Enumerating bacterial cells on bioadhesive coated slides

Franklin, Rima B.; Campbell, Alaina H.; Higgins, Colleen B.; Barker, M. Kensey; Brown, Bonnie L.

doi:10.1016/j.mimet.2011.08.013

Cited by 10 publications

(5 citation statements)

References 26 publications

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“…Using epifluorescence microscopy (Olympus BX-51), bacterial cells were enumerated at 1000 × under oil immersion in at least 20–40 randomly chosen microscope fields in triplicate for each sample. The total cell count in each sample was calculated by applying the formula (equation 1 ) 45 :…”

Section: Methodsmentioning

confidence: 99%

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface

Ueno

Shimizu

Tamamura

et al. 2016

Sci Rep

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Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface.

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Section: Methodsmentioning

confidence: 99%

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface

Ueno

Shimizu

Tamamura

et al. 2016

Sci Rep

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“…This relationship was significant only for the biofilm samples, a finding which could be associated with EPS presence (Flemming and Wingender 2010), allowing more cells to remain with intact membranes even after the fixation process (death). For this reason, PI has been commonly used to label dead cells , Falcioni et al 2008, Franklin et al 2011, although fast-growing cells can also show ruptured membranes, allowing PI marking (Shi et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Comparison of techniques for counting prokaryotes in marine planktonic and biofilm samples

Agostini

Rodrigues

Macêdo

et al. 2021

scimar

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Though a large number of techniques are available for the study of aquatic bacteria, the aim of this study was to establish a technique for analysing free-living and biofilm prokaryotic cells through laboratory assays. In particular, we wished to analyse the efficiency of ultrasound to detach and disrupt biofilm, to obtain an efficient stain treatment for quantifying free-living and biofilm prokaryotes in flow cytometry (FC), and to compare epifluorescence microscopy (EFM), scanning electron microscopy (SEM) and FC for quantifying free-living and biofilm prokaryotes#. Marine-grade plywood substrates were immersed in natural marine water that was conditioned for 12 days. At 6 and 12 days, water aliquots and substrates were removed to estimate free-living and biofilm prokaryote density. Ultrasound efficiently removed marine biofilm from substrates (up to 94%) without cell damage. FC analysis (unstained) reliably quantified marine plankton and young or mature biofilm prokaryotes compared with other staining (acridine orange, 4′,6-diamidino-2-phenylindole, propidium iodide and green fluorescent nucleic acid), EFM or SEM techniques. FC and SEM achieved similar results, while a high variability was observed in the EFM technique. FC was faster and more precise than SEM because the count is not dependent on the observer.

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“…An alternative fixation method has been reported by other, bioadhesive slides (Excell AdhesionTM, Fisher Scientific) which showed good cell trapping, but compounds present on the slides caused mortality or affected permeability of the cell membrane, thus inducing mortality of some pure cultures and river water microbes (5). Recently, a low-melting point agarose gel supplemented with Bac Light stains was used to rehydrate and determine viability of dried cell particles deposited on Anopore chips (19).…”

Section: Discussionmentioning

confidence: 99%

“…Using 0.22 μm polycarbonate (PC) black membranes to trap bacterial cells has been reported by some studies (7, 10, 18, 21). However this filter membrane method could underestimate total cells when environmental samples (5) and non-dairy drink (13) were determined. Because PI fluorescence dye causes background fluorescence and non-specific binding to the matrix of samples (3).…”

Section: Introductionmentioning

confidence: 99%

Novel Method for Viability Assessment ofBifidobacterium longumATCC 15707 on Non-dairy Foods during drying

Min

Mason

Bennett

et al. 2018

Preprint

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16This study demonstrates a new technique for separating and purifying viable microbes from 17 samples that interfere with viability staining. The viability of Bifidobacterium longum ATCC 18 15707 was assessed using PBDC to separate bacteria from complex non-dairy food matrices and 19 Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEAD 20 BacLight bacterial viability staining. Water agar (3%) was used to retain cells of B. longum and 21 offered a lower fluorescence background with BacLight viability staining, compared with fixation 22 on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods 23 on viability of B. longum was assessed. B. longum coated on oat, peanut or raisin was separated 24 by filtration, low-and high-speed centrifugation, flotation and sedimentation buoyant density 25 centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed 26 by LIVE/DEAD BacLight viability staining and enumeration. Conventional plate counting was 27 also conducted to compare viability results. Finally, the applicability of this novel method for 28 viability assessment was demonstrated and informative information of cell membrane damages of 29 B. longum incorporated onto non-dairy foods during 24 h drying was observed. Viability 30 assessment of B. longum coated onto oat, peanut, or raisin was much lower by plate counting 31 compared to viability staining. Drying appeared to have a greater impact when viability was 32 assessed by plate counting compared to viability staining. 33 IMPORTANCE Enumeration of viable beneficial bacteria from function foods presents a 34 significant bottle neck for product development and quality control. Interference with 35 3 microscopic and/or fluorescent techniques by ingredients, time required to incubate plated 36 microbes, and the transient nature of the colony forming unit make rapid assessment of viable 37 bacteria difficult. Viability assessment of Bifidobacterium longum ATCC 15707 by Percoll 38 Buoyant Density Gradient Centrifugation with LIVE/DEAD BacLight viability staining on water 39 agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation 40 viability assessment by staining provided a more rapid means to assess the impact of drying on 41 the viability of B. longum coated onto oat, peanut or raisin. 42 43 44Viability of probiotics in food products plays a critical role for delivering health benefits to 45 consumers. Drying is an old method that has been diversely applied to preserve food, disinfect 46 surfaces, prevent pathogen transmission, and to produce and prepare probiotic culture powders 47 (17). During drying, the removal of water can cause desiccation damage to proteins, nucleic 48 acids, lipids and membranes depending on the different levels of drying (20). As a conventional 49 viability determination method, plate counting of colony forming units (CFU) may underestimate 50 numbers of bacteria if bacterial cells are viable but uncu...

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Enumerating bacterial cells on bioadhesive coated slides

Cited by 10 publications

References 26 publications

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface

Comparison of techniques for counting prokaryotes in marine planktonic and biofilm samples

Novel Method for Viability Assessment ofBifidobacterium longumATCC 15707 on Non-dairy Foods during drying

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