Ink Lab Report

3.6 EXTRACTION OF INK FROM THE SQUID

The squid was dissected using a knife on the aluminum dissecting board. The ink was squeezed out from the ink sac which is behind the eyes of the squid. The fresh ink from the squid was collected on a sterile beaker.

3.7 SPREAD PLATE TECHNIQUE

The collected fresh ink from the squid was aseptically transferred a drop of inoculum onto the both LA and SWC solidified media. Then, the sterilized hockey stick was used to spread over the ink onto the solidified media. The glass hockey stick was pass through flame before using to ensure aseptic transfer. Then, the petri dish was sealed with parafilm, wrapped with aluminum foil and kept at room temperature for 24 hours. The production of bioluminescent was

On a glass slide, 1 drop of distilled water was placed. Then, a loop-full of culture was transferred on the slide and it must be spread. It is then allowed to dry. Then, the smear must be heat fixed by exposing it to flame for few times until it got fixed. It is to prevent the cell from washing away during the staining and washing process. Then, it is air dried and followed by fixing it with flame from Bunsen burner. After fixing the smear, it must be stained using Gram staining solution, firstly crystal violet solution was flood onto it, and allowed for 1 minute, then wash off with tap water. Then, flood the slide with iodine solution for 1 minute and wash it off with tap water again. The formation of a dye-iodine complex will occur in the cytoplasm. Then, it was flooded with ethanol and washed immediately. It is where the process of decolorization occurs. It should not be prolonged as it can over decolorized and immediate washing would sometimes cause under decolorized smear and. Finally safranins were flooded on slide for 30 seconds and rinse it with tap water and the slide must be dried using a blotting paper before viewing and examine in the

Lightly smear it on the glass slide. Then, add a drop of 3% hydrogen peroxide (H202) on the bacterial smear on the glass slide. The formation of bubble indicates it is positive for catalase test. If no formation of bubbles, it is a negative test.

3.12.2 Oxidase Test

On a filter paper or cotton bud, pick the desired colonies from the agar plate. Then, using a dropper, take the oxidase reagent and drop it on to the filter paper and observe for colour changes. The blue colour appearance indicates positive reaction whereas if there are no changes, then it’s a negative reaction. 3.12.3 Genus Verification using TCBS agar

The TCBS medium, known as Thiosulfate Citrate Bile Salts Sucrose Agar is a recommended selective medium which allows the growth of bacteria belonging to the genera Vibrio. TCBS agar was prepared for about 100ml and poured into petri plates, after solidifying, pure colonies of bioluminescent bacteria was streaked. The petri plates were observed after 24 hours. If there is presence of yellow color colonies, it is concluded as Vibrio spp.

3.12.4 Species Verification by Sub-culturing at 4°C using SWC agar media

The pure culture of Bioluminescent bacteria was streaked on the agar media and refrigerate at 4°C. The Photobacterium phosphoreum will exhibit slow growth at