ENVIRONMENTAL MICROBIOLOGY:

Suzanne Segner

K a r e n - B e T H g . S c H o lT H o f

he near daily news reports on food-borne diseases caused by contaminated produce, dairy, or meats suggests to the public that the safety of the U .S . food supply is in jeopardy . These reports, as well as a general distrust in federal agencies due in part to mad cow disease and toxigenic forms of E. coli in ground beef, have resulted in an increasing demand for organic foods and an interest in sustainable agriculture (Macilwain, 2004) . It is, therefore, timely and appropriate to provide classroom lectures and laboratory experiences to show that microbes are ubiquitous in our environment, including in the foods that we eat (Scholthof, 2001; Scholthof, 2003) . In presenting such material, it is important that we distinguish between common microbes and potentially harmful pathogens . In particular, it is imperative to place an emphasis on the fact that many bacteria and fungi are both beneficial and necessary for crop production and robust ecosystems . This hands-on laboratory experience has the added benefit of reinforcing safe food storage and preparation . The lab described in this article was specifically adopted for an undergraduate course Pathogens, the Environment, and Society for the Bioenvironmental Sciences undergraduate degree program at Texas A&M University . This three-credit hour course includes several lectures on food safety and agriculture . For our specific needs we decided to develop a laboratory exercise using fruits and vegetables with an emphasis on identifying cosmopolitan bacteria and fungi associated with fresh produce we eat . This laboratory exercise uses simple microbiology techniques and can be completed in three one-hour sessions . The objective is to reveal the intimate relationships between plant pathogens and environmental and food safety issues in agriculture (Scholthof, 2001; Scholthof, 2003) .

were selected because there have been several reports warning about food-borne illnesses that are linked to consuming fresh sprouts (Breuer et al ., 2001; Taormina et al ., 1999) .

Preparation
Standard microbiology protocols were used for the preparation of growth media . The details for preparing materials and identifying bacteria were based on protocols provided by the Bacteriological Analytical Manual Online (www . cfsan .fda .gov/~ebam/bam-toc .html) and the supplier of the dehydrated culture media (Carolina Biological Supply Co .) . Two growth media were used: nutrient broth-yeast extract agar (NBY) and potato dextrose agar (PDA) supplemented with two antibiotics, ampicillin and streptomycin (PDA-AS) . NBY was used for bacteria because it allows for good growth of most bacteria with characteristic colony morphology . PDA is a common growth media for isolating fungi . The addition of the antibiotics in PDA-AS is to inhibit bacterial growth, allowing for better development of the fungi . The fungi and bacteria were cultured at 28 C in a standard laboratory incubator . Alternatively, they can be kept at room temperature, although it will require a longer incubation period . Bacterial growth was apparent after 24 hours . Most fungi were visible on PDA-AS plates within 72 hours . For this laboratory session, two to four students were assigned to a group working with fruits or vegetables . For example, both organic and conventionally farmed produce were tested by Group 1 (apples and peaches), Group 2 (carrots), and Group 3 (tomatoes) . Group 4 was assigned button mushrooms (Agaricus bisporus), including packaged mushrooms; washed, sliced and packaged mushrooms; and bulk mushrooms . Group 5 sampled celery and green peppers; Group 6 worked with alfalfa sprouts and spring lettuce mixes (bagged, washed lettuces, and bulk mix); Group 7 sampled several small fruits, including strawberries, raspberries, and blueberries . Group 8 was assigned green and red table grapes from Chile and Argentina, respectively; Group 9 sampled cilantro and green onions; and Group 10 tested green beans and snow peas .

Materials & Methods

The goal of this student laboratory experience is to use basic microbiological techniques to examine common bacteria and fungi on fruits and vegetables . Inexpensive conventionally grown produce (see Table 1) that is available year-round was selected from a supermarket . We also purchased organically grown carrots, apples, and tomatoes from the supermarket to determine if there were differences in the types of bacteria and fungi when compared to conventionally produced fruits and vegetables . Alfalfa sprouts

Sample Preparation
Each group of students followed the same general protocol for preparing its samples and streaking the fruits or vegetables to Petri dishes with either NBY or PDA-AS . We also prepared individual protocol sheets for each group; some minor variations in sample preparation are indicated later . The following detailed protocol illustrates how grapes were prepared using four treatments: a) unwashed, b) washed, c) one minute bleach and wash, and c) five minute bleach and wash .

