scholarly journals Revisiting soil bacterial counting methods: Optimal soil storage and pretreatment methods and comparison of culture-dependent and -independent methods

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246142
Author(s):  
Jeonggil Lee ◽  
Han-Suk Kim ◽  
Ho Young Jo ◽  
Man Jae Kwon
Keyword(s):  
Soil Bacteria ◽  
Epifluorescence Microscopy ◽  
Most Probable Number ◽  
Soil Matrix ◽  
Probable Number ◽  
Soil Particles ◽  
Soil Storage ◽  
Dna Quantitation ◽  
Soil Bacterial ◽  
Culture Dependent

Although a number of different methods have been used to quantify soil bacteria, identifying the optimal method(s) for soil bacterial abundance is still in question. No single method exists for undertaking an absolute microbial count using culture-dependent methods (CDMs) or even culture-independent methods (CIMs). This study investigated soil storage and pretreatment methods for optimal bacterial counts. Appropriate storage temperature (4°C) and optimal pretreatment methods (sonication time for 3 min and centrifugation at 1400 g) were necessary to preserve bacterial cell viability and eliminate interference from soil particles. To better estimate soil bacterial numbers under various cellular state and respiration, this study also evaluated three CDMs (i.e., colony forming unit, spotting, and most probable number (MPN) and three CIMs (i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA quantitation). Each counting method was tested using 72 soil samples collected from a local arable farm site at three different depths (i.e., 10–20, 90–100, and 180–190 cm). Among all CDMs, MPN was found to be rapid, simple, and reliable. However, the number of bacteria quantified by MPN was 1–2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA quantitation method appeared to overestimate soil bacterial numbers, which may be attributed to DNA from dead bacteria and free DNA in the soil matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead).

scholarly journals Comparison of Antibody-Direct Epifluorescent Filter Technique with the Most Probable Number Procedure for Rapid Enumeration of Listeria in Fresh Vegetables

1997 ◽  
Vol 80 (6) ◽  
pp. 1208-1214 ◽  
Author(s):  
Mary L Tortorello ◽  
Karl F Reineke ◽  
Diana S Stewart
Keyword(s):  
Fluorescent Antibody ◽  
Epifluorescence Microscopy ◽  
Most Probable Number ◽  
Probable Number ◽  
Filter Surface ◽  
Colony Forming Units ◽  
Fresh Vegetables ◽  
Rapid Enumeration ◽  
Plate Counts ◽  
Direct Epifluorescent Filter Technique

Abstract The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 μm pore nylon filter, and passage of filtrate through a 0.4 μm pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 107 colony forming units (CFU)/mL. Microbial backgrounds as high as 108 CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.

scholarly journals Sensitive Determination of Microbial Growth by Nucleic Acid Staining in Aqueous Suspension

2006 ◽  
Vol 72 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Willm Martens-Habbena ◽  
Henrik Sass
Keyword(s):  
Nucleic Acid ◽  
Sybr Green ◽  
Sybr Green I ◽  
Epifluorescence Microscopy ◽  
Most Probable Number ◽  
Staining Procedure ◽  
Fluorescence Measurement ◽  
Probable Number ◽  
Microbial Cells

ABSTRACT The determination of cell numbers or biomass in laboratory cultures or environmental samples is usually based on turbidity measurements, viable counts, biochemical determinations (e.g., protein and lipid measurements), microscopic counting, or recently, flow cytometric analysis. In the present study, we developed a novel procedure for the sensitive quantification of microbial cells in cultures and most-probable-number series. The assay combines fluorescent nucleic acid staining and subsequent fluorescence measurement in suspension. Six different fluorescent dyes (acridine orange, DAPI [4′,6′-diamidino-2-phenylindole], ethidium bromide, PicoGreen, and SYBR green I and II) were evaluated. SYBR green I was found to be the most sensitive dye and allowed the quantification of 50,000 to up to 1.5 × 108 Escherichia coli cells per ml sample. The rapid staining procedure was robust against interference from rRNA, sample fixation by the addition of glutaric dialdehyde, and reducing agents such as sodium dithionite, sodium sulfide, and ferrous sulfide. It worked well with phylogenetically distant bacterial and archaeal strains. Excellent agreement with optical density measurements of cell increases was achieved during growth experiments performed with aerobic and sulfate-reducing bacteria. The assay offers a time-saving, more sensitive alternative to epifluorescence microscopy analysis of most-probable-number dilution series. This method simplifies the quantification of microbial cells in pure cultures as well as enrichments and is particularly suited for low cell densities.

