Common Laboratory and Specimen Coilection Procedures for

Appe~ctix A

Common Laboratory and Specimen CoilectionProcedures for Disease Diagnosis

Diagnoshc Specimen Fixation and Submission forHistopathology Procedure

Purpose

Histopathology is a standard laboratory procedure widely used in diagnosis of diseases of animalsand man. Histopathology methods have been adapted for and are essential in the diagnosis ofmany of the described diseases of marine shrimp.

For a histopathology examination, preserved tissues of shrimp are carefully prepared, sectionedin thin layers, placed on g!ass slides, stained and examined with a microscope. The trainedpathologist is then able to direct!y observe and evaluate major internal organs and cells withinthese organs for evidence of pathological abnorma!! changes. Specific pathological changes inthe tissues of shrimp are characteristic for many shrimp diseases and allow the pathologist todetermine if the disease is affecting the shrimp population. Once a particular disease has beenidentified, appropriate control measures can be recommended.

Equipment and Supplies

~ Davidson's Fixative

~ 50 - 70% ethyl alcohol

~ Syringes k needles. 1 cc tuberculin and 5 - 20 cc syringes;27 and ! 8 - 20 gauge needles

~ Tissue dissection scissors, waterproof gloves, glass orplastic containers for fixation and plastic bags/paper towe! s if tissues will be shipped!.

Composition of Davidson's Fixative

Procedure

Preparation of Samples

1t is important to be aware that immediately after the shrimp die, the processes of tissuedegeneration can cause severe and widespread changes which may obscure any true histopatho! ogylesions present in the tissues. For this reason, shrimp that die before preservation make very poor

Appendix A: Laboratory Procedures for Disease Diagnosis170

The number of shrimp specimens that should be submitted to allow for a meaningful histologicalevaluation of a disease problem of shrimp wil! vary with the life stage affected, and the particulardisease in the population. A guideline for the number of specimens to prepare and send to thelaboratory for diagnostic examination is given below.

specimens for diagnostic histopathology examination. Therefore, for diagnostic histopathologyexamination ONLY SUBMIT SHRIMP THAT HAVE BEFN SACRIFICED BY INJECTIONOR IMMERSION IN PRESERVATIVE SOLUTION.

Preserving Shrimp

Larval or postlawal shrimp less than I cin in total length! can be immersed directly into Davidson'sfixative. Remember ALWAYS WEAR GLOVES WHEN WORKING WITH TISSUE FIXA-TIVES, Larger shrimp should be injected with fixative and then the cuticle opened to allowadequate, rapid penetration of fixative into the tissues. To insure proper exposure of organs tothe fixative, inject Davidson's fixative into the hepatopancreas and abdomen be liberal with theamount of fixative solution injected!, and open the shell longitudinally from the orbit to the tailalong one side smaller shrimp! or both sides larger shrimp! of the body. Large shrimp > 20grams! should also be bisected or trisected.

Preserve shrimp in a 10: I volume ratio of Davidson's fixative to shrimp tissues for 24 to 72 hours larger shrimp require longer fixation!. Once presewed, store shrimp tissues in 50-70% ethanol�; I volume ratio of ethanol to tissues! until further processing of the samples can be done.

Davidson's fixative can be reused several times before being discarded. If preserved shrimp areto be stored for any length of time, these samples should be kept in airtight containers to preventevaporation of ethyl alcohol

Suggestecf Numbers of Shrimp to Prepare for Submission

Nauplius through PL20.

Preserve and submit 200 to 500 individuals from each group population! to be evaluated.

PL25 through 50-gram shrimp if showing disease signs!:

Preserve and submit 5 to l 0 individual shrimp per sample group.

PL25 through 50-gram shrimp if not showing disease signs [e.g., a random sample]!.

Preserve and submit 30 to 60 individual shrimp per sample group.

Shipment o1 Specimens

Shrimp specimens can be prepared and submitted to a diagnostic laboratory according to thefollowing procedure. Once presewed and transferred to 50-70% ethyl alcohol leave thespecimens in alcohol for at least several hours!, the shrimp specimens can be removed from thealcohol solution, wrapped in paper or cloth soaked in the alcohol solution, placed in two or threelayers of plastic bags seal each bag individually so the alcohol solution does not leak or evaporate!and packaged for shipment to a diagnostic laboratory. Shrimp specimens should arrive in goodcondition for further laboratory work as long as they do not dry out. Be sure to label the outsideof the box: "preserved biological specimens for scientific study."

Appends A: Laboratory Procedures for Disease Diagnosis 171

Reference

Hurnason, G,L. 1979, Animal Tissue Techruques. 4th Edition�W.H, Freeman and Company,San Francisco.

Appendix A: Laboratory Procedures for Disease Diagnosis172

Bell, T.A. and D.V. Lightner 1988. A Handbook af Normal Penaei d Shnmp Hi itolngy, WorldAquaculture Society, Baton Rouge, Louisiana.

Diagnostic Specimen Fixation and Submission forTransmission Electron Microscopy TEM!

Purpose

In shrimp disease work the purpose of TFM evaluation is usually to assess microscopic tissuelesions for the presence or absence of very small infectious agents.


i Phosphate Buffer - O. l5M Appendix 0!

~ Glutaraldehyde - 6% in 0 15 phosphate buffer

~ 3. 50% EM-grade ampules

Procedure

Remember ALWAYS WEAR GLOVES WHEN WORKING WITH TISSUE FIXATIVES.Using 50% EM-grade ampules, add l0 rnl glutaraldehyde to 76 ml 0 l5M phosphate buffer tomake approximately a 6% working solution. Store and use at 4 C Once prepared usebuffer/glutaraldehyde solution within 90 days and discard unused fixative.

As with samples prepared for diagnostic hi stopathology, tissue preservation should be done rapidlyand at cool temperatures to minimize changes due to degeneration Preserve shrimp tissues byinjection of cold � C!, 6% glutaraldehyde solution into a live animal followed by quick dissectionand removal of the tissue s! of interest. Tissue sections should be immediately cut with a razorblade, in a few milliliters of cold, 6% glutaraldehyde into pieces no larger than Ix2 mrn. Transfertissues to vials and fix in a 10. I ratio of fixative to tissue volume for 24 to 4S hours, pour off the6% glutaraldehyde, and replace with 0.15M phosphate buffer. Fix and store tissues at 4 degreescentigrade. Further processing can be done using standard methods for routine EM workup,Shrimp tissues can be stored for several weeks in 6% phosphate-buffered glutaraldehyde atrefrigerator temperatures Make up new fixative and buffer solutions every three months.

Reference

Lightner, D.V., R.M. Redman and T.A Bell. 1983. Infectious hypodermal and hematopoieticnecrosis, a newly recognized virus disease of penaeid shrimp. J. 1nverr. Pathol 42:62-70

Apperidix A: Laboratory Procedures for Disease Diagnosis f73

Transmission Electron Microscopy TEM! is a powerful tool in the diagnosis of specific viruses,rickettsia, etc., which are associated with and cause particular cellular lesions. In TEMpreparations, the pathologist examines the internal structure of cells and cell organelles forabnormalities Improper fixation or storage of tissues for TEM will cause major artifacts orchanges. The preparation, sectioning, staining and interpretation of TEM material is a highlyspecialized activity

Collecting Samples for Microbiological Tests

Pur~Under certain circumstances, enumeration and identification of bacteria, or confirmation of theidentification of a fungal pathogen, may need to be conducted on samples of water, feeds or shrimp.This section provides general instructions on collecting, handling and submitting samples to alaboratory with facilities to carry out microbiological culture/isolation or enumeration tests.

Materials and ~Su ijllieS

~ Sterile, sealable plastic bags

~ Marking pen

~ Sterile pipette for collection of water and/or water and larvae

~ Sterile forceps for handling shrimp

~ Ice or other coolant and cooler for specimen transport

procedure

Specimens for bacteriological testing should be collected and processed with as little delay aspossible. If possible, inoculation of media and dilutions should be conducted while in the field.However, if field inoculation is not practical, water, dead shrimp or other moist substrates willneed to be transported. Under these circumstances the samples should be held at refrigeratortemperature e,g., 4 C in a cooler with ice! until the specimens can be processed.

