Detection of Escherichia coli in Foods: Indole Staining Methods for Cellulosic and Polysulfone Membrane Filters

Abstract

Optimized procedures for staining Escherichia coli colonies on cellulosic and polysulfone membrane filters are described. An explanation for the behavior of the Ehrlich reaction on membrane filters is suggested.A recent ICMSF (Intemational Commission on the Microbiological Specifications for Foods) study (4) recommends use of the direct membrane filter (MF) plating method (2) of Anderson and Baird-Parker (ABP) for enumeration of Escherichia coli in foods. The technique calls for incubating MF at 44.5°C for 24 h on a tryptophan-rich medium. Indole in colonies is detected by its reaction with 4-dimethylaminobenzaldehyde (DAB) in acid to produce a red color. The stain produces diffuse red zones instead of sharply defined colonies; at the same time, a general browning of indole-negative colonies weakens the differentiation. Because of this, an upper counting limit of 150 colonies on 85-mm membranes was recommended in the preliminary protocol to the ICMSF study (4).We recently described a promising modification to this method, using hydrophobic gridmembrane filters (HGMF) (6). However, two problems arose when the new technique was used in other laboratories: (i) on cellulosic MF (Oxoid or Millipore), the above-mentioned problem caused difficulties in scoring HGMF which were densely packed with mixed flora; and (ii) on polysulfone MF (Gelman Tuffryn), indolepositive colonies did not stain.It quickly became evident that the accumulation and retention of indole were very different for cellulosic and polysulfone MF and that it would be necessary to develop staining procedures specific for each material. After preliminary experiments with a number of the complex color reactions of indole (7), we concluded that the acidic DAB (Ehrlich) reaction remained the best basis for demonstrating indole. MATERIALS AND METILODSHGMF. Cellulose ester HGMF (47-mm diameter, 0.45-pm pore size, 1,984 grid cells) were printed in our laboratory from HAWP 04700 MF (Millipore Ltd., Mississauga, Ontario, Canada), using a cross-country ski wax as the hydrophobic material (6). Polysulfone HGMF (60 by 60 mm, 0.45-pm pore size, 2,500 grid cells) were purchased from QA Laboratories Ltd. (Etobicoke, Ontario, Canada). The base material for these was Tuffryn HT-450 (Gelman Instrument Co., Montreal, Canada).Indole staining procedures. The following two staining procedures were used, together with the ABP staining method (2). For method A, cellulosic HGMF were placed on filter paper wetted with 0.5% DAB in 1 N HCl and then (still on the filter paper) irradiated under an ultraviolet (UV) lamp (254 nm, 10,000 !W/ cm2 at 6 cm) for 30 min. For method B, polysulfone HGMF were placed on a filter paper wetted with a reagent prepared from equal volumes of 2.5% DAB in 1 N HCI-90% ethanol (solution 1) and 1% potassium persulfate (solution 2) mixed just before use. Still on the filter paper, they were heated under an infrared lamp or in an 80°C oven for 5 min. After color development by both methods, HGMF were returned to the agar surface for counting.Microbial count...

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