Dr. Rubin's investigation

1. Soil Collection of Cultured bacteria on
R2A and TSA media for Antibiotic
Properties
Pérez-Ayala, Michelle C.1, Figueroa-Monsanto, Héctor L.2,
Department of Biology, University of Puerto Rico at Cayey1, Department
of Chemistry, University of Puerto Rico at Cayey2

2. Soil Collection from Rhizosphere:
First Soil Collection
• Figure 1 and 2. Surrounding lake and flora. Animals commonly pass
through (e.g. chickens, ducks and dogs).
• Figure 3. Whole from where the soil was collected about an inch
deep with roots and grass.
Figure 1. Figure 3.Figure 2.

3. Soil collection area :
Second Soil Collection
Figure 4. Collection Area
• Moist terrain
• 25˚C
• Grass
• Animals: dogs, cats,
chickens & “iguanas”
• Plants: cannon tree &
coriander plants
(“cilantro”).
Figure 4.

4. Soil Collection Area Characteristics
• Table 1. Shows the characteristics of both soil collections areas.
• It shows that there is a probability of finding bacteria in the gathered soil.
Table 1.

5. Weight and Dilution of Soil
• Figure 5. Approximately, 1.033±0.001 grams of the soil sample was weighted
and diluted in six tubes (100, 10-1, 10-2, 10-3, 10-4 and 10-5).
Figure 5.

6. Weight and Dilution of Soil:
First Soil Collection
Figure 6. a, Shows the TSA 100 colonies. b, R2A 100 bacteria colonies are shown.
Figure 6. a, b

7. Purification of Bacteria
From the first soil collection, The TSA 100 colony morphology is shown in Table 2.
Table 2.

8. Purification of Bacteria
Table 3. shows the morphology for R2A 100 bacteria.
Table 3.

9. Weight and Dilution of Soil:
Second Soil Collection
Figure 7. TSA Morphology
1) Gray apparel- dull
2) Cloudy
3) Irregular shape
4) Flat volume
Figure 7. Shows TSA Colony

10. Weight and Dilution of Soil
Figure 8. R2A Morphology
1) Circle like(donut)
2) Dull yellow
3) Lifted volume
Figure 8. Shows R2A Colony

11. Gram Staining:
First Soil Collection
Figure 9. a, b, Shows that the R2A 100 colonies are gram positive and Rods.
Figure 9. a, b,

12. Gram Staining
Figure 10. c, d, Shows the Rods gram positive bacteria for TSA 100 .
Figure 10. c, d,

13. Gram Staining:
Second Soil Collection
Figure 11. a, b, Shows the Rods gram positive bacteria for TSA 100 .
Figure 11. a, b,

14. Freezing:
First Soil Collection
a, b,
c, d,
• Figure 12. a, Freezing of TSA bacteria. b, TSA
back up. c, R2A frozen bacteria. d, R2A back
up.
• Each tube had 1.5 mL of broth, 0.2 mL of
glycerol and the R2A/ TSA colonies.
Figure 12. a, b, c, d,

15. Electrophoresis
• The R2A copies of the PCR were positive for the 16S gene, according to the
electrophoresis gel in Figure13. a, b, This shows that the DNA copies
belonged to a bacteria.
• a, HLFM30R01A b, MCPA30R01A
Figure 13.a,b,

16. Antibiotic production test
M. Luteous was inhibited only by TSA 100, according to Figure 14 a,b,. E. coli on
Figure 12. c,d, was not inhibited neither by TSA 100 nor by R2A 100. This shows
that TSA bacteria might have an antibiotic property on M. Luteous.
Figure 14. a, b,

17. Acknowledgments
• Dr. Michael Rubin
• Giovanni Cruz
• Edwin Alvarado
• Mariceli Fernández
• Dr. Enida Díaz
• Dr. Elena González
• Dr. Robert Ross
• Raquel Montañez