Improved detection of acid mine water stressed coliform bacteria on media containing catalase and sodium pyruvate

Pure culture suspensions of two strains of exponential and stationary phase Escherichia coli exhibited significant reductions in catalase activity following exposure to acid mine water (AMW). The exogenous addition of catalase (500-2000 U) or sodium pyruvate (0.05-5%) to a nonselective recovery medium resulted in enhanced detection (12- to 465-fold) of AMW-stressed E. coli as compared with recovery on the medium lacking these supplements, whereas addition of 3,3'-thiodipropionic acid failed to improve recovery. Additional in vitro experiments utilizing selective M-FC, mT7, and M-Endo media containing 1000 U catalase or 1.0% pyruvate similarly resulted in improved detection of AMW-stressed cells, with the exception of M-Endo containing pyruvate. Appropriately modified media were then used to analyze an AMW-impacted stream by the membrane filtration technique. Addition of catalase, pyruvate, or a combination of both significantly improved recovery of fecal and total coliforms without promoting growth of noncoliforms. Supplementation of plate count agar with pyruvate and (or) catalase enhanced detection of total heterotrophs. These findings suggest that addition of catalase or pyruvate to standard recovery media may improve detection of coliform and total heterotrophic bacteria in AMW-impacted waters.     

The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide

B olton , F.J. C oates D. & H utchinson , D.N. 1984. The ability of campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. Journal of Applied Bacteriology 56 , 151–157.
Nutrient agar plates stored in light and air for 48 h became inhibitory for Campylobacter jejuni, C. coli and nalidixic acid-resistant, therrnophilic campylobacter (NARTC) strains. All five campylobacter test strains showed > 5 log reduction in counts on media which had been stored in light and air. Media stored in the dark and/or in a reduced atmosphere did not become inhibitory and supported the growth of campylobacters. Ferrous sulphate, sodium pyruvate, blood, charcoal or sodium metabisulphite, compounds frequently used as supplements in campylobacter media, were added to nutrient agar prior to storage of media in light and air. All additives except sodium metabisulphite prevented the accumulation of photochemically generated toxic oxygen derivatives and allowed growth of test strains. In qualitative tests to determine the ability of supplements to neutralize hydrogen peroxide, blood was the most active, charcoal and sodium pyruvate slightly less active and ferrous sulphate and sodium metabisulphite the least active. The results of this study confirm that supplements in campylobacter media act as quenching or detoxifying agents and not as enrichment factors.     

A comparison of solid and liquid media for measuring the sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media, and the time needed to recover resistance

Sensitivity of heat-injured Salmonella typhimurium to selenite and tetrathionate media was measured by viable counts in liquid and on agar-solidified versions of these media and on nutrient media. All solid media, including the supposedly non-inhibitory nutrient agar, were more inhibitory to injured cells than the corresponding liquid media. Catalase or pyruvate increased counts on nutrient agar to the level obtained in nutrient broth. Therefore nutrient agar plus pyruvate was the most suitable reference medium against which to compare recoveries on other media. Although recoveries of injured cells varied widely depending on the composition and physical state of the medium, this had a minor effect on estimates of repair time because resistance to all selective media was regained by the end of the lag phase.     

The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.     

The Effect of Recovery Medium on the Isolation of Staphylococcus aureus after Heat Treatment and after the Storage of Frozen or Dried Cells

The recovery of heated or dried cells of Staphylococcus aureus was best on media containing blood or sodium pyruvate. It is suggested that the catalase of S. aureus may be destroyed or its activity reduced by heating or drying and that one of the main reasons for the better recoveries on these media is that they are able to destroy peroxide. All media tested adequately supported the recovery of frozen cells.     

自然界中难分离培养微生物的分离和应用

采用在分离培养基中添加自然来源的抽提液,或加入一些特殊化合物,使其中处于Viable but non-culturable（VBNC）状态的微生物恢复其生长繁殖能力,从而得到分离。实验结果发现甜菜碱、丙酮酸钠、SOD以及过氧化氢酶可使分离到的微生物种类及菌落总数明显增加。还采用固液结合的方法来分离那些在普通平板培养基上不能形成肉眼可见菌落的那些微小菌落的微生物。采用这几种方法从4份土壤样品中共分离得到52株放线菌,103株细菌,17株真菌。对其中的放线菌和真菌进行了生物活性的测定,得到多株具有抗菌活性的微生物,经过多次复筛的平均阳性率为4.325％,略高于用常规方法分离得到的微生物。因此证明有效的分离方法将为今后微生物药物的筛选和药用微生物菌种的保藏提供更丰富的来源。     