scholarly journals Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

2013 ◽  
Vol 80 (1) ◽  
pp. 138-145 ◽  
Author(s):  
Masaki Shintani ◽  
Kazuhiro Matsui ◽  
Jun-ichi Inoue ◽  
Akira Hosoyama ◽  
Shoko Ohji ◽  
...  
Keyword(s):  
Single Cell ◽  
Soil Bacteria ◽  
Fluorescent Protein ◽  
Independent Method ◽  
Rrna Gene ◽  
Reporter Protein ◽  
Content Type ◽  
Culture Independent ◽  
Soil Bacterial ◽  
Culture Dependent

ABSTRACTThe conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods.Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylumProteobacteriawas identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phylaActinobacteria,Bacteroidetes, andFirmicuteswere detected only by the culture-independent method. Members of the genusPseudomonas(classGammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereasDelftiaspecies (classBetaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containingDelftiastrains could not be cultivated after a one-to-one filter mating assay between the donor and cultivableDelftiastrains as recipients, fluorescencein situhybridization detected pCAR1-containingDelftiacells, suggesting thatDelftiawas a “transient” host of pCAR1.

scholarly journals The Impact of a Fish Cannery Wastewater Discharge on the Bacterial Community Structure and Sanitary Conditions of Marine Coastal Sediments

Water ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2566
Author(s):  
Paolo Paliaga ◽  
Igor Felja ◽  
Andrea Budiša ◽  
Ingrid Ivančić
Keyword(s):  
Bacterial Community ◽  
Pathogenic Bacteria ◽  
Bacterial Community Composition ◽  
Coastal Sediments ◽  
Epifluorescence Microscopy ◽  
Most Probable Number ◽  
Redox Potentials ◽  
Probable Number ◽  
Prokaryotic Abundance ◽  
The Impact

The effects of fish cannery discharge (FCD) on bacteria in marine coastal sediments were investigated. Redox potentials were measured, and granulometry was determined by wet ASTM sieving, and with the Sedigraph method. Prokaryotic abundance (PA) was determined by epifluorescence microscopy (DAPI staining), and faecal indicator bacteria (FIB) enumerated with the multiple test tube and most probable number method. Total lipids were determined gravimetrically, and sterols analysed by GC/MSD. Bacterial community composition was determined after total DNA isolation, Illumina MiSeq amplification, and SILVAngs processing pipeline. The FCD was rich in lipids, heterotrophic prokaryotes and FIB. The bacterial community of the FCD was dominated by Firmicutes and Gammaproteobacteria and many potentially pathogenic bacteria. Highly porosusgravelly sands clogged with fish remains transitioned to less permeable sandy muds away from the FCD. All sediments were anoxic with extremely negative potentials around the outfall. High surface PA and FIB spread 300 m from the outfall. Gammaproteobacteria and Deltaproteobacteria appeared in all sediments. Sulfurovum and Anaerolineaceae characterized the most polluted locations where gammaproteobacterial Woeseiaceae/JTB255 marine benthic group declined. Gammaproteobacteria and Bacteroidetes characterized surface sediments, while Chloroflexi and Deltaproteobacteria prevailed in deeper layers. The FCD enriched sediments in lipids and allochthonous bacteria degrading sanitary quality, lowering the permeability, redox potential, and bacterial diversity.

