Vacuum Probe: New Approach to the Microbiological Sampling of Surfaces

The need for a device to sample large areas that are lightly contaminated with microorganisms motivated the development of the vacuum probe. The intended use of the instrument is to sample clean surfaces in laminar flow clean rooms, but the device could be used for sampling surfaces in other clean environments. Such a device was designed, fabricated, and tested at Sandia Laboratories, Albuquerque, N.M. In these tests, the vacuum probe removed a mean of 89% and assayed a mean of 67% of bacterial spores, approximately 1 mum in length, settled on smooth surfaces which were free from viscous films.     

Effectiveness of Rice Agricultural Waste,Microbes and Wetland Plants in the Removal of Reactive Black-5 Azo Dye in Microcosm Constructed Wetlands

Azo dyes are commonly generated as effluent pollutants by dye using industries, causing contamination of surface and ground water. Various strategies are employed to treat such wastewater; however, a multi-faceted treatment strategy could be more effective for complete removal of azo dyes from industrial effluent than any single treatment. In the present study, rice husk material was used as a substratum in two constructed wetlands (CWs) and augmented with microorganisms in the presence of wetland plants to effectively treat dye-polluted water. To evaluate the efficiency of each process the study was divided into three levels, i.e., adsorption of dye onto the substratum, phytoremediation within the CW and then bioremediation along with the previous two processes in the augmented CW. The adsorption process was helpful in removing 50% dye in presence of rice husk while 80% in presence of rice husk biocahr. Augmentation of microorganisms in CW systems has improved dye removal efficiency to 90%. Similarly presence of microorganisms enhanced removal of total nitrogen (68% 0 and Total phosphorus (75%). A significant improvement in plant growth was also observed by measuring plant height, number of leaves and leave area. These findings suggest the use of agricultural waste as part of a CW substratum can provide enhanced removal of textile dyes.     

Freeze-fracture autoradiography. Progress towards a routine technique

Freeze-fracture autoradiography was introduced in 1976 as a new technique for the autoradiography of diffusible compounds at the electron microscope level. With the original approach coating of the frozen replicated specimens was performed in a cryostat at atmospheric pressure. Ice contamination of the specimen surface acting as an outstanding source of artifacts was thereby not excluded. With the use of a specially designed coating device and volatile spreading substances it was made possible to coat the frozen replicated specimens in the maintained vacuum of the freeze-fracture plant. In this complicated technique we have recently extended the freeze-fracture autoradiography to labeled frozen-dried "half" membranes of red blood cells.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

First-trimester diagnosis on chorionic villi obtained by direct vision technique

Summary An improved technique for direct vision chrionic biopsy that gives a clear view of the amniotic sac was developed. With this technique, used in 48 women prior to vacuum aspiration and in six cases for diagnosis (karyotyping or enzyme analysis), it was possible to obtain chorionic villi free from contamination by maternal tissue. It was also possible to pick out villi (rich in blood vessels and with abundant buds on their surface) found to be most capable of growing in vitro. In the diagnostic cases, the pregnancies have continued uneventfully since the sampling; one pregnancy is now in the 32nd week.     

Biological Evaluation of Carpeting

Studies of microbial contamination were carried out on a carpet strip (acrilan) installed in a laboratory corridor and on two carpet strips (acrilan and wool) installed in two rooms in a pediatric hospital. A sampling procedure of randomly removing 8-mm plugs from the carpets was used for subsequent enumeration and identification of contaminating microorganisms. Microbial counts increased with time, reached a "plateau" at about 4 weeks, and appeared to be related to the amount of activity present in the carpeted area. Vacuuming of carpets showed only a slight reduction in the number of recoverable microorganisms. Qualitative studies on a strip of acrilan carpet installed in a hospital room indicated that Staphylococcus epidermidis and gram-positive nonsporeforming rods were the predominating microorganisms. Coagulase-positive S. aureus organisms were isolated during every sampling period, and gram-negative rods were also regularly isolated. Most nitrate-reducing gram-negative rods belonged to the Enterobacter and Escherichia groups. Survival studies conducted on carpet strips after hospital installation showed relatively constant levels of contamination for about 6 weeks, followed by a gradual reduction in numbers; coagulase-positive S. aureus were found for 35 days, whereas the proportions of most organisms remained relatively constant with time.     

