A comparison of contact plate and calcium alginate swab techniques for quantitative assessment of bacteriological contamination of environmental surfaces

A contact plate method for enumeration of bacteriological contamination at 9 environmental sites in 64 homes was compared with swab sampling techniques. Contamination levels of 100 or more organisms/21-25 cm2 were demonstrated more frequently using swab methods, but for some sites where low numbers of organisms were present, higher recovery rates were obtained using contact plates. When contamination levels from contact plate and swab techniques were compared according to rank order a good correlation was obtained. Results of this investigation indicate that the contact plate method is satisfactory for differentiation of hygiene levels at environmental sites whilst facilitating handling of large numbers of samples in a field survey.     

A bacteriological investigation of the effectiveness of cleaning and disinfection procedures for toilet hygiene

S cott , E. & B loomfield , S.F. 1985. A bacteriological investigation of the effectiveness of cleaning and disinfection procedures for toilet hygiene, Journal of Applied Bacteriology 59 , 291–297.
The bacterial contamination of hospital and institutional toilets and toilet areas which were cleaned daily was investigated. The effect of daily disinfection with hypochlorite or a quaternary ammonium product, or with a continuous-release hypochlorite disinfectant system, based on the chlorine-releasing agent trichloro-isocyanuric acid, was determined. The continuous release system produced substantial and sustained reduction in contamination of the toilet itself (water, toilet bowl and rim) and some reduction in contamination of sites surrounding the toilet (seat, floor, and air). By contrast, although daily disinfection produced some reduction in contamination compared with daily cleaning, the reductions were less than that associated with the continuous release system and indicated the inadequacy of daily disinfection and/or cleaning for toilets where effective procedures are required.     

Most-Probable-Number Rapid Viability PCR method to detect viable spores of Bacillus anthracis in swab samples

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1 × 10⁴, 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.     

Quick Counting Method for Estimating the Number of Viable Microbes on Food and Food Processing Equipment

A rapid method for estimating the extent of microbial contamination on food and on food processing equipment is described. Microbial cells are rinsed from food or swab samples with sterile diluent and concentrated on the surface of membrane filters. The filters are incubated on a suitable bacteriological medium for 4 hr at 30 C, heated at 105 C for 5 min, and stained. The membranes are then dried at 60 C for 15 min, rendered transparent with immersion oil, and examined microscopically. Data obtained by the rapid method were compared with counts of the same samples determined by the standard plate count method. Over 60 comparisons resulted in a correlation coefficient of 0.906. Because the rapid technique can provide reliable microbiological count information in extremely short times, it can be a most useful tool in the routine evaluation of microbial contamination of food processing facilities and for some foods.     

The hygienic efficiency of conventional and inverted lamb dressing systems

R.G. BELL AND S.C. HATHAWAY. 1996. Aerobic plate counts (APC 37°C and APC 25°C) and Escherichia coli enumerations (Petrifilm) were used to determine sources of bacterial contamination during sheep dressing, determine the hygienic efficacy of hand wash and knife 'sterilization'procedures and compare the hygiene efficiency of conventional and inverted sheep dressing systems. The major slaughterline sources of microbial contamination were: fleece > workers' hands > faecal pellets > knife blades. Aerobic plate counts (APC 37°C) exceeding log 4.4 cfu cm^-2 were considered indicative of direct fleece contact, whereas E. coli numbers exceeding log 3.3 cfu cm^-2 were considered indicative of direct faecal contact. A 44°C water hand rinse removed 90% of the microbial contamination from workers' hands, but rinsed hands, particularly those contacting the fleece, still carried a microbial population exceeding log 4.0 cfu cm^-2. A 44°C rinse followed by an 82°C water dip reduced the contamination on knife blades to less than log 3.0 cfu cm^-2. Inverted dressing systems produced carcasses with a lower contamination level than conventional systems. With both systems little increase in contamination occurred after pelt removal. The areas of highest contamination were the forequarter region with inverted dressing and the hindquarter with conventional dressing. In both cases these regions are the sites where cuts are made through the skin. With both systems contamination around these cuts was entirely consistent with direct fleece contact resulting from 'rollback'.     

Detection of the site of contamination by Staphylococcus aureus within the defeathering machinery of a poultry processing plant

Counts of Staphylococcus aureus from samples of neck skin taken from poultry carcasses at different stages of processing showed that the numbers decreased approximately 10-fold during scalding but increased by almost 1000-fold during plucking, reaching up to 10⁴/g. Swab samples taken from the defeathering machinery yielded counts of ca. 10³/swab at the entry to the first plucker but these increased to ca. 10⁷at the exit, with a subsequent decrease through the second and third machines. Counts of other organisms growing on the Baird-Parker isolation medium showed much lower levels at the exit to the first plucker, suggesting that this was the major site of contamination at which S. aureus grew preferentially. Data obtained from four visits to the processing plant over a period of 10 months suggested that the incidence of S. aureus on the birds is affected by the season (summer or winter) whereas levels in the plucking machines depended on the day of sampling.     

