Emergency Section Koi Health Sticky 2010
The Koi Health Sticky has been updated August 9, 2010.
Welcome to the Koiphen Emergency Section! If you are reading this, chances are you have a sick fish or worse; several of them. I hope you will take the time to seriously study their problems and find a way to successfully treat them. That is what this section and this "Sticky" is all about.
The information given here is based on many year's experience; the experience of koi keepers, wholesalers, breeders and importers. In most cases you will be given some treatment options. You must do your homework, form your own opinion and do what is best for your fish in your situation. Once you have established a treatment program follow it through to the end unless it becomes obvious that a change is needed. Keep an open mind. Treatments have and will continue to change as we travel the journey of the koi kichi.
If you have not done so, it would also be helpful to establish a relationship with your local veterinarian. Doing so immediately may help in obtaining some local advice as well as antibiotics if needed. Once again, welcome. You've come to a very interesting place!
Water Quality
Water quality is your first line of defense in keeping your fish(koi/goldfish)healthy.
All fish have immune systems and will protect themselves when the water quality is excellent and consistent. Your pond and quarantine tank water should be tested frequently to insure the quality of the water and thus the health of the fish.
Stability of Ph:
Test the Ph of your tap or well water. Test the Ph of your pond. Make comparisons over a few days to establish what Ph your pond water should be on average. Pond Ph may be 7.0 to 9.0 but should be stable in a range of .3. If your pond tests at 7.5 it should maintain that level or similar to a low of 7.2 and a high of 7.8 over 24 to 48 hours.
Ph crashes are generally caused by a low level of carbonate hardness. Carbonate hardness or Kh levels above 100 will prevent such Ph crashes and protect the health of your fish. Kh levels of 200 are required for bead filters.
Stability of carbonate hardness(Kh):
Carbonate Hardness = Total Alkalinity. Test the Kh of the tap or well water. Test the Kh of the pond. Make comparisons over a few weeks to establish your Kh average. Kh is the buffering ability of your water to hold the Ph at a safe level. Kh should test no less that 100. In the event of low Kh readings baking soda works exceedingly well to maintain proper levels, and can be added without fear of harming the fish. In addition crushed oyster shells or crushed coral can be bagged and placed in filters or in water current areas to buffer the water on a long term basis. Just remember that shells and coral are "slow release" and a fairly large quanitiy must be used for any effect at all. Baking soda: See instructions below.
One further note: If you have a bead filter in your filtration system, the Kh must be kept at 200 ppm or more for it to function properly.
Baking soda: 2 to 3lbs per 1000 gallons will raise and stabilize Kh levels and keep the Ph at 8.3 to 8.4. Raise your Ph/Kh slowly over several days. Use maintenance doses of baking soda to keep them at a desired level. Lowering Ph shouldn't be necessary.
Water quality in general insures pond health. Establish a baseline by testing your "raw or tap water". Then periodically test your pond water. In the event of a koi behavioral problem or fatality testing is very important. In addition, water samples may need to be sent to an outside lab for heavy metal testing.
Water test kits should include Ammonia, Nitrite, Nitrate, Ph, General Hardness, and Carbonate Hardness at a minimum. If you post on this forum with sick fish issues you will be asked for your test results. We will need them to make a proper recommendation for treatment.
Water Tests and Associated Information
You will be asked for:
Raw water test parameters(water you use to change or add water to the pond):
Ammonia
Nitrite
Nitrate
GH-general hardness
Kh-carbonate hardness or total alkalinity
PH
City or well water
Pond Parameters:
Ammonia
Nitrite
Nitrate
GH-general hardness
KH-carbonate hardness or total alkalinity
PH
Pond temperature.
Ideal Water Parameter Guidelines
Source water varies greatly so make sure that it is tested to give you a base line to work with. Pond water can vary depending on the load, the filtration type and how well the water is managed including water changes.
Here are some guidelines: Although your pond may not fit exactly within them it is important to note that consistency is equal if not more important than the test results themselves.
pH: Between 7.2 to 8.3 but consistent and not a lot of swing.
Ammonia: "0". Always. If you are using a formaldehyde product to treat your koi the ammonia reading will be high as formaldehyde reads as ammonia for about 24 hours. Ammonia can cause the fins to become frayed, and the fish may begin to gasp at the surface. Ammonia can cause bacterial infections to develop and of course eventually will kill the koi. It needs to be monitored closely.
Nitrite: "0". Always. Small amounts of nitrite is not dangerous, but as a filter system cycles it may become intolerable to the koi and they will develop "brown blood disease" as they will "absorb it" rather than the oxygen they need so nitrite needs to be monitored closely.
Nitrate: "0". Almost never is. A trace is fine. More than that do a water change. Nitrate is called the silent killer. It is usually not monitored very well by the average koi keeper. If the levels slowly become high due to lack of water changes and testing it will cause ulcer disease and death.
KH: Carbonate hardness/Total Alkalinity: 100 to 200ppm. About 200ppm if using bead filters. KH stabilizes(buffers) the pH and prevents any crashes. Baking soda works well for this if dosed regularly.
GH: General Hardness is measured in grains or ppm. "0" hardness is soft water. Although there are those who might disagree, any hardness IS hardwater. It's just a matter of degree. Although hardness can be altered, it can be expensive and it can reduce the overall stability of the pond. Better to monitor the GH level and refrain from trying to tinker with it.
Other tests worth looking at:
Dissolved Oxygen
Iron
Phosphates
I'm sure there are others.
In addition you'll need to tell us about your filtration system and anything else you feel might help. New fish? Heavy rains?
What behaviors makes you think there is a problem?
Please post pictures of you pond and fish.
Hydrogen Peroxide for Algae Control
Algae control in ornamental ponds took a turn for the better with the introduction of 27% hydrogen peroxide used as an oxidizer for all types of algae. Used correctly, it safely controls algae growth giving pond owners a simple inexpensive method to use even with koi present.
27% Hydrogen peroxide is dangerous! Wear protective gloves and clothing! Wear goggles/eye protection! Touching a drop of this material will turn your skin white! It will burn! Store this chemical in a cool dry place, secure the container cover and place the container in a metal can with secure cover. Read all label safety instructions! Used properly and stored safely 27% hydrogen peroxide does the job on algae.
Some guidelines:
Algae must be present. (any type of algae which you want to control)
Dosing is one cup (8 ounces) per 1000 gallons. This is a starting point. Increase dosage slightly only if algae remains present. Dosage is every three days. Once you have established the amount needed for your pond, then back off until the algae begins to reappear. The objective is not to eradicate it but to keep it in check.
Where streams or water fall areas need to be treated mix one quart of hydrogen peroxide with two quarts of water and place in a pump up sprayer that has not been used for weed killer. Turn off the water flow and spray the areas thoroughly. Let stand for 20 minutes. Restart water.
Hydrogen peroxide is available at local pool stores. It is known as Baquacil. The pool people will tell you that it will kill the fish. This is true only if you overdose or treat a pond that has little if any algae.
I have used and continue to use Baquacil with a very expensive collection of koi. I know of several hobbiests using it successfully. But these are all careful people who pay attention to the presence of algae and the proper dosage. Use this method at your own risk. Done correctly it is indeed safe for fish.
