a guide to perservation techniques for amphibians and reptiles

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uide to PresenJationTechniques for mphibiansReptiles

byGeorge R iu

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OITOR S NOTEThis is the ffrst of a new series of Miscellaneous Public.tlons

entitled "Herpetological Circulars". desfgned to provfde b.sic lnfonnationfor the amateur or beginning herpetologist. Museums. zoos, and laboratorieswl1l find 1t useful to distribute In response to Inquiries concerning: A GUIDE TOcurrent procedures u ~ in preserv1 ng amphibians and reptiles t maintenance PRESERV T JON TECHN IOUESof collections; what lnfo_t lon is recorded In field notes; how to ship rORspecimens; color preservation. Brand names (and conmon names) of reagents, AMPHIBIANS ND REPTILESfixatives, paper a.nd the address of the source of supply, are cited when.ppUcable. A Sp.nlsh edition Is available.

Single copies are available at US SI.0eeach (SO.75e.ch for orders of25 or re) including postage. Orders moy be sent to Or. Henri SeIbert,OeparUlent of Zoology. Ohio Universfty. Athens, Ohio 45701. U.S.A.

PubUshed in 1973 by theSOCIETY FOR THE STUDY OF AMPHIBIANS ND REPTILESSecond Printing (1977)

MISCElLANEOUS PUBLICATIONSHerpetologlc.l Clrcullr 1 George R. PisaniGeorge R. PI51nl. Editor Biology DepartmentBiology Deportolenr-UniversIty of Kan5ls. L ....ence. Kansas 66045 (U.S.A.) University of KansasA Hst of other Society publications. including those of The Ohio lawrence, Kansas 66045Herpetological Soci ety. racsimlle Reprlmth in Herpetology. Herpetol0gtcalReview nd the ~ t l ~ o g p e of Mierlcan and Reptiles. w1l1 esent on request y t e ubITcatlons Secretary. Henri C. Seibert.DeportMnt of Zoology. Ohio University. Athens. Ohio 4S701, U.S.A.

_rship in the Society Includes subscription to the Circullr sertes,r.csilll1e Reprint series, the Society's technlcel joumal (.loum.1 ofHirpetol ff)' .nd newsletter (Herr,tol091c.l ReYiew). u r ~ g u l l r dues are 0.00 (S8.00 for s t u e n t s ~ SUStllnlng 15.00, ContrIbuting S20.DO. 111ustratlonsInstitutlon.1 subscriptions are S20.00. All 'nquirles about _ e r sh ip or bysubscrIptIons should be addressed to the TrelSurer, Henri C. SeIbert(.ddress aboYe).Patricia May and om Swearingen(C) 1973 Soci . t for the Study o A8ph1b1.a.u and Repti le Museum of atural History

University of Kansas


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INTRODUCTION

Olier the years. several infonnative works descr1bing the preservatof amphibians and reptiles have been published. Host of these have beeTable of Contents intended for relatively limited distribution by the institutions or indviduals publishing them. This fact . coupled with new laws pertainingto syringes and certain drugs used for 1c.111ing specimens, warrants anIntroduction additional treatment of the subject. This article is an attempt to com

Field Notes 2 bine a complete survey of current techniques with a page size that theKi 11 i ng of Specimens 5 individual collector can conveniently carry in the f ie ld .Fixing . ) In an age when so many wl1d species and areas of suitable habitatDomestic. Shipment. IJ tne th.reshold of extermination, t seems advisable at the outset to incInternational Shipment 15 plea for conservation in this booklet. Current museum collections conStorage and Labelling 17 ex.cellent samples of various North American species of reptiles and amColor Presenation 20 from certain areas within their ranges. In these instances, t is a neLitera ture C; ted 22 was te to co 11 ect and preserve add i ana 1 rna tel i a1 when thi s will not adINDEX 23 apprecia.bly to our k.nowledge of these creatures. I am not referring tosuch collectlng as may be associated with the compiling of a synopticteaching collection by a school or to collection of specimens needed fod particular aspect of research. but rather to the capture and preservaof animals simply to amass a collection which may never be used for scor educational purposes. There are numerous geographic areas, includinin N.orth America, 1n which the amphibians and reptiles are poorly knowCollections from these areas can add measurably to our herpetologicalknowledge. Persons wishing to learn of the desirabll i ty of specimensfrom par t icular areas Should consult with herpetologists at nearbyuniversities, museums, zoos, etc.

l would lik.e to express ::J thanks to Woodrow W Barber, Hobart HWilliam E. Ouellman, Joseph 1 . Collins, Clarence J. McCoy and GeorgeIannarone for furnishing helpful material and/or advice and to Phyllis Judy Hamilton, leanne Johnson, and Ginger Stiggins for typing assistancThanks are also expressed to Jaime Vf1la for preparing much of the Intional Shipment section.


