Microcolony epifluorescence microscopy for selective enumeration of injured bacteria in frozen and heat-treated foods

Abstract

A rapid (<6 h) method for selectively enumerating coliforms, pseudomonads, and staphylococci has been developed which involves counting microcolonies grown on the surface of polycarbonate membranes under selective conditions. The method was not directly applicable to foods containing injured bacteria due to the poor formation of or an inability to form microcolonies under selective conditions. However, the introduction of a 3to 5-h resuscitation step in tryptone soya broth allowed the method to give reliable estimates of these organisms in a variety of frozen and heat-processed foods. Under nonselective conditions, i.e., for total counts, the microcolony method enabled a rapid count to be made of viable bacteria in heat-treated foods, but these results were also made more consistent by the introduction of a resuscitation step. This method makes results from these foods available far faster than conventional enumeration methods.

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods

“…These include assay of microbial ATP (Webster et al. 1988), direct counting of single cells or microcolonies by fluorescent microscopy or flow cytometry (Pettipher & Rodrigues 1982;Rodrigues & Kroll 1989;Suhren 1989;Pinder et al 1990), incorporating fluorescent antibodies (Donelly & Baigent 1986 ; Rodrigues & Kroll 1990), enzyme-labelled immunoassays (Beckers et al 1988), DNA or rRNA probes (Fitts et al 1983;Romanuik & Trust 1987; Klinger &Johnson 1988) and the polymerase chain reaction (Steffan & Atlas 1988;Olive 1989) or specific phages containing cloned genes for bio-when applied to foods. When they are used alone, however, these physical methods may have limited application because the filters may be blocked by food particles/ components or organisms partitioned at interfaces during centrifugation.…”

Application of rapid methods and ultrasound imaging in the assessment of the microbiological quality of aseptically packed starch soup

“…g-' sample. A resuscitation step has also been recommended for the DEFT microcolony method in cases where there might be injured cells (Rodrigues & Kroll, 1988. It seems that the pre-incubation step used in the quality control of aseptic packaging corresponds with this resuscitation step (Mattila & Alivehmas, 1987;Ahvenainen et al, 1989c;Manninen et al, 1990), and therefore further incubation of dilutions in, e.g., tryptone soy broth was not used.…”

“…The incubated membranes were remounted in the filter manifold tower unit (Bio-Foss, UK) for staining with acridine orange (Difco, USA). Twenty fields of the mounted membranes were examined and properly formed microcolonies were counted in an Olympus BH-2 BHT epifluorescence microscope (Olympus Optical Co. Ltd, Japan) using a x50 objective and a X 10 ocular (Rodrigues & Kroll, 1989).…”

“…The membrane is then transferred to the agar surface to develop microcolonies. The results of the microcolony technique are obtained towards the end of the working day, depending on how long it takes for the colonies to develop (Rodrigues & Kroll, 1988). The lowest detected contamination level by DEFT is about 103c.f.u.…”

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

“…g-' Kroll 1988, 1989). The addition of a 3-5 h resuscitation step in tryptone soy broth permitted reliable estimates of injured bacteria in frozen and heat-processed foods (Roderigues and Kroll 1989). For this microcolony epifluorescence microscopy (MEILI) method, microcolonies on membranes were stained with acridine orange before examination by epifluorescence microscopy.…”