Water changes, filtration and aeration:
Consistent and frequent water changes are required for nearly every pond, quarantine tank or aquarium. 10% to 20% weekly water changes are considered customary depending on the number and size of the fish and the size of the pond. This is also the only way besides filtration to reduce the concentration of contaminants in the pond, especially dissolved organics and nitrates. Recently, several types of freshwater foam fractionators have been introduced which do a nice job of reducing DOCs. Whether DIY or commercial, they all seem to work pretty well.
Almost any pond with fish needs adequate filtration as well as aeration for the health of the fish. These two issues must be addressed but the type of filtration and how aeration is accomplished is better considered on the Pond Construction Section here at Koiphen. However, if you have sick fish issues and you post on this forum you will be asked to describe your system.
Water changes usually means the addition of at least a dechlorinator as most mains water contains at least some chlorine and/or chloramine. Treat accordingly and dose it at the same time in the same manner every time so that it becomes a habit and is not forgotten. Sodium thiosulfate crystals are a very inexpensive way to dechlorinate. Mixing 1 and 1/2 lbs of the crystals with one gallon of water will yield enough dechlorinator to do about 85,000 gallons.
Water changes means actually pumping or siphoning out a percentage of the existing pond water-not just adding to it-and making sure the new water TEMPERATURE matches that of the pond or tank involved.
TEST KIT MANUFACTURERS AND RESELLERS:
Hach Chemical Company
http://www.Hach.com
Hach Company
P.O. Box 389
Loveland, CO. 80539
1-800-227-4224
1-970-669-3050
LaMotte Company
P.O. Box 329
802 Washington Avenue
Chestertown, MD. 26120
1-800-344-3100
1-410-778-3100
Taylor Technologies, Inc.
31 Loveton Circle
Sparks, MD. 21152-9206
1-800-TESTKIT
All three of the above named companies are known for their excellent test kits. If you want to use the best and protect your wet investments call one of them and "get it started". They will all use total alkalinity test kits as Kh or carbonate hardness.
Aquarium Pharmaceuticals and Tetra test kits are available at most pet shops and pond suppliers.
Note: If the test kit looks old or dusty, it has probably been on the shelf far too long and is outdated. Check for dating before you buy. Liquid reagents have a shelf life of one year. Powdered reagents have a shelf life of up to four years.
Some Common Abbreviations:
Kh=carbonate hardness, or for many hobbiests, total alkalinity. It is usually measured in PPM.
PPM=parts per million.
GH=general hardness of your tap or pond water. It is measured in grains or in PPM
PP=potassium permangante(KMn04). A strong oxidizer. It is used for parasite control and reduction of organics.
FMG=formaldehyde and malachite green. ProformC is a common trade name. It is used for parasite control. The product will read as "ammonia" if you test the water.
ANN=ammonia, nitrite and nitrate. The primary tests for water problems.
IP=intraperitoneal injection. The injection site in front of the vent behind the ventral fins and just off to the side of an imaginary "centerline", at approximately 15 degrees with the needle pointed toward the head.
IM=Injection site in the muscular region of the body either side of the dorsal. Not for the untrained.
SAP=Saprolegnia. The genus of water molds responsible for significant fungal infections of freshwater fish and eggs.
ST=sodium thiosulfate. A dechlorinator commonly in crystal form but also in liquid. Goes by many trade names but most do about the same thing.
DIY=do it yourself.
GFCI=A moisture sensitive circuit breaker. A very good thing to have in the electrical box for pond use. In most areas it is code. Another device commonly used is a GFCI receptacle. This is the wall mounted one which you will commonly find in the bathrooms of many homes. It has a reset button on it. These are known for their short lifespan and the inability to hold a circuit with age.
Scrape and Scope=A procedure which can help identify parasites with a microscope. A microscope is equally as valuable as medications, perfect water conditions, and your most expensive fish. Expect to pay about $400.00 for one which can be used to identify parasites.
FPH=Frogs Per Hour. Skimmers trap them and the pump can grind them up. Try to screen your pumps adequately to prevent his from happening!
Behavioral Symptoms Which Should Send The Alarm:
There are many unusual behaviors which can cause concern. Observe for those which repeatedly occur. Isolating from the rest of the herd, laying on the bottom especially if the fins are clamped to the body, a head down attutude, a headup attitude, dark pigmentation, sandpaper-like skin, breaching(partially jumping above the water surface), repeatedly flashing or rubbing against the pond floor or walls, a slimy-white excess slime coat, not eating, spitting, coughing, shaking of the head while swimming in "reverse" and so forth. These observations must be relayed along with any other pertinent information. Be assured we'll ask whether you've acquired any new fish and if they were quarantined.
Quarantine?
If you haven't and you're reading this it's probably too late. The next time you acquire a new fish it should be quarantined for at least six to eight weeks in water from your pond or from your mains supply. In addition it is always good to place one of your present collection with the newcomer. A buddy is always good, and it insures that the existing population will not be adversly affected by the newcomer's bugs. I assure you, they will have bugs.
Quarantine tanks are a necessity. Many people sadly relate their experiences of putting a new fish in with the existing population and watching as they all begin to behave in unusual ways and one by one begin to die. During quarantine, many collectors wisely treat their new arrivals as if they have every parasite in the book. By doing this, they reduce if not eliminate introducing parasites to their collection and more importantly reduce if not eliminate the potential for bacterial ulcer disease.
Here's a setup which can go in the garage, or the basement. Pearls of Paradise has two sizes of collapsible show tanks. Setting up one of these takes just a few minutes, and it can be left up until the new fish(and a buddy from the existing population)are introduced back into the main herd. It needs to have a filtration system, and I've had very good luck simply using a 55gallon drum as a trickle filter filled with floor stripping pads or spiral cut pvc.(EasyClean). Bulkhead assemblies, valves and pvc pipe for piping through the drum as well as fernco adapters(rubber clamp devices) are available at any farm store and most of the big box hardware stores and Aquatic Eco Systems(Florida). A 500 to 1000gpm submersible pump will handle that size filter very well, turning over the system frequently. Some of the big box stores have very inexpensive collapsible swimming pools which also work well as quarantine tanks. Just remember to cover them.
If you'd like something a bit more permanent as well as very strong, the bulk water tanks at the farm stores work very well also. In either case make sure you cover them securely with netting or plastic. Fish in unfamiliar territory and water will jump!
20 x 48" 4 Mil Industrial Poly Bags for transporting your fish are available through Uline at 1-800-958-5463. All sizes available.
Parasites Common To Koi And Other Ornamental Fish:
A microscope is needed to view these:
Ichthyobodo Necatrix - Costia----------300X-400X:
Costia reproduces quickly when conditions are right. Reproduction is by binary fission. The symptoms are varied. Lethargic, disoriented, dark pigmentation, clamped fins, redening at the base of the dorsal, thin slime coat or sandpaper like feel are all indicators. More advanced cases will produce a notch above the mouth and the eyes will become sunken. Infections of costia commonly occur around the base of the dorsal fin.
Under the microscope it looks somewhat teardrop shaped and moves with the irregularity of bumper cars at the county fair. Cell streams are sometimes mistaken for costia. Cell streams all move in the same direction. They vary in speed but are not to be mistaken for costia.
