Daniel Y. C. Fung, Ph.D.—
Pioneering Methods for the Rapid Detection of Pathogens

[For information on attending the International Rapid Methods and Automation in Microbiology Workshop go to http://www.dce.ksu.edu/dce/conf/microbiology or call 1-800-432-8222.—ed.]

FoodTechSource: Your summer workshop is world renowned. How did you get it started?

Daniel Y. C. Fung, Ph.D.: In 1980 I realized there were lots of people like myself who wanted to know about all these diagnostic systems but who had no time to study them individually in their own lab. And when you go to big national conventions, you see companies with their instruments, but they can’t let you do experiments there. So I decided to invite these two groups of people to the same place and let them interact. That was the starting point of my workshop in 1980.

FTS: Was it a success?

Fung: Yes. About 15 companies showed up, which surprised me because that first year it was developed as a graduate course for two academic credits and about 30 students attended. But companies came even though they knew they wouldn’t make a sale. They came to demonstrate and teach our students for about two weeks. And it was very successful from an academic standpoint. So in 1981 I opened up the course to the public and sent out about 2,000 letters. I got about 40 replies and ended up with 16 “students” from several countries. And the next year we got 18....

FTS: A very intimate affair.

Fung: It is a hands-on workshop; you don’t really want too many people. We started with 16, then 18, then 20, 25, 35, 40, 60...and for the past 10 years we have been floating between 45 and 60. When you add in the people who come to demonstrate their systems it turns out to be 120-150 people in all.

“The newest
technology all
comes to Kansas State.
That week we
have the highest
concentration
of rapid method
microbiologists
in the world.”

FTS: Are the attendees all graduate students?

Fung: Oh, no. Even though we say students, many of the participants are business people or faculty. In fact, for our 20th anniversary workshop last July, we invited 15 of the past participants to come back and lecture.

FTS: Have you been able to track your alumni?

Fung: We track all of them. I have a book with everybody’s name from 1981 until now.

FTS: Big names?

Fung: Yes. These people are all directors of laboratories, some are deans, professors, lab techs. My directory is a Who’s Who in applied microbiology. And they are very happy to come. I have all kinds of testimonials saying that not only did they learn a lot, they had a lot of fun. And more importantly they get to do a lot of networking. We have a big family of microbiologists. We have almost 3,000 participants from 55 countries and 46 states who have come through the workshop in one way or another.

FTS: What exactly goes on at the workshop? When you say hands-on, do you break down into small groups of five or ten?

Fung: It’s a “Big Production.” The regular workshop starts Friday with three or four lectures, and then we have exhibits brought in by the companies. We have upwards of 15 companies present with their instruments and manufacturer reps. At night we have a banquet with a keynote speaker. The second day, I give several lectures in the morning to set the stage for the workshop, then in the afternoon we all leave the hotel for the laboratory, where we conduct the experiments. We give the students unknown cultures and they have to inoculate these cultures through conventional test tube methods and inoculate into the miniaturized methods I developed. Then they inoculate the cultures into all the diagnostic kits that happen to be in the workshop, and we ask them to identify each culture. Every day they have new systems to try out. At the end of the week they have to hand in a big report with all the cultures, and all the bar chemical reactions indentified. They have to do this before they are allowed to leave Manhattan, Kansas. So they actually do experiments.

FTS: I bet they love it.

Fung: Recently we began devoting a full day to molecular food microbiology. It’s exciting. We let them study all the molecular microbiology diagnostic tests PCR systems, ribo printing...they study all those things in one day. People really like that day because they are really on the cutting edge of microbiology. The newest technology all comes to Kansas State. That week we have the highest concentration of rapid method microbiologists in the world.

FTS: What are some of the newest methods?

Fung: First let me mention how important the conventional methods are. Even the newest methods need about one day of letting the bacteria grow. In a lot of tests, you need one million cells present in order to be able to detect the bacteria. Even the most sensitive methods, like ATP, require 10⁴ cells in order to register. So, you still need to encourage the cells to grow or centrifuge it down or filter it through something to concentrate it. One new technique for doing this is called immuno-magnetic separation. It utilizes paramagnetic beads. You put antibodies on the beads so that the antibody can trap the target bacteria. Then these beads are magnetized—you put a magnet there and it will suck all the beads to the side of the tube and you dump the junk out and wash it and suck it up and capture only the things you want. It’s a very powerful tool.

