Guar gum: a cheap substitute for agar in microbial culture media

AIMS: To determine the possibility of using guar gum, a colloidal polysaccharide, as a cheap alternative to agar for gelling microbial culture media. METHODS AND RESULTS: As illustrative examples, 12 fungi and 11 bacteria were cultured on media solidified with either guar gum or agar. All fungi and bacteria exhibited normal growth and differentiation on the media gelled with guar gum. Microscopic examination of the fungi and bacteria grown on agar or guar gum gelled media did not reveal any structural differences. However, growth of most of the fungi was better on guar gum media than agar, and correspondingly, sporulation was also more advanced on the former. Bacterial enumeration studies carried out for Serratia sp. and Pseudomonas sp. by serial dilution and pour-plate method yielded similar counts on both agar and guar gum. Likewise, a selective medium, succinate medium used for growth of Pseudomonas sp. did not support growth of Bacillus sp. when inoculated along with Pseudomonas on both agar or guar gum supplemented medium. CONCLUSIONS: Guar gum, a galactomannan, which is 50 times cheaper than Difco-bacto agar, can be used as a gelling agent in place of agar in microbial culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: As the media gelled with guar gum do not melt at temperature as high as 70 degrees C, these can be used for isolation and maintenance of thermophiles.     

Guar gum as a gelling agent for plant tissue culture media

Summary Guar gum, a galactomannan derived from the endosperms of Cyamposis tetragonoloba, has been successfully used as a sole gelling agent for plant tissue culture media. Its suitability as a gelling agent was demonstrated by using guar gumgelled media for in vitro seed germination of Linum usitatissimum and Brassica juncea, in vitro axillary shoot proliferation in nodal explants of Crataeva nurvala, rooting of regenerated shoots of the same, in vitro androgenesis in anther cultures of Nicotiana tabacum, and somatic embryogenesis in callus cultures of Calliandra tweedii. The media used for these were gelled with either guar gum (2, 3, or 4%) or agar (0.9%). Guar gum-gelled media, like agar media, supported all these morphogenic responses. Rather, axillary shoot proliferation, rhizogenic and embryogenic responses were better on guar gum-gelled media than on agar media.     

Gum katira – a cheap gelling agent for plant tissue culture media

Gum katira, an insoluble gum derived from the bark of Cochlospermum religiosum, has been successfully used as a gelling agent in tissue culture media for in vitro shoot formation and rooting in Syzygium cuminii and somatic embryogenesis in Albizzia lebbeck. The epicotyl segments, excised from in vitro grown seedlings of S. cuminii, developed shoots when cultured on MS medium (Murashige and Skoog, 1962), supplemented with 4% sucrose and 1 mg l^–1 BA. The so-developed shoots were rooted on Knop's medium, augmented with 2% sucrose and 1 mg l^–1 IAA. For somatic embryogenesis, hypocotyl segments derived from in vitro developed seedlings of A. lebbeck were cultured on B5 medium containing 2% sucrose. Media were gelled with either 3% gum or 0.9% agar. The quantitative response obtained on media fortified with either of the gelling agents was not significantly different. The media gelled with gum katira were almost as transparent as the liquid medium. However, viscosity of gum katira gelled medium was less than one-sixth of the viscosity of agar-gelled media, and therefore, shaking ofthe culture vessel often resulted in submersion of the explants. Nevertheless, even these submerged explants responded positively. To increase the firmness of the gum katira-gelled medium, various combinations of agar (0.2–0.6%) and gum (1–3%) were used. However, the viscosities of the media gelled with 3% gum katira as well as different concentrations of agar (0.2–0.6%) were lower than that of the medium containing only gum katira (3%). Moreover, the explant productivity obtained in neither of these combinations was more than those recorded on the control media, which were gelled either with 0.9% agar or 3% gum alone.     

Isubgol as an Alternative Gelling Agent for Microbial Culture Media

Isubgol, the mucilaginous husk derived from the seeds of Plantago ovata, has been successfully used as a gelling agent for microbial culture media. As illustrative examples, fast growing symbiotic bacterium, Rhizobium meliloti and saprophytic fungi, Aspergillus flavus and Penicillium chrysogenum were cultured on media gelled with either Isubgol or agar. All the three microbes employed in the study exhibited normal growth when cultured on their respective media gelled with Isubgol. Rather, Isubgol gelled medium appears to promote the growth of bacterial cultures as the colonies on this medium were denser than the corresponding ones on the medium gelled with agar. Likewise, on Isubgol gelled medium, sporulation in both the fungi took place earlier than on the medium gelled with agar, thus indicating the promotive influence of the former gelling agent.     

