Rapid Confirmation of Suspect Salmonella Colonies by Use of the Minitek System in Conjunction with Serological Tests

Colonies of 40 members of the Enterobacteriaceae family (26 Salmonella serotypes and 14 other organisms) were picked from selective agar plates and inoculated into Minitek inoculum broth (BBL) and then onto Minitek discs of dextrose, lactose, sucrose, mannitol, maltose, dulcitol, lysine and H₂S. After incubation for 6 h, the inoculum broth was tested with salmonella Poly O and after 24 h with salmonella Poly H antisera. The results of the biochemical tests were read after 24 h incubation. With this procedure, all the salmonella cultures used in this study were confirmed as salmonella and differentiated from all the other organisms, which were rejected. This procedure provides an alternative to the time consuming conventional procedures for the biochemical and serological confirmation of suspect salmonella colonies on selective agar plates.     

Agar medium for differential enumeration of lactic streptococci

An agar medium containing arginine and calcium citrate as specific substrates, diffusible (K₂HPO₄) and undiffusible (CaCO₃) buffer systems, and bromocresol purple as the pH indicator was developed to differentiate among lactic streptococci in pure and mixed cultures. Milk was added as the sole source of carbohydrate (lactose) and to provide growth-stimulating factors. Production of acid from lactose caused developing bacterial colonies to seem yellow. Subsequent arginine utilization by Streptococcus lactis and S. diacetilactis liberated ammonia, resulting in a localized pH shift back toward neutrality and a return of the original purple indicator hue. The effects of production of acid from lactose and ammonia were fixed around individual colonies by the buffering capacity of CaCO₃. After 36 hr at 32 C in a candle oats jar, colonies of S. cremoris were yellow, whereas colonies of S. lactis and S. diacetilactis were white. S. diacetilactis, on further incubation, utilized suspended calcium citrate, and, after 6 days, the citrate-degrading colonies exhibited clear zoning against a turbid background, making them easily distinguishable from the colonies of the other two species. The medium proved suitable for quantitative differential enumeration when compared with another widely used general agar medium for lactic streptococci.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Sugar preferences of "black rats", Rattus rattus L.

The choice of "black rats", Rattus rattus L ., for common sugars - sucrose, jaggery, glucose, fructose and lactose are described. In laboratory colonies, the sugars were preferred in the order glucose > sucrose > jaggery > fructose > lactose; but in another sequence - sucrose > jaggery > glucose > fructose > lactose, in the free living colonies. The discrepancy is accounted for by the characteristics of sugars which influence consummatory behaviour in the two situations.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

The Examination of Foods for Enterobacteriaceae using a Test of the Type Generally Adopted for the Detection of Salmonellae

S ummary : In countries that have recently increased their bacteriological inspection of foods, many products have shown considerable improvement in microbiological quality; simultaneously, however, discrepancies between salmonella and coli-aerogenes tests, especially on dehydrated and frozen foods, have tended to become more frequent. These discrepancies have been eliminated by applying the following measures: incorporating glucose in culture media so as to reveal all Enterobacteriaceae; placing reliance on growth rather than gas formation so as to avoid missing anaerogenic organisms, and, especially, examining quantities of foods commensurate with those used in salmonella tests.
For this purpose a procedure is recommended in which 10 g of well homogenized food are enriched in 100 ml of buffered brilliant green-bile-glucose broth, with no attention being paid to gas formation; the enrichment cultures are then streaked on to MacConkey's glucose agar. Single colonies so obtained are tested for fermentative attack on glucose and may be further examined for other characteristics. The same enrichment fluid can be used for the so-called 'nonselective pre-enrichment' of samples of food containing salmonellae impaired by periods spent in conditions of low water activity, low pH, etc.     

Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures

Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal organisms eventually took over the culture with all four strains examined. Gal cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-beta-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal-Gal cultures were repeatedly transferred in milk, the Gal cells became the dominant cell type. The Gal phenotype of stock cultures probably reflects their prolonged maintenance in milk.     

Lysine-Iron Agar in the Detection of Arizona Cultures

A lysine-iron agar is described and recommended for the detection of Arizona strains which ferment lactose rapidly. Black colonies which appear on bismuth sulfite agar should be transferred to the medium. Salmonellae and Arizona cultures produce a distinctive reaction since they are the only recognized groups of enteric bacteria which regularly produce lysine decarboxylase rapidly and form large amounts of hydrogen sulfide. Use of the medium is particularly recommended in the examination of specimens from enteric infections in which shigellae and salmonellae are not detected.     

Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.     

