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We describe a simple test for the evaluation of phagocytosis and provide a chart of reference values to evaluate normal phagocytosis by age. We assessed the postnatal maturation of phagocytic function of neutrophils and monocytes. Phagocytosis was evaluated in newborn children delivered vaginally or by cesarean section, infants, preschool children, schoolchildren, and adult subjects. Two drops of blood were placed on a microscope slide and incubated with Saccharomyces cerevisiae yeasts, and phagocytosis was evaluated by microscopy. Our technique showed results comparable to or better than those obtained by other usual techniques. The neutrophils of newborn children delivered by cesarean section showed a phagocytic capacity 45% higher than those of neonates delivered vaginally, whereas neutrophils from children in the latter group showed the lowest phagocytic capacity of all age groups. Phagocytosis by neutrophils reached the levels seen in adults at about the first year of life, while there were no important variations in phagocytosis by monocytes in the different age groups. The technique described is reliable and fast, uses only a few drops of blood, and allows better preservation of cell function due to the minimal manipulation to which the cells are submitted. The delayed maturation of the phagocytic function by neutrophils may account for the high levels of susceptibility of newborn and infant children to bacterial infections. This practical method of assessment of phagocytosis may allow the diagnosis of primary or secondary phagocytic deficiencies to be made more easily and may allow better monitoring and treatment of those with dysfunctions of these cells.

A new microtechnique has been developed for the detection of leptospiral antibodies in serum by the microscopic agglutination test. The test was set up in a microtiter transfer plate held in a transfer plate holder, resting on a transfer plate cover. Live leptospiral antigen was added and a second transfer plate cover was placed over the transfer plate during 2 h of incubation at 32 C. After incubation the bottom cover was removed and the complete unit was placed in a specially designed base plate containing microscope slides (50 by 75 mm). The serum/antigen mixture was ejected on to the microscope slides by means of a sharp tap. The agglutination was then read using a 10 X objective, 10 X eyepieces, and a dry, dark field condenser.

A hemolysis-in-gel test for the demonstration of antibodies to mumps virus is described. The results were compared with those of neutralization tests using a modified microtechnique. In the neutralization test viral replication was demonstrated by the hemadsorption of guinea pig erythrocytes, the visibility of which could be further enhanced by the use of o-tolidine. Good correlation was found between the results of the two techniques. The hemolysis-in-gel test was simple to perform, rapid, sensitive, and shown to be a useful test for the demonstration of mumps antibodies.

A study has been carried out on the Moselle River by means of a microtechnique based on the most-probable-number method for fecal coliform enumeration. This microtechnique, in which each serial dilution of a sample is inoculated into all 96 wells of a microplate, was compared with the standard membrane filter method. It showed a marked overestimation of about 14% due, probably, to the lack of absolute specificity of the method. The high precision of the microtechnique (13%, in terms of the coefficient of variation for log most probable number) and its relative independence from the influence of bacterial density allowed the use of analysis of variance to investigate the effects of spatial and temporal bacterial heterogeneity on the estimation of coliforms. Variability among replicate samples, subsamples, handling, and analytical errors were considered as the major sources of variation in bacterial titration. Variances associated with individual components of the sampling procedure were isolated, and optimal replications of each step were determined. Temporal variation was shown to be more influential than the other three components (most probable number, subsample, sample to sample), which were approximately equal in effect. However, the incidence of sample-to-sample variability (16%, in terms of the coefficient of variation for log most probable number) caused by spatial heterogeneity of bacterial populations in the Moselle River is shown and emphasized. Consequently, we recommend that replicate samples be taken on each occasion when conducting a sampling program for a stream pollution survey.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.

A microtiter technique was used to measure lymphocyte stimulation by antigen-pulsed monocytes. Monocytes from human blood were isolated on polyester film disks. The disks were exposed to antigen and incubated together with lymphocytes in the wells of a microtiter plate. The method was technically simple and required no expensive materials.

The use of a microtechnique (modified nitrous acid extraction) to test samples from 150 school children and from patients with acute follicular tonsillitis has indicated that group A streptococci in the throat can be identified from tonsillar scrapings in 30 min. The results are comparable to the grouping results obtained by standard throat culture techniques and the Lancefield procedure for grouping. No cross-reaction with other bacteria or cellular material occurs. Study has also shown that the nitrous acid extraction yields three- to fourfold more polysaccharides than the Lancefield hot-HCl of Fuller formamide techniques. The use of the microtechnique leads to another 20-fold concentration of the antigen. Immune salting-out effect could be obtained with 1.00 M sodium acetate. Such molarity is too low to cause nonspecific slating out. It leads to a strong ampliciation of the precipitin reaction.

