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

A rapid (less than 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 3- to 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.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of microcolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0.89), pseudomonads (r = 0.93) and staphylococci (r = 0.92) after incubation at 30 degrees C for 3 or 6 h (staphylococci) at contamination levels of above 10(3) bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique

The growth patterns of macrocolonies of 59 different pure cultures were studied on eight selective solid media. A method of growing microcolonies on the surface of polycarbonate membrane filters, placed on the selective agar media, followed by staining and examination by epifluorescent microscopy was developed. The patterns of growth of the pure cultures as microcolonies were studied on the eight selective media. Only four media proved to be reliable for this purpose and the relationship between the microcolony count and plate count was studied on these media together with nutrient agar. Microcolony counts using three of these media (enriched lauryl sulphate aniline blue, pseudomonas selective agar (C-F-C) and Baird-Parker medium) were capable of giving reliable estimates of coliforms (r = 0·89), pseudomonads (r = 0·93) and staphylococci (r = 0·92) after incubation at 30°C for 3 or 6 h (staphylococci) at contamination levels of above 10³ bacteria/g in a variety of foods. The results are available within a working day and should allow the more efficient management of food supplies.     

Microcolony morphology as a criterion in bacterial diagnosis. The differentiation of Pneumococcus from alpha-hemolytic Streptococcus

The authors have developed a method permitting the microscopic study of the morphology of bacteria and their relative position in microcolonies. Thus, the use of this method has made it possible to distinguish Streptococcus pneumoniae from other bacteria by the morphology of their microcolonies. In the study of 75 streptococcal strains, all strains yielding positive results in two or three tests, similarly to all strains pathogenic for mice, formed microcolonies with granular (pneumococcal) morphology, while all strains yielding negative results have been found to form microcolonies with catenulate ("nonpneumococcal") morphology. The authors suggest that the morphology of microcolonies is a more reliable criterion for the identification of S. pneumoniae than other tests used separately.     

Bacteria can form interconnected microcolonies when a self-excreted product reduces their surface motility: evidence from individual-based model simulations

Recent experimental observations of Pseudomonas aeruginosa, a model bacterium in biofilm research, reveal that, under specific growth conditions, bacterial cells form patterns of interconnected microcolonies. In the present work, we use an individual-based model to assess the involvement of bacteria motility and self-produced extracellular substance in the formation of these patterns. In our simulations, the pattern of interconnected microcolonies appears only when bacteria motility is reduced by excreted extracellular macromolecules. Immotile bacteria form isolated microcolonies and constantly motile bacteria form flat biofilms. Based on experimental data and computer simulations, we suggest a mechanism that could be responsible for these interconnected microcolonies.     

A method for estimating viability of aquatic bacteria by slide culture

To estimate the viability of freshwater bacteria, slide cultures were prepared by spreading 10 μl of water, concentrated by centrifugation, over 1 cm² of agar. After drying, a cover-slip was placed on the agar. Following incubation, microcolonies and single cells were counted under a phase contrast microscope, and the viability estimated. Incubation at 10°C on casein-peptone-starch agar for 24 or 72 h provided the highest estimates of viability ( ca 45%). Most microcolonies developed under these conditions and the total number of microcolonies and single cells did not decrease significantly during incubation. Several morphological types of bacteria were observed as both microcolonies and single cells. Most microcolonies consisted of two cells, although some larger ones were present. The method could be used as an alternative to the spread plate and other methods which assess viability from bacterial growth on agar.     

Bacterial microcolony--a possible approach for a rapid differentiation of bacteria

A method is developed for cultivating and observing bacteria in an early phase of their growth, when microcolonies were formed. The morphology of the microcolonies, including form, structure, characteristics of its periphery and center, as well as the mode of arrangement of bacteria in it and to each other proved to be typical for a species and often permitted its differentiation from the other species. Photographs are presented of typical microcolonies of S. aureus and of E. coli. A series of photographs is presented also as an illustration of the possibility to differentiate some species in the genus Streptococcus. The microcolonies observation is made 3 hours after material inoculation, that may permit a rapid bacteriological diagnosis. It is believed also, that the microcolony technique could be useful in the characterisation and identification of the species in the general bacteriology and taxonomy.     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.     

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred.     

Growth of silicone-immobilized bacteria on polycarbonate membrane filters, a technique to study microcolony formation under anaerobic conditions.

A technique was developed to study microcolony formation by silicone-immobilized bacteria on polycarbonate membrane filters under anaerobic conditions. A sudden shift to anaerobiosis was obtained by submerging the filters in medium which was depleted for oxygen by a pure culture of bacteria. The technique was used to demonstrate that preinduction of nitrate reductase under low-oxygen conditions was necessary for nonfermenting, nitrate-respiring bacteria, e.g., Pseudomonas spp., to cope with a sudden lack of oxygen. In contrast, nitrate-respiring, fermenting bacteria, e.g., Bacillus and Escherichia spp., formed microcolonies under anaerobic conditions with or without the presence of nitrate and irrespective of aerobic or anaerobic preculture conditions.     

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred. and accepted 22 June 1989     

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

Role of sublethal injury in decline of bacterial populations in lake water.