Understanding the cause of false negative β‐d‐glucuronidase reactions in culture media containing fermentable carbohydrate

Aims:  To explain the basis for false negative β‐glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Methods and Results:  Escherichia coli strains were assessed for their reactions in culture media containing a β‐d ‐glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of β‐d ‐glucuronidase at pH 5·0 and pH 7·2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme β‐d ‐glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose‐containing media expressed virtually no β‐d ‐glucuronidase activity at pH 5·0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. Conclusions:  E. coli that failed to produce green colonies on MLGA produced lower levels of β‐d ‐glucuronidase than did strains that formed green colonies, the difference being greater at pH 5·0 than pH 7·2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Significance and Impact of the Study:  Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme β‐d ‐glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7·2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).     

Information content of amino acid residues in putative helix VIII of the lac permease from Escherichia coli

Mutants in putative helix VIII of lactose permease that retain the ability to accumulate lactose were created by cassette mutagenesis. A mutagenic insert encoding amino acid residues 259-278 was synthesized chemically by using reagents contaminated with 1% each of the other three bases and ligated into a KpnI/BclI site in the lacY gene in plasmid pGEM-4. Mutants that retain transport activity were selected by transforming a strain of Escherichia coli containing a wild-type lacZ gene, but deleted in lacY, with the mutant library and identifying colonies that transport lactose on indicator plates. Sequencing of the mutated region in lacY in 129 positive colonies reveals 43 single amino acid mutations at 26 sites and 26 multiple mutations. The variable amino acid positions are largely on one side of the putative alpha-helix, a stripe opposite Glu269. This mutable stripe of low information content is probably in contact with the membrane phospholipids.     

Cellulase production by Penicillium echinulatum on lactose

The inducer effect of lactose on cellulase activity in Penicillium echinulatum 9A02S1 was studied. Submerged cultivation was performed using different concentrations of lactose and cellulose, in which the pH, mycelial mass, soluble proteins, filter paper activity (FPA), and activity of β-glucosidases were measured. The cultures containing lactose only presented low FPAs (0.1 FPU/ml). The cultures with associated cellulose and lactose and those containing cellulose only presented similar enzymatic activities (1.5 FPU/ml), suggesting the possibility of up to 75% reduction in the cellulose concentration. In relation to the β-glucosidases, increasing the lactose/cellulose ratio results in a proportional increase of enzymatic activity. In the cultures using both inducers, there is a longer duration of the acid phase in relation to treatments using only cellulose or lactose, indicating diauxia and catabolic repression.     

Immunocytochemical identification of murine and human megakaryocyte colonies in soft-agar cultures

Summary Murine megakaryocyte (MK) colonies in soft-agar cultures were immunocytochemically stained with platelet antiserum and an immuno-alkaline phosphatase procedure. Subsequently, cytochemical staining for acetylcholinesterase was used to confirm the specificity of the immunolabelling technique. The correlation of numbers of megakaryocyte colonies enumerated by independent observers was excellent. A comparable platelet antiserum directed against human platelet epitopes was utilized to identify human MK colonies in soft-agar cultures of human bone marrow. Using this method, we determined that the frequency of detectable human MK colonies in our agar culture system was maximal between days 10 and 12. The immunocytochemical staining technique we have developed for identification of MK colonies in soft-agar cultures yielded good cellular morphology and produced an intensely specific label against a clear background; it therefore facilitated accurate enumeration of MK colonies. This non-fluorescent method avoids dependence upon a non-permanent marker, and allows the simultaneous enumeration of positive and negative colonies.     

Rapid decline of clonogenic hemopoietic progenitors in semisolid cultures of bone marrow samples derived from patients with chronic myeloid leukemia

Bone marrow samples, from newly diagnosed patients with chronic myeloid leukemia (CML) and normal individuals, were grown in methylcellulose and serially recultured under identical conditions. Specimens derived from normal individuals gave rise to multilineage and megakaryocyte colonies for one to two sequential cultures. Erythroid bursts and granulocyte-macrophage colonies were observed for three to five sequential cultures. Cultures initiated from samples of patients with CML showed a rapid decline of all types of colonies. Colonies were rarely seen for more than two sequential cultures. When pooled colonies and background cells were recloned separately, secondary colonies were mainly seen in cultures of background cells. This observation is consistent with the view that secondary colonies are more likely to arise from dormant clonogenic progenitors, rather than through cells that have formed primary colonies through a self-renewal process.     