Numerical abundance of myxomycetes (myxogastrids) in soils in the West of England

Abstract A most-probable-number technique was used to quantify the abundance of myxomycetes (myxogastrids) in soil samples taken from 23 'Sites of Special Scientific Interest' and one other site in the West of England. Associated organisms and soil conditions were also recorded. Sixteen of the 24 sites yielded myxomycete plasmodium-forming units (PFUs) in numbers averaging 230 cm⁻³ fresh soil. Where detectable in samples of grassland soil, the numbers ranged from 20–2750 cm⁻³, in woodland soil from 20–800 cm⁻³, and in sand dunes from 80–400 cm⁻³. Repeated sampling revealed changing numbers at single sites. The abundance of PFUs was correlated positively with numbers of soil amoebae, ciliates and nematodes, and with levels of magnesium and soil density, and negatively with organic matter content and levels of NH₄-nitrogen and phosphate. Each PFU probably corresponded to one or several uninucleate cells in the soil rather than to a plasmodium.     

Bacteriological and epidemiological findings during examination of the uterine content of ewes with retention of fetal membranes

We included 92 pairs of ewes with or without retention of fetal membranes in a cohort study of 25 flocks in Southern Greece. We obtained two uterine content samples under aseptic conditions, by introducing a swab into the uterus of these ewes, on the 2nd-4th and the 5th-9th day after lambing. We used conventional bacteriological techniques to isolate and identify bacteria and to carry out antimicrobial agents susceptibility testing. The prevalence of bacterial intrauterine contamination among ewes with retention was 24% on the first and 46% on the second sampling (P < 0.0001) and that among ewes without retention was 8 and 2% (P > 0.05), respectively. Clinical signs accompanying the retention of fetal membranes were more frequently observed among ewes with intrauterine contamination than among those without (P = 0.0007). The odds of an ewe having an intrauterine contamination increased multiplicatively by 1.06 when the median duration of retention in the flock increased by 6 h. The principal bacteria isolated from the ewes with retention were Arcanobacterium pyogenes and Escherichia coli; 21% of 73 isolates tested were found resistant to at least one antimicrobial agent.     

Comparison of multiplex PCR and standard bacteriological methods of detecting Salmonella on chicken skin

A multiplex PCR technique was compared with standard bacteriological techniques for the detection of Salmonella , and Salm. enteritidis in particular, in samples of chicken skin obtained from retail outlets. Five of 68 samples were positive by both systems (7.4%), 58 of 68 were negative by both systems (85%), five were positive by PCR only (7.4%) but none were positive by culture only. The PCR was quicker and more sensitive than the bacteriological methods, finding 15% of samples positive for Salmonella compared to 7.4%, and taking less than 24 h to reach a result as opposed to 2–3 d.     

Microbiological Sampling of Poultry Carcasses

S ummary : The skin contamination of poultry carcasses from 3 different processing plants was found to vary considerably on the 6 different sites sampled. The neck skin and to a less extent the back and near-vent sites were more heavily contaminated than 2 sites on the leg and 1 under the wing. Significantly ( P < 0·001) lower counts were obtained when the organisms present were recovered at 4° than at 15 or 22°. Swabbing of the skin did not give as good recovery as when the skin sample was removed and shaken in a diluent with an abrasive material, but is adequate to indicate contamination where a nondestructive method is essential. When defrosting frozen carcasses, 2 h on the bench gave as good recovery as carcasses defrosted for 4 h.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Microbial Spoilage of Fresh Refrigerated Beef Liver

The types and numbers of micro-organisms involved in the spoilage of refrigerated beef liver were studied together with pH, hydration and organoleptic changes of the material. Fresh liver harboured a mixed population ( c . 1 × 10⁵ organisms/g) of Gram positive cocci, chromogens and non-chromogens, sporeformers, presumptive coliforms and Gram negative rods. When samples were rejected organoleptically, after 7–10 days at 5°, the contamination attained levels of c . 7–8 × 10⁷ organisms/g. Spoilage was due to souring; the pH fell from 6·3 (fresh liver) to c . 5·9. Lactic acid bacteria were predominant and Gram negative bacteria did not exceed 1·0 × 10⁶ organisms/g. This type of spoilage is explained by the carbohydrate content of c . 5% in liver. The value of pH appears to be a reliable indicator of liver freshness, with a pH of 6·1 indicating incipient spoilage.     

A Study of the Breakdown of Organic Phosphates by Micro-organisms from the Root Region of Certain Pasture Grasses

A survey of micro-organisms isolated from the root surface, rhizosphere soil and nonrhizosphere soil of pasture grasses (Ryegrass, S23; Timothy, S50; Cocksfoot, S143) was made to determine their ability to attack phenolphthalein diphosphate, sodium glycerophosphate, sodium phytate, lecithin, ribonucleic and deoxyribonucleic acids. The total numbers of organisms capable of decomposing these compounds were higher on the root surface and in the rhizosphere soil of the grasses when compared with the numbers in nonrhizosphere soil. Occasionally, preferential stimulation of organisms hydrolysing some of the compounds was observed in the root regions. The addition of deoxyribonucleic acid to medium inhibited the numbers of colonies developing in dilution plate counts. This inhibition was generally more pronounced on root surface organisms than on those from nonrhizosphere soil.     

