This article describes an inexpensive and safe experiment using citrus fruit to demonstrate Koch's Postulates. Citrus fruit purchased from grocery stores can be experimentally infected with Penicillium digitatum, a common mold found on citrus. Students isolate the mold from naturally infected fruit into a pure culture, then reintroduce it to uninfected fruit. The mold can then be recovered, completing all four steps of Koch's Postulates. This exercise allows students to associate a disease with its causal agent and practice sterile technique. It has been performed by hundreds of students without allergic reactions, making it a very safe demonstration for high school and college biology labs.

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An inexpensive and safe experiment to demonstrate
Koch's Postulates using citrus fruit
a
Steven Jakobi Ph.D.
a
Alfred State College, USA
Available online: 13 Dec 2010
To cite this article: Steven Jakobi Ph.D. (2010): An inexpensive and safe experiment to demonstrate Koch's Postulates
using citrus fruit, Journal of Biological Education, 44:4, 190-192
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2. Practical
An inexpensive and safe experiment
to demonstrate Koch's Postulates
using citrus fruit
Steven Jakobi, Ph.D.
Alfred State College, USA
Citrus fruit (oranges, tangerines, grapefruit or lemons) purchased in a grocery store can be experimentally infected
with readily-available sources of Pénicillium digitatum to demonstrate the four basic steps of Koch's postulates, also
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known as proof of pathogenicity. The mould is isolated from naturally-infected citrus fruit into pure culture on artificial
agar medium. The mycelium is then reintroduced into uninfected fruit from which the pathogen can be recovered.
The entire set of procedures can be completed in as little as four weeks, using easily available equipment. In
addition to demonstrating the importance of associating a disease with its causal agent, the exercise also allows for
practicing sterile technique. None of the hundreds of students who performed these steps in college lab settings has
ever experienced an allergic reaction to the fungus, making this a very safe, quick and inexpensive demonstration.
Key words: proof of pathogenicity; Koch's postulate; pedagogical tool; Pénicillium mould; citrus fruit; high
school/college biology lab experiments.
Introduction postulates are as important and useful today as they
The second half of the 19th century witnessed the were revolutionary when Koch proposed them in 1876.
emergence of microbiology as a serious and credible Thus, for example, these steps were used to identify the
science. Among the dozens of notable scientists causal agent of Legionnaires disease of humans as
responsible for the advancement of this discipline, few Legionella pneumopbila in the 1970s (Krause, 2001), and
are more important than Robert Koch, the German in assigning the pathogen involved in the recently
physician who identified the causal agent of discovered tree and shrub disease, "sudden oak death",
tuberculosis, and used the deadly disease anthrax to to the Oomycete mould, Pbytopbthora ramorum (Rizzo
formulate the germ theory of disease, now called Koch's et al, 2002). Some organisms, such as Treponema
postulates (Madigan, et al, 2008). The latter involved a pallidum, can not be grown in artificial media, and most
set of three steps to show that a particular micro­ viruses require living host cells for their demonstration.
organism may be the cause of a specific disease. Koch's Species of the genus Borrelia have specialized growth
proof of pathogenicity may be summarized as follows: requirements. Therefore, Koch's postulates are either not
applicable, or must be used in a modified form for the
1. The suspected disease-causing agent should be identification of some disease-causing agents.
present in all cases of a diseased host, and absent Many laboratory exercises have been devised to
from a healthy organism; demonstrate Koch's postulates to high school and
2. The suspected agent must be grown in pure culture college-level students (Fulton, 1981; Hill, 1981; Stewart,
and exhibit a consistent, characteristic growth pattern 1990). However, some of these are complicated
in the growth medium; procedures, or call for materials and or organisms that
3. Cells from the suspected agent must be taken from are not easily available or are expensive to purchase
the pure culture and introduced into a healthy host, from biological supply companies. The exercise
which then must develop the disease. described here is a simple, inexpensive procedure, using
citrus fruit as host, and the green mould, Pénicillium
A fourth step was added to Koch's original procedure by digitatum as the pathogen.
the American microbiologist, Erwin Smith (1905). Step 4
then states that the pathogen must be able to be re- Materials and Methods
isolated from the experimentally infected host and must Step 1 of Koch's postulates: Mouldy oranges, tangerines
be identical to the original disease-causing agent. Koch's or grapefruit are easy to find in grocery stores. Look for
190 JBE Vol 44 No 4 Autumn 2010

3. Koch's Postulates Jakobi
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damaged or bruised fruit that has the typical white Citrus fruit, especially when it has gone slightly mouldy, can be easily
mycelial growth in the peel and/or already has the and cheaply purchased and makes an ideal medium to demonstrate
the four basic steps of Koch's postulate - or proof of pathogenicity.
green spore- producing stage of Pénicillium digitatum.
In stores where the produce manager is especially
vigilant and fastidious, ask him to save the culled fruit. to 1 L distilled water in a 2 L flask and autoclaved under
In the 10 plus years of conducting this exercise, there standard 121 C, 15 psi, 15 min. conditions) and stores
has never been a time a Penicillium-infected fruit could well in poured plates in the refrigerator for several
not be found. weeks. The autoclaved liquid PDA is allowed to cool to
about 50 C and poured into disposable Petri dishes at
Step 2 of Koch's postulates: The mycelium and/or spores about 25 ml per dish. If an autoclave is not available, the
are scraped from the skin of the fruit with a flame- PDA-water solution can be boiled carefully on a hot
sterilized transfer tool or scalpel onto potato dextrose plate until it becomes an amber-colored liquid (care
agar (PDA; Difco, Becton, Dickinson and Co., Sparks, should be taken to avoid over-cooking to prevent the
MD) medium. This medium is preferable to others medium to become caramelized). Other media (e.g.,
because it is easy to make (39 gm PDA powder added glucose yeast extract, Sabouraud, etc.,) are equally
JBE Vol 44 No 4 Autumn 2010 191