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2. Oate- Always write out the name of the month, or indicate month bya Roman numeral; 6-10-71 could refer either to June 10th or October 6th.

3, Name s) of all collector(s) present.

4. Time of collecting.

The following is a representative outline of data included in fieldnotes numbers refer to those in f1gure la):

1. locality- o not record locality with reference to businessestablishments. Use towns or mapped roadways; determine distancefrom an automobile odometer if available). or estimate distancecarefully from maps. I t ; s not unusual for roads to be rerouted.r e n ~ b e r e d or both. It is therefore advisable to refer to roadsindicated in a good atlas to which future reference can be made.In the U S. . the Amer1can Highway Atlas Gousha Co . Chicago) issuitable. If collecting in areas remote from roadways, locate thecollecting site as accurately as possible from U S. GeologicalSurvey topographic maps. Collectors in foreign countries shouldtry to obtain accurate, detailed maps of the areas in which theyare working. Elevation of the locality should also be recordedwhenever possible.

FIELD NOTES

Specimens not accompanied by data identify1ng the collect10n local i tyare virtually useless to scientific investigators. The more data availablefor I specimen, the greater i ts value in research. Hence, k.eeping accurate,complete field notes is necessary. Many times. data felt to be trivial atthe time of collection may prove to be quite useful when many observationsare pooled. Field notes should be written in waterproof ink (IlPelikan l brands preferred by many) using only one side of each page. Several brands of

waterproof 1nk will run i alcohol 1s acc1dentally spilled on the page,hence care should be used in selecting ink.. A worthwhile technique ;s tocarry a small notebook for o n ~ t h e s p o t data taking. Then transfer thesedata 1nto the pennanent field notes as soon after-ward as possible.


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Of the above data, numbers 1 (Io,ality), 2 (date). 3 (name(s) of5. Air temperature and other appropriate weather notes- t s often collector(slJ and 8 (field number) represent the minimum data Which sho

useful to note existing cloud cover and moisture conditions, as be recorded. f specimens are donated to an institution, the field nowell as general weather conditions preceding the collection. should be donated with them. Do not include field notes in the same co

used to hold specimens.6. Speeles- List all speeles collected plus the number collected of

each, followed by species which may be observed but not collected.Accurate color notes are a worthwhile inclusion. especially when KIlllllG Q[ SPECIMENScollecting in regions having a poorly known herpetofauna. t isalso worthwhlle to take accurate color notes when an atypically It is essential that live herpetological specimens be killed in sucolored individual of a well-known species s encountered. manner as to leave the musc1es in a relaxed state. Following this, the

be fixed, or hardened, in standardized positions which enables research7. Microhabitat of species collected and any significant beha."ior examine them conveniently and most accurately (refer to Plates J and 2

(courtship, defensive display. etc.) observed. bOOkS reconmend that reptiles be k.illed by hypodermic injection of aquesodi urn pentobarbl ta 1 Nembuta 1) ; nto the hear t. Th 1S techn 1que is inde

8. Field number- t 15 useful to carry a series of numbered field tags excellent. but the reader should be aware that Nembutal is not a generon collecting trips. These should be printed on heavy paper in available drug. i ts possession being closely regulated by the Federal Bpennanent ink. Satisfactory tags can also be made with one of of Narcotics and Dangerous Drugs. It 15 possible for qualified personsthe col111lt!rclally available label makers that i ~ r n t plastic obtain a permit to purchase Nembutal, but the application procedure istape. Thread can be sewn through the numbered tags. but the begun several months in advance of anticipated need. For additional dbacking on the tape should not be removed . Avoid using colored concerning the permtt, the reader is urged to consult r e ~ r e s e n t t i v e s othread. or thread made from synthetics such as nylon, which may above-mentioned Bureau at the Federal centers in most large cities. Sbe destroyed by preservatives. White, cotton carpet thread is and local regulations should also be check.ed. Corrrnercial Nembutal ;s ssuitable for tagging. After threading the tag, t ie a small ,not a concentration of 50 mg/cc The fOnll of Nembutal sold as a syrupy elin the string as shown in figure lb. Each specimen should be should be avoi ded. Convnercial Nembutal may be used directly for largeassigned i ts own number, which greatly s1tJ1>lifies the task of specimens (over 5 pounds body we1ght), and diluted 1:5 with water for skeeping specimens and locali tfes associated. Testes. stomach reptiles;. for very small specimens such as or small Scincel1acontents. photos, tape recordings. etc. are ass igned the same possible to dilute to 1:10 and retain effectiveness. Nembutal dilutednumber to increase efficiency in future analyses. Field tags also be used on larger specimens. but death will be delayed. One cc (ushould be securely tied (with a square knot) to specimens as ccmnercial strength) injected into the heart is generally sufflcient toi l lustrated in Plates 1 and 2. lizards possessing femoral pores kill an animal of the bulk (volume) of a 3 foot timber rattlesnake (Crshould be tagged by knotting the string below the knee; this horridus). Position of the heart in snakes can often be judged by closavoids covering the pores with string. Tags in the field series watching the ventral plates on the anterior 1/3 of the body to detect hshould be numbered independently from the catalog series discussed Injection anywhere into the anterior 1/3 of the body cavity 1S also effin a later section. but death is not as rapid as frorr. heart injection. Other reptiles can