Suzanne Segner is the Laboratory Coordinator and KarenBeTH g. ScHolTHof (e-mail: kbgs@tamu.edu) is Professor, both in the Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843.

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Bacteria & Fungi on the Foods We Eat

Table 1. identification of bacteria isolated from fruits and vegetables either washed with water (yes) or unwashed (no).
produce Washed: Apple (organic) Apple peach grapes (green) grapes (red) strawberry raspberry Blueberry carrots (organic) carrots tomato (organic) tomato lettuce mix, prewashed package lettuce mix, bulk Bacillus no d + + (Bm) + + ++++ (Bm) +++ (Bs) + + (Bm) + (Bm) + ++ (Bc) ++ (Bs) + +++ (Bs) + +++ (Bm) +++ (Bm) + + ++ (Bm) ++++ ++++ +++ (Bc)
a

yes + + (Bm) + + +++ (Bm) +++ (Bs) + + (Bm) + (Bm) + ++ (Bc) ++ (Bs) + +++ (Bs) + +++ (Bm) +++ (Bm) + + ++ (Bm) ++++ +++ +++ (Bc)

Chryseobacterium no yes + +

Pantoea no yes

no

Pseudomonas yes

Unknown no yes + +

+++

+++

+++ +++ (pf) ++ (ps) +++ +++ (pf) +++ +++ (pf) + (ps)

++ ++ (pf) ++ (ps) +++ +++ (pf) +++ +++ (pf) + (ps) + (ps) + (ps) ++ ++ (pf) + +++ (pf) +++ (ps) + (ps) + (ps) +++ +++ (pf) ++++ (ps) ++++ +++ (pa) ++++ (pf) ++++ (ps) ++++ +++ (pa) +++++ (pf) +++++ (ps) +++++ (ps) +++ (pf) ++++ (pf) ++ (ps)

+ ++ + + ++ + +

+ +

+ +

+ (ps) + (ps) +++ +++ (pf) + +++ (pf) +++ (ps) + (ps) + (ps) +++ +++ (pf) ++++ (ps) ++++ +++ (pa) ++++ (pf) ++++ (ps) ++++ +++ (pa) +++++ (pf) +++++ (ps) +++++ (ps) +++ (pf)

+ + +

+ + +

++

++

+ +

+ +

++

++

Alfalfa sprouts cilantro green onions green Beans snow pea celery Bell pepper mushrooms, sliced, packaged mushrooms, packaged mushrooms, bulk

+++ (Bc) +++++ (Bm) +++ +++ (Bm) +++ ++ (Bs) ++ +++ (Bm) ++ (Bs) ++++ + (Bs) ++++ + (Bm) + (Bs) ++ (Bm) + + (Bs)

+++ (Bc) ++++ (Bm) +++ +++ (Bm) ++ ++ (Bs) ++ +++ (Bm) ++ (Bs) +++ + (Bs) +++ + (Bm) + (Bs) ++ (Bm) + + (Bs) + + +++ +++

+++ + +

+++ + +

++ ++

++ ++

++ ++

++ ++

++++ (pf) ++ (ps)

++ ++ ++ ++ (pf) ++ (ps) +++ (ps) + (pf) + (ps) ++ (pf) ++ (ps) +++ (ps) + (pf) + (ps) +

++

a. further identification to species is indicated as follows: B. cereus (Bc); B. megaterium (Bm); and B. subtilis (Bs). b. further identification to species is indicated as follows: P. aureofaciens (pa); P. fluorescens (pf); P. syringae (ps). c. unidentified bacterium, based on fatty acid analysis and a clinical microbiology database. d. increasing numbers of (+)-signs indicate the relative greater numbers of colonies on the original nBy or pdA-As plates. the lack of a (+)-sign indicates the bacterium was not detected on the fruit or vegetable.

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Prior to streaking the fruit or vegetable onto the plate, all of the Petri dishes were labeled with the: 1) name of fruit or vegetable, 2) treatment applied to the fruit or vegetable, and 3) date . Printed labels were made prior to the beginning of the laboratory period to decrease the amount of time that the students needed to complete their group assignment and to reduce the chance of labeling errors .

