Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 379
Table 1. Characteristics of Bacterial Isolates of C. binotalis of Rurukan Tomohon
No Isolate
Characteristic Form of
Colonies Colour of Colonies
Gram Stain Motility
D-glukosa 1
Isolate C1 cocus
White translucent +
- +
2 Isolate C2
cocus Yellow
+ -
+ 3
Isolate C3 cocus
White brownish +
- +
4 Isolate C4
td White translucent
- +
+ 5
Isolate C5 cocus
White translucent
yellowish _
- +
Spore staining results showed that the five isolates of bacteria do not form spores. It can be seen as forms of all isolates cells are cocci. According Fardiaz 1992, generally bacteria
spore-forming rod shape. Bacterial spores are heat-resistant structures and chemicals. Spores formed by certain bacteria to overcome the unfavorable environment. Spores are formed in
the cell so often referred to as endospores. In bacterial cells there is only one spore. This spore does not work for reproduction. Examples are the spore-forming bacterium Bacillus,
Clostridium, Thermoactinomyces and Sporosarcina Lay 1994. The test results of cell shape and gram staining bacterial isolates can be seen in Figure 5.
The result of bacterial movement motility, it appears that all five isolates of bacteria from non motile. It can be seen from the growth that does not spread on agar soft NA Figure 3.
Because of the shape of the cell isolates cocci and non motile, it can be said that the five isolates has no flagellum. This is supported by the opinion Pelczar and Chan 1986 which
states that not all bacteria have a flagellum, many species of Bacillus and Spirilum have it, but rarely found in cocci. Flagellum is one of the main structures outside the bacterial cells that
cause movement motility in bacterial cells. The test results of bacterial motility can be seen in Figure 4.
Figure 4. Isolate bacteria on nutrient media broth used for DNA extraction. Notes: a: isolates 1, b: isolate 2, c: isolates 3, d: isolates 4, e: isolates 5
3.2. Amplification of the gene 16 S RNA
Extracted DNA was amplified 16S RNA gene. Primers used in this study was designed by co Integrated DNA Technologies IDT of Singapore. PCR Kit is used Biodye Solis Biometra.
Amplification machine used is Master Cyler Eppendorf, with PCR conditions, namely an initial denaturation: 940 Celsius for 5 minutes. Denaturation: 94 Celsius for 30 seconds,
annealing: 50 Celsius for 50 seconds, Extension: 72 Celsius for 50 seconds and the Final Extension: 72 Celsius for 5 minutes. Amplification was carried out as many as 45 cycles.
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 380
A B
Figure 5. A. Master Cycler for amplification PCR and the PCR conditions were applied B.
3.3. Electrophoresis
Amplicon PCR amplicons next-gen 16S RNA visualized using electrophoresis techniques Qiaxel Aautomatic Qiaxel kit DNA Electrophoresis with Screning Kit Qiagen.
Figure 6. Qiaxel Aautomatic electrophoresis Qiagen
Selection of 16 S RNA genes for the purpose of identification of the organism is based on several reasons. By functional and evolutionary homologue gene has properties of a variety of
different organisms, the primordial molecules with structures and sequence nucleotides are very conservative, very much in the cell, big enough to allow a statistical test their differences
with one another. The results of gel electrophoresis of 16S-rRNA gene amplification are presented in the following.
Figure 7. Isolate electrophoresis chromatogram entomopathogenic Col. Dai plant pests. Description: M = DNA marker, I = isolates, C = control
M I
C
Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 381
In the chromatogram image 16 S RNA isolates formed band at 1500 bp this shows that the primers used to amplify the gene can have 16 S RNA. Evident band formed thus the migration
process went well.Bacterial identification methods are widely recommended is the analysis of bacterial DNA through the reading of the nucleotide sequence of nitrogenous bases making up
the gene 16S rDNA fragments bacteria. Judging from several considerations, this method is considered better than the phenotypic analysis method. The first consideration is the 16S
rDNA gene of almost all species of bacteria have been determined sequence of nitrogenous bases that can be used as guidelines if found new species. The second consideration is the
base sequence of 16S rDNA gene nitrogen has a lower intraspecific diversity than other protein-coding genes, as well as the nature of 16S rDNA fragments were sustainable. To
obtain the base sequence or sequences should be in sequencing PCR products. Product sequencing will be used for the analysis of kinship phylogeny isolates obtained compared
with the existing data in the NCBI or other molecular data center.
4. Conclusion
