Mitsuokella jalaludinii sp. nov.,

norsyafiqah shukor
Mind Map by norsyafiqah shukor, updated more than 1 year ago
norsyafiqah shukor
Created by norsyafiqah shukor about 4 years ago
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Mitsuokella jalaludinii sp. nov.,
1 Characteristic
1.1 5 strains of phytase-producing
1.2 Gram negative
1.3 Non spore forming
1.4 Non motile
1.5 Small
1.6 Stout
1.7 Rod shape
1.8 Strictly anaerobic
1.9 Fermentative bacteria
2 Identification
2.1 Determine and production of carbohydrate by API kit system
2.1.1 Fermentation of glucose by formation of gas
2.1.1.1 Detected in PYG broth (bubbles in Durham tube)
2.1.1.1.1 Measure the final pH by incubating the staining for 48 hours
2.1.1.1.1.1 Fermentation produced volatile and non volatile fatty acids
2.1.1.1.1.1.1 Growth stimulation bile/glucose
2.1.1.1.1.1.1.1 Determine by adding 2% bile or 1% glucose to peptore/yeast (PY medium)
2.1.1.1.1.1.1.1.1 Monitor the growth by measuring the optical density (OD600)
2.1.1.1.1.1.1.1.1.1 Stimulation effect are determine by comparing optical density and control tube
2.1.1.1.1.1.1.1.1.1.1 All tests in physiological study repeate 3x with duplicate
2.1.1.1.1.1.1.1.1.1.1.1 Nucleotide sequence of 16S rRNA gene (MgT) determine
2.1.1.1.1.1.1.1.1.1.1.1.1 Genomic DNA extraction
2.1.1.1.1.1.1.1.1.1.1.1.1.1 PCR mediated amplification of the 16S rRNA
2.1.1.1.1.1.1.1.1.1.1.1.1.2 Purification PCR product
2.1.1.1.1.1.1.1.1.1.1.1.1.3 PCR product purified with ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1 Sequence reaction products electrop horesed using Applied Biosystem 373A DNA Sequencer
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1 Sequencing data by Alignment Editor ae2
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1 Compare with same sequence to the gram positive bacteria
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1 lbs rRNA gene sequence obtained from EMBL database for comparison
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1 lbs rRNA same similarity values were calculated by pairwise comparison
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1 Phylogeny Inference Package was used for construction phylogenetic dendogram
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1 Completed from percentage by the correction of Jukes and Cantor and by neighbour-joining method
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1 Root of tree determined including the 16S rRNA gene sequene of Bacillus subtilis
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1 DNA was purified on hydroxyapatite by Cashion al.(1977) and Visuvanathan et al. (1989) procedure
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1 DNA was hydrolysed with P nuclease
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1.1 The nucleotides were elephosphorylized with bovine alkali phosphatase by using procedure of Mesbah et al. 1989
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1.1.1 resulting deoxyribonucleosides were analysed by HPLC
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1.1.1.1 Used chromatography method
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1.1.1.1.1 Non-methylated Lambda-DNA with G+C used for calibration
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 DNA was isolated by chromatography on hydroxyapatite
2.1.1.1.1.1.1.1.1.1.1.1.1.3.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1 Observed by light microscope and scanning electron microscope
3 The way to get pure culture
3.1 Cattle
3.1.1 Feed with 60% commercial conc. and 40% oil palm for 15 days
3.1.2 Sample was taken 4 hour after breakfast
3.2 Serial dilution
3.2.1 10^-1 -- 10^-9
3.3 Inoculated using MPS medium (modified phytase-screening)
3.3.1 + 1g peptone and 37.5ml free casein
3.4 Incubate, 39'C (6 days)
3.5 Result: Clear Zone
3.6 Incubate, 39'C (24h)
3.6.1 medium 10 modified to MM10
3.6.1.1 replace K2HPO4 & KH2PO4 with Sodium phytate
3.7 Reinoculate & Reisolation
3.7.1 IN MPS medium
3.8 The pure culture were stored at -70'C in MM!0
3.9 Subculture every 4 month
3.10 Collect M. multacida at ATCC
3.10.1 For reference for morphology and physiology
3.11 Morphology
3.11.1 Steak on peptone/yeast agar medium PYG in 100% CO2 at 39'C, 48h
3.12 Observe the cell , determine the G(-)
3.12.1 used light microscope
3.13 Record size, shape and elevation characteristic
3.13.1 used scanning electron microscope
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