Evaluation of kappa carrageenan as a substitute for agar in microbiological media

Seventy-one samples of the colloid kappacarrageenan extracted from 12 seaweed species were subjected to a number of standard physical demands of solid bacteriological culture media. All samples had a lower melting temperature (less than 67° C) than agar and a gelling (setting) temperature between 16° C and 51° C, some the same and others lower or higher than agar. Temperature spreads were narrow (ca 10° C) to broad (ca 30° C), depending on the seaweed source, but none were as broad as that of agar (ca 40° C). The majority of commercially prepared samples held a slant when incubated at 37° C, but California seaweed colloids were best at 28° C in this test. The majority of samples released little to no water of syneresis in slant tests as well as in plates. Some plates prepared with the colloid were crystal clear as compared to agar plates. All test microorganisms grew as well on kappa-carrageenan media as on agar media. Some media responses could be attributable to the seaweed species, but others could be traced to chemical extraction methods and modification of the colloid.     

Comparison of Media for Detection of Fungi on Spacecraft

Five media, including Trypticase soy agar (TSA; BBL) pour plates, spread plates of TSA, Mycophil agar with chloromycetin, Mycophil agar with chloromycetin and Actidione, and cornmeal agar with chloromycetin were quantitatively and qualitatively compared for the detection of fungi on spacecraft. Cornmeal agar with chloromycetin yielded the highest number of fungal colonies, although not always significantly higher than Mycophil agar with chloromycetin or TSA spread plates. Cornmeal agar with chloromycetin also gave the best qualitative representation of fungi on the spacecraft, recovering 68% of the genera found from all media. This medium yielded 10 times the number of fungal colonies and 3 times the number of genera found on TSA pour plates as currently used for spacecraft assay.     

盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27^T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.     

Effect of sealing and Tween 80 on the antifungal susceptibility testing of essential oils

Concentrations of essential oils showing high volatility decreased substantially in broth and agar media when incubated under open conditions. The decrease in the half life was from 0.7 to 38 hr in broth medium at 27 C. When evaporation was prevented by sealing, MIC values against Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes by broth or agar dilution assay were lowered two to eight-fold, as compared with those obtained under open conditions. Addition of Tween 80 caused a rise of the MICs against A. fumigatus by two to four-fold in broth dilution assay, but little affected the MICs in agar dilution assay.     

Effect of selective media on loss of congo red binding inShigella flexneri

Summary The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Effect of culture conditions on microorganism identification by matrix-assisted laser desorption ionization mass spectrometry

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures. This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K. H. Jarman, S. T. Cebula, A. J. Saenz, C. E. Petersen, N. B. Valentine, M. T. Kingsley, and K. L. Wahl, Anal. Chem. 72:1217-1223, 2000). A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.     

Comparison of two techniques for obtaining endometrial bacteriologic cultures in the mare

The endometria of 39 mares were cultured simultaneously using a swab guarded with a double cannula and distal, teflon plug and an unguarded swab with a single, open cannula. Sheep blood (5%) agar, Mac-Conkey's agar, and Sabourad's agar media were innoculated with each swab. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. There were significantly more plates that failed to yield bacterial or fungal growth when streaked with swab specimens obtained with the guarded cannula than when streaked with those obtained with the unguarded cannula. It was concluded that while culturing the endometrium of mares for bacteria or fungi, the use of a guarded instrument consisting of a double cannula with a closed end will result in the recovery of fewer contaminants; therefore, it will be more likely to result in a more accurate representation of uterine bacterial and fungal flora.     

Effect of Culture Conditions on Microorganism Identification by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify bacteria based upon protein signatures. This research shows that while some different proteins are produced by vegetative bacteria when they are cultured in different growth media, positive identification with MALDI-TOF MS is still possible with the protocol established at the Pacific Northwest National Laboratory (K. H. Jarman, S. T. Cebula, A. J. Saenz, C. E. Petersen, N. B. Valentine, M. T. Kingsley, and K. L. Wahl, Anal. Chem. 72:1217-1223, 2000). A core set of small proteins remain constant under at least four different culture media conditions and blood agar plates, including minimal medium M9, rich media, tryptic soy broth (TSB) or Luria-Bertani (LB) broth, and blood agar plates, such that analysis of the intact cells by matrix-assisted laser desorption/ionization mass spectrometry allows for consistent identification.     

Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed BALB/c3T3 cells

Clones were derived in culture from a tumor initiated by spontaneously transformed 3T3 cells and tested for their colony-forming efficiency in agar (CFEag). Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination. There was great variation in CFEag between clones but also, under certain conditions, within clones. The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of a mass population from a single cell. Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates. If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer. The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration. Damaged cells were restored to their full growth potential after 3 d in isotonic medium. When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced. With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice.     

Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions.     

