This article was downloaded by: [83.33.247.204]On: 17 February 2012, At: 14:57Publisher: RoutledgeInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Biological EducationPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/rjbe20

An inexpensive and safe experiment to demonstrateKoch's Postulates using citrus fruitSteven Jakobi Ph.D. aa 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 Postulatesusing citrus fruit, Journal of Biological Education, 44:4, 190-192

To link to this article: http://dx.doi.org/10.1080/00219266.2010.9656221

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arisingdirectly or indirectly in connection with or arising out of the use of this material.

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 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 The second half of the 19th century witnessed the emergence of microbiology as a serious and credible science. Among the dozens of notable scientists responsible for the advancement of this discipline, few are more important than Robert Koch, the German physician who identified the causal agent of tuberculosis, and used the deadly disease anthrax to formulate the germ theory of disease, now called Koch's postulates (Madigan, et al, 2008). The latter involved a set of three steps to show that a particular micro­organism may be the cause of a specific disease. Koch's proof of pathogenicity may be summarized as follows:

1. The suspected disease-causing agent should be present in all cases of a diseased host, and absent from a healthy organism;

2. The suspected agent must be grown in pure culture and exhibit a consistent, characteristic growth pattern in the growth medium;

3. Cells from the suspected agent must be taken from the pure culture and introduced into a healthy host, which then must develop the disease.

A fourth step was added to Koch's original procedure by the American microbiologist, Erwin Smith (1905). Step 4 then states that the pathogen must be able to be re-isolated from the experimentally infected host and must be identical to the original disease-causing agent. Koch's

postulates are as important and useful today as they were revolutionary when Koch proposed them in 1876. Thus, for example, these steps were used to identify the causal agent of Legionnaires disease of humans as Legionella pneumopbila in the 1970s (Krause, 2001), and in assigning the pathogen involved in the recently discovered tree and shrub disease, "sudden oak death", to the Oomycete mould, Pbytopbthora ramorum (Rizzo et al, 2002). Some organisms, such as Treponema pallidum, can not be grown in artificial media, and most viruses require living host cells for their demonstration. Species of the genus Borrelia have specialized growth requirements. Therefore, Koch's postulates are either not applicable, or must be used in a modified form for the identification of some disease-causing agents.

Many laboratory exercises have been devised to demonstrate Koch's postulates to high school and college-level students (Fulton, 1981; Hill, 1981; Stewart, 1990). However, some of these are complicated procedures, or call for materials and or organisms that are not easily available or are expensive to purchase from biological supply companies. The exercise described here is a simple, inexpensive procedure, using citrus fruit as host, and the green mould, Pénicillium digitatum as the pathogen.

Materials and Methods Step 1 of Koch's postulates: Mouldy oranges, tangerines or grapefruit are easy to find in grocery stores. Look for

190 JBE Vol 44 No 4 Autumn 2010

Dow

nloa

ded

by [

83.3

3.24

7.20

4] a

t 14:

57 1

7 Fe

brua

ry 2

012

Koch's Postulates Jakobi

damaged or bruised fruit that has the typical white mycelial growth in the peel and/or already has the 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. In the 10 plus years of conducting this exercise, there has never been a time a Penicillium-infected fruit could not be found.

Step 2 of Koch's postulates: The mycelium and/or spores are scraped from the skin of the fruit with a flame-sterilized transfer tool or scalpel onto potato dextrose agar (PDA; Difco, Becton, Dickinson and Co., Sparks, MD) medium. This medium is preferable to others because it is easy to make (39 gm PDA powder added

Citrus fruit, especially when it has gone slightly mouldy, can be easily and cheaply purchased and makes an ideal medium to demonstrate the four basic steps of Koch's postulate - or proof of pathogenicity.

to 1 L distilled water in a 2 L flask and autoclaved under standard 121 C, 15 psi, 15 min. conditions) and stores well in poured plates in the refrigerator for several weeks. The autoclaved liquid PDA is allowed to cool to about 50 C and poured into disposable Petri dishes at about 25 ml per dish. If an autoclave is not available, the PDA-water solution can be boiled carefully on a hot plate until it becomes an amber-colored liquid (care should be taken to avoid over-cooking to prevent the medium to become caramelized). Other media (e.g., glucose yeast extract, Sabouraud, etc.,) are equally

J B E Vol 44 No 4 Autumn 2010 191

Dow

nloa

ded

by [

83.3

3.24

7.20

4] a

t 14:

57 1

7 Fe

brua

ry 2

012

Jakobi Koch's Postulates

suitable for the growth of this fungus, and the Difco Manual (1977) can be consulted for one of these alternatives. Students may carry out the plate inoculation step under a laminar flow hood but this is not necessary for the success of the experiment, as long as basic laboratory hygiene is practiced. As an overabundant precaution, students with known allergies to moulds may not want to use the laminar flow hood, even if one is available. This is a good time to introduce students the importance of hand washing and the use of dilute (10 percent) bleach solutions as routine laboratory hygienic practices.

