A handy method to remove bacterial contamination from fungal cultures

Autoři: Xiao-Xiao Shi aff001;  Hai-Ping Qiu aff001;  Jiao-yu Wang aff001;  Zhen Zhang aff001;  Yan-Li Wang aff001;  Guo-Chang Sun aff001
Působiště autorů: State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Protection Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China aff001;  College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua, China aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224635


Contamination control and removal are very important technical aspects of microbiological research. Bacterial contamination is very common in fungal cultures. Currently, the commonly used approach for inhibiting bacteria is antibiotic treatment; however, there are drawbacks to using antibiotics, including incomplete removal, limited antibacterial spectra, tendency toward recontamination, effects to fungal strains, and potential risks to the environment. Therefore, in the present work, we developed a new method for bacterial removal from fungi cultured on solid medium, the Cabin-Sequestering (CS) method, based on the different culture characteristics between fungi and bacteria. First, 3–5 mm round or square holes (the “cabin”) are excavated on a solid medium plate. The fungal strain containing possible bacterial contamination is inoculated into the cabin. The cabin is then covered with a sterilized coverslip, followed by incubation at the appropriate temperature. After 7–10 days of culturing, fungal hyphae grow out along the edge of the coverslip; however, the contaminating bacteria cannot pass through the space formed between the medium and the coverslip and, thus, remain in the cabin. The newly grown fungal hyphae around the coverslip are re-inoculated into fresh culture plates, where they form bacteria-free fungal colonies. The CS method is easy handling, with a short experimental cycle and rare recontamination. When necessary, it can also be used in combination with antibiotics in bacterial removal operations.

Klíčová slova:

Agrobacterium tumefaciens – Animal sociality – Antibiotics – Bacteria – Fungal pathogens – Fungi – Plant fungal pathogens – Polymerase chain reaction


1. Zhou MY, Yong Q, Xu L. Cell culture aseptic technique and effective measures to control pollution. Exp Lab Med. 2016; 34(4): 477–479.

2. Reed BM, Buckley PM., Dewilde TN. Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Dev-Pl. 1995; 31: 53–57.

3. Kritzinger EM, Vuuren RJ, Woodward B, Rong IH, Spreeth MH, Slabbert MM. Elimination of external and internal contaminants inrhizomes of Zantedeschia aethiopica with commercial fungicides and antibiotics. Plant Cell. 1998; 52(2): 61–65.

4. Yang X, Xia TQ, Liu CL, Hang HP, Xing WH, Zhang LQ, et al. Analysis and Control of Pollution Problem in Process of Wheat Anther Culture. Hortic Seed. 2013; 06:1–3+29.

5. Zhang XM, Jin SJ, Xiang JH. Sea squirt blood cell culture and bacterial contamination control. Mar Sci. 2009; 11: 26–33.

6. Leifert C, Camotta H, Waites WM. Effect of combinations of antibiotics on micropropagated Clematics, Delphinium, Hosta, Iris and Photinia. Plant Cell. 1992; 29: 153–160.

7. Deng XM, Xi RC, Fu SG, Cai ZG. Advances in research on pollution phenomena in plant tissue culture. Jiangxi Forestry Sci Tech. 2004; 6: 33–36.

8. Wang LB, Li XY. Application of antibiotics to control pollution in plant tissue culture. Liaoning Agric Sci. 2007; 3: 69–70.

9. Cai X H, Li J, Zhang XS, Zhang HY, Wang XY. Control of pollution in plant tissue culture. Chin J Trop Agric. 2003; 23(6): 40–43.

10. Xiao YL. The techniques of sugar-free micropropagation production. 1st ed. Kunming: Yunnan Science and Technology Press; 2003.

11. Davis RH, de Serres FJ. Genetic and microbiological research techniques for Neurospora crassa. Method Enzymol. 1970; 17A: 79–143.

12. Robson GD, Bell SD, Kuhn PJ, Trinci AP. Glucose and penicillin concentrations in agar medium below fungal colonies. J Gen Microbiol. 1987; 133(2): 361–7. doi: 10.1099/00221287-133-2-361 3116159

13. Talbot NJ, Ebbole DJ, Hamer JE. Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell. 1993; 5(11): 1575–90. doi: 10.1105/tpc.5.11.1575 8312740

14. Machacek JE. A simple method of obtaining Pythium cultures free from bacteria. Phytopathology. 1934; 24: 301–303.

15. Ko SS, Kunimoto RK, Ko WH. A simple technique for purifying fungal cultures contaminated with bacteria and mites. J Phytopathol. 2001; 149: 509–510.

16. Cother NJ, Priest MJ. A simple and effective method for the elimination of bacteria from fungal cultures. Australas Plant Path. 2009; 38:132–134.

17. Ryan MJ, Smith D. Fungal genetic resource centers and the genomic challenge. Mycol Res. 2004; 108(12): 1351–1362.

18. Mora C, Tittensor DP, Adl S, Simpson AGB, Worm B. How many species are there on Earth and in the ocean. PLOS Biol. 2011; 9: e1001127. doi: 10.1371/journal.pbio.1001127 21886479

19. Jones MDM, Forn I, Gadelha C, Gadelha C, Egan MJ, Bass D, et al. Discovery of novel intermediate forms redefines the fungal tree of life. Nature. 2011; 474: 200–203. doi: 10.1038/nature09984 21562490

20. Tang XQ, Fan MZ, Li ZZ. Study on the colony phenomenon and environmental factors in the subculture of Beauveria bassiana. Acta Mycol Sin. 1996; 15(3): 188–243.

Článek vyšel v časopise


2019 Číslo 11