Effect of different grades of agar-agar on the nature of the test microbe growth inhibition zones in controlling antibiotic activity by means of agar diffusion]

The effect of various agar grades on the size and margin character of the inhibition growth zones in assay of antibiotic activity by the agar diffusion method was studied. It was shown that not all the agar grades could be used in antibiotic activity assay. Depending on the agar type the size of the inhibition growth zones produced by the same antibiotic concentration significantly varied. The variations in the size of the inhibition growth zones depended on the agar ability to bind antibiotics and were mainly defined by the agar purity. The agars with low content of nitrogen admixtures bound the antibiotics to a low extent. The commerical grades of the agars of the South-Sea and Korsakov Plants, the experimental grade of the TINRO agar with additional purification, as well as the agars imported from Argentina and France proved to be most useful for determination of the antibiotic activity by the agar diffusion method.     

Mannose inhibition as a significant marker for differentiating among novobiocin-resistant staphylococci of relevance in clinical microbiology

A specific method for the identification of Staphylococcus saprophyticus among novobiocin-resistant species isolated from man is described. The test is based on novobiocin resistance, non-fermentation of D-mannose and early inhibition with late secondary growth on glucose/mannose + novobiocin agar plates. On this medium novobiocin-resistant Staphylococcus cohnii showed a confluent, continuous and homogeneous growth after 24 h which remained unchanged at 48 h whether or not it fermented D-mannose, whereas novobiocin-resistant Staphylococcus xylosus fermented D-mannose. These results are discussed in relation to a possible causal role of PTS enzymes and phosphomannose isomerase deficiency in mannose inhibition.     

Mode of Action of an Inhibitor from Agar on Growth and Hemagglutination of Group A Arboviruses.

Colón, Julio I. (Fort Detrick, Frederick, Md.), Jane B. Idoine, Orville M. Brand, and Richard D. Costlow. Mode of action of an inhibitor from agar on growth and hemagglutination of group A arboviruses. J. Bacteriol. 90:172-179. 1965.-A polysaccharide obtained from agar, and having properties similar to a previously described sulfated polysaccharide, was observed to inhibit growth and hemagglutination of some group A arboviruses. The evidence presented confirms that the inhibitory activity, in part, is the result of direct interaction between the agar polysaccharide (AP) and free virus particles. Additional evidence indicates that inhibition of viral growth also occurs as the result of interaction between AP and the chick-fibroblast cells used for propagation of the virus. The possibility was considered, therefore, that at least two different inhibitors could be present in AP-one that reacts directly with the virus particle and another that reacts with host cells. AP does not induce the production of interferon in the test system used.     

Agar Diffusion Method for the Assay of Colicins

An agar diffusion method for the assay of colicins A, B, D, E(2), E(3), and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about +/- 10%.     

Effects on winter wheat seedling growth by toxin-producing rhizobacteria

Summary Root-colonizing pseudomonads capable of inhibiting seedling winter wheat (Triticum aestivum L.) root growth in an agar seedling bioassay also significantly inhibited wheat root growth in vermiculite; however, the inhibitory trait is quite labile in laboratory culturing. The extent of inhibition in both the agar and vermiculite medium depended on inoculum level. These pseudomonads were found to produce a toxin capable of inhibiting growth ofEscherichia coli C-la andBacillus subtilis. Field isolates that strongly inhibit growth of indicator bacteria also inhibited root growth. Toxin production by the bacteria appeared necessary for inhibition of root growth and indicator bacteria as toxin-negative (TOX^–) mutants no longer inhibited either. Antibiosis towardsE. coli as well as wheat seedling root inhibition in agar was reversed by L-methionine, providing further evidence that a toxin, produced by these organisms, is involved in growth retardation.Contribution in cooperation with the College of Agric. Res. Center, Washington State Univ., Pullman, WA 99164. Scientific Paper No. 6837.     

Biphasic resin system for growing concentrated cultures of microorganisms and its application to the cultivation of Neisseria gonorrhoeae.

A technique is described in which a polyester casting resin is used instead of agar for the growth of Neisseria gonorrhoeae in a biphasic system. In complex medium, higher concentrations and stability of T1 colony type were obtained with resin than with either agar or broth alone after 24 h of incubation. In defined medium, the growth of gonococci and colony type stability were similar in single-phase and resin systems and superior to the growth and stability occurring in the biphasic agar system.     