Soil contamination with Toxocara sp. eggs in squares and public places from the city of La Plata. Buenos Aires, Argentina

This study consisted of a stratified sampling, randomly taken, of the soil from the squares and parks of the city of La Plata, Province of Buenos Aires, in order to establish the prevalence of contamination caused by Toxocara sp. A total 242 soil samples was examined. From each sample a 10 grams aliquot was taken, washed in a 0.2% Tween 80 solution, and processed using the technique of concentration by flotation with sugar solution. There was a prevalence of 13.2%. In each positive sample, the quantity of eggs varied from 1 to 4. Toxocara sp. eggs were observed in 15 out of 22 squares and parks investigated. The sampling design and the processing method employed were satisfactory for the recovering and identification of Toxocara sp. eggs.     

NO(x) contamination in laboratory ware and effect of countermeasures.

Contamination of various types of laboratory wares with NO(x) (NO(-)(2) and NO(-)(3)) was assessed systematically and the effect of extensive washing as a countermeasure was evaluated. Mean NO(x) contamination arising from a model procedure for NO(x) determination in plasma was 0.93 microM (range, 0.35-1.49 microM). The major source of contamination included conical tubes (54.8%) and pipette tips used for transfer of solution (12.3-16.3%). Except for soft glassware, most NO(x) contamination could be washed out by pure water. Although NO(x) contamination in respective laboratory wares could be reduced below detection levels by extensive washing, summation of the contamination through the model procedure could not be completely abolished (but the effect of washing persisted at least 10 days). Heavy contamination was noted in glassware (especially soft glass) and ultrafiltration units, which was difficult to remove. Several types of vacuum blood sampling tubes contained various levels of NO(x). Our results indicated that a small but significant amount of contamination remained in laboratory ware even after extensive washing, and that it is advisable to avoid the use of glassware (soft glass), ultrafiltration units, and vacuum blood sampling tubes during the processing of clinical sampling for the measurement of NO(x).     

Use of ultrasonic energy in assessing microbial contamination on surfaces

Ultrasonic tanks were evaluated for their ability to remove viable microorganisms from various surfaces for subsequent enumeration. Test surfaces were polished stainless steel, smooth glass, frosted glass, and electronic components. The position of contaminated surfaces in relation to the ultrasonic energy source, distance of the ultrasonic source from the test surfaces, and temperature of the rinse fluid were some of the factors which influenced recovery. Experimental systems included both naturally occurring microbial contamination and artificial contamination with spores of Bacillus subtilis var. niger. The results showed that ultrasonic energy was more reliable and efficient than mechanical agitation for recovering surface contaminants. Conditions which increased the number and percentage of microorganisms recovered by ultrasonic energy were: using a cold rinse fluid, placing the sample bottle on the bottom of the ultrasonic tank, and facing the contaminated surfaces toward the energy source. It was also demonstrated that ultrasonic energy could be effectively used for eluting microorganisms from cotton swabs.     

Maximizing collection and minimizing risk: does vacuum suction sampling increase the likelihood for misinterpretation of food web connections?

