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

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.

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.

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.

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.

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

Purification of Bacteria

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

Table 2.

Purification of Bacteria

Table 3. shows the morphology for R2A 100 bacteria.

Table 3.

Weight and Dilution of Soil:Second Soil Collection

Figure 7. TSA Morphology

1) Gray apparel- dull2) Cloudy3) Irregular shape4) Flat volume

Figure 7. Shows TSA Colony

Weight and Dilution of Soil

Figure 8. R2A Morphology

1) Circle like(donut)2) Dull yellow3) Lifted volume

Figure 8. Shows R2A Colony

Gram Staining:First Soil Collection

Figure 9. a, b, Shows that the R2A 100 colonies are gram positive and Rods.

Figure 9. a, b,

Gram Staining

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

Figure 10. c, d,

Gram Staining:Second Soil Collection

Figure 11. a, b, Shows the Rods gram positive bacteria for TSA 100 .

Figure 11. a, b,

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,

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,

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,

Acknowledgments

• Dr. Michael Rubin• Giovanni Cruz• Edwin Alvarado• Mariceli Fernández

• Dr. Enida Díaz• Dr. Elena González• Dr. Robert Ross• Raquel Montañez