Epifluorescence Microscopy Method Research Articles

Application of real-time PCR for total airborne bacterial assessment: Comparison with epifluorescence microscopy and culture-dependent methods

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count non-culturable or non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescence microscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270–290 fold greater than those obtained by the “impaction on nutrient agar” method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria.

Characterisation of arbuscular mycorrhizal fungi in roots by means of epifluorescence microscopy and molecular methods

In this work we characterised different species of the arbuscular mycorrhizal (AM) fungal genusGlomus by using Restriction Fragment Length Polymorphisms (RFLPs), combining epifluorescence microscopy and nested PCR, for the easy retrieval of AM fungal DNAin planta. Epifluorescence microscopy allowed us to select highly colonised root segments for DNA extraction, enhancing the chance of fungal DNA amplification by PCR. The primer pair used in nested PCR after a first reaction performed by using NS31/AM1 primers, showed a high specificity forGlomus DNA amplification and yielded enough fungal DNA for RFLP analyses. RFLP patterns of PCR products from colonised roots matched with reference profiles obtained from spores and discriminated the fourGlomus species tested. The method described may be useful for checking the establishment of mycorrhizal colonisation by the inoculated fungal species in controlled experimental conditions.

Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line

Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in different malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact that it is a fluorescent molecule, and its uptake and distribution in cells can be studied by flow cytometry and epifluorescence microscopy. To test the relationships between berberine uptake, distribution and cellular effect in melanoma cells, K1735-M2 mouse and WM793 human melanoma cells were treated with different concentrations of berberine, and alterations in cell cycle progression, DNA synthesis, cell proliferation, and cell death measured. Cell proliferation was measured by sulforhodamine B assays, cell death by flow cytometry, berberine uptake and distribution by laser scanning confocal microscopy and flow cytometry, cell cycle progression by flow cytometry, and DNA synthesis, M-phase, and mitochondrial effects by immunolabeling and epifluorescence microscopy methods. In these melanoma cell lines, berberine at low doses (12.5-50 muM) is concentrated in mitochondria and promotes G1 arrest. In contrast, higher doses (over 50 muM) result in cytoplasmic and nuclear berberine accumulation, and G2 arrest. DNA synthesis is not markedly affected by low doses of berberine, but 100 muM is strongly inhibitory. Even at 100 muM, berberine inhibits cell growth with relatively little induction of apoptosis. Berberine displays multiphasic effects in these malignant cell lines, which are correlated with the concentration and intracellular distribution of this alkaloid. These results help explain some of the conflicting information in the literature regarding the effects of berberine, and suggest that its use in clinical development may be more as a cytostatic agent than a cytotoxic compound.

Effect of different environmental conditions on the bacteria survival on stainless steel surfaces

The effect of different environmental conditions on the survival of Enterobacter cloacae, Pseudomonas aeruginosa and Staphylococcus aureus on stainless steel surfaces was determined using direct epifluorescence microscopy (DEM) and plate count method. A strong linear relation ( R 2 = 0.97; P < 0.01) between DEM and plate counts over the range of 10 4–10 7 CFU/cm 2 was observed when counts by DEM were performed over viable microorganisms. However, when microorganisms were subjected to adverse conditions, such as slow air-drying, the correlation between both methods diminished considerably ( R 2 = 0.225). This may be due to microorganisms subjected to adverse conditions can be injured, so some bacteria can not be detected by cultivation, but they still present a danger. Microorganisms on wet surfaces had the ability to produce biofilm after 24 h. However, air-drying on surfaces, reduced considerably the bacterial count. E. cloacae was the most susceptible to slow air-drying on surfaces at room temperature however, S. aureus, remained viable for at least 72 h. The characteristics of an organism and its surrounding environment are among the important factors that may affect the survival of bacterial cells on surfaces.

Microbial Communities on Glacier Surfaces in Svalbard: Impact of Physical and Chemical Properties on Abundance and Structure of Cyanobacteria and Algae

