How Do Labs Identify Karnal Bunt?-- APSnet Karnal bunt Forums

Symposium Discussion Forum

How Do Labs Identify Karnal Bunt?

If you are one of those lucky individuals identifying Karnal Bunt in your laboratory, tell us what's going on. Most of us do not have to deal directly with laboratory testing but would be very interested in how it is done and how much time it takes. Is everyone using a protocol established by USDA APHIS? What is it?

What percent of your time in now spent in checking for KB?

Robert Johnston - 12:05pm Jun 26, 1996 EDT (#1 of 23)

Montana State University-Bozeman

Message posted by Bob Johnston at Montana State University

Our lab is running KB tests on grain produced in Montana in 1995. We are using the procedure outlined by APHIS for isolation of teliospores using 53 and 20 micron screens. This technique works fine for clean seed. However, if one has to test millings, screening or dust samples the amount of material left on the 20 micron screen makes it very difficult to observe the sample for teliospores.

Has anyone come up with a lab assay to help cleanup these kinds of samples? Unfortunately, dilution also reduces the sensitivity of the test. Any help will be appreciated.

Elizabeth Vavricka - 04:06pm Jun 26, 1996 EDT (#3 of 23)

We've been testing both large lots of wheat (ie. several truck loads) and small breeders lots. We use the APHIS protocol, but have found the time to look at a sample varies from 45min to 2hr. I would like to know if anyone samplling large shipments has come up with a sampling protocol. Right now we take 1lb for every 10,000 lbs in the lot and then test 50 gm from that 1lb sample. We've also adopted the new policy for positives, where less than 5 spores in a 50gm sample means the sample has to be retested up to two more times, depending on wether any spores have been found the second time. Since doing this we have declared two lots negative that we have found spores in. Any comments?

Dennis Mayhew - 05:38pm Jun 26, 1996 EDT (#4 of 23)

To Elizabeth Vavricka: in California, 1 spore is a positive. It has been shown that in any given subsample of a heavily infected lot, you can get 0 to hundreds of spores. We are not testing for disease, just the presence of T. indica. Since we cannot tell the source of the spores (infected seed, contamination from ?, etc.), 1 spore is positive.

Natalie Goldberg - 11:13am Jun 27, 1996 EDT (#5 of 23)

To: Peggy Sellers From: Natalie Goldberg, New Mexico State University

I think we have a positive slide we can send you that we obtained from Arizona. It will be rather clean compared to what you will probably be looking at, but there are lots of spores - mature and immature. I also have some pictures I told you about earlier. I'll get it in the mail today. Also for those interested....in New Mexico, one spore is positive. We might run the sample again, but even if no spores were found the result would be positive.

Robert Johnston - 12:45pm Jun 28, 1996 EDT (#6 of 23)

Montana State University-Bozeman

I have found that the 53 micron nylon mesh from Spectra will become brittle after extended use. Filters which I had made became brittle after approx 300-400 samples with 15 minutes of 30% chlorox soak time between samples. In contrast, the 20 micron mesh remains in good shape and microscopic examination showed no difference in pore size between old-used mesh and new mesh.

Douglas Prasher - 11:28am Jul 2, 1996 EDT (#7 of 23)

USDA, APHIS

KB Fans, I am trying to interest a variety of commercial firms to develop an imaging system for the detection of Karnal bunt teliospores on microscope slides. The State of Kansas is also doing the same. There seems to be a fair amount of interest from at least four firms in such development.

Do you know of anyone else pursuing such a system? I would like to pull our effors if possible.

Douglas Prasher USDA, APHIS, Otis Plant Protection Lab dprasher@capecod.net

Douglas Prasher - 03:24pm Jul 2, 1996 EDT (#8 of 23)

USDA, APHIS

KB Fans:

I have a question regarding the following citation that Mary Palm listed in her Symposium Paper: Ferreira, M. A. S. V., P. W. Tooley, E. Hatziloukas, C. Castro, and N. W. Schaad. 1996.Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

Is this in preparation? in press?

Such a sequence would be useful for identification, especially in the presence of interferring fungal spores, if it could serve as an in situ hybridization probe. My question: is there a method to make Tilletia spores permeable to a tagged oligonucleotide? Can you provide a citation(s) or personal contact who might provide an answer.