killed by injection into the heart region. o not attempt to inject s;which dre so small Cd thin as to be hed\lily damaged by the needles at h


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Recent drug laws have greatly increased the difficulty of obtainingA number of other effectlve killing means are available. Turtles may be for preserving pUt'poses. State laws maya 1so vary in the regulat ion of

chlorafonned i care is taken not to allow them to st i ffen . Confining the above-men ti oned chemi Cll 1s. I t is often pass i ble to obta in necessary suptur t le with a chloroform moistened rag or cotton wad in a closed container for through insti tutions. par t icular ly in return for depositing desired spec15-30 minutes (Cook, 1965) should suffice. The use of chloroform on otherreptiles is definitely not reconmended. as severe contortion usually results.Trichloroethylene or ether melY be substituted for chloroform with good results, FIXINGand can be used on most reptlles. Most specimens can be killed by confinementwith either tr ichloroethylene or ether for 5 minutes beyond the time the anlmell The purpose of fixation is to preserve the actual morpholugical staloses the abi l i ty to right i tself ,hen turned over. These l iquids are available color of the specimen. and to prepare the tissues for microscopic eXelm;to the public from either biological supply houses or certain drugstores. Their Hence, the fixative should kill tissue quickly; penetrate i t l:niformly ause may be superior to Nembuta 1 when working with sma 11, fraglle animal s 1ike prevent postmortem decomposition; not distort the tiSSUE:. and shou1ci presome tropical geclcos. Caution should be observed with ether , as i t is highly tissue for staining. No single fixative will do all of these things. soflanmable and can. under certain storage conditions. explode. Read labels compromises must be made.carefully.

The most widely accepted and suitable general fixatives for field uAll amphibians and a number of smaller reptiles . 9 small. tropical

geckos) are eas i ly killed by inmersing them in a solution of Chloretone l Formalin ( Formal or Fonnalina in Spanish; das Fonnaldehyd(hydrous chlorobutanol). A stock supply is cOrmJonly prepared as a saturated German)- Sold comercially as a solution of approximately 40 pesolution of Chloretone in 95% ethanol. This stock solution may be conveniently fo't'Tllaldehyde gas in water, formalin is the most widely used fiecarried in a small vial 2 cc of i t added to a pint of water is effective. fixative. For purposes of dilution. cOJTmei"Cial formalin is usuThe solution should be kept t ightly covered when not in use, and can be used considered as 100%, and can be used in 10% strength (1 part forover and o v e r ; t . ~ e diluted solution's strength will diminish with use. 9 parts water) for fixation. Formalin may be buffered ( ,hich h

reduce discoloration of spec1mens) by mixing 1 tablespoon of baVarious other means are suitable for ki 11 ing rept i les and amphibians. soda or borax with each pint of 102: formalin. G e n e r a n ~ sold a

Securing the animal (s) in a cloth sack. and irTJnersing the sack in warm (llOQ liquid (often in drugstores) . i t is also available as a solid pl F ~ 43-4JOC water is effective, but specimens should be removed immediately (parafonnaldehyde), which is convenient for saving weight and saf ter death. Specimens may also be imersed in alcohol (15-25% for amphibians; transport. Huheey (1963) reco TJTlends sealing 16 grams of parafo50-60% for reptiles). Though the method is not reco:rmended, bags containing hyde and 4 grams of anhydrous sodium carbonate in packets for freptiles may also be lef t exposed to direct sunlight until death from transport; 1 packet added to 400 ml (about 1/2 quart) of ,ateroverheating occurs. Great care must be used howeller. as dehydration and 10% solution of buffered formalin. Premixed. buffered paraformaccompanying contortion can happen quickly; amphibians should never be killed hyde powder is available from Carolina Biological Supply House.in this way. Both procaine hydrochloride (Livezey, 1958) and succinylcholine Paraformaldehyde alone can be obtained from Eastman Organic Chechloride (Anectine) (Lambert. 1967) have been used effect ively as killing Rochester. New York. Formalin. while an excellent general fixaagents; however. their availabili ty is usually restricted like that of Nembutal. is highly i r r i tat ing to the user' 5 skin and (as a vapor) to muc