Contamination of the samples with non-sterile water or other materials should be avoided. Ifshrimp are to be tested, they should be kept alive until the tissues are collected for bacteriologicalprocedures. Sample bags should be clearly and individually labeled for easy identification.

Reference

Plumb, J,A, and P.A. Bowser. 1983. Microbial Fish Disease Laboratory Manual. AlabamaAgricultural Experiment Station, Auburn University.

174 Appendix A: Laboratory Procedures for Dhease Diagriosls

Collecting and Submitting Specimens for IHHNV Gene Probe Testing

P~ur se

A gene probe test kit ShrirnProbe.! to diagnose HHNV is commercially available for use bylaboratory personnel or shrimp culturists DiagXostics, 27 Cannon Road, Wilton, CT 06897!,These kits provide a sensitive, accurate means for rapid diagnosis of IHHNV infection and arefor experimental use only.

Gene probe kits are used to detect IHHNV nucleic acid in l! in situ hybridization with preservedtissue substrate e.g., shrimp collected and processed for histopathology examination! and 2!dot-blot reaction for use with fresh tissue smears or hemo'lymph.

Gene probes provide highly sensitive and selective means for pathogen diagnosis. We advise thatthese diagnostic tools be applied only when the quality control standards are followed for the kits.In cases where farms or hatcheries do not have suitably trained personnel or adequate facilities,specimens for diagnosis should be submitted to a reputable shrimp disease diagnostic laboratory.

Equuiment and Sup~lies

in sitv hybridization

~ Davidson's fixative and 50'!o ethanol page 170!

Dot-blat Method

~ Tuberculin syringes and one- to two-ml sample vials with leak proof caps

~ Refrigeration, ice or dry ice to keep the specimens chilled to frozen until tested

Procedure

Samples for in simba hybridization are collected using the procedures for histopathology samplepreservation Hemolymph or pleopod specimens are often used as tissue substrates for the dot-blotmethod, Hemolymph specimens �.1 ml! should be collected by aseptic puncture into ahemolymph sinus usually the pericardial or ventral sinus! with a 27-gauge needle and syringe.Alternatively, the outer tip of a pleopod is collected. Hemolyrnph or pleopod samples are placedinto a labeled container and held chilled or stored frozen until analysis can be performed.

Apr.radix A: Laboratory ProcedureS fOr Disease Diagnosis

Specimen Collection and Submission for Indicator ShrimpSioassay Testing

Purpose

To evaluate shrimp populations for TSV or IHHNV. Note. Gene probe diagnostic test procedureshave largely replaced use of bioassay for IHHNV.

Equipment and SupplieS

~ Cast net or seine for collection of shrimp from ponds or tanks

~ Sealable plastic bags, marking pens

~ Ice or refrigeration during collection, freezer for storage

Procedure

Shrimp for indicator shrimp bioassay tests should be collected and handled to minimize decom-position of the tissue. Shrimp should be iced irnrnediately after removal from the pond and frozenas soon as possible ln transport to a diagnostic laboratory, the shrimp tissues should remaincompletely frozen.

Appendix A. Laboratory Procedures for Disease Diagnosis176

The number of shrimp collected varies on the circumstances. For TSV diagnosis for shrimppopulations with active to chronic discase, samples of five to ten symptomatic shrimp i s sufficient.For populations lacking gross evidence of TSV, random sampling procedures should be followed See Appendix B!.

A ppencfix 8

Shrimp Evaluation Procedures

Wet-Mount Procedure

P~ur use

A common technique used to diagnose diseases in marine shrimp is the microscopic examinationof wet-mount preparations of tissue Figure 99! for specific abnormalities associated with thedisease. For example, larval mycosis is easily diagnosed in wet-mount preparations of whole

shrimp larvae. Figure 99, Preparation of a wet-mountslide.Materials and Supplies

~ Clean microscope slides

~ Cover slips

~ Pipette

~ Tweezers

~ Light microscope

Procedure

~ Place a drop of clean saltwater on aslide

i Put the whole larvae or tissue i e.,gill filament! in the water

e Place the edge of a coverslip on theslide so that it touches the edge of thewater

~ Slowly lower the coverslip to preventforming and trapping air bubbles

~ Place the slide under the microscopeand proceed from the lowest-power tothe highest-power objectives makingminor corrections in focus and lightas necessary

Reference

AbramotT, P. and R. G. Thomson 19S6. Light Microscopy. Laboratory Outlines inBiology IV.

178 Appendix B; Shrimp Evaluation Procedures

Larvae and Postlarvae Fitness:General Behavior and Physical Apped«n«

Purpose

To assess the behavioral and physical condition of larvae or Pos' rv


~ Beaker 100 to 500 ml

~ Clean glass slides and cover slips cover slips that are 22x30 mrn are Preferred!

~ Clean, filtered seawater

~ A compound microscope with 10x eyepieces and 4x, 10x, 40x and 100x objectives

s Ocular micrometer

~ Record forms to record observations.

Procedure

~ Collect a sample of 20 to 100 larvae or postlarvae in a 100 to 500 rnl clear glass beaker

~ Observe and grade the swimming behavior of the animals. Record observations andcomment if dead larvae are present in the sample, For protozoea note if fecal strandspresent and well formed?

~ For microscopic evaluation, examine a minimum of 10 animals, Collect each larvaewith a pipette and place between five to 10 on a glass slide. Add filtered seawater andsecure with a cover slip �2 x 30 mm! Remove excess water with a clean paper toweland place slide on the stage of a compound microscope. Study each larvae for thefollowing features and record results on a data sheet

Features

Ap~ricllx g Shnrrip Evaluation Procedures 179

Larval stage, fullness of the gut, level of hepatopancreas lipid vacuolation, grade of bacterialinfestation on the appendages, eyes, gills, body and abdomen; grade of infestation of attachedprotozoa; grade of melanized cuticle lesions; grade of dark, grainy muscle lesions; presence ofinternal melanized nodules; grade of BP polyhedra in the hepatopancreas; the condition of thechromatophores along the ventral abdomen or other microscopic physical abnormalities. Inaddition, postlarval determination of the muscle-to-gut ratio is a predictor of larval fitness:

~ Measure the diameter of the mid-gut and the vertical height of the muscle in the 6thabdominal tail! segment.

~ Divide the muscle height by the rnid-gut diameter to determine the Muscle:Gut M G!ratio, Healthy postlarvae have a M:G ratio of > 4: 1 while unthrifty animals have aratio of < 4:1.

Postlowal Fitness: Stress Test

Purpose

To measure the fitness of a population of postlarvae prior to sale or stocking into a nursery orgrowout pond.

Equipment and Materials

~ 100 Postlarvae

~ Centigrade thermometer

~ Refractometer

~ Saltwater 32-34 ppt

~ Freshwater 0-1 ppt

e 10-15 L bucket

Procedure

~ Two parameter test temperature and salinity!> Fill a 10-15 L bucket with water at 22 C and 5 ppt salinity.

> Measure and record temperature and salinity from the larval rearing or postlarvaeholding tank. Conditions are usually 27-28 C and 32-34 ppt,

> Randomly collect 100 postlarvae from the larval rearing or postlarvae holding tank andtransfer into the test bucket.

> Hold for 1 hour and count the number of survivors those that respond and swim normallywhen stimulated!. Eighty percent or more active indicates the population has passedthe test.

~ Single parameter test salinity!> Fill a 10-15 L bucket with water at 27-28 C and salinity of 0-1 ppt.

> Measure and record temperature and salinity from the larval rearing or postlarvaeholding tank. Conditions are usually 27-28 C and 32-34 ppt.

> Randomly collect 100 postlarvae from the larval rearing or postlarvae holding tank andtransfer into the test bucket.

> Hold for 1 hour and count the number of survivors those that respond and swim normallywhen stitnulated!. Eighty percent or more active indicates the population has passedthe test.

If the postlarvae do not pass the stress test, the population is oAen held for several days withimproved conditions and retested at a later date.