FTS: Why?

“Conventional
methods are
still important.
Even today’s
most sensitive
methods,
like ATP,
require 10⁴
cells in order
to register.”

Fung: It is a very good system. After you use immuno-magnetic separation, you can do a lot of things, like perform an Enzyme-Linked Immuno-Sorbent Assay (ELISA). And there are systems today which have the process completely automated. Even five years ago you had to wash the tubes by hand. Now you take a sample, enrich it overnight, take that sample and put it in a machine, press a button, walk away and an hour and a half later it will tell you “Salmonella Positive,” or “Listeria Positive.” Lateral Immuno Assay is also very exciting. You drop a drop of culture that has grown overnight into a well containing antibodies. If the target organism is present they will trap the organism and migrate it by capillary action. If it is positive they will be immobilized by another antibody to form a line. If you see a line it means you are presumptive positive for the pathogen. That test takes only ten minutes.

FTS: How long has that been available?

Fung: There are several versions of that test system out right now. They have been available for maybe five years. In ten minutes you know presumptive whether you have contamination or not. Then it must go to the lab for confirmation. There are also many instruments that can monitor biochemical or biophysical activities of the cells. For example, when the cells start growing it changes the electrical conductivity of the liquid. So you can have an instrument that can measure conductance or impedance. These are machines that are very simple to use. You press a button and walk away and you know approximately how many organisms you have. The newest technology in molecular biology is called Polymerase Chain Reaction, or PCR. It is rapid enough where, if you have one piece of one strand of DNA, in less than two hours the system can make that into one billion pieces and detect it. It is very fast and accurate for the detection of organisms.

FTS: Is that a preliminary, presumptive test?

Fung: They are arguing right now as to whether it is a confirmative or presumptive test. But my impression is it is still presumptive. If it registers a positive you still need to use the conventional method that takes about seven days to confirm it. But then hopefully 99% of your food tests negative, so you can already sell that food.

FTS: Are most advances coming in the presumptive arena?

Fung: All these tests will give you a preliminary screen first. Then you have to confirm it. So if you have a negative screen you’re okay.

FTS: Do you foresee a time when they will have immediate confirmation tests, where they will be able to detect on premise within an hour for a confirmed screen?

Fung: Very hard to tell right now. The problem is we have set very stringent requirements. For example, right now the law is you should not have one Salmonella bacteria in 25 grams of food. If you think about it, one bacterium is 10^-12 grams. One little particle in 25 grams. I don’t care what instrument you use, even if a single bacteria can be detected, you cannot find it in these 25 grams.

“The problem
is we
have set
very stringent
requirements.
The law is
you should not
have one Salmonella
bacteria in
25 grams of
food; one
bacterium
is 10^-12 grams!”

FTS: What does the future hold?

Fung: Right now in molecular biology there are two lines of development: PCR that will tell you whether or not you have a target pathogen, and ribo printing which doesn’t detect pathogens but can tell you precisely where that culture came from. For example, even e. coli O157:H7, if you find one in chicken and you find one in a vegetable you don’t know if they are the same e. coli O157:H7 or not. But if you use ribo-printing you can type the bacteria in detail. That is a very exciting development. By using ribo printing technology people can pinpoint where the organism came from. Many researchers are also working in the biosensor field. For that you need a lot of ingenious capturing of the cells and you have to multiply the cell, filter the cells, capture the cells, and then do the biosensor. That is perhaps the new wave.

FTS: What are you developing right now?

Fung: I am working on all kinds of quick methods for early detection. I have a test called the catalyst test; in about five minutes I can tell you if food is loaded with bacteria. It’s a technology that can give you an answer quickly for sanitation check or spoilage check. For single pathogen tests I am combining several technologies. I am also studying ways of improving fermentation methods. And I am studying how to kill the organisms by microwave.

FTS: I read that you are doing work with spices.

Fung: Oh, yes. I have been able to determine that spices can be used to kill pathogens. We have tested garlic and cinnamon and other spices against pathogens and they are pretty effective. For example, if you put garlic in your hamburger, you will have 90% fewer pathogens than without the garlic. And I was just asked by a group in California to study prune extract to see if prunes can suppress pathogens. And it turns out they can!

FTS: That’s nice, I guess, if you like prunes.

Fung: Yes, very nice.