Part I:  Preparation of Media

1. Prepare NaCl Agar, and seawater complete liquid medium.
2. For NaCl Agar, double recipe, add ingredients to flask and fill to 2.0L with distilled water. Autoclave for 20 minutes and pour into plates.
3.  For seawater complete liquid medium, add ingredients to flask, autoclave for 20 minutes, and pour into sterile flasks.
4.  Prepare motility stab and carbohydrate utilization tests by adding salt to components of glucose and mannitol tubes.


Table 4.  Media Recipes

NaCl Agar	Seawater Complete Liquid Medium
o    NaCl- 70.0g o    Distilled water- 1000.0ml o    Nutrient Agar- 15.0g            Beef extract- 3.0g            Peptone- 5.0g            Agar-agar- 15.0g            Distilled water to 1000ml	o    Pancreatic digest of casein- 5.0g o    Yeast extract- 3.0g o    Synthetic seawater- 750.0ml            NaCl- 27.0g            CaCl2- 0.3g            KCl- 0.6g            MgSO4 7H2O- 7.0g            Na-glycerophosphate- 0.2g            Tris buffer- 2.0g            Monosodium glutamate- 5.0g            Glucose- 1.0g            Vitamin B12- 1.0mcg o    Distilled water- 1.0L o    Glycerol- 3.0ml

Part II:  Isolation and Maintenance

1.    Obtain 1 squid that has not been frozen or washed with freshwater.
2.    Incubate squid in 3% salt solution for 24 hours at room temperature.  Be sure not to fully submerge the squid because luminescent bacteria exhibit optimal growth in the presence of oxygen.  In addition, be sure that the container allows air to enter.
3.    To obtain a primary culture, view in a dark room.  After eyes adjust, luminous colonies will be visible.  Use an sterile swab to transfer the brightest colonies onto NaCl agar by the streak plate technique.
4.    The following day, observe plates in a dark room.  Check for expected luminescent, convex, smooth, creamy white, entire colonies.  Re-streak to obtain pure colonies.
5.    The following day, observe plates in a dark room and re-streak onto NaCl Agar in order to ensure successful isolation.
6.    Observe plates in a dark room the following day.  Re-streak if necessary
7.    Re-streak plates every few days until isolated colonies are obtained.
8.    Once luminescent bacteria has been isolated, maintain growth by inoculating seawater complete liquid medium and incubating at room temperature for 5 days.


Part III:  Tests to Characterize Luminescent Bacteria

1.    Perform gram stain to confirm gram negative and characterize bacteria based on shape and arrangement.
2.    Perform motility stab and wet mount to determine whether isolated organism is motile.
3.    Perform oxidase test to examine oxygen requirements.
4.    Use mannitol and glucose carbohydrate utilization tests to determine fermentative properties including acidic end products and evolution of gas.
5.    Once results are obtained, see table 5 to distinguish between Photobacterium and Vibrio species.


Table 5. Distinguishing Characteristics of Vibrio and Photobacterium

Tests Performed	Expected Results for Vibrio species	Expected Results for Photobacterium species
Gram Stain	pink, short rods	pink, short plump rods
Motility Stab/Wet Mount	motile	may or may not be motile
Oxidase Test	oxidase positive	oxidase negative
Utilization of Mannitol and Glucose	utilizes both mannitol and glucose, no gas production	utilizes glucose, does not utilize mannitol, may or may not emit gas

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