LAVENBERG ET AL.: PRESERVATION AND CURATION 



59 



causing some degree of specimen damage. A pH of less than 

 6.4 begins the process of decalcification, mineral loss in bone, 

 whereas a pH in excess of 7.0 initiates the clearing process that 

 results in translucency. 



Tucker and Chester (in press) recommend that sodium borate 

 not be used as a buffer on the basis that it results in high pH, 

 i.e., loss of pigment may occur. Calcium carbonate also is not 

 recommended because it tends to precipitate out of solution and 

 onto the larvae. Hexamine should not be used at all because it 

 tends to clear specimens independent of pH, and to damage 

 them (Steedman, 1976) 



Markle (1984) summarized five years of data for phosphate 

 buffered formalin solutions used as a preservative. He used the 

 standard ichthyoplankton protocol for fixation of his samples. 

 He gives compelling reasons for using a phosphate buffer to 

 control pH of formalin solutions used as a preservative for fish 

 larvae, on the basis that the amount of the buffer can be adjusted 

 to control pH. 



A review of the ichthyoplankton protocols indicates that so- 

 dium borate (borax) and calcium carbonate (marble chips) are 

 the preferred buffers, although Tucker and Chester (in press) 

 recommend sodium acetate. We wish to stress that our knowl- 

 edge is inadequate, particularly in understanding the chemistry 

 of these processes. Clearly, a study of the chemistry of fixation 

 and preservation must occur before a recommendation of an 

 acceptable buffer can be made. However, we agree with Markle 

 (1984) that phosphate buffers offer the best alternative to borax 

 and marble chips for long-term preservation on the basis of 

 their versatility in adjusting pH. 



Presenarives. — Afler the fixation process is completed, the zoo- 

 plankton collections are processed to obtain data on plankton 

 volumes. Then the samples are sorted to remove the ichthyo- 

 plankton component, the eggs and larvae of fishes. After the 

 identification, enumeration, and measurements offish eggs and 

 larvae, they are ready for long-term archival preservation. 

 Through this process the collections are usually maintained in 

 a buffered formalin solution. However, Ahlstrom (1976) indi- 

 cated that if an investigator was sensitive to formalin then eth- 

 anol or a similar preservative was acceptable. 



For final long-term archival preservation Ahlstrom (1976) 

 indicated that fish eggs and larvae were separately vialed, and 

 placed in fresh preservative. This fresh preservative was a one 

 percent buffered formalin solution made with freshwater. Ac- 

 cording to Ahlstrom (1976) the larvae remained in excellent 

 condition for a period of 15-20 years. Tucker and Chester (in 

 press) recommend a long-term preservative consisting of a 4% 

 formalin solution made from distilled water with sodium acetate 

 used as a buffer. Whenever formalin is used as the basis for a 

 long-term preservation fluid for fish eggs and larvae there will 

 be problems of pH. Phosphate buffers apparently control pH 

 best as they are capable of maintaining pH within a narrow 

 range between 6.4 and 7.0. Unfortunately the use of formalin 

 as a final preservative has the potential to incur considerable 

 curatorial expenses just to monitor pH levels. 



We recommend that 70% ethanol be used as the final pres- 

 ervation fluid on the basis that it renders the pH problem moot, 

 eliminates working with the fumes of formalin, and eliminates 

 problems associated with the staining process. In recommending 

 ethanol we wish to reduce or eliminate the bufliering problems 

 and their associated pH problems in formalin solutions. After 

 fixation, the concentration of formalin can be reduced to a 1% 



solution, then this fluid can be drained off during the volume 

 determination process and replaced with ethanol. It is important 

 to transfer the collections directly from the I % formalin solution 

 into ethanol without washing them through a water bath. Thus 

 a small concentration of formalin fixative will be retained in 

 the ethanol preservative. Also, the transfer should be a staged 

 one through a series of ethanol solutions, from 1% formalin to 

 20% ethanol to 45% ethanol to 70% ethanol, rather than a direct 

 transfer. Zooplankton collections should be stored in the dark, 

 specifically avoiding light. Also, the storage facility should be 

 as cold as possible, and it should avoid fluctuating temperatures. 

 In summary, we recommend that formalin be the fixative of 

 record until a suitable alternative can be established. Buffers 

 should be investigated to determine how they affect long-term 

 effects of fixation and preservation. Phosphate buflfered formalin 

 is recommended as the most suitable one to control pH within 

 a narrow range to prevent melanistic pigment loss and deminer- 

 alization. We recommend that ethanol replace formalin as a 

 preservative fluid. Finally, the chemistry of fixation and pres- 

 ervation should be addressed by a chemist to establish a suitable 

 protocol for processing zooplankton samples. 



Curation.— The chief problems with storage and curation of 

 larval fish collections are to prevent fluid loss, stabilize collec- 

 tions, and to allow for retrieval availability. 



Fluid losses through evaporation in small containers, such as 

 vials, can be disastrous. There are means to reduce evaporation. 

 We propose that a double vialing procedure be established (Fig. 

 27). First, evaporation may be significantly reduced, and second, 

 a double vialing system provides a mechanism to eliminate 

 abrasion and damage to fish eggs and larvae. The procedure 

 calls for an inner vial containing the specimens and preservation 

 fluid sealed with a poly-seal closure. This vial is inserted into 

 another glass vial, which leaves sufficient space for labels and 

 specimen documentation. The second vial is sealed with a plas- 

 tic closure. The outer vial is placed upside down over the inner 

 one. The procedure here is to allow gravity to work on vapor 

 evaporating from the inner vial in such a manner that it must 

 be compressed before escaping from the outer vial. Essentially 

 an equilibrium would be achieved that would act to prevent 

 further evaporation. In addition, a means for specimen docu- 

 mentation can be achieved that allows for maximizing these 

 data for curation without causing abrasion or damage to the 

 delicate specimens. 



Another important aspect of this curation technique would 

 be its contribution to retrieval availability. The vials can be 

 integrated into an existing ichthyological system so as to make 

 them immediately available to researchers while offering to 

 maximize long-term archival preservation protection. 



We would like to thank all of our colleagues who provided 

 us with information relative to the fixation, preservation and 

 curation of the early life history stages of fishes. 



On behalf of the steering committee of the Ahlstrom Sym- 

 posium we would like to recommend that the National Museum 

 of Natural History in Washington, D.C., the Museum of Com- 

 parative Zoology (Harvard University), and the Natural History 

 Museum of Los Angeles County in Los Angeles be considered 

 for the deposition of the early life history stages of fishes for 

 long-term archival care. 



Section of Fishes, Natural History Museum of Los Ange- 

 les County, 900 Exposition Boulevard, Los Angeles, 

 California 90007. 