Costia is easily eradicated by applying heat. At 87*F. Costia is immobilized and can no longer reproduce. It only takes a few days for all to die. Unfortunately most pond settings do not allow for heat to applied to this degree. For those situations ProformC will work well. It is a formaldehyde and malachite green solution safe enough to be shipped without HazMat fees and is quite effective. The dosage may be increased slightly without fear to allow for errors in calculating the gallonage of the pond. Prior to dosing a 25 to 50% water change is advised to reduce the organics and improve performance of the chemical.
Formaldehyde(37%)and malachite green may also be made from stock ingredients. But beware! This is not something to take lightly. Neither chemical are particularly friendly to your eyes, nose, mouth or skin. The formula is listed below. Formaldehyde will read as ammonia during the first 24 hours of treatment.
Chilodonella -Chilo--------------------200X:
Chilodonella has a distinctive round shape. It moves like the mother ship in space. Fairly slow under the scope, and cilliates wave from it's outer ring.
Chilo is easily eradicated with salt at .6%. This salt treatment has an overall duration of 14 days. There are no ill effects using salt at this level. After the treatment is complete a series of water changes should be completed to slowly reduce the salt level to just that of the raw(incoming)water.
Chilo may also be treated as above (ProformC) for Costia. Either treatment will be effective but in the case of formaldehyde it is important that the organics(dirty water-poor water quality)be removed somewhat with a minimum 25% water change to improve the performance of the chemical.
Trichodina -tricky---------------------200X:
ProformC/malachite green treatments work well.
Ichthyophthirius multifilils -ick----------200X:
Known as white spot. Easily seen on the body with the naked eye. The problem often experienced with koi is that it is only on the gills and so is not observed except for symptoms of flashing like so many other parasite infestations. Salt at .6% along with an increase in temperatures usually is enough to eradicate them. ProformC also is an effective treatment with an increase in temperature. Ich also is known to become quite active during barometric swings. The Florida fish farmers nearly always treat their fish for Ich following a strong thunderstorm.
Epistylis-------------------------------200x Under Construction
Oodinium------------------------------200X Under Construction
Hexamita------------------------------200X:
Hexamita Specific Treatment
Make your own Hexamita specific medicated food using Metronidazole:
Using a medium size bowl, mix 1/2 teaspoon of knox Gelatin in 1/2 cup of warm water. Then add 1/2 teaspoon of Metronidazole powder. Mix thoroughly. Then add one pound koi food pellets into the mixing bowl. Mix this concoction thoroughly. Pour this mixture onto waxpaper and let completely air dry. Overnight drying should be sufficient. Store your new medicated food in air tight bags and refrigerate.
Feed this medicated food ONLY(no other foods) for 10 days. Feed generously, but make sure the food is gone in about five minutes or less.
Note: If you wish you can make a fecal squash for microscopy both before and after the feeding protocol to insure success. Otherwise, regard this as a shotgun type treatment and observe the fecal waste of the fish for brown (normal) segments.
Metronidazole powder is available at most of your koi dealers.
Dactylogyrus - gill flukes---------------100X:
This parasite as well as skin flukes rarely kills koi. However it is a known carrier of aeomonas and pseudmonas bacteria which it injects while feeding on the gills and body of the fish, and if left untreated that WILL kill koi.
It is easily seen at minimum magnification and is distinctive due to it's eye spots and it's stretching and contracting of the body while feeding. Gill flukes are egglayers. Colder water means a longer gestation period. Most of the time I find that treating for a period of four weeks is not out of the question and in fact is necessary to "catch" those eggs which must be hatched to accomplish control if not complete eradication.
Treatments are indicated below. Praziquantel is very effective but the water must be in the mid-seventies to improve the medication's abilities. Some say that potassium permanganate will work. I believe it is more correct to say that it may temporarily knock them back but they will recover. Salt has little effect.
Gyrodactylus - skin flukes--------------100X:
Skin flukes are similar to gill flukes but lack the distinctive eye spots. They may be named skin flukes but they often appear in the gills as well. These are live bearers and so their life cycle is somewhat shorter and they seem to be easier to eradicate. Nevertheless they are dangerous simply because they are carriers just like gill flukes.
Treatments are as above with gill flukes and that is further described below.
A scrape and scope is needed to find flukes. The most likely area to find them is on the gills. Take two samples from each side of the gills. One from the upper edge and one from the lower edge. It is not uncommon to find them only on one side and sometimes on the upper or lower area only. To say that you've only found one or two so the infestation is not severe is simply not correct. When you consider the tiny sample if you find a couple, just imagine how many more there probably are.
Argulus - fish lice---------------close visual inspection/Diflubenzuron, several trade names for this.
Lernaea - anchor worm---------close visual inspection/Diflubenzuron
At best, parasites open the doors for bacterial infection. At worst, they are carriers of it. That is why it is so important to try to identify them and treat for them properly.
While treating for parasites is very important, treating the resultant ulcerated area is equally important. Some will say to use hydrogen peroxide, then iodine, then dental adhesive to cover the wound. I have no argument with using all three, if they are available and you're proficient treating with all of them so the fish is not left on the table too long. Most people are not that proficient. I've treated many many koi and until recently the only topical I've ever used was iodine. It is extremely effective. Swab the wound thoroughly making sure to get underneath the skin and/or scaled edges. Remove any loose scales during this treatment as they will fall off anyway. This is generally a one time treatment. Further swabbing if any healing is evident will injure the new tissue which is slowly forming around the wound(pale pink to white in color). Further deterioration of the ulcered area after a thorough swabbing indicates the need for a more accurate parasite discovery or a more accurate dose of antibiotic or a change in the antibiotic used.
An alternative to iodine is gentian violet. This seems to work equally well and since it initially colors the area and then fades as it wears off is equally as valuable as iodine as an indicator of healing. Gentian Violet is available as a non-prescription med at most Walgreen/CVS pharmacies.
The third method which I have used and have had some good success is a mix of Tricide Neo and Silverdene(a burn cream). This mixture seems to work quite well. Silverdene is a prescription product. Work this mixture in slowly and allow it to "stand" for several minutes. Then rinse before reviving.
A fourth method is a drop of water on a tiny number of potassium permanganate crystals is only suggested if everything else seems to have failed. This not only kills any bacteria in the area but has a detrimental effect on the flesh which it touches. It kills tissue in the area, so be careful how far you swab with this. I have used it in extremely difficult cases where it becomes a kill or cure situation. I have had success with this. I have only used it after I've tried everything else. The area treated takes up to several months to recover where areas treated with iodine or gentian violet are a few weeks.
Potassium Permanganate is an Oxidizer! It is a dangerous chemical is used improperly! Use protective clothing and gloves.Wear Goggles/eye protection!
1. Iodine first.
2. Gentian Violet second.
3. Tricide Neo and Silverdene third.
4. Potassium Permanganate fourth.
Swabbing is generally done with Q-tips. I have used a super sharp sewing tweezers to apply PP under the scales where there were pus blisters evident.
STOCKING THE MEDICINE CHEST FOR GENERAL PARASITE CONTROL:
To start with, a plastic tool box from a big box store will do for keeping all your supplies in one place.
