Dilution Plate Count Method Research Articles

Screening of Different Media and Heat Shock Treatment Regimens for Enhancing Sporulation in Bacillus licheniformis

Bacillus licheniformis is a spore-producing bacteria. The present study aims to identify maximum sporulation in Bacillus licheniformis through induced heat shock conditions using different solidified media and broth media. Suspensions of Bacillus licheniformis spores were spread-plated on nutrient agar plates before and after heat shock treatments of 60°C, 80°C and 100°C for 30 minutes. The number of spores was determined by the dilution plate count method. The result indicates that the highest spore production for Bacillus licheniformis was observed when the spores were induced in AK (Arret and Kirshbaum) Agar for seven days under a heat shock treatment of 60℃ for 30 minutes. Whereas, in the case of Nutrient Broth media, the spore production was highest after four days at heat shock treatment of 80℃ for 30 minutes. This study will help find the optimal production of spores from Bacillus licheniformis, an industrially beneficial microorganism.

Population Distribution of Phosphate-solubilizing Microorganisms in Agricultural Soil

Phosphorus (P) is an essential macronutrient for plant growth and is mainly present in agricultural soil in unavailable forms. Phosphate-solubilizing microorganisms (PSMs) increase soil P availability. The objective of the present study was to assess the population and type of PSMs and their relationships with soil characteristics in the agricultural soil of Manokwari. Twenty-one composite soil samples (0–20‍ ‍cm) were collected at the rhizospheres of plants in the Prafi and Masni Districts. A dilution technique and plate count method on Pikovskayas agar medium were used to examine the PSM population, phosphate-solubilizing index (PSI), and various soil properties. The results obtained showed that the total population of phosphate-solubilizing bacteria ranged between 25×103 and 550×103 CFU g–1 of soil, while that of phosphate-solubilizing fungi was between 2.0×103 and 5.0×103 CFU g–1 of soil at all locations. The PSI of the isolates ranged between 1.1 to 3.6‍ ‍mm, with the most efficient and highest PSI being obtained for Bacillus sp. (strain 8) and the lowest for Pseudomonas sp. (strain 15). Six isolates found at all locations were identified at the genus level: Chromobacterium sp., Pseudomonas sp., Bacillus sp., Micrococcus sp., Caulobacter sp., and Aspergillus sp. A correlation was observed between the number of PSMs and the level of soil P availability and moisture content, indicating an increase in soil P availability with a greater abundance of PSMs in soil.

Effect of Raw-SWCNTs, SWCNTs/Ag-TiO2 nanoparticles for control the biological contaminates in water Treatment

In this study, hybrid material like Ag-TiO2 coated single-walled carbon nanotubes (Ag–titania hybrid/SWCNTs) was formation by a multistep chemical procedure and evaluated for their application potentials as an antimicrobial agent for slowing down bacterial growth as bacteriostatic and inactivating pathogenic. The structural and morphological properties of Raw-SWCNTs and SWCNTs/TiO2-Ag nanoparticles were investigated by using X-ray diffraction (XRD), Field Emission Scanning Electron Microscope (FESEM) and Raman spectroscopy improving the polycrystalline structure of Ag and TiO2 NPs decorating SWCNTs due to chemical method. As well as, Raman spectroscopy, reveal the formation of nanocomposite by increasing the intensity of D to G and disorder of carbon structure with Ag-TiO2 interaction. In addition, the antimicrobial properties of nanocomposite material (SWCNTs/TiO2-Ag) were tested against bacterial strains, including E. coli as Gram negative by using standard agar dilution (plate count) method to inhibit microbial growth in water treatment.

Synthesis of Multi-Walled Carbon Nanotubes Decorated with Zinc Oxide Nanoparticles for Removal of Pathogenic Bacterial

A series of Zinc Oxide Nanoparticles (ZnONPs)-functionalized Multi-Walled Carbon Nanotubes (F-MWCNTs) Nano composites were developed as antibacterial. In this study, chemical oxidation of pristine MWCNTs were carried out with a mixture of strong acids (3H2SO4 98%:1 HNO3 65%).The F-MWCNTs were used as templates to prepare hybrid material like ZnONPs decorated F-MWCNTs. Pristine MWCNTs, F-MWCNTs and (ZnONPs/F-MWCNTs) Nano composites powder were investigated using Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Anti-bacterial activity has been carried out using standard agar dilution (plate count) method against Escherichia coli (E. coli). This study demonstrated that (ZnONPs/F-MWCNTs) Nano composite has a powerful bactericidal effect against Escherichia coli (E. coli) at concentration 0.5 mg/ml after 3 hr, which led to speculation that the combination of ZnONPs and F-MWCNTs altered their toxicity and improved antibacterial property of Nano composite.

