tion. Once solidified, the gelatin in each plate was 

 cut in half with a sterile needle along the cheese- 

 cloth division, thus making two mock fish for use 

 in experimental procedures. The mock fish were 

 gently pried loose from the Petri dish with the aid 

 of a sterile spatula or large, blunt forceps and 

 placed into a beaker containing crushed ice. The 

 cheesecloth provides ample structural support to 

 the solidified gelatin. 



Preparation of Crushed Ice 



To minimize contamination, distilled water, 

 glassware, ice cube trays, and an ice cube crusher 

 were sterilized prior to use in the preparation of 

 solutions and crushed ice. Using distilled water to 

 minimize the presence of chlorine, minerals, etc., 

 the following solutions were prepared: 1) 1% 

 EDTA; 2) 0.1% EDTA; 3) 1% EDTA plus 1% 

 CaCl 2 ; 4) 0.1% EDTA plus 1% CaCl; and 5) 0.1% 

 EDTA plus 10 ppm of Accord (an iodophore man- 

 ufactured by BASF Wyandotte Corp., Wyandotte, 

 Mich.). 



In order to demonstrate the applicability of this 

 mock fish method, we tested the effect of EDTA 

 embedded in ice on typical Pseudomonas species 

 found associated with iced fish. Interest in EDTA 

 for use as a microbial inhibitor has been cited by 

 Levin ( 1967), Winarino et al. (1971), and Maunder 

 et al. 2 The addition of calcium ions was to interfere 

 with the chelating property of EDTA. The addition 

 of an iodophor was to observe for a possible greater 

 effect. 



The control ice contained no added ingredients. 

 These solutions were poured into ice cube trays 

 and frozen. A hand operated individual ice cube 

 crusher was used to prepare crushed ice to fill 

 800-ml beakers. From 8 to 10 mock fish were 

 placed into each beaker containing crushed ice 

 and stored at 0°C for the duration of the experi- 

 ment. 



Bacterial Assays 



At each time interval (0, 1, 3, 6, and 11 days), 

 mock fish were removed from each beaker and 

 placed in a sterile plastic petri dish. The Petri 

 dishes were floated on a 31°-32°C water bath to 

 melt the gelatin. Aliquots of the melted, well- 



2 Maunder, D. T., W. P. Segner, C. F. Schmidt, and J. K. Boltz. 

 1966. Growth characteristics of Type E Clostridium botulinum 

 in the temperature range of 34 to 50°F. Annu. Rep. to U.S. At. 

 Energy Comm. (now ERDA), Contract No. ATI 11-1)1 183. 



stirred gelatin were decimally diluted and plated 

 using Eugon Agar (BBL) with 0.1% yeast extract 

 (BBL) added. Plates were incubated at 20°C for 5 

 days prior to counting. 



Results and Discussion 



The results of the experiments are shown in 

 Figure 1 . The initial starting population was 4.5 x 

 10 4 pseudomonads/ml of gelatin medium. The re- 

 sulting growth patterns reflect the effect of agents 

 contained in the ice and melt water. By the 5th 

 day, melt water entirely surrounded the mock fish 

 in each beaker. By about the 10th day, the floating 

 ice composed one-half to one-third of the beaker 

 contents. 



The mock fish held together throughout the ex- 

 periment with only occasional slivers, not sup- 

 ported by the cheesecloth, breaking off. 



The mock fish method permits an evaluation of 

 the effects of microbial inhibiting additives, used 

 singly or in combination, to yield relatively accu- 

 rate results. Thus, the method may be used to 

 screen a wide variety of antibiotic systems before 

 going into efficacy studies. The value of the mock 

 fish system is that it not only permits a broad 

 screening of additives, but it also permits one to 

 determine, in efficacy studies, whether microbial 

 inhibition is due to additives alone or partly to 

 substrate antibiotic components such as certain 

 polypeptides (J. T. R. Nickerson pers. commun.). It 

 affords a method of controlling some variables 

 and/or allowing the study of effects upon specific 

 microorganisms. We have employed versions of 



2- 



EDTA  Ethylenediominetetracetic acid 

 Co*" = Calcium ions 



Control 



-O 

 0.1% EDTA 



0.1% EDTA 



plus 10 ppm iodophore 



_i i i i_ 



2 3 4 5 6 



DAYS 



7 8 



10 



FIGURE 1. — Survival of Pseudomonas spp. in mock fish. 



881 



