Mindmap
Mitsuokella jalaludinii sp. nov.,
Inhalt der Mindmap
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
- DNA was isolated by chromatography on hydroxyapatite
- Non-methylated Lambda-DNA with G+C used for calibration
- Used chromatography method
- resulting deoxyribonucleosides were analysed by HPLC
- The nucleotides were elephosphorylized with bovine alkali phosphatase by using procedure of Mesbah et al. 1989
- DNA was hydrolysed with P nuclease
- DNA was purified on hydroxyapatite by Cashion al.(1977) and Visuvanathan et al. (1989) procedure
- Root of tree determined including the 16S rRNA gene sequene of Bacillus subtilis
- Completed from percentage by the correction of Jukes and Cantor and by neighbour-joining method
- Phylogeny Inference Package was used for construction phylogenetic dendogram
- lbs rRNA same similarity values were calculated by pairwise comparison
- lbs rRNA gene sequence obtained from EMBL database for comparison
- Compare with same sequence to the gram positive bacteria
- Sequencing data by Alignment Editor ae2
- Sequence reaction products electrop horesed using Applied Biosystem 373A DNA Sequencer
- Genomic DNA extraction
- Nucleotide sequence of 16S rRNA gene (MgT) determine
- All tests in physiological study repeate 3x with duplicate
- Stimulation effect are determine by comparing optical density and control tube
- Monitor the growth by measuring the optical density (OD600)
- Determine by adding 2% bile or 1% glucose to peptore/yeast (PY medium)
- Growth stimulation bile/glucose
- Fermentation produced volatile and non volatile fatty acids
- Measure the final pH by incubating the staining for 48 hours
- Detected in PYG broth (bubbles in Durham tube)
- Fermentation of glucose by formation of gas
- Determine and production of carbohydrate by API kit system
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
- medium 10 modified to MM10
- 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
- Cattle