Method for Staining and Preserving Agar Gel Diffusion Plates

An improved method for the preservation of agar gel precipitin lines has been developed.     

Stability of Antibiotics and Chemotherapeutics in Agar Plates

The stability of chemotherapeutic agents incorporated into agar plates was studied by comparison of minimum inhibitory concentrations on fresh and stored plates and by direct bioassay of the chemotherapeutic agar plates. Plates were stored in sealed bags at 4 C. No loss of bioactivity was demonstrated after 30 days of storage in plates containing methicillin, erythromycin, cephalothin, tetracycline, chloramphenicol, kanamycin, streptomycin, polymyxin B, or nalidixic acid. Penicillin G, ampicillin, and nitrofurantoin showed statistically significant losses of activity after 4 weeks. None of the chemotherapeutics tested showed significant loss in activity after 1 week.     

A Simple Method for Isolation and Photography of Clostridium Botulinum Colonies from Surface Cultures on Agar Plates

Reflected light gives a very good contrast using blood agar plates in an obliquous position under a stereoscopic microscope. Thus it is possible to isolate pure colonies of Cl. botulinum in the early stage of the growth on blood agar plates.     

Evaluation of Agar Plates for Direct Enumeration of Campylobacter spp. from Poultry Carcass Rinses

Campy-Cefex, a modification of Campy-Cefex, modified charcoal cefoperazone deoxycholate (mCCDA), Karmali, CAMPY, and Campy-Line agars were evaluated for their efficiency to isolate and enumerate Campylobacter spp. from poultry carcass rinses. Campy-Cefex and its modification produced the best results but were statistically similar to CAMPY, mCCDA, and Karmali.     

A Method for the Detection of Weak Lipolysis of Dairy Lactic Acid Bacteria on Double-Layered Agar Plates

A marine bacterium, strain 7-246-6, isolated from sea water was found to elaborate aerobically an extracellular enzyme specific for native collagen and gelatin. The strain was identified as Pseudomonas marinoglutinosa from the morphological and physiological characteristics, though the strain 7-246-6 slightly differs from the latter in the following points; the shape of the colony on the plate culture, the optimum growth temperature, the presence of granule by granular staining, the utilization of carbohydrates and the number of flagellum.     

益生乳酸菌的纸片扩散法药敏性试验评价

乳酸菌菌株的药敏性评价是目前国际上益生乳酸菌安全性评价的首要内容, 然而用于乳酸菌药敏性测试的相关国际通引标准还未出台。本文就药敏试验使用的培养基进行了探讨, 并从中选取了合适的商品化培养基——RCA用于乳酸菌的药敏性测试。选取的培养基适合目前市场上常用益生菌的生长, 经方法优化后可得到满意的结果, 为今后制定针对乳酸菌的抗菌药物敏感性试验执行标准提供了一定依据。随后采用纸片扩散法对分属于4个属的9株乳酸菌进行了19类51种抗生素的敏感性测定, 较全面地了解了这些菌株的药敏谱。     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Use of Hydrogen Peroxide Treatment and Crystal Violet Agar Plates for Selective Recovery of Bacteriophages from Natural Environments

Hydrogen peroxide inactivated bacteriophages and bacteria at different rates. A concentration of 0.1% hydrogen peroxide reduced the numbers of several bacteria by an average of 94% but caused an average of 25% inactivation in the numbers of bacteriophages tested. Treating natural samples with hydrogen peroxide selectively reduced the indigenous bacterial flora and permitted better visualization of plaques of lawns of Escherichia coli C-3000. In some cases indigenous gram-positive bacteria were relatively resistant to hydrogen peroxide, but their growth could be limited by incorporation of crystal violet into the bottom agar used for plaque assays. The use of hydrogen peroxide treatment and crystal violet-containing plates permitted recovery of more phages from natural samples than did other procedures, such as chloroform pretreatment or the use of selective plating agar such as EC medium.     

Punched-Out Agar Petri Plates for Systematic Fixation and Embedding of Multiple Small Tissue Samples

A 5% agar solution is poured into Petri dishes, jelled, and a pattern of cylindrical holes punched out according to the number and size of tissue samples to be processed. These punched-out Petri plates withstand neutral formalin, water, benzene, and paraffin immersion at 60 C; thus providing an adaptable container for processing small tissue samples. Reduced handling of the tissues, minimized risk of loss, and easy identification of samples by means of the map-like arrangement of the holes are among the virtues of such plates. An optimal fixative-volume: tissue-weight ratio can be attained by using wells of varying diameter and depth according to the sample size.     

