The differentiation of Staphylococcus aureus from other Micrococcaceae isolated from bovine mammary glands

Haemolysin production, the slide coagulase test and the tube coagulase test were assessed for their capability to differentiate Staphylococcus aureus among other Micrococcaceae in 199 isolates from udders of cows in herds with a low bulk milk somatic cell count. The API-Staph test was used as a reference.
Haemolysin production was less effective in identifying Staph. aureus among Micrococcaceae than a combination of other tests. Differences were found in the predictive values of results from diagnostic protocols in which the slide coagulase test was performed on all Micrococcaceae, or on β-haemolysin-negative Micrococcaceae only. Diagnostic protocols in which haemolysin production was combined with the results of the other tests resulted in excellent diagnostic performance and a reduction in diagnostic procedures. Recommendations for routine Staph. aureus identification in bovine mastitis bacteriology are given.     

A time-saving thin-layer chromatography plate-scraping system

A system for efficiently scraping thin-layer chromatography plates is described. A height-adjustable rack for holding the thin-layer chromatography plate is constructed of transparent acrylic plate. The base provides a surface on which to slide vial holders and also gives stability to the rack. The plate is scraped with a single-edge razor blade cut to an appropriate width and the scrapings fall into a polypropylene funnel which sits on a vial that is in either a test tube rack or a cassette from a scintillation counter. The plate-scraping system reduces scraping time by more than 60% and increases the accuracy of results.     

Microagglutination Procedures for Febrile Agglutination Tests

Febrile agglutination tests were done by using as antigens Brucella abortus, Salmonella group D, Proteus OX19, and Pasteurella tularensis. Comparison of results from 23 sera showed that the microtechnique, rapid slide, and test tube methods gave similar titers, although those from the microtechnique were generally higher. The sensitivity of the microtechnique depended upon the concentration of antigen, and, to obtain reproducible results, the optimal concentration of antigens had to be determined by preliminary titrations against specific, positive control antisera. Readability of reactions in the microtechnique was enhanced by adding the dye Safranin O to diluent for antigen and by use of V-type, rather than U-type, microtiter plates. Tests were also done to determine the effects of dye and salt concentrations, pH, and temperature of incubation upon the titer of agglutinations by the microtechnique. Our results indicated that the microtechnique could be used for agglutination tests involving febrile antigens. The procedure is less time-consuming than the tube method and requires less antigen and serum than the latter method or the rapid slide method.     

Use of microplate cell culture and enzyme immunoassay in titration of serum neutralizing antibody against Hochi strain of serotype 4 human rotavirus

The tube neutralization test read by enzyme immunoassay developed by Wyatt et al. (1983) for serotype determination of human rotavirus was modified so as to use stationary cultures of MA104 cells in a microtiter plate instead of roller tube cultures. Sera obtained from different age groups were titrated for neutralizing antibody against serotype 4 human rotavirus Hochi strain by this test and the results were compared with those obtained by the plaque neutralization test. There was a good correlation between the titers obtained by the two tests and the age distribution pattern of serotype 4 neutralizing antibody was similar to those of serotype 1 and 3 antibodies previously reported.     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

An improved micromethod for infectivity assays and neutralization tests of dengue viruses

An improved micromethod for infectivity assays and neutralization (N) tests of dengue (DEN) type 1-4 viruses was developed, using 96-well plates and the PAP (peroxidase-antiperoxidase) staining technique. The foci formed on BHK-21 cell monolayers in wells of the plate were readily countable under an ordinary stereomicroscope. This micromethod has the advantages over the micromethod of the Lab-Tek 8 chamber slide system of lower cost, requirement for smaller volumes of test sera and applicability to larger number of serum specimens for N tests of DEN viruses.     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

Evaluation of four slide test kits for the detection of human chorionic gonadotropin in urine

Three “indirect-type” slide tests utilizing the principle of hemagglutination inhibition and one new “direct-type” slide test employing direct agglutination were evaluated for their sensitivity in detecting human chorionic gonadotropin (HCG) in urine. The results of positive tests in a group of woman in very early pregnancy were correlated with the “days after last menses”. In this series the direct slide test was the most accurate. A control must be used with each direct test to indicate interfering substances and when such are present a different test must be used. All tests were found to be of the relative sensitivity stated by the manufacturer.     

