A COMPARISON OF SOME MILK REJECTION TESTS

SUMMARY: Examination of 202 samples of raw milk showed that the combination of a pH paper strip test with a second sorting test (80 or 76% alcohol or rapid acidity) provided a basis to select milk which required a more time-consuming test before grading accurately.
Samples of pasteurized milk at the yellow-green stage of fluorescence when examined in ultraviolet light did not clot on boiling and possessed long methylene blue reduction times, whereas at the purple stage of fluorescence the samples clotted on boiling and reduced methylene blue almost instantaneously.     

ADVISORY MICROBIOLOGICAL STANDARDS AND TOLERANCES FOR RAW MILK

SUMMARY: To study the value of recent modifications of microbiological tests used for advisory purposes, samples of tuberculin tested milk, taken at weekly intervals over a 5 year period from the Trawscoed Experimental Husbandry Farm and selected at random during a 12 months period from farms in Wales, were examined by a temperature compensated keeping quality test at 22°, colony count on Yeastrel milk agar in 3 days at 30° and coli-aerogenes colony count on violet red bile agar in 20–24 hr at 30°.
The results show that milk produced and handled under hygienic conditions can be expected to have colony counts of less than 2 × 10⁴/ml and coli-aerogenes colony counts less than 10/ml when examined within 18 hr of milking.     

Comparison of radioimmunoassay and gas-liquid chromatography analyses of progesterone concentrations in cow's milk.

Progesterone concentrations in milk were not significantly different when quantitated by different methods (RIA vs. GLC). There was a significant day effect (P less than 0.05) on milk progesterone level due apparently to gradually decreasing values as pregnancy advanced over days 30, 120 and 210. The means for the percent fat content were different (P less than 0.05) for all comparisons among four times of collection (immediately premilking, milking pool, immediately postmilking, and 3 hr postmilking). For progesterone concentration, the main effect of time and the three-way interaction (time times antiserum times purification method) were significant (P less than 0.005); all other main effects and interactions were not significant. Within each of 4 assay groups (2 antisera times 2 purifications), the concentration of progesterone was greater (P less than 0.05) for the samples which were collected immediately postmilking than for any of the other times of collection. The three-way interaction seemed due primarily to difference in progesterone determinations among the four assay groups in the samples which were taken immediately postmilking. Over all milk samples within each assay group, the percent fat content and the concentration of progesterone were positively correlated (r = 0.71, P less than 0.01).     

Cellulose acetate electrophoresis of casein proteins.

Samples of the milk proteins α_s1-casein and β-casein partially dephosphorylated by means of bovine spleen phosphoprotein phosphatase have been electrophoretically analysed using cellulose acetate as the supporting medium and Procion blue as the protein dye. Sufficient resolution was obtained in 1 hr to allow quantification of the proteins present. Skimmed-milk samples and acid-precipitated whole casein samples have been analysed by the same technique. The advantages of the method are discussed in relation to the more conventional electrophoretic techniques normally used to analyse these milk proteins.     

Reproductive activity in Finnish Landrace x Rambouillet ewes exposed to various daylength cycles applied over periods of six months

Twenty-six Finnish Landrace x Rambouillet ewes were exposed to a series of artificial daylength cycles over periods of 6 mo. The cycles in general consisted of 3 mo. of long or increasing daylength, followed by 3 mo. of short or decreasing daylength. Increasing daylength to 22 hr.; 17.5 hr. or 15.75 hr. but not 14 hr. resulted in the onset of anestrus in all ewes. Only four of eight ewes in the 14 hr. cycle became anestrus. Abrupt decreases in daylength resulted in a more rapid return to reproductive cyclicity than a gradual decline. In addition, there appeared to be an inverse relationship between the length of the longest day of a treatment cycle and the time taken for ewes to return to reproductive cyclicity, following a subsequent abrupt decrease in daylength. Reducing daylength form 22 hr. to 17.5 hr. did not induce reproductive cyclicity. Reducing daylength from 17.5 hr. to 15 hr.; 12.75 hr. or 7.5 hr. did result in the onset of reproductive cyclicity. Finally, extending the period of long day exposure in a daylength cycle decreased the response time to a subsequent reduction in daylength. It was concluded that reproductive seasonality may be initiated by the interaction of darkness with a daily photosensitive phase in the ewe.     

