Ward's World Working with Guides

Page 9 Care of Cultures When you receive your live cultures, they should be refrigerated to slow metabolic rates. The recom- mended medium and the optimum temperature for each bacterium is given on the tubes of the cultures supplied. The above technique is used for all but the following organisms: 1. Vibrio fischeri marine bacterium, is one of the simplest light producing organisms and also one of the easiest to work with. a. Since V. fischeri will only produce light in fresh cultures, it is necessary to subculture it in a dark environment (you can use a cardboard box for this purpose) in order to observe its luminescent characteristics. This should be done 18 – 48 hours before the luminescence is to be observed. b. For best results, the room in which the observations are to occur should be completely darkened, and the eyes of the people who observe the experiments should be allowed to acclimate to the darkness. If this is not feasible, the room should be made as dark as possible and the experiments may be viewed in the bottom of a double paper bag with the opening of the bag held tightly about the viewer's eyes. 2. Halobacterium salinarium requires a 25 – 35% salt medium for growth. It is a slow growing organ- ism, taking 5 – 7 days at 37°C for agar slants. Twelve drops of Halobacterium solution (in 25% salt water) added to the tube and incubated horizontally in a slant rack will provide ample bacteria. Halobacterium can be stored at room temperature and can survive at temperatures up to 40°C. 3. Chromobacterium violaceum (pathogen) should not be refrigerated or subjected to cold tempera- tures as the culture will quickly die. Chromobacterium prefers to stay at 30°C and will keep for months this way. 4. Aquaspirillum prefers room temperature storage, although refrigeration will not hurt the culture. Spirillum volutans prefers to be held at 30°C. Rhodospirillum rubrum is photosynthetic and needs room temperature and a light source. Lyophilized (freeze-dried) Bacteria or Fungi Cultures Under this mode of culture preservation, bacteria cultures will remain true-to-type for at least two years. It is recommended that these cultures be stored at 5 – 6°C, which is normal refrigeration temperature. Culture re-establishment requires no sophisticated equipment or special technical experience. A serological pipet, along with general sterile technique, is all that is required. Please follow the instructions to be assured of success. 1. Using a sterile serological pipet, aseptically add to the lyophilized material no more than 0.5 mL of the appropriate sterile liquid transfer medium. 2. Mix well by drawing the hydrated cell suspension up and down through the pipet at least ten times. 3. Using a sterile swab, inoculate the agar. 4. Put remaining culture in broth tube. Re-established culture may then be sub-cultured onto growth medium. 5. All materials used (shell vial, pipet, etc.) should be autoclaved prior to disposal. 6. Incubate agar tubes horizontally and agar plates agar side up. Given proper treatment and conditions, freeze-dried bacterial cultures will grow out in 24 – 48 hours, and fungal cultures will grow in 3 – 5 days. Some strains may exhibit a prolonged lag phase and should be given twice the normal incubation period before discarding as not viable.

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