Thanks in advance. Douglas Prasher USDA, APHIS Otis Plant Protection Lab

dprasher@capecod.net

Mary Palm - 03:47pm Jul 2, 1996 EDT (#9 of 23)

To Peggy If you have not received reference slides please call Coanne O'Hearn at 301-734-8717. A set of slides is available and many were sent approximately a week ago. To Doug The paper you asked about is published and the reference is listed in my symposium paper. Its Appl. Environ. Microbiol. 1996(Jan.):87-93. You might want to contact Paul Tooley, research scientist at Ft. Detrick for further information.

Peggy Sellers - 12:42pm Jul 7, 1996 EDT (#10 of 23)

Purdue University

To all who responded to Peggy Sellers about positive KB microscope slides. I have enough. Thank you very much!!!!

Peggy Sellers - 12:47pm Jul 7, 1996 EDT (#11 of 23)

Purdue University

We have only received a few samples thus far. We have been practicing our lab protocol with wheat "spiked" with common bunt. When we followed the protocol (using the centrifuge suggested), a pellet did not form within three minutes. My first thought was to increase the amount of time for centrifuging. Has anyone else experienced this problem? For those of you that have been doing this for a while, how long do you centrifuge the samples and at what RPM?

Diane M. Karasevicz - 11:02am Jul 9, 1996 EDT (#12 of 23)

I have read messages that mention a positive control slide for KB. I would be VERY interested in obtaining such a slide. We are just starting to look at our result slides. Also, does anyone know of a good way of preventing air bubbles from forming in the slides? The mounting medium (we're following the APHIS protocol) has "evaporated away" on several slides sealed with nail polish. The protocol calls for a huge coverslip (22 x 50). We are careful to slowly lower the coverslip. We've had to re-run several samples because of this problem. Thanks!

GARY L. PETERSON - 04:26pm Jul 9, 1996 EDT (#13 of 23)

To Peggy Sellers: Centrifuge speed is 1000 RPM for 5 minutes. If you are using size-selective sieving to extract the spore you may be lucky enough to be working with a clean sample. In that case you may not see a pellet.

GARY L. PETERSON - 04:30pm Jul 9, 1996 EDT (#14 of 23)

To Diane Karasevica: using nail polish to seal slides is only temporary, basically to prevent movement of the coverslip. We found that Cytoseal was much better for longterm storage, two coats is best. You need to use this in a fume hood.

Jennifer Dartez - 08:42pm Jul 10, 1996 EDT (#16 of 23)

Houston, Texas

Is it necessary to use such a huge coverslip(22x50)? I suppose if your sample is dirty, it would be advantageous to spread the "junk" out :). But I have had pretty clean slides and find that I can still see if there is anything in them when using the small slips. I use Shear's solution to mount slides and it seems to keep the slide pretty well preserved with fingernail polish around it. I also centrifuge for 5 min at 3000rpms. About the air bubbles, for me it was "practice, practice, practice" :)

Betsy Hudgins - 12:02pm Jul 11, 1996 EDT (#17 of 23)

To Diane Karasevicz: We also had trouble with the mounting medium evaporating. Air bubbles don't seem to be the problem (we can't get rid of them either!) but using 2 coats of coloured nail polished has at least reduced the amount of fluid lost. The clear nail polish we were using may have been the problem. Some people have suggested using toluene and formaldehyde free nail polish to seal the coverslips.

Diane M. Karasevicz - 02:25pm Jul 11, 1996 EDT (#18 of 23)

To Gary Peterson and others. Does anyone have a catalogue source for Cytoseal? I have come across similar sounding sealants (Cytotech?) but not Cytoseal. I assume this is used as a replacement to the nail polish, and not in addition to? Maybe we'll also try switching to colored nail polish. I am using the 22 x 50 coverslips because the APHIS protocol calls for them (it also calls for centrifuging at 200 cycles per minute for 3 minutes). I did receive some positive control KB slides. I was happy to see "air bubbles" in them, too.

Betsy Hudgins - 04:11pm Jul 11, 1996 EDT (#19 of 23)

We've had good luck with the cheapest nail polish we could find. At this point, it looks like the color "Champagne Frost" of the "Love My Nails" product has worked well (although most of the colors we've tried are fine). To correct what I said earlier, the lab now only using one thick coat of polish which has prevented some of the coverslip cracking that we noticed earlier.