After transfer of the fungus from the fruit and the PDA medium is accomplished, the plates are sealed with Parafilm (American National Can, Greenwich, CT), labeled, and incubated upside down (to prevent the condensation that builds up on the inside of the lid from dripping onto the medium) for 1 week at room (20-25 C) temperature.

Step 3 of Koch's postulates: Fresh, uninfected citrus fruit (typically the least expensive oranges) is purchased from the grocery store. Oranges with thick rind are preferable because these are less likely to be bruised or injured and, therefore, least likely to carry latent infections. P. digitatum spores may be present on the surfaces of uninjured fruit, but these are easily washed off from the peel surfaces. Depending on the numbers of students in the labs, have them working individually, in pairs, or even groups of three. Each group is given two oranges, which they wash in a mild dish detergent solution, rinse with distilled water, and then spray with a 10 percent bleach solution from a spray bottle to remove most surface contaminants. The oranges are then dried with a paper towel from a newly opened roll, and kept in the hood, or are kept covered until used. One orange receives a sterile agar square and serves a control for the experiment. The experimental orange is inoculated with Pénicillium. The procedure is to use a flame-sterilized scalpel to remove a lcm x 1cm piece of skin from the fruit, and insert a comparably-sized square of Penicillium-colonized agar into the experimental fruit, or sterile agar into the control orange. Sterile technique is once again demonstrated and its importance in the process is reiterated. The agar squares are then covered with masking tape to prevent desiccation. The experimental and control oranges are placed into two separate metal bins. These containers have been prepared ahead of time by adding a thin layer of vermiculite or sand to the bottom of the container along with 200 ml of distilled water to keep the material moist, covered with heavy-duty aluminum foil and autoclaved at 121 C and 15 psi pressure for 20 minutes. If an autoclave is not available, the sealed containers can be 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 poultry, can be used if other metal containers are not available. The oranges are incubated in their respective bins for 1 week.

Step 4 of Koch's postulates: The final step is to re-isolate the pathogen from the experimentally infected fruit. Once again, mycelium and spores are scraped from the surfaces of the inoculated mouldy oranges and grown on agar-based media for a week. Students then compare the colonies from Steps 2 and 4 microscopically and identify the hyphae, conidiophores, and conidia characteristic of this fungus.

Discussion I have used this exercise in introductory biology classes, microbiology labs and in my plant pathology course for about ten years. The technique is perfectly safe and none of the hundreds of students who performed this procedure had ever exhibited an allergic reaction to inhaled spores of Pénicillium. Never-the-less, students should be informed that there is a small potential for an adverse hypersensitivity reaction. Students with known allergies, asthma, or immunocompromised pupils may be excused from performing the experiment and given an alternate task. The procedures described here utilize inexpensive and readily available materials and a ubiquitous organism, and can be completed in about 4 weeks' time. Of the hundreds of oranges used in these experimental protocols, the inoculated oranges always developed the infection, and there was only one instance of a single control orange that became infected.

In addition to demonstrating the importance of the association between a causal agent and a disease, this exercise can be used to introduce the concept of using controls in experimental protocols. The procedures are also useful for introducing students to fungi, their morphology and reproduction, as well as discussion of post-harvest pathogens as spoilage agents. With respect to the latter, basic hygienic steps and food preservation techniques in the home environment also can be described and discussed.

References Difco manual. 9th. edn (1977). Detroit, Ml: Difco Laboratories, Inc. Fulton, M.M. (1981). A simple exercise demonstrating Koch's

postulates. The American Biology Teacher. 43(7), 3-8 Hill, E.C. (1981 ). A microbiology experiment for schools. Journal of

Biological Education 15(2), 96-99 Krause, R.M. (2001). Microbes and emerging infections:

The compulsion to become something new. American Society of Microbiology News 67(1), 15-20

Madigan, M.T.; Martinko, J.M.; Dunlap, P.V.; and Clark, DP. (2008). Brock Biology of Microorganisms. San Francisco: Benjamin Cummings

Rizzo, D.M.; Garbeletto, M.; Davidson, J.M.; Slaughter, G.W.; and Koike, ST. (2002). Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant Disease 86, 205-214

Smith, E. F. (1905). Bacteria in relation to diseases. Vol. 1. Methods of work and general literature of bacteriology exclusive of plant diseases. Washington, D.C.: Carnegie Institution.

Stewart, P. (1990). Safely teaching Koch's postulates on the causation of infectious diseases. Journal of Biological Education 24(2), 117-123.

Steven Jakobi, Ph.D., Associate Professor of Biology, Alfred State College, Alfred, NY 14802 USA. Email: jakobisr@alfredstate.edu

192 JBE Vol 44 No 4 Autumn 2010

Dow

nloa

ded

by [

83.3

3.24

7.20

4] a

t 14:

57 1

7 Fe

brua

ry 2

012