Spiral Plate Method for Bacterial Determination

A method is described for determining the number of bacteria in a solution by the use of a machine which deposits a known volume of sample on a rotating agar plate in an ever decreasing amount in the form of an Archimedes spiral. After the sample is incubated, different colony densities are apparent on the surface of the plate. A modified counting grid is described which relates area of the plate of volume of sample. By counting an appropriate area of the plate, the number of bacteria in the sample is estimated. This method was compared to the pour plate procedure with the use of pure and mixed cultures in water and milk. The results did not demonstrate a significant difference in variance between duplicates at the α = 0.01 level when concentrations of 600 to 12 × 10⁵ bacteria per ml were used, but the spiral plate method gave counts that were higher than counts obtained by the pour plate method. The time and materials required for this method are substantially less than those required for the conventional aerobic pour plate procedure.     

Heat resistance of microorganisms: an improved method for survival counting

A modification of the plate count method is described which brings important advantages upon the normal plate-counting system as used in heat resistance determinations (D_t). It saves cost and time, and makes less liable the appearance of handling errors. Precision of counting in the range of 70–20000 colonies per plate was studied and D_t values obtained by survival counting at three ranges of number of colonies per plate were compared. Precision of this system was shown to be as high as that of the pour plate method as normally used, and no significant difference was found among thermal death rates (D₁₀₅ values) obtained at the three ranges of number of colonies per plate tested.     

Evaluation of a radiometric method for studying bacterial activity in the presence of antimicrobial agents

In a study involving 2760 tests, the BACTEC semi-automatic radiometric method which measures bacterial metabolic activity and produces a BACTEC growth index, was compared with two conventional methods commonly used for determining growth, absorbance and viable counts. In 92% of radiometry tests the suppression of growth was inversely related to the antibiotic concentration. This compared with 83% for absorbance and 63% for viable counts. The radiometric method was found to be more rapid, easier to use and more reproducible in determining the effect of antibiotics on the activity of bacteria than viable counting or absorbance methods.     

Use of Growth Indices from Radiometric Culture for Quantification of Sheep Strains of Mycobacterium avium subsp. paratuberculosis

A simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains of Mycobacterium avium subsp. paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log₁₀ inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration of M. avium subsp. paratuberculosis from field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of ±1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.     

Cost-effectiveness of blood agar for isolation of mycobacteria

Background

Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture.

Methodology

We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques.

Conclusions

Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.     

Statistical evaluation of electronic and plate counts for estimating bacterial populations

Bacteria from cultures of Staphylococcus aureus, Pseudomonas geniculata, and Rhodotorula glutenis were counted by use of an electronic counter and by plate counts from broth cultures at selected periods from 0 to 24 hr. Variations in the two methods were noted, and the results were compared after calculating correlations, coefficients of variation, and nested analyses of variance. It was not possible to determine the absolute accuracy of the two methods; however, the precision of the results obtained with an electronic counter was better than that obtained with plate counting. It appears that electronic counting, because of ease and convenience, provides better comparative counts at a particular stage of growth, especially during the early stages of the growth period, whereas plate counting yields the best results in determining growth curves.     

Impact of reduced vancomycin susceptibility on the therapeutic outcome of MRSA bloodstream infections

Background

Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.

Methods

In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.

Results

Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.

Conclusion

The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.     

Application of Amido black staining to enumerating bacteria grown on membrane filters

A method to detect and enumerate bacterial colonies grown on membrane filters (MF) was described. The colonies were stained with an ethanolic solution of 0.1% Amido black 10B. The procedure yielded the rapid detection of colonies as compared to a conventional plate counting method.     

Species-Specific Oligonucleotides for Enumeration of Pseudomonas putida F1, Burkholderia sp. Strain JS150, and Bacillus subtilis ATCC 7003 in Biodegradation Experiments

Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism, Pseudomonas putida F1 and Burkholderia sp. strain JS150, and a third, Bacillus subtilis ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.     

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ~10⁸ bacteria/ml (equivalent to ~10⁷ CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Spoilage Bacteria in Canned Foods: II. Sulfide Spoilage Bacteria in Canned Mushrooms and a Versatile Medium for the Enumeration of Clostridium nigrificans

Clostridium nigrificans was found to be a spoilage organism of canned mushrooms in Taiwan. A modified beef extract tryptone iron medium, both in broth and agar form, was designed for the detection and recovery of the organisms. A procedure of simple plate counting method of C. nigrificans was established.     

