sterilization is available at drugstores, supermarkets, convenience stores, etc.

99-100% ethanol is available as pure grain alchohol from liquor stores. It is often sold under the name Everclear. Ethanol is worth the effort to acquire, though it costs more than rubbing alcohol. It is an extremely good surface sterilizer, evaporates very quickly, is much more ideal for fixation of tissues, and is relatively non-toxic to humans and organisms in the aquarium.

Sterile swabs, syringes and containers are available at some drugstores and laboratory supply houses. Most veterinarians or your physician will likely be willing to part with some, too, if you ask them.

Please contact me by email for further inquiries.

Support for the study

If you cannot contribute coral material but want to support the study by helping to pay for the costs involved in the study of the condition, you may make donations to the Elegance Coral Project Fund. Please send donations in the form of check, money order, or electronic payment to the following address. Funds will be maintained and used as needed by drawing from the account. Any unused funds over 10% of the donation total will be refunded if the research is completed without using all the funding. Refunding 10% of donation totals will be cost and labor prohibitive, and this remaining money will either be saved for any future projects or donated to the investigator for his efforts in the project, to be designated by the request of the donating party.

I am currently working on establishing the fund, and will post the information shortly.

Expected costs

The costs of this initial phase of the project will be directly related to the amount of sample material obtained. Materials costs may vary depending on what is already available to the party. My estimates of average costs per sample are:

1. Cost of obtaining the coral if it is purchased. 25-75
2. Materials required to collect and ship the coral 2-20
3. Shipping costs 15-20
4. Aquarium facilities for gross etiological description 500
5. Chemicals and disposable supplies for sample preparation 5-10
6. Histological services
(2.00 per slide, 6-10 sections stained and unstained) 12-20
7. Prepared sample shipping costs 3-5
7. Laboratory supplies and equipment for analyses of samples (may be
highly variable depending on what is found after microscopic
examination) 10-50
9. Fees for additional researcher expertise/consultation 0-???

It is conceivable, though very unlikely, that the per sample cost if a coral is provided for free, all materials and shipping are available or free, and that the cause of this condition is relatively obvious, that the project could be completed for the cost of the aquarium facilities (unless donated) plus $500.00.

I suspect this initial phase of the project, hopefully successful and conclusive, and excluding the costs associated with obtaining the material for a good sample size of 50 corals, will cost around $3500.00. I think this would be a reasonable goal to try and reach in terms of initial funding.

Absolutely Required Information on All Contributions
(except financial only, which is voluntary)

1. Contact information including name, address, email (phone number optional but highly recommended if I need to talk with you quickly)
2. Tank Information
a. Size
b. Equipment used
c. Maintenance routine, including water changes and salt brand, and any major changes in brands or routines implemented during the ownership of the affected coral.
d. Additives, including food, supplements, and medications, and any major changes in brands or routines implemented during the ownership of the affected coral.
e. Water parameters, including but not limited to pH, alkalinity, ammonia, nitrate, phosphate, calcium, temperature, salinity or specific gravity, and any major changes in brands or routines implemented during the ownership of the affected coral.
f. Length of time the tank has been set-up
g. Any unusual problems or events that may have contributed to this condition.
3. Coral Information
a. How long the coral has been in the current tank (date of acquisition)
b. If alive, how long did it take before signs of the condition were noticed
c. If dead, how long did the coral live before it died. Please include the length of time it appeared healthy, and the length of time it looked diseased.
d. Location where it was purchased or obtained
e. A written description of the condition and any accompanying photographs
f. Any changes in the signs of the condition over the time the coral was in the tank.
g. Any other background information on the coral that is known (Fiji, Indonesia, in three other tanks before the current one, was dropped on the floor by accident, etc.).
4. If this is not the first Catalaphyllia owned, please provide the information above for others and their fate.
5. The date when the coral was removed and sent in for this study.

Example:

Joe Aquarist
15 Griggs Street
Houston, TX 77252
713-555-1111
email: jaqua@earthlink.net

- 75 gallon tank
- 2 years 7 months in operation
- 4 MaxiJet 1200 powerheads, 20 gallon sump, ETS Gemini skimmer, Mag 5 return, 2 x 175w 6500K metal halide, 4” CaribSea oolitic sand bed, 65lbs live rock, Red Sea ozonizer, 1 liter ESV carbon changed every six months
- Kent Calcium 100 ml/week
- Kent Strontium 20 ml/week
- Brine shrimp – approx 1 tablespoon per day
- DT phytoplankton, 50 ml/week
- Red Slime Remover used once, 50 mg, 10/2003
- 10% water change every week using Instant Ocean salt. Used Reef Crystals for the first year.
- Ph stable 8.0-8.2
- Alkalinity stable at 3.5 meq/l
- Calcium varies between 350-450ppm
- Ammonia unmeasurable since week 4 of the tank
- Nitrate was 5.0 ppm but has been 1.0ppm for past three months
- Phosphate levels stable at 0.50 ppm
- Specific gravity always maintained at 1.025
- Temperature varies: 78F in winter, 84F in summer
Notes: this coral was stung by an anemone six months ago, and bleached four months ago. The condition appeared prior to either of these events. I also added a lot of sand to the tank three months ago and lost three other corals at the time.

This elegance coral was acquired from Joe’s Fish Store in Memphis Tennessee in June, 2003. It looked normal when I bought it, but developed a swollen disk and shrunken tentacles two weeks after I put it in the tank. I dipped it in freshwater but it didn’t change the conditions. I tried treating it with Maracyn, but again no effect. In August, 2003 the entire coral appeared shrunken, and a white web formed on the surface about two weeks after it began to shrink. The coral died in the first week of September, 2003. I purchased another one from online corals, inc (www.sickcorals.com) two weeks ago, and it arrived with the swollen condition. They told me this coral came from an exporter in Jakarta. It is still alive and its appearance is unchanged. I am sending this coral alive to you according to the information provided on the project page. I have emailed you photos of the coral taken on January 25, 2004 and provided the information requested. I will remove and send this coral to you on February 6, 2004 as per instructions.