Molecular tools that characterize the structure of complex food webs and identify trophic connectedness in the field have become widely adopted in recent years. However, characterizing the intensity of predator-prey interactions can be prone to error. Maximizing collection success of small, fast-moving predators with vacuum suction samplers has the potential to increase the likelihood of prey DNA detection either through surface-level contamination with damaged prey or direct consumption within the sampling device. In this study, we used PCR to test the hypothesis that vacuum suction sampling will not cause an erroneous increase in the detection of 'predation', thereby incorrectly assigning trophic linkages when evaluating food web structure. We utilized general (1) Aphidoidea and (2) Collembola primers to measure the predation rates of Glenognatha foxi (Araneae: Tetragnathidae) on these prey collected by hand versus those sampled with a vacuum suction device. With both primer pairs, there was no significant increase in predators screening positive for prey DNA when sampled by vacuum suction versus those predators collected, in parallel, by hand. These results clearly validate the application of vacuum suction sampling during molecular gut-content analysis of predator-prey feeding linkages in the field. Furthermore, we found no evidence that predation was occurring inside the suction sampler because specimens collected were never observed to be feeding nor did they screen positive at greater frequencies than hand-collected individuals. Therefore, it can be concluded that the use of vacuum suction sampling devices (in this case a Modified CDC Backpack Aspirator Model 1412) is suitable for molecular gut-content analysis.     

Freeze-fracture autoradiography: The in-vacuo coating technique

Summary Freeze-fracture autoradiography (FFA) was introduced in 1976 as a new method for electron microscopic autoradiography of diffusible compounds (Fisher and Branton, Rix et al.). With the original technique, the film monolayer was applied to the cold specimen in a cryostat at atmospheric pressure. Coating under these conditions did not exclude the risk of artifacts, mainly due to uncontrolled ice contamination of the cold specimen surface.A new method has been developed for coating the frozen specimen, immediately after replication, in the maintained vacuum of the freeze-fracture unit. Two main components of the new technique are described in detail, a specially designed coating device, and the use of spreading substances promoting adhesion of the film in vacuo. Using this technique artifacts so far inherent in the FFA method can be eliminated.Supported by the Deutsche Forschungsgemeinschaft     

Studies of microbiological contamination of ultrasonic apparatus used in pediatric aerosol therapy

It was found that inflating tube is most rarely contaminated with microorganisms during the use of ultrasonic inhalator TUR USI 70. Glass cylinder is contaminated more frequently whereas a diaphragm, aerosol preparation, inhaling mask and a pipe joining it with the device are contaminated most frequently. Sporadic contamination of the inflating tube indicate an efficient work of air filters while frequent contamination of the diaphragm, aerosol preparation and glass cylinder prove that the contamination is caused by a coupling fluid. It was also found that ultrasound exerts a destructive effect on microorganisms in the aerosol preparation. The investigations have shown that the inhaling mask and tubes joining it with the device should be changed before each use while the other parts of an inhalator and aerosol preparation may be changed once per 15 inhalations. It was also noted that disinfection of different parts of the device by a 2% aqueous glutaric aldehyde (30 minutes at room temperature) is efficient in about 95%.     

Proposal for an integrated approach to microbial environmental monitoring in cultural heritage: experience at the Correggio exhibition in Parma

Microbial environmental monitoring represents one of the most useful methods to assess potential risks related to the integrity of cultural heritage and people’s health. The monitoring plan described in the present work is based on standardized techniques for measuring microbial air and surface contamination. Air contamination is assessed through both active and passive samplings, measuring the concentration of microbes in air (in colony forming units per cubic metre, CFU/m³) and the rate at which microorganisms settle on surfaces (expressed by the Index of Microbial Air Contamination, IMA, CFU/dm²/h). For surface contamination, two parameters are measured using nitrocellulose membranes: the Microbial Buildup (MB, the total number of microorganisms accumulated on a surface in an unknown period of time prior to the sampling) and the Hourly Microbial Fallout (HMF, the number of microorganisms that settle on a specific surface during 1 h). The monitoring plan was implemented at the Pilotta Palace in Parma, Italy, during the Correggio exhibition in 2009. Samplings were taken before and during opening times. Some microbial contamination was already detected before the arrival of visitors: air contamination mean values of 99.1 CFU/m³ and 5.2 CFU/dm²/h were recorded, while MB and HMF mean values for surfaces were 92 and 7 CFU/dm², respectively. A significant increase was recorded in air contamination during opening times, with mean values of 323.7 CFU/m³ and 19.4 CFU/dm²/h; surface contamination values increased as well. This monitoring plan represents a contribution towards the definition of a much needed standardized methodology.     