Enhanced Killing of Biliary Bacterial Biofilm Using Electroporation: An In Vitro Evaluation

Background: Bacterial biofilm is a known cause of chronic device related infections requiring removal of implanted device such as a biliary stent. Biofilm bacteria are resistant to antibiotic therapy requiring 100 to 1000 times minimum inhibitory concentration (MIC) for complete eradication. Electroporation (EP) - a pulsed electrical field induces cell membrane permeability and enhances penetration of drugs into bacterial cells. Aim: To determine effects of EP and antibiotics therapy in eradication of biofilm bacteria. Method: Mixed biofilm were formed on micro TFA filters (Applied Biosystems, CA) by incubation with E. coli (E) and Enterococcus (En) in ox bile for 72 hours. The established biofilm was tested against Ciprofloxacin at 100xMIC for E. coli (Cipro) or 4% tetra sodium EDTA (TE) with or without EP. The filters were inserted into cuvettes and electroporated (ECM 630, Harvard Apparatus, MA) with constant voltage, capacitance, pulse duration, frequency and number of pulses for 1 minute. The experiments were divided into 2 groups with 10 or 15 EP pulses and exposure to individual antimicrobials for 4 hours. Experiments included: 1. Control; 2. 10 pulses of EP at 1500 volts; 3. 4% TE; 4. Cipro at 100xMIC; 5. EP plus Cipro; 6. EP plus TE. Experiments were repeated using 15 EP pulses. The filters were rinsed and then sonicated in 5 ml sterile PBS to release the bacteria. Bacterial counts in colony forming units (CFU/cm2) were determined by serial dilution and viable plate count method. The experiments were repeated and the mean bacterial count (n = 2) was calculated and expressed as mean (log10 CFU/cm2) ± SD. Results: Mean control bacterial counts were (E 5.6-5.8, En 4.8-4.4). Compared with controls, EP at 10 pulses or Cipro alone produced a 100-fold (2 log) reduction in E. coli but no significant effects on Enterococcus; 2. EP plus Cipro produced a 100-fold decrease in E. coli and 10-fold decrease in Enterococcus. 3. TE alone killed all E. coli but not Enterococcus; 4. EP plus 4% TE produced a 100-fold decrease in Enterococcus. With 15 EP pulses, all E. coli were killed by Cipro. A combination of TE and 15 EP pulses killed ALL bacteria in the biofilm. The differences were statistically significant (Box plot, p < 0.001). Conclusion: Electroporation enhanced the killing effects of antibiotics/antimicrobials against established biofilm bacteria and may offer an alternative to prevent biliary biofilm and stent blockage. Background: Bacterial biofilm is a known cause of chronic device related infections requiring removal of implanted device such as a biliary stent. Biofilm bacteria are resistant to antibiotic therapy requiring 100 to 1000 times minimum inhibitory concentration (MIC) for complete eradication. Electroporation (EP) - a pulsed electrical field induces cell membrane permeability and enhances penetration of drugs into bacterial cells. Aim: To determine effects of EP and antibiotics therapy in eradication of biofilm bacteria. Method: Mixed biofilm were formed on micro TFA filters (Applied Biosystems, CA) by incubation with E. coli (E) and Enterococcus (En) in ox bile for 72 hours. The established biofilm was tested against Ciprofloxacin at 100xMIC for E. coli (Cipro) or 4% tetra sodium EDTA (TE) with or without EP. The filters were inserted into cuvettes and electroporated (ECM 630, Harvard Apparatus, MA) with constant voltage, capacitance, pulse duration, frequency and number of pulses for 1 minute. The experiments were divided into 2 groups with 10 or 15 EP pulses and exposure to individual antimicrobials for 4 hours. Experiments included: 1. Control; 2. 10 pulses of EP at 1500 volts; 3. 4% TE; 4. Cipro at 100xMIC; 5. EP plus Cipro; 6. EP plus TE. Experiments were repeated using 15 EP pulses. The filters were rinsed and then sonicated in 5 ml sterile PBS to release the bacteria. Bacterial counts in colony forming units (CFU/cm2) were determined by serial dilution and viable plate count method. The experiments were repeated and the mean bacterial count (n = 2) was calculated and expressed as mean (log10 CFU/cm2) ± SD. Results: Mean control bacterial counts were (E 5.6-5.8, En 4.8-4.4). Compared with controls, EP at 10 pulses or Cipro alone produced a 100-fold (2 log) reduction in E. coli but no significant effects on Enterococcus; 2. EP plus Cipro produced a 100-fold decrease in E. coli and 10-fold decrease in Enterococcus. 3. TE alone killed all E. coli but not Enterococcus; 4. EP plus 4% TE produced a 100-fold decrease in Enterococcus. With 15 EP pulses, all E. coli were killed by Cipro. A combination of TE and 15 EP pulses killed ALL bacteria in the biofilm. The differences were statistically significant (Box plot, p < 0.001). Conclusion: Electroporation enhanced the killing effects of antibiotics/antimicrobials against established biofilm bacteria and may offer an alternative to prevent biliary biofilm and stent blockage.

