This technique can be used to examine any kind of freshwater or marine fish, from an inch long Neon Tetra, to a 24-inch Koi. It is probably the most common technique used in fish medicine, and is applicable to the hobbyist as there is only a minimal amount of apparatus required. Performing a microscopic examination of tissue mucus of a sick fish will allow correct diagnosis and thus use of the most appropriate treatment for a rapid recovery and prevention of spread or the infection.

Apparatus required:

• Microscope capable of magnifying to at least 400 times (400X)
• Microscope slide and cover slip
• Bath capable of holding the sick fish (two baths are required if anaesthesia is used)
• Fish anaesthetic, e.g., Koi-calm (THIS IS OPTIONAL)
• Plastic gloves
• Paper towel 

Preparation of apparatus:

The key to taking a good smear, is to take the sample as quickly and painlessly as possible, so the patient has a minimal amount of stress, and the infection is not worsened. To do this preparation of all surfaces and equipment is essential. Clear a bench or suitable working surface and, set up your microscope ensuring all components of it are working correctly.

Place a smooth, non-absorbent surface such as laminated wood or plastic onto the bench and clean it using an antibacterial cleaner such as Gill Wash diluted 5 mls to 4.5 L. Onto this place a moistened paper towel long enough for the fish to be placed on. Have another moistened towel ready to cover the fish's head.

Get a new clean slide and cover slip to hand, alternatively clean an old slide and cover slip with isopropyl alcohol.

A bath must be prepared to hold the fish before the actual sample is taken, this may not be essential in all situations, but does allow more time to be taken, and also allows for anaesthesia of larger fish if desired. To prepare a bath, take a vessel suitable for the fish's size, and place enough pond/aquarium water into it so that the fish will be comfortable.

Finally put on plastic gloves to protect the fish during handling.

Preparation of the fish:

If only one fish is showing symptoms, catch it as quickly and with the minimal amount of stress possible. If a population of individuals is affected, select the most debilitated fish, or that with the most obvious symptoms.

Once the fish has been caught and placed in the bath it can be anaesthetized. This is optional, but with larger fish such as Koi carp, but it makes the whole operation considerably easier. If anaesthesia is to be employed, add 10 drops of Koi-Calm to every 5 litres of water in the holding bath.

Koi-Calm is a natural anaesthetic based on clove oil. It is best used by placing the drops of oil into warm water before adding it to the bath. This emulsifies the oil, and most effectively distributes it throughout the bath. The fish will become placid and easy to handle after around 7 - 10 minutes depending on size. Prepare another bath without Koi- calm to hold the fish during recovery.

Once the fish is in the holding bath and is anaesthetized, the next stage is to take the mucus smear.

Taking the mucus smear:

Wearing the plastic gloves, gently lift the fish out of the water, (figure 1) and place it onto the wet towels, using the spare wet towel, cover its head, as the darkness created reduces the photophobic response (fear of light).

Figure one: Gently lift the fish out of the holding bath

If the fish has an obvious area of infection it is simplest to take a localised mucus smear. To do this, take the microscope slide, and press it onto the area of interest, a small amount of mucus will be impressed onto the slide, and any parasites present will be transferred. Many wounds may be present over the fish, if this is the case, the best site to examine would be a smaller developing wound. This would not be overgrown with secondary bacterial and fungal invaders that would hinder the diagnosis.

If there is no obvious lesion, then a general mucus smear should be taken. To do this, scrape the edge of the slide from just behind the head to the base of the tail. Do not scrape too heavily, as this will damage the fish. Even light motions will remove sufficient mucus and even skin cells from the fish. Alternatively scrape off the mucus with a scalpel, and place it on the slide (Figure 2)

Figure 2: gently scrape some of the mucus from the fish

Return the fish to the holding bath, unless anaesthesia was used, where an anaesthetic free holding vessel should be ready.

Once the mucus smear is on the slide, a drop of tank, or pondwater should be added to the smear, and then a cover slip added. Tap water should not be used as the chlorine will kill the parasites before they may have been observed. Hold the cover slip by the sides at a 45 (angle touching the slide, and then lower it over the mucus. This ensures no air bubbles are trapped under the cover slip.

Dry the slide with paper towel without disturbing the cover slip, and then place it on the stage of the microscope, ensuring it does not get covered in fingerprints.The smear should be examined immediately as the fish parasites will not live long on the slide, as it dries out.