Following their addition to lake water, the populations of Escherichia coli and of antibiotic-resistant strains of Pseudomonas fluorescens, Agrobacterium tumefaciens, Micrococcus flavus, Rhizobium meliloti, and Klebsiella pneumoniae declined rapidly, as determined by counting on media containing antibacterial compounds. The estimates of population sizes were occasionally higher if procedures were used that permitted possible resuscitation of injured cells. No resuscitation procedure yielded consistently higher estimates of populations of surviving cells than the use of selective media alone. The patterns of survival of the test bacteria in lake water amended with eucaryotic inhibitors were essentially the same whether a resuscitation procedure was used or not, and the patterns of survival in sterile lake water or buffer were the same whether counts were made on selective media or on media without antibacterial agents. On the basis of the methods used to show sublethal injury caused by stress, we suggest that such injury to the test bacteria is not a significant factor involved in their decline in lake water.     

Role of sublethal injury in decline of bacterial populations in lake water

Following their addition to lake water, the populations of Escherichia coli and of antibiotic-resistant strains of Pseudomonas fluorescens, Agrobacterium tumefaciens, Micrococcus flavus, Rhizobium meliloti, and Klebsiella pneumoniae declined rapidly, as determined by counting on media containing antibacterial compounds. The estimates of population sizes were occasionally higher if procedures were used that permitted possible resuscitation of injured cells. No resuscitation procedure yielded consistently higher estimates of populations of surviving cells than the use of selective media alone. The patterns of survival of the test bacteria in lake water amended with eucaryotic inhibitors were essentially the same whether a resuscitation procedure was used or not, and the patterns of survival in sterile lake water or buffer were the same whether counts were made on selective media or on media without antibacterial agents. On the basis of the methods used to show sublethal injury caused by stress, we suggest that such injury to the test bacteria is not a significant factor involved in their decline in lake water.     

Evaluation of a cup scrub technique for quantification of the microbial flora on bovine skin

A cup-scrub technique devised for sampling the human skin surface microflora was evaluated in cattle. Scrub samples from bovine skin contained clumps of squama and bacterial microcolonies which were progressively broken down by shaking. This was accelerated in the presence of ballotini beads but aggregations of bacteria were still present after prolonged agitation. Vigorous shaking, particularly with beads, decreased the viability of the bacteria and optimum viable counts were obtained after manual shaking for half a minute. Immersion in buffered detergent, wash and diluting fluids for up to 2 h promoted release of bacteria from microcolonies but decreased the viability of aerobic and anaerobic pleomorphic rods and a Bacillus strain. There was no significant effect on strains of Micrococcaceae. Prolonged exposure of bacteria from scrub samples to these fluids can thus lead to both quantitative and qualitative alterations in the counts obtained, although these effects may be masked by the continuing release of bacteria from microcolonies. The cup-scrub technique provides a convenient means of quantifying changes in the bovine skin microflora but results obtained from different studies should only be compared if closely similar techniques are used.     

RAPID DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

The aim of this study was to determine the prevalence of Listeria monocytogenes in packaged fresh ground turkey in Turkey using immunomagnetic separation (IMS) as a selective enrichment step in method and polymerase chain reaction (PCR). A total of 180 ground turkey samples were collected during a 1-year period. Thirty-two (17.7%) of the samples contained L. monocytogenes, 24 (13.3%) contained Listeria innocua, 7 (3.8%) had Listeria ivanovii and 5 (2.7%) had Listeria seeligeri by means of IMS-based cultivation method. A PCR assay was performed, based on hlyA gene-specific primers. In all L. monocytogenes isolates, hlyA gene was confirmed, indicating that the correlation between IMS-based cultivation and PCR methods was 100%. The results suggest that the prevalence of L. monocytogenes in ground turkey is relatively high in Turkey and that ground turkey should be produced under appropriate hygienic and technological conditions for the prevention of public health hazards.

PRACTICAL APPLICATIONS

Using fast and reliable methods to detect and identify foodborne pathogenic bacteria, including Listeria monocytogenes , is important to detect the risk of contaminated product and protect public health. In some ways it is time-consuming to isolate and identify the pathogenic microorganisms from food products using conventional techniques. Different methods or techniques can be used both for redounding the isolation chance and to gain time for this purpose. Immunomagnetic separation (IMS) and polymerase chain reaction (PCR) techniques are effective and rapid methods for separation, detection and confirmation of Listeria spp. from foods. In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates.     

Characterization of monospecies biofilm formation by Helicobacter pylori

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.     

多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously “unculturable” organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.     

FT-IR microspectroscopy for early identification of some clinically relevant pathogens

AIMS: To investigate the potentials and limitations of Fourier transform-infrared (FT-IR) microspectroscopy as a tool to identify, at the level of microcolonies, pathogenic bacteria frequently isolated in the clinical environment. METHODS AND RESULTS: A total of 1570 FT-IR spectra from 164 gram-positive and gram-negative bacteria isolated from patients were recorded from 6 to 10-h old microcolonies of 50-150 microm size. A classification of 100% was obtained for the most frequent gram-positive bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Enterococcus faecium at the species level. An average accuracy of about 80% was reached with Gram negative bacteria from the Enterobacteriaceae and Pseudomonaceae families; Enterobacter aerogenes, Enterobacter cloacae, Klebsiella spp., and Citrobacter koseri; and Proteus mirabilis and Escherichia coli. Results were comparable with FT-IR measurements on dried suspensions from 18-h cultures. CONCLUSIONS: Early identification of young microcolonies is feasible with FT-IR microscopy with a very high accuracy for gram-positive bacteria. Some improvement in the transfer of microcolonies is necessary to increase the accuracy for gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Combination of FT-IR microscopy and multivariate data analysis could be a complementary, rapid, and reliable tool for screening and discriminating, at species and subspecies level, micro-organisms of clinical, food-borne, or environmental origins.