A simple and effective plating method to screen polycyclic aromatic hydrocarbon-degrading bacteria under various redox conditions

Agar plates with a polycyclic aromatic hydrocarbon (PAH) layer have been used to screen for microorganisms that degrade PAHs, leaving clear zones around colonies; however, there are several problems with previous methods such as undesired contamination in the fume hood and difficulty in controlling the amount of PAH on the plates. In this study, we developed a modified screening method to address the drawbacks encountered with previous screening methods. A uniform white layer of PAHs was generated by spreading PAHs dissolved in volatile solvents over a surface of solidified agar medium, followed by the evaporation of the solvents. An inoculation was then performed by spreading a molten agar medium containing microbial samples over the solidified agar medium with a PAH layer. Subsequently, the white PAH layer migrated to the surface of the molten agar medium. This essential modification enabled us not only to solve problems of the previous screening methods but also to prepare an agar plate with a PAH layer without a complicated experimental scheme in the anaerobic chamber. After solidification of the molten agar medium and incubation of the plates, clear zones were successfully detected around colonies with aerobic and anaerobic PAH-degrading microbial cultures.     

Selective and Differential Media for Isolation and Tentative Identification of Each Species of Pityrosporum Residing on Normal or Diseased Human Skin

Selective and differential media were designed for each species of Pityrosporum; P. pachydermatis, P. ovale, and P. orbiculare in order to make feasible a quantitative cultivation. Medium for P. pachydermatis (medium A) was composed of 1% trypticase peptone (BBL), 0.5% yeast extract (BBL), 0.3% glucose, 0.2% NaCl, 1.2% KH₂ PO₄ (anhydrous), 1.5% agar, 0.01% ampicillin, and 0.025% cycloheximide with a pH of 5.5. Medium for P. ovale (medium B) was medium A supplemented with 0.05% sodium acetate (anhydrous), 0.2% Tween 80, and 0.025% (selective medium) or 0.075% (differential medium) sodium laurate. Medium for P. orbiculare was medium B (devoid of laurate) supplemented with 2% olive oil, 0.25% glycerol, 0.25% gall powder, 0.05% sodium palmitate, 0.05% sodium stearate, 0.05% sodium oleate and 8% (selective medium) or 10% (differential medium) sodium lactate and an increase in Tween to 1%. For isolation of Pityrosporum, specimens were suspended in 0.1% Tween 80 solution and inoculated onto agar plates of three selective media. The plates were incubated aerobically at 37 C for 8–10 days under conditions of prevention of water loss from the media. The plating efficiency of each selective medium, expressed as a ratio of cultural counts to microscopic counts was generally over 70%. Species of Pityrosporum could also be identified when we inoculated the cell suspension onto differential agar plates and incubated the preparations at 37 C for 7 days.     

Arabidopsis Alcohol Dehydrogenase Expression in Both Shoots and Roots Is Conditioned by Root Growth Environment

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.     

Comparison of Mycobacterial Cultures on Agar and Lowenstein Medium

In a comparison of Lowenstein's medium with two agar media, 7H10 without and with the addition of Triton WR 1339, 4,100 sputa were planted on all three, yielding 803 (19.6%) positive cultures. The addition of Triton WR 1339 to the 7H10 medium made it possible to observe the cord formation directly on the isolation plates and did not alter, in any respect, the results obtained with the basic 7H10 medium. The agar media produced much earlier and more positive cultures than Lowenstein's medium. A greater number of cultures showed drug resistance on Lowenstein's medium as compared with the agar media.     

Value of the K+ salt of carageenan as an agar substitute in routine bacteriological media

The K+ salt of carageenan has no distinct advantages as a gelling agent, but it compared favorably with agar in most of the media tested. The difficulty involved in the preparation of blood plates and the results obtained with this medium prohibit its complete acceptance as a substitute for agar in routine solid media. However, it could be a suitable substitute for agar in all other routine bacteriological media.     

Value of the K+ salt of carageenan as an agar substitute in routine bacteriological media.

The K+ salt of carageenan has no distinct advantages as a gelling agent, but it compared favorably with agar in most of the media tested. The difficulty involved in the preparation of blood plates and the results obtained with this medium prohibit its complete acceptance as a substitute for agar in routine solid media. However, it could be a suitable substitute for agar in all other routine bacteriological media.     

A simple method for counting Staphylococcus aureus in swimming pool water

Staphylococcus aureus counts from swimming pool water were determined by the membrane filtration technique. Water samples were passed through a membrane filter and then put on Baird-Parker media. After incubation, the filters were transferred to nutrient agar, and incubated at 37 degrees C, for 3 h. After removal of the filters, the plates were incubated at 60 degrees C for 2 h. An overlay of toluidine blue agar was added and the plates reincubated for 4 h at 37 degrees C. The formation of thermonuclease correlated with the formation of coagulase, and the results indicated that Staphylococcus aureus could be present in swimming pool water without the presence of either coliform or faecal coliform bacteria.     

Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.