Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater

S ummary . Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fermenting colonies from seawater, developing at 44° on a 4% enriched Teepol medium, are E. coli type I. A total of 1352 colonies from seawater was tested for production of indole and for gas from lactose at 44°. In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonies tested ranged from 10–100%, depending on the number of colonies on the membrane. Many of the colonies (81.9%) to which IMVEC tests were applied were E. coli type I; a further 10.9% were Irregular type I. The practical implications of these findings are discussed.     

Dichromate-Chlorate Perfusion Prior to Staining Degeneration in Brain and Spinal Cord

A method of fixation compatible with both the Nauta-Gygax and Swank-Davenport procedures for degenerating nerve fibers, which shortens the time required by the former procedure, is as follows: The central nervous system is perfused with a 0.9% aqueous solution of NaCl followed by an aqueous solution containing 5% K₂Cr₂O₇ and 2.5% KClO₃. The central nervous system is then hardened in 10% formalin for 1-3 days. Tissue for Marchi-type staining can be taken at this stage. For silver staining, the processing is continued by immersion overnight in 10% formalin in 20% alcohol, and frozen sections cut the next day. Sections, up to 50μ in thickness, are collected in 10% formalin and impregnated by the Nauta-Gygax technique. Best results are obtained by impregnating within 24-48 hr after sectioning.     

Paraffin-Beeswax-Stearic Acid: An Embedding Mass for thin Sections

It is possible to cut 1-3 μ sections of rat tissue after passing it through ethanol and chloroform and infiltrating in a wax mixture consisting of 95% Shell Chemical Co. paraffin (MP 125-130° F) and 5% commercial beeswax (clarified by boiling with water and decanting), to which is finally added 10% of technical stearic acid (melted, and clarified by filtration). A potential disadvantage is the slow expansion of the section on the water flotation bath, due to a surface spreading effect of the contained stearic acid. This expansion can be minimised as follows: by adding 0.5% concentration of a secondary alkyl-aryl sulfonate detergent, such as Shell Chemical Co. Teepol, to the notation water; by keeping the temperature of the water at 45° C; by making sure that no section is left on the water for more than 30 sec; and by drying on chemically cleaned slides for 4-18 hr at 45° C., controlled to ±2°. The spreading effect is advantageous in reticulo-endothelial studies, where overlap of cells needs to be reduced to a minimum, and thinly layered cytoplasm expanded.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Concentrated Formalin Versus A 10% Solution as a Fixative Preceding Silver Staining

The effect of concentrated and 10% formalin as fixatives for the silver staining of axons in the central nervous system in a series of mammals has been compared. It is felt that concentrated formalin constitutes a better fixative for this purpose in several respects: it has a greater speed of penetration without causing additional distortion or artifacts, the staining time can be reduced somewhat and the normal configuration of the axons is better preserved particularly in the largest ones. Satisfactory results were obtained following 51/2hours fixation in concentrated formalin. Further, concentrated formalin is at least equal to a 10% solution in the ease in which paraffin and frozen blocks can be sectioned, in the clearness of the background of stained sections and in the consistency of results.     

Stability of ectromelia virus strain NIH-79 under various laboratory conditions

Ectromelia virus strain NIH-79 was suspended in fetal bovine serum (FBS), minimum essential medium, Hanks' base plus 10% FBS (MEMH + FBS), phosphate-buffered saline (PBS) or PBS plus 50% glycerol (PBS + G). Suspensions were held as liquids or as dry spots at various temperatures. Virus was most stable in FBS and least stable in PBS + G at 4 degrees C, room temperature (23-25 degrees C) or 37 degrees C. Virus held at 4 degrees C was more stable than virus held at higher temperatures, irrespective of supporting medium. Dried spots of blood or serum from ectromelia virus-infected mice remained infectious at room temperature for 11 days and 4 days, respectively. Dried spots of FBS that contained virus were infectious for 5 days, whereas virus retained infectivity for 1 day after drying in other media. Virus was inactivated completely in 10% serum in PBS exposed to 60 degrees C for 30 minutes. Virus was inactivated completely in slices of infected liver and spleen immersed in 10% neutral buffered formalin for 20 hours. These results show that the stability of ectromelia virus strain NIH-79 is medium and temperature dependent and that rapid inactivation occurs after treatments routinely used in diagnostic and research procedures.     

Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.     

Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods.

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.     

Enrichment of Vibrio parahaemolyticus in a Simple Medium

A medium which contained 3% NaCl and 0.2% Teepol in 1/15 M phosphate buffer was prepared and was evaluated to be a useful enrichment medium for the isolation of Vibrio parahaemolyticus in marine specimens. Glucose salt Teepol broth produced a poorer result than direct culture.     