Here's a list which will get you started:
1. Sharp tweasers.
2. Blunt heavy duty tweasers.
3. Small sharp scissors.
4. Scraping device such as exacto knives.
4a. Cotton bath/hand towels used wet in a flat pan or tote lid-holds the fish nicely.
5. Q-tips.
6. Bottle of Iodine.
6a. Clove Oil for sedation.
7. Bottle of Gentian Violet.
8. Small packet of Tricide Neo used as a paste with 8a.
8a. Silverdene-a burn cream which aids healing when mixed with Tricide Neo as a paste.
9. Nitrile, plastic or latex gloves.
9a. Suturing kits for split fins.
10. Praziquantel (flukes treatment).
11. ProformC (other microscopic parasite treatment).
12. Dimilin/diflubenzuron (fish lice and anchor worm treatment).
13. Digital Gram scale (eBay-Oldwillknott site).
14. Small cups for measuring.
15. Assorted syringes(no needles)for measuring(10-60CC).
16. Thermometers(aquarium style-at least two).
17. Test kits-Ammonia, Nitrite, Nitrate, Salt, Ph, GH, Kh.
18. Hydrogen peroxide.
19. Oxolinic acid for short baths.
19a. ChloramineT for bacterial gill infections used as a short bath.
20. Flashlight and a new pack of batteries.
21. Anything else....a microscope would be very valuable.
Medications:
Be aware that there are companies and individuals who are marketing remedies which purport immediate healing just by dumping it into the pond. The labels will say it will cure just about anything and smell good doing it. Label claims such as this should make you wary; for good reason. These products generally don't work. Yet they are packaged and sold in nearly every garden store across this country. Go figure. If you find a product which states it will do just about everything, don't count on it.
KMno4 - Potassium Permanganate:
Potassium permanganate is capable of doing great things in terms of parasite control, but it is also capable of rapidly killing all your fish if used improperly. This is a poison. The "dip", which is described further on in the Sticky is very effective and is fairly safe because of it's precise nature. PP, when used in a pond setting is only as good as the person using it. Too strong and the fish will suffer and die. Too weak and the parasites will laugh at it. PP is an oxidizer! Wear protective clothing/gloves! Wear eye protection! Wear breathing mask!
For it to be effective in a pond the pond itself must be in fairly good shape, clean and free from organics(waste-plant debris etc.). The pond capacity(gallons) must be known. No guesswork on this.
For microscopic parasites excluding flukes use 2ppm KMNo4(potassium permanganate) in a clean pond. Two consecutive days and do not reverse the chemical. Add aeration. Be prepared to reverse the treatment with dechlorinator if you notice the fish in obvious discomfort. Before you do this treatment for the first time it would be wise to discuss it on this forum. Start a thread.
The Dip
A dip of 8 grams KMN04 per 40 litres of mid-seventy degree water can be used but for no more than 2.5 minutes. Precise measurement is critical. Aerate. This dip requires several containers as well as an uncontaminated pond or q-tank to place the treated specimens. Pre-dip and post-dip containers are a must. Make sure they can be covered securely. This is a bit over 200ppm! Dips only work if the container(pond)the treated fish are being returned to is parasite free.
A gram scale is required to measure this chemical. Please calibrate it frequently.
For 1000 US gallons here is a brief chart:
PPM---------------Grams needed:
1.0----------------3.785
2.0----------------7.570
3.0---------------11.355
4.0---------------15.140
Any dechlorinator or hydrogen peroxide will reverse this chemical. Make sure you have enough to neutralize at least twice the amount used.
ProformC or Formaldehyde and Malachite Green:
This treatment is safe at temperatures of 50*F to 80*F.
FMG is effective on all microscopic parasites except flukes. This is a poison. It is dosed in amounts which will effectively kill the parasites without killing the host. Overdosing may damage or kill your fish. Formaldehyde is generally available from farm supply stores. It is 37% formaldehyde and 11~12% methanol. Commercial mixes such as ProformC are available from pond and koi suppliers. While it is a bit weaker it still works well. It also gives the end user a larger margin for error. It is 22% formaldehyde and about 7.5% methanol. Formaldehyde is a dangerous chemical. Wear protective clothing/gloves! Wear eye protection!
The following treatment is suggested by many: Three treatments, daily with 25% water changes before each treatment. On the third dose combine ProformC and Praziquantel and allow to stand for seven days Another treatment should be considered after seven days AFTER a 25% water change. See the paragraph below for Fluke Specific treatment. A small percentage of salt in the water will not affect the treatment or the fish. This treatment is for all parasites and it is temperature/life cycle dependent. Flukes can take 30 days to eradicate if the water is in the low sixties. This takes several retreatments. Warmer waters reduces the time frame.
Malachite Green Stock Solution
15 grams makes one gallon. Thereafter use one drop per gallon. This is a great Ich treatment when combined with increased temperature for a few days. It has been used for many years by accomplished aquarists on their collections as well as fish farmers in Florida. For those with planted aquaria or ponds the plants seem to suffer no harm. Malachite Green should only be used/mixed when wearing eye protection, a good breathing mask and protective clothing! Malachite Green dust easily migrates with air movement. Be careful!
Praziquantel: Gill and Skin Flukes:
A trematodicide. Praziquantel induces a rapid contraction of schistosomes(a worm or fluke)by a specific effect on the permeability of the cell membrane. The drug further causes vacuolization(causes more and larger spaces or cavities within each individual cell)and disintegration of the cells as well as the Schistosome tegument(the fluke body covering). Bursting or breakdown of the cell walls might be a much simpler way of describing it and may be equally correct.
Praziquantel must be ingested by the fluke to work. The dosage is one gram per 100 gallons of water. Currently the directions suggest using level tablespoons to measure the product. That is a mistake. In my experience a level tablespoon may hold anywhere from 2.5 to 4.5 grams of product. Not only is this wasteful and inaccurate; it is also very expensive. Please use a gram scale. Calibrate it often.
Since it does not mix readily with water a label dosage ProformC may be used, or it can be dosed with the ProformC as part of the treatment described above. Once it is fully dissolved it does not seem to filter out or precipitate. It simply degrades over time. When using Praziquantel, the water temperature should be in the mid-seventies(US). The first and all succeeding treatments should last seven days each whether treating for gill flukes(Dactylogyrus) or skin flukes(Gyrodactylus). 25% water changes should be made between treatments. Redosing will immediately bring it to the strength needed to kill flukes. I strongly recommend a third or even fourth application of this product due to the life cycle of the flukes as well as the thickness of the slime coat on Koi. In colder waters (60*F to 70*F) a fourth application is usually necessary when treating for gill flukes. Because of the cost of Praziquantel and the reduced treatment time it is well worth the effort to bring the temperature up to optimum. Another way to reduce cost is to simply lower the water in the container or pond to be treated.
Praziquantel "It doesn't seem to be working":
Koi are bottom feeders. They excrete an abundance of slime to the gill area which protects the gills, as well as the parasitic gill flukes, from the debris passing through them. So repetition of the treatment is necessary to allow time for gill flukes, in particular, to ingest the "poison".
