Neutralization efficacy of Dey-Engley medium in testing of contact lens disinfecting solutions

A quantitative assay for the demonstration of neutralizer efficacy was developed to monitor contact lens disinfecting solutions. Adequate neutralization of disinfecting agents is essential to the accurate determination of disinfecting activity with time. This method employed the recovery of small numbers of micro-organisms from neutralizing medium containing a disinfectant. A statistical estimation of significance between treatments demonstrated that Dey-Engley medium (DE; Difco) was generally effective when tested as an agar growth medium with several bacterial test organisms. DE medium from another vendor was less effective, underscoring the need for laboratory quality control and monitoring. DE agar (Difco) adequately neutralized all solutions tested at a 1:20 dilution. The solutions included those containing Dymed (polyaminopropyl biguanide, 0.00005%), chlorhexidine (0.005%), Polyquad (0.001%), chlorhexidine (0.005%) and thimerosal (BP, 0.001%), thimerosal (BP, 0.002%) and Tris(2-hydroxyethyl) tallow ammonium chloride (0.013%), and a solution preserved with 115 ppm benzalkonium chloride (BAK). A modification of this medium was developed which retained virtually all of the neutralizing efficacy for the solutions tested while allowing the use of automated testing procedures.     

Neutralization efficacy of Dey-Engley medium in testing of contact lens disinfecting solutions

A quantitative assay for the demonstration of neutralizer efficacy was developed to monitor contact lens disinfecting solutions. Adequate neutralization of disinfecting agents is essential to the accurate determination of disinfecting activity with time. This method employed the recovery of small numbers of micro-organisms from neutralizing medium containing a disinfectant. A statistical estimation of significance between treatments demonstrated that Dey-Engley medium (DE; Difco) was generally effective when tested as an agar growth medium with several bacterial test organisms. DE medium from another vendor was less effective, underscoring the need for laboratory quality control and monitoring. DE agar (Difco) adequately neutralized all solutions tested at a 1:20 dilution. The solutions included those containing Dymed^TM (polyaminopropyl biguanide, 0·00005%), chlorhexidine (0·005%), Polyquad® (0·001%), chlorhexidine (0·005%) and thimerosal (BP, 0·001%), thimerosal (BP, 0·002%) and Tris(2-hydroxyethyl) tallow ammonium chloride (0·013%), and a solution preserved with 115 ppm benzalkonium chloride (BAK). A modification of this medium was developed which retained virtually all of the neutralizing efficacy for the solutions tested while allowing the use of automated testing procedures.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

A comparison between total viable count by spread plating and AquaPlak for enumeration of bacteria in water from a shrimp farm

A new system named AquaPlak^® designed for evaluating heterotrophic bacteria and those belonging to the genus Vibrio was compared to the standard method of total viable count by spread plating. Water samples from a shrimp farm were evaluated by the two procedures over a 3-month period. The spread plating technique gave significantly higher viable counts for both Vibrio and heterotrophic bacteria than the AquaPlak^® system.     

Microtiter Method for the Evaluation of Viable Cells in Bacterial Cultures

A microtiter technique was investigated as a means of evaluating viable cells in bacterial cultures. Parallel experiments were performed employing the conventional agar plate method along with the microtiter procedure. Statistical analyses showed that the correlation between the two methods was highly significant. With this new method, many samples were analyzed simultaneously, and readable results were obtained in 12 to 15 hr. Other advantages of this method were substantial savings of time, space, and materials. Also, the applicability of this method to estimates of mixed bacterial populations was demonstrated by studying the associative growth of two bacterial cultures.     

Fluorescence microscopy procedure for quantitation of yeasts in beverages.

Existing methods for quantitating yeasts in beverages include time-consuming plate counts that detect only living cells and hemacytometer counts that are reliable only at very high concentrations (e.g., 10(6) to 20 X 10(6) cells per ml). The new method described here involves the use of fluorescence microscopy with the fluorescent stain aniline blue to differentiate yeasts (and other fungi) from backgrounds for easy counting and also may be used in conjunction with membrane filtration to concentrate yeasts from liquids before cell enumeration. Recoveries averaged 91.5% for beverages spiked with levels of 500 to 600,000 organisms per ml. The correlation coefficient of count to spike level was 0.996.     

Fluorescence microscopy procedure for quantitation of yeasts in beverages

Existing methods for quantitating yeasts in beverages include time-consuming plate counts that detect only living cells and hemacytometer counts that are reliable only at very high concentrations (e.g., 10(6) to 20 X 10(6) cells per ml). The new method described here involves the use of fluorescence microscopy with the fluorescent stain aniline blue to differentiate yeasts (and other fungi) from backgrounds for easy counting and also may be used in conjunction with membrane filtration to concentrate yeasts from liquids before cell enumeration. Recoveries averaged 91.5% for beverages spiked with levels of 500 to 600,000 organisms per ml. The correlation coefficient of count to spike level was 0.996.     

Longitudinal analysis of early HIV-1-specific neutralizing activity in an elite neutralizer and in five patients who developed cross-reactive neutralizing activity

We previously established that at 3 years postseroconversion, ~30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.     

