The Preservation of Bacteria and Fungi on anhydrous Silica Gel: an Assessment of Survival over Four years

The preservation method of Perkins (1962) using suspensions in skim-milk was used to preserve 33 bacteria and 22 fungi on anhydrous silica gel. During storage at room temperature, 64% of the bacteria and 77% of the fungi survived 1 year or more. Storage at 4° often increased the survival period c. 2- to 3-fold: 73% of the bacteria and all 12 of the fungi tested at 4° survived > 1 year. At the last testing, 60% of the bacteria and 36% of the fungi were still viable after storage at 4° for periods between 3 and 4 years. The Gram positive bacilli tended to survive the silica gel preservation process better than most Gram negative bacilli. Some factors influencing survival after preservation on silica gel are discussed; the results support the use of a closed storage tube.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4 degrees C, as well as in silica gel granules at 20 and -60 degrees C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at -60 degrees C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage temperatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at -60 degrees C.     

The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link

AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum. METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying. Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation. Citrinin was detected in all cultures for all preservation techniques on YES. The patulin profiles obtained differed with strain and culture media used. CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains. There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4°C, as well as in silica gel granules at 20 and ?60°C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at ?60°C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage tempeatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at ?60°C.     

Resistance of bacterial strains to dry conditions: use of anhydrous silica gel in a desiccation model system

The Viability of 18 bacterial strains desiccated on anhydrous silica gel and stored at a temperature of 22°C for at least 3 months was determined. According to their stability in the dried state, these strains could be classified into three typical groups. Group 1, containing Gram-positive strains and Salmonella serotypes, was marked by a very slow decrease of the concentration of culturable cells from day 14 on (respectively day 21 for Salmonella thompson . The rate of decrease expressed as regression coefficient (b) ranged from —0.000389 to —0.00521 log (cfp ml^-1) per d. The Group 2 strains Enterobacter cloacae and Escherichia coli did not reach a comparable slow decrease in the dry material within the indicated time period. Regression coefficients were respectively —0.04406 and —0.03412 log (cfp ml^-1) per d. The reciprocal values —(1/b) were respectively 23 d per log (cfp ml^-1) and 29 d per log (cfp ml^-1), indicating the time periods in which a reduction of 1 log unit of culturable cells occurred. Group 3 strains Pseudomonas aeruginosa, Aeromonas hydrophila and Aer. sobria were marked by a significant susceptibility to cell damage caused during desiccation and reconstitution. A high initial decrease (ID) of the concentration of culturable organisms seems to be a characteristic property of these bacterial strains: culturable organisms could not be detected after storage for 1 d ( Aer. hydrophila, Aer. sobria ) or 7 d ( Ps. aeruginosa ). The wide range of resistance of the different bacterial strains tested indicated that the silica gel model system is a suitable tool for microbiological challenge tests to investigate the survival of micro-organisms exposed to desiccation and their stability in dry materials.     

Immobilization of bacteria in silica matrices using citric acid in the sol–gel process

The aim of this work was to use citric acid in the sol–gel process to generate an inorganic polymer that allows bacterial survival for long periods of time and to study the influence of different storage temperatures. We compared gram-negative Escherichia coli and gram-positive Staphylococcus aureus, immobilized and preserved at different storage temperatures in silica matrices prepared by the method proposed. Immobilized E. coli and S. aureus in silica matrices were stored in sealed tubes at 20, 4, −20, and −70°C for 4 months during which the number of viable cells was analyzed. Results show that the immobilization in silica matrices using citric acid, to neutralize the alkalinity of the silica precursors, makes the technique not only biocompatible but also easier to perform since polymerization does not occur immediately as it does when hydrochloric acid is utilized.     

Viability of ectomycorrhizal fungi following cryopreservation

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at −130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.     

Viability and genetic stability of the bacterium Escherichia coli HB101 with the recombinant plasmid during preservation by various methods.

The effects of various methods of preservation (freeze-drying and storage at 4 degrees C; low-temperature freezing on desiccated silica gel and storage at -70 and -150 degrees C; cryopreservation and storage at -196 degrees C) on viability and genetic stability of the bacterium Escherichia coli HB101 carrying the recombinant plasmid with the human DNA fragment were studied. Genetic stability was estimated by maintenance of the selective markers of antibiotic resistance and the stability of the restriction sites in recombinant DNA. Freezing on silica gel and cryopreservation were shown to be the optimal preservation methods providing a high level of survival and genetic stability.     

The preservation of medically important fungi.

Details are given of a method, first described in 1962, for preservation of fungi in anhydrous silica gel. It is shown that the procedure is suitable for use with dermatophytes, yeasts and other fungi of medical importance and that they may be preserved for periods of 5 years or more.     