Agar/galactomannan gels applied to shoot regeneration from tobacco leaves

This study concerns the efficacy of partial agar substitution by galactomannans as support in plant regeneration media for Nicotiana tabacum. The production of multiple shoots from leaf-derived callus and their rooting were evaluated. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum — a commercial galactomannan) seeds. The results obtained on media solidified with mixtures of agar/galactomannan (3 g dm⁻³ each) gels were compared with those on media gelled with a standard concentration of agar (6 g dm⁻³). The in vitro performance allowed to conclude that the use of galactomannans raised the number of shoots and improved their quality. Furthermore, the length of roots and the size of leaves were significantly higher in the media solidified with agar/guar galactomannan mixtures.     

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

Xanthan gum: an economical substitute for agar in plant tissue culture media

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.     

Micropropagation of ‘Durondeau’ pear in modified-gelled medium

Summary The influence of partial substitution of agar by galactomannans (GMs) in culture media was studied in pear (Pyrus communis L. cv. ‘Durondeau’) micropropagation. GMs. extracted from seeds of Cassia fastuosa (cassia) or Cyamopsis tetragonolobus (guar gum, a commercial GM), were mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent. The production of multiple shoots and the formation of roots from shoots were compared with the control solidified with agar alone at a concentration of 0.6% (w/v). In the media solidified with the mixtures of agar/guar and agar/cassia GMs, an, increase of 32 and 17%, respectively, was obtained in the number of regenerated shoots. The modified media promoted a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM and containing 9.80 μM indole-3-butyric acid. Less callus formation at the base of the shoot was also observed on this medium. The improved in vitro performance of shoot formation and rooting, combined with a significantly lower cost, suggests a potential use of agar/GM gels in plant tissue culture.     

Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment

Since Robert Koch and colleagues found agar to be an effective gelling agent over a century ago, the pure culture method using agar plates has long been a standard of microbiology. Agar is undoubtedly easy to handle and useful for culture of microorganisms, but recent discovery of the ubiquity of microorganisms that cannot be cultured on agar raises a question: is agar really the best agent? In this study, we investigated the effect of two gelling agents, agar and gellan gum, on colony formation of a diverse array of microorganisms (total 108 strains) newly isolated from freshwater sediments and a representative microorganism as a slow grower on agar medium, Gemmatimonas aurantiaca, to clarify (i) whether they can grow on both agar and gellan gum plates, and (ii) the difference in time required for colony formation between the two gelling agents. Interestingly, 22 of 108 isolates showed no ability to form any visible colonies on the agar medium but did so on the gellan gum medium, and showed low 16S rRNA gene sequence similarities to their closest species. The remaining 86 isolates grew on both agar and gellan gum, but 52 of them grew much faster on gellan gum than on agar. Moreover, gellan gum also significantly stimulated the colony formation of the representative slow‐growing microorganism G. aurantiaca. Our results demonstrate that the gelling agent is a crucial factor for the growth of bacteria on plate media, and that alternatives to agar will be very important for increasing the culturability of yet‐to‐be cultured microorganisms.     

Variability in airborne bacterial and fungal population in the tertiary health care centre

An investigation of the air quality and the quantity of airborne microbes was conducted in a private and a government tertiary health care centre of Davanagere in the month of November 2011 to assess the level of air borne pathogens. Using a Merck Microbial Air Sampler MAS-100NT, samples were collected in the morning and in the evening from the different environs of the private and government tertiary health care centre. The media used for the study of fungi was sabouraud dextrose agar. Aspergillus spp, Curvularia spp, Alternaria spp, Penicillium spp, Rhizopus spp, Nigrospora spp, and Fusarium spp were found in either of the tertiary health care centre. Aspergillus spp was dominant in the Government tertiary health care centre, and Alternaria spp and Curvularia spp were dominant in the private tertiary health care centre. For the bacteria, quantitative enumeration was done using soyabean casein digest agar and selective media like Escherichia coli and coliform agar and urinary tract infection agar were used in qualitative enumeration. Selected pathogens like E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Proteus mirabilis, and Entirococcus faecalis were found in either of the tertiary health care centre. Maximum number of fungi and bacteria were isolated from emergency ward and general ward of government and private health care centre. There was also considerable difference in the morning and in the evening.     

Inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> and coliform bacteria in traditional brass and earthernware water storage vessels

The detection and enumeration of indicator bacteria such as Escherichia coli is used to assess the extent of faecal contamination of drinking water. On the basis of this approach, the effectiveness of storing water contaminated with faecal indicator bacteria in brass or earthern vessels (mutkas) of the type used in rural India have been investigated. Suspensions of bacteria in sterile distilled water were maintained for up to 48 h in each vessel and enumerated by surface plate counts on nutrient agar (non-selective) and several selective coliform media at 37 °C either under standard aerobic conditions, or under conditions designed to neutralise reactive oxygen species (ROS), e.g. using an anaerobic cabinet to prepare plates of pre-reduced growth medium or by inclusion of sodium pyruvate in the growth medium, with incubation of aerobically-prepared plates in an anaerobic jar. The counts obtained for E. coli decreased on short-term storage in a brass mutka; counts for selective media were lower than for equivalent counts for non-selective medium, with ROS-neutralised conditions giving consistently higher counts than aerobic incubation. However, after 48 h, no bacteria were cultivable under any conditions. Similar results were obtained using water from environmental sources in the Panjab, and from rural households where brass and earthern mutkas are used for storage of drinking water, with enumeration on selective coliform media (presumptive total coliforms). In all cases results indicated that, while storage of water in a brass mutka can inactivate E. coli and coliforms over a 48 h period, standard aerobic plate counting using selective media may not be fully effective in enumerating sub-lethally damaged bacteria.     

A new solid medium for the isolation and enumeration of Thiobacillus ferrooxidans and acidophilic heterotrophic bacteria

A solid medium (FeTSB) was developed for the simultaneous isolation and enumeration of the iron-oxidising bacterium Thiobacilluls ferrooxidans and acidophilic heterotrophic bacteria. The medium consisted of ferrous sulfate, tryptone soya broth and basal salts, solidified with agarose, although bacteriological agar could be substituted for some strains. The medium has been used to isolate bacteria from natural environments and also in laboratory studies of characterised strains. The factors which influence the success of colony growth on solid media are discussed.     

Use of hydrophilic natural gums in formulation of sustained-release matrix tablets of tramadol hydrochloride

The objective of this work was to develop matrix sustained-release tablets of highly water-soluble tramadol HCl using natural gums (xanthan [X gum] and guar [G gum]) as cost-effective, nontoxic, easily available, and suitable hydrophilic matrix systems compared with the extensively investigated hydrophilic matrices (ie, hydroxypropyl methylcellulose [HPMC]/carboxymethyl cellulose [CMC] with respect to in vitro drug release rate) and hydration rate of the polymers. Matrix tablets of tramadol (dose 100 mg) were produced by direct compression method. Different ratios, of 100∶0, 80∶20, 60∶40, 20∶80, 0∶100 of G gum (or X):HPMC, X gum:G gum, and triple mixture of these polymers (G gum, X gum, HPMC) were applied. After evaluation of physical characteristics of tablets, the dissolution test was, performed in the phosphate buffer media (pH 7.4) up to 8 hours. Tablets with only X had the highest mean dissolution time (MDT), the least dissolution efficiency (DE₈%), and released the drug following a zero-order model via swelling, diffusion, and erosion mechanisms. Guar gum alone could not efficiently control the drug release, while X and all combinations of natural gums with HPMC could retard tramadol HCl release. However, according to the similarity factor (f ₂), pure HPMC and H₈G₂ were the most similar formulations to Topalgic-LP as the reference standard. Published: March 17, 2006     

Application of microelectrode technique to measure pH and oxidoreduction potential gradients in gelled systems as model food

A microelectrode system is used in order to simultaneously measure pH and oxidoreduction potential (Eh) gradients developed during growth by Escherichia coli and Lactobacillus plantarum immobilized in gelled media used as model food. Unlike E. coli, L. plantarum steadily decreased medium pH independently of depth of measurement and time of incubation. Both bacteria brought about the creation of an Eh gradient throughout the gelled medium. This gradient was much more important for E. coli (700 mV) than for L. plantarum (80 mV) but more transitory.     

Determination of oxygen profiles in agar-based gelled in vitro plant tissue culture media

Keeping account of the limited knowledge concerning the relevance of the oxygen status of the medium during in vitro culture, a technique was elaborated to systematically study the oxygen concentration in gelled media. In a first series of experiments, the Oxygen Diffusion Rate (ODR) technique was used to investigate the dissolved oxygen concentration as a function of time at different depths. The results obtained demonstrated that the oxygen concentration in agar media was reduced by 80% during the heating steps included in the preparation procedure. It took about one week to reach an oxygen concentration equal to 90% of the equilibrium value at a depth of 1 cm, irrespective of the brand of agar used. As the recovery of the oxygen concentration at various depths could be nicely modelled by Fick's law, it follows that this process is diffusion limited. In this respect, fluorescence recovery after photobleaching (FRAP) measurements revealed that the diffusion coefficient of oxygen in gelled media was only affected to a very small extent by the presence of up to 2% (w/v) agar. In a final experiment, explants of Ficus benjamina were cultured on a rooting medium. As the oxygen concentration in the gelled medium was not significantly affected by the presence of the biological material, it was concluded that the oxygen uptake by explants from gelled media is negligibly small and hence cannot be considered as being a growth-limiting factor during in vitro micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