Detection of salmonellas in animal feeds by electrical conductance

A comparison was made between standard culture methods and electrical conductance using a Malthus AT Microbiological Analyser for the examination of animal feeds for salmonella. Conductance testing with a selenite cystine/trimethylamine-N-oxide/dulcitol medium resulted in the detection of salmonellas in 49 of 55 known positive animal feeds, 13 of 19 spiked feed samples and 36 of 47 salmonella cultures. Testing with a lysine decarboxylase/glucose medium gave significantly better results (P less than 0.05) than with selenite cystine medium but five lysine decarboxylase negative strains of salmonella were undetected. When both media were used in parallel all salmonella positive samples were detected. No difference was found between preenrichment in buffered peptone water containing trimethylamine/mannitol and that containing lysine/glucose. Positive detection criteria for selenite medium of conductance peak at greater than or equal to 500 microsiemens (microS) with a rate of change of greater than or equal to 60 microS/h or 400-499 microS with a rate of change of greater than or equal to 40 microS/h and for lysine medium with a peak of greater than or equal to 100 microS have been established. The method offers savings in media and operating costs over conventional standard culture methods, provides results within 48 h and is recommended for statutory feed monitoring purposes.     

Occurence of lactose-negative mutants in chemostat cultures of lactic streptococci.

Batch and chemostat cultures of Streptococcus cremoris HP and Streptococcus lactis 829 were examined for lactose-hegative (lac-)mutants on indicator agar. In batch cultures, S. cremoris HP gave less than 1% of the total count as lac- colonies while S. lactis 829 consistently contained about 15% of the total as lac- colonies. In chemostat cultures of S. cremoris HP in 2% skim milk containing casamino acids and yeast extract (0.1% each), the percentage of lac- colonies increased markedly when the temperature of growth was 18 degrees C but not when the temperature of growth was 25 degrees C. The percentage of lac- colonies in chemostat cultures in the skim milk medium at 25 degrees C was about the same as that in batch cultures. On the other hand, when chemostat cultures of S. lactis 829 in the skim milk medium were grown at several temperatures between 18 and 33 degrees C, the percentage of lac- colonies was markedly lower than that found in batch cultures of this organism. Cultivation of S. cremoris HP in chemostats with yeast extract-glucose broth at low temperature (18 degrees C) resulted in a selection of cells giving lac- colonies and atypical (small) lac+ colonies. The results show that cultivation of S. cremoris HP and S. lactis 829 in chemostats sometimes gave rise to altered populations. Conditions causing a change in one organism did not necessarily cause a similar change in the other. The results indicate that the successful propagation of lactic streptococci in chemostats for use as starter cultures in the dairy industry will require the careful establishment of optimum conditions for every strain so as to minimize the possible selection of undesirable populations.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Rapid detection of salmonellae by immunoassays with titanous hydroxide as the solid phase.

Radioimmunometric and enzyme-immunometric assays were developed for the detection of salmonellae in pure and mixed cultures as well as in 59 food samples. The performances of titanous hydroxide suspension and microtiter plates as the solid phase for the immobilization of microorganisms were compared in these immunoassays. Detection of populations of salmonella cells in pure culture, diluted with saline, was 4- to 10-fold more sensitive with the microtiter plates. However, with mixed culture of salmonella and other enterobacterial species, the detection sensitivity with titanous hydroxide was 100- to 160-fold more sensitive than with microtiter plates. Good correlation existed between results of a standard cultural method for the detection of salmonellae in foods and those obtained from radioimmunometric and enzyme-immunometric assays utilizing titanous hydroxide. However, a high incidence of false-positive and false-negative results with food samples occurred with the enzyme-immunometric assay utilizing microtiter plates. The results provided strong evidence for the merits of substituting titanous hydroxide for microtiter plates as the solid phase for the immobilization of salmonellae for their detection by immunoassays. The immunoassays were rapid and enabled the analysis of a large number of selective enrichment cultures of food samples for salmonellae within 8 h.     

Effect of Type of Enrichment and Duration of Incubation on Salmonella Recovery from Meat-and-Bone Meal

Twenty-five meat-and-bone meal samples were enriched with either selenite-cystine or tetrathionate and incubated for 1 and 2 days. Seven were previously found to be positive; of the other 18, 16 were positive for salmonella. The number of somatic serogroups per sample ranged from 1 to 11 with a mean of 3.8. Significantly more (P < 0.01) group C₁ salmonellae were isolated using tetrathionate than selenite, whereas significantly more of groups G, 35, and Difco poly-valent D were isolated from selenite than tetrathionate. Seventy-six percent of the presumptive colonies from Brilliant Green agar showed a positive lysine decarboxylase reaction, and there were no differences between media or times of incubation. Ninety-four per cent of the lysine decarboxylase-positive cultures showed a positive somatic antiserum response; again there were no differences between times or enrichments although there were significantly more total positive serogroups at 2 days than at 1 day from tetrathionate but not from selenite. There were indications that certain serogroups preferred either one or the other enrichment. There were no differences in total positive samples with the two enrichments although neither alone was sufficient to identify all positives. Several lactose-positive salmonellae were recovered.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.