Incubate Plates from Treatments A-D

1 . Place all of the streaked Petri dishes (NBY and PDA-AS) in a 28 C incubator for 48 hours . 2 . After 48 hours, remove plates from incubator and observe for microbial growth . 3 . Discard all of the grapes into the trash containers provided .

Unwashed

1 . Take two grapes from the bunch and streak one each across a nutrient broth yeast-extract agar (NBY) plate and a potato dextrose agar plate with ampicillin (50 mg/mL) and streptomycin (50 mg/mL), indicated as PDA-AS . The streaking strategy is shown in Figure 1A . 2 .

Wash
1 .

2 . 3 .

The protocols for the other fruits and vegetables were essentially the same as described for grapes . Minor variations for apples, peaches, and tomatoes included using an entire fruit for each treatment and slicing the fruits into four pieces . For the carrots, two additional treatment types included blanching (immersion in boiling water for one minute) and peeling . The lettuce mixes and cilantro leaves were streaked 1) without Repeat with a third and fourth grape, streaking onto washing or 2) following a rinse with tap new NBY and PDA-AS plates . water, then blotted dry with paper towels . Figure 1. streaking and sampling (Note: The FDA specifically recommends that with Tap Water techniques for bacteria and fungi. A). foods NOT be treated with bleach . Therefore, Take four grapes from the bunch, start at the solid dot and follow the it should be emphasized that the bleach treatwash thoroughly with tap water, and pattern. Keep fruit or vegetable in ments used for this lab, are for demonstration pat dry with paper towels . purposes only .) We were not able to obtain contact with the media on the plate, Streak one grape across an NBY plate, produce washes, but this would be another and streak to arrow. B). to transfer bacand a second across a PDA-AS plate . treatment option, or a replacement for the teria, short-streak to a fresh plate. use bleach protocols . Repeat with grapes three and four on a sterile toothpick for each transfer. A new NBY and PDA-AS plates .

Bleach for One Minute Followed by Rinse in Running Tap Water

1 . Take four grapes from the bunch and place in 5% bleach solution . 2 . Keep the grapes in the 5% bleach solution for one minute, moving them around to expose all surfaces to the bleach . 3 . Remove the grapes from the bleach solution, wash thoroughly under running water, and pat dry with a paper towel . 4 . Streak one grape across an NBY plate, and a second across a PDA-AS plate . 5 . Repeat with grapes three and four on new NBY and PDA-AS plates .

single nBy plate can be used to streak 10-12 representative bacterial colony types. c). to transfer fungi, use a sterile toothpick to excise a small piece of each different type of fungal colony. place one specimen on the center of a fresh pdA-As plate.

Microbe Isolation

For the second lab period, the students counted and selected bacteria and fungi to transfer to new NBY and PDA-AS plates, respectively . This process took 30 minutes and was followed by a 20-minute slide presentation on food safety . Sufficient time was allowed for each group to discuss its observations and to examine the plates from each of its peer groups . Each group made notes on the gross morphology of the microbes . The students were instructed to use a sterile toothpick to select representative bacterial colony types . These were streaked to a fresh NBY plate (Figure 1B), followed by incubation at 28 C for 48 hours (Lelliott & Stead, 1987; Schaad et al ., 2001) . Several bacterial streaks must be made to a single plate, and the students are reminded to use a new sterile toothpick for each transfer . For fungal colonies, each group was instructed to use a sterile toothpick to individually select characteristic fungi from the original plate and place each selection on a separate, fresh PDA plate (Figure 1C) . The plates were labeled (produce type, treatment type, date) and incubated at 28 C for 72 hours . Petri dishes were inverted in the incubator (or during storage in the refrigerator) in order to prevent condensation from forming on the agar-side of the plate .

Bleach for Five Minutes Followed by Rinse in Running Tap Water

1 . Take four grapes from the bunch and place in 5% bleach solution for five minutes, occasionally moving the fruit around to expose the entire surface to bleach . 2 . Remove the grapes from the bleach solution, wash thoroughly with tap water, and pat dry with a paper towel . 3 . Streak one grape across an NBY plate, and another across a PDA-AS plate . 4 . Repeat with grapes three and four on new NBY and PDA-AS plates .

Identification & Discussion

The Petri dishes were removed from the incubator after 48-72 hours and the bacterial and fungal isolates were identified by

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Table 2. filamentous fungi and yeast isolated from produce washed with water prior to plating on pdA-As medium.

