Laboratory testing, reveals Syntol, a probiotic/enzyme preparation is effective at crowding out yeast colonies.
1. Growth curves
A "yeast growth curve" -- The growth and division of the yeast cells can be followed by optical density (light scattering). A graph depicting stages of population growth of Candida albicans in an environment that mimics conditions the yeast would experience in the body: The common yeast culture shown below goes from linear growth to logarithmic, then to stationary phase. Experiment was done in triplicate.
A "yeast growth curve" -- The growth and division of the yeast cells can be followed by optical density (light scattering). A graph depicting stages of population growth of Candida albicans in an environment that mimics conditions the yeast would experience in the body: The common yeast culture shown below goes from linear growth to logarithmic, then to stationary phase. Experiment was done in triplicate.
Example:
Results and Conclusion:
Addition of Syntol at an O.D. 0.4 did not change the growth rate of Candida compared to the control. However, O.D. measures growth of any organism in the culture and Syntol has several organisms including Saccharomyces and B. subtilis that could lead to false positive growth curve results. Additional experiments are needed to look directly at the Candida count from the above experiment. This leads us to the next set of experiments.
2. Growth points dilution plating
Samples were taken 4 hours after addition of Syntol (or buffer control) in the above experiment and plated at dilutions of one thousand, one million and one billion on 3M yeast and mold plates which inhibit bacteria growth while differentiating between Saccharomyces (tan, light blue, large diameter colonies) and Candia Albicans (round, small blue green colonies). Experiment was done in triplicate and averaged.
2. Growth points dilution plating
Samples were taken 4 hours after addition of Syntol (or buffer control) in the above experiment and plated at dilutions of one thousand, one million and one billion on 3M yeast and mold plates which inhibit bacteria growth while differentiating between Saccharomyces (tan, light blue, large diameter colonies) and Candia Albicans (round, small blue green colonies). Experiment was done in triplicate and averaged.
A.
Serial dilution
involves repeatedly mixing known amounts of source culture with (sterilized)
liquid. 1 ml added to 9 ml gives a 10-fold dilution; 1 ml added to 99ml gives a
100-fold dilution.
B.
When fixed
amounts of this dilution series are mixed with an appropriate agar and
incubated, then different numbers of colonies will be obtained.
C.
By working back
from an easily counted plate and using the appropriate dilution factor, the
number of micro-organisms in the original source culture can be calculated.
Example:
Results and Conclusion:
A candida count after 4 hours of growth with and without Syntol is shown above. In the presence of Syntol the Candida count was reduced 75% (from 80 million to 20 million) from the control culture. This result showed that Syntol components can reduce Candida growth. Reasons behind this result could be that Syntol components are out-competing or inhibiting Candida that has established itself under physiological conditions. Syntol in the above experiment shows it does effect the growth of established Candida but does it function as well or better as a preventive measure to Candida growth? To answer this question the next set of experiments were performed.
3. Inhibition plates
A modification of the Kirby-Bauer disk-diffusion method was set up. One set of experiments mixes yeast at O.D. of 0.5 with a recommended dosage of Syntol under physiological conditions for 30 minutes and plated without dilution. The initial major population was Candida which grew over (coat) the entire plate, unless the ingredient added inhibits growth. If it inhibits growth immediately, the plate will be clear (no growth), if the ingredients require time to establish inhibition, clear zones will appear, size of the zone will depend on how quick and strong the inhibition was.
A modification of the Kirby-Bauer disk-diffusion method was set up. One set of experiments mixes yeast at O.D. of 0.5 with a recommended dosage of Syntol under physiological conditions for 30 minutes and plated without dilution. The initial major population was Candida which grew over (coat) the entire plate, unless the ingredient added inhibits growth. If it inhibits growth immediately, the plate will be clear (no growth), if the ingredients require time to establish inhibition, clear zones will appear, size of the zone will depend on how quick and strong the inhibition was.
Results and Conclusions:
(Featured Above) Nutrient plate with no growth
(Featured Above) Nutrient plate coated with Candida albicans
(Featured Above) Nutrient plate coated with Candida albicans and Syntol.
Syntol did not immediately inhibit growth; however, it did cause small zones of inhibition (example circled in red above). This showed that material in the product prevented Candida growth given time. These results suggested that Syntol may work even better as a preventive measure. This leads to the second set of experiments based off this technique.
4. Zones of Center inhibition:
The ingredient of interest was plated at the center of the plate and allowed to grow either 10 minutes or 6 hours, and then the yeast of interest was swabbed uniformly across the same culture plate. The compound diffused out from the center of the agar. If the compound was effective against Candida at a certain concentration, no colonies will grow wherever the concentration in the agar was greater than or equal to that effective concentration. This region is called the "zone of inhibition."
Example:
Results & Conclusions:
(Featured Above) Syntol was placed at the center of the plate and allowed to grow for 10 minutes. Candida was then swabbed on the plate and incubated for 72 hours. No zones of inhibition were observed.
(Featured Above) Syntol was placed at the center of the plate and allowed to grow for 6 hours. Candida was then swabbed on the plate and incubated for 72 hours. A zone of inhibition was observed. Colonies from the middle of the plate were picked and tested on 3M TVC and Yeast&Mold plates under aerobic conditions (data not shown). The main growth in the center of the plate was determined to be B. subtilis. Based on these results B. subtilis alone was tested using the same experiment and the results were as follows.
(Featured Above) B. subtilis was placed at the center of the plate and allowed to grow for 6 hours. Candida was then swabbed on the plate and incubated 72 hours. A zone of inhibition was observed. Colonies from the middle of the plate were picked and tested on 3M TVC and Yeast&Mold plates under aerobic conditions (data not shown). The main growth in the center of the plate was determined to be B. subtilis.
Final Conclusions:
Growth curves and dilution plating showed that Syntol reduced/inhibited growth of Candida albicans under physiological conditions by as much as 75%. Zone inhibition plating showed that Syntol inhibited Candida and was especially good when allowed to establish itself first. Further analysis of the composition of the Syntol colony formed on the plate confirms that B. subtilis (one of the active ingredients in Syntol) caused the zone of inhibition.
Growth curves and dilution plating showed that Syntol reduced/inhibited growth of Candida albicans under physiological conditions by as much as 75%. Zone inhibition plating showed that Syntol inhibited Candida and was especially good when allowed to establish itself first. Further analysis of the composition of the Syntol colony formed on the plate confirms that B. subtilis (one of the active ingredients in Syntol) caused the zone of inhibition.