Identification procedure for Pasteurella pneumotropica in microbiologic monitoring of laboratory animals.

Discrepancies have been recognized in the identification of Pasteurella pneumotropica between testing laboratories. To determine the causes of the differences and to propose a reliable identification procedure for P. pneumotropica, a working group was organized and 69 isolates identified or suspected as P. pneumotropica were collected from 8 laboratories in Japan. These isolates were examined by colony morphology, Gram-staining, the slide agglutination test using two antisera (ATCC35149 and MaR), two commercially available biochemical test kits (ID test, API20NE) and two primer sets of PCR tests (Wang PCR, CIEA PCR). The 69 isolates and two reference strains were divided into 10 groups by test results. No single procedure for P. pneumotropica identification was found. Among tested isolates, large differences were not observed by colony morphology and Gram-straining except for colony colors that depended on their biotypes. Sixty-eight out of 69 isolates were positive by the slide agglutination test using two antisera except for one isolate that tested with one antiserum. The ID test identified 61 out of 69 isolates as P. pneumotropica and there was no large difference from the results of CIEA PCR. From these results, we recommend the combination of colony observation, Gram-straining, the slide agglutination tests with two antisera and biochemical test using the ID test for practical and reliable identification of this organism.     

氧化型革兰氏阴性杆菌一管多用鉴定培养基的研制和应用

研制出一种一管多用鉴定培养基。在一支单管培养基上,可根据高层是否产气,斜面有无荧光,高层、斜面与凝固水附近的颜色变化五个特征,结合氧化酶试验与鞭毛染色结果,同步将临床常见的氧化型革兰氏阴性杆菌鉴定到属或种,具有广泛的实用性。     

Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

A study in test reproducibility between laboratories: Report of a Pseudomonas Working Party

The results are reported of a collaborative study in laboratories of 17 tests commonly used for pseudomonads, together with statistical analysis of the results in the form of analyses of variance. The studies involved 59 strains. The following tests showed good or reasonably good consistency between different laboratories: motility if checked by a Craigie tube, growth at 4 C, production of fluorescin, oxidation of glucose and sucrose, gluconate oxidation, hydrolysis of aesculin, arginine and casein, and gelatin hydrolysis by the tube method. The following were less satisfactory: shape, size and arrangement of organismis, growth at 42 C, oxidase, acid from lactose, reduction of nitrate, acetic acid from ethanol, gelatin hydrolysis by the plate method, and the egg yolk reaction. The following showed poor consistency or noticeable difficulties in performing or interpreting results of the test: growth at 37 C, the denitrification test of Stanier, Palleroni and Doudoroff, and hydrolysis of urea. However, with several of the unsatisfactory tests the replicates within a laboratory showed much better reproducibility. This (together with other evidence on the importance of exact control of variables such as temperature, and time of reading, and the occurrence of mutants in some strains) suggests that careful attention to standardization may give much better testing methods. The considerable value of statistical analyses in such work is discussed.     

Practical Confocal Microscopy and the Evaluation of System Performance

The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by the user's laboratory. To achieve better performance from the equipment, it is necessary to run a series of tests to ensure that the optical machine is functioning properly. We have devised these methods on the Leica TCS-SP and TCS-4D systems. Tests measuring field illumination, lens clarity, laser power output, dichroic functioning, spectral alignment, axial resolution, laser power stability, machine performance, and system noise were derived to test the Leica laser scanning confocal microscopy system. These tests should be applicable to other manufacturers' systems as well. The relationship between photomultiplier tube (PMT) voltage, laser power, and averaging using a 10-microm-diameter test bead has shown that the noise (coefficient of variation of bead intensity, CV) in an image increases as the PMT increases. Therefore increasing the PMT setting results in increased noise. For ideal image quality, it appears that it is better to decrease the PMT setting and increase laser power, as noise generated by high PMT settings will reduce the image quality far more than the bleaching caused by higher laser power. Averaging can be used to improve the image at high PMT values, provided the sample is not bleached by repeated passes of the laser.     

