R Kuddus, J Oakes, C Sharp, J Scott, K Slater, J Kirsi, O Kopp, W Burt
complementary and alternative medicine cam, ginkgo tea, microbial contamination
R Kuddus, J Oakes, C Sharp, J Scott, K Slater, J Kirsi, O Kopp, W Burt. Isolation of medically important fungi from Ginkgo biloba leaves and crude ginkgo supplements. The Internet Journal of Microbiology. 2007 Volume 5 Number 2.
We observed fungal growth in
Materials and methods
Tissue Staining for Detection and isolation of fungi
Affected leaves, tea flakes and healthy leaves (as control) were cleared of superficial microbes by boiling in 2.5% KOH. The treated leaves were stained with trypan blue and counter-stained with Sudan IV to detect intercellular fungi as described previously (Barrow and Aaltonen, 2004). The treated samples were examined using a Labomed CXR3 microscope at 400-1,000x magnification and photographed. At least 20 fields were examined for each leaf sample.
To isolate fungi, leaves or tea flakes were initially cleaned using sodium hypochlorite, distilled water and ethyl alcohol as described by Tuite (1969). Tissue explants were harvested aseptically under a dissection microscope and then placed on potato dextrose agar (PDA) plates. Fungi in brewed loose Ginkgo tea were examined as described previously (Halt, 1998; Wilson et al., 2004). Pre-packed tea bags were brewed in hot (rolling-boiled) distilled water in sterilized cups following standard brewing methods (Rombauer et al., 2006). Tea was steeped for 2-3 minutes and the brew was serially diluted with sterile water and 0.1 ml was plated in two PDA plates. Similarly treated sterile distilled or tap water was plated as the controls. The plates were incubated at room temperature for 4-10 days. All experiments were done at least twice. Fungal (and bacterial) colonies were counted as colony forming units (cfu)/gm of tea using the formula- (number of colonies multiplied by dilution factor multiplied by total volume of water used in brewing)/grams of tea used.
Identification of Fungi
Fungal hyphae were transferred to new plates to establish pure culture and then identified to the level of genera using conventional methods (data not shown).
Several adult trees (about 20 meters in height) in Pittsburgh were found with drying leaves in June and July (midway of the growth season). The affected female trees had green seeds yet the plants were attempting to sprout new buds (Fig. 1A). Normally
We observed the presence of brown spots in many healthy adult green leaves of every
To test if microbes are present in the brewed Ginkgo tea, cold- and hot-brewed tea samples were plated in PDA plates. Fungi grew out from cold-brewed tea derived from one of the three flake tea samples and the fungal load was estimated to 8x103 fungal cfu/gm of tea. All water controls for cold brewing experiments were negative for fungal growth. The sporulating fungi were identified as
We observed that brown spots are common in adult healthy Ginkgo leaves particularly during the later half (August-September) of the growing season. Brown spots were also common in Ginkgo tea flakes sold in health food stores. We isolated several species of fungi from aseptically explanted tissues of such brown spots and cold-brewed Ginkgo tea derived from one of the three flake tea brands tested. Cold-brewed teas from two other samples were negative for fungal growth although staining showed presence of fungus in both of tea flake samples. No fungi grew from hot-brewed tea. This data indicated that the colonizing fungi are not readily released by the standard brewing conditions.
We also noticed that bacteria (and occasionally yeast) grew from both cold and hot-brewed tea derived from all three flake Ginkgo tea samples and two of the three bagged ginkgo tea samples with bacterial count ranging 0.1x103-1.1x106 bacterial cfu/gm of tea (data not shown). No bacterial growth was observed in cold or hot brewed tea derived from the powdered loose Ginkgo tea and one of the three brands of bagged ginkgo tea (data not shown). Wilson et al. (2004) reported the presence of bacteria at 1x101-1.6x106 cfu/100 ml of herbal tea brewed for 5 minutes at 90°C. Bacteria we isolated were mostly spore formers (
All of the fungal species we isolated from live
This work supported in part by UVU Presidential and Foundation grants to RHK. The authors are grateful to Drs. S. Rushforth, R. Van Buren, M. Bracken, and R. Robbins for helpful discussions and Dr. B. Bargeron for help with photography.