membranes. t is not uncotllOOn for users to develop strong alle


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to farmalln. Also, fannalin has a tendency to cause swelling of 5) Miscellaneous- If a valuable specimen must be saved and no otherseveral types of t issue, rendering them unsuitable for some histologi are available, a number of emergency measures are possible. Theca purposes. may be frozen Or packed in strong brine until preservative can be

Liquor is generally not a suitable source of alcohol, as llf) pro2) F (formalin-alcohol-acetlc ac1d)- Prepared by mixing 10 parts is only 55 ethanol. However, strong tequila (about 160 proof) mconrnerc1al formalin, 50 parts of 95 alcohol (ethyl or isopropyl), 40 useful; rubbing alcohol can also be used. These. however. are oparts water and 2 parts glacial acetic acid. F penetrates tissue far desperation measures and it is usually more beneficial to get thebetter than fonnalin alone, and has less tendency to cause ce1l distortion. into a proper fixative (hospitals, local schools, etc. are suggeThe rapid tissue penetration can also be an aid to preserving valuable possible sources).specir.Jens found dead and, perhaps, partially decomposed. The primarydisadvantages of F are: the need to mix several components; and. the It is always preferable to introduce fixative into the body cavity.necessary alcohol and acetic acid may not be available in certain specimens (particularly reptiles and larger amphibians) can decompose intelocalities. F is not avaflable in powder form. but can be premixed if simply pla.ced in fixative. Enough fixative should be injected to iillwithout the water to reduce volume in transport; water may be added but not distend, the animal. Care should also be taken not to damage thelater. f F is to be used extensively in hot regions. it is recolllTlended femoral pores of many lizards by puncturing them with the needle. The nethat the acetic acid be added jus t prior to actua 1 use, as i t quickly of turtles should be completely extended and the mouth held open with wooevaporates from the solution. containers may be cooled by wrapping or tightly wadded paper prior to fixation. Excellent neck extension canthem in wet rags and shading them to retard evaporation of acetic obtained by hooking the dead turtle S upper jaw over a nail or broken branacid. letting the animal s hanging weight pull the neck out straight prior to in

i t . The upper jaw can also be hooked over a paper clip placed over tne ed3) Alcohol- If neither formalin nor F are avai lable, alcohol may be the fixing tray, and the neck then drawn out. One henipenis of male lizarused as a fixative. Cook (1965) reconmends ethanol (951: for reptiles; snakes should be partially everted with thumb pressure on the base of the

70 for amphibians) or isopropanol to fix in the absence of other followed by injection to completely evert it as indicated on the front cosolutions, but the latter is not desirable. The hemipenis should not be perm; tted to remain incompletely everted; th

may be tied around the base of the fully everted hemipenis to help retain4) Special- A large number of other fixatives exist, each being useful .nthln i t . I t is also an acceptable practice to evert the hemipenis byfor different types. of t issues, and studies. 8ouin s solution (75 injection of fixative alone. Typical sites for injection of preservativeparts saturated aqueous picric acid. 25 parts comnercial formalin, are starred in Plate 2. Tails of lizards and snakes should be s l i t lengt5 parts glacial acetic acid) is especially useful for field preservation being very careful not to break the tail off; sharp instruments are a muof testes to be used in spennatogenesis studies. Testes may be placed Large amounts of fixative can be conveniently handled in the injection apin vials of Bouin s and safely kept there for 1 l1g periods of time designed by Jackson (1971). although the author has never felt at a disadvwithout distortion of cells; the remainder of the specimen may be fixed tage using larger syringes. f no injection apparatus is available. thewith F or fonnalin. For a complete discussion of special fixatives, men should be deeply s l i t in several places ventrally and placed belly-upthe reader is referred to Guyer (1961) and similar texts. fixative. Spread the sides of the s l i t s to admit fixative more easily.

cutting the anal plates of snakes and lizllrds and femoral pores of lizard


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10Once the animal is injected or sl1t. 1t 1s most conveniently fixed by Plate 1 Amphibiansplacing i t (after proper positioning) between pieces of whi te paper toweling

mo1stened liberally with fixative. This can be done in shallow. covered plasticor rustproof metal pans. Surgical 1nstrument pesos with sl fding metal covers arevery handy for this. Avoid colored towels, as the colors dissolve in the fixativeand stain the specimen.