The Numbers and Biomass of Bacteria and Fungi Obtained by the Dilution Plate Count Method and the Direct Count Method by Microscopy (Program for 2005 Annual Meeting of Japanese Society of Soil Science and Plant Nutrition)

The Numbers and Biomass of Bacteria and Fungi Obtained by the Dilution Plate Count Method and the Direct Count Method by Microscopy (Program for 2005 Annual Meeting of Japanese Society of Soil Science and Plant Nutrition)

Chromium-Resistant Bacterial Populations from a Site Heavily Contaminated with Hexavalent Chromium

Chromium-containing industrial effluents are primarily responsible for environmental contamination by toxic and highly mobile, hexavalent chromium. The dilution plate-count method, using media amended with Cr(VI) at concentrations ranging from 0 to 1000 mg L-1, was used to compare the sizes of Cr(VI)-resistant bacterial populations from a soil contaminated with 25 100 mg kg-1 total Cr [12 400 mg kg-1 Cr(VI)] to those isolated from a slightly contaminated soil (99.6 mg kg-1 total Cr) and two other soils without any history of Cr contamination. Bacterial populations resistant to 500 mg L-1 Cr(VI) were isolated from all soils except the heavily contaminated soil. To determine whether Cr-resistant bacterial populations were indigenous to both the contaminated and the uncontaminated soils, enrichment cultures containing Cr(VI) at concentrations ranging from 0 to 1000 mg L-1 were employed. Bacterial populations, as high as 105 (colony forming units) CFU g-1 soil, tolerant of 500 mg L-1 Cr(VI) were isolated from all soils within 48 h of enrichment suggesting that the presence of aerobic Cr(VI)-resistant bacterial populations is unrelated to contamination levels or contamination history. However, identification of these resistant bacteria using fatty acid profiles was unsuccessful suggesting that these populations may have unique characteristics. Fungal colonies resistant to 1000 mg L-1 Cr(VI) were routinely isolated from both uncontaminated and contaminated soils. The results suggest that Cr-resistant microorganisms may be present in soils, even those with no history of Cr contamination.

In-vitro activity of azithromycin in against intracellular Helicobacter pylori.

We studied the effect in vitro of azithromycin on the clinical strain Helicobacter pylori H:72 growing intracellularly in monolayers of HEp-2 epithelial cells. After using gentamicin to eradicate extracellular bacteria, different concentrations of azithromycin were added to the infected cells and samples were taken after 0, 4, 8 and 24 h. Infected cells not exposed to antibiotic were included as controls. The MIC of azithromycin to the H. pylori was 0.25 mg/L and the MBC 0.5 mg/L in a broth dilution plate count method. A bactericidal effect was observed on intracellular H. pylori, with inhibition increasing with increasing azithromycin concentrations. However, extracellular concentrations of 200 x MBC were necessary to achieve intracellular killing. Our results show that azithromycin is active against intracellular H. pylori suggesting that it might be possible to exploit this activity when treating infections due to the organism.

Comparative studies on microbial activity in Azotobacter chroococcum inoculated wheat (Triticum aestivum) rhizosphere by plate count and manometric methods

The serial dilution plate count method has been generally used to estimate the number of microorganisms in the rhizosphere soil. However, this method gives a partial view of the range of organisms and the microbial biomass in the soil. In view of finding an alternate method to estimate the microbial activity, in the present study the plate count method was compared with the respiratory activity of rhizosphere soil measured by a manometric technique. Two strains of Azotobacter chroococcum were used for seed inoculation of three varieties of wheat. Periodically, the titres of different groups of microorganisms, viz. bacteria, actinomycetes, fungi and Azotobacter were determined by the plate count method and the results obtained were correlated with the respiratory activity of rhizosphere soil.