Alternaria P (b) P

P P

P P P P P

A third class session was used to examine the bacteria and fungi on the covered Petri plates using dissecting microscopes (40 X magnification) . Instructor-prepared microscopy slides and commercially prepared slidesets (Carolina Biological Supply Co .) were used to show details of fungal

Aspergillus

P (c)

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Apple (organic) Apple peach grapes (green) grapes (red) strawberry raspberry Blueberry carrots (organic) carrots tomato (organic) tomato Bag lettuce mix Bulk lettuce mix Alfalfa sprouts cilantro green onion green Bean snow pea celery green pepper mushrooms, washed, packaged mushrooms, packaged mushrooms, bulk

P (d)

We were fortunate to have access to the Texas Plant Disease Diagnostic Laboratory (http://plant p a t h o l o g y . t a m u . e d u / extension/tpddl/tpddl . asp) for fatty acid analysis of the bacteria to identify the samples that the students isolated from the produce . This type of analysis revealed that the bacteria were common plant pathogens, with one exception (Tables 1 and 3) . Fungi were identified to genus by microscopy and the use of mycology texts and online reference guides (Barnett & Hunter, 1998; Larone, 1995) . The identified fungi are common (Tables 2 and 3), known saprophytes or plant pathogens . Illustrative materials on fungi and bacteria that are common to plants can be found at the American Phytopathological Society Education Center (http:// www .apsnet .org) .

a. the identification of Rhodotorula is likely based on morphological features on potato dextrose agar (pdA); it has not been further characterized. b. the check-mark (P) indicates that fungi were observed. for the yeasts (Saccharomyces and Rhodotorula), increasing numbers of check-marks indicate the relative number of cells on the original pdA-As plates. c. identified to species as A. niger. d. identified to species as A. terreus.

Phoma

the instructors (Barnett & Hunter, 1998; Larone, 1995; Lelliott & Stead, 1987; Schaad et al ., 2001) (Tables 1 and 2) . In some cases, several transfers of the fungi were made by the instructors to ensure that pure cultures would be used to identify the microbes to genus and species . This step resulted in a 10-day gap between the initial plating of the microbes and the follow-up lab and discussion for this section . However, characterization to genus can be completed without subculturingthis would reduce materials costs .

Rhodotorula (a) P P PP

PPP PPP

P PPP PPP

Saccharomyces PP (b) P PP

P PPP PP

P P P

P P

P P P P P P P P P P P P

Trichoderma

Rhizopus

P P

Penicillium P P P P P P P P

Nigrospora

Fusarium

P P

P P

Epicoccum

Colletotrichum

Cladosporium

P P P

P P

Botrytis

P P P P

P P P P

P P P

P P

P P P P P P P

P P P

P P P P P P

morphology with a compound microscope (100 - 400 X magnification) . A question-and-answer discussion period followed a slide presentation that illustrated diseases (Table 3) and crop losses caused by plant pathogens as well as food production and food safety processes (Table 4) .

could be completed with as few as 50 plates . The students could bring in produce from home . Produce managers at the local grocery have also been helpfuloccasionally providing slightly-blemished fruits and vegetables for this lab . The morphological features of the fungi are distinctive and easy to characterize with a dissecting and light microscope,

Follow-Up
An in-class writing assignment was used to test the students understanding of the topics and issues related to this laboratory exercise . Scenarios from published food-safety outbreaks were presented and the students had one hour to dissect the potential chain of events from farm to fork that could have caused an outbreak and to suggest how to prevent a recurrence of the event . This allowed the students to synthesize the information and their observations in order to define practical approaches to food handling (Table 4) . This lab can be easily integrated into the previous How-To-Do-It component on microbial assays of chicken wings (Deutch, 2001) . (This lab can also be adapted for upper level undergraduate students, providing them with an opportunity to prepare pure cultures followed by the identification of the isolated fungi and bacteria .) Anonymous course evaluation comments were extremely positive and the students urged that the lab be developed as a standard component of the lecture course on Pathogens, the Environment, and Society . The cost to prepare this lab for 25 students, with all of the indicated fruits and vegetables was $200 . However, the lab costs can easily be reduced if each student group had two treatments, unwashed and washed . This would require four plates each of NBY and PDA-AS; therefore, for three students/group the entire exercise