Preferred positions for fixing and sites for field tag attachment arel1lustrated in Plates 1 and 2. Amphisbaenids and caecilians should be fhed inthe saJfle pos1t1on as snakes ; t is useful to fix these with the mouth open. asthis greatly facilitates examination of oral characters later on. lizards withlong t ns should be fixed ""ith the ta11s bent as shown. Frogs and toads may bepositioned with the sole of the foot down (Ouellman. 1962). However, becausethis position obscures many hind 11mb and anal characters. others feel thatanurans are best fixed with the hind limbs in the position shown in Plate lc.Toes and fingers should always be straight and spread apart. Small 4lf4Jhfbiansneed not be injec.ted or s l i t prior to positioning, as the fixative will penetrateto the body caytty quite easily. Small amphibians and lizards may haye the fieldtag t ied around the body jus t anterior to the pelvic region.

Amphibian eggs and laryae are best fixed and stored by dropping the", directlyinto jars of formal1n, preserve entire egg clutches whenever possible. Manyamphibians attach their eggs to leaves, twigs. etc. Whenever it is practical.these items should be preserved with the eggs n ~ as the latter arE oftenseverely damaged by attempts to disengage them. Change the formalin on eggs andlaryae after about 12 hours. Reptile eggs should be measured (length and width,; n millimeters), then injected. J

All specimens should be allowed to remain in fixative for 24 hours. JLarge Specimens (too large to be conveniently stored entire in liquid)1. Snakes- Record the snout to yent and tail lengths (1n rrm). Then skin

by making a long ventral incision to the side of the mid-line; leavethe head and tail attached to the skin. severing these from the carcass(avoid cutting the .nal plate). and then inject he.d and tall (eyerth"",ipenis i male) with fixative. With boids,seYer the hind part jus t

. \ .\

1


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12ahead of the bony, Vestigial pel'Vic elements, The skin may npreserved by cOver;"9 the f1 es h So de wi th cloth or absorben tPlate 2. Reptiles ro11109 loosely and ifJ1Tlersing in f ixative, or by rubbing witharsenical soap, rolling and drying. In this latter instance,best to preserve the head and tail separately in liquid. If

.)

specimen is a male. a testis should also be preserved. Reprocondition of females should be noted U., .- number of ova presize of the largest ovum etc.) . Embryos. especially those oknown sped es s hou 1d be preserved in l QU i d t is preferrab 1this by preserving the entire oviduct rather than by removing

2. Turtles- Avoid cutting the shell . It is preferable to cut thneck and forelegs out as one unit. the hlnd legs and tail as unit and preserve these in liquid. The stomach and teprOductshould also be preserved in liquid. Carefully clean out andshell .

3. Crocodilians- Measure and skin the specimen as for snakes, exthe ta ils should be Skinned as well. Feet lIlay be le f t attachewith fi.xative). instead of skinned out. Rub the skin with boarsenical soap and dry.

SHIPPING

Once they have been properly fixed, /TIOst herpetological specimensconveniently be tr,Msported by wrapping them loosely in cheesecloth orpaper towel which has been l iberally moistened with alcohol 70X ethanisopropanol), or the fixative, then sealing them in two plastic bags (the other. individually closed by twisting the end and knotting or secwith a rubber band). Sevetal small specimens may be wrapped in a singof cheesecloth by laying the cloth out f la t spacing the specimens dowlength of i t folding the sides over the animals and rolling the clothl ikea rug .

Thus packed, the specimens occupy m;nimum space and weight, impowhen they must be ttansported any distance or mailed. Specimens will


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good condition o r several week.s, so long as the bags are well sealed to re tard leakage and evaporation. Cotton in sheet fonn may be substituted for cheesecloth,but i s bulkier and may adhere to the scales of some rough-scaledspecies of l izards,e en 'fIIMen i t s rnoi s t.

Specimens beingshipped (parcel post is a convenient means) should be carefullypackaged and cle r ly marked PRESERVED SCIENTIFIC 5PECIt1EN5. Packages often aresubject tomuch "wear aod tear," so, effort in preparation pays off! New paint cansof varying s izes make leakproof, sturdy mailing containers. Plastic bags containingspecimens may be simply placed in the can and extra space f i l led with wadded ragsor paper. Bags \.,rith heavy specimens should never be place,:i un top of l ighter ones. hree address labels should be typed or written with permanent ink. Place oneof these in the can with the ~ p e c i m e n s . tape a second to the s ide of the can. Affix the third l a be l t o t he paper used to wrap the parcel.