Microbial communities occurring in three types of supraglacial habitats--cryoconite holes, medial moraines, and supraglacial kames--at several glaciers in the Arctic archipelago of Svalbard were investigated. Abundance, biovolume, and community structure were evaluated by using epifluorescence microscopy and culturing methods. Particular emphasis was laid on distinctions in the chemical and physical properties of the supraglacial habitats and their relation to the microbial communities, and quantitative multivariate analyses were used to assess potential relationships. Varying pH (4.8 in cryoconite; 8.5 in a moraine) and texture (the proportion of coarse fraction 2% of dry weight in cryoconite; 99% dw in a kame) were found, and rather low concentrations of organic matter (0.3% of dry weight in a kame; 22% dw in cryoconite) and nutrients (nitrogen up to 0.4% dw, phosphorus up to 0.8% dw) were determined in the samples. In cryoconite sediment, the highest numbers of bacteria, cyanobacteria, and algae were found, whereas relatively low microbial abundances were recorded in moraines and kames. Cyanobacterial cells were significantly more abundant than microalgal ones in cryoconite and supraglacial kames. Different species of the cyanobacterial genus Leptolyngbya were by far the most represented in all samples, and cyanobacteria of the genera Phormidium and Nostoc prevailed in cultures isolated from cryoconite samples. These species are considered opportunistic organisms with wide ecological valency and strong colonizing potential rather than glacial specialists. Statistical analyses suggest that fine sediment with higher water content is the most suitable condition for bacteria, cyanobacteria, and algae. Also, a positive impact of lower pH on microbial growth was found. The fate of a microbial cell deposited on the glacier surface seems therefore predetermined by the physical and chemical factors such as texture of sediment and water content rather than spatial factors or the origin of sediment.

Use of an automatic cell-counting system with LED illumination for enumeration of marine bacteria

The concentration of a quatic bacteria is basic information required to evaluate the status of environments and to assess bacterial contribution to material cycles. However, the standard direct counting method using epifluorescence microscopy (EFM) is tedious and there is variation in the counts among workers. Here an automatic counting system that consists of Bioplorer (BP) and image analysis has been applied to marine bacteria. BP is composed of a light-emitting diode (LED) illuminant, an optical unit, a driving stage and a charge-coupled device camera. In combination with fluorescent labeling and simplified membrane filtration, bacteria are enumerated auto matically. The reproducibility, sensitivity and accuracy of the system were tested for natural marine bacteria, in comparison with EFM and flow cytometry (FCM). The counts obtained by BP showed good correlation with those obtained by EFM and FCM methods. The counts were significantly higher in inshore and oceanic samples, indicating high sensitivity with low background noise. Considering its reproducibility, objectivity, ease of use and compact size, BP can be used as a routine tool for counting aquatic bacteria in substitution for EFM or FCM.

Fluorochrome in Monitoring Indoor Bioaerosols

In this study total concentration, viability, culturability of indoor and outdoor bioaerosols in ten residential environments were studied by using a cultivating method and an epifluorescence microscopy method with fluorochrome (EFM/FL) using four different dyes (AO, DAPI, PI, and YOPRO-1). Results from the EFM/FL were then compared with those from the traditional cultivating method. The total indoor microbial cell concentrations measured by EFM/FL was 630 to 5200 times higher than that measured by the cultivating method; ranging from 1.38 × 106 to 4.48 × 106cells/m3 (microbial cells per cubic meter) with AO staining, and from 7.84 × 105 to 2.75 × 106 cells/m3 with DAPI staining, compared with the average concentration of 721 CFU/m3 for bacteria and 587 CFU/m3 for fungi by the cultivating methods. The viability of bioaerosols ranged from 0.63 to 0.92 by EFM/FL with PI staining, from 0.19 to 0.72 by EFM/FL with YOPRO-1 staining, and from 0.0002 to 0.0015 by the cultivating method. The viability determined ...

Time series observation based InfraRed Epifluorescence Microscopic (TIREM) approach for accurate enumeration of bacteriochlorophyll-containing microbes in marine environments

Bacteriochlorophyll a Containing Microbes (BCM) are a unique group of microorganisms in the marine environment. Accurate determination of their abundance is critical for understanding their role in energy flow and carbon cycle in the ecosystem. The InfraRed Epifluorescence Microscopy (IREM) method, using infrared fluorescence as the diagnostic signal of BCM, is the most convenient means to date for enumeration of BCM in seawater, but IREM methodology suffers from serious errors introduced by cyanobacteria, which also can emit infrared fluorescence and whose abundance is of the same order of magnitude as BCM. In the present study, an advanced “Time-series observation based cyanobacteria-calibrated InfraRed Epifluorescence Microscopy (TIREM)” approach is established for accurate enumeration of BCM in marine environments. The protocol is distinguished by its use of time series observation, auto-imaging and digital analysis. In principle, the correct count of BCM can be obtained by subtracting the cyanobacterial count from the total infrared positive count. The challenge, however, is that Prochlorococcus, the most abundant cyanobacterium in the sea, is readily visible in infrared images but not visible in the initial cyanobacterial images obtained by epifluorescence microscopy because its emission signals are masked by brighter fluorescence from larger cells like Synechococcus coexisting in seawater samples. Prochlorococcus cells become gradually visible when the fluorescence from Synechococcus cells declines after a period of exposure to excitation light. Therefore the plateau (maximum) count of the cyanobacterial cells in time series images rather than in the initial ones, as previously believed, represents the correct count for the total number of cyanobacteria ( Synechococcus plus Prochlorococcus cells). Thus, the accurate estimation of BCM abundance can only be calculated from the formula: [BCM cells] = [plateau count of infrared positive cells] − [plateau count of cyanobacterial cells]. The conceptual advance of the TIREM protocol is that in classical epifluorescence microscopy or in IREM protocols, quick observation is recommended to avoid quenching the fluorescence, but in the TIREM protocol, instead, time series observation is the key for obtaining reliable data. The TIREM protocol is validated by studies using BCM and cyanobacterial pure cultures as well as by examination of samples from various marine environments.