Supaverm:
Know that this product has been accused of burning fins and suppressing the immune system. This accusation has been substantiated by several members of Koiphen. In the UK, there are more reports surfacing regarding the long range effect on the immune system as they have been using it for several years longer than here in the US. Therefore I would strongly urge that it be avoided. There are several methods for reducing flukes which are much safer. It is not recommended here.
Organphosphates-Fluke Tabs:
Fluke Tabs work well in waters where the Kh is less than 170ppm. Above that the poison is bound by the water and becomes ineffective. Attempts at double and triple dosing in high Kh waters results in killing the flukes AND the fish.
Fenbendazole:
An older type dewormer which seems to work at times, the dosage is 1 gram per 100 gallons and is used the same way as Praziquantel.
Chloramine T Dip:
For bacterial gill disease. This is very effective as a dip. Dosing is 1 gram per 10 gallons, five times consecutively for one hour. Do not mix this with any other treatments. Use old (conditioned) tank or pond water with aeration. I have not seen any printed directions for this, nor am I aware of any others who have used it this way. I have used it on several ocassions including a shipment of 180 small Japanese imports. It has proven to be very effective yet appears to be completely safe.
Diflubenzuron 25% wettable powder:
Actually this is an insecticide for greenhouse use. This powder works well in eradicating anchor worm(Lernea)and fish lice(Argulus). One gram will treat 3000 gallons. It is quite forgiving and an overdose usually will not cause irrepairable harm. The parasites should be removed by soaking them in iodine and pulling them carefully with a tweezers. Sedating(described below)the fish will prevent uncontrolled thrashing and further damage to the fish during this process. No ill effect is noted from sedation.
There are liquid forms (Dimilin) on the market. Follow the label directions.
Salt:
Salt has been used on such a large scale and usually for no reason that most parasites are resistant to it. However it still has it's place. Using it at a .6% - .7% level is excellent when bringing in new fish to a quarantine tank. If your dealer suggests using salt then follow his or her directions. Salt is effective on all microscopic parasites at this level. At least it knocks them back a bit giving you some time to determine what further treatments, if any, which you might use. See statements below for overview on salt:
Salt is known to protect fish from nitrites. The percentage of salt in this case is .15%. When adding fish to a pond or q-tank pay attention to ammonia and nitrites!
Salt is required, at least so the story goes, when using Elbagin(a Japanese water column antbacterial agent very similar to Furazone Green). The percentage of salt in this case is .6%.
Salt will aid in osmotic regulation of an immuno-depressed fish. New imports, as well as fish with bacterial infections are sometimes treated to aid their recovery. However this practice is no longer so widespread as it is recognized that overuse of any medicine leads to resistance and diminished effect. The percentage of salt used this way varies. Be aware that salt may adversely affect some medications.
Several parasites can still be controlled successfully with salt. The percentage of salt in this case is .6%. The treatment is not too temperature dependent and the duration of the treatment should be 14 days. Some strains of microscopic parasites are no longer deterred by salt.
Since salt occurs naturally in most waters, it is unnecessary to add it to pond water for ANY health reasons except those listed above and prevention of nitrite poisoning during the cycling of a filter. For informational purposes here is how to calculate salt:
.1% salt in 100 US gallons is .8333 pounds.
.1% salt in 1000 US gallons is 8.333 pounds.
.3% salt in 100 US gallons is 2.5 pounds.
.3% salt in 1000 US gallons is 25 pounds.
.6% salt in 100 US gallons is 5.0 pounds.
.6% salt in 1000 US gallons is 50 pounds.
.15% salt in one US ton(264 gallons) is 3.313 pounds.
Aquariums or qt - Apply one teaspoon of non iodized table salt per gallon of water every 12 hours for three treatments:
3 tsp per gallon for .3%.
6 tsp per gallon for .6%
Salt and Nitrites:
Dosage is .15%. Salt will slow if not stop the uptake of nitrites. Yes, it is listed and described here twice.[/COLOR]
Fungal Infections
Sap or Saprolegnia is the main genus of water molds which can bring about significant fungal infections in fish and eggs. It's safe to say that all ponds have water molds in them. Unless the fish are compromised the slime coat will, in most cases, protect the fish from an invasion. Sap invades epidermal tissues In common with all molds, it does so by secreting digestive enzymes into the surrounding areas. Since enzymes in general break things down it's easy to understand that this secretion enables the fungi to more easily absorb nutrients from the affected area. SAP slowly is digesting the fish! at this point it is called saprolegniasis. It sometimes has a green haze which is the fungal infection combined with algae. It is best to scrape and remove this growth before topical treatments. A surgical scalpel works well for this purpose.
SAP can spread over the entire body, but is usually spotted as pale white or grey patches. Sometimes it will be green if algae has been caught in it. It is usually considered to be a secondary infection seen after damage to the fish skin or gills. However it can be primary as it is temperature dependant, occuring in cooler waters as a consquence of immune system shutdown.
SAP Treatment:
It is generally agreed that Malachite Green is by far the most effective treatment. Be careful with this stuff! Eye, breathing and skin protection is required. A stock solution can be made with it and it is applied topically to thoroughly and deeply clean the wound. A dental adhesive may be applied afterward if desired.
Stock solution: 15 grams makes one gallon. To treat directly use the stock solution straight. For other treatments use one drop per gallon.
Treatment is should be made as soon as possible along with determining the actual cause of the invasion and dealing with that as well.
Gentian Violet may also be used as it is antifungal. GV is available over the counter at most pharmacies. It is very inexpensive and easily applied without the mess of mixing or the dangers of Malachite Green.
Columnaris: Mouth Fungus; Fin Rot, Cottonmouth Disease:
This is not a fungus or a mold. This is a bacterial infection-gram negative. The problem is that it can somewhat resemble SAP. Symptoms include grayish white spot on the head, gills or body sometimes surrounded by a reddish tinge. It can be topically treated with Iodine and then given antibiotic injections per the charts listed in that section.
Dropsy:
The term "Dropsy" describes the symptoms of more than one kind of disease, but all of which share the same symptoms of a bloated body with protruding scales. The accumulations of fluid within the body causes bloating, that becomes more severe in a short time. The scales stand away from the body and the term "pine-coning" is used to describe it . As the internal bacterial disease progresses the eyes will be affected, causing them to protrude or bulge as well. The recovery rate under these conditions is very low, and death is inevitable.
Sedating a fish for treatment:
Clove oil at a rate of 8 drops per gallon will calm a fish in a matter of minutes. Larger fish may require a bit more and so 10 drops per gallon may be used without harm to the fish. Add the clove oil to a small container of water which can be shaken thoroughly. Then add it to the sedating bowl. DO NOT LEAVE THE FISH UNATTENDED DURING THIS TIME. Use only enough water to cover the fish. Aerate. When the fish lays over on your hand without flopping and while still having gill movement it is ready for treatment. Practice this a couple of times keeping a supply of fresh but aged water close by. If for any reason you feel the fish has become sedated too deeply, simply move it to fresh aerated water and assist the fish in using the gills and hold near aeration. Assist the gills by moving the fish back and forth forcing water to pass over the gills.
MS-222: Tricaine Methanesulfonate. This can be fairly tricky at first so practice is important. Using a gram scale measure out two grams of MS-222 and two grams of baking soda. MS-222 MUST be buffered. Without the buffer, the Ph of the water to be used will drop as far as 3.3! That is ACID. In a matter of a few minutes the gills will be compromised and after several more minutes the fish skin and gills may be permanently damaged.