Appendix B: Shrimp Evaluation Procedures

Sampling Shrimp Popuiations for Pathogen or Disease Detection

Sampling is the process of obtaining information about a large group of items e.g., a population!by examining only a small number of them. Within the context of shrimp health and diseasemanagement, sampling shrimp populations is undertaken for some of the following reasons

~ To determine the individual and average size or larval stage! of animals in thepopulation

~ To evaluate the physical appearance of shrimp in a tank or pond population~ To evaluate the product quality of shrimp in the population

~ To test the population for the presence of a known pathogen or disease using alaboratory diagnostic procedure

The initial step in the sampling process is to clearly understand the purpose of sampling. Forexample, if the average weight and size distribution of a shrimp population are the criteria forsampling, then it is important to obtain something that resembles a realistic "random sample" ofthe population and to include a sufficient number of animals. On the basis of practical experience,approximately 200 animals, collected from four to six locations in the pond, is usually sufficientIf the purpose of the sample is to submit physically or behaviorally abnormal shrimp to a diagnosticlaboratory for a pathological examination, then five to t 0 ~mtomatic individuals is usually anadequate number of specimens for di agnostic evaluation.

However, if the purpose is to examine a population for the prevalence of a certain subclinicaldisease condition or to assess for the presence of carrier or latent infection by a pathogen e. g,IHHNV, BP or TSV!, then the number of evaluated animals should follow statistical criteria.Simon and Schill �984! detail the required sample sizes for a variety of fish disease samplingapplications. The needed sample size is dependent upon the following variables:

~ Confidence level desired for the test

i Prevalence of the pathogen/disease in the population

~ Number of shrimp in the population

Generally, a 95% confidence level is acceptable. Detection of tow population prevalences of theagent/pathological condition e.g., 10% or less! is used in cases where, for example, the purposeof the test is to insure the absence of a pathogen prior to export of a group of shrimp to anothergeographical region. Generally, the prevalence of infected shrimp by a pathogen has been foundto increase in the population following initial exposure to the agent. On a practical level, theprevalence of infected individual shrimp in cultured populations can be assumed to be > 2 y'0 withinseveral tnonths from initial exposure. Thus, a reasonable sample size for detection of these agentscan be set for a population prevalence of 2% or more, We often work with the assumption thatthe level of infection is 5% or more.

Also, an important feature to understand is that according to statistical theory, the number ofindividual shrimp that need to be examined for detection of a pathogen/disease for a givenprevalence and confidence level does not change much for populations over 1,000 animals. For

181Appendix B; Shrimp Evaluation Procedures

example, the number of shrimp examined to detect a pathogen at a 5% prevalence, with 95%confidence, is similar if the population size is 1,000, 10,000 or 100,000 ln this instance, thenumber of animals to examine is 60.

Number of Shrimp to Testevalence of Pathogen/Diseasein the Population

1% or higher

2% or higher+

5% or higher

10% or higher 30

Therefore, for a 95% confidence level, to detect one infected individual for the pathogen/diseasethe test numbers and prevalance levels for a population over 1,000 animals are given below.Finally, these sample criteria are valid when sampling is randomly conducted. This aspect shouldbe considered in the interpretation of the results. Repeating a sample and testing procedure canalways be used to improve confidence in the validity of the results

Reference

Siinon, R.C. and W.B. Schill. 19S4. Tables of sample size requirements for detection of fishinfected by pathogens: Three confidence levels for different infection prevalence and variouspopulation sizes. J. Fish Disease. 7: 5 l5-520,

Appendix B: Shrimp Evaiucition Procedures182

The Use and Construction of Histograms

Introduction to Histotrcrn

A histogram is a graph made up of vertical bars, commonly referred to as a bar chart. The usualway to graph a continuous distribution is in the form of a histogram. Each of the graphed segmentsis a rectangle of the same width and the bars touch each other to show that the actual limits of theclasses are contiguous or connected in an unbroken sequence. The midpoint of the bar correspondsto the class interval and the height of the bars represent the frequency of each class,

Histograms are used by shrimp farm managers to look at the variation in size distribution withina pond or group of ponds The individual shritnp weights are used to create a bar chart showingthe size distribution and amount of variation in the sample.

How to Construct a Histogram The vertical axis represents the frequency or nurn-ber of occurrences and the horizontal axis repre-sents the dass intewal or size. The frequenciesare represented by vertical bars, Each bar is oneclass interval wide, and is centered over its classmidpoint. The height of the bar is equal to thefrequency of its corresponding class intewal. Thevertical axis always starts with zero to illustratethe relative size of the bars.

~FFcam fe Draw a frequency histogram for thefoll owing data:

Step 1. Divide the horizontal scale into equallengths, one for each class interval. There arefour class intervals in this example.

Step 2. Divide the vertical scale into equal lengthsto represent all the frequencies starting with zeroand going to the maxitnum value in the data.

Step 3. Plot vertical bars of the data to show howthe various weights are distributed.

Appendix 8: Shrimp Evaluation Procedures

References

Henrik, R. 1965. Stattstics. Monarch Press, inc., New York. 1 lop

Sokal, R.R. and F.J, Rohlf. !969. Bi<>metry. W.H. Freeman and Company, San Francisco776p.


Purpose

Procedure

GilI Examination Procedure

To assess the physical condition of thegills Figure 100!.

KcCuIpment and Sup~t1es

~ Scissors and forceps

~ Clean glass slides and coverslips �2x30 mm!

~ 2.5% saline solution

~ A compound microscopewith 10x, 40x and 100xeyepieces and 40x and 100xobjectives

~ Record forms to record observations

~ Collect 10 shrimp from the pond ortank and hold live in the source water

~ Sacrifice each shriinp by severingthe ventral nerve cord between thebody and abdomen tail!

e Gently remove the cuticle over oneside of the gill chamber, clip offseveral gill filaments, place on aclean glass slide in a drop of salineand cover with a coverslip

~ Examine under 10x and 400x mag-nification for the following:


Figure 100. Gills exposed for examination drawing by Norton Chan 1994!.

Figure 10l a. Different grades of gillfouling, Grade 0 or 'no fouling',

Figure 101b. Different grades of gillfouling, Grade 1.5 or'some fouling' byfilamentous bacteria and diatorns,

Figure 101c. Different grades of gillfouling: Grade 4 or "severe fouling" byfilamentous bacteria and diatoms.

> Grade of filamentous bacterial infesta-tion Figure 101 a, lolb, ]Olc!

> Grade of peritrich protozoan infesta-tion

> Grade of detrital and algal fouling

> Grade of melanized cutic/e lesions

> Grade of internal melanized tissue ornodules

e Record results on the data form Appen-dix C!

Appendix 8; Shrimp Evaluation Procedures

Analysis of Stomach Contents Procedure

Purpose

To determine what the shrimp are eating.


~ Scissors and forceps

~ Clean glass slides and cover slips �2x30 mm!

~ 2.5% saline solution

~ A compound microscope with l0x eyepieces and 4x and 10x objectives

~ Record forms to record observations

Procedure

~ Collect 10 shrimp from the pond or tank and hold live in the source water

~ Sacrifice each shrimp by severing the ventral nerve cord between the body andabdomen tail!

~ Gently remove the dorsal cuticle from just behind the eyes to the end of the carapaceto expose the stomach and hepatopancreas. With the forceps grasp the posterior ofthe stomach and gently lift up and separate from the hepatopancreas. Sever theesophagus and remove the stomach. Open the stomach along the dorsal mid-line andtransfer a drop of the stomach contents to a clean glass slide. Add saline and securewith a coverslip.

~ Examine under l Ox and 40x magnification for the following;

! the relative percent of feed pellet present

! the relative percent of detritus present

> the relative percent of algae present

! other observations

~ Record results on the record sheets

' 87Appendix B; Shrimp Evaluation Procedures

Mid-gut intestine Fullness Examination

Purpose

To assess shrimp feeding activity.

Eq r ~dS

~ Cast net or seine to collect shrimp from the pond or tank

~ Bucket with clean water for holding shrimp

~ Record form to record observations

Procedure

Do mid-gut fullness examination Figure 102! on several ponds each month to develop baselinenormal values for the procedure and personnel making the assessment.

