Media Recipes

Includes:
Enzyme activity plates
HrpMM
Selective media for isolation of soft rot enterobacteria
Motility media

Semi-quantitative Enzyme Activity Plates

Make wells in the plates with a #2 cork borer and seal the well bottoms with molten agarose (0.8 % wt/vol)

Dilute culture supernatants in 10mM Tris-HCl buffer (pH 7.0) to a final concentration of 15% vol/vol. Apply 10 ul of sample to the each well and incubate at 28C.

After 16-18hr:
Pel/Peh plants: pour 4 N HCl on plate and look for a halo
Cel: Pour 0.1% Congo red and develop the plate for 30 minutes to 45 minutes. Wash with 1M NaCl several times until a clear zone is visible around the sample.
Prt: halos will be visible after 24-36 hrs without further treatment

We have found that the results from the skim milk protease plates are clearer than those from gelatin plates.

Chatterjee, A., Y. Cui, Y. Liu, C. K. Dumenyo, and A. K. Chatterjee. 1995. Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Appl. Environ. Microbiol. 61:1959-1967. (for all media except the skim milk protease plates)

Wery, N., U. Gerike, A. Sharman, J. B. Chaudhuri, D. W. Hough, and M. J. Danson. 2003. Use of a packed-column bioreactor for isolation of diverse protease-producing bacteria from Antarctic soil. Appl. Environ. Microbiol. 69:1457-1464. (for skim milk protease plates)

Hrp Minimal Medium (plus polypectate)

10 x HrpMM salts
To 450 ml of H2O, add
KH2PO4 27.5 g
K2HPO4 7.5 g
(NH4)2SO4 5.0 g
MgCl2 1.72 g
NaCl .5 g

Adjust pH to 5.5 and bring to a final volume of 500 ml. Filter sterilize and store at RT.

20% Fructose
To 70 ml of H2O, add 20 g of fructose. Bring volume to 100 ml with water. Filter sterilize in aliquots of 5-10 mls and store at 4C.

HrpMM 250 ml
Autoclave 222.5 (225) ml of H2O with .375 g of polygalacturonic acid (sodium polypectate)
Add 25 ml of HrpMM salts
Add 2.5 ml of 20% fructose. Note- if omitting fructose use the amount of H2O in parenthesis. For agar add 18 g/L before autoclaving.

Cupples, D.and A. Kelman. 1974. Evaluation of selective media for isolation of soft-rot bacteria from soil and plant titssue. Phytopathol. 64:468-475.

Pour 300 ml of boiling water into a preheated blender jar, then add the first 4 ingredients. Blend at high speed for 15 seconds while adding the polypectate. Blend for another 15 seconds while adding 200 ml of hot water. Pour the medium into a 1 L bottle and add the SDS and crystal violet. Autoclave for 25 min.

The medium will be very soft and translucent with a brown-violet tint. Great care must be usedto not disturb the medium when spreading or streaking bacteria onto CVP plates.

Motility Assays – PNAS 2000. 97:4885-4890

Swimming Medium – 1 liter
Tryptone 10 g
NaCl 5 g
Agar 3 g

Inoculate swim plants with bacteria from an overnight LB culture. The swim plates can either be inoculated with a toothpick swabbed across an LB agar plate or with a few microliters from a liquid culture. The liquid culture is preferable since the inoculum level can be measured and standardized. Keep the plates in a humid chamber for 12-14 hrs, then record the results. Temperature has a large effect on this assay, so record the temperature used for incubation.

Swarming Medium – 1 liter
Nutrient broth 8 g
Agar 5 g

After autoclaving add 100 ml of a 50 g/L filter sterilized glucose stock solution

Swarm plates should be dried overnight before using. Swarming efficiency is improved when cells were inoculated onto swarm plates from swim agar incubated overnight at 30C, although other inoculation methods can be used. Keep the plates in a humid chamber for 12-14 hrs, then record the results. Temperature has a large effect on this assay, so record the temperature used for incubation.

Twitching Medium – 1 liter

Tryptone 10g
Yeast extract 5 g
NaCl 10 g
Agar 10 g

Stab assay – twitch plates are briefly dried and strains are stab inoculated with a sharp toothpick to the bottom of the Petri plate from an overnight culture grown on LB agar. After incubation for 24 hours, the zone of motility at the agar-Petri plate interface is measured.

Slide culture assay. Strains are point inoculated with a toothpick onto the surface of a slab of LB agar placed on a microscope slide. The inoculated LB agar is then covered with a glass cover slip and the slide cultures are incubated for 4-5 hours in a Petri plate.

NGM Medium - used to identify Erwinia chrysanthemi (Dickeya sp.) based on production of indigoidine.

Lee, Y.-A. and C.-P. Yu. A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi. J. Microbiol. Methods 64:200-206.

23 g nutrient agar
10 ml glycerol
0.4 g MnCl2-4H2O

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