Use of Fast Green in Agar-Diffusion Microbiological Assays

Microbiological assay plates containing agar stained with fast green and inoculated with test microorganisms could be readily distinguished from unstained seeded agar plates. The boundaries of zones of growth inhibition were more sharply defined in those plates which contained stained agar.     

Development of a method for the quantitative assessment of microorganism sensitivity to antibiotics using discs. The study of different nutrient media for determining microorganism sensitivity to antibiotics

Five nutrient media used for determination of microbial sensitivity to antibiotics, i.e. beaf-peptone agar, Hottinger pancreatic beaf infusion agar, sprat hydrolysate nutrient agar of the Dagestan Research Institute of Nutrient Media, Muller-Chintone agar from Bulgaria and "Oxoid" agar for determination of microbial sensitivity were studied comparatively. The media were compared with respect to the growth density with the use of different test-cultures and the clearance of the inhibition growth zones around the discs containing different antibiotics. The best results were obtained with the use of sprat hydrolysate nutrient agar. Further studies on the medium standardization are necessary.     

CAS平板覆盖法检测氢氧化细菌铁载体

【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。     

Medium- and Light-dependence of Growth and Conidiomata Production in Phaeocytosporella zeae Pathogenic to Maize

Culture appearance, mycelium growth and pycnidial conidiomata formation in Phaeocytosporella zeae cultivated on potato dextrose agar (PDA), Leonian agar and carnation leaf agar (CLA) during permanent dark (25 C) or a 12-h photoperiod (24/18°C) are described. Different media and light conditions significantly affected fungus growth and the occurrence of conidiomata.
Dark conditions favoured more mycelium growth of the fungus than alternating light and dark. Moreover, fungus growth was more rapid on PDA and CLA media than on Leonian agar. Carnation leaf agar and a 12-h photoperiod provided excellent conditions for the promotion of rapid conidiomata formation in a number of P. zeae isolates.     

A peculiar example of bacterial antagonism

Summary An example of antagonism has been described. The properties of the growth inhibiting bacterium have been investigated more closely. These appear to agree nearly fully with those of the genusGaffkya.The development ofPasteurella avicida, Bacillus orpheus andStaphylococcus was strongly inhibited by this bacterium. Other species are less sensitive or insensitive.After the theoretical problems have been discussed, which such investigations give rise to, it has been studied under which conditions Staphylococci are inhibited.The occurrence of the inhibition effect depends on the stage of development either bacterium is in;Staphylococcus having developed completely it will not notably be acted upon. Initially inhibition of growth occurs along with dying off ofStaphylococcus. In a somewhat later stage the vitality remains unimpaired.The inhibiting bacteria produce inhibiting substances merely in the first 24 hours. In the filtrate of a broth culture these are hard to detect. Their behaviour in agar plates makes it apparent that their action depends strongly on the concentration of the antagonist.The durability of the inhibiting substances in the agar medium is not great. In all probability this depends on a volatibility of these substances.     

Modulation of growth of human carcinoma SW-13 cells by heparin and growth factors

This study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type-e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW-13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin-Sepharose column with 2 M NaCl and 1.6 M NaCl, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin-Sepharose column with 0.5 M NaCl. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW-13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW-13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW-13 cells. A dose-dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) were unable to overcome heparin-induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR-2B and partially inhibited AKR-2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW-13 cells and possibly of other epithelial cells in complex ways and that heparin-like substances present in the extracellular matrix play an important role in the control of epithelial growth.     

A microbiological test system for determining dioxidine levels in biological fluids

A microbiological procedure for determining dioxidine concentrations in biological fluids with using E. coli AB 2472 rec A 16, a reparation deficient strain as a test organism is described. Cell suspension of the strain 24-hour culture is added to 1.2 per cent agar with Hottinger digest (140 mg per cent of amine nitrogen), 3 g/l of disubstituted sodium phosphate and 0.4 per cent of glucose cooled to 50 degrees C. 10 ml of the medium are added to every Petri dish with metallic cylinders put on the agar. After the medium solidification the cylinders are removed and 0.1 ml of the solution being tested is added to every well. The dishes are incubated for 24 hours under anaerobic conditions. The test system sensitivity is 0.2 microgram/ml of dioxidine. The relationship between the growth inhibition zone and the drug concentration is linear within dioxidine concentrations of 0.2 to 3.2 micrograms/ml.     

