Use of the Minitek system for characterizing lactobacilli.

Incubation of inoculated substrates of the Minitek system in anaerobic GasPak jars provided a method that produced results comparable to those of conventional tubed media for characterizing species of Lactobacillus. The use of sterile mineral oil to overlay inoculated substrate disks was responsible for erroneous results.     

Evaluation of the Minitek system for characterization of Bacillus species.

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Evaluation of the Minitek system for characterization of Bacillus species

Minitek (BBL Microbiology Systems, Cockeysville, Md.) substrate disks were evaluated as alternatives to conventional tests for the characterization of Bacillus species. Results were compared for 10 reference isolates and 87 isolates from food sources. The overall agreement of results between the Minitek and conventional tests was 92% for reference strains and 86% for food isolates.     

Use of the oblique illumination method for characterizing N. meningitidis cultures]

The authors used the method of oblique illumination for detection of the state of the population of meningococcus cultures. In studying 48 strains there were revealed three types of fluorescence of the colonies: bright orange with a transition into greenish-light blue along the lower margin (the I type), bright light blue with a narrow orange or green upper margin (the II type) and greyish-light blue colonies (the III type). The type of fluorescence was not associated with the sero-group specificity. Populations of meningococcus cultures, depending on conditions and duration of growth on the nutrient medium could consist both of the colonies with the same type of fluorescence, and also represent a combination of colonies with a different type of fluorescence. The colonies of the I and II types of fluorescence had marked group specificity, but mutation occurred during passage and the III type of fluorescence formed with a loss of serological specificity. Continuous selection of the colonies of the I and the II types of fluorescence permitted to preserve the population with specific properties necessary for production of diagnostic and vaccine preparations for a long time.     

Use of the EpiAirway model for characterizing long-term host-pathogen interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa (1; 2). To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods(3). For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function(4). For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.     

Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels.     

Use of a quantitative oxidase test for characterizing oxidative metabolism in bacteria.

It was possible to quantitate the terminal oxidase(s) reaction using bacterial resting-cell suspensions and demonstrate the usefulness of this reaction for taxonomic purposes. Resting-cell suspensions of physiologically diverse bacteria were examined for their capabilities of oxidizing N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using a manometric assay. For organisms having this capability, it was possible to calculate the conventional TMPD oxidase Q(O2) value (microliters of O2 consumed per hour per milligram [dry weight]). All cultures were grown heterotrophically at 30 C, under identical nutritional conditions, and were harvested at the late-logarithmic growth phase. The TMPD oxidase Q(O2) values showed perfect correlation with the Kovacs oxidase test and, in addition, it was possible to define quantitatively that point which separated oxidase-positive from oxidase-negative bacteria. Oxidase-negative bacteria exhibited a TMPD oxidase Q(O2) value (after correcting for the endogenous by substraction) of less than or equal 33 and had an uncorrected TMPD/endogenous ratio of less than or equal 5. The TMPD oxidase Q(O2) values were also correlated with the data obtained for the Hugh-Leifson Oxferm test. In general, bacteria that exhibited a respiratory mechanism had high TMPD oxidase values, whereas fermentative organsims had low TMPD oxidase activity. All exceptions to this are noted. This quantitative study also demonstrated that organisms that (i) lack a type c cytochrome, or (ii) lack a cytochrome-containing electron transport system, like the lactic acid bacteria, exhibited low or negligible TMPD oxidase Q(O2) values. From the 79 bacterial species (36 genera) examined, it appears that this quantitative oxidase test has taxonomic value that can differentiate the oxidative relationships between bacteria at the subspecies, species, and genera levels.     

Evaluation of the Minitek System for Identification of Enterobacteriaceae

Clinical isolates (869) and stock cultures (35) of Enterobacteriaceae were tested in parallel with the Minitek and conventional systems. The Minitek correctly identified 822 of 904 cultures. When a deoxyribonuclease plate was inoculated along with the Minitek, it was possible to speciate Enterobacteriaceae within 24 h. False-positive hydrogen sulfide reactions were the major fault with this system. Reactions were clear-cut and easy for technologists to read.     

Evaluation of the BBL Minitek System for the Identification of Enterobacteriaceae

Thirty-one different substrate disks were tested in parallel with comparable, prepared media (BBL) against a minimum of 300 cultures of Enterobacteriaceae. An overall correlation of 98% was observed with all the disks tested. In addition, the system was used to identify 461 fresh isolates of Enterobacteriaceae in parallel with conventional media using the schema used at the Veterans Administration Hospital, Baltimore. An overall correlation of 97% was observed. Minitek is a time and space saving system. It is accurate and easily adapted to the clinical laboratory. A wide variety of substrates are available, allowing most laboratories to use their own schema. The long shelf life of most disks is a definite advantage.     

Integrative expression system for delivery of antibody fragments by lactobacilli

A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection.     

Modulation of immune system cells by lactobacilli

The influence of lactobacilli on the proliferative potential of immune system cells after the intragastral administration of viable microbial cells and their native filtrates to mice CBA is evaluated. The data have been obtained on the modulating influence of lactobacilli on the formation of T- and B-cell immune response--their role in maintaining homeostatsis and specific features of cell mediated immune reactions after the intragastral administration of virulent Shigella dysenteriae for modeling experimental infection in CBA mice. The mechanisms of the immunomodulating action of lactobacilli on local and systemic reactions of the host as well as realization of the protective properties of lactobacilli against the causative agents of acute enteric infections are discussed.     

A computer program for characterizing root system branching patterns

A computer program is presented which measures the length, branching patterns and distribution of link length within a root system. The program skeletonizes digitized images of root systems, loads these images into a binary tree data structure and uses this data structure to characterize the root systems. Measurements of the root length and topological parameters of root systems of Senecio vulgaris made by hand and by computer program were linearly related, with r² values greater than 0.99 in all cases.     

A fast method for monitoring the colonization rate of lactobacilli in a meat model system

A random amplified polymorphic DNA (RAPD) assay coupled to a fast and reproducible cell lysis method from Lactobacillus colonies were developed to type lactobacilli of different strains and species, with the aim of precisely enumerating each of the different Lactobacillus strains inoculated in a nutrient-rich environment, such as sausage meat batter. Colonization assays were carried out in an aseptic meat fermentation system for up to 14 d and the inoculated strains were challenged with mixtures of wild lactobacilli. The proportion of inoculated strains remaining at different times was compared with the total number of lactobacilli grown on MRS agar by RAPD. The colonization rate of the different strains tested was very different. The RAPD-fast lysis method developed is simple and, with a low cost per assay, could also be applied to other food fermentations.     

A general-purpose system for characterizing medically important bacteria to genus level

A computer program and accompanying data matrix have been prepared for bacteria of medical interest, to assist the assignment of an unidentified bacterium to the most likely genus. The results on a set of relatively simple tests are entered. The program prints the more likely genera, followed by a list of diagnostic tables in Cowan & Steel (1974) and Buchanan & Gibbons (1974). Where available, identification matrices for further computer-assisted study, are presented. This program may be of particular help in laboratories where a wide range of bacteria have to be identified.