Analysis of Coliform and Colifecal Total Pollution Test on Various Types of Drinking Water Using the MPN (Most Probable Number) Method

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Wanda Aulya ◽  
Fadhliani Fadhliani ◽  
Vivi Mardina
Keyword(s):  
Drinking Water ◽  
Fecal Coliform ◽  
Pathogenic Bacteria ◽  
Microbiological Quality ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Inspection Method ◽  
Sample Number ◽  
Probable Number ◽  
Fecal Coliform Bacteria

Water is the main source for life and also the most severe substance caused by pollution. The mandatory parameters for determining microbiological quality of drinking water are total non-fecal Coliform bacteria and Coliform fecal (Escherichia coli). Coliform bacteria are a group of microorganisms commonly used as indicators, where these bacteria can be a signal to determine whether a water source has been contaminated by bacteria or not, while fecal Coliform bacteria are indicator bacteria polluting pathogenic bacteria originating from human feces and warm-blooded animals (mammals) . The water inspection method in this study uses the MPN (Most Probable Number) method which consists of 3 tests, namely, the presumption test, the affirmation test, and the reinforcement test. The results showed that of 15 drinking water samples 8 samples were tested positive for Coliform bacteria with the highest total bacterial value of sample number 1, 15 (210/100 ml), while 7 other samples were negative. From 8 positive Coliform samples only 1 sample was stated to be negative fecal Coliform bacteria and 7 other samples were positive for Coliform fecal bacteria with the highest total bacterial value of sample number 1 (210/100 ml).

Cemaran Eshericia coli dalam makanan laut yang beredar di pasar tradisional Kota Pontian

2015 ◽  
Vol 1 (1) ◽  
pp. 44
Author(s):  
Rafika Sari ◽  
Pratiwi Apridamayanti
Keyword(s):  
Most Probable Number ◽  
Probable Number

Latar Belakang: Makanan laut merupakan salah satu jenis makanan yang banyak dikonsumsi oleh masyarakat selain sebagai komoditi ekspor. Mengkonsumsi makanan laut yang telah terkontaminasi bakteri hidup atau toksin yang dihasilkannya dapat menyebabkan keracunan makanan. Tujuan penelitian ini adalah untuk mengetahui adanya kontaminasi bakteri koliform E.coli sebagai indikator pencemaran pada makanan laut dan memberikan informasi kelayakan dan keamanan konsumsi dari makanan laut di dua pasar tradisional terbesar di daerah Pontianak. Metode: Sampel yang digunakan adalah ikan, sotong dan udang. Penelitian terhadap sampel dilakukan menggunakan uji Most Probable Number (MPN) yang dilengkapi dengan uji biokimia untuk mengidentifikasi jenis bakteri pada sampel melalui penanaman bakteri pada media agar Lactose Broth (LB) dan Briliant Green Lactose Bile Broth (BGLB). Hasil: Hasil penelitian menunjukkan bakteri koliform E.coli terdeteksi pada 100% sampel dengan nilai MPN yang tidak memenuhi kriteria kelayakan konsumsi, yakni >3/g. Kesimpulan: Makanan yang ada tidak memenuhi kriteria kelayakan konsumsi.

The Occurrence of Legionella Bacteria in Cooling Towers in South Africa

1991 ◽  
Vol 24 (2) ◽  
pp. 149-152 ◽  
Author(s):  
N. A. Grabow ◽  
E. J. Pienaar ◽  
R. Kfir
Keyword(s):  
South Africa ◽  
Most Probable Number ◽  
Cooling Towers ◽  
Probable Number ◽  
Service Water ◽  
Standard Plate ◽  
Viable Bacteria ◽  
Plate Counts ◽  
High Level ◽  
Biocide Treatment

A total of 510 service water samples from cooling towers throughout South Africa were analysed for the presence of Legionella bacteria. Legionella was detected using an immuno-labelling technique based on the most probable number principle. Only cultural (viable) bacteria were counted. Legionellae were found in most of the samples tested. However, in only 4% of the samples a high level of legionellae was recorded. No correlation was found between the numbers of legionellae and those of standard plate counts. Biocide treatment was shown to be effective in the removal of the bacteria from cooling towers after a 3-month treatment period.

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