A novel nonchemical method for quarantine treatment of fruits: California red scale on citrus

A process for removing or killing California red scale, Aonidiella aurantii (Maskell), from citrus fruit as a postharvest treatment was evaluated. The process subjects the fruit to vacuum, steam, and vacuum that physically removes red scale from the fruit and kills those scales that are not removed from the fruit. Different numbers of cycles and steam temperatures were compared for efficacy in removing scale from lemons or killing those that remained. Multiple (two to three) cycles removed up to 96% of first molt scales on the fruit, but they were much less effective in removing other stages, especially those that had advanced beyond the second instar. However, it was extremely effective in killing the scales remaining on the fruit. Although this process does not eliminate cosmetic damage caused by scale presence, it might be used in combination with high-pressure washers currently used in packing houses to allow importers and exporters to meet the most stringent quarantine requirements. Because of its killing power, this technique should be tried on other insects and commodities to see whether it can be substituted for certain uses of methyl bromide.     

Exogenous bacterial contamination of donor blood.

The Canadian Red Cross blood transfusion service has followed a set protocol for phlebotomy and collection of a unit of blood. Recent requirements for automated testing have necessitated that a second tube of blood be obtained from the blood line following collection of the unit. Evaluation of the techniques used, however, has indicated the possibility of bacterial contamination from the skin of donors, from insertion of the needle through an unsterile rubber stopper, and through backflow from a nonsterile vacuum tube. To test these possibilities swabs were taken from skin and stoppers of vacuum tubes. Further, vacuum tubes were deliberately contaminated with Escherichia coli. The normal sampling procedure, which involves stripping the donor line to refill and mix the blood, was then followed. This resulted in contamination of the segments and even the blood bag. These findings led to modification of the standard bleeding technique, whereby stripping was eliminated and sterile vacuum tubes were to be used at all times.     

Techniques for Contamination Assessment During Drilling for Terrestrial Subsurface Sediments

Details about the procedures for drilling a ca. 150 m long drill core in a terrestrial setting under contamination controlled conditions are presented. Different to previous studies we only used commercially available drilling equipment to reduce the cost of operation significantly. The goals were (1) to minimize, (2) to monitor and, if possible, to quantify the contamination of the recovered sediments, and (3) to identify the different sources of contamination. Both the potential contamination of the sample material by surface microorganisms and non-indigenous material was assessed. To estimate the infiltration of drill mud into the core, fluorescent microspheres, having about half the size as microorganisms, were added to the mud. The drilling technique used was mud rotary drilling. With the exception of the very beginning of the drilling operations, the drill mud was devoid of any allochthonous hydrocarbons potentially derived from the drilling equipment or drill additives, and its biomarker composition reflected the varying organo-facies that were penetrated. Due to the lack of allochthonous hydrocarbons in the drill mud, its infiltration into the sediment cannot be traced by organic geochemical biomarker analysis. Microspheres proved to be a sensitive tool for the assessment of infiltration of drill mud into the core. The concentration of microspheres in the drill mud decreased continuously during the drilling, most probably caused by seepage of mud through leaks and attachment of spheres to the surface scum in the mud pit. Microscopic enumeration of the microspheres showed great variability in the depth of penetration of mud into the core, apparently unaffected of lithology. The sampling of the core material in the laboratory was carried out inside an anaerobic chamber. Several techniques for subsampling were used, according to sediment properties. The overall results indicate that, if strict contamination control protocols are employed, it is possible to recover uncontaminated samples at reasonable cost with commercially available drilling equipment.     

