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Setting Up a Karnal Bunt
Identification Laboratory
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| Step 1-Field samples are logged into a computer and id numbers generated. Adhesive labels are printed and attached to a 250ml wide mouth Nalgene bottle and a centrifuge tube (Figure 1). |  Figure 1 |
| Step 2- Labeled samples are taken into a containment room (Fig. 2). Water with a surfactant (Tween) is added to the bottle in a negative flow hood that is equipped with a HEPA filter (Fig 3). |  Figure 2  Figure 3 |
| Gloves and pre-measured paper cups used are disposed of between each sample(Fig. 4). |  Figure 4 |
| Step 3- The Nalgene bottle is taken to another room and placed on a shaker (Fig 5). |  Figure 5 |
| Step 4- The work bench where extraction takes place is marked off in sections for each sample (Fig 6). All glassware is duplicated for each location. |  Figure 6 |
| The sample bottle and the centrifuge tube are kept together (Fig. 7). |  Figure 7 |
| Each section also has a 53 micron sieve and a 20 micron sieve (Fig 8). |  Figure 8 |
| The samples are run through the sieves; the 54 allows Karnal bunt spores but not most chaff and seed to pass; the 20 micron sieve catches the KB spores (Fig 9). |  Figure 9 |
| Residue on the 20 micron screen is washed into the centrifuge tube (Fig. 10). |  Figure 10 |
| All other residue is put in Bio-Hazard bags and incinerated (Fig. 11). |  Figure 11 |
| The Nalgene bottles are discarded and taken to a biohazard incinerator. Glassware and sieves are placed in separate carts to go into a 30% bleach wash for sterilization and subsequent washing (Fig. 12). |  Figure 12 |
| Bleach washes are kept in fume hoods to prevent negative effects on personnel (Fig 13). |  Figure 13 |
| Step 5- Contents of the centrifuge tubes are spun down and the supernatant is discarded in a biohazard container. Shear's solution is added and tubes are agitated to re-suspend the sample pellet (Fig. 14). |  Figure 14 |
| Step 6- Centrifuge tube is taken to the 'slide making station' where it is centrifuged again, supernatant discarded and slides prepared from the pellet. Gloves, pipettes, blotter paper and cover cement applicators are discarded into a biohazard bag after each sample (Fig. 15). |  Figure 15 |
| Step 7- Microscopic examination of the complete sample area on the slide (22mm X 50mm) is carried out. This is the most time-consuming activity (Fig. 16). (See the paper presentation by Mary Palm for pictures of teliospores.) |  Figure 16 |
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Identify Karnal Bunt discussion
© Copyright 1996 by the American Phytopathological Society