Comparison of supplements* to enhance recovery of heat‐injured Salmonella from egg albumen

Aims: The purpose of this study was to determine the proficiency of supplements to enhance the recovery of Salmonella from heat‐treated liquid egg albumen on solid agar media. Methods and Results: Salmonella‐inoculated albumen, heated at 53·3°C for 4 min, was plated on 39 combinations of solid media with or without the addition of 12 supplements. Greater numbers of Salmonella (P < 0·05) recovered with the addition of 1·0 g l^?1 ferrous sulfate (FeSO₄) than with any other supplements, except for 0·5 or 1·0 g l^?1 3′3′‐thiodipropionic acid (TDP), which recovered equivalent populations. Addition of 1·0 g l^?1 sodium pyruvate or 6·0 g l^?1 yeast extract plus 1·0 g l^?1 sodium pyruvate supported greater resuscitation than unsupplemented tryptic soy agar (TSA) or supplementing with 0·01 or 0·1 g l^?1 N‐propyl gallate, 10 g l^?1 activated charcoal, 0·1 g l^?1 KMnO₄ or 50 mg l^?1 ethoxyquin. The remaining supplements supported recovery of equivalent numbers of Salmonella, which were fewer cells than recovered with 1·0 g l^?1 FeSO₄, yet greater populations than recovered with 50 mg l^?1 ethoxyquin. Conclusion: Supplementation of plating media with FeSO₄, TDP or sodium pyruvate enhanced recovery of sublethally injured Salmonella from albumen. Significance and Impact of the Study: Pasteurizing albumen impedes recovery of pathogens. These results suggest that the addition of supplements to plating media may assist resuscitation and colony development of heat‐injured salmonellae.     

The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes

The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS). Survival and growth of some follicles was maintained for 23 d.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools.

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation.

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Variable cellular composition of Chromatium in growing cultures

Summary A strictly anaerobic spirochete was isolated from a sample of marine mud. The organism possessed two axial fibrils entwined with the regularly coiled protoplasmic cylinder. An outer envelope or sheath enclosed both protoplasmic cylinder and axial fibrils. The spirochete grew in chemically defined media containing glucose, amino acids or NH₄Cl, sulfide, NaCl, vitamins, coenzyme A, and in-organic salts. A reducing agent, such as sodium sulfide or l-cysteine, as well as exogenous supplements of biotin, niacin and coenzyme A were required for growth. Pantothenate replaced coenzyme A as an exogenous growth factor, but the resulting cell yields were low. The spirochete grew in media prepared with sea water, but not in fresh water media containing less than 0.05 M NaCl (optimum concentration 0.35 M). Both Na⁺ and Cl^- were required. Carbohydrates served as fermentable substrates. Amino acids, sugar alcohols, tricarboxylic acid cycle intermediates, and other organic acids and alcohols were not fermented. Glucose was fermented to ethyl alcohol, acetate, CO₂, H₂, and small amounts of lactate, formate and pyruvate. The guanine + cytosine content of the DNA of the spirochete was 50.5 moles-% (buoyant density). It is proposed that the marine isolate be considered a new species and that it be named Spirochaeta litoralis.     

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds

Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 °C. Plate counts declined from 3 × 10⁶ to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or α-ketoglutaric acid, plate counts increased to 10⁴–10⁵ CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H₂O₂, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells. Received: 15 January 1999 / Accepted: 8 April 1999     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

建立维生素K_1微生物限度测定方法

目的:根据计数培养基适用性检查和计数方法适用性试验结果,建立维生素K1微生物限度测定方法。方法:以聚山梨酯80作为乳化剂,使维生素K1在p H 7.0无菌氯化钠-蛋白胨缓冲液中充分乳化。取1:20供试液1 m L,按平皿法分别用营养琼脂培养基和玫瑰红钠琼脂培养基培养,以计数细菌、霉菌和酵母菌。结果:验证所用培养基的菌落平均数均大于对照培养基上的70%,且菌落形态大小一致。稀释液对照组和试验组培养基上菌落平均数的回收率均大于70%,且菌落形态大小一致。三批维生素K1及其加速和长期稳定性样品中均未见菌落。结论:所选培养基适宜大肠埃希菌、金黄色葡萄球菌和白色念珠菌生长。且含聚山梨酯80的p H 7.0无菌氯化钠-蛋白胨缓冲液和维生素K1对所含大肠埃希菌、金黄色葡萄球菌和白色念珠菌无抑菌性。     

Effect of cereal brans on Lentinula edodes growth and enzyme activities during cultivation on forestry waste

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.