Collect shrimp from several locations in the pond/tank. Evaluate a minimum of 30 specimens foreach pond and sampling period.

Evaluate shrimp immediately after removal from the pond. Hold each specimen so that the mid-gutabdominal intestine, from the first abdominal segment to the hind-gut dorsal mid-line!, is clearlyvisible. Grade the fullness of the intestine as follows: empty = 0; 1/4 full � � 1; 1/2 full = 2; 3/4full = 3; and full = 4 Make a note of the molt stage of each animal by feeling the rigidity andfeel of the exoskeleton. Grade shrimp either as molting soft or flexible exoskeleton! or inintermolt rigid exoskeleton!.

Record results on the record sheet.

Int~er cetation

Appendix B; Shrimp Evaluation Procedures

Shrimp feed more or less continuouslyexcept during late pre-molt through molt-ing ecdysis!. If shrimp are feeding, theaverage intestine fullness for a group sam-ple will be greater than 0.75. Loweraverage values suggest the depressedfeeding activity by the population. Thiswould warrant further evaluation to deter-mine the cause.

Figure l02. Photo showing shrimp with full bot-tom! and empty top! mid-gut intestine.

Environmental Sioassay Procedure

Purpose

Aquatic environmental bioassays involving shrimp or phytoplankton have been used to determine�! appropriate locations for shrimp hatcheries or farms, and �! the presence of pesticides or toxiccompounds in the water or soil

There are two types of environmental bioassays - acute and chronic bioassay tests Ziemann 1990!.Acute bioassays measure survival at the end of a fixed time period i.e., 24 or 96 hours! wherethe test organism remains in a test solution, but is not fed. Chronic bioassays last as long as oneweek and are generally used to test sublethal effects on growth rate or development and survival.The results of an acute bi oassay are described in terms of an "LC50" for the chemical being tested.This is the concentration at which 50% of the organisms remain alive at the end of the test period.

Procedure

To determine an appropriate location for a shrimp hatchery, an environmental bioassay wouldinvolve rearing small groups of larvae and batches of larval feeds particularly algae! at theproposed site in the available water. Survival, growth and appearance of larvae and algae areused to assess the suitability of the conditions. To determine the appropriate location for a growoutfarm, scaled-down pond growout trials should be conducted and then successful animal perform-ance evaluated.

Acute bioassays can be conducted in beakers using shrimp nauplii/protozoea to identify thepresence of toxic cotnpounds. Ziemann �990! conducted a 96-hour bioassay using glass beakerscontaining 10 P. vansramei nauplii, 200 ml of seawater at 35 ppt salinity, Chaeloceror a marinediatom! at 100,000 cells/ml as food for the shrimp protozoea and a test toxic compound i.e.,cadmium at a concentration of 10 ppb! The nauplii were counted and the water quality of thecontrol beakers was monitored daily.

Chronic bioassays can also be conducted in beakers to determine the effect of toxic compoundson shrimp growth and survival. Ziernann �990! conducted a seven-day bioassay using 10postlarval P. vannamei PL6-8! in 1000 ml plastic beakers containing 500 ml of seawater at 35ppt salinity and a test toxic compound i. e., copper at a concentration of 1000 ppb!. Once a day,the postlarvae were counted, fed Arremia, beakers were cleaned and test solutions were replaced.Dry weight of the shrimp at the end of the seven-day period was used to determine the effect ofthe toxicant on growth rates.

Reference

Ziemann, D.A, 1990. Acute and chronic toxicity testing for water quality management � FinalReport to the Hawaii State Department of Health 50 pp.

Appendix B: Shnrnp Evaluation Procedures 189

Shrimp Bioassay Procedures

Pu~r>se

To investigate the possibility of apparently healthy P. Lannamei populations asymptomaticallycarrying pathogens such as IHHNV or TSV. To indicate the presence of IHHNV, the bioassaystudy is conducted using IHHNV-free J'. stylirosrris For TSV, the indicator shrimp are TSV-freeP. vannamei.

Loboratory Procedure to Identify IHMNV-Infected Shrimp

Apparently healthy P. vannamei can be tested for IHHNV infection in glass aquariums.e In direct or oral exposure, 10-20 P. stylin~<ins are fed macerated carcasses of 10 to

20 IHHNV-suspect P. vannamei for 8 separate days during the initial 21 days of theexperiment.

e Samples for histopathology and 1HHYV gene probe evaluation should be randomlycollected at approximately five-day intervals after initiation of the experiinent,beginning with day 15 and ending with day 30

Pond Procedure to Identiffr IHHNV-Infected Shr~lm

Apparently healthy P. vannamei can also be tested for IHHNV using bioassay methods in earthenponds. Thirty P. styIIrostris are stocked into a 0.2 m bioassay cage that is placed in an earthen3

pond. P. srylirosiris are then fed an artificial pelleted diet and macerated P. vannamei carcasses heads only - two per feeding! from the pond every other day over a 29-day exposure period.Shrimp should be randoinly sampled for histopathology evaluation/IHHNV gene probe startingon day 15 of the bioassay and thereafter, at approximately five-day intervals.

Laboratory Procedure to Identity TSV-intected Shrimp

Apparently healthy or obviously Taura syndrome TS! affected P. vannamci or other biologicalsubstrates e.g., potential insect reservoir hosts, etc.! can be tested for Taura syndrome virus TSV! by this procedure. Collect the tissue sample to be evaluated and either process as freshmaterial or hold frozen for a period. TSV remains viable in frozen tissues for an extended period.

Stock 10-liter aquariums or fiberglass tanks with 10 to 20 known TSV-free, juvenile �. 5-2 g! P,vannamei. A control group must always be used in these trials. The optimally sensitive animalsare the Mexican strain SPF stock. Collect and macerate epidermis e.g., pleopods, gills, etc,!from the shrimp to be tested, macerate and mix thoroughly If insects are used, feed wholeorganisms to the shriinp, Feed exposure groups of shrimp test tissues for two to five days, thenswitch to commercial shrimp pellets. The control group is fed pellets. Alternatively, an injectionexposure can be used to test the tissue for TSV. In this approach, the tissues can be maceratedin sterile saline, centrifuged, filtered �.45 %! and injected into the abdominal musculature of testshrimp.

ApperidlX 8; Shnrnp EValuahon ProCedures

In feeding trials, the onset of Taura syndrome mortality occurs usually within three to seven daysfrom initial exposure to the test substrate, Sacrifice by injection and immersion in Davidson'sfixative test shrimp that display abnormal behavior, if these are observed. If mortality reachesor exceeds 50%, the entire tank group can be sacrificed by injection and immersion in Davidson'sfixative. At days l0-12 all shrimp in each test group should be sacrificed and preserved for ahistopathological examination.

For injection exposure, the onset of Taura syndrome will be oAen within 24 to 36 hours followinginoculation. Mortality from TSV will peak oAen by 4S to 72 hours from exposure. Sacrificesymptomatic shrimp for histopathology, if these are observed. Otherwise harvest the test groupsfor histopathology exam on day five from initial exposure.

Subtnit all specimens for a histopathological examination to a shrimp pathology laboratory withexperience in recognition of TS. For the test to be valid, tissue exposure groups must haveindividuals with classical TSV lesions and the control shrimp must be free of TS histopathologychanges.

References

Lightner, D.V., R.M. Redman, T.A. Bell and J.A. Brock. 19S3. Detection of IHHN Virus inPetirteus stylirosrris and P. vannamei Imported into Hawaii. J. world Maricul. %~, 14:212-225.

Appends B: Shnrrip Evaluation Procedures

Appendix C

Hatchery and GrowoutManagement Procedures

Disinfection Procedures in the Hatchery and Growout System

Purpose

Periodic disinfection is part of the routine in shrimp hatcheries. On the other hand, disinfectionin shrimp ponds is unusual.

The following provides a brief outline of disinfection procedures that can be applied in shrimpculture. The use of disinfection methods will vary among hatcheries and the information givenhere only lists a general outline of methods.