Paint cans are not too costly. and a Source of supply can generally be foundby consulting local paint stores. Watching auction notices sometimes turns up apaint s to re tha t is going out of business and 1 ,03y have cans. Remove handles fromcans before use. ln l ieu of cans, specimens may be packed in any durablecon tainer. Check postal regulations for s ize and we'ight rest r ic t ions before packing extremelyheavy or uO\'fieldy parcels. Specimens such as large tur t le shells or tile skins of large crocodilians may have to be sent via fre ight . and again, secure packing is a m us t. S hi pm en t of al l crocodilian specimens is subject to stringent regulation,as many of these spec ies a re endanger anirnals. Collectors planning to tak.ethese should careful ly check customs regUlations for import rest r ic t ions as wellas checking capture laws in countries where the animals occur. Proper arrangementscan often be made through the inst i tu t ion " here one proposes to deposit the specimens.

Generally, specimens should never be sent in glass containers. Obviousexcept ions to th is are amphibian eggs (and sometimes, larvae) and very fragi le specimens. These should be placed in the smallest containers needed to hold thespecimens plus fluid to maintain them; fluid should f i l l the containers, whichmust be heavily padded with cardboard or cotton. f rigid ph s t i c tUbing of suffic ient diameter is available, break resis tant containers may be fashionedfrom i t by cutt ing an appropriate length. stoppering one end, enclosing specimensand fluid, then sealing ths other end. The tube may be wrapped lengthwise with

wire to secure the stoppers. Plast ic v ia ls are a v a i l . ~ ? l e from somesupplyhouses; larger drugstores may also furnish the names of suppBe sure to only use vials which can be securely closed (screw-on orAgain, pay special at tent ion to wrapping such containers.

INTERNATIONAL SHIPMENTS

Collectors should be aware of proper methods for sh1pping speciinternatlonally. Donation of al l or par t of a collect ion to instituoutSide one's own country serves to:

make synoptic herpetofaunal collections of different areasavailable to as many researchers as possible,

2. prevent the loss (througi1 ~ a r neglect, earthquakes andother damage) of valuable collect ions deposited entirelyin a single inst i tu t ion , and

3. place the herpetologist in contact with colleagues in foreiinst i tu t ions; this f requent ly leads to a most beneficial exchange of ideas and data, thus advancing herpetology as af i e ld o f study.

The private hoarding of specimens by any pp.rson is a waste of valuabbiological data. and can lead to overcollecting i . ~ . r e s e a r c h e r s mgather specimens fror.l areas already represented, though inaccessibleprivate collections). I t is witt) f ~ abol/e points in mind. and thethat more collectors wil l decide to enter into donation, exchange orrelationships with foreign inst i tu t ions, that the following guidelinpresented.

The methods of racking described in the precedlng sec t ion a re afor international shipment. Generally. mail is the most convenientsending packages which are not too heavy or bulky. Parcels sent via("ordinary") mai should have e>-tra preservative added to the specimas they may tak.e as long as 4 m o n - ~ r r s to !"each their des t inat ion. Pe


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16mailing specimens internationally should check local mail re ,jl lations onparcel s z ~ weight and any spechl packing prol,dsions. shipper may alsobe requ;l ed to aff ix various postdl and customs declarat ion tags to parcels,These tags vary with parcel destination and are g e n e r ~ I I : provided by thepostal service.

Very large or heavy packages will have to be sent via freight air ,train. ship). The sender will be required to complete a waybill (available from the carrier) listing, among other things. the nature and value ofthe contents. To a..-oid excess charges, package and waybill should be marked" Q. COlllTlercial Value. Postal services in all countries have the legal r ightto inspect all packages. Intensive effor ts to curtail the t ra ff ic of narcot i cs and other res tr icted drugs has led to the extensive exercising of th isr ight , and the fact that se eral persons ha e attempted to smuggle drugs within specimen containers has not aided the s i tuat ion. Inspectors often openplas t ic bags of specimens. and may be unaware of the need to reseal them.This causes loss of f luid and dehydration and probable loss of the specimens.t ;s therefore advisable to include two copies of the foll( :;ng statement with

the parcel (one pasted on the outside and one sealed within):

INSPECTION OfFICER: This package contains dead, preserved amphibians and/orrepti les packed in plastic bags. As the specimens have great scientificvalue and will be ruined i not k.ept moist in their preservative, i t isimperative that the bags be tightly resealed afte r inspection to avoidevaporatlon or lealc.age of preservative. Thank you.

