It very simple
technique which is based on the principle of isolation of micro-organism on a
solid nutrient media and then selective growing isolate colony for further
analysis.
REQUIREMENTS:-
Media required :
Sterile Sea salt
saline(5ml saline in 10ml tube) - 3
Sterile Sea salt
nutrient Agar plate(20ml media in a large petri plate for isolation) - 5
Sterile Sea salt
nutrient Agar slant(5ml media in a 10ml tube for monoculturing) - 10
Sterile Sea salt
nutrient broth(100 ml in 250ml flask) -
4
Sample Required :
Fresh sea water fish - 1
Miscellaneous :
Dark Room - 1
Procedure :
Step 1: Disect the fish and supend its gills, intestine
and scales in three separate sea salt saline tube then shake it well or vortex
it.
Step 2: Streak the three different samples on three
different sea salt nutrient agar plate and then incubate it at Room
Temperature(R.T.) for 12 to 18 hour.(Note: over incubation may lead to loss of
biolumenesce due to depletion of nutrients)
Step 3: Check for growth on the plate and look for well
isolated biolumnesce colony in the dark room. Carefully mark the colony on the
plate using permanent marker under dark.
Step 4: Carefully pick the selected biolumencent colony
and streak them on sterile Sea salt slant individually. Incubate at R.T. for 12
to 18 hours.
Step 5: Select those
slant which shows uniform biolumescent on the slant were growth has appeared.
(Note: if uniform bioluminescence is not observed on the slant then touch the
sterile loop on the glowing area and continue from step 2 or step 4.)
Step 6: Scrape out loopful growth and inoculate in
Sterile sea salt broth and incubate at R.T. at static condition for 18 hours.
Step 7: Flask glow
when it is shaked in a dark room, this happens because the organism requires
dissolved oxygen to glow.
Media Composition:
Sea Salt Saline
Solution:
3.5 gram sea salt in 100ml distilled water.
Sea Salt Nutrient
Broth:
Peptone 1
gram
Beef extract 0.3 gram
Sea salt 3 grams
Distilled
water 100mL
pH 7.0
Sea Salt Nutrient
Agar:
Peptone 1
gram
Beef extract 0.3 gram
Sea salt 3 grams
Agar 2 grams
Distilled
water 100mL
pH 7.0
(Note: Sea salt is
easily available at aquarium shops who maintain sea fishes. It is also
available at local marker known as uniodized sea salt. In Hindi, it is known
as "jada namak" which means thick salt .Market cost is 8 rupees per
kilogram. )