Table 3. general features of bacteria, fungi, and yeasta identified on fresh produce.
B Ac t e r i A Bacillus cereus Bacillus megaterium Bacillus subtilis Chryseobacterium Pantoea Pseudomonas aureofaciens P. fluorescens P. syringae fungi & yeAst Alternaria Aspergillus Botrytis Cladosporium Colletotrichum Epicoccum Fusarium Nigrospora Penicillium Phoma Rhizopus Trichoderma Saccharomyces Rhodotorula type gram (+) gram (+) gram (+) gram (-) gram (-) gram (-) gram (-) gram (-) type deuteromycete deuteromycete deuteromycete deuteromycete deuteromycete deuteromycete deuteromycete deuteromycete deuteromycete deuteromycete Zygomycete deuteromycete yeast yeast B i o lo g y gastrointestinal illness in humans common in soil and vegetation common in soil and vegetation, used as seed treatment to protect plants against root pathogens common in soils, plants, food stuffs, and water systems Biocontrol of storage fungi on citrus, can cause bacterial brown spot on melons Biocontrol of fungi on turf Biocontol of fungi and fireblight; to reduce frost damage on fruit trees Bacterial speck (tomato); halo blight of bean B i o lo g y leaf and flower rots; indoor molds; mycotoxin Bread molds, seed decay, mycotoxin grey mold (strawberry); noble rot (grape) postharvest and ear (corn) mold Anthracnoses in many plants saphrophytes, ubiquitous & cosmopolitan fusarium head blight (small grains); mycotoxins found in air, soil, plant materials. Allergen Blue mold (oranges); Antibiotic; mycotoxins fruit rots Bread mold; postharvest fruit disease Biocontrol to inhibit growth of other fungi fermentation Allergen, indoor mold, carotenoid production

a. helpful references for the identification of fungi included Barnett & hunter (1998) and larone (1995).

Table 4. methods for remediation or prevention of microbial contamination on produce from farm to fork. (Adapted from scholthof, 2003).
AgriculturAl eVent production and harvest initial processing distribution final processing eVent feAtures growing, picking, bundling, boxing Washing, waxing, sorting, boxing trucking slicing, shredding, peeling, juicing contAminAtion sources irrigation water, manure, lack of field sanitation Wash water, handling, cutting equipment ice, dirty trucks, improper storage temperature Wash water, handling, cross-contamination, improper storage temperature handling, cross-contamination, improper storage remediAtion clean water for irrigation; segregate animals from production fields use clean, cool water. Wash and/or bleach tools often use ice from clean water, monitor shipping temperatures clean wash and misting water, hand-washing with soap if handling produce, climate controlled storage, irradiation, pasteurize juices. purchase fruits and vegetables from reliable source, good quality produce (no obvious post-harvest fungi), hand-washing, thorough wash and/or blanch produce, storage conditions BActeriA & fungi 153

in the kitchen

food preparation

either from the original Petri plate or following a single passage to fresh plates . Bacteria can be generally classified by Gramstain and light microscopy . Extra costs would be incurred for fatty acid analysis ($45/sample), but these fees are often reduced or waived for teaching demonstrations .

Internet Resources
There is a wealth of information on the Internet to prepare lectures and presentations . Of particular importance are the Fight Bac site (www .fightbac .org) with tutorials and general information on food safety; the Centers for Disease Control and Prevention (www .cdc .gov); the United States Department of Agriculture (USDA; www .usda .gov); and the Food and Drug Administration (www .fda .gov) . Other general information to identify microbes has been posted online by the American Phytopathological Society (www .apsnet .org) and Microbe World (www .microbeworld .org) .