Heat Resistance of Salmonellae in Concentrated Milk

The heat resistance of Escherichia coli, Salmonella typhimurium, and Salmonella alachua in milk solutions containing 10, 30, 42, and 51% (w/w) skim milk for total solids was determined. Increased milk-solids level effected a significant increase in the heat resistance of each organism. Although E. coli was more heat-resistant than both strains of Salmonella in 10% milk, the situation was reversed in 42 and 51% milk. Prior growth temperature was found to exert a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vaccum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

The anti-inflammatory mechanism of MK-447 in rat carrageenin-induced pleurisy

Intrapleural injection of 2% λ-carrageenin caused the accumulation of exudate up to 19 hr. The rate of plasma exudation, measured by the exuded dye amounts for 20 min in the pleural cavity after intravenous injection of pontamine sky blue, showed a peak at 5 hr. Aspirin (100 mg/kg, i. p.) suppressed the dye exudation up to 5 hr, but did not at 7 hr. This inhibition coincided with the decrease of the PG and TXB₂ levels, which were measured by gas chromatography-mass spectrometry, in the pleural exudate. In _vitro experiments, MK-447, a phenolic compound, stimulates PG endoperoxide biosynthesis at lower doses and inhibits it at higher doses, acting as a tryptophan-like cofactor required by PG endoperoxide synthetase. This drug (0.3, 1.0 and 3.0 mg/kg, i. p.) suppressed the dye exudation dose-dependently up to 5 hr, but did not at 7 hr even at a higher dose, in combination with the dose-dependent decrease of the pleural level of PGE₂, which was reported to be a major PG among PGs and TXB₂ in the exudate in inducing the plasma exudation (Harada ; Prostaglandins, : 881, 1982). Thus, the anti-inflammatory action of MK-447 can be explained by inhibition of PGE₂ generation, giving no consideration to the role of oxygen-derived free radicals as a prime mediator in inflammation.     

Dynamics of vitellogenin uptake in Aedes aegypti as demonstrated by trypan blue

Trypan blue has been shown to be a reliable indicator of the micropinocytotic uptake of vitellogenin by developing oöcytes. Trypan blue was injected into the mosquito Aedes aegypti to determine at what times after the blood meal vitellogenin was taken up. Histological sections examined by light microscopy showed that trypan blue began to be sequestered from 2 to 5 hr after the blood meal. Any association between dye and ovariole ended from 39 to 42 hr after the blood meal, in which period no dye was incorporated into spheres of yolk protein. Of the times investigated in this experiment, the greatest amount of dye was seen in the oöcyte at 24 hr after the blood meal. The onset and conclusion of trypan blue uptake correspond with the related events in the synthesis of vitellogenin by the fat body. The presence of trypan blue in occasional interfollicular spaces suggests that the route of entry of vitellogenin in Aedes aegypti is indeed an interfollicular one.     

Applicability of Aeration and Delayed Addition of Selenite to the Isolation of Salmonellae

The effect of aeration combined with the delayed addition of selenite on the lag period of several strains of salmonellae and other enterobacteria is reported. A procedure has been developed involving shaking of the sample in a basal medium for 4 hr at 37 C, adding selenite and cystine, and continuing shaking for 20 hr. Confirmation by selective plating, biochemical tests, and serology gave results comparable to the standard lactose pre-enrichment method with the saving of 24 hr and elimination of one set of media. Confirmation by fluorescent-antibody tests showed that fewer positive fluorescent stains were obtained from the aerated procedure than from the lactose pre-enrichment procedure. Precautions in the application of this procedure are discussed.     