4. Jakobi Koch's Postulates
suitable for the growth of this fungus, and the Difco Step 4 of Koch's postulates: The final step is to re-isolate
Manual (1977) can be consulted for one of these the pathogen from the experimentally infected fruit.
alternatives. Students may carry out the plate inoculation Once again, mycelium and spores are scraped from the
step under a laminar flow hood but this is not necessary surfaces of the inoculated mouldy oranges and grown
for the success of the experiment, as long as basic on agar-based media for a week. Students then compare
laboratory hygiene is practiced. As an overabundant the colonies from Steps 2 and 4 microscopically and
precaution, students with known allergies to moulds identify the hyphae, conidiophores, and conidia
may not want to use the laminar flow hood, even if one characteristic of this fungus.
is available. This is a good time to introduce students
the importance of hand washing and the use of dilute Discussion
(10 percent) bleach solutions as routine laboratory I have used this exercise in introductory biology classes,
hygienic practices. microbiology labs and in my plant pathology course for
After transfer of the fungus from the fruit and the about ten years. The technique is perfectly safe and
PDA medium is accomplished, the plates are sealed none of the hundreds of students who performed this
with Parafilm (American National Can, Greenwich, CT), procedure had ever exhibited an allergic reaction to
labeled, and incubated upside down (to prevent the inhaled spores of Pénicillium. Never-the-less, students
condensation that builds up on the inside of the lid should be informed that there is a small potential for an
from dripping onto the medium) for 1 week at room adverse hypersensitivity reaction. Students with known
(20-25 C) temperature. allergies, asthma, or immunocompromised pupils may
be excused from performing the experiment and given
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Step 3 of Koch's postulates: Fresh, uninfected citrus fruit an alternate task. The procedures described here utilize
(typically the least expensive oranges) is purchased from inexpensive and readily available materials and a
the grocery store. Oranges with thick rind are preferable ubiquitous organism, and can be completed in about 4
because these are less likely to be bruised or injured weeks' time. Of the hundreds of oranges used in these
and, therefore, least likely to carry latent infections. P. experimental protocols, the inoculated oranges always
digitatum spores may be present on the surfaces of developed the infection, and there was only one
uninjured fruit, but these are easily washed off from the instance of a single control orange that became infected.
peel surfaces. Depending on the numbers of students in In addition to demonstrating the importance of the
the labs, have them working individually, in pairs, or association between a causal agent and a disease, this
even groups of three. Each group is given two oranges, exercise can be used to introduce the concept of using
which they wash in a mild dish detergent solution, rinse controls in experimental protocols. The procedures are
with distilled water, and then spray with a 10 percent also useful for introducing students to fungi, their
bleach solution from a spray bottle to remove most morphology and reproduction, as well as discussion of
surface contaminants. The oranges are then dried with a post-harvest pathogens as spoilage agents. With respect
paper towel from a newly opened roll, and kept in the to the latter, basic hygienic steps and food preservation
hood, or are kept covered until used. One orange techniques in the home environment also can be
receives a sterile agar square and serves a control for the described and discussed.
experiment. The experimental orange is inoculated with
Pénicillium. The procedure is to use a flame-sterilized References
scalpel to remove a lcm x 1cm piece of skin from the Difco manual. 9th. edn (1977). Detroit, Ml: Difco Laboratories, Inc.
fruit, and insert a comparably-sized square of Fulton, M.M. (1981). A simple exercise demonstrating Koch's
postulates. The American Biology Teacher. 43(7), 3-8
Penicillium-colonized agar into the experimental fruit, Hill, E.C. (1981 ). A microbiology experiment for schools. Journal of
or sterile agar into the control orange. Sterile technique Biological Education 15(2), 96-99
is once again demonstrated and its importance in the Krause, R.M. (2001). Microbes and emerging infections:
process is reiterated. The agar squares are then covered The compulsion to become something new. American Society of
Microbiology News 67(1), 15-20
with masking tape to prevent desiccation. The Madigan, M.T.; Martinko, J.M.; Dunlap, P.V.; and Clark, DP. (2008).
experimental and control oranges are placed into two Brock Biology of Microorganisms. San Francisco: Benjamin Cummings
separate metal bins. These containers have been Rizzo, D.M.; Garbeletto, M.; Davidson, J.M.; Slaughter, G.W.; and
prepared ahead of time by adding a thin layer of Koike, ST. (2002). Phytophthora ramorum as the cause of extensive
mortality of Quercus spp. and Lithocarpus densiflorus in California.
vermiculite or sand to the bottom of the container along Plant Disease 86, 205-214
with 200 ml of distilled water to keep the material moist, Smith, E. F. (1905). Bacteria in relation to diseases. Vol. 1. Methods of
covered with heavy-duty aluminum foil and autoclaved work and general literature of bacteriology exclusive of plant diseases.
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Stewart, P. (1990). Safely teaching Koch's postulates on the causation
autoclave is not available, the sealed containers can be of infectious diseases. Journal of Biological Education 24(2), 117-123.
sterilized by placing them into a kitchen oven and
baked at 375 F for 1 hour. Disposable aluminum
cooking or baking pans, such as those used for cooking Steven Jakobi, Ph.D., Associate Professor of Biology,
poultry, can be used if other metal containers are not Alfred State College, Alfred, NY 14802 USA.
available. The oranges are incubated in their respective Email: jakobisr@alfredstate.edu
bins for 1 week.
192 JBE Vol 44 No 4 Autumn 2010