Evaluation of procedures to determine absolute density of Plasmodium vivax ookinetes

The ookinete is the key determinant of infection within the mosquito vector, yet there are few population studies of ookinetes in nature. This investigation compared different techniques used to estimate ookinete densities in mosquitoes. Laboratory-reared Anopheles dirus mosquitoes were fed on gametocytemic blood drawn from 7 Plasmodium vivax patients at a malaria clinic in Mae Sot, Thailand. At 20-26 hr, bloodmeals were excised. Three techniques were evaluated, i.e., hemacytometer counts under phase-contrast microscope, Giemsa staining of bloodmeal smears, and immunofluorescent staining with a monoclonal antibody specific against the 25-kDa antigen expressed on the surface of P. vivax zygotes and ookinetes. Additional mosquitoes were dissected at day 10 for oocysts. The hemacytometer method was the simplest and quickest method but lacked precision at low ookinete densities. Immunofluorescent staining was the most sensitive, accurate, and the only method that enabled unequivocal detection of zygotes. Bloodmeals contained a mixture of zygotes, retorts, and mature ookinetes, indicating that postzygotic development of P. vivax in A. dirus was asynchronous. The conversion efficiency of zygotes/ookinetes to oocysts varied among patients and was independent of zygote-ookinete density, suggesting that variations in host blood composition, e.g., antibodies, drugs, etc., may influence the success of zygote-ookinete development.     

Survival and growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

A rapid twofold dilution method for microbial enumeration and resuscitation of uninjured and sublethally injured bacteria

AIMS: A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the twofold dilution method (2FD), was developed and evaluated. METHODS AND RESULTS: Following twofold serial dilution of samples in a 96 well microtiter plate, double strength selective broth or nonselective broth was added to each well. For resuscitation of heat-injured (55 degrees C for 10 min) coliforms, the selective broth was added to the wells after 3 h preresuscitation time in buffered peptone water. The results of the 2FD were compared to plating methods for total and coliform plate counts from mixed cultures and beef carcass surface tissue samples. CONCLUSION: The 2FD method results were not significantly different for uninjured cells (P > 0.05) from those obtained using Petrifilm and standard plating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R(2)=0.98 for total counts and R(2)=0.96 for coliforms from mixed cultures; R(2)=0.98 for total cell counts and R(2)=0.94 for coliforms from faeces inoculated beef carcasses). SIGNIFICANCE AND IMPACT OF THE STUDY: The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to conventional plating methods (P < 0.05).     

Sources of Variability in the Measurement of Fungal Spore Yields

Variability in the production of fungal spores and in the measurement of spore yields was investigated in four species of fungi: Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides, and Acremonium strictum. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean ± 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.     

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.     

Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii

Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at -70 degrees C using dimethyl sulfoxide (DMSO; 5 or 10%) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg2+ at 4 degrees C remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at -70 degrees C with DMSO (5 or 10%) or 40% glycerol, but not 80% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at -70 degrees C.     

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.     

Bioluminescent Most-Probable-Number Method To Enumerate lux-Marked Pseudomonas aeruginosa UG2Lr in Soil

Bioluminescence measured with a luminometer and charge-coupled device was an effective marker in most-probable-number assays for luxAB-marked Pseudomonas aeruginosa UG2Lr in soil. Most-probable-number assays with microtiter plate wells and luminometer tubes gave estimates for UG2Lr that were similar to viable colony counts. Both methods detected five cells per g of soil.     

MINIATURIZED ANAEROBIC CULTIVATION METHODS FOR RECOVERY OF CLOSTRIDIUM SPOROGENES FROM MEAT

Two new miniaturized methods (sandwiched microtiter plate [SMP] and mini-tube) for recovery of Clostridium sporogenes from food samples were developed and evaluated. One hundred microliter of SFP (Shahidi Ferguson Perfringens) agar and 10 üL of diluted food sample were used in the SMP anaerobic system. The samples were sandwiched between two sterile microtiter plates to create an anaerobic environment. Black colonies in the sandwiched wells were counted as C. sporogenes. In case of mini-tube system, 1 mL of SFP agar (45C) containing diluted food sample was aspirated into a 1.2 mL sterile pipet and allowed to solidify in place. The two ends are sealed to create an anaerobic environment. Black colonies were counted directly through the plastic tube. C. sporogenes was inoculated into ground beef samples for recovery study. The recovery rates in SFP agar, using the SMP and mini-tube method were compared with the double-tube method. Organisms were recovered more in the double-tube than with SMP method and mini-tube method after 24–30 h incubation. However, the final counts at 48 h were similar in all methods from the food samples. These new simple methods have potential use for recovery of Clostridium spp.     

BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm

The medical device-related infections are frequently a consequence of Staphylococcus biofilm, a lifestyle enhancing bacterial resistance to antibiotics. Antibiotic susceptibility tests are usually performed on planktonic forms of clinical isolates. Some methods have been developed to perform antibiotic susceptibility tests on biofilm. However, none of them counts bacterial inoculum. As antibiotic susceptibility is related to bacterial inoculum, the test results could be mistaken. Here, a new method, BioTimer Assay (BTA), able to count bacteria in biofilm without any manipulation of samples, is presented. Moreover, the BTA method is applied to analyze antibiotic susceptibility of six Staphylococcus strains in biofilm and to determine the number of viable bacteria in the presence of sub-inhibitory doses of four different antibiotics. To validate BTA, the new method was compared to reference methods both for counting and antibiotic susceptibility tests. A high agreement between BTA and reference methods is found on planktonic forms. Therefore, BTA was employed to count bacteria in biofilm and to analyze biofilm antibiotic susceptibility. Results confirm the high resistance to antibiotics of Staphylococcus biofilm. Moreover, BTA counts the number of viable bacteria in the presence of sub-inhibitory doses of antibiotics. The results show that the number of viable bacteria depends on sub-inhibitory doses, age of biofilm and type of antibiotic. In particular, differently to gentamicin and ampicillin, sub-inhibitory doses of ofloxacin and azithromycin reduce the number of viable bacteria at lower extent in young than in old biofilm. In conclusion, BTA is a reliable, rapid, easy-to-perform, and versatile method, and it can be considered a useful tool to analyze antibiotic susceptibility of Staphylococcus spp. in biofilm.     

A New Method for the Detection of Neutralizing Antibodies against Mumps Virus

Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.