Enrichment and Isolation of Rhizobium



Protocol

Materials: Agar, K2HPO4, Mannitol, Yeast extract, MgSO4.7H2O, NaCl, beakers, flasks, graduated cylinders, innoculating loops, test tubes, aluminium foil, pair of scissors, distilled water, bleach, alcohol, ziploc bags, tweezers, scaples, glass rods, petri dishes, slides, cover slips, Iodine, Safranin, Crystal violet, Maneval's solution, Congo red, Oxidase, Hydrogen Peroxide and dextrose tubes.

A. Making the medium

A total of 3 liters of Yeast Mannitol Agar (YMA) [Microbial Media, p. 1007] was made:

Agar 15.0g
Mannitol 10.0g
K2HPO4 0.5g
Yeast Extract 0.4g
Mg2SO4.7H2O 0.2g
NaCl 0.1g

Components were poured into a large flask and the volume was brought to 1 L by adding distilled water. The flask opening was covered with foil, and medium was gently heated to boiling with the stir bar mixing the solution. The flask was autoclaved for 15 minutes at 15 psi at 121 C. Plates were poured and allowed to cool overnight.

B. Preparation of bacteria samples from root nodules

  1. Soybean plant inoculated with Rhizobium last year was obtained from the greenhouse with the permission of Dr. Kenworthy of at UMCP. Brown nodules ~7mm wide and a little bit of root were cut from the root system of the soybean plant.
  2. Clover with white nodules growing on roots were cut from a clover patch outside of the Microbiology Building on UMCP campus. These samples were kept in some soil in a Ziploc bag punctured with several holes for air, stored in a dark place for two days before nodules were processed.
  3. The roots and nodules were rinsed with water
  4. About 5-6 1 cm long sections, each with one or two nodules, were cut from the clover roots, and 2 soybean root sections, each with a nodule, were immersed in 1% chlorine bleach solution in sterile petri dishes for 15 minutes.
  5. (All tweezers, scalpels, and glass rods used to manipulate the nodules were contained in 70% alcohol for sterlilization.)
  6. Bleach solution was poured off, and nodules were immersed in 70% alcohol. The lid was closed and the dish was swirled for about a minute.
  7. The solution was again poured off, and nodules were immersed in distilled water. The dish was swirled with the lid closed for a minute. This step was repeated two more times for rinsing.
  8. The tweezer sitting in alcohol was passed over flame for sterilization. The largest root nodules from the clover were transferred to separate drops of water on a petri dish and crushed. The two soybean nodules were also crushed in water.
  9. A large number of Yeast Mannitol Agar plates were streaked with either soybean or clover samples, using sterile technique. The plates were inverted and left to grow at room temperature (20-25 C) for two or three days.
C. Growth, Isolation and Confirmation of the samples

  1. Impure colonies on a single plate were restreaked on separate plates. Several innocula of soybean colonies were restreaked, and several innocula of clover samples looking like soybean colonies were restreaked. Soybean and clover samples were restreaked every two or three days.
  2. Unused media plates and observed plates were stored in the cold room.
  3. Gram stains were performed on the two different clover colonies and the soybean colonies. Gram stains were done after the first and second streaking of the plates.
  4. Capsule stains were performed on the soybean and clover samples.
  5. Wet mounts were prepared for soybean and clover samples. Movie clips showing motility were made for the clover sample.
  6. Catalase and oxidase tests were performed for soybean and clover samples.
  7. Dextrose tests were performed for a soybean and a clover sample.
  8. One soybean plate and two clover plates (one yellow and white clover colony sample) were placed in the anaerobic chamber for three days.