Microbial biomass and activity distribution in an anoxic, hypersaline basin

The evaluation of an improved wipe-rinse technique for the bioassay of large areas was undertaken due to inherent inadequacies in the cotton swab-rinse technique to which assay of spacecraft is currently restricted. Four types of contamination control cloths were initially tested. A polyester-bonded cloth (PBC) was selected for further evaluation because of its superior efficiency and handling characteristics. Results from comparative tests with PBC and cotton swabs on simulated spacecraft surfaces indicated a significantly higher recovery efficiency for the PBC than for the cotton (90.4 versus 75.2%). Of the sampling areas sites studied, PBC was found to be most effective on surface areas not exceeding 0.74 m2 (8.0 feet 2).     

Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms.

The ability of different aerobic groundwater microorganisms to cometabolically degrade trichloroethylene (TCE), 1,2-cis-dichloroethylene (c-DCE), and 1,2-trans-dichloroethylene (t-DCE) was evaluated both in groundwater-fed microcosms and in situ in a shallow aquifer. Microcosms amended with phenol or toulene were equally effective in removing c-DCE (> 90%) followed by TCE (60 to 70%), while the microcosm fed methane was most effective in removing t-DCE (> 90%). The microcosm fed ammonia was the least effective. None of the microcosms effectively degraded 1,1,1-trichloroethane. At the Moffett Field groundwater test site, in situ removal of c-DCE and TCE coincided with biostimulation through phenol and oxygen injection and utilization, with c-DCE removed more rapidly than TCE. Greater TCE and c-DCE removal was observed when the phenol concentration was increased. Over 90% removal of c-DCE and TCE was observed in the 2-m biostimulated zone. This compares with 40 to 50% removal of c-DCE and 15 to 25% removal of TCE achieved by methane-grown microorganisms previously evaluated in an adjacent in situ test zone. The in situ removal with phenol-grown microorganisms agrees qualitatively with the microcosm studies, with the rates and extents of removal ranked as follows: c-DCE > TCE > t-DCE. These studies demonstrate the potential for in situ TCE bioremediation using microorganisms grown on phenol.     

Contamination of mural paintings by indoor airborne fungal spores

Summary The processes of biodeterioration on mural paintings have often been discussed, whereas the causes of contamination have seldom been examined.Many microorganisms responsible for the biodeterioration of paintings are of airborne origin. It follows that an investigation on the aerial microbial concentration and air movements in painted indoors is very useful.This paper reviews the literature of mural painting biodeterioration and the aerobiological studies of painted indoors. Hypogean environments, for their particular microclimatic conditions, are not considered.The fungal species most frequently found in the biodeterioration of wall-paintings are reported, as well as comparisons of surface contamination and aerobiological investigation.This review shows the necessity of finding the best sampling methodologies for cultural heritage studies. The control of airborne contamination and proper sampling methods are highly important in determining treatment strategies for the conservation and prevention of microbial attack on painted surfaces.     

Evaluation of Membrane Filter Field Monitors for Microbiological Air Sampling

Due to area constraints encountered in assembly and testing areas of spacecraft, the membrane filter field monitor (MF) and the National Aeronautics and Space Administration-accepted Reyniers slit air sampler were compared for recovery of airborne microbial contamination. The intramural air in a microbiological laboratory area and a clean room environment used for the assembly and testing of the Apollo spacecraft was studied. A significantly higher number of microorganisms was recovered by the Reyniers sampler. A high degree of consistency between the two sampling methods was shown by a regression analysis, with a correlation coefficient of 0.93. The MF samplers detected 79% of the concentration measured by the Reyniers slit samplers. The types of microorganisms identified from both sampling methods were similar. Variables in the MF samplers, such as pore size, relative humidity, and flow rates, have been studied, but no effect was noted on recovery. The results show that the MF method could be used to estimate the number and types of microorganisms found in the air.