An Optimized SYBR Green I/PI Assay for Rapid Viability Assessment and Antibiotic Susceptibility Testing for Borrelia burgdorferi

Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.     

Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BackgroundAutomated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell–based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed.MethodsMicroorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry.ResultsThe BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h).ConclusionsWhen a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).     

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [³H]benzo(a)pyrene and 7,12-[12-¹⁴C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.     

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.     

Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P < 0.05) with a high correlation coefficient (R² = 0.82) between the E. coli counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques.     

Effects of Calcium on Sugar Transport in Azotobacter vinelandii

A fast and environmentally safe procedure was used to study sugar uptake by Azotobacter vinelandii. Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10⁸ cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in A. vinelandii. Results showed that Ca²⁺ at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent K_m of sucrose transport. Higher Ca²⁺ concentrations inhibited sucrose uptake in this organism.     

Necrotrophic Growth of Legionella pneumophila

This study examined whether Legionella pneumophila is able to thrive on heat-killed microbial cells (necrotrophy) present in biofilms or heat-treated water systems. Quantification by means of plate counting, real-time PCR, and flow cytometry demonstrated necrotrophic growth of L. pneumophila in water after 96 h, when at least 100 dead cells are available to one L. pneumophila cell. Compared to the starting concentration of L. pneumophila, the maximum observed necrotrophic growth was 1.89 log units for real-time PCR and 1.49 log units for plate counting. The average growth was 1.57 ± 0.32 log units (n = 5) for real-time PCR and 1.14 ± 0.35 log units (n = 5) for plate counting. Viability staining and flow cytometry showed that the fraction of living cells in the L. pneumophila population rose from the initial 54% to 82% after 96 h. Growth was measured on heat-killed Pseudomonas putida, Escherichia coli, Acanthamoeba castellanii, Saccharomyces boulardii, and a biofilm sample. Gram-positive organisms did not result in significant growth of L. pneumophila, probably due to their robust cell wall structure. Although necrotrophy showed lower growth yields compared to replication within protozoan hosts, these findings indicate that it may be of major importance in the environmental persistence of L. pneumophila. Techniques aimed at the elimination of protozoa or biofilm from water systems will not necessarily result in a subsequent removal of L. pneumophila unless the formation of dead microbial cells is minimized.     

Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture

The BACTEC MGIT 960 system is increasingly used to culture Mycobacterium tuberculosis. We evaluated the performance of the new immunochromatographic assay BD MGIT TBc Identification Test (TBc ID) for the rapid identification of M. tuberculosis complex in clinical samples when performed directly from BACTEC MGIT 960 culture positive for acid-fast bacilli (AFB).Of 92 cultures evaluated, the sensitivity and specificity of the TBc ID test was 98.5% and 100%, respectively compared to sequencing of the 16S rRNA gene. One culture that was TBc ID test negative but that was identified as M. tuberculosis by 16S rRNA sequencing was confirmed to have a mutation in the mpt64 gene.The TBc ID test is an easy and sensitive method for the identification of M. tuberculosis complex in liquid culture medium, does not require a high level of skills, neither any additional specific equipment and gives results in 15 min, which provide a good alternative for the rapid identification of M. tuberculosis complex in liquid medium.     

Collaborative trial of the direct epifluorescent filter technique (DEFT), a rapid method for counting bacteria in milk

The direct epifluorescent filter technique (DEFT) is a new rapid method which uses membrane filtration and epifluorescent microscopy for counting bacteria in milk. A collaborative trial of the DEFT was conducted between six laboratories. Each laboratory obtained a highly significant relationship between the DEFT count and plate count with a correlation coefficient generally > 0.9 but there were significant differences between these relationships. The repeatability of the DEFT, although ca 1·5 times worse than that of the plate count, was of a level acceptable in practice. Reproducibility of the DEFT was ca 3 times that of the plate count. This poor reproducibility was probably mainly due to counting errors. Possible reasons for this and ways of reducing counting errors are discussed.