However, the slide can be stored for short periods in a humidified container to prevent it drying out. This means the sample can then be transported to the microscope, if the hobbyist does not personally own one.

To make a humidified container simply place some wet paper towel into a sealable plastic food container.

Examination of the mucus smear:

The level of magnification is determined by the product of the objective lens magnification and the eyepiece magnification. For example if the eyepiece = 10X and the objective = 40 X then the magnification = 400X

Starting at the lowest magnification (usually around 40X), systematically scan the slide for moving parasites. The best method is to locate the corner of the cover slip, and then move to the adjacent corner, move up slightly, and return to the original side.

A parasite will be recognized by rapid movement around the mucus. Be aware of general water flow around the slide, as this will make inanimate objects appear to move. A parasite will move in a variety of directions, movement along one direction only means water flow. Also look out for large circular objects, like rubber grommets, these are simply air bubbles.

Large parasites such as the flukes and the white spot organism will be visible at this magnification.

Figure 3: Rotate the turret to the next level of magnification

Once the smear has been thoroughly explored at the lowest magnification, rotate the turret (figure 3) to the next level of magnification, usually around 100X. Again systematically explore the smear for moving parasites. Once this level of magnification has been explored, step up to 400X. If a parasite is seen at 100X and it needs to be seen in greater detail, move up to 400X. For fish parasitology, this is highest magnification required. The smallest, common, fish parasite, Icthyobodo (formerly Costia) and some larger bacteria, are clearly visible at this level.

Many microscopes have a further objective lens capable of magnifying up to 1250X. This lens cannot be used for wet preparations, only stained slides, as it requires a drop of special immersion oil to be placed between the tip of the lens and the slide; this oil further magnifies the image. At this level more bacteria become visible, and their general morphology can be determined. However for fish mucus biopsies this technique is not really relevant.

Identification of common fish parasites: Dactlyogyrus and Gyrodactylus

Figure 4: Gyrodactylus Sp.

These are monogenetic trematode flukes. They infect the mucus layer of the fish and graze on the skin cells of the epidermis. The flukes are recognized best under the microscope at 40X to 100X (figure 4).

Dactylogyrus is recognized by having 4 eyespots and a 4 pointed caudal (rear) end. This species affects the area predominantly around the gills. Symptoms include rapid respiration, flicking, and flashing followed by periods of lethargy.

Gyrodactylus has two eyespots, and a two pointed caudal end. This species mainly affects the skin. Symptoms include excessive mucous secretion, rubbing and scratching, leading to ulceration in long term cases.

Both species can cause severe debilitation in heavily infected fish.

The ideal treatment is Formaldehyde. Many flukes carry pathogenic bacteria on their attachment organs and mouthparts. These bacteria will invade the fish wounds created by the flukes. Therefore it is wise to use an antibacterial treatment (such as Acriflavin) in association with the antifluke treatment. Bacterad / Finrot is a mixture of formaldehyde, and antibacterial agents, to provide the optimal Dactylogyriasis, and Gyrodactlyiasis treatment for ponds and Koi. Use Bactocide in Freshwater aquaria. Bactocide is safe to use in marine aquaria as it will not harm invertebrates, but it has been known to make them look dull for around one day. Use Bactocide at half dose in marine aquaria.

Icthyopthirius multifilis

This single celled protozoan parasite causes one of the most common freshwater fish diseases, White-Spot or Ick. This pear shaped parasite, shown in figure 6 has a characteristic horseshoe shaped nucleus. It swims in a characteristically slow motion, and cilia (hair-like projections the cell uses for propulsion) can clearly be seen at higher magnifications.

This visible stage is called a theront, or `swarmer'. The encysted stage that forms the visible white spot on the fish is called a trophont. (The prefix `troph' means feeding: the parasite is feeding on the fish.) From the trophont free-swimming 'tomonts' emerge, these form a tough resistant cyst at the bottom of the pond or tank, from which hundreds of infective theronts emerge.

Symptoms are most obviously white spots on the fish, general debilitation, and secondary infections are common.

The best treatment is a mixture of Malachite Green and Formaldehyde. For Koi use FMG mixture, for more sensitive fish such as Sterlet, Rudd, Tench, and Orfe use Eradick / White Spot. In aquaria use either Ichcide or Aquarium White Spot.

The Trichodinids

Members of this group of parasites include Trichodina, Trichodonella and Tripartiella.