A microtechnique based on the most-probable-number (MPN) method has been developed for the enumeration of the ammonium-oxidizing population in soil samples. An MPN table for a research design ([8 by 12] i.e., 12 dilutions, 8 replicates per dilution) is presented. A correlation of 0.68 was found between MPNs determined by the microtechnique and the standard tube technique. Higher MPNs were obtained with the microtechnique with increased accuracy in endpoint determinations being a possible cause. Considerable savings of time, space, equipment, and reagents are observed using this method. The microtechnique described may be adapted to other microbial populations using various types of media and endpoint determinations.

Direct bacterial hemagglutination was investigated with 20 clinical isolates of Neisseria gonorrhoeae. The hemagglutination tests were performed by both a macrotechnique with glass slides and a microtechnique with autotrays. Only organisms from form type 1 or 2 colonies caused hemagglutination. There was no statistical difference at a 10% or higher level in hemagglutination powers of type 1 and type 2 organisms, of male urethral and female cervical isolates, and of the eight major human blood types (ABO-Rh). Of seven erythrocyte species tested, only human cells were agglutinated. D-Mannose did not prevent the agglutination. Rabbit antigonococcal serum and high-titer antigonococcal human sera inhibited the hemagglutination. The results suggest the pili are the mediators of hemagglutination and that their specific agglutination of human erythrocytes may be a correlate of their adherence to human mucosal cells in natural infection. Also, although the procedure is presently insensitive, it is possible to detect human antigonococcal antibody by inhibition of direct bacterial hemagglutination.

We have developed a microtechnique for detection of streptococcal serum opacity factor (OF) and for typing of group A streptococci by inhibition of OF. This technique, which involves the use of single wells of standard 96-well tissue culture plates, offers several advantages over previous methods: no advance test preparation is required, allowing tests to be quickly and easily performed; only small quantities of reagents are required; results can be determined visually (qualitative) or by using a photometric enzyme-linked immunosorbent assay plate reader (quantitative); and human serum samples may be quickly and easily screened for OF-inhibitory antibody and subsequently used in place of difficult-to-produce and expensive hyperimmune animal sera for OF characterization of group A streptococci. Fifty-eight samples of normal adult human serum were tested by this new microtechnique for anti-OF antibodies, and 49 (84%) were found to have antibody against 1 or more of the 27 recognized OF-positive serotypes. OF antibodies to M-4, M-2, M-75, and M-48 were most common in these individuals. These 58 human serum samples collectively contained antibody to 25 of the 27 different OF-producing serotypes. Serum samples from four individuals were tested for persistence of OF antibody. OF antibodies to eight different serotypes present in the serum samples collected 7 to 12 years previously were present in the freshly collected sera, indicating that OF antibody persists in human antisera for many years. This new technique has distinct advantages and makes it possible for many laboratories to use this technique to characterize group A streptococci.

A mechanized microtechnique for the serological typing of Escherichia coli cultures is described.

A microtechnique has been developed and adapted successfully to the microscopic agglutination test with live antigens for detection of leptospiral antibodies. Simultaneous titrations were performed by the conventional microscopic agglutination test and the microtechnique. When the microtechnique was used to screen 50 unknown leptospiral strains with a battery of hyperimmune sera, 98% agreement was obtained with the conventional procedure. Comparative data on 635 tests on these 50 cultures established the reliability of the microtechnique. Results with the two tests on 46 human sera revealed 93% agreement in the detection of leptospiral antibodies. The validity and reliability of the microtechnique obtained in these comparative studies suggests that it can be used as a valuable screening procedure for the microscopic agglutination test for preliminary cross agglutination studies on unknown strains and for the detection of leptospiral antibodies in human and animal sera.

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, p H, 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.

A direct extraction of the antigen of group A beta-hemolytic streptococci from 373 throat swabs was done with a microtechnique of the nitrous acid extraction-Phadebact coagglutination method. The technique yielded a 96% agreement with the results obtained by a standard throat culture method in which the Phadebact grouping procedure with overnight broth supernatants was employed.

A microtechnique for determining antibiotic susceptibilities by the serial dilution method was evaluated in a clinical diagnostic microbiology laboratory. As compared with the standard tube method, an agreement of 94% was achieved for determining minimal inhibitory concentration with ± one tube dilution as the criterion of comparison. The experience with this system suggests that it could easily be incorporated into diagnostic laboratories as a routine procedure.

A Limulus amoebocyte lysate microtechnique performed in petrolatum wells on a microscope slide is described. Injection of a dye solution in ethanol directly into the wells leads to an unambiguous interpretation of the results. Twelve samples can be tested on a single slide, and compact storing of the samples is possible.

A microtechnique is described for agglutination typing of Haemophilus influenzae. It also provides a further means for classification and study of noncapsular type-specific strains.

A microtechnique for staining preparations of cells grown in vitro for indirect immunofluorescence tests is described. Smaller amounts of materials, as well as less handling of cover slips, greatly facilitates the process.