Equi ment and Supplies

Procedure

Disinfection of Tan@, Pipes and Fquipment

Tanks, pipes, equipment and the inside surfaces of all buildings should be thoroughly cleaned inpreparation for disinfection. Tanks and pipes should be filled with water and treated with chlorineat one of the foll owing treatment levels:

~ 100 � 150 ppm �5 g of 65% active chlorine/25 gat! for 30 minutes to 1 hour. If pHof water is greater than 7.5, it may be desirable to pre-treat water with acetic acid �fl oz �9.6 ml! per 100 gal �80 L!!, AtN s add aerd ro water. Never add acid Iod chlorure or water ro acid

~ 10 - 25 pprn �-3 g of 65% active chlorine/25 gal! chlorine for 12 hours or longer.

~ For the control of l~geuldium, chlorine disinfection should be at a treatment level of500 ppm �5 g of 65% active chlorine/25 gal! or greater for a 24 hour period.

The tank exteriors, walls and floors should be washed with a 50 to 100 ppm solution of chlorineand rinsed with freshwater. Alternatively, in special cases where complete disinfection of anenclosed and sealed area is desired, formaldehyde gas can be used according to methods given inHundemann and Holbrook �959!,

Following di sinfection, access to "clean "areas should be restricted. Iodophore �0 to 100 ppm!or chlorine �00 to 200 ppm, pH 7! footbath stations may be established at the entry areas, It isbest to inaintain specific footwear for use only in the limited-access area. If this is not possible,individuals should dip the bottom of their footwear in the footbath upon each entry or exit fromthe area.

lodophore �0 to 100 ppm! or chlorine �00 ppm, pH 7! equiptnent baths should be used andmaintained in each limited-access area. The disinfection solution should be changed frequently.

194

~ Chlorine

~ lodophore

~ Acetic Acid

~ Hydrated Lime

Appendix C: Hatchery and Growout Management Procedures

If chlorine i s used, be sure to acidify the water to improve the killing power of chlorine. Equipmentshould be dipped and shaken dry prior to, and following, each use. Separate sets of equipmentshould be labeled and used only within designated areas. Transferring equipment between water,culture or tank areas in the facility should be prohibited. Nets and brushes should be stored offthe floor on a drying rack.

Oisirifection of Pooch

Ponds should be completely drained and all animals removed prior to disinfection. Hydrated limeshould be evenly dispersed over the entire pond bottom at 1,000 to 2, 500 kg per hectare �13-2S2g/m !. Ponds should be left dry for a minimum of 14 days before being refilled.2

Reference

Hundemann, A.S. and A.A. Holbrook. 1959. A practical method for the decontamination ofmicrobiological laboratories by use of formaldehyde gas. J. Amer. Veter. Axvoc. December,1959:549-552

195Appendix C: Hatchery and Gtowout Managerrient Procedures

Washing Eggs

~Pur use

Spawned eggs of I'. va>niamei are washed to remove debris, reduce bacteria, protozoa and fungi,and minimize the transmission of BanrIrpvirw» yenaei if the broodstock are harboring this agent!from spawner to the oAspring.

Equiprrtent and~Su plies

~ 50' Nitex screen net

~ 5 � 10 liter clean plastic bucket

Source of clean, nearly bacteria-free seawater

Procedure

The steps involved in collecting and washing shrimp eggs prior to incubation are illustrated inFigure 103. After the spawned brood stock have been removed from the tank, aeration is terminatedand the tank is partially drained, The eggs, which then sink to the bottom, are collected by a drainpipe Figure 103! and filtered through an inner 300it-mesh net and an outer 100@-mesh net. Theyare then washed with clean seawater. The rinsing and washing water temperatures should be thesame as that in the spawning tank. The eggs are incubated in a temperature-controlled rearingtank, which contains 40-50 cm of seawater.

Figure 103. Egg washing procedure.

Reference

Sano, T, and K. Momoyama.1992. Baculovirus infection of

penaeid shrimp in Japan. In:Diseases of Cultured Penaeid

Shrimp in Asia and the UnitedStates. W. Fulks and K.L.

Main Eds.!. pp. 169-174.

Appendix C: Watchery and Growout Management Procedures

Figure IO4, Nauplii rinsingprocedure,

Rinsing Naupiii

Purpose

Nauplii of]'. vrvvicvnei are washed to remove debris, reducebacteria, protozoa and I'ungi, and minimize the transmissionof Bacrd<n/ruspciatel if the broodstock are harboring thisagent! from spawner to the offspring


~ l4-L piastic bucket with large slits cut into the

sides, which are covered with 100it Nitex screen

~ Formalin

e Seawater rinse = 30 ml/sec I 8 L/min!

Procedure

The procedure for rinsing nauplii is described in Figure l04

Reference

Mrs Carol Cozzi-Schrnar, Amorient Aquafarm, IncKahuku, Hawaii

Appendix C: Hatchery and Growout Management Procedures 197

~ Iodophore stock solution - 10 m! Iodophore into I L of d}stilled or tap water. Aworking solution of IO ppm =- I ml stock solution per liter, a working solution of 50ppm =- 5 ml stock solution per liter!.

Treatments for Diseases in penaeid Shrimp La~ac

Pur~ee

The dosage levels of the chemicals that have been used to control disease in the various stages ofpenaeid shrimp larvae are listed below,

Appendix C: Hatchery and Growout Management Procedures198

Treatments for Vibriosis in penaeid Shrimp

purpose

Ufe Stage

l UPL

UPL

UPL

UPLJ/A

L/PL

UPLJ/A

UPLol

J/A

stiarvae; J=juvenile; A=adult

T18 +met-30!

Procedure

In the hatchery environment, treatments for bacterial management are usually administered as abath. In a bath treatment, the chemical is added to the water at a designated concentration andthe exposure period can vary from 30 minutes to overnight.

Reference

Dosages adapted from Lightner, D. V. 1988. Disease in cultured penaeid shrimp and prawns. In:C. Sindermann and D. Lightner Eds.!. Disease Diagnosis and control in North AmericanMarine Aquaculture. Elsevier, Amsterdam. pp, 8- l37

AppendiX C: Hatchery arid &owOut Marlagement Procedures

The dosage levels of chemicals that have been used to control vibriosis in the various stages ofpenaeid shrimp listed below

Incorporation of Medications into Growout Feeds

Antibiotics or other oral medications may need to be fed to shrimp to control vibriosis or otherbacterial diseases. Ideally these substances should be incorporated directly into the diet prior toextrusion of the pelleted feed. In some cases, it may not be practical to do this, and alternatively,medications can be coated onto a feed prior to its being fed to shrimp using one of the followingmethods. Gelatin or oil can be used as a drug carrier for coating feed pellets.

Procedure

Gelatin: 125 g gelatin in 3.0 quarts water per 100 lbs of pellets.

~ Slowly dissolve gelatin in hot tap water.

e Mix the drug into the gelatin until it is evenly dispersed.

~ Slowly add, with even constant stirring, the drug/gelatin solution to the pellets. Stironly long enough to insure even drug coating. A cement mixer can be used for largerbatches.

Soy oil: 2-3 lbs per 100 lbs of pellets.

~ Mix into warm �00-120 F! oil.

~ Pour or spray mixture over pellets.

Reference

Piper, et al 1982. Fish Hatchery Management. United States Department of the Interior. Fishand Wildlife Service. Washington D.C. 517 pp.


oo

!gi oogoogo ZSRSR88

n oW VOoo

O A 0 Vl oCh

ohio ogooo oo

oo

Ul

O.E

Z EA

P I I

2' pe

O ATE

80 ~

g Eaez g.

o<p<o a os+ox oFlmm&MC

RRRR88888888

oeomootngeogogosooo oooo o

8 o o o ~ ~ ~ R cv w w K N w 00 R 8oo oooooooooooooo�

~<~ewaaa o~~o~ooo oo Kt

Posttarval Acclimation for Salinity

~PUr ioso

I'ostlarval I', vannamei are often stocked into nursery or growout ponds in which the salinity isdifferent from that of full seawater e.g., 33 ppt!. In a broad sense, acclimation should be viewedas the postlarval period of adjustment from the hatchery environment to shrimp farm waterconditions. Also, the acclimation period allows time for the postlarvae to recuperate from thestress of shipment. As postlarvae develop they improve their ability to adapt to changes in salinityNevertheless, postlarval stocking survival is strengthened if they are adjusted gradually to a loweror higher salinity environment. Thus, the purpose of salinity acclimation is to enhance the survivalof newly stocked postlarvae.