Morphology and Oxygen Sensor Response of Luminescent Ir-Labeled Poly(dimethylsiloxane)/Polystyrene Polymer Blend Films

Polymer films consisting of a linear poly(dimethylsiloxane) end-functionalized with a luminescent Ir(III) complex (Ir-PDMS), blended with polystyrene (PS), function as optical oxygen sensors. The sensor response arises by quenching of the luminescence from the Ir(III) chromophore by oxygen that permeates into the polymer film. The morphology and luminescence oxygen sensor properties of blend films consisting of Ir-PDMS and PS have been characterized by fluorescence microscopy, atomic force microscopy, and scanning electron microscopy. The investigations demonstrate that microscale phase segregation occurs in the films. In blends that contain a relatively small amount of Ir-PDMS in PS (ca. 10 wt %), the Ir-PDMS exists as circular domains, with diameters ranging from 2 to 5 mum, surrounded by the majority PS phase. For larger weight fractions of Ir-PDMS in the blends, the film morphology becomes bicontinuous. A novel epifluorescence microscopy method is applied that allows the construction of Stern-Volmer quenching images that quantify the oxygen sensor response of the blend films with micrometer spatial resolution. These images provide a map of the oxygen permeability of the polymer blend films with a spatial resolution of ca. 1 mum. The results of this investigation show that the micrometer-sized Ir-PMDS domains display a 2-3-fold higher oxygen sensor response compared to the surrounding PS matrix. This result is consistent with the fact that PDMS is considerably more gas permeable compared to PS. The relationship of the microscale morphology of the blends to their performance as macroscale optical oxygen sensors is discussed.

Influence of Langmuir–Blodgett nanolayers on microbial adhesion

Langmuir–Blodgett (LB) nanolayers of alkyl hydroxamic and phosphonic acids on glass, iron and copper surfaces were studied in the presence of different, corrosion relevant micro-organisms. The quality of monomolecular and multimolecular LB layers was characterised by dynamic contact angle measurement, atomic force microscopy and the sum frequency vibration technique. The inhibition efficiency of microbial adhesion and biofilm formation, which was analysed by the surface visualising technique, epifluorescence microscopy and microbiological methods, increased with layer thickness. There were some differences in the blocking activity of the two amphiphiles (octadecylphosphonic acid C18P and octadecanoyl hydroxamic acid C18N) applied in LB films. Their repellent activity did not differ in cooling water but, in the presence of Acidithiobacillus ferrooxidans, the C18N nanolayer inhibited the biofilm formation much better.

Bacterial adherence to different inert surfaces evaluated by epifluorescence microscopy and plate count method

The adherence of Listeria inoccua L6a and Staphylococcus aureus ATCC6538 was evaluated on stainless steel (SS), polycarbonate (PC) and polypropylene (PP) chips. The EP results showed a significant difference (p&lt;0.05) among the microorganism species interactions, the surfaces and the contact time. The number of adhered cells on the chip surfaces increased as contact time increased and the number of L. innocua or S. aureus on the surface of SS, PC and PP chips reached 1.0x10(5) CFU/cm² after 12 h of contact, in both methods. The CP method showed a better sensitivity to detect low number of adhered cells. The EP was better when the average number of adhered cells was between 10 and 100 per microscopy field.

Bioactive Packaging Materials from Edible Chitosan Polymer—Antimicrobial Activity Assessment on Dairy‐Related Contaminants

ABSTRACT The inhibitory activity of chitosan‐based edible coatings was assessed against 2 food pathogens (Staphylococcus aureus and Listeria monocytogenes) and 1 strain involved in food alteration (Pseudomonas aeruginosa) on model agar medium and on a real cheese food product. Colony counting and epifluorescence microscopy methods were conducted, and the results show a nonsignificant influence of the components of the food matrix in the protection of the microbial population against chitosan activity. Numeration on model agar medium showed 100% inhibition of the development of selected Gram‐positive bacteria and 77% inhibition on Pseudomonas growth. Chitosan is thought to act through binding to the cytoplasmic membrane surface, and it is possible that the outer membrane protects the Gram‐negative cells. Moreover, epifluorescence microscopic results showed a possible chitosan action during a short time duration on the synthesis of nucleic acids and especially on the relative proportion of RNA compared with DNA. This impact was followed by an adaptative mechanism of the cells. Edible chitosan coating could thus be used to increase the microbial lag phase while decreasing the maximum density of selected microorganisms and could have potential application for dairy products preservation.