Trichodinids are very common ciliated protozoan parasites of freshwater and marine fish. One species is shown in figure 7. It can be seen to be a very characteristic parasite. Viewed from above it has intricate radiating patterns called the denticular rings that give the parasite a cartwheel-like appearance. Viewed from the side it can be seen to be dome shaped. The Trichodinids have a very characteristic rotating, swimming motion.

These parasites can live freely in the substrate of the pond or tank. Thus they are more commonly found in those systems with much accumulated detritus.

Symptoms of Trichodinid infection include excess mucus production, as the fish tries to slough off the infection. Skin ulceration is common in heavy infestation. Gill involvement is frequent, leading to respiratory distress.

The ideal treatment is an antiparasitic such as formaldehyde and malachite green. Use FMG Mixture for Koi, Eradick / White Spot for more sensitive pond fish, and Ichcide or Aquarium White Spot in freshwater tanks, and copper-based Paracide for marine fish. Always treat in a hospital tank, as copper is deadly to marine invertebrates. 

Tetrahymena

Figure 8: Tetrahymena Sp.

This ciliated protozoan parasite is usually free-living, but can become parasitic. It is most often found in tropical fish such as livebearers, Tetras and Dwarf Cichlids. Shown in Figure 8, it can be seen to be an oval shaped parasite covered in minute hair-like projections from the cell called cillia. Symptoms include general debilitation, hemorrhage of the skin, and occasionally bulging eyes.

As this organism occupies the detritus of the tank, infection can be prevented by maintaining good tank hygiene. Formaldehyde is a useful treatment for Koi, use Eradick / White Spot in ponds, or Ichcide in freshwater aquaria , but this will not cure internal infections. A policy of prevention through good water quality and nutrition is best.

Cryptocaryon (Marine white spot)

Figure 9: Cryptocarypon irritans,exflagellate form

This ciliated parasite shown in figure 9 resembles Ichthyopthirius mulitifilis, the cause of white spot in freshwater fish. The life cycle is similar to I. Multifilis. The parasite can be seen to be pear shaped, but has a 4 lobed nucleus instead of the horseshoe shape of the freshwater parasite. The infective stage aggressively burrows into the fish, causing it to flash and flick against objects in the tank. The fish may even swallow sand as they attempt to relieve the infection. The most obvious symptom is however the white spots.

Treatment is via Paracide, which contains copper and formaldehyde. Copper is lethal to invertebrates, so fish in reef systems must be treated in a hospital tank. The infective stage can only last 48 hours in the absence of a host. Reef systems can be treated with Bactocide at 1/2 dose. This will not harm invertebrates, but it has been known to make them look dull for around one day.

An alternative treatment is hyposalinity. A dip in a bath of 16 parts per thousand salinity will kill the infective stages, thus fish only systems, can be purged of Cryptocaryon infective stages by temporarily lowering the salinity. 

.Ichthyobodo necatrix (formerly Costia necatrix)

This is one of the smallest parasites commonly encountered in freshwater fish, and is best viewed at 400X. It can be seen to be a comma shaped parasite, and usually occurs either attached to an epithelial surface, or swimming by frantic beating of hair-like projections from one end, called flagella. Symptoms of infection include excess mucous production, respiratory distress, and general debilitation, as the parasites feed on the gill and skin cells. Treatment is via antiparasitics such as Malachite green and Formaldehyde. Use FMG Mixture for Koi, Eradick / White Spot for more sensitive species and pondfish. Use Ichcide or Aquarium White Spot in freshwater aquaria. Icthyobodo is not believed to infect marine fish.

An alternative is to dip the fish in a salt bath of up to 3 Oz per gallon for 6 - 10 minutes. For smaller fish use 1 1/2 Oz salt per gallon for 30 minutes. After the salt bath it can be beneficial to place the fish in strong running water to wash of the excess mucus produced by the salt.

NB: Some strains of Ichthyobodo are known to be resistant to salt treatment. Thus if this cure is used, check its affectivity by performing regular mucous biopsies on the fish to monitor re-infection.

Heteropolaria (AKA Epistylis)

Figure 11: Heteropolaria Sp.

These parasites are called sessile peritrichs, and are found in freshwater fish. Shown in figure 11 they can be seen to be a large body rooted to the epidermis by a stalk (hence `sessile'). They are very numerous in water with a high level of organic matter. If a fish becomes stressed, its immunity to these parasites declines and they invade the fish leading to white-grey patches, and red sores in heavy infections. Interestingly, the parasite only uses the fish for attachment; it does not feed on the host, so it is not a true parasite. However through the attachment, wounding is created and secondary bacterial and fungal infection often follows. Treatment can be through salt baths as in Icthyobodo above, or antiparasitics. Use FMG mixture for Koi, Eradick / White Spot for more sensitive fish and pond fish, Use Ichcide or Aquarium White spot in freshwater aquaria.