Add the MS-222 and baking soda to five gallons of aged pond or q-tank water. Mix thoroughly and you are ready. DO NOT LEAVE THE FISH UNATTENDED DURING THIS TIME. As the fish becomes sedated it will lay over and become quiet and easy to handle. If at any time you think it is too far sedated revive it at once by holding it in a tub of fresh untreated water and move it back and forth assisting it in using the gills. Once sedated, a fish can be tabled for 5-10 minutes without worry. The first time is usually far more stressful on the owner than the fish.
Injectable antibiotics and bacterial infections(ulcer disease):
Two things will bring on ulcer disease: poor water conditions and parasites.
Ulcer disease displays itself in many ways. Fin reddening, erosion, body sores, reddening and swelling of areas of the body, mushy or grey gills to name a few. Catching them in their early stages and injecting them with antibiotics is almost always successful and is actualy quite easy. Swabbing the fins or any ulcerated area with iodine is also recommended at this time.
If you've never injected anything with a syringe, grab an orange and practice injecting with water. It only takes a couple times to get the hang of it. For ease of injections, as well as any topical treatments, always sedate the fish.
Intraperatoneal injections: IP: Load the syringe by holding the antibiotic bottle upside down. Pull the plunger of the syringe to add the same amount of air to the bottle as antibiotic to be removed. Holding the body of the syringe, pierce the rubber stopper, release the air in to the bottle and remove what antibiotic is needed for injection.
To properly inject: IP injections are in inserted midway between the vent and the ventral fins either side of an imaginary line between them. The needle should be inserted-not stabbed-at about a 10 degree angle pointing toward the head. Slowly inject by pressing the plunger. Keep finger pressure on the injection site while removing the needle. This is primarily to stop accidental scale removal. However if one does come off it usually will grow back.
The most commonly used syringes are 3CC 25gauge 5/8". Smaller ones are used on fish which are 2" to 8".
The following are injectable tables. These are set forth to help koi keepers help themselves, and to get help from their family veterinarian. Injections should never be given unless a veterinarian has approved the use, or knows the koi keeper well. I deal with two veterinarians(husband and wife team)and keep them informed from time to time about my collection.
Baytril injectable antibiotic is available in three strengths: 2.27%. 5% and 10%. The table below is calculated for 5% Baytril. Baytril in most cases should be the first injectable to use. It is powerful, forgiving, and in almost every instance healing is visible after three injections. The protocol for Baytril is three injections on consecutive days, then two more every other day(EOD) for a total of five injections. Like antibiotics for pets or yourself, it is best to follow through completely. Short-circuiting the injections will lead to Baytril resistant strains. Intraperitoneal injections are usually suggested and are the easiest and safest. Keep this product cool or refrigerate. Baytril is dated and has a fairly long shelf life.
One more thing about Baytril. There have been some cases of new imports being resistant to Baytril. While I know this to be true, it can also look like Baytril resistance when it is actually under dosing of the antibiotic. Measure carefully and refer to the charts for accuracy. If 2.27% Baytril is all that is available, the dosage on this Baytril chart can be doubled.
Naxcel, Nuflor and Azactam antibiotics may be used in place of Baytril, if Baytril does not promote healing after the third injection, or if it is not available. The injection routine is the same, but some will need to be reconstituted with a saline solution or injectable water. The table reflects the different dosage amounts for each antibiotic. Please note: Other charts on other sites and in published books and periodicals may differ from this chart. This is a compilation of information from actual use by members of Koiphen and from suggested doses found in books and periodicals.
1ml = 1cc
Koi length(inches)----5% Baytril-----Naxcel----Nuflor-----Cefotan-----Azactam
4"-----------------------0.1-------------0.2-------0.2---------0.2---------.08
8"-----------------------0.2-------------0.5-------0.5---------0.5---------.15
12"----------------------0.3-------------0.9-------0.9---------0.9---------.2
14"----------------------0.5-------------1.0-------1.0---------1.0---------.4
16"----------------------0.6-------------1.3-------1.3---------1.3---------.5
18"----------------------0.8-------------1.6-------1.6---------1.6---------.6
20"----------------------0.9-------------1.7-------1.7---------1.7---------.8
22"----------------------1.0-------------1.9-------1.9---------1.9---------.9
24"----------------------1.2-------------2.2-------2.2---------2.2--------1.0
26"----------------------1.3-------------2.4-------2.4---------2.4--------1.1
28"----------------------1.5-------------2.8-------2.8---------2.8--------1.3
30"----------------------1.7-------------3.2-------3.2---------3.2--------1.5
Amikacin: Diluted:
Amikacin is bactericidal. It kills on contact. When injected daily it can also cause irreparable if not fatal damage to koi. As with all antibiotics use at your own risk. *One suggested protocol is twice with a four day interval(animal grade)*. There are reports that a single injection of Amikacin did such an amazing job that no further injections were given. I have also found this to be true.(See below)
I have used the human grade with success. *My interval is one injection every seven days*(human grade). a maximum of three injections. In many cases I have used a single injection along with injectible VitaminC as an additional injection the first time.
Amikacin comes in two strengths either of which must be diluted:
Human use: 250mg/ml, and
Veterinary use: 50mg/ml.
Both should be diluted. The following chart is for Veterinary grade Amikacin:
Koi length (in inches)---50mg/ml Amikacin (diluted)--sterile saline IP:
4-----------------------------.08-------------+------------.3
8-----------------------------.2--------------+------------.5
12----------------------------.4--------------+------------1.0
16----------------------------.6--------------+------------1.4
18----------------------------.7--------------+------------1.5
20----------------------------.85-------------+------------1.75
22----------------------------1.0-------------+------------2.0
24----------------------------1.3-------------+------------2.25
26----------------------------1.5-------------+------------2.5
28----------------------------1.75------------+------------3.3
30----------------------------2.0-------------+------------4.0
FORTAZ
Fortaz is a third generation cephalosporin. It is a broad spectrum bacteriocidal antibiotic. Fortaz is active against many strains of gram positive and gram negative organisms which are resistant to other cephalosporins.
Fortaz is very forgiving: the dosage has a wide range. Although somewhat expensive, once it is reconstituted it can be pre-loaded in syringes and frozen. The shelf life of the dry material is many years, and the frozen pre-loaded product about a year.
Fortaz is available in a one gram vial. To this vial is slowly added 10 ml of bacteriostatic water for injection. DO NOT SHAKE! When this solution is mixed it releases carbon dioxide and the vial is then under pressure. Gently roll the vial between your palms and let it sit for a few minutes. You may vent the carbon dioxide with a syringe if you wish.
Yield: 100mg/ml
For large fish(the big 25+" girls)a more concentrated solution can be made by reducing the bacteriostatic water to 3ml. Injections can be given IM or IP. I strongly suggest IP.
Yield: 280mg/ml
Dosage table: These are suggested dosages based on length but have also been compared to those using weight. It is fairly close. Because Fortaz is relatively forgiving is it suggested that the heavier females receive a slightly higher dosage of the 100mg.ml solution or be switched to the 280mg/ml concentrate.
