Fish Diseases and Management

Obtaining samples for microbiological diagnosis from shrimp

Nauplii, Larvae, PL

1. Use the whole animal after rinsing in sterile seawater or 2.5% NaCl saline. Put the animal to broth culture medium and incubate.
2. Place whole animal on agar medium surface, crush with loop, streak plate with exudate and incubate
3. If transport to distant diagnostic lab is required, place sample in small sterile vial with sterile seawater, cover with sterile mineral oil. Keep transport sample vial cool, but do not freeze, as Vibrio species are sensitive to cold. Upon receipt at lab, transfer specimens to media.
   

Juveniles

1. Disinfect the surface by dipping in 1% calcium hypochlorite or sodium hypochlorite for 10 to 60 second.
2. Rinse in sterile seawater or 2.5% NaCl saline.
3. Using alcohol flamed dissecting tools, remove the cuticle of an abdominal segment or of the carapace, excise the organ or a sample tissue of interest.
4. For systemic infections, excise a block of abdominal muscle or the heart, touch it to the surface of an agar plate, streak and incubate.
5. For enteric infections, excise the HP, midgut, fore gut and touch the exposed inner surfaces of the excised organ to the surface of an agar plate. Streak and incubate.
   

Animals large enough to bleed

1. Remove haemolymph with a sterile tuberculin syringe and needle. A different syringe and needle must be taken for each set of shrimp. Place a drop of the haemolymph on an agar plate and inoculate with a sterile loop.
2. An alternate method is to disinfect the antennae with alcohol then cut them. Place the drop of haemolymph appearing on an agar plate and streak with a sterile loop.
   

Culture media for initial isolation

1. Marine agar (Zobell marine agar): A common medium used for bacteriological examination of marine bacteria. 
2. TCBS agar (selective agar): for Vibrio species
3. Trypticase soy agar TSA with added NaCl 2%: can be used as both an isolation and purification medium
   

Isolation

1. Using aseptic technique, inoculate appropriate isolation agar(s) with sample to be tested.
2. Each sample should be on a separate plate to avoid cross contamination
3. Incubate the agar plates for 24h at room temperature or 25 to 30⁰ C
4. Check the plates at 12 to 18 h for luminescent colonies as the luminescence may fade quickly by 24 h.
5. The organism should be purified on a general medium and further identified using staining, morphology, motility and biochemical tests.
   

Stuart transport medium

Stuart’s transport medium can be used for transporting samples to distant laboratories. The composition of the medium is given below:

Sodium chloride - 3 g

Potassium chloride - 0.2 g

Disodium phosphate - 1.15 g

Monopotasium phosphate - 0.2 g

Sodium thioglycollate -1g

Calcium chloride 1% aqueous -10 g

Magnesium chloride 1% aqueous -10 g

Agar -4 g

Distilled water - 1000 ml

pH -7.3

The medium is essentially a solution of buffers, with carbohydrates, peptones, and other nutrients and growth factors excluded, designed to preserve the viability of bacteria during transport without significant multiplication of the microorganisms. Sodium thioglycollate is added as a reducing agent to improve recovery of anaerobic bacteria, and the small amount of agar provides a semi solid consistency to prevent oxygenation and spillage during transport. Sterile cotton swabs can be used for collecting the pathogens from any specific sites in the body of the fish after cleaning the surface with a disinfectant or chlorinated water and then wash with sterile saline. The ulcers or lesions could be swabbed and placed in the tube containing sterile medium. In case of small fishes they can be put directly into the medium after washing in sterile saline.