students were impressed with these observations . In particular, they had not anticipated that both washed and bagged salad mixes would have high bacterial counts . In all cases, under the stated conditions, there was little advantage in washing produce, as only minor differences in the number of bacteria were observed (Table 1) . However, our type of washing protocol may be less rigorous than washing techniques used for commercial (restaurant) food preparation . We did not determine if washing was as effective at reducing other microbes including viruses . In contrast to the lettuce experiment, the apples had very little microbial contamination . When apples are harvested, they are washed with clean water under high pressure, and coated with a non-petroleum based FDA-approved food wax . This process effectively reduces postharvest contamination by microbes and increases shelf-life, mostly by reducing water loss (dehydration or shriveling); it also has the beneficial effect of reducing microbial contamination (Table 3) . Post-harvest losses are economically important considerations for growers and wholesale shippers . Reducing microbial contamination has the obvious benefit of increasing the shelf-life of fruits and vegetables, which reduces costs to grocers and customers . In contrast to apples, alfalfa sprouts, cilantro, mushrooms, and salad mixes (lettuces) have little or no post-harvest treatments (Breuer et al ., 2001; Taormina et al ., 1999) . Of particular interest are alfalfa sprouts . Alfalfa seed is harvested from plants grown in pastures that may be accessible to roaming deer or cattle . Feces from these animals may harbor pathogenic strains of E. coli or Salmonella spp . that can contaminate alfalfa seed that is used as seed for sprouts and forage pastures . Recent recommendations by the FDA (www .cfsan .fda .gov/~dms/sprougd1 .

Results & Discussion

Washing Produce
This laboratory exercise examined microbial contamination in produce purchased from the supermarket and the effect of washing on the microbial contamination (Tables 1 and 2) . Our experiments showed that commercially washed and bagged salad lettuces had lower levels of bacteria and fungi than bulk spring mix lettuces (Tables 1 and 2) . We also found that washing bagged salad mixes with tap water did not further reduce (or increase) the type or amounts of bacteria . The

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html) for commercial sprout production also discuss handling to ensure low microbial contamination, including soaking seed in 20,000 ppm calcium hypochlorite and monitoring bacterial counts in water supplies . Although chlorine treatment is not completely effective in abolishing bacteria (some of which are known agents of food-borne diseases), it reduced, but did not eliminate, the incidence of sproutassociated food-borne outbreaks in the past five years . Heat treatment and irradiation are effective strategies for reducing bacterial counts . However, both approaches have detrimental effects on seed germination, resulting in increased operating costs for sprout growers . It is important to note that even low bacteria titers can rapidly increase when water is recycled between sprouting trays . For this reason, the CDC recommends that both the sprouts and the water be tested, and that water not be allowed to (re-)circulate between sprouting trays . (This also suggests another classroom laboratory experience: to obtain alfalfa seed, determine the bacterial contamination of the seed, wash the seed, treat the seed with bleach, and again count colonies, to determine the amount of microbial contamination .) We performed several other tests and found that peeling was very effective at removing bacteria (not shown) . Blanching effectively removed all bacteria . We found that spraying vegetables with hydrogen peroxide, followed by spraying with vinegar, and then rinsing with water, did not result in any significant reductions in total colony counts or types of microbes isolated when compared to unwashed fruits and vegetables . Bleach or sodium hypochlorite (which is not approved for general use with foods by the FDA) also reduced the total amounts of bacteria and fungi (not shown) . However, in response to recent known outbreaks of pathogenic E. coli and Salmonella spp ., the EPA has approved the used of calcium hypochlorite as a disinfectant for commercial sprout production .

dangerous microbes such as E. coli and Salmonella . We were surprised to find that there was little difference between the tomatoes produced by conventional vs . organic growers (Table 1) . Organic and conventionally grown carrots harbored several species of bacteria (Table 1), as might be expected since they are exposed to soil as root vegetables . Both types of carrots were the easiest produce to clean, perhaps because they are so amenable to rough washing .

Identification of Bacteria
Most of the bacteria identified in this lab were Pseudomonads, bacteria common to many plants (Tables 1 and 3) . Some species of Pseudomonas are plant pathogens, others are used for biological control of plant diseases caused by bacteria or fungi (Table 3) . Pseudomonas fluorescens is easily identified in the classroom by exposing cultures to long-wavelength ultraviolet light (365 nm) resulting in emission of a greenish-yellow fluorescence . P. syringae is a common plant pathogen causing bacterial speck on tomato . An unidentified yellow-color gram-negative bacterium lacking flagella was consistently found on strawberry, carrots, and tomato (but not on organically grown carrots or tomato) . Bacillus spp . are widely distributed in the environment, so it was not unexpected to find it on produce (Table 1 and 3) . Bacillus cereus, an aerobic, spore-forming, gram-positive rod-shaped bacterium (Table 3) was identified on strawberry, salad mix, and alfalfa sprouts (Table 1) . B. cereus is a commonly isolated environmental microbe and it has been implicated in gastrointestinal illness, accounting for 2% of all reported food-borne illnesses from 1973-1987 (Bean & Griffin, 1990) . B. subtilis is used as a biological control agent to control pathogens that invade plant roots .