The following chart is calculated with a 10ml dilution. Divide by three for the stronger 3ml dilution.
Length in inches:--------------------Fortaz 100mg/ml
7................................................. .045ml
8................................................. .06ml
9................................................. .09ml
12...............................................0 .3ml
14...............................................0 .375ml
16...............................................0 .55ml
18...............................................1 ml
20...............................................1 .2ml
22...............................................1 .5ml
24...............................................2 .0ml
26...............................................2 .5ml
28...............................................3 .15ml
30...............................................4 .0ml
Note the increase in antibiotic as the length increases. This corresponds favorably with several length/weight charts which study the weight increase versus length.
Fortaz dosage can be adjusted upward based on heavier females without fear. I would suggest an injection protocol of three injections - one every other day.
There have been situations where I have used two antibiotics in seperate injections on the same fish. Amikacin and Fortaz used at the same time has worked well but I would caution by saying this should be a "last try" effort in the event nothing else has worked.
Chloramphenicol:
This antibiotic is mentioned favorably from time to time. Please do not use it! This antibiotic will cause Grey's Syndrome in humans.
Dexamethasone:
This is not an antibiotic but a treatment/steroid for shock. For a variety of reasons fish may go into shock. A single injection of "dex" will bring them out of it safely and quickly. The concentration generally used for fish is 4mg/ml. The chart to use is the 5% Baytril chart. A single injection is all that is necessary. The life of this steroid in the body is about two days.
Example:
12"koi .3cc
16"koi .6cc
20"koi .9cc
24"koi 1.2cc
28"koi 1.5cc
Injectible Vitamin C
An immune system booster. I have used Injectible Vitamin C on several koi and all have responded very well indeed. I used it on a fish with recurring ulcers once a week side by side with human grade amikacin with a final single injection of C on the third week. The fish is doing well and has been placed in a new home with top notch care.
Use the 5% Baytril chart. A single injection given as a secondary injection with the first antibiotic injection. Injections of VitaminC may be continued but space the injections weekly and I have reason to believe two injections over a period of two weeks is probably more than enough.
Do not mix injectibles in a single syringe. Inject a small distance apart.
When using these charts remember that the use of an injectible does not guarantee success. Many things can go wrong in the treatment efforts so proceed with caution.
Water column antibiotics/antibacterials:
There are many medications which lay claim to doing wonders in the water column. The truth of the matter is that few do very much, and few can be used alone. One exception seems to be Elbagin. This is a water column antibacterial which is gaining an excellent reputation. If you have need for a small quantity some of the better importers seem to have it available from time to time. Many of the Japanese breeders treat with Elbagin and salt almost immediately after their fish are harvested from the mud ponds.
Microscopes and supplies:
For microscopes the best place is EBAY. You'll need magnification of 100X to 600X to see the microscopic bugs associated with koi.
For supplies go to:
http://www.carolina.com
1-800-334-5551
Koi Herpes Virus
This section is a series of articles, pictures and discussions about Koi Herpes Virus. It is written by a number of people inclined to take interest in the problem and discuss it at length. While there is a great deal yet to be learned about this virus this is the best information available at the present time.
From koihealth.org:
Koi Herpesvirus (KHV) represents a third cyprinid herpesvirus formally designated Cyprinid herpesvirus 3(CyHV-3). Other nomenclature (Carp Interstitial Nephritis and Gill Necrosis Virus (CNGV)) has been suggested. For purposes of this web site the causative agent of Koi Herpesvirus disease will be be abbreviated KHV and the semantics left to others.
We hope to provide enough practical information regarding KHV such that the reader will be able to make informed choices regarding the acquisition of koi and their subsequent quarantine and testing. Some of the material is admittedly oversimplified for clarity. A partial listing of references is included.
There are now approximately one hundred known herpesviruses isolated, with at least one herpesvirus for each species studied. There are eight known human herpesviruses, of which most of us have been infected by six. One of the characteristics of herpesvirus infection is the ability to cause recurrent infection by one of several mechanisms including reactivation of latent virus or persistent infections (chronic low grade virus multiplication). As a whole, the herpesviruses studied have evolved a number of mechanisms for "hiding" from the "immune system" during their latent or persistent infectious states. These places of hiding vary between herpesviruses but have been found to include nucleated nerve cells, lymphoid cells, salivary glands, kidney, etc. The ability of herpesviruses to avoid the immune response and remain latent as an intracellular virus makes it very difficult for the host animal to clear the virus. Once infected, many animals have the virus for life and may intermittently shed virus without showing signs of disease. The site of latency of KHV is unknown.
There are direct and indirect tests to demonstrate viral infections. Direct tests detect the presence of the virus or of a portion of a virus. Growing the virus in cell culture or a PCR test to a portion of the viral DNA would be examples of direct tests. Generally these are most useful when the virus is present in large numbers such as in a KHV disease outbreak. Indirect tests measure antibodies to a particular virus. They are most useful in detecting previous infection or the stage of infection.
Diagnosis of KHV disease outbreaks is usually made based on epdemiology, characteristic histopathology, and the presence of the virus in internal organs demonstrated by cell culture or PCR assay. Periodic KHV surveillance may be done in koi growing countries by periodically sampling fish from ponds and doing PCR testing. Some health certificates and certifications will state "KHV specific amplification was not detected when genomic DNA from specimens were used as template". Others may just say they are free of KHV. PCR technology has proven very useful for confirming diagnosis in acute disease outbreaks. Unfortunately, PCR has not been found sensitive enough to detect those KHV survivors which may carry the virus in a latent or persistent infective state (carriers). It currently takes measurement of anti KHV antibodies to determine if a koi has previously been infected with KHV and has the potential of periodic virus shedding, infecting other fish.
No KHV quarantine or testing procedure offers 100% assurance a koi previously infected with KHV will be detected. Through quarantine and serological testing we can greatly reduce the odds of infecting other koi. . Serological technology is available to measure antibodies to the KHV virus. Both Enzyme Linked Immunosorbant Assay (ELISA) and the Virus Neutralization Antibody Assay (VN) serologies are available at modest cost, the latter being the most sensitive. The KHV disease outbreak illustrated in the photos should have been prevented by testing the fish for the carrier state while in quarantine. Serology done on survivors strongly suggests some imports had survived a previous KHV infection as they carried low antibody titers. It is unclear how long a KHV survivor will maintain detectable antibodies but studies are in progress. The CEFAS group has demonstrated detectable antibodies a year or more following infection. Using serology on fish in quarantine is another tool we have available for protecting our collections. If a fish has detectable levels of antibodies to KHV, it has been previously infected and must be considered a carrier. Carriers have the potential to shed the virus at a later time, infecting other fish. Materials and procedures we find useful for drawing blood for anti KHV antibody testing are outlined.
PCR testing is useful when fish are sick and dying but is usually not of benefit when fish appear healthy. PCR testing is extremely helpful in confirming histopathology when fish are dying. Again, PCR testing is extremely unlikely to detect disease in healthy appearing fish. All PCR techniques are not equally sensitive. Current PCR technology is inadequate for detecting the latent or carrier state of Koi herpesvirus.
A safe and effective vaccine will be required to control KHV disease. Currently the alternative is quarantine and testing each and every koi for antibodies to KHV before allowing new fish to enter our ponds.