Lateral phase separation in adsorbed binary protein films at the air-water interface.

Lateral phase separation in two-dimensional mixed films of soy 11S/beta-casein, acidic subunits of soy 11 (AS11S)/beta-casein, and alpha-lactalbumin/beta-casein adsorbed at the air-water interface has been studied using an epifluorescence microscopy method. No distinct lateral phase separation was observed in the mixed protein films when they were examined after 24 h of adsorption from the bulk phase. However, when the soy 11S/beta-casein and AS11S/beta-casein films were aged at the air-water interface for 96 h, phase-separated regions of the constituent proteins were evident, indicating that the phase separation process was kinetically limited by a viscosity barrier against lateral diffusion. In these films, beta-casein always formed the continuous phase and the other globular protein the dispersed phase. The morphology of the dispersed patches was affected by the protein composition in the film. In contrast with soy 11S/beta-casein and AS11S/beta-casein films, no lateral phase separation was observed in the alpha-lactalbumin/beta-casein film at both low and high concentration ratios in the film. The results of these studies proved that proteins in adsorbed binary films exhibit limited miscibility, and the deviation of competitive adsorption behavior of proteins at the air-water interface from that predicted by the ideal Langmuir model (Razumovsky, L.; Damodaran, S. J. Agric. Food Chem. 2001, 49, 3080-3086) is in fact due to thermodynamic incompatibility of mixing of the proteins in the binary film. It is hypothesized that phase separation in adsorbed mixed protein films at the air-water and possibly oil-water interfaces of foams and emulsions might be a source of instability in these dispersed systems.

Microbiological control of wine. The application of epifluorescence microscopy method as a rapid technique

In this study, epifluorescence microscopy (vital staining) was used for microbiological control of wine, the results being compared with plate counts of colony forming units (cfu). 4′,6-diamidino-2-phenylindole (DAPI) combined with propidium iodide (PI) were the fluorochroms used. The correlation between the two methods (plate counts and vital staining) was 0.52 and 0.83 for the control of finished and non-finished wines, respectively. According to the results obtained the vital staining with DAPI combined with PI, is trustworthy as a fast control for wine during the technological process, and it can be used by industrial wine producing units as an indicator of wine microbiological quality, with the consequent savings in time and money. For finished wines, the results obtained by the vital staining technique, were 103-fold higher than the ones obtained with the plate count method, which is in accordance with what should be expected, since under stressful conditions the number of metabolically active bacteria exceeds by far the number of culturable bacteria.

Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.

Total bacterial abundances estimated with different epifluorescence microscopy methods (4',6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, "live" bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and "dead" bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of "live" cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.

Diurnal Distribution of Total and Culturable Atmospheric Bacteria at a Rural Site

During the summer and fall of 1996 ambient air samples were collected using a high-volume wet cyclone sampler positioned 2 m above ground level in an agricultural area near Corvallis, OR. Samples were collected for approximately 2-hr periods from 0500 to 2100 hr on 11 days. Total and culturable atmospheric bacterial (TAB and CAB) concentrations in the samples were determined using epifluorescence microscopy and filtration-culture methods. It was found that the CAB concentration in the atmosphere was the lowest at dawn, gradually increased from sunrise to reach a maximum in the afternoon, and finally decreased in the evening. The TAB load had a similar but much less pronounced trend. The geometric mean of TAB concentration was about 27 to 222 times greater than that of CAB concentration in the atmosphere. A much higher TAB determination suggests that atmospheric bacteria might play a greater role than expected when the culturable determination is used as an investigation method in allergic disease and ecol...

Immunoelectrochemical assays for bacteria: use of epifluorescence microscopy and rapid-scan electrochemical techniques in development of an assay for Salmonella.

