Methodology for recognition of invasive potential of Escherichia coli.
Surveillance for dysentery-related invasive potential in bacteria using the Sereny keratoconjunctivitis test is restricted by expense, time factor, and necessity for confirmation. Primary screening of isolates in a standardized mammalian cell culture system is recommended. Bacteria are grown 20 hr in veal infusion, washed, and resuspended in 20% heat-inactivated fetal bovine serum (FBS) supplemented with 0.12% brain heart infusion and 0.1% bile salts. The HeLa culture is grown 20 hr as a monolayer in chamber slides with 90% minimal essential medium (MEM)-10% FBS. The host culture is infected at a ratio of 10 bacteria/mammalian cell for 3 hr at 35 degrees C. The infection medium is replaced with MEM-FBS supplemented with 300 microng lysozyme and 5 microng gentamycin/ml. The infected monolayer is incubated 5 hr at 35 degrees C to permit intracellular multiplication. Specimens are washed, fixed with methanol, and stained successively with May-Grunwald and Giemsa dyes. Bacteria occur within the cytoplasm if invasion has occurred. The criterion for a positive test is that 1% of the host cells possesses at least 5 bacteria in 2 of 3 trials. Invasiveness is correlated with and possibly preconditioned by cytotoxic principle(s). Infectivity rates vary from 0 to 30%. The cytopathic effect is noted in 5-50% of HeLa cells. Positive results must be confirmed by the Sereny test.
D001681:Biological Assay; D003231:Conjunctivitis / Q000382:microbiology; D003470:Culture Media; D004403:Dysentery / Q000382:microbiology; D004926:Escherichia coli / Q000302:isolation & purification / Q000472:pathogenicity*; D005516:Food Microbiology / Q000379:methods; D006367:HeLa Cells
D003470:Culture Media