When dealing with a plant sample, the first step is generally to determine the causal agent of the problem: abiotic factor, fungi, bacteria, or a virus. Once this is determined, each agent has a different method of approach. Today we will be dealing with a bacterial pathogen, so the first step is going to be the search for the illustrious bacterial ooze. Plant pathogenic bacteria have an interesting property in that they will “ooze” out of tissue when sliced. With a large enough (and well infected) sample, this can actually be seen in a beaker or glass of water. With a smaller sample, a small section of infected tissue is sliced, preferably at a transition zone, and then mounted onto a slide. Under the scope, along the edges of the cut tissue, bacteria can be seen streaming out in what can be best described as a mist or a cloud. Once this is found, we can move onto the next step, the best step, the culturing step.
The transition zones mentioned previously are next going to be used to make up our nasty and kind of creamy bacterial broth. These zones are selected, cut, and then surface sterilized with a quick dip in a 10% bleach solution. Following this is a quick dip in distilled water, they are then left to dry in the crease of a paper towel in our flow hood. Once air dried the sample material is placed in a test tube of sterile water, shaken up, and left to sit for at least 30 minutes. This is when the bacteria start streaming out into the water, making what can be best described as a “broth” of bacteria. This nasty little tube of liquid is what will be used to culture out our bacteria.
In this case, we suspect a Pseudomonas bacteria, so we have chosen Kings B or Pseudomonas Agar. This medium is pretty cool in that most bacteria will grow just fine on it, but if a Pseudomonas bacteria is cultured on it. This agar will glow under UV or blacklight, once there has been adequate bacterial growth. The Plant Clinic cultures its bacteria in two ways, either by spread plates or streak plates. Both techniques are quite easy, with each having its own benefits. In one technique, an inoculating loop (a small wire loop) is used to grab a droplet of bacteria solution which is then “streaked” over the agar plate. The other technique is called a spread plate. In this a larger amount of the liquid bacteria solution, 1/10 of a mL, is pipetted onto the plate. A bent glass rod is then used to “spread” it evenly over the surface of the media.
Hopefully you found this quick look behind the scenes at the U of I Plant Clinic to be informative (and hopefully somewhat interesting too).