INSPECTOR POSTAL: Este paquete contiene ejemplares de anfibios y/o rept i lesmuertos. preservados y empacados en balsas plas t icas . Puesto que losejemplares son de valor cientif ico y se arruinan si no permanecen E >n suliquldo preservativo, suplica que, despues de abr ir las balsas parainspeccionarlas. las cierre hermeticamente para evitar que el l iquido seevapore 0 se derrame. Gracias.

As,AuTORIDADES ALSANDEGARIAS: Este volume contem anfibios e repteis mortos,conservados em sacos plasticos. Como 0 conteudo tern valor cientif ico e see'stragara se nao for mantido humido no preservativo, Qedese Que apos abr iras sac os para inspecao os mesmos sejam firmemente fech dos para evitarevaporacao ou derramafTIento do liQuido. Obrigado.

Biologists should also be aware that the international shioment ofspec imens (a 1ive or preserved) is bei ng ever more closely regu 1a ted f orservation reasons. Shlp-nents of preserved animals sent to the USA muaccompanied by a l i s t bearing the number and scientific name of all spemens included. The importer (1n the USA) must obtain a special permitthe Bureau of Sport Fisheries and Wildlife (Dept. of the Interior) in oto recei e foreign shipments of preserved or l ive specimens.

Live shipments are additionally regulated by the Dept. of Agricultand the Public Health Service. In a l l cases, endangered species are coby regulations separate from species not currently conSidered endangereYou are urged to ~ ~ _ f l . l Y _ investigate 8 aspects f. internationShipment before ~ Q send animals.

STORAGE AND LABELLING

This section is not intended to be a complete guide to curatorialRather, it is meant to serve as a set of capsule dlrectlons for those ws tar t a preserved herpetological collection. A detailed discussion oftechnique may be found in Slevin (1927).

Preserved collections are best maintained in alcohOl. Suitable algenerally costs about the same (per gallon) as formaldehyde. and alCOhspecimens are far eas ier to work. with. Formaldehyde also tends to corrl ids and containers. Most collectors will be deterred from using ethanhigh tax imposed upon i ts sales. Isopropanol is far cheaper, and is ensatisfactory for storage of specimens. Methanol should never be used.of 50% is sui table for rept i les , while 40% is better for amphibians. Band 1sopropanol are general ly sold a t 95% concentrat ion; 526 ml of this474 ml of water m.'Jke one l i t e r of SOCk concentration (421 ml alcoholwater for 40%). Specimens being transferred from formalin or FAA f ixatalcohol must f i r s t be soaked ;n water for 48 hours. Failure to soak thspecimen often results in i ts being severely dp.hydrated by the alcohol.fixed specimens will not be harmed by this method. If material is desiuse in histological ~ I o r k . seiected pieces of t i ssue should rf!main in 3D


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18for 24 hours, then 24 hours of 50t alcoh'ol before going to final storage (omitthe water soak). Do not pack soecimens tightly in the jar . Snakes fixed inthe position i l lustrated earlier will readily coil in jars for storage.

Each specimen retained in tl'\e collection should be assigned a catalognumber (In addition to the aforementioned I+field number ). Amphibian eggsand larvae and reptile eggs may be cataloged with a single tag designatingone clutch or lot . This number should be entered in a pernanent catalog (usingwa terproo 10k). along wi th the spec; es name. da te of capturel preserva t; on. sex,local i ty , ecological notes and name of collector . iags may be t ied 1n the sameregion as the field tay. Collector 's field number should also be entered. Asa cross-reference. i t is useful to maintain a card f i le (by taxonomic family)in which a single card is used for each species. On this card may be enterednurlbers from the catalog that apply to these specles.

l t is convenient to place a label bearing species name, catalog numbersand locality data with each container. These should be written in permanentink on heavy. durable paper. I n : ~ produced by Byron-Weston Mills under thename "linen Record Ledger". 100 cotton and linen fiber. 36 lb. and DennisonPaper Company's product "Resistall Index Bristol". rag, 110 lb. wt. areboth excellent and are available through printing shops. The label may eitherbe pl aced withi n transparent conta i ners, or a ttached to the outs i de of opaqueones with masking tape. f moderate cost can be withstood. external labelscan be placed within tie-on, plas t ic label-hOlders. A typical museum labelis shown in Figure 2.

Specimen jars should be stored in cool places to help retard evaporationof preservative, and should never be e'''posed to sunlight, as specimen colorsare rapidly faded by such e ~ p o s u r e . Placing a piece of Parafilrn sheet (availablefrom Carolina Biological Supply House, Burlington, North Carolina) over j a rmouths before screwing on the cap '.:111 also reduce evaporation. Containersshould be checked periodically and f luid level maintained. ~ e l l preservedand cared for collections make v a l u ~ l e teaching and research tools.