`Isubgol' as an alternative gelling agent in plant tissue culture media

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar. Received: 9 October 1996 / Revision received: 22 July 1996 / Accepted: 30 July 1997     

Comparison of the rheological and diffusion properties of some gelling agents and blends and their effects on shoot multiplication

The rheological and diffusion properties of blends of agar/guar gum, agar/Phytagel and Phytagel/guar gum were analysed and compared to those properties of agar or Phytagel applied alone at two different gelling concentrations. Moreover, their effects on the shoot multiplication of the apple scion Galaxy and two black locust clones (SF63, SF82) were studied, and their cost benefits over agar were calculated. Elastic hydrogel formation was demonstrated for each blend by rheological measurements, but the gel strength depended on the types and concentrations of the applied gelling agents and blends. Guar gum was able to speed the diffusion in the different blends, and diffusion was independent of gel strength. The rate of shoot multiplication increased (to 8.9 shoots per explant) and the percent of hyperhydrated shoots decreased (to 12%) when the blend of agar/guar gum was used for the shoot multiplication of apple. Similarly, the highest multiplication rates of black locust clones (between 3.9 and 4.1) were obtained on media solidified by blends containing guar gum. The best shoot performance with the lowest percent of hyperhydrated shoots (11–12% in SF63 and 2–23% in SF82) was achieved using agar alone or the agar/guar gum blend. The shoot multiplication was improved of both species and the production cost was reduced by 42% by using the agar/guar gum blend.     

Partial Characterization of the Antimicrobial Activity of Streptomyces antimycoticus FZB53

The paper describes experiments aimed at evaluating the sensitivity of different fungi, most of them plant pathogens and bacteria towards Streptomyces antimycoticus FZB53, a biocontrol agent that, when applied as a seed treatment, in previous studies has shown good activity against different seed‐borne fungal diseases. When incorporated into agar media, the filtrate from shake cultures of S. antimycoticus FZB53 inhibited the mycelial growth or spore germination, respectively, of a broad spectrum of fungi. The most sensitive of the fungi tested was Fusarium culmorum. The inhibitory activity could be removed from the culture filtrate by extraction with ethyl acetate. When ethyl acetate extracts of the pellet and supernatant obtained by centrifugation of the shake culture were added to the agar medium, inhibition of mycelial growth of F. culmorum was restored, especially with the extracts of the pelleted biomass. Autoclaving of the culture filtrate reduced the inhibition of F. culmorum but completely eliminated the inhibitory activity against Fusarium graminearum. Among the bunt fungi tested, spore germination of Tilletia tritici was more sensitive to the culture filtrate of S. antimycoticus FZB53 than spore germination of Ustilago avenae and U. tritici. Separation by thin layer chromatography (tlc) and spraying with different reagents showed that ethyl acetate extracts from shake cultures or biomass scraped from agar media contained several hydrophobic metabolites. When eluted from the tlc‐plates, the material from one of the spots had strong antifungal activity against spore germination of T. tritici and mycelial growth of F. culmorum, respectively. Ethyl acetate extracts from biomass of S. antimycoticus FZB53 prevented the growth of the tested Gram‐positive bacteria, namely Clavibacter michiganensis and different species of Bacillus. The results indicated that these bacteria were at least as sensitive towards the metabolites of S. antimycoticus FZB53 as F. culmorum. The tested Gram‐negative bacteria were not affected.     

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.     

TEMPO-mediated selective oxidation of substituted polysaccharides--an efficient approach for the determination of the degree of substitution at C-6

2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidations of substituted polysaccharides were studied at pH 10.2 and at a temperature of 0 °C with NaOCl as the oxidant. The reaction is highly selective, and it was shown that the oxidation can proceed to a yield of nearly 100%. The oxidation process was investigated for several substituted polysaccharides, especially for a series of hydroxypropyl guar gums with different molar degrees of substitution. It was shown that this oxidation can be used for the determination of the degree of substitution at C-6 of the polysaccharide by comparing the difference in oxidation yield between substituted and natural polysaccharides. Studies on several hydroxypropyl guar gums showed that the degrees of substitution at C-6—for MS of 0.08, 0.34, 0.62, and 1.08—are 0.06, 0.24, 0.40, and 0.44, respectively. The results were extended to other polysaccharides such as carboxymethyl cellulose, cationic guar gum, carboxymethyl pullulan, and methyl cellulose. It can be concluded that the TEMPO-mediated oxidation is a useful method for the determination of the DS at the substituted C-6 position for different kinds of modified polysaccharides.