How to get a “worthless positive sample ” for a meaningless Virus Neutralization test:
Remember the test is only as good as the sample submitted. Remember a positive test is one in which virus growth is inhibited by addition of the sample. There are several ways in which a sample can be accidentally altered or contaminated to inhibit viral growth. The Virus Neutralization assay is repeatable and measures inhibition of the viral growth. If the sample is not properly prepared or contaminated, one can get inhibition of viral growth at low titers or dilutions. The result may be a positive test at low titers even though there is no antibody to virus present. Those in the medical field understand the need for perfect samples and take it for granted but perhaps it would be useful to review some ways in which blood specimens can be altered to provide worthless results.
Four ways to contaminate or alter your blood sample to inhibit viral growth.
Heat up your sterile sample. The process may well cause enough changes to inhibit viral growth. If your carefully prepared sample of serum or plasma sits over a weekend on a delivery truck in the heat, it may well be worthless. From a practical viewpoint, one must pack samples with some form of ice packs in an insulated container and ship by a traceable overnight service such as FedEx. For International samples requiring longer shipment times, more heavily insulated Styrofoam containers with more ice packs are usually used. A call to the receiving lab to let them know to expect your shipment or verify receipt may be in order. We prefer our samples to arrive in the lab frozen.
Allow your specimen to hemolyze red cell rupture. If the specimen is handled in such a way that a significant number of the red cells rupture, viral growth can be inhibited. There are a number of powerful intracellular substances which can inhibit viral growth. If sufficient quantities of these are accidentally released into the serum or plasma, they can inhibit viral growth without the presence of antiviral antibodies. This is usually pretty easy to detect. When the red cells lyse, hemoglobin which is red is released into the serum or plasma specimen. The specimen will be red stained after centrifugation. A small amount of hemolysis is usually not critical but larger amounts can and do inhibit viral growth. Four of the more common and preventable means of hemolyzing specimens include:
Allowing the blood to rapidly transfer through a tiny needle into the vacutainer. When the blood flows rapidly through the needle, some red cells rupture. If the syringe plunger is forcibly pushed putting the cells under increased pressure, hemolyis occurs.
If the needle is inserted into the tube and blood is allowed to rapidly splash into the bottom of the tube, some hemolysis occurs. This can largely be prevented by holding the tip of the needle against the inside wall of the wall of tube when transferring blood.
If the blood is not almost immediately transferred from the collecting syringe to the tube, blood may clot in the syringe. Although this clotting may be prevented by using heparin in the syringe, use of heparin adds another potential contaminate and is not necessary when proficiency in veinipuncture is acquired. The increased difficulty in transferring blood from a partially clotted syringe may introduce other unwanted components or produce hemolysis in the sample.
After centrifugation, if the red cells and red cell fragments below the gel migrate above the gel, they can contaminate the sample. Shipment could potentially jostle the sample such that the sample is contaminated. Indeed laboratory couriers have repeatedly demonstrated the ability to “shake up”and contaminate centrifuged samples in gel tubes transporting samples across town from physician offices to the reference lab. Imagine what cross country shipping can do to samples. If the samples are frozen and arrive at the lab frozen, rough handling during shipment is usually not an issue. An alternative is to transfer the plasma or serum to a second sterile transport tube (about $.30) prior to shipment. The tops can be removed from the vacutainer and the sterile tube (top carefully placed with sterile side up to avoid contamination) and the sample is carefully dumped from one tube to the other and recapped with the sterile top for shipment. The top is taped in place or wrapped in Parafilm. Alternatively the sample can be withdrawn into a small syringe, the needle capped and the plunger taped to prevent movement. The receiving lab technicians usually hate the latter. If samples are being sent internationally, it is recommended the serum or plasma be separated into transport tubes. If the sample is being transferred to transport tubes, it can be divided such that some sample remains in the freezer in the event of shipping mishap.
Allow “tissue juice” or tissue to mix with your sample. “Tissue juices” and ruptured tissue contains some very potent antiviral substances. They cannot be mixed with your sample. Frequently, when an individual is learning to draw blood from a fish, there is a tendency to withdraw the plunger of the syringe while moving the needle. This may result in small amounts of tissue or various “tissue juices” being drawn into the hub of the syringe. The correct technique involves placing the needle in the vein and then withdrawing blood by gentle backpressure on the plunger. Blood should slowly flow into the syringe. If the tip of the needle is not appropriately in the vein, pressure is released on the plunger, the needle is repositioned, and the plunger is again gently withdrawn. Very little suction is required if the needle is properly positioned. Novices will often continue to hold back pressure on the plunger when repositioning the needle. This frequently will result in tissue or tissue juice being withdrawn into the syringe. If there is any doubt about the syringe being contaminated with tissue or “tissue juice”, it is tossed and a fresh needle and syringe is used. A larger than needed blood sample will effectively dilute a small amount of “tissue juice”. We prefer to withdraw at least one ml of blood in the syringe to minimize potential tissue contamination.
Use inappropriate tubes for your sample. There are many types and sizes of blood and serum collection tubes. Most are a form of sterile plastic with additives in a vacumn . Gold and red topped serum collection tubes frequently have a “clot activator”. Plasma collection tubes usually have heparin. Purple top tubes contain EDTA to prevent clots, etc. For example, in a vaccine study, avian blood samples were collected by a professional in a 4 ml purple top tube ( which contains 7.2 mg of K2 EDTA to prevent blood clotting). A small amount of collected blood would be transferred to the tube, and the tube subsequently spun down in the centrifuge. This resulted in the blood cells being spun to the bottom of the tube with the clear supernatant on the surface. The later was transferred to lab in transport tubes. At this point the sample includes most of the 7.2 mg of K2 EDTA. As you might imagine, the EDTA in the sample consistently inhibited viral growth. You would not want to use a 10 ml serum collection tube containing a clot activator to hold 1 ml of blood before centrifugation. The excess clot activator can be transferred to your sample and potentially inhibit cell growth. A tube half that size is more appriate. We do know that a light green capped 4.5 ml vacutainer containing gel and 76 units of lithium heparin works well with samples of 1 ml or greater. The microtainers do well from a size stand point but they are not sterile and that may cause contamination problems.
Again, the virus neutralization test is worthless if the sample is not good. As BODAD says “Garbage in, Garbage out."
Shipment
Once the samples have been obtained and the plasma or serum separated, they are usually wrapped and shipped in a small insulated container to the labratory by means of a trackable overnight service. Shipping containers may include cardboard boxes holding small styrofoam containers, insulated freezer sacks, insulated childrens lunch sacks, etc. One or more ice packs are usually included. Care should be taken to avoid shipment arrival on weekends or holidays. If in doubt, it is recommended to contact the receiving labratory by phone before shipping. A completed sample submission form, a page indicating the numbering system utilized, and a check for the processing cost for each sample should be included with the samples.
Contents may be freely reproduced in their entirety for educational purposes with appropriate credit.
Koihealth.org is sponsored by a small private foundation for educational purposes.
__________________
Disclosure statement: The opinions, suggestions, directions, recommendations, advice are just that. Nothing more.
Reply With Quote
Originally Posted by cindy