Prevention is the best policy by maintaining good pond or tank cleanliness. 

Oodinium and Amyloodinium

These are Dinoflagellate parasites (Flagella are long hair-like structures the cell uses for propulsion), which contain the green photosynthetic pigment chlorophyll. In freshwater fish, Oodinium causes velvet disease, where the fish appears to be covered in gold coloured dust, which can be enhanced by shining light on the fish in a darkened room. In marine fish the closely related Amyloodinium causes Velvet disease or Coral Fish Disease, whose appearance is the same as above.

The parasite is flagellated in the free-swimming stage, but once attached to the fish it loses the flagella. (Figure 12).

The parasite can be recognized as it has a pigmented body inside the cell, which gives it a slight green colour. In both freshwater and marine fish, the parasite attaches to the skin and gill epithelium. Root like attachment organs called rhiziods penetrate deep into the fish, causing severe pathology. Symptoms include distressed breathing in gill infections, flashing, scratching, and skin infection. Secondary bacterial infection is common.

Figure 12: Oodinium Sp. exflagellate form.

Both Amyloodinium and Oodinium are best treated using a copper-based medication such as Paracide. Marine fish should be treated in the absence of invertebrates, as these are very intolerant of copper, and freshwater snails will also be killed by the treatment.

An alternative to the copper-based treatment is Bactocide. This can be used in marine aquaria at half the dose of that applied to freshwater systems. The treatment will not harm marine invertebrates, but has been known to make them look dull for around one day.

These parasites are notoriously difficult to treat, as the parasite forms a resistant cystic structure in the substrate of the aquarium, and this can lie dormant, reappear and then cause relapses of infection.

Other disease organisms frequently seen in mucus biopsies:

Saprolegnia

Figure 13: Saprolegnia fungus.

Saprolegnia is not a parasite; it is actually a fungus, which grows on freshwater fish's wounds and ulcers where it feeds on the dead tissue present. The fungus appears on the fish as a dense cotton wool like growth. Under the microscope, at low magnification (40X), it appears as a dense mat of strands called hyphae, some with dense black bodies at the ends (Figure 13). These black bodies are called sporangiospores, and contain thousands of new fungal spores.

Saprolegnia is a secondary invader of fish wounds, so its presence usually indicates an underlying parasite or water quality problem.

This fungal infection is best treated with Malachite Green. Use Aquarium Fungus in freshwater aquaria, pond Fungus for ponds and Malachite Green for Koi. The wounds created should be treated with the Paramedic Kit.

For particularly stubborn cases of fungus, the fish can be dipped in Malachite Green: use 0.5 ml per gallon for one hour, or in extreme cases, 30 ml per gallon for 1 minute.

Infection with Saprolegnia superficially resembles a similar condition called Cotton Mouth. This is actually caused by a bacterium and is described in more detail below. 

Flexibacter columnaris (Cotton Mouth)

Figure 14: Flexibacter forming stacks on the epidermis.

This is a bacterial disease of freshwater fish, whose symptoms greatly resemble that of infection with the fungus Saprolegnia. The fish becomes covered in patches of whitish grey cotton wool like patches, most commonly, but by no means uniquely, around the mouth.

A mucus biopsy around the offending area will not reveal the large dense mat of growth seen in the fungal infection. Flexibacter is seen at much higher magnification (400X), and appears as minute thin rods, that wriggle through the slide. These frequently aggregate into large stacks of bacteria that writhe around the mucus.

This bacterium invades wounds created by parasites, poor water quality or physical damage. These wounds should be treated using the Paramedic Kit. Flexibacter infection itself can be treated by bathing the infected fish in a dip of Gill-Wash, which has a marked antibacterial action. For smaller pond fish use Cleargills.

Whenever cotton wool like growths are noticed on a fish, a microscope diagnosis of the infection should be conducted, so the correct treatment is used. If the wrong cure is applied, the infection is likely to be worsened.

NOTES:

* When using salt as a dip: 4 Oz/gallon is the absolute maximum, for smaller fish maxiumum is 1 1/2 Oz/gallon

** In soft water areas use Gillwash / Cleargills @ 1/2 dose