Identification of Fungi
Most of the fungi that were isolated are considered cosmopolitan . For example, postharvest fungi often contaminate bread, produce, and dairy products . These include grey mold on strawberries (Botrytis), green mold on oranges (Penicillium), and black mold on bread (Rhizopus) . Rotting molds on peaches were identified to genus as Phoma and tomatoes often had soft black spots caused by infections with the fungus Alternaria (Table 3) .

Organic vs. Conventional Produce

We hypothesized that the organic tomatoes would have much more bacteria and fungi contamination for two reasons: fungicides are not used for organically grown crops, and manure (compost) is used which may contain

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Fungi such as Fusarium, Aspergillus, and Penicillium can produce mycotoxins that are harmful to livestock and humans . Today the U .S . government strictly regulates the amount of toxins in grain that are acceptable for human food or animal feed . Genetically engineered plants also hold promise of reducing the accumulation of toxigenic fungi in grain during cultivation and harvest (Scholthof, 2003) . It is important to emphasize that many fungi (molds) and bacteria are beneficial microbes . For example, yeasts (Saccharomyces) are well known and essential components of the fermentation processes to make bread, beer, and wine . Other beneficial fungi produce antibiotics that have saved millions of lives since the first commercial preparations in the 1940s . This includes the antibiotic penicillin, which was originally produced from fermentation culture of Penicillium spp . A common thickening agent is xanthan gum, a polysaccharide isolated from bacteria (Xanthomanas campestris .) that is used in prepared foods, such as salad dressing . Lactic acid-forming bacteria such as Lactobacillus spp . are used to make yogurt . That microbes are both beneficial and detrimental to crops and man provides a entry into fascinating discussions to reveal the links between agriculture, public health, and the history of mankind (Hudler, 1998; Large, 2003) .

many university faculty are interested in helping teachers and in developing closer ties with local public schools; it is part of their expected service to the community .

Laboratory Safety
This lab exercise was developed after successfully using the Chicken Wing lab (Deutch, 2001) that ended with plating of bacteria, but lacked species identification . Considering that raw poultry often harbors bacteria that are known human pathogens, we felt that it was important to develop an alternative laboratory using fruits and vegetablesfoods that are typically consumed without cooking . It is important to reiterate that the laboratory materials are from the grocery storefruits and vegetables that we consume daily . We thought it would be useful to determine if there would be any health and safety concerns that could be anticipated by identifying bacteria to species . In our experiments we did not identify microbes that would obviate the use of this exercise in our classroom . Of course, as part of our routine lab safety procedures, the students are not allowed to eat or drink in the lab . For this lab they washed their hands with soap and water when they arrived and before leaving the lab . We also autoclaved all cultured microbes before putting them in the trash . The extra produce was considered lab material once the experiments were initiated, and therefore the students were not allowed to eat it or remove it from the classroom . The fungi were kept on covered Petri plates: our primary concern was cross-contamination of samples, but this also prevented the spores from circulating in the classroom . We also found that an additional benefit of this lab is the opportunity it presents for promoting basic food safety habits . This includes routine hand-washing, separating produce and meats when preparing foods, and storing foods at proper temperatures (www . fightbac .org) .

Lessons in Food Safety

There are at least two strategies that can be employed to reduce the level of food-borne illness and postharvest contamination of fruits and vegetables (Table 4) . The first is irradiation, which has been approved by the USDA and can be used for wrapped or prepared foods to kill pathogens, and increase shelf life (Tauxe, 2001) . Irradiation also allows better quality (ripe) produce to be shipped to market as it reduces the potential for mold contamination (as is commonly observed on strawberries), increases shelf life of produce, and reduces waste . Irradiation also allows for transportation of fruits and vegetables from outside the U .S . that may otherwise harbor unwanted pathogens or insects . Irradiation can reduce the use of chemicals, such as those used to control sprouting in potatoes, and also kills insects that are in spices and flour (i .e ., flour beetles) . Beginning with a straightforward lab exercise, it is possible to make more detailed studies of issues intersecting with agricultural production, food safety, and plant-pathogenic microbes . The lab exercise gives the students a realistic perspective of microbes in an environment that they experience daily . This lab also brings up several themes including cultural and social issues, the media and advertising, public health, gardening, pest control, agribusiness, farmers markets, and global food production (Table 4) . It can readily be integrated into a multidisciplinary-systems approach to study microbes in our environment (Scholthof, 2007) . In summary, this lab has been