“Freezing artifacts” in human tissue samples: Their formation and prevention

The mechanism underlying the formation of the so-called “freezing artifacts” in biopsies exposed to low outdoor temperatures is discussed.Assuming that this mechanism primarily consists of osmotic damage derived from ice formation in the fixative solution in which the biopsies are customarily dispensed (rather than of actual freezing of the samples as it is currently assumed), a 70/30 (v/v) buffered neutral formalin/methanol mixture was developed, which can be held for 15 hr at ?20 °C without ice development.This mixture, whose fixative properties are for practical purposes similar to those of regular buffered neutral formalin, used as a substitute for the latter fixative medium, suppressed the expected incidence of about 1–3% cases of freezing artifacts among the several thousand biopsies processed in this laboratory during the winter months of 1979/1980.     

Blood Serum as an Adhesive for Paraffin Sections

Diluted human and other mammalian blood serum (15 ml. of fresh blood serum diluted with 10 ml. of freshly distilled water, with 6 ml. of 5% formalin solution in distilled water added) can be used as a good adhesive for paraffin sections. It is preferable to Mayer's egg albumin-glycerol mixture because it is easily obtainable, can be quickly prepared, and sections are less subject to loosening after its use.     

Embedding and Fixation Techniques for Immunohistochemical Staining with Anti-DNA Polymerase α and Ki-67 Monoclonal Antibodies to Analyze the Proliferative Potential of Tumors

Anti-DNA polymerase a and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.     

Effect of Fixatives on Silver Impregnation of Plant Cells

The kind of fixative and duration of fixation modify the affinity of plant cell structures, as shown by a 10-15 hr impregnation at 70 C in 2% aqueous AgNO₂, and a 1-2 hr reduction at room temperature by a 1:1 mixture of 10% formalin and 1% hydroquinone. Cytoplasmic staining was enhanced by fixing in salts of heavy metals, in buffered 6.5% glutaraldehyde, and in 0.5% picric acid. Nuclear staining was prominent after mixtures of glutaraldehyde and hydroquinone, after formalin and pyrogallol, and after acetone, propylene glycol or ether. Nucleolar staining was favored by fixing in 10% formalin, in 5% formalin containing 0.5% hydroquinone, in 50% ethanol containing 0.5% pyrogallol, or in ethylene glycol. Chromosome staining was favored by fixation in 50% acetic or propionic acid, in 2% trichloroacetic acid, and in methanol or ethanol. The best morphological preservations were seen after 50% acetic acid, 6.5% glutaraldehyde, or the 5% formalin-0.5% hydroquinone mixture.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

A simplified aluminum stain in paraffin sections of bone from hemodialysis patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

A new technique for long preservation of 14C-labelled Cladocerans

Three ways of preserving labelled Cladocerans fed with ¹⁴C-Chlorella for 7.5–10 min were tested. Tracer leakage in 4% formalin at room temperature is rapid and extensive (half of the label was found in the animals after 1 hour of preservation). Even when individuals are frozen and sorting is made quickly in a liquid, losses nevertheless occur (substantial decrease of animal activity after only 4–5 min in the water in one of the two experiments performed). Results obtained after freezing in 4% formalin and sorting exactly 2 hours after thawing gave consistent losses: 16 separate experiments with Daphnia pulex, Ceriodaphnia spp. and Diaphanosoma brachyurum gave apparent filtering rates underestimated from 35% to 63% for freezing periods of up to 45 days. The good agreement in in situ community filtering rates between measured values and estimated ones from individual data confirmed the validity of a correction factor of x 2 applied to animals frozen in formalin.     

Effects of ethanol on deep pain evoked by formalin injected in TMJ of rat

It has been reported that ethanol can alter nociceptive sensitivity from superficial tissues, such as skin and subcutaneous region. However, the influence of ethanol on deep pain conditions is not understood. The aim of this study was to demonstrate the acute, chronic and ethanol withdrawal effects on nociceptive behavioral responses induced by the injection of formalin into the temporomandibular joint (TMJ) region of rats. In experiment 1, rats were injected with ethanol (2,5 g/Kg, i.p.) or an equal volume of saline 15 min before the administration of formalin (1.5%) into the TMJ. Rats pretreated with ethanol showed a decrease in nociceptive behavioral responses. In experiment 2, rats were given an ethanol solution (6.5%) or tap water to drink for 4 and 10 days. On day 4, the animals (ethanol group) showed amounts of analgesia when submitted to the TMJ formalin test. Tolerance to the antinociceptive effects was observed on day 10. Behavioral hyperalgesia was verified 12 hr after withdrawal in another group that drank ethanol for 10 days. These results show that ethanol can affect the nociceptive responses related to deep pain evoked by the TMJ formalin test.