C O L O ~ PRESERVATION

While preserving the morphological s tate of herpetological specimensn v r presented any severe hurdles to colleLtl)fs. preservation of color 1Squite another matter. All currently used. popular preserving f luids are aand/or acidic to some degree. Therefore. t usually 1S not too long beforpigments are dissolved by such f luids and extracted from the specimens. Aseem particularly vulnerable in this regard. though the effect on reptilesnot; ceab 1e.

Previously, the only acceptable method of retaining amphibian skin cowas that described in Cook (1965). Basically, thlS C(lnsists of skinning tspecimen, confining all cuts to the ventral surfaces of body and limbs. Tskin is then floated f lesh-side up in a pan of water and remaining particlof t issue are removed. The skin nex.t is floated f lesh-side down and spreain a second pan. A wet piece of cardboard may then be brought up beneathskin, which is rubbed lightly to flatten i t and remove trapped a i r . Thecardboard-skin preparation may be dried on blottir,g pdper until no;s!. theplaced between layers of blotting paper and thoroughly dried wiUI heavy w(such as books) on top of i t ; i t may also be placed in a plant press. Helllay be similarily orepared. In all cases, the carcass should be preservedfluid and tagged with the same nUl1ber as the skin. Skins thus prepared shbe stored in the dark and not e ~ p o s e d to prolonged 119ht .

The above technique, While useful, is tedious. Windsor (197l) has da technique for using 50 saturated, aqueous ammonium sulfate solution aspreservative of frogs. As the compound is an aqueous, neutral s a l t no pwas dissolved and natural color was s t i l l evident in the specimens 6 montpreparation. Total f ixing time should be at least 36 hours.

Specimens may be stored in buffered formal in H ~ . or isopropanol (4wl1ich l iquid 10nol-40 R has been added (White and Peters, 1969). Storagebe ; n dark places which are not subjected to heat much above 70F. Theformalin/Ionol method has been successful ly used with herpetological mateby Mr. Woodrow Barber, Biology Department University of Kentucky at Moreand by Or. George lannarone, Chicago Academy of Sciences (personal COJTJT


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20Powdered lono1 should not be used, as i t is difficult to prepare a stablesolut ion of it in preservative. lonol is sold by the Shell Oil Company(Chemicals D1Vis.;on).

While the two chemical methods discussed above have not been widely usedi th herpetological material. their success on a limited scale coupled with

the value of accurate color preservat ion suggests. tha t they should be morethoroughly invest igated.

LITERATURE CITEDCook Francis R 1965. Collecting and preserving amphibians and rep t i le

p. 128-151,. .fl. R M Anderson ed)., Methods of collecting andpreserving vertebrate animals. Nat. Mus. Canada. Bull. No. 69. Bio1Ser. 1-199.

Ouellman, Iftilliam o 1962. Directions for preserving amphibians and repp. 37-40 . . >. E. Raymond Hall (ed). , Collect in9 and prep..-ing studyspecimens of vertebrates. Misc. Publ. Univ Kansas Mus Nat. Hist.1-46.

GIJyer Michael F. 1961. Animal micrology, 5th lmpress;on. Uni ChicagoPress, I l l . ; xVll l 327 pp.

Huheey James E 1953. Concerning the use of paraformaldehyde as a fielpreservat ive. Copeia.1963(1):192-193.

Jackson, Morris K 1971. A new syringe for ;nject1ng herptiles. HerpeRev . 3(4): 75.

Lanbert. lewis H t Jr . 1967. 'Anect ine': a herpetological ki l l ing agenHerpetolo9 i ca.23(2):135-136.

Livezey, Robert L. 1958. Procaine hydrochloride as a 1:: 111109 agent forreptiles and amphibians. Herpetologica, 13(4):280.

Slevin. Joseph R 1927. The making of a sc ien t i f ic collection of reptiland amphibians. Proc. Calif . Acad. Sci . 16(9):231-259.

White. David A and Edward J. Peters. 1969. A method of preserving coloaquatic vertebrates and invertebrates_ Turtox Newsy 47(9);296-297.

Windsor Donald A 1971. Ammonium sulfate as a preservative which doesnot remove color from frogs. Copeia.1971(2):356-357.


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22

INDEXHDPITOLOGICAL CIRCUu.a

Acid, acet ic (see fAApicr ic (see Souin 's solut ion) Hydr ous ch 1orobutano1. 6Injection, apparatus, 9 A Guido to P 1an Tod... . . Amphib_ ond RopoII byAlcohol, ethyl. 6,8.13 s i te (ki 11 ing), 5 Goo

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