Ampicillin killing curve patterns for ampicillin-susceptible nontypeable Haemophilus influenzae strains by the agar dilution plate count method

Ampicillin killing curve patterns for 20 strains of ampicillin-susceptible nontypeable Haemophilus influenzae were determined by the agar dilution plate count method. The paradoxical effect was detected in the 24-h killing curve patterns for each strain. For the biphasic effect, minimum survivor percentages (maximum killing) occurred over a narrow range of ampicillin concentrations immediately above the MIC, with survivor percentages then rising rapidly to peak at approximately 1-log10-unit increment higher. The 24-h minimum survivor percentages for the 20 strains ranged from approximately 0.01% (rapid killing) to greater than 10% (slow killing). In comparison with the previous results for typeable strains, the present findings suggest that nontypeable stains are, on average, killed much more slowly. Based on the initial 24-h killing curve patterns for the 20 strains, 4 strains were selected as putative representatives of the range of bactericidal responses encountered. These strains were then studied to examine the reproducibility of the 24-h patterns and to determine sequential killing curves. These patterns were found to be reproducible and served to characterize the relative killing responses of the strains. In the sequential studies of three of the four strains, tiny colonies having the gross and microscopic characteristics of L-forms were found to be present on the agar dilution plate count plates prior to the application of penicillinase at 48 and 72 h. Such colonies reverted to vegetative forms within 24 to 48 h after application of penicillinase to the panels. Of particular interest was the observation that the paradoxical effect was manifested both by the L-form colonies and by the reverted vegetative colonies. The late development of L-forms was observed for both rapidly and slowly killed strains.

Effect of temperature on inoculum as a potential source of error in agar dilution plate count bactericidal measurements.

The effect of increased temperature on Staphylococcus aureus during the inoculation step of the agar dilution plate count method was investigated as a possible cause of artificially high persister counts. For some isolates, exposure of the inoculum to increased temperature resulted in higher persister counts and diminution or loss of the paradoxical effect. The persister patterns for three representative S. aureus isolates are presented to illustrate the strain- and temperature-dependent nature of the phenomenon. For any isolate, the net effect appears to be caused by an interplay of temperature-induced inoculum loss and temperature-induced cell division cycle blockage. A modification of the agar dilution plate count inoculation step to circumvent such problems is described.

Ampicillin killing curve patterns of Haemophilus influenzae type b isolates by agar dilution plate count method.

The responses of 20 ampicillin-susceptible Haemophilus influenzae type b clinical isolates to the bactericidal action of ampicillin were studied by using a modified agar dilution plate count method. A well-defined paradoxical effect was observed in each of the 24-h killing curve patterns for 19 of the 20 isolates, the remaining isolate showing a less-well-defined but suggestive paradoxical effect after 48 h of ampicillin exposure. For each isolate, the lowest 24-h persister percentage representing maximum killing (paradoxical trough percentage) occurred over a narrow range of concentrations immediately above the MIC, with such paradoxical trough percentages for the 20 isolates ranging from greater than 0.1 to less than 0.001%. Three isolates selected to represent slow, intermediate, and rapid responses were investigated by repetition of 24-h studies and by determination of expanded killing curve patterns. Resultant agar dilution plate count killing curve patterns were found to be reproducible and strain dependent and served to characterize each isolate. The paradoxical effect became more distinct with the prolongation of ampicillin action. Maximum killing was again evident for a narrow range of ampicillin concentrations immediately above the MIC, with persister percentages rising rapidly over the next few ampicillin concentrations to peak at 1 to 2 log10 increments higher than trough percentages. Based on the broad range of responses observed for the 20 isolates, the consistent presence of the paradoxical effect, and the time-dependent nature of bactericidal action, we suggest that the MBC and MBC/MIC ratios are inadequate indices of bactericidal action and that the all-or-none concept of "antimicrobial tolerance" should be abandoned.

Oxacillin killing curve patterns of Staphylococcus aureus isolates by agar dilution plate count method.

The bactericidal dynamics of oxacillin against four Staphylococcus aureus isolates with known 24-h "persister" percentages were studied by using the agar dilution plate count method. Isolates were selected to provide a representative spectrum whose individual 24-h trough intrinsic persister percentages ranged from greater than 1 to less than 0.01%. Resultant agar dilution plate count method killing curve patterns were found to be reproducible and served to characterize each isolate. The paradoxical effect was observed for each isolate, with paradoxical peaks tending to develop and diminish sequentially during the course of oxacillin action. The observed strain-dependent dynamics of oxacillin killing underscore the artifactual nature of the so-called tolerance phenomenon and negate the usefulness of MBCs and MBC/MIC ratios for the characterization of S. aureus isolates.