Equ~ltmetN

Adequate volumes of fresh and saltwater are needed to carry out the acclimation procedure. Acovered postlarval receiving station with water reservoirs and acclimation tanks is common onmost shrimp farms. An operational dissolved oxygen meter, salinity refractometer, thermometerand light microscope are also needed The water delivery system should be set upas a flow-throughsystem in which fresh and saltwater flow can be adjusted. Temperature control can be achievedwith ice-filled plastic bags placed in reservoir tanks.

Procedure

< The number of postlarvae should not exceed 500 per L in acclimation tanks. Watertemperature should be maintained at a moderate level e.g., 25 + 1 C! and remainconstant during the acclimation procedure If temperature acclimation is required, thisshould be done after salinity acclimation has been accomplished. At this time,temperature can be increased or decreased at a rate of 2 C per hour.

~ Dissolved oxygen should be maintained at saturation levels throughout the acclimationperiod. Dissolved oxygen readings should be monitored twice per hour in each tankthroughout the acclimation period. If low DO conditions arise, increase aeration oruse supplemental oxygen to raise DO as fast as possible.

~ Feed the postlarvae during the acclimation period. Frozen Arremia can be addedperiodically at 5 nauplii/ml and supplemented with a commercial formulated feed.Avoid overfeeding during the acclimation period.


Table 28. Acclimation schedule for healthy postlarvaestage 6 or older.

~ Day six or older postlar-vae that are in goodhealth will tolerate a

change in salinity accord-ing to the guidelines inTable 28. However,poor condi tion oryounger postlarvae mayrequire longer periods to

Figure 105, Example of an acclimation data lo

achieve acclimation. It is the responsibility of the person in charge of acclimation toadjust the schedule, which depends on an assessment of postlarval condition uponarrival to the acclimation station Salinity should be measured at least twice per hourduring the acclimation period.

~ Postlarval condition should be visually evaluated at least hourly throughout theacclimation period. A microscopic examination of postlarval samples from shipmentbags is also recommended. If postlarval behavior changes or if a mortality problemis encountered, the acclimation process should be halted and conditions stabilized untilthe problem can be corrected.

e Water parameter measurement and visual monitoring data must be recorded on a logsheet Figure 105! immediately aAer the measurements have been made.

Reference

Villalon, !.R. 1991. Postlarval Receiving. 1n: Practical Manual for Semi-intensive CommercialProduction of Marine Shrimp. TAlVIU-SG-91-501. Galveston, Texas. pp. 21-32,

A~ndix C: Hatchery and Growout Management Proc~>r

General Guidelines for Major Cations, Anions and TraceMinerals in Seawater

Reference

Modified froni: Todd, D. K. 1970. The Water Encyclopedia, U. S. Geological Survey, Waterlnforrnation Center, Port Washington, New Vork. 559 pp


Record Keeping Forms

Table 29. Daily MatUration Tank Log.


U!

0 U>C C

Table 31. Larval Raaring: Daily Larval Rearing Tank Log.

Appendix g Hatchery and Growout Managefnent Procedures

Table 32. Captive Reproduction; Broodstock Feeding Schedule and Quantity Log.

Tank ¹ I Tank ¹ 2 Tank ¹ 3

AM PM AM PIVI AM i PMt

ok ¹5

FreshFeed

Formulated'Feed

Formulated, Feed

FreshFeed

FormulatedFeed

FreshFeed

Formulated

FreshFeed

FreshFeed


I 'Date

I Fresh~ Feed

! FormulatedFeed

T

LTank ¹4

AM PM

I

7

0

l34r

0!

0 U8 0 U0

B

P-CQ

CD

C D<D

U 0

J3

0 Z

C0 !gk 'g 8

d0 ~g8

5 j'U

ef

VCh tt5

ce ~5 00CJ

Y!gj Q

Au.a,

5 'U C D 5

Table 34. Transfer and Receiving, Postlarvae Harvest Log.

Date:

Time:

Lcsval Rearing Tank Harvested:

Harvest Chamber Voi~; liters!

e Number Sample Volume, ml!

Number of

Postlarvae

irt Sample

Sample PopulctEstimate

2

Mean population estimate:

Number of postlarvae per liter in harvest chamber.

Hauphi&tt& X 100= % Survival From NaupliiPostlarvae Harvested to Postlarvae

/liter

Bagging:

Shipping temperature:

Destination:

Number of postlarvae per bag desired for shipping:

Comments aud Calculations:


Adapted fram: The Oceanic Instttute Shrimp Program Postlarvae Harvest Log

Table 35, Transfer and R'eceiving: Nursery Harvest/Growout Stocking Log.

Appendix C: Hatchery and GroNrout Management Procedu~es

C50 0 0CL

0 08

0 0

P38Zt0I�

E' 0

l3C

pp g> g Q!p LZ Ch~ me>=c~~ ~ Eo~~av+OQ+u

0

C

0 0CE0X

8E0CD0

CL0.E

ID

C 0 C 0 ID00ID

0

0

8

E0

0 IDO.0

0 CD0

C 00LE

8 O

o

zli II

Q

~e ~OQ CI5s

oil II II

Table 38. Growout: Growout Pond Sample Log,

Date;

Sampler

Pond ¹ Throws mmentsotal Wt. ¹ Shrimp

214

L I

T Co


0 08

Table 40. Growout, Shrimp Growout Report Log,

tock ed testwt, g!Count

Harvesting:

tt Pos Mean Ltss. ' Remaining! ' harv. pcs. Feedlbs.arv. wt. g


Pon } ¹

Stocking:

Total

From lbs.

TAvg tal

bs.!Total I Bag Condition

Appendix D

Microbiological Proceduresand Recipes for Solutions

Total Plate Count

PurposeTo provide quantitative data on bacterial nuinber in water or live-feed substrates.

Equuiment and Sup~lies

~ Sterile bottles or plastic leak-proof bags

~ Sterile petri dishes, inoculation loops; Marine, TCBS or other agar, incubator orcontrolled temperature chamber; bunsenburner or alcohol/propane flame; and generalfacilities, equipment and supplies for routine microbiology

~ Sterile dilution tubes; sterile 2% saline; one O. 1-ml and ten 1-ml sterile pipette;calibrated 0. 01-ml inoculati on loops

~ Tissue grinder

Procedure

Water, larvae or wet-feed substrates should be collected to avoid contamination in a sterilecontainer. Usually, 100 ml of water per sample is adequate. Sample size for larvae or live feedsdepends on the circumstances. A pool of 30 larvae or 0.1 ml �.1 gram! of packed live feed isoAen used. Hold samples iced until plating procedures can be preformed. Time between collectionand processing should not be more than 3-4 hours. Note time of sample collection on the datasheet prepared for the samples.Ready dilution tubes by adding 10 ml of 2% saline to each tube, cover and autoclave. Each samplewill need a set of dilution tubes. If shrimp larvae or live feeds are to be tested, add tissue substrateto tissue grinder with 1 ml of sterile saline and thoroughly macerate and mix the specimen in thesaline solution

just prior to testing, mix water specimens by agitation for one minute. Depending on the needsfor the test add 0. 1 or 1 ml of test water to the first dilution tube. Mix by swirling the dilutiontube for 30 seconds With a sterile pipette collect 1 inl of the first dilution solution and transferto the second dilution tube. Mix and transfer 1 ml to the third tube, etc., until the desired di]utionshave been obtained. Note that if 1 ml is used, each step provides a O. 1 �0 ! dilution step of theinitial sample.

