Growth and Plating Efficiency of Methanococci on Agar Media

Plating techniques for cultivation of methanogenic bacteria have been optimized for two members of the genus Methanococcus. Methanococcus maripaludis and Methanococcus voltae were cultivated on aerobically and anaerobically prepared agar media. Maintenance of O₂ levels below 5 μl/liter within an anaerobic glove box was necessary during plating and incubation for 90% recovery of plated cells. Under an atmosphere of H₂, CO₂, and H₂S (79:20:1), 2 to 3 days of incubation at 37°C were sufficient for the formation of visible colonies. The viability of plated cells was significantly affected by the growth phase of the culture, H₂S concentration, and the volume of medium per plate. In addition, colony size of methanococci was affected by agar type, as well as by the concentrations of agar, H₂S, and bicarbonate.     

Modeling Surface Growth of Escherichia coli on Agar Plates

Surface growth of Escherichia coli cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H.Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34°C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves.     

Ultrastructure of Nocardia Cell Growth and Development on Defined and Complex Agar Media

The cell growth of Nocardia strain 721-A on Brain Heart Infusion Agar (BHIA), nutrient agar, and chemically defined agar media was studied by light and electron microscopy. Light microscopy revealed a change in cell morphology induced by growth on BHIA. Electron microscopy demonstrated a concurrent change in intracellular complexity. On BHIA, the cells became bulbous and developed irregularly branched filaments which fragmented by multiple and random septation. These fragments appeared to undergo a secondary stage of development similar to that described for Arthrobacter. Cells grown on defined or nutrient agar did not become bulbous and lacked the unusual complexity found in cells grown on BHIA. Intracytoplasmic membranes were altered by the nutritional state of the cell and changed during cell development.     

Automated Device for Preparing Agar Media

An automated device which facilitates safe and effective agar media preparation without constant supervision is described.     

Natamycin as a Fungicide in Agar Media

Fungal inhibition in four commonly used agar media was improved by substituting natamycin (pimaricin) for cycloheximide. The recovery of bacteria was not affected by natamycin, whereas fungal contamination from a variety of samples was significantly suppressed. Furthermore, natamycin lacks the occupational health hazards of cycloheximide. Medium-dependent natamycin degradation occurred during the preparation and refrigerated storage of agar plates, but the addition of natamycin at 21.6 μg/ml resulted in effective residual activity.     

Lysine Decarboxylase Activity in Broth and Agar Media

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.     

GELRITE as an Agar Substitute in Bacteriological Media

GELRITE gellan gum (formerly known as PS-60 and S-60) is a new naturally derived, highly purified polysaccharide which displays several interesting properties, including selfgelling. The suitability of GELRITE as an agar substitute was tested by evaluating the performance of several media selected from among those most commonly used in the isolation, identification, and enumeration of microorganisms in clinical laboratories. Fifty different bacterial species previously implicated in human infections served as test strains. On the basis of the various parameters considered, namely, colony characteristics, biochemical reactions, hemolytic patterns, and plating efficiency, media gelled by agar and by GELRITE compared quite favorably.     

Nutrient Requirements of Renibacterium salmoninarum on Agar and in Broth Media

In well-aerated broth cultures, good growth of Renibacterium salmoninarum was obtained in a serum-free medium consisting of 1% peptone, 1% yeast extract, and 0.1% l-cysteine (PYC broth). In contrast, serum or charcoal is required for growth on agar medium. Charcoal treatment of broth media, either before bacterial inoculation or during growth, increased the growth of R. salmoninarum, whereas the surfactants Tween 20 and Tween 80 inhibited growth. l-Cysteine was essential for optimal growth. Other organic sulfur compounds, such as d-cysteine, l-methionine, homocysteine, homocysteine thiolactone, and reduced glutathione, supported only lower levels of growth, while cystine and dithiothreitol did not allow growth.     

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.     

Effect of Diethylaminoethyl Dextran on the Growth of Mycoplasma in Agar

The growth of certain strains of Mycoplasma is inhibited by substances present in commercial agar preparations. The addition of diethylaminoethyl (DEAE) dextran (10 mg per 100 ml) to agar media appears to enhance the growth of some strains. Of eight strains initially tested, the presence of DEAE dextran grossly enhanced the growth of three strains. One strain appeared not to be affected, and a clearly enhancing effect was not evident with four strains. Quantitative studies revealed that growth enhancement varied from 10 colony-forming units (CFU) for M. hominis type II (strain Campo) to 10(3.3) CFU for M. pulmonis (strain 880). The growth-enhancing effect is probably due to the ability of DEAE dextran to bind the sulfated polysaccharide moieties in agar and not to the DEAE dextran, per se.     

Method for Measuring Changes in Surface Tension on Agar

The surface tension of agar surfaces was determined by measuring the contact angles formed by drops of various hydrophobic liquids on the surface and then calculating the composite surface free energy function by solving a series of simultaneous equations derived from these data. This method was used to measure the change in the surface tension of agar produced by the addition of various concentrations of albumin. The resulting curve was typical of the effect of increasing concentrations of surfactants on surface tension. The method was compared with other methods of determining surface tension of solids, and it was concluded that the technique used here provided the most reliable results.     

Effect of Method of Agar Addition on Post-autoclave pH of the Tissue Culture Media

Tissue culture media (MS, GB-5 and N6) were gelled with agarby employing different methods When agar was dissolved directlyin media by autoclaving at 120 °C for 1 mm followed by sterilization,the media pH dropped markedly — more so at the lower concentrationsof agar On the other hand, when agar was first dissolved indistilled water and added to the culture media the pH loss wasminimized considerably pH measurements carried out as a functionof time showed a gradual decline in media pH The media supplementedwith Panax ginseng callus became more acidic than the mediawith no callus Tentative reasons for the post autoclave pH fallassociated with various methods of agar addition are described Culture media, agar addition, post-autoclave pH, Panax gingseng