By David Smith
Despite the Danger: Biohazard signs posted on the door,
John Burg feels comfortable in 118 Borland Lab.
"The
pathogens in here are deadly, but they're food-borne diseases. You
have to eat them to get sick," Burg said. "It's not like I'm working
with anthrax or anything that's airborne."
Burg, a microbiology major at Penn State, is working with Listeria
monocytogenes. This bacteria, which can be found in ready-to-eat
foods such as hotdogs and soft cheeses, milk, and raw meats, cause
listeriosis, a disease with such flu-like symptoms as headache,
fever, and diarrhea. In the most serious cases, the bacteria attack
the nervous system, infecting the brain and causing meningitis.
In 1998, hotdogs made at a Michigan plant caused an outbreak of
listeriosis that killed 21 people and sickened 80.
Yet it can be several weeks after a person has eaten food contaminated
by Listeria monocytogenes before symptoms appear. This long
incubation period makes it difficult for doctors to determine the
cause of an outbreak and makes accurate testing within the food
industry very important.
Burg's project has been to test a test, one that former Penn State
graduate student Alex Teo designed to detect Listeria in
milk. Teo called his test "oPSU broth," or "optimized PSU broth."
The oPSU broth is jelly-like, brownish yellow at the bottom and
slightly pink at the top.
If Listeria is present in the sample added to it, the oPSU
broth turns black. Here's how it works: A sugar called esculin gets
hydrolyzed, split in half, by an enzyme that Listeria produces.
One of the products of that split reacts with the iron in oPSU broth;
the compound formed is black.
Burg spends most of his time mixing reagents together and putting
them through the autoclave, a kind of pressure cooker used to sterilize
the mixture. But the heat of the autoclave would destroy some of
the amino acids used in the broth, so Burg sterilizes these by putting
them through a filter with holes only 0.2 micrometers wide. Those
holes are too small for bacteria to pass through.
Once the broth is ready, Burg prepares a sample by taking a hotdog
(bought at a local supermarket) and putting it and some water through
the Stomacher, a machine that pounds the hotdog for about five minutes.
Burg draws 0.5 ml samples from the resulting pulp and adds them
to test tubes filled with oPSU broth. He then lets the tubes sit
in a warm incubator for anywhere from a day to a few weeks. If the
broth turns black in that time, chances are Listeria are
present.
In the course of his research Burg has discovered that two other
bacteria — ones that are not a type of Listeria — will
also turn the broth black. Burg hopes to improve on the broth's
design so that it only detects strains of Listeria.
Once he has ruled out these two bacteria, he still can't be sure
that the bacteria in the tube are dangerous. The oPSU broth also
reacts to the harmless types of Listeria: L. grayi, L.
innucua, L. seeligeri, and L. ivanovii. Only by testing
the DNA of the bacteria he finds in the hotdog can Burg be sure
which strain is present. So far, Burg has only tested the DNA of
one sample and the test was positive for L. monocytogenes.
While Burg was surprised to find Listeria monocytogenes
in his sample, he adds that it "isn't really news because there
are occasional outbreaks. What's important to me is making sure
this broth works well with hotdogs and trying to improve on it."
The USDA's Food Safety and Inspection Service (FSIS) laboratory
guidebook, explains the "official" method for isolating and identifying
Listeria in hotdogs. The FSIS method uses two broths followed
by streaking the sample onto a solid medium and seeing if the medium
changes color. Burg and his adviser, food scientist Stephen Knabel,
believe oPSU broth followed by a DNA test is more sensitive.
"The current methods used in industry don't work as well with
low numbers of cells," Knabel said.
No one is sure how much bacteria it takes for a person to get sick
but scientists have estimated it at 1000 cells. So to protect himself,
Burg always wears gloves while working. Only after Burg had worked
for the lab for some time was he permitted to work unsupervised.
"We wanted to make sure he knew what he was doing, because we do
work with dangerous bacteria," Knabel said. "But once we were confident
in him we let him pursue this project on his own."
John Burg is a senior honors student majoring in microbiology.
His research adviser is Stephen J. Knabel, Ph.D., associate professor
of food science in the College of Agricultural Sciences, 101 Borland
Laboratory, University Park, PA 16802; 814-863-1372; sjk9@psu.edu.
Writer David Smith graduated in May 2000 with a B.A. in journalism.