Appendix D: Recipes for S60tions218

Add a 1-ml sample undiluted and each dilution, if necessary! to a petri dish and overlay withliquid agar keep liquid agar held in a water bath at 45 C until plates are poured!. Gently swirlin a circular motion to disperse the sample through the agar and allow agar to cool with the petridish cover opened slightly to prevent excessive condensation. Alternatively, inoculate the I-mlsamples onto pre-poured agar in petri dishes and spread evenly with a sterile glass rod orinoculation loop. Because Vibrro alginolylicus is a commonly found bacteria in shrimp hatcherywater and this organisin swarms, it may be necessary to overlay with nutrient agar the spreadplate water satnples. It is recommended that each dilution plate be replicated in triplicate. Also,water can be diluted using a calibrated inoculation loop e.g., 0.01 ml!. To check the sterility ofthe agar and plates, always pour additional plates which have no specimen added.

In some cases the "threshold level" of bacteria wi1 l be sufficient for management to make decisions,That is, it may be adequate to know if the bacterial load is above or below a particular concentration e.g., bacterial count is > or < IO /ml!. A modified dilution and agar inoculation schedule can4

be used so that only the necessary dilution is plated. This approach can save time and reduce theexpense for the test.

Inoculated dilution plates should be incubated at 28-30 C for 24 hours. On plates with 30 to 300bacterial colonies, count and record the number for each replicate of the dilution. Report as cellforming units CFU! the average number and the range per ml for the sample.

Appendix D: Recipes for SoluNoris 219

Incorporation of Antibiotics into Bacteriological Media

Pur~se

There are several reasons to incorporate an antibiotic into bacteriological media. For example,defined antibiotic concentrations in a culture media can be used to measure the minimum inhibitoryconcentration MIC! and assess the level of drug resistance by specific bacterial isolates.Alternatively, antibiotic fortified isolation media can be used as a selective media to recoverbacteria resistant to the drug in environmental or tissue samples. An antibiotic can be addeddirectly to priinary isolation media to make the media selective for bacteria that are resistant tothe incorporated antibiotic.

Materials and Sup~lies

~ Use a media such as Tryptic Soy Agar + 2% NaCl and avoid more complex orselective medium, which seem to interfere with the activity of some antibiotics.However, chlorarnphenicol can be used with TCBS media.

~ Dissolve the antibiotic in an appropriate solvent. Suggested solvents for the followingantibiotics are:

> Triethylene glycol TEG! - chloramphenicol, oxytetracycline, anderythromycin

> Water - nalidixic acid and nitrofurazone

e Make a stock solution of the antibiotic at a concentration so that 1.0 ml of the stocksolution will provide the desired final concentration of the antibiotic in 1000 rnl of theisolation media. For example, for 10 mg of antibiotic in the media, make the stocksolution by adding 1 0 g of antibiotic to 100 ml of the diluent.

~ Make the primary isolation media according to instructions. Allow the media to coolto 50'C and add the antibiotic/diluent to the liquid media and swirl to get a completemixture of the antibiotic in the media.

~ Store the prepared media in the refrigerator. Discard media if not used within 2months.

Procedure

~ Use standard plate dilution methods for water or for tissue homogenate samples. Somewater samples contain very low numbers of resistant bacteria. In these cases, collectseveral liters of water and filter through a 0.45 nitrocellulose filter. Apply the filterdirectly to the agar media.

~ Incubate plates at room temperature and read results within 72 hours. Growth after72 hours can be due to non-resistant bacteria on the plates,

Appendix D: Recipes for Solutions

References

«ume, Carl. 1992 personal Communication. Department of Microbiology, University ofHawaii, Honolulu, Hawaii

«angpan, L. and T Kitao. 1992. Minimal inhibitory concentration of 19 chemotherapeutantsagainst Vihriubacteria of shrimp, J'enaetrs moncxfon. Irt'. Shariff, M., R. Subasinghe and J,R,Arthur '%4s.!. Diseases in Asian Aquaculture, Asian Fisheries Society, The Philippines. pp.132-142,

Bacteriological Media

General Purpose Media for the Recovery of Marine Bacteria

~ Marine Agar 2216 Dlfco 0 0979-01!

Pu~rse

Marine Agar is a general purpose media for isolating or counting marine bacteria.

Com~siNon and Procedure

Reference

Anon 19S4. Difco Manual of Dehydrated Culture Media and Reagents for Microbiology andClinical Laboratory Procedures. Tenth Edition!. Difco Laboratories, Inc., Detroit, Michi-gan.


~ Brain Heart Infusion BHl! Agar + 2'Yo NaCI

P~ur >se

BHI Agar Difco Laboratories ff 041S-01-5! supplemented with salt is a general purpose agar forisolating or counting marine bacteria.

Compos Non

Reference

Anon 1984. Difco Manual of Dehydrated Culture Media and Reagents for Microbiology andClinical Laboratory Procedures. Tenth Edition. Difco laboratories, Inc., Detroit, Michigan

~ Tryptic Soy Agar TSA! + 2 '4 NaCI

P~ur 48

TSA Agar BBL ff 11043! supplemented with salt is a general purpose agar for isolating or countingmarine bacteria.

Co~mmsition

Reference

Anon 1973. BBL Manual of Products and Laboratory Procedures. Fifth Edition!. BBL Divisionof Becton, Dickinson and Company

Appendix D; Recipes for Sc4Nons

Selective Media for the Recovery of Vibrio sp.

~ Thiosulfate Citrate Bile Salts TCBS! Agar

P~ur se

TCBS Agar Difco Laboratories ¹ 0650-Ol -2! is useful as a semi-selective media for the recoveryof Vibrio spp.

Composition

Expected Results

Colony color characteristics + sucrose reaction! of selected Pi 'bri0 spp. on TCBS Agar

References

Anon l984. Difco Manual of Dehydrated Culture Media and Reagents for Microbiology andClinical Laboratory Procedures Tenth Edition!. Difco Laboratories, Inc,, Detroit, Michi-gan.

224 Appendix D: Recipes for Solutions

~ ~ ~

Massad, G, and J.D. Oliver, 1987. New and differential medium for l'ihrio cholerae and Vibrioivrlnificrr.s. Appl Frtvir. Microbiol. 53 9!: 2262-2264.

e Celloblose Agar

Purpase

Selective media for the isolation and differentiation of Vibrio cholerae and V. vulnrfrctrs on CPCAgar.

V. vtrlnrjicrrs = yellow colonies, yellow zone around the colony

V, cholerue = purple colonies, blue zone around the colony

e Caution: Make and use CPC agar fresh; old preparations may have a history of V. cholerae

C~om sition and Procedure

Procedure

~ Innoculate cellobiose with a previously isolated colony or with a substrate of mixedbacterial composition,

~ Incubate plates at 25 to 28 C for 24-48 hours.

~ Vibrio vulnificus will produce yellow colonies with a yellow zone around the colony.Vibrio cholerae will have a purple colony rimmed by a blue zone.

Appendix D: Recipes for Solufions

~ Rarely will other species of bacteria grow on CPC media Thus, colonies appearingas described above can be presumptively identified as either V. vulnipcu» or V.cholerae.

Reference

Massad, g. and J.D. deliver. l987. New selective and differential medium for Vibrio choleraeand Vrbrio vulnifscu» Applied and EnvironmernalMscrohioiogy. 53 9!; 2262-2264.

Appendix D: Recipes for SoluHons

Other Selective Media

~ Synthetic Seawater Media

~Pur ose

M d' used to culture l~'@co>hriy mucor a pofilamentous marine bacteria.

Com~sition and Procedure

Reference

Brock, I D. 1966 The habitat of Lelcorhrixmuoor, a widespread marine microorganism. LimnoI.Oceanogr. i i. 303-307.


Complex Seawater Media CSM!

Purpose

Complex Seawater Media CSM! is used to enhance the recovery and recognition of biolurnines-cent strains of marine bacteria.

Composition and Procedure

Reference

Ruby, E.G, and K H. Nealson 1978. Seasonal changes in the species composition of luminousbacteria in nearshore seawater. Limnol. Oceanogr. 23�!, 530.

