Jaime Porter Biology 493 June 14, 2002 The Antimicrobial Affects of the Coelomic Fluid of Holothuria atra Against Candida albicans Jaime Porter Biology 493 June 14, 2002

Introduction Sea cucumbers are believed to have medicinal properties Sold as an aphrodisiac in Asian markets Possess chemicals of biomedical interest

Various Toxic Properties of Holothuria atra Reddish pigment released from skin is highly toxic to fish (Richmond 1999) Pigment flushes octopus from lairs in tide pools (Richmond 1999) Phosphate Buffered Saline from inner layer inhibited growth of all gram-negative and gram-positive bacteria (Ridzwan et al. 1995)

Coelomic fluid of Holothuria polii Contains nonspecific hemolytic activity against red blood cells of several vertebrate species (Parrinello et al 1979) Lysed rabbit erythrocytes (Canicatti 1987) Contains a low level lysozyme (Canicatti & Roch 1989) Phagocitic coelomocytes that show positive reaction for acid phosphatase (Dybas & Frankboner 1986)

Fungi and Holothurians Holothurians contain triterpene glycosides that show fungitoxic activity (Pivkin 2000) Of the 27 species of marine fungi isolated from organs and surface, only 2 were present in the coelomic fluid (Pivkin 2000) Yeast and tumor cells are sensitive to action of triterpene oligosides of Holothurians (Kuanetsova et al. 1982)

Purpose To aseptically determine the antifungal affects of the coelomic fluid of Holothuria atra against the yeast, Candida albicans

Materials and Methods 10 sea cucumbers collected from Laie Bay at depth of 1-2 meters 2 mL of coelomic fluid from each test subject removed Sterility control set up using 0.2 mL of coelomic fluid from each sea cucumber Sterility control incubated at 250C for 48 hours

Materials and Methods C. albicans was serially diluted 1,000 CFU dilution selected 1.8 mL of coelomic fluid inoculated with 0.2 mL of diluted C. albicans and labeled test system

Making the Sterility Control and the Test System 2.0 mL coelomic fluid 500-800 CFU 0.2 mL coelomic fluid 1.8 mL coelomic fluid 0.2 mL C. albicans Sterility Control Test System

Materials and Methods 0.2 mL removed from test system and plated out on Sabouraud Dextrose agar at time 0,1,2,3,6,12,24,and 48 hours Plates incubated at 350C for 48 hours

Plating the Test System All plates contain 0.2 mL removed from Test System T=3 T=24 T=48 T=0 T=1 T=2 T=6 T=12

Materials and Methods Growth control system using 1.8 mL of sterile Sabouraud broth made with distilled water was inoculated with 0.2 mL of C. albicans 0.2 mL removed from growth control and plated out on Sabouraud Dextrose agar at time 0,1,2,3,6,12,24,and 48 hours Plates incubated at 350C for 48 hours

Materials and Methods Osmotic Control System using 1.8 mL of sterile Sabouraud broth made with sterile seawater was inoculated with 0.2 mL of C. albicans 0.2 mL removed from growth control and plated out on Sabouraud Dextrose agar at time 0,1,2,3,6,12,24 and 48 hours Plates incubated at 350C for 48 hours

Growth Control Systems 500-800 CFU T=0 T=0 T=1 T=1 0.2 mL of C. albicans 0.2 mL of C. albicans T=2 T=2 T=3 T=3 Growth Control T=6 T=6 Osmotic Control T=12 T=12 T=24 T=24 T=48 T=48

Results --Controls All ten sea cucmbers displayed zero growth for sterility controls Both osmotic and growth controls were not significantly different from each other Both controls showed general increase in CFU with jump to uncountable numbers after six hours

Table 1. The plate counts for growth and osmotic controls for the coelomic fluid of Holothuria atra against Candida albicans incubated on Sabouraud Dextrose agar at 350C for 48 hours. Control Time 1 Hr 2 Hrs 3 Hrs 6 Hrs 12 Hrs 24 Hrs 48 Hrs Osmotic 1112 880 1212 878 1208 TNTC Growth 1164 944 1008 904 1376

Results Test subjects one and two showed general decrease in number of colonies Test subjects three, four and nine all showed gradual decrease followed by immediate increase at the 24-hour mark

Results 50% of sea cucumbers were significantly different from both controls (p <0.05) Test subjects one, two, four, nine, and ten all significant (p <0.05) Test subject two was significantly different from test subjects five and seven

Results Mean plate counts were significantly different from both controls (p <0.05) Average decline in CFU present in first 12 hours

Discussion Vortexing problems Possible yeast settlement Plating techniques Counting methods Life expectancy of coelomocytes

Conclusions Coelomic fluid displays at least some antifungal activity Further experimentation needed to determine specific antifungal capabilities

Acknowledgments Dr. Goodwill Dr. Winget Dr. Oba BYUH Biology faculty Heidi Hansen Alisa Heitmann Dave Neville Cari Ballard

QUESTIONS