Outreach & Collaborative Projects

We strongly encourage middle- and high school teachers to contact university faculty in microbiology or plant pathology departments, clinical diagnostic labs, or plant disease clinics, to pursue the fatty-acid analysis . It provides an excellent opportunity to develop fruitful contacts with research scientists who can help students with summer projects or internships . Such interactions naturally lead to longer-term projects including Science Fair, and in providing suggestions for labs that students might find useful and interesting as they become more experienced with basic microbiology techniques . Furthermore,

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useful both from an educators perspective in that it provides a jumping off point for other lectures and reading, as well as demonstrating the fascinating journey that food takes from farm to fork . It also serves to emphasize that not all microbes are bad and in fact are omnipresent in nature, including on the foods that we eat .

Acknowledgments
We appreciate the helpful suggestions and criticism provided by Drs . Herman Scholthof, Jeff Batten, and Julie Hicks . The comments and critiques provided by the students enrolled in BESC 314 Pathogens, the Environment, and Society the past three years, especially during the spring semester of 2004, were greatly appreciated . We also thank Dr . Larry Barnes of the Plant Disease Diagnostic Laboratory at Texas A&M University for fatty acid analysis of the bacteria . Funding for this project was supported by the Program for Scholarly and Creative Activities at Texas A&M University (K .-B .G .S .) .

References
Barnett, H .L . & Hunter, B .B . (1998) . Illustrated Genera of Imperfect Fungi . St . Paul, MN: APS Press . Bean, N .H . & Griffin, P .M . (1990) . Foodborne disease outbreaks in the United States, 1973-1987: Pathogens, vehicles, and trends . Journal of Food Protection, 53, 804-817 . Breuer, T ., Benkel, D .H ., Shapiro, R .L ., Hall, W .N ., Winnett, M .M ., Linn, M .J ., Neimann, J ., Barrett, T .J ., Dietrich, S ., Downes, F .P ., Toney, D .M ., Pearson, J .L ., Rolka, H ., Slutsker, L ., Griffin, P .M . & Investigation-Team . (2001) . A multistate outbreak of Escherichia coli O157:H7 infections linked to alfalfa sprouts grown from contaminated seeds . Emerging Infectious Diseases, 7, 977-982 . Deutch, C .E . (2001) . Microbial contamination of chicken wings: An open-ended laboratory project . The American Biology Teacher, 63, 262-266 . Hudler, G .W . (1998) . Magical Mushrooms, Mischievous Molds . Princeton, NJ: Princeton University Press . Large, E .C . (2003) . The Advance of the Fungi . St . Paul, MN: American Phytopathological Society Press . Larone, D .H . (1995) . Medically Important Fungi . Washington, DC: ASM Press . Lelliott, R .A . & Stead, D .E . (1987) . Methods for the Diagnosis of Bacterial Diseases in Plants . London, UK: Blackwell Scientific Publications . Macilwain, C . (2004) . Organic: Is it the future of farming? Nature, 428, 792-293 . Schaad, N .W ., Jones, J .B . & Chun, W . (Editors) . (2001) . Laboratory Guide for Identification of Plant Pathogenic Bacteria . St . Paul, MN: APS Press . Scholthof, K .-B .G . (2001) . The chimerical world of agricultural biotechnology: Food allergens, labeling, and communication . Phytopathology, 91, 524-526 . Scholthof, K .-B .G . (2003) . One foot in the furrow: Linkages between agriculture, plant pathology, and public health . Annual Review of Public Health, 24, 153-174 . Scholthof, K .-B .G . (2007) . The disease triangle: pathogens, the environment, and society . Nature Reviews Microbiology, 5, 152-156 . Taormina, P .J ., Beuchat, L .R . & Slutsker, L . (1999) . Infections associated with eating seed sprouts: An international concern . Emerging Infectious Diseases, 5, 626-634 . Tauxe, R .V . (2001) . Food safety and irradiation: protecting the public from foodborne infections . Emerging Infectious Diseases, 7, 516521 .

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