Considerations 1. We used bacteria from both groups : the Gram-positive and Gram-negative bacteria. Gram-positive and Gram-negative bacteria differ from each other in structure, and venom may have different effects on them. These bacteria are also opportunistic pathogens which are found on and around humans.
2. Other pathogenic microbes, namely yeast and fungal are also used in this experiment. These are also commonly found on human skin. We differentiated between yeast and fungus as yeasts have been identified as a separate class of its own in terms of physiology. .
Objectives:
To test for anitimicrobial activities of scorpion (Heterometrus longimanus) venom. 1. To test for effectiveness of inhibitory action in dilutions of scorpion venom (i.e. to see if diluted venom has any effect on microbes)
2. To find out if there are any microbes that are resistant to action by scorpion venom.Aparatus:
· Sterile Petri dishes
· Agar medium (refer to Appendix 9.3 "Preparation of Agar Plates")
· Microbe colonies (S.aureus, S.faecalis, E.coli, B.subtilis, Yeast 1, S.cerevisiae, orasae, orasae f., Unknown (fungus) and Niger)
· Scorpion venom (Heterometrus longimanus venom)
· Photo-spectroscope
· Inoculating loop
· Blotting paper discs
· Micropipettes (0.5ul - 10ul, and 10ul-100ul)
· Autoclave
· Incubator (37C for bacteria, and 28C for yeasts and fungi)Procedures:
1. Agar plates were prepared using 3 different nutrient agars :
Nutrient Agar (NA) for bacteria
SDA for fungi
YPD for yeasts2. The bacteria and yeast were measured to an optical density of 0.5 using the photo-spectroscope to standardize the number of microbes on the agar plate. The agar plates were flooded with the bacteria or yeast respectively. Fungi were inoculated on SDA plates and left to grow for 24 hours.
3. Autoclaved blotting paper discs were placed on the agar surface, and 10ul of crude scorpion (Heterometrus longimanus) venom was deposited on the paper discs using miscropipettes. The cultures were then placed in their respective incubation areas (37C for bacteria, and 28C for yeasts and fungi) and left for 24 hours.
4. Measurements of the diameter of halos (cleared area) around the paper discs were measured, and photographs were taken. A tabulated presentation of the results is shown overleaf.
Results of antimicrobial test
1. E.coli
E.coli Dilution (%) Test 1 # Test 2 Test 3 Average of 2 closest results 12.5 6.0 6.0 5.9 6.0 25 7.5 6.3 7.0 7.3 50 8+9/2 = 8.5 6.4 6.7 6.6 100 10.0 7.0 80+66/2 = 7.3 7.2
# - Diameter of halo in mm.
2.S.faecalis
S.faecalis Dilution(%) Test1# Test 2 Test 3 Average of two closest results 12.5 7.0 5.7 5.9 5.8 25 8.0 6.1 5.8 6.0 50 8+10/2=9 6.0 6.2 6.1 100 9.0 6.3 6.4 6.4
# - Diameter of halo in mm.
3.B.subtilis
B.subtilis Dilution(%) Test1# Test 2 Test 3 Average of two closest results 12.5 7.0 5.8 6.0 5.9 25 8.0 6.2 6.1 6.2 50 9.0 6.3 6.3 6.3 100 9.0 6.6 6.2 6.4
# - Diameter of halo in mm.
4.S.aureus
S.aureus Dilution(%) Test1# Test 2 Test 3 Average of two closest results 12.5 8.0 6.5 6.6 6.6 25 9.0 6.5* 6.4* 6.5 50 12+8/2=10 6.0 8.2* 9.1 100 9.4 7.0 7.5 7.3
# - Diameter of halo in mm.
* - Partial clearing.
6. Orasae (fungus)
Orasae Dilution (%) Diameter of halo (mm) / Observations 12.5 0 No halo 25 0 Filter paper disc overridden by mycelium 50 0 Filter paper disc overridden by mycelium 100 0 Filter paper disc overridden by mycelium
7. Orase f.(fungus)
Orasae f. Dilution (%) Diameter of halo (mm) / Observations 12.5 0 No halo 25 0 No halo 50 0 Filter paper disc overridden by mycelium 100 0 Filter paper disc overridden by mycelium
8.Unknown(fungus)
Unknown Dilution (%) Diameter of halo (mm) / Observations 12.5 0 No halo 25 0 Plate entirely covered with mycelium 50 0 Filter paper disc overridden by mycelium 100 0 No halo
9.Niger(fungus)
Niger Dilution (%) Diameter of halo (mm) / Observations 12.5 0 No halo 25 0 No halo 50 0 Plate entirely covered with mycelium 100 0 Plate entirely covered with mycelium
CONCLUSIONS
1. Crude scorpion venom was able to inhibit the growth of both Gram-positive and Gram-negative bacteria, as it showed inhibition of all 4 strains of bacteria, especially for S.aureus.2. The venom was also effective against both species of yeasts, resulting in clearings in the colonies of yeasts.
3. The venom has no antifungal effect against all the fungi tested - some of the fungi even grew on the paper discs soaked with venom.
DISCUSSION
1. In all cases except for S.cerevisiae, S.aureus and E.coli, the diameter of the halos showed a progressive increase with the increase in concentration of venom. This was expected, since the more concentrated the venom, the more active agents it contains to interfere with bacterial growth.2. The slight discrepancies in results for S.cerevisiae, S.aureus and E.coli were probably due to experimental error. After analysis, we identified 2 possible sources of error.
· Differences in rate of diffusion: The Kirby-Bauer disc diffusion method that we used for the antimicrobial tests is dependent on diffusion of the agent, in this case, the scorpion venom. However, the thickness of the agar medium in all the plates was non-uniform. This was due to a difficulty in trying to measure out a precise amount of medium in a sterile environment - using containers and such to measure the medium would highly increase the chances of contamination. We therefore tried to keep the level of medium as uniform as possible through visual judgement. Due to the non-uniformity of the medium, the diffusion rate may vary, causing the slight discrepancies in readings.
· Venom containing large amounts of insoluble solids : Crude H.longimanus venom contained a large amount of solid material that were produced together with the more familiar liquid venom and existed as a sticky mucus. During the application of venom onto the paper discs, some of these solids may have been deposited on the paper discs as well. As the solids do not diffuse out into the agar, some of the compounds may be lost. This might, too, contribute to the non-uniformity of the readings.