SOURCES OF ERROR AND THE NO-PREFERENCE OPTION IN DAIRY PRODUCT TESTING

When products are physically identical, consumers will choose a no‐preference option in a paired preference test only about 30% of the time, giving a nonintuitive result. We evaluated the no‐preference false alarm rate in dairy products and the potential sources of error in panelist behavior, including discrimination problems and poor repeatability of judgments. Paired preference tests were conducted with milk (2% versus nonfat or skim), with cottage cheese (4% versus nonfat) and with identical samples. Triangle, dual standard and same/different discrimination tests were also conducted. The no‐preference option was chosen by about 30% of the consumers when samples were identical, and less frequently when samples were different. Discrimination performance, although statistically significant, was poorer than expected with about 30% discriminators (d′ of 1.7) in most tests. Even in the dual standard tests on milk (visually obvious and with references present), performance was only 88% correct (76% discriminators). Repeatability in preference tests was also poorer than expected, with only 56% choosing the same preference option for different samples on repeat trials although the group preferences were stable. Same/different tests indicated that over 50% of the consumers called identical samples different, although this fell short of explaining the 70% average false alarm rate in preferences. Even with apparently discriminable consumer products, accurate discrimination of the samples and repeatability of preferences should not be assumed. Random responding may contribute to error variance and produce nonsensical results such as the expression of choice between identical samples.     

Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.

We show here the identity of Alamar Blue as resazurin. The 'resazurin reduction test' has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.     

Unique differences in infectivity and seroreactivity of Toxoplasma harvested from mice infected for different lengths of time

For use in experiments, Toxoplasma of the RH strain are usually harvested from mouse peritoneal cavities 48 hr (2-day Toxoplasma) or more after intraperitoneal inoculation. In this report we show that Toxoplasma harvested at 24 hr (1-day Toxoplasma) after inoculation are much more infective for and replicate to a greater degree within mouse resident peritoneal macrophages in vitro and are much more resistant to the cidal activity of activated mouse peritoneal macrophages and resident rat peritoneal macrophages than are 2-day Toxoplasma. Ingestion of 1-day Toxoplasma by macrophages did not trigger the respiratory burst as measured by reduction of nitroblue tetrazolium (NBT), but coating 1-day Toxoplasma with specific antibody did result in reduced NBT. However, coating 1-day Toxoplasma with specific antibody did not markedly decrease infectivity for macrophages in vitro, unlike decreased infectivity observed when 2-day Toxoplasma are coated with specific antibody. Use of 1-day Toxoplasma in the dye test resulted in a 5-fold decrease in titer of specific antibody in human sera. Use of Toxoplasma harvested 24 hr after infection may serve as a new tool to probe virulence factors of Toxoplasma and of host cells' antimicrobial mechanisms.     

NIGHT BLUE AND VICTORIA BLUE AS INDICATORS IN LIPOLYSIS MEDIA

SUMMARY: Optimum conditions for the use of night blue and victoria blue as lipolysis indicators in fat emulsion agar medium required a dye strenth of 1: 15,000 in a medium of pH 8·0 containing 5% fat dispersed by hand shaking. Pour plates should contain 6 ml. and streak plates 10 ml. of the medium in a standard Petri dish. Incubation should be for 5 days at 30°. The pinkish-mauve medium with clear blue-zoned lipolytic colonies gave the same results as butter fat agar without dye but treated with CuSo₄, when tested with 962 pure cultures. The inhibitory powers of the dye were assessed and although strongly toxic in the aqueous phase to Gram-positive bacteria, victoria blue appears to have none to slight inhibitory power in the fat agar medium: night blue suppressed growth to about the same extent as tributyrin. The lipolytic flora of butter and to some extent milk shows a remarkable dominance of micorcocci. Organisms lipolytic on fat agar media are able to produce appreciable acid in a fat emulsion in a liquid medium.     