Tissue storage and primer selection influence pyrosequencing‐based inferences of diversity and community composition of endolichenic and endophytic fungi

Next‐generation sequencing technologies have provided unprecedented insights into fungal diversity and ecology. However, intrinsic biases and insufficient quality control in next‐generation methods can lead to difficult‐to‐detect errors in estimating fungal community richness, distributions and composition. The aim of this study was to examine how tissue storage prior to DNA extraction, primer design and various quality‐control approaches commonly used in 454 amplicon pyrosequencing might influence ecological inferences in studies of endophytic and endolichenic fungi. We first contrast 454 data sets generated contemporaneously from subsets of the same plant and lichen tissues that were stored in CTAB buffer, dried in silica gel or freshly frozen prior to DNA extraction. We show that storage in silica gel markedly limits the recovery of sequence data and yields a small fraction of the diversity observed by the other two methods. Using lichen mycobiont sequences as internal positive controls, we next show that despite careful filtering of raw reads and utilization of current best‐practice OTU clustering methods, homopolymer errors in sequences representing rare taxa artificially increased estimates of richness c. 15‐fold in a model data set. Third, we show that inferences regarding endolichenic diversity can be improved using a novel primer that reduces amplification of the mycobiont. Together, our results provide a rationale for selecting tissue treatment regimes prior to DNA extraction, demonstrate the efficacy of reducing mycobiont amplification in studies of the fungal microbiomes of lichen thalli and highlight the difficulties in differentiating true information about fungal biodiversity from methodological artefacts.     

Preservation of Mycoplasmas on Anhydrous Silica Gel

1. 26 strains of mycoplasmas were successfully preserved on anhydrous silica gel crystals for 80 weeks at +4°C.
2. There was a sharp decline in viability of the strains if they were stored at room temperature.
3. This preservation technique is simple to perform and requires only inexpensive equipment.     

Reversed-phase HPLC separation of proteins on chemically bonded silica gel columns

Reversed-phase high-performance liquid chromatographic (RP-HPLC) separation of proteins on chemically bonded silica gel columns is described. Efficiency of nonporous alkylsilyl bonded silica gel is compared with that of a macroporous gel that has been widely used for the purpose. A comparative study of the separation under conventional and fast separation conditions is also given. The fast separation technique on the nonporous reversed-phase column has the advantage of improving the recovery of late-eluting hydrophobic and large proteins, such as ovalbumin and apoferritin.     

Detection of antifungal activity in Belamcanda chinensis by a single-cell bioassay method and isolation of its active compound, tectorigenin

The antifungal activity of Belamcanda chinensis was evaluated by a single-cell bioassay method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The isolated compound was found to be identical to tectorigenin (5,7-dihydroxy-3-(4-hydroxy phenyl)-6-methoxy-4H-1-benzopyran-4-one) which has formerly appeared in the literature without any remarks on its antimicrobial activity. Antimicrobial activity was investigated against 17 strains of fungi and 6 strains of bacteria. This compound showed marked antifungal activity against dermatophytes of the genera Trichophyton, the minimum inhibitory concentration (MIC) being in the range of 3.12-6.25 mg/ml.     

Silica Gel as a Preserving Agent for the Cellular Slime Mold Acrasis rosea

SYNOPSIS. Modifications of the silica gel technic of Perkins and Ogata have been successfully used to preserve Acrasis rosea for 6–13 months and Dictyostelium purpureum for 31 months. The technic is simple, reliable and inexpensive and is recommended for its possible application for the general preservation of certain fungi and protozoa.     

Sequence-related amplified polymorphism (SRAP) marker as a new method for identification of endophytic fungi from Taxus

A total of 20 endophytic fungi stains were classified into four groups using traditional morphological identification method, and were studied for genetic diversity by sequence-related amplified polymorphism (SRAP) technique. Genomic DNA (deoxyribonucleic acid) of these strains was extracted with CTAB method. SRAP analysis was done with 24 pairs of primers. All strains could be uniquely distinguished with 584 bands and 446 polymorphism bands which generated 76.4% of polymorphic ratio. Unweighted pair-group method with arithmetical averages cluster analysis enabled construction of a dendrogram for estimating genetic distances between different strains. All strains, which were just divided into four groups by traditional morphology identification, were clustered into four major groups at GS = 0.603 and further separated into eight sub-groups at GS = 0.921. Dendrogram also revealed a large genetic variation in 20 strains; different primer combinations allowed them distinctly distinguished one from others with relatively low genetic similarity. The results show that the SRAP technology is more efficient than traditional morphology identification. It is found that SRAP markers could more really reflect the genetic diversity of endophytic fungi strains from Taxus, and also could be used as a method for identification of endophytic fungi from Taxus. It also suggests that SRAP can be used to establish foundation for further screening of taxol-producing endophytic fungi strains which can produce high levels of paclitaxel.     