Detection of post-pasteurization contamination of cream by impedimetric methods

Impedimetric methods for evaluating post-pasteurization contamination and shelf-life of cream were assessed. Over 94% of the samples tested were in agreement, using selected cut-offs of 20 h for detection time measured at 21°C with creams containing inhibitors for the growth of Gram positive bacteria on standard plate count agar as growth media, and 3.2 × 10⁷ cfu/g for plate counts obtained on cream which had been pre-incubated in the presence of inhibitors for the growth of Gram positive organisms, and on cream stored at 6°C for 7 d. Agreement between the impedimetric method and plate count was not as good if either Brain Heart Infusion or Milk Agar was used in place of Plate Count Agar in the former technique. A poor correlation was obtained between plate count methods for enumerating post-pasteurization contamination and keeping quality with impedimetric measurements on cream alone. It was possible, with a reasonable degree of certainty, to determine if cream had suffered post-pasteurization contamination within 20 h of production.     

Distribution and sources of microbial contamination on beef carcasses

Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.     

Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.     

Microbiological Evaluation of Protected Environments During Patient Occupancy

Microbiological monitoring has been conducted in two life island (LI) units and two laminar airflow (LAF) rooms while they were occupied by patients undergoing cancer chemotherapy. There were only 5 organisms per 1,000 ft(3) of air sampled in LAF rooms, 31 organisms in LI units, and over 3,000 organisms in regular hospital rooms. None of the floor samples obtained from hospital rooms was sterile, compared to over 70% in LAF rooms. The rate of deposition of organisms onto settling plates was one organism per 4.5 hr in LAF rooms compared to one organism per 0.08 hr in hospital rooms. Potential pathogens were isolated much more frequently from environmental samples obtained from hospital rooms than from LI units or LAF rooms. Two sites of persistent contamination arose in the LAF rooms: the vinyl tile flooring and the water supply system. Over half of the potential pathogens cultured from the protected environment units were cultured initially from the patients who occupied the units.     

Comparison of selective procedures for isolation and enumeration of Legionella species from hot water systems

E. LEONI AND P.P. LEGNANI. 2001 . Various sample pre-treatment techniques and different growth media for the isolation of Legionellae from hot water supplies in public buildings were compared. A total of 102 hot water samples from taps and showers was examined. The highest recovery frequency was obtained with the heat pre-treatment method and using the selective medium GVPC. However, the results differed according to the concentration of legionellas. In the case of low plate counts (≤5000 cfu l⁻¹), the heat pre-treatment technique gave a significantly higher percentage of positive samples compared with other techniques ( P  < 0·05). With increasing concentration, the differences between the procedures decreased until they became statistically not significant for concentrations above 50 000 cfu l⁻¹. The direct inoculum method allowed a significantly higher detection of concentrations ( P  < 0·001) compared with heat and acid decontamination methods, which brought about a 67–68% reduction in detectable Legionellae. Heat decontamination techniques show greater sensitivity and specificity. However, they underestimate the number of legionellas. In environmental surveillance programmes, this underestimate must be taken into consideration when assessing the health risk.     

Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis

Analysis of microbial community structure in complex environmental samples using nucleic acid techniques requires efficient unbiased DNA extraction procedures; however, humic acids and other contaminants complicate the isolation of PCR-amplifiable DNA from compost and other organic-rich samples. In this study, combinations of DNA extraction and purification methods were compared based on DNA yield, humic acid contamination, PCR amplifiability, and microbial community structure assessed by terminal restriction fragment length polymorphisms (TRFLP) of amplified 16S rRNA genes. DNA yield and humic acid contamination, determined by A230, varied significantly between extraction methods. Humic acid contamination of DNA obtained from compost decreased with increasing salt concentration in the lysis buffer. DNA purified by gel permeation chromatography (Sepharose 4B columns) gave satisfactory PCR amplification with universal eubacterial 16S rRNA gene primers only when A260/A280 ratios exceeded 1.5. DNA purified with affinity chromatography (hydroxyapatite columns), and showing A260/A280 ratios as high as 1.8, did not show consistently satisfactory PCR amplification using the same 16S rRNA primers. Almost all DNA samples purified by agarose gel electrophoresis showed satisfactory PCR amplification. Principal components analysis (PCA) of TRFLP patterns differentiated compost types based on the presence/absence of peaks and on the height of the peaks, but differences in TRFLP patterns were not appreciable between extraction methods that yielded relatively pure DNA. High levels of humic acid contamination in extracted DNA resulted in TRFLP patterns that were not consistent and introduced a bias towards lower estimates of diversity.     

Internal quality control samples for water bacteriology

A method is described for the preparation and application of a bacteriological quality control system for use with tests for plate counts and the enumeration of indicator organisms in water. Statistical limits are obtained and control charts prepared which are analagous to those used in analytical chemistry and clinical biochemistry laboratories. This approach has been applied successfully in this laboratory but needs to be tested more widely. Such numerically-based systems have seldom been attempted in microbiology, especially where multiple organisms have been included for the simultaneous control of several tests.