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UAZ Sample Submission; date of this form: April 29, 2010; Page
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This general information is requested to facilitate rapid
processing of the samples, provide
background information to the diagnostician who will analyze the
samples, and for use in
documenting the epidemiology and geographic distribution of
pathogens within specific penaeid
species. The main purpose for requesting this information is to
give us some idea of the disease
problem, which will assist us in our final diagnosis and in
identification of the causative agent(s).
If the findings of a particular case should be used as part of
an epizootiological report concerninga given pathogen, no reference
to the submitting company will be made. Failure to provide the
above mentioned information may result in delayed processing
until the information is obtained.
For those companies using brokers or governmental agencies,
please inform the appropriate
customs or agriculture inspection officials to leave all the
case submission information,
regarding the samples, within or securely attached to the box
being shipped. If the official
requires a copy, please include two copies - one for them and
one for the UAZ laboratory.
SAMPLES FOR HISTOLOGY
Shrimp for Histological microscopy to be fixed in Davidsons
fixative should be fixed live bythe injection/immersion method.
1. Larvae and early postlarvae - fix by immersion in fixative
with fixative volume to shrimp
volume exceeding 10 to 1. Fix for 12 to 24 hours; transfer to
50% alcohol for storage or
shipment in screw-cap glass or plastic vials.
2. Larger postlarvae, juveniles and adults: Inject fixative into
hepatopancreas, stomach, and
midgut region in ~2-4thabdominal segment; then on small shrimp
open shell
longitudinally for the length of the animal or bisect or trisect
larger shrimp as well as
opening the shell. When opening the shell cut only through the
cuticle as to not harm the
underlying organs. Special precautions should be made when
cutting the cephalothorax
area to only cut just through the carapace and not into the
underlying organs.
The fixative should be made up as follows:
Davidsons Fixative (for 1 liter):
95% ethyl alcohol* 330 ml
37% formaldehyde (technical grade) 220 ml DO NOT USE10%
FORMALIN
Glacial Acetic Acid** 115 ml
Tap Water 335 ml
Notes:
* Isopropyl alcohol may be used as a substitute for ethyl
alcohol, but the results may be lessthan optimal.
** DO NOT USE OTHER ACIDS SUCH AS HCl (Hydrochloric acid) AND
H2S04(Sulfuric
acid) AS THESE ACIDS INTERFERE WITH TISSUE PROCESSING, STAINING,
AND
MOLECULAR OR ANTIBODY TESTS.
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2. Larger specimens (juveniles to adults) should be shipped by
wrapping in paper towels
moistened with 95% alcohol and packed in double plastic bags. Do
not send large
specimens in jars with alcohol.
3. Hemolymph samples may also be fixed in 95% ethanol. We
require at least 200 :l of
hemolymph to perform DNA and RNA extractions for PCR. Expel the
hemolymphsample into a sterile 1.5 ml microcentrifuge tube and add
one volume of 95% ethanol for
shipment. Be sure to use a new syringe and needle for each
sample to avoid cross-
contamination of samples or animals.
4. Other tissues such as gill tissue, pleopods, abdominal tissue
or stomach which are
biopsied or excised from moribund shrimp should be submersed
into 95% ethanol for
PCR analysis.