Influence of technical factor variations during inoculum preparation on the agar dilution plate-count method for quantitation of Staphylococcus aureus oxacillin persisters

The possible introduction of error into Staphylococcus aureus oxacillin intrinsic persister measurements by the agar dilution plate-count method resulting from technical factor variations associated with inoculum preparation was investigated. pH stability and exponential growth were shown to be present at the time of inoculum standardization. The use of glass or plastic tubes and the contamination of tube walls during inoculum broth culture produced no differences in test results. For S. aureus and oxacillin, the agar dilution plate-count method is a new and reliable approach to the quantitative study of bacterial inhibition and killing.

The effects of grazing or cutting a perennial ryegrass and white clover sward on the microflora of the soil

An investigation was carried out on soils from under perennial ryegrass and white clover swards comparing the effect of grazing and cutting on the soil microflora. The dilution plate count method was used with three different media: soil extract, cellulose and plant protein agars. Over a 3-yr period changes occurred in the microflora of the surface 2.5 cm layer of the soil; numbers of actinomycetes and bacteria generally increased under grazing whereas fungi increased under cutting. An increase in the numbers of propagules of actinomycetes and bacteria as shown by colony counts and in biological activity as revealed by the determination of carbon dioxide and mineral-nitrogen produced during incubation of the soil may have been due to increases in the amount of organic matter in the soil under grazed swards.

NITRIFICATION UNDER AND AFTER ALFALFA, BROME, TIMOTHY, AND WESTERN RYE GRASS: II. SOIL MICROBIOLOGICAL ACTIVITY

Soil microbiological activity was measured for eight seasons, 1927 to 1934, in order to study some underlying causes of the comparative effects of alfalfa, brome, timothy, and western rye grass on the yield and nitrogen content of succeeding wheat crops.When previously fallowed soil was seeded to alfalfa and grasses, the moisture and nitrate content of the soil were reduced, and generally remained at a relatively low level until the sods were plowed up. In the drier seasons the nitrates were reduced to a very low level or disappeared entirely in the grass and alfalfa plots. The nitrate content of the alfalfa plot soils was generally greater than that of the grass plots, and the brome grass plots were generally lower in nitrates than the timothy and western rye grass plots. The wheat plot soils generally contained more nitrate than the grass and alfalfa plots, especially in the drier seasons. When the sods were plowed up, nitrates accumulated in the alfalfa plots to a greater extent than in the grass plots and to a lesser extent generally in the brome plots than in the timothy and western rye plots. The greater nitrate content of the soil under wheat following alfalfa was observed for a period of three or four years in separate sets of plots plowed up two years apart. The nitrate level of the soil under wheat had a tendency to drop in mid-summer, often reaching its lowest point in July. The fallow plot soils were always higher in moisture than any of the cropped plots at the end of each season, and higher in nitrates in the latter half of each season.The concentration of water-soluble phosphorus was greatest in the surface soil and seemed to be slightly higher under alfalfa and grasses than under wheat, but the total concentration was small and there was no very definite seasonal trend.The numbers of fungi and bacteria, as determined by the plate count method for five seasons, 1929 to 1933, did not fluctuate very much in certain plots and seasons, but fluctuated greatly in others. The greatest fluctuations in fungal counts were observed under the first crop of wheat following brome grass, and in bacterial counts also under the first crop of wheat following sods, in the relatively moist season of 1931. Plate counts of actinomycetes did not fluctuate very greatly during the one season in which they were determined. The numbers of fungi were generally higher in the alfalfa plots than in the grass plots, but the differences between the grasses were apparently insignificant. Under the first crop of wheat following sods, large Mucor colonies predominated in the alfalfa plot soil plates and the counts were relatively low. Brome grass plot soils gave by far the highest counts of fungi, which consisted mainly of small Penicillium colonies, under the first crop of wheat following sods in 1931. The differences between numbers of bacteria in the alfalfa and grass plots were not very significant. The moisture content of the surface soil fluctuated greatly during most of the seasons. There was evidence of correlation between fluctuations in bacterial numbers and moisture, especially in certain seasons, in all the cropped soils. There was less evidence of such correlation in the case of fungi, except under the first crop of wheat following brome grass in 1931. Fallow soil, though normally higher in moisture content in the latter part of each season, did not differ significantly from the grass-cropped soils in counts of fungi and bacteria. Although surface samples usually gave the highest counts, the deeper soil samples (to a depth of three feet) gave fairly high counts of both fungi and bacteria. During the season of 1930, amoebae were determined by the dilution plate count method; more than 1,000 and less than 10,000 per gram were nearly always found in both cropped and fallow soils.The total nitrogen content of the plot soils showed considerable variation (owing to random sampling) from year to year, but no definite trend downwards or upwards during this period of seven years. The surface soil in every case contained most nitrogen and the subsoil least.