Tolypocladium cylindrosporum (Deuteromycotina:Moniliales), a fungal pathogen of the mosquito Aedes australis

A laboratory fermenter was used to produce up to 12 l of infective Tolypocladium cylindrosporum blastoconidia in Sabouraud dextrose broth. Two media derived from coconuts were also demonstrated as suitable alternative systems for the production of viable blastoconidia. T. cylindrosporum conidia when dried at 37 degrees C and stored at 4 degrees C retained their viability for 10 months, but, when stored at 25 degrees C, the conidia lost viability after 2 months and blastoconidia did not survive the drying process. Distilled water suspensions were a simple, economic technique for the long-term storage of spores at both 4 and 25 degrees C. The adsorption of conidia onto silica gel crystals was a very suitable technique for the storage of stock culture material at 4 degrees C. The virulence, production and storage capabilities of both spore types were examined.     

Influence of different storage methods on the predatory capacity of the fungi Arthrobotrys robusta and Monacrosporium thaumasium after passage through the bovine gastrointestinal tract

The biological control of helminth parasites of bovines by nematophagous fungi is an alternative to the use of drugs with the principal objective of reducing the source of infection available on pastureland. The maintenance of predatory activity of the fungal isolates is one of the basic prerequisites to ensure the success of this form of control. In this study behaviour of the isolates I31 of Arthrobotrys robusta and NF34a of Monacrosporium thaumasium was investigated following three storage methods: stored at 4 °C, cryopreserved with or without cryoprotectants or preserved in silica gel. All samples were subsequently passed through the gastrointestinal tract of calves. The latter involved the administration of 20 g of mycelia to the animals. This quantity was sufficient to recover fungal material from the faeces. The peak reduction in the number of infective larvae in the faeces occurred 24 h after administration of the samples (P < 0.05). The storage at 4 °C was the treatment that produced the greatest reduction in larvae for NF34a (81.3%) and I31 (65.1%) isolates. Nf34a isolate was responsible for the highest percentage reduction of larval helminth populations (P < 0.05). Cryopreservation appears to be an efficient method of preserving isolates, although diminished predatory capacity compared to storage at 4 °C was seen only for isolate NF34a (73.2%). Cryopreservation did not interfere in predatory activity of I31 isolate (P < 0.05). Maintenance of isolates in silica gel showed the lowest reduction throughout the experiment (P < 0.05).     

Recovery of Infective 3Rd Stage Larvae of Haemonchus Contortus and Ostertagia Ostertagi Ry Migration in Agar Gel

A laboratory technique is described for the recovery of Haemonchus contortus and Ostertagia ostertagi infective larvae by migration in agar gel. The addition of bile increased the recovery rate of the haemonchus larvae, but had a somewhat depressive effect on the number of ostertagia larvae recovered. Similarly, storage at 4°G lowered the yield of larvae of both species, compared to freshly harvested larvae. However, the recovery rates for both species were sufficiently high to recommend the technique for isolation of the infective stages from field samples.     

Preservation of Listeria monocytogenes and other pathogenic foodborne bacteria on silica gel

Preservation of Listeria monocytogenes on silica gel was examined as a means of storing and transporting bacterial cultures. Listeria monocytogenes remained viable through 168 d of storage on silica gel at room and refrigeration temperatures. The viability of Escherichia coli O157 : H7, Salmonella typhimurium and Staphylococcus aureus after storage on silica gel was determined and compared with earlier reports.     

大孔吸附树脂结合硅胶柱层析纯化环孢菌素A

采用大孔吸附树脂层析结合硅胶柱层析,对环孢菌素A的分离纯化进行研究,确定了最佳层析条件,建立了工业化制备环孢菌素A的工艺。大孔吸附树脂层析选用D101树脂作为吸附介质,提取液丙酮含量控制在50%,最大吸附量为35 mg/g湿树脂,洗脱剂选用丙酮;硅胶柱层析选用42~64μm硅胶作为层析介质,最优层析条件为柱床高径比10∶1,流动相配比V(石油醚)∶V(丙酮)=70∶30,流速80 mL/m in,环孢菌素A上样质量浓度100 g/L,硅胶层析平均收率为84.2%,环孢菌素A纯度可达到97%以上,整个工艺总收率为65%~70%。