Chromogenic plate assay distinguishing bacteriolytic from bacteriostatic activity of an antibiotic agent

A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. The assay uses a bacterial culture harboring β-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, β-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds consistently did not display blue halo at the inhibition zone.     

Permanent mounts of fungus cultures grown on media optimal for production of characteristic structures

Summary 1. Dermatophyte cultures in plastic flasks were compared on four different media for total growth, sporulation, and pigment production. Czapek's agar favored more rapid and more abundant sporulation in most cultures than Sabouraud's dextrose agar, potato dextrose agar, or wort agar.2. A simple technique for embedding agar island cultures of fungi is described. These plastic-embedded cultures form permanent mounts useful for study of microscopic morphology.3. A relatively durable slant culture in a similar plastic flask is suggested as a companion for study of gross morphologic features of the cultures.     

Tryptic Soy Bile-Kanamycin Test for the Identification of Bacteroides fragilis

A simple and practical test for the identification of Bacteroides fragilis is described. It utilizes two well-known properties of this species, i.e., stimulation of growth by bile and resistance to kanamycin. The test media are a tryptic-soy bile agar plate and a supplemented blood agar plate on which a kanamycin 1,000-mug/ml disk is placed. Incubation is for 24 h at 37 C in GasPak. The results of screening 190 strains, mostly clinical isolates, indicate that B. fragilis can be easily and reliably distinguished from other Bacteroides and from Fusobacterium species by its growth on tryptic-soy bile agar and resistance to kanamycin.     

A new screening method for investigating microbial interactions

Interactions among Candida albicans, Staphylococcus aureus and Escherichia coli were investigated using a screening system in which test micro-organisms were incorporated in agar discs and effector micro-organisms in fluid growth media. Total as well as partial inhibition of test micro-organisms was observed in agar discs when these were incubated in broths containing effector micro-organisms. The ratio of numbers of test to effector micro-organisms was found to be of importance in the inhibition effect. The technique was found to be cheap, simple and versatile.     

Induction of Curvature in Maize Roots by Calcium or by Thigmostimulation : Role of the Postmitotic Isodiametric Growth Zone

We examined the response of primary roots of maize (Zea mays L. cv Merit) to unilateral application of calcium with particular attention to the site of application, the dependence on growth rate, and possible contributions of thigmotropic stimulation during application. Unilateral application of agar to the root cap induced negative curvature whether or not the agar contained calcium. This apparent thigmotropic response was enhanced by including calcium in the agar. Curvature away from objects applied unilaterally to the extreme root tip occurred both in intact and detipped roots. When agar containing calcium chloride was applied to one side of the postmitotic isodiametric growth zone (a region between the apical meristem and the elongation zone), the root curved toward the side of application. This response could not be induced by plain agar. We conclude that curvature away from calcium applied to the root tip results from a thigmotropic response to stimulation during application. In contrast, curvature toward calcium applied to the postmitotic isodiametric growth zone results from direct calcium-induced inhibition of growth.     

An Agar Diffusion Method for the Determination Of Antibodies Against Staphylococcus Aureus Deoxyribonuclease

On the addition of small concentrations of deoxyribonuclease, produced by Staphylococcus aureus, to Toluidine Blue DNA agar, a medium is produced on which antibodies against S. aureus deoxyribonuclease may be detected. When samples of milk, or blood serum, containing antibodies against S. aureus are applied into wells in the agar, the deoxyribonuclease activity is inhibited by the antibodies diffusing into the agar. As a result of this inhibition, blue zones are produced around the wells in the otherwise bluish-red agar. The diameters of the zones correspond to the concentrations of antibodies, and the method may consequently be used for qualitative and quantitative examinations of antibodies against S. aureus deoxyribonuclease in milk and serum. The procedure and certain limitations of the method are described.     

Studies on pre-incubation conditions in the seeded agar plate well inhibition assay

Pre-incubation at 4°C increases the zone of bacterial growth inhibition produced by methanolic extracts of freshwater algae in the agar diffusion assay. Preloading of wells with methanol increases the inhibition zone produced by water-insoluble antibiotics.