1. 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

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

3. 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)

4. 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)

5. 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)

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

7. 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

8. 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

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

10. 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

11. 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

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

13. 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

14. Growth Control Systems
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

15. 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

16. 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

17. 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

19. 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

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

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

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

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

26. QUESTIONS