Appendix D: Recipes for Soluhons

~ ~ ~

General Media for the Isolation of Fungi

~ Sabouraud Dextros Agar SAS! + Antibiotics and NaCI

Purpose

C p gati dP d

Reference

Anon 1953. Difco Manual of Dehydrated Culture Media and Reagents for Microbiological andClinical Laboratory Procedures. Ninth Edition!. Difco Laboratories, Inc,, Detroit, Michigan.


SAB media Difco Laboratories A- '0109-l 7-l! is suitable for thein vitro culture of marine fungimedically important to shrimp health.

~ Peptone-yeast Extract - Glucose Agar PYG!

~Pur:use

P YG Agar is suitable for the in vitro culture of marine fungi medically important to shrimp health

C~om sition and Procedure

Reference

Anon. l953. Difco manual of dehydrated culture media and reagents for microbiological andclinical laboratory procedures. Ninth Edition. Difco Laboratories, Inc., Detroit, Michigan.


~~'pcs for Solutions 231

~ Quillard's F/1 Medium Niociified! � �,000 X Stock Solutions!

Purpose

Guillard's F Medium is a nutrient mixture that is used to culture algae for shrimp larvae.

Composition

Procedure

Add sterile distilled water to parts A, B and C as follows:

i Part A: 300 mi; Part B: 300ml; Part C: l,000ml.

~ Combine parts A and Badd sterile distilled water to l L and mix thoroughly solution l!.

Dispense solution in sterile screw-cap bottles of desired size and autoclave at 121 Cand l0 pounds of'pressure for 10 minutes,

~ Filter Part C solution 2! through a 0,47'. membrane filter and distribute in sterilescrew-cap bottles of desired size.

To Use: Add l ml of solutions 1 and 2 per liter of sterile or pasteurized seawater.

Note: Preparation modifications are from Guillard �967!

F/2 medium = l/2 strength or l:2,000 dilution of stock solutions.

Appendix D: Recipes for Solutions232

Solution l can be refrigerated indefinitely. Solution 2 should be stored frozen if more than a30-day supply is kept on hand.

Reference

Guillard, R.R.L. 1967. Media for isolation and maintenance of marine algae. In: Conferenceon Marine invertebrate Larvae, Duke University Marine Lab. Mimeo!

Tabb, D.C., W T. Yang, Y. Hirono and J. Heinen. l972. A manual for culture ofpink shrimp,Penaeus chioranin, for eggs to postlarvae suitable for stocking. Sea Grant Special BulletirtNo. 7.


Appendix E

Conversion Factors and Formulas

Standard Measurement and Conversion Factors forLength, Area, Volume and Weight

lengthUNITS in cm

1 foot

Area

UNITS crn mydin ff1 square inch 1

1 square foot 144

1 square yard 1296

1 square centimeter 1550

1 square meter 1550.003

Volume

UNITS cmin ff CI31 cubic inch 1

1 cubic foot 1728

1 cubic yard 46,656

1 cubic centimeter .0610

1 cubic meter 61,024

16.3871

28,317

764,55 5

1

1,000,0001.308

Weight

UNITS Ib metric tonOZ

1 ounce

Appendix E: Conversion Factors and Formuias

1 yard

1 centimeter

1 meter

1 pound

1 kilogram

1 metric ton

1

12

36

.3937

39.3701

16

35.274

35,274

0,0833

1

3

,0328

3.2608

,0069

1

9

.0011

10,7639

.0006

1

27

,OOOM

35.3147

0.0625'i

2.2046

2204.623

0.0278

0.3333

1

,0109

1.0936

,0008

,1111

1

.0001

1,196

,00002

,0370

1

.0283

.4536

1

1 LEO

2.54

30.48

91.44

1

100

6.4516

929.0304

8361,2736

1

10,000

.0006

.0929

,8361

0,0001

1

.00003

.00045

.001

1

0.0254

0.3048

0.9'i 44

0,01

.00002

,0283

,7646

Additional Measurement and Volume Conversion factors

Abbreviations not defined in the table

~ g = gram

~ gal = gallon

e L =- liter

~ pprn = parts per million

A>~rictbt E: Conversion Factors anct Formulas237

Dosage Calculations, Volumes and Capacities

The following formula may be useful in calculating the desired amount of chemicals needed fortreatment or disinfection.

Volumes an4 Capacities

References

Plumb, J.A and P R. Bowser. 1983. Microbial Fish Disease Laboratory Manual. ConversionFactors, p 67. Alabama Agricultural Experiment Station, Auburn University, Alabama. p.95,

Wellborn, T.L. 1979. Control and Therapy. In: Principal Diseases of Farm Raised Catfish!.Southern Cooperative Series, No. 225. pp. 61-89.

Appendix E: Conversion Factors and Formulas

Definitions oncf Formulas for Statistical Terms

Mean

X = ZX/n

where X = individual value

Z = summation sign all values are added!

n = number of values added together to get ZX.

lmampie: To determine the mean weight of a group of 5 shrimp

I! Measure the weight of each individual shrimp

e g., 12 05 g, 13.75 g, 10.45 g, 12,55 g, 14.25 g!

�! Add all 5 weights together e.g., 63.05!

�! Divide the total by 5 e.g, 63.05/5 = 12.61! to get the mean weight

Standard Deviation SD!

Standard deviation is a measure of the amount of variation in a set of data. It is the difference ordeviation between an individual value and the mean. The formula to calculate standard deviationis:

SD = ~K X - X! in-1~ Use the symbols and values defined above

Note: 8%en using a comptrter program lo calculate standard deviation, be sere to check thefrrrmulu thai i» used by lhe program, The»landard deviali on oja sample l s calculated using "n-l"nol "n"; "n" i» used to calculate lhe»landard deviation of a popttlation.

Appencibt E: Corwershn Factors anci Formulas 239

The average value of a group of individual values is represented by the symbol X The formulato calculate the mean is

Example: To determine the standard deviation for the sample of shrimp weig>hts presented above:

I! Construct a table and calculate the difference between the individual weights and the meanweight. Then calculate the squared value of the difference.

%eight X Keigttt ~ A=-2012.05 12,61 -0 56 0 3136

e.g,, 8 972!.

e.g., ~2.243 = l.4976! to get the standard deviation of the sample.

Coefficient af Variation

The coeAicient of vari ation CV! is used to compare the amount of variati on in samples that havedifTerent mean values. It therefore, gives you an estimate of the relative, not the absolute, amountof variation in a sample. It is calculated by dividing the standard deviation of the individual valuesby the mean of the individual values. The formula to calculate CV is:

CV = SD/X00

For example, the CV for weight in a individual pond can be calculated by dividing the standarddeviation for that pond by the mean weight and multiplying that value by 100,

References

Herink, R, 1965. College Level Statistics. Monarch Press, Inc., New York, NY. I I 1 pp.

Sokal, R.R. and F.J. Rohlf. 1981. Btometry, W H Freeman and Co. San Francisco, CA. 776PP.

Appendix f: Conversion Factors and Formulas

13. 75 12.61 + 1,14

10. 45 12. 61 -2. 16

12. 55 12. 61 -0. 06

14.25 12.61 + 1.64

�! Add the values in the last column to get the sum

�! Divide the total by 1 less than the sample size

e.g., 8.972/5-1 = 2 243!

�! Calculate the square root of that number

1, 2996

4, 6656

0. 0036

2, 6896

P oI s o Calculate Stotisticol Farmuias any graph DcgaFollowing is a list of corn uter ro amP P ograms that can be used to calculate statistical formulas andgraph data. The list does not include aude all possible progranis and does not represent productendor sement.

Computer Programs to Calculate Statistical Formulas

Borland Qua rro Pro Borland International, inc

I.orus l-2-3, Version 3. l or greater. Lotus Development CorporationMrcrosofr Fwcel. Microsoft Corporation,

SigmaSlar. Jandel Scientific SoAware.

SPXS SlaIi sti cal Package for hi,%<xi al 5ci cures!

SKYTAT. SYSTAT, Inc

Computer Programs to Graph Data

SigmaPlor. Jandel Scientific Software.

SYGRAPH. SYSTAT, inc.

241Appendix E: Conversion Factors anct Farrnula'

Nates

Documents

Common Laboratory and Specimen Coilection Procedures for