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4709426
Mitsuokella jalaludinii sp. nov.,
Descripción
A mindmap
Sin etiquetas
farrahnabilah
sayfiqah
batrisyia
zarith
farhana
amelia
puteri
Mapa Mental por
norsyafiqah shukor
, actualizado hace más de 1 año
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Creado por
norsyafiqah shukor
hace alrededor de 8 años
19
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Resumen del Recurso
Mitsuokella jalaludinii sp. nov.,
Characteristic
5 strains of phytase-producing
Gram negative
Non spore forming
Non motile
Small
Stout
Rod shape
Strictly anaerobic
Fermentative bacteria
Identification
Determine and production of carbohydrate by API kit system
Fermentation of glucose by formation of gas
Detected in PYG broth (bubbles in Durham tube)
Measure the final pH by incubating the staining for 48 hours
Fermentation produced volatile and non volatile fatty acids
Growth stimulation bile/glucose
Determine by adding 2% bile or 1% glucose to peptore/yeast (PY medium)
Monitor the growth by measuring the optical density (OD600)
Stimulation effect are determine by comparing optical density and control tube
All tests in physiological study repeate 3x with duplicate
Nucleotide sequence of 16S rRNA gene (MgT) determine
Genomic DNA extraction
PCR mediated amplification of the 16S rRNA
Purification PCR product
PCR product purified with ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
Sequence reaction products electrop horesed using Applied Biosystem 373A DNA Sequencer
Sequencing data by Alignment Editor ae2
Compare with same sequence to the gram positive bacteria
lbs rRNA gene sequence obtained from EMBL database for comparison
lbs rRNA same similarity values were calculated by pairwise comparison
Phylogeny Inference Package was used for construction phylogenetic dendogram
Completed from percentage by the correction of Jukes and Cantor and by neighbour-joining method
Root of tree determined including the 16S rRNA gene sequene of Bacillus subtilis
DNA was purified on hydroxyapatite by Cashion al.(1977) and Visuvanathan et al. (1989) procedure
DNA was hydrolysed with P nuclease
The nucleotides were elephosphorylized with bovine alkali phosphatase by using procedure of Mesbah et al. 1989
resulting deoxyribonucleosides were analysed by HPLC
Used chromatography method
Non-methylated Lambda-DNA with G+C used for calibration
DNA was isolated by chromatography on hydroxyapatite
Observed by light microscope and scanning electron microscope
The way to get pure culture
Cattle
Feed with 60% commercial conc. and 40% oil palm for 15 days
Sample was taken 4 hour after breakfast
Serial dilution
10^-1 -- 10^-9
Inoculated using MPS medium (modified phytase-screening)
+ 1g peptone and 37.5ml free casein
Incubate, 39'C (6 days)
Result: Clear Zone
Incubate, 39'C (24h)
medium 10 modified to MM10
replace K2HPO4 & KH2PO4 with Sodium phytate
Reinoculate & Reisolation
IN MPS medium
The pure culture were stored at -70'C in MM!0
Subculture every 4 month
Collect M. multacida at ATCC
For reference for morphology and physiology
Morphology
Steak on peptone/yeast agar medium PYG in 100% CO2 at 39'C, 48h
Observe the cell , determine the G(-)
used light microscope
Record size, shape and elevation characteristic
used scanning electron microscope
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