Quantification of metabolically active biomass using Methylene Blue dye Reduction Test (MBRT): measurement of CFU in about 200 s

Quantification of viable cells is a critical step in almost all biological experiments. Despite its importance, the methods developed so far to differentiate between viable and non-viable cells suffer from major limitations such as being time intensive, inaccurate and expensive. Here, we present a method to quantify viable cells based on reduction of methylene blue dye in cell cultures. Although the methylene blue reduction method is well known to check the bacterial load in milk, its application in the quantification of viable cells has not been reported. We have developed and standardized this method by monitoring the dye reduction rate at each time point for growth of Escherichia coli. The standard growth curve was monitored using this technique. The Methylene Blue dye Reduction Test (MBRT) correlates very well with Colony Forming Units (CFU) up to a 800 live cells as established by plating. The test developed is simple, accurate and fast (200 s) as compared to available techniques. We demonstrate the utility of the developed assay to monitor CFU rapidly and accurately for E. coli, Bacillus subtilis and a mixed culture of E. coli and B. subtilis. This assay, thus, has a wide applicability to all types of aerobic organisms.     

Time dependent degradation of mixture of structurally different azo and non azo dyes by using Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360, a yeast species showed 88% ADMI (American dye manufacturing institute) removal of mixture of structurally different dyes (Remazol red, Golden yellow HER, Rubine GFL, Scarlet RR, Methyl red, Brown 3 REL, Brilliant blue) (70 mg l⁻¹) within 24 h at 30 °C and pH 7.0 under shaking condition (120 rpm). Glucose (0.5%) as a carbon source was found to be more effective than other sources used. The medium with metal salt (CaCl₂, ZnSO₄, FeCl₃, MgCl₂, CuSO₄) (0.5 mM) showed less ADMI removal as compared to control, but did not inhibit complete decolorization. The presence of tyrosinase, NADH-DCIP reductase and induction in laccase activity during decolorization indicated their role in degradation. HPTLC (High performance thin layer chromatography) analysis revealed the removal of individual dyes at different time intervals from dye mixture, indicating preferential degradation of dyes. FTIR (Fourier transform infrared spectroscopy) and HPLC (High performance liquid chromatography) analysis of samples before and after decolorization confirmed the biotransformation of dye. The reduction of COD (Chemical oxygen demand) (69%), TOC (Total organic carbon) (43%), and phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products having less toxic nature.     

Immediate effects of thermal shocks on the plasma level of various components in the Rainbow trout: compounds indicating stress and protein fraction]

Rainbow trout were subjected to thermal shocks (9 degrees water temperature increase, 1 hr stay at 21 degrees and return to initial temperature) at the rate of 2 shocks a day during 1 day or 3 successive days. The observed changes only show a moderate reaction. cAMP does not vary; lactate slightly increases at 17 hr after the end of the shocks. Glucose seems to be the most reliable stress indicator; it increases at 2 hr and remains again above control value at 17 hr after a 3 day shock time. Fibrinogen increases after a 3 day shock time. Lastly, a strong decrease in low density lipoprotein level is seen at 17 hr following both shock duration times and from 2 hr in case of a 3 day shock time.     

An investigation of dye reduction by food-borne bacteria

S.A. LEAROYD, R.G. KROLL AND C.F. THURSTON. 1992. The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli , dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis , dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens , cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.     

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

Summary It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10^–3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.     

Iontophoretic injection of lucifer yellow CH into zygotes and blastomeres of the hydroidHydractinia echinata

The dye Lucifer yellow CH was iontophoresed into recently fertilized eggs and early blastomeres ofHydractinia echinata. Iontophoresis was carried out on the stage of an inverted microscope in order to follow filling of the injected cells by short pulses of epifluorescent illumination. Lucifer yellow proved to be nontoxic and development in embryos with injected blastomeres proceeded normally. When zygotes were injected all the cells of the forming embryo contained dye. When one of the first two blastomeres was injected all the progeny of the injected cell also contained dye. Dye-coupling between injected and uninjected blastomeres did not occur in two-cell embryos nor between descendants of either line. Development of Lucifer-yellow-filled blastomeres or zygotes could be stopped by blue light irradiation. In a number of injected cells, the dye tended to accumulate forming brightly shining spots. The dye did not penetrate the nuclear envelope of injected cells.