Diagnostic Aquatic Laboratory
Since very precious time is often lost in sending samples to the lab and waiting for the results of analyses, I made the investment myself to thoroughly expand my lab activities and the “Diagnostic Aquatic Laboratory” was established.
– Water analysis : chemical and bacteriological :
Photometric determination of parameters, analysis for heavy metals, measurement of bacterial infection pressure, sensitivity tests for water disinfection
– Bacteriological examination of tissues and organs :
Isolation of mainly Aeromonas/Pseudomonas from ulcers, kidney/milt, abdominal fluid. Sensitivity tests for determination of efficacy various antibiotics
– Blood analysis : hematology, biochemistry and cytology.
Minor or major determination of blood parameters with contiguous cytological examination of a blood smear for interpretation of an inflammatory or non-inflammatory finding
– PCR : Viral/Bacterial DNA analysis of tissues and organs.
Screening for KHV (Koi Herpes Virus) and CEV (Sleeping Disease Virus) are the 2 most common tests available but a whole battery of aquaculture analyses are possible such as SVCV, WSSV, Streptococcus,Flavobacterium, Aeromonas Salmonicida….
Water Analysis
The principle of this is that reagents are added to a measured amount of water and the device will then use a light beam to measure the scattering and convert it into a numerical number.
Pond water also contains bacteria. The more bad, the higher the infection pressure for the fish, or in other words chance of infections. Good bacteria are indispensable in a pond, helping to form a biological balance and preferably as diverse as possible. In bacteriological water analysis, several series of dilutions of pond water are inoculated on various nutrient media and the colonies formed are counted : total bacterial count, Aermonas, Pseudomonas, Vibrios, Coliforms and other Enterobacteriaceae, Shewanella Putrefaciens.
Bacteriological examination
flank. This further increases infection pressure and tends to make other fish sick as well. Bacterial infections are highly contagious in nature.
1. Using a sterile swab, a sample is taken from the edge of the lesion, preferably deep within the skin. This swab can be stored in transport medium, which is a special sterile tube that contains just enough nutrition for the bacteria to survive for a while. This is kept cool. If you want to send samples to our lab, this type of swab with transport medium is essential in order for the pathogens to reach the lab alive.
2. As soon as possible, the swab is spread on a special nutrient medium. This contains nutrients to grow just these bacteria you wish to examine (as long as they are present in the sample, of course). Through a special technique, you get a dilution effect so that the bacteria lie freely in loose colonies and can be judged by shape, smell, color, thickness,…
3. It takes about 24 to 36h for the colonies to be visible. Pseudomonas spp. are slower growers than Aeromonas Hydrophila species. Aeromonas Salmonicida, on the other hand, grow even slower. The inoculated nutrient media go into a special incubator for this purpose. Since fish are cold-blooded they will not be incubated at 37°C but at a colder temperature.
4. When the bacteria have grown well, the next step is to inoculate them into a so-called pure culture (a pure culture). At the same time, some biochemical tests can already be done to start determining the type of bacteria.
5. Finally, the sprouts are spread evenly on a special nutrient medium that is well penetrating. Several antibiotic tablets are then placed on top of this, which disperse a substance throughout the growth medium. Where the bacteria do not grow that means they are sensitive to the drug, where the bacteria do grow the diameter of the area around the antibiotic tablet is decisive in deciding whether the drug is effective or not.
Blood analysis
A blood analysis consists of several steps.
1. First there is the blood draw, for this the fish must be fully anesthetized. A puncture is made in the blood vessel that runs just below the spine. So a delicate job!
2. Fish can only miss 0.5% of their body weight in blood, otherwise there is a chance they will go into shock. That is, we will not draw blood until the fish is sufficiently large and the volume of blood we draw will be only 0.5 to 1 ml.
3. Next, first and foremost, the fish must be cared for so that the bleeding is stemmed and that the fish can begin to absorb oxygen again and remain moist. On the other hand, the blood must be processed as quickly as possible because once the blood is out of the body it will clot.
4. 1 drop of blood is spread onto a slide for direct microscopic examination (cytology)
5. A small amount of blood is drawn into a capillary tube for determination of hematocrit (which is the ratio of blood cells to plasma). This is done in the practice lab.
6. The remaining blood is stored in a heparin tube and taken to the practice or pipetted directly into the Fat Scan for analysis.
7. Fish blood is very fragile and must be handled with care. If you don’t, the cells are going to burst (hemolysis) and you’ll get disrupted results
PCR
PCR stands for Polymerase Chain Reaction. Simply explained, it is a technique in which specific pieces or sequences of DNA from pathogens are propagated to a detectable level. This test is so specific that a positive result has a very high diagnostic value. Viruses, bacteria as well as fungi or parasites can be tested as long as a test is available for the thing you wish to test. For simplicity, in the following paragraphs we only talk about viruses.
With a negative result, it can have several meanings:
1. No virus is present in the fish as a totality and it is virus-free.
2. Too little virus was present in the sample and the result is false negative. With Herpes viruses, this can be the case with the so-called “carriers.” The virus is then hidden in nerve nodes, for example, and is barely detectable. Sometimes a negative result can also be obtained for infection when the sample is taken from the wrong place. For example, sampled the wrong organ or if you happened not to take an inflammatory foci with active virus. I have already experienced that in fish with KHV, one gill half can test negative while the other is positive!
The practice has an iiPCR device, or “isothermal insulated PCR.” This technique allows you to get a result in a short period of time. Within 24 hours of sampling, the result may already be known. The test is OIE* validated and certified! (*Office Internationale des Epizooties)