Immunoelectrochemical sensors in which the sensor surface functions as both analyte capture phase and electrochemical detector have recently been developed for bacteria analysis. The speed and sensitivity of these devices make them very attractive for applications such as the detection of pathogenic microorganisms in food and water. However, the development and optimization of assays utilizing these sensors can be complicated by undesired interactions between the capture and detection functions. Modification of the sensor to achieve improvements in one function can have deleterious effects on the other function, and such effects can be difficult to diagnose and correct. In the course of investigations on immunoelectrochemical detection of Salmonella, we developed a rapid, nondestructive epifluorescence microscopy method to determine bacteria capture efficiency. This method enabled us to study capture and detection functions independently and efficiently identify performance-limiting factors. Rapid-scan electrochemical methods were used to optimize detection sensitivity and to provide diagnostic information on detection performance.

An epifluorescence microscopy method for direct detection and enumeration of the fungilike marine protists, the thraustochytrids

An epifluorescence microscopy method using the fluorescent dye acriflavine is described for direct detection and enumeration of the fungilike protists, the thraustochytrids, in natural samples. The technique is based on the principles that the fluorochrome stains thraustochytrid cell walls containing sulfated polysaccharides red and the nucleus yellow‐green. Photosynthetic protists were distinguished by autofluorescence of pigments and protozoans by absence of cell‐wall fluorescence. Several natural phytoplankton samples from the North Sea were examined with this technique, where up to 5.4 × 106 cells of thraustochytrids were found per gram dry weight of sample. The results suggest that thraustochytrids might be much more abundant in natural samples than previously suspected.

Changes in densities of bacteria and microfungi during gut transit in Lumbricus rubellus and Aporrectodea caliginosa (Oligochaeta: Lumbricidae)

Numbers of bacteria, actinomycetes and micromycetes were estimated in gut content samples of specimens of two earthworm species using the plate count technique and the epifluorescence microscopy method. An increase in numbers of all three microbial groups was observed in the intestine of Lumbricus rubellus during the food passage. The total number of palatable aerobic and facultatively anaerobic bacteria was 7 × 10 6 g −1 dry gut content in the foregut, but it increased to 16 × 10 6 and 29 × 10 6 in the midgut and hindgut, respectively. A similar increasing tendency was also observed in numbers of actinomycetes and micromycetes. In contrast higher numbers of bacteria and actinomycetes were detected in the foregut (62 × 10 6, 6 × 10 6 g −1 dry gut content) than in midgut (37 × 10 6, 4 × 10 6) of Aporrectodea caliginosa, and their numbers did not change significantly from midgut to hindgut. Micromycetes were relatively stable in number in all three gut sections. Number of living bacterial cells, estimated by the epifluorescence microscopy method, in general corresponds well with plate counts, although an increase in number of bacteria was detected from midgut to hindgut of A. caliginosa. Factors which would be responsible for differences in the composition of intestinal communities between two earthworm species are discussed.

Distribution and activity of microorganisms in coastal waters off the Netherlands and Germany

Coastal waters up to 90 km off the coast of the Netherlands and Germany were investigated with the RV ‘Friedrich Heincke’ at 21 stations in July 1987. Hydrographic, chemical and biological parameters were measured simultaneously, such as temperature, salinity, oxygen saturation, adenosine triphosphate (ATP) concentrations and electron transport system (ETS) activities. The mean surface temperature was 16°C, salinity distribution showed a tongue of high salinity in the Southern Bight and coastal areas of low values by river input from the Rhine and Elbe. The mean oxygen concentration was 102.4%, with the highest percentages northwest of Helgoland and the lowest near Scheveningen and Cuxhaven. The ecosystem parameters of ATP-biomass and respiratory ETS-activity showed a distribution pattern corresponding to the inflow of clear Channel water and nutrient-rich river water. Highest biomass values and respiratory activities were found near Texel and northwest of Helgoland. The distribution patterns of microplankton species were more patchy. Distribution patterns are shown of phytoplankton species such as Ceratium fusus, Phaeocystis spec., Rhizosolenia shrubsolei, and Mesodinium rubrum and various groups of microplankton. The highest numbers of Ceratium were found near Helgoland, of Phaeocystis near Terschelling and Cuxhaven, of Rhizosolenia near Texel and Langeoog and of Mesodinium near the Dutch coast and north of Borkum. Concentrations of bacteria were determined both by cultivation and epifluorescence microscopy methods. The numbers obtained by the pour-plate method varied much more than those determined by the direct-count method. At all stations, oil-degrading bacteria were found. The numbers of bacteria able to degrade mineral oil were higher than the numbers of bacteria able to degrade a tetradecane-pentadecane mixture. Biological oxygen demand (BOD) was measured with and without oil additions after incubation of 5, 10 and 15 days and a potency to degrade oil was shown. Beside the mapping of respiratory activity by the ETS-method, the real oxygen uptake at in situ temperatures was measured in a few samples with short incubation. An ETS/R ratio of 4.1 was estimated.