Isolation Of Bacteria And Testing For Gelatinase Enzymatic Activity

~Sagnik Biswas, Dipannita Bhattacharya (presenting author), Suman Chakraborty, Poulami Pal, Udit Majumdar, Sayantan Sarkar, Ananya Baghchi, Subhechchha Majhi, Anwesha Goldar, Somalika Pramanik,
Amina khatun, Hishma Fareah, Nisae A Memon, Ananya Debnath, Raju Mondal, Swastik Maity, Sanjana Guho Neogi.
Semister 1, Department Of Microbiology, Maulana Azad Collage,
8 Rafi Ahmed Kidwai Road, Kolkata, 700013

Objective: Five potential strains with suspected gelatinase activity were pure-cultured, and their gram-nature and morphology were determined.
Their biochemical characterization also carried out with respect to nitrate reductase, catalase and urease activity.

We first collected 5 bacterial colony from collage campus.

We have prepared pure culture of those strains by
streaking. And proceeded through below mention staps as
follows-

strains Cell shape Colonial colour Gram Colony

shape charecter number
1 spherical filamentous white +ve 14
2 Rod shaped circular orange +ve 9
3 spherical irregular Creamish yellow +ve 6

4 spherical circular Light yellow +ve 4

5 spherical circular Yellowish orange +ve 17

Gelatinase producing bacteria isolation

• Media =>Minimal media +15% gelatin+0.5% agar.
• Stabbing in testube and incubated for 1 week.
• rechecked and confirmed by HgCl2 precipitaton process.
Strain no. Observation- HgCl2 precipitation Gelatinase enzyme
Media +/- Gelatinase? Interesting!!!!
• Cancer therapy-exhibit proteolytic activity against
1 solid no - tumour associated ECM proteins,potentially aiding in
tumour invasion and metastasis.
2 liquified yes + • Bioremedian application-degrades
microplastics,hydrocarbons,dyes,pesticides.
3 solid no -
• Wound healing-can degrade collagen,a major
component of extra cellular matrix(ECM) facilitating
4 solid no - tissue remodeling and wound healing
• Enzyme engineering-enhancing the activity,specificity
5 liquified yes +
and stability of enzymes.

Chemical tests:
Urease test- strain no. Observation-Change Urease +/-
• Principle-identification of urease producing strains which convert of media colour
urea to ammonia and co2. 2 no -
• Media-Christensen’s urea agar + phenol red
• Method-1.slants using prepared broth are prepared,2.streaking 5 no -
of thick pure culture,3.incubation.

Catalase test- Strain no. Observation-

bubble Catalase +/-
• Principle- detection of preascence of catalase enzyme in formation after
bacteria,by addition of H2O2. adding H2O2
• Media-1.L.B.agar,2.3% H2O2 solution.
• Method-1.a drop of water +inoculum,smear formation,2. 2
no -
1 drop H2O2 added.
5 no -
Strain no. Observation- Observation-
Nitrate reductase test- red colour red colour
after adding after adding
• Principle- Detection of nitrate reductase enzyme producing A and B zinc powder
strains which convert NO3- >NO2->NH3+ OFR N2.
2 no no
• Media-tripticase nitrate broth(ph-7.2)
• Other requirements- sulphanilic acid(A), alpha
napthylamine(B),zinc powder. 5 no no
• Method- 5 drops od A+ 5 drops of B+zinc powder.

Current status- currently working on antibiotic resistance checking by amphicillin, kanamycin,methicillin,tetracycline; gelatin quantitative
analysis;16s rRNA profiling.