Final Protocol

Materials
-Forceps                    -Pestle
-Scalpel                     -Petri plates
-Inoculating loops       -Microscope slides, coverslips
-Erlenmeyer flask       -Ethanol (70%, 95%)
-Cholrine (1%)           -Crystal violet
-Gram's iodine            -Safranin
-Congo red                -Meneval's solution
-Malachite green        -Blotting paper
-Immersion oil           -Inoculation needle
-Test tubes, lids         -motility stab agar
-Dextrose, Mannitol utilization tubes
-Sterile mineral oil     -tubes of O/F broth (2)

Media preparation (1 liter of Yeast Mannitol agar (YMA)
1. Combine in a 2 liter Erlenmeyer flask
        -agar (15.0 g),
        -mannitol (10.0 g)
        -K2HPO4 (0.5 g)
        -yeast extract (0.4 g)
        -Mg2SO4 * 7 H2O (0.2 g)
        - NaCl (0.1 g)
2. Dilute with distilled water to 1 liter.
3. Autoclave this mixture
4. Pour liquid form of YMA media into Petri plates to the appropriate level marked on side of plate
5. Allow media plates to cool for approximately 20 minutes

Obtain environmental isolate
1. Obtain white clover with roots intact
2. Rinse roots with tap water to remove excess soil
3. Remove and discard leaf and stem parts of clover
4. Sterilize forceps and a scalpel in 95% ethanol
5. Flame with Bunsen burner to remove alcohol.
6.  Section roots containing nodules into 1 cm length using the scalpel in the lid of a Petri plates
7. Immerse root sections in 1% chlorine solution for 15 minutes in Petri plate
8. Keep Petri plates covered from this point on to prevent contamination using the lid.
9. Pour off chlorine solution
10. Immerse root nodules in 20 ml of 70% ethanol for 1 minute with swirling action.
11. Pour off ethanol solution
12.  Immerse root nodules in sterile water
13. Swirl for 1 minute.
14. Repeat the water rinse 3 times replacing the lid for each rinsing.
15. Transfer roots with nodules to new Petri plates
16. Place root nodules in1 ml of sterile water.
17. Grind roots with nodules using a sterilized pestle in the Petri dish.
18. Transfer Rhizobium containing solution toYeast Mannitol agar (YMA) plate using a sterilized microbiological inoculation loop.
19. Repeat this inoculation for 4 other YMA plates
20. Incubate plates at room temperature (25◦C) for 4 days.

Confirmation of Pure Culture1. Examine morphological characteristics.  Culture should appear white and opaque, have irregular edges and be extremely mucilaginous.
2. Perform a simple stain
        - flood slide with crystal violet
        - allow stain to remain on culture for one minute
        - gently rinse slide with water, drain
        - place slide into the interior of a blotting paper tablet. Apply pressure on blot, do not rub
        - use brightfield microscope to observe slide
3. Perform a Gram stain
        - prepare slide as in the simple stain
        - flood slide with crystal violet, stain for 30 seconds
        - rinse slide with water and drain
        - flood slide with Gram’s iodine. Let stand for 10 seconds to one minute
        - rinse slide with water and drain
        - flood slide with 95% alcohol for 30 seconds or until stain no longer washes from slide
        - quickly rinse slide with water
        - flood slide with safranin. Allow stain to remain for 30 seconds
        - rinse with water and drain
        - blot slide with a pad of blotting paper. Do not rub.
        - use brightfield microscope to observe slide
4. Perform a capsule stain
        - place a few drops of Congo Red on a clean slide
        - mix in a small amount of the culture
        - air dry, do not heat fix
        - place slide on rack of staining tray. Gently flood smear with Maneval’s solution. Wait 5 minutes.
        - rinse with water with great care. Place slide on bottom of staining tray
        - add water to tray in opposite corner of the slide, tilt tray and allow water to rinse the slide
        - remove slide from tray, allow sample to air dry. Do not blot
        - use brightfield microscope to observe slide
5. Perform a motility stab
        - obtain a motility agar test tube
        - flame a wire needle, transfer a small inoculum to the motility stab agar
        - inoculate the agar piercing vertically through the agar in a non wavering motion. Remove the needle along the same stab line.
        - incubate tubes at 25 degrees C for 48 hours
        - observe growth in tubes
6. Oxidation/Fermentation Test
        - obtain 2 tubes of OF broth
        - inoculate each tube using a sterile inoculating needle.  To inoculate, stab to approximately one cm from the tube bottom
        - to one of the tubes add a 1 cm layer of mineral oil
        - incubate at 25 degrees C for 24 hours to allow for growth
        - observe growth in tubes
7. Carbohydrate utilization test
        - obtain 2 carbohydrate utilization tubes; one with mannitol and one with dextrose
        - using a sterile inoculating loop, transfer culture from the plates of Rhizobium to the sugar tubes
        - incubate at 25 degrees C for 24 hours to allow for growth
        - observe growth in tubes

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