To Peggy Sellers: Our lab also had some trouble getting a pellet to form. Much of our trouble was using a fixed rotor; once we switched to a swinging bucket rotor, the pellets formed nicely. We use 5000 RPM for 5 minutes and have had success using 15 ml conical tubes.

GARY L. PETERSON - 10:25am Jul 14, 1996 EDT (#20 of 23)

To:Diane M. Karasevicz

Found the supplier through a net search using Yahoo, copied this discription. CYTOSEAL MOUNTING MEDIUM Formulated from the highest quality advanced acrylic resin, CytoSeal will not become brittle and crack, nor will it dis-colour or yellow with age. It dries rapidly, allowing exam-ination soon after application. CytoSeal can be used with oil immersion objectives and in fluorescent procedures. Slides will not stick together and the medium will not "cold flow" to the edge of the slide during long term storage. Dissolve in toluene or xylene. An antioxidant has been added to prevent fading of slides. Available in two viscosities: CytoSeal 60, with a viscosity of 60cps is intended primarily for use with cover glass. CytoSeal 280 with a viscosity of 280cps is intended for use where minimal spread of medium is required. IA013 CytoSeal 60, 120ml IA014 CytoSeal 280, 120ml Price Probing & Structure, PO Box 111; Thuringawa Qld 4817; Austrailia. Tele +61 77 740-370;

e-mail p&s@ultra.net.au

Cytoseal can be ordered directly from the net using a VISA

Cynthia L. Ash - 10:07am Jul 17, 1996 EDT (#21 of 23)

Director of Scientific Services

Have you seen the new paper by Bill Brown, 'How to Set up a Karnal Bunt Identification Lab: An Arizona Experience'. What are your reactions to having this detail available? Do you like the photographs? Would you like to keep this diagnositic discussion up after the symposium ends?

What protocols are you using that respond to Randy Clear's question under the 'Look-alikes and the Risk of False Positives in Northern Wheat States' discussion. He asks "...what one would do if they recovered a non-viable spore matching the description of Tilletia indica", since they apparently can not be used in genetic testing.

Douglas Prasher - 09:17am Jul 19, 1996 EDT (#22 of 23)

USDA, APHIS

KB Netters:

Please HELP: I need some numbers regarding costs of scanning slides for KB spores.

If you have read my previous postings, you may remember I am trying to 'attract' imaging experts to develop imaging systems that could potentially automate the slide examination for KB spores. Fortunately, four private firms are pursuing the problem in addition to a DOE group (working primarily with the Kansas DA). An important aspect of this development will, of course, be cost which, BTW, will not be cheap. Last month, I have acquired some costs of the Arizona 'operation' and I am hoping other ID labs might be willing to provide similar information. If you are 'in charge' of an ID group I hope you might provide answers to the following questions regarding 'viewing KB slides'. 1. Average cost to keep one person at a microscope per shift (define # hours per shift). 2. Average number of slides viewed by an identifier per shift. 3. Total number of hours logged in by identifiers to date. 4. Expected number of slides that will be examined during the next 12 months.

Thanks in advance.

Douglas Prasher, APHIS, Otis Plant Protection Lab (Cape Cod, MA) dprasher@capecod.net

Sheldon L. Epstein - 03:37pm Aug 2, 1996 EDT (#23 of 23)

Epstein Associates - K9APE

In response to Doug Prasher's Message #22, above, we are one of the four private businesses building an Image Analysis Inspection System For Automatic Identification of KB (T. Indica) Teliospores on Microscope Slides.

Doug comments that the "cost will not be cheap". It has been our experience that good automatic inspection systems are far more economical than manual inspection systems when they can meet customer technical requirements. As in the case of KB which may have many varieties or stages, inspection systems can be very hard to engineer and this translates into their prices. However, their operating costs are generally very low. I look forward to seeing the results of Doug's survey. BTW, it was Doug's posting on a newsgroup that we read that initiated our interest in KB - so thank you Doug.

If you have an interest in our work, have questions or wish to contribute suggestions, please contact me at ++1 847 853 9292 or FAX at ..93. We're located on Chicago's North Shore in the CDST time zone -6 Hrs. GMT.

You bring up some very good points and I think that if all steps are followed we will be able to avoid any false positives (or negatives for that matter!). If you have any questions about the National Survey or about how to obtain sample slides you can contact Coanne O'Hern or Dave McNeal at 301-734-8247 or Arne Tschanz at 301-504-8141.