A successful attempt in standardising the ratio of catechu:slaked lime required for media formulation.
After interactive sessions with various paanwalas we learned that the amount of catechu applied to paan is high whereas the amount of slaked lime applied is quite low keeping in mind its inflammatory properties.
With the above information we tried out various ratios of catechu:slaked lime so as to get the maximum intensity of the distinct red colour which is characteristic to the paan stains prevalent across the country.
Experiment 2:
Media Formulation and Enrichment
Various media formulations for enriching natural samples with the aim of finding catechu degrading isolates.
We narrowed down to four variants of the minimal medium ,M9, for processing samples collected from different sources. The four different media used were:
M9
M9+Glucose
M9+CS (catechu+slaked lime stock)
M9+Glucose+CS (catechu+slaked lime stock)
The samples were processed (passed through bacterial proof filters of 0.22µ) before inoculation so as to eliminate chances of growth due to presence of carbon source in the collected sample.
Hence a flask with M9 medium was also inoculated and checked for growth so as to ensure that the bacteria are not growing due to the carbon source in the sample but are using the carbon source provided in the medium.The choice of minimal medium was also made keeping in mind the single carbon source utilisation by the organisms.
Growth was observed in all the flasks, except the flasks containing only M9 medium without any carbon source, in less than 72 hours of shaker incubation at room temperature.
No growth/turbidity in the flasks containing only M9 medium proves that the carbon source from sample was successfully filtered & thus has proven to be an efficient negative control.
Moreover, reduction in the intensity of the red colour (due to catechu extract in the medium) was observed after 24 hours.
Experiment 3:
Isolation of organisms and identification by 16S rRNA sequencing
Isolation on solid media after enrichment of samples and identification of the isolates by 16s rRNA sequencing
Isolation was carried out by taking inoculum from the enrichment broths using various combinations (same as used for enrichment of samples) of solid media. Growth was seen on all the plates except on M9 minimal medium plates.
Isolates which were showing prominent degradation of colour on the plates were sent for identification by 16s rRNA sequencing.
Results from National Centre for Microbial Resource-NCCS for 16s rRNA sequencing:
Experiment 4:
In search of potential enzymes that degrade catechu!
Content not given
Experiment 5:
DEGRADATION - Chewing Catechu
Content not given
Experiment 6:
Cloning - Brings out the best in you!
Molecular cloning of the gblock fragments in pSB1C3 backbone (digestion, ligation, transformation) and their confirmation by diagnostic digest agarose gel run.
Content not given
Experiment 7:
SDS-PAGE Protein Characterisation
After analysing a successful cloning, the plasmid DNA was then transferred into DH5a cells to have growing transformants for characterising protein expression.
Content not given
Experiment 8:
Content not given
Content not given
Content not given
Experiment 9:
Versatility - Testing on dyes
Degradation of common lab dyes. Clearance around the colony observed on plates containing several dyes.