New Method of Grouping Beta-Hemolytic Streptococci Directly on Sheep Blood Agar Plates by Coagglutination of Specifically Sensitized Protein A-Containing Staphylococci

A technique is described that allows the grouping of beta-hemolytic streptococci directly upon the primary colony. This was accomplished by applying a small drop of specifically sensitized protein A-containing Staphylococcus aureus over a colony of streptococci, rocking the plate to allow mixing of the particles with the soluble group-specific polysaccharide, which in the case of beta-hemolytic streptocicci was produced in abundance during colony formation, and observing for agglutination of the sensitized particles. Such a simple test for group A beta-hemolytic streptococci should allow accurate identification of group A streptococci in small laboratories, such as in clinics or physicians' offices, as well as in the larger public health and private laboratories.     

Use of Sodium Polyanethol Sulfonate in the Preparation of 5% Sheep Blood Agar Plates

An alternative method to defibrinating sheep blood for use in bacteriological media is described. The new procedure incorporates sodium polyanethol sulfonate in a concentration of 0.05% (vol/vol). In testing 117 bacterial and fungal isolates, no significant differences were found with respect to adequate growth, pigment production, hemolytic reactions, and other physical attributes. Further tests demonstrate that the sodium polyanethol sulfonate in sheep blood agar plates does not cause any aberrations in zone sizes around disks used in antibiotic susceptibility tests. Consequently, the method represents a suitable alternative to the use of defibrinated sheep blood in the preparation of bacteriological media.     

Alternative Procedure to Screening a Large Number of Plates for a Low Number of Mutants

An apparatus is described in which tygon tubing replaces the use of petri dishes in the screening of a large volume of agar for an expected low number of mutants.     

Agar Slush as a Method to Insure Homogeneity of Monocentric Fungi in Continuous Culture

Agar granules suspended in the growth medium reduce clumping, which permits the continuous culture of monocentric chytridiomycete fungi.     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar.     

Agar Plate Method for Detecting Microorganisms Which Produce Equilin and Other Estrogens from Various Steroids

The intense red color produced by a reagent specific for xi(7)-estrogens is used to directly detect microorganisms which produce these estrogens from various steroids.     

A Non-metric Procedure for Transforming Dissimilarities to Euclidean Distances Useful in Numerical Taxonomy and Ecology

Because (a) in agglomerative cluster analysis, it is the smallest dissimilarities which are used, (b) in numerical ecology, little or no information is given about the resemblance between two localities if there are very few (or no) species in common, (c) for a given dissimilarity, the number of ways two objects can differ in the states shown by their attributes is small for small dissimilarities, and disproportionately larger for larger dissimilarities, (d) the major axes in principal coordinates analysis are determined by the largest dissimilarities, which from (a–c) are the least informative, and (e) the ‘orsesho’ pattern often shown by two-dimensional scalings of dissimilarities, it is concluded that the largest dissimilarities should be replaced by values determined from the smallest. The proposal is to retain the dissimilarities corresponding with the edges in the relative neighbourhood graph, and to replace the remaining by the shortest paths on the dissimilarity-weighted graph. The new dissimilarities, which are linearly related with the retained subset, are shown to be Euclidean, and usually the effective dimensionality is reduced in comparison with the original set. The internal disposition of distinct subsets of objects is little affected, but any distinct subsets tend to be further separated. By contrast, if the smallest dissimilarities represent random differences, while the larger subset represents those which are systematic, it is the latter which should be retained. The notion of the relative external graph is introduced, and the procedures required to obtain this, and to replace the deleted empirical smallest dissimilarities briefly described. However, this results in an increase in the effective dimension, changes in the internal disposition of distinct subsets, and reduction in their separation.–Two numerical examples, based on empirical data, illustrate some of the consequences of the transformations.     

A New Method for Estimation of the Mycelial Weight in Koji

A New method was devised for the estimation of the mycelial weight in rice-koji. In this method, the content of glucosamine in koji was used for the calculation of mycelial weight. The content of glucosamine in the mycelia of Aspergillus oryzae, koji, and rice was determined by a colorimetry after hydrolysis of these materials with sulfuric acid and purification of glucosamine fraction with a Dowex 50 W column. And the values of glucosamine were 114.5 mg/g in mycelia, 3.03 in the koji for amazake,* 1.34 in the koji for sake, and 0.0 in rice. The mycelial contents calculated from these data were 2.6% dry weight in amazake-koji and 1.2% in sake-koji.