Microtiter Plate Agglutination Test for Salmonella Antibodies

Similar results were obtained when testing human sera for Salmonella antibodies by the tube agglutination test and by the Microtiter plate agglutination test. The plate test was easier to perform and saved time, space, antigen, and serum.     

Locating Iodine in Tissues Autographically, Especially after Fixation by Freezing and Drying

The ability of radioactive elements to affect photographic emulsions enables the detection of radioactive iodine in the thyroid. By placing unstained histological sections of a thyroid (from an animal treated with radioactive iodine) in contact with the gelatin side of medium lantern slide plates, each accumulation of radioactive iodine in the section affects the photographic plate. After exposures prolonged for several days to several weeks depending on the amount of radioactivity in the tissues, the plate is developed and fixed by routine photographic methods. The histological section is stained and may be compared under the microscope to the reactions on the plate or “autographs”.

In an attempt to detect the location of the inorganic iodine which is displaced during fixation and embedding by ordinary methods because of its solubility, a simplified freezing-drying technic for fixation was devised which, at least with the thyroid, yielded well fixed sections. The quick freezing was obtained with acetone-dry-ice mixtures; and the drying was performed at -25° to -30° C. Preliminary addition of paraffin to the tube in which the drying was performed made possible the inclusion in vacuum by heating the tube when drying was completed. The tissue could then be sectioned at 10ju on the microtome. The slides were placed on photographic plates for detection of radioactive iodine as indicated above. Before staining, the sections were treated with absolute alcohol for denaturation of the proteins.     

A simple modification of the Baermann method for diagnosis of strongyloidiasis

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37 degrees C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.     

The Use of Acenaphthene in Pollen Tube Technic

A procedure is described for growing pollen tubes in such a manner that a large number of clearly analyzed figures can be obtained. The pollen grains are sown on an artificial medium of sugar, agar, gelatin, and water, the proportions of each varying with the species of pollen grain used. The medium is smeared on the slide while still hot to insure a thin covering, and the pollen grains are dusted on when the medium has sufficiently cooled and hardened. The slides are placed in a staining dish provided with slide slots and a cover, the inside of the cover and the bottom of the dish being lined with moist, but not wet, filter paper. Acenapthene crystals are lightly sprinkled on the bottom of the dish. The developing pollen tubes are thus exposed to the fumes given off by these crystals with consequent disturbance to the spindle mechanism. As a result, the chromosomes are not crowded on a metaphase plate but are widely separated in the tube facilitating any observations to be made.     

Improvement of the micromethod for the limulus lysate test.

Frauch's micro-slide method was improved to facilitate the endpoint-determination of the Limulus test. Two precise observations, by inverted phase contrast microscopy and with a staining procedure, were newly performed as additions to the slide test. The staining procedure was proposed as an improved method for the Limulus test since it is simple and convenient. In the staining method, bromophenol blue (BPB) solution was used as the staining solution. A negative (-), a strong positive (++) and a weak positive reaction (+) were characterized by a "ring" formation, a "cloud-like" spread of gel and a "spot" in the "cloud" respectively. Since the distinction between (-) and (+) reactions was obvious in the proposed method, determination of the endpoint was easier than in the ordinary tube and Frauch's method. The sensitivity of the present method was equal to or higher than that of other methods. Inverted phase contrast microscopy was utilized to confirm the findings obtained by the staining method. The volume of the lysate used in this method was as little as 1/10 of that used in the tube method.     

Rapid identification of yeasts by serological methods: a combined serological and biological method.

A total of 387 yeasts from the contents of the digestive tracts of domestic animals and poultry were identified by slide agglutination tests using factor antisera and urease tests. The results of this serological test were very satisfactory with respect to accuracy and rapidity, particularly when performed in combination with concomitant physiological tests only for assimilation of inositol and potassium nitrate. It may be concluded that such a combination of serological and biological tests is very useful for identifying yeast strains from various sources.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.