Simple Method for Culturing Anaerobes

A simple, effective method is needed for growing obligate anaerobes in the clinical laboratory. This report describes a pre-reduced anaerobic bottle that can be taken to the bedside for direct inoculation, provides a flat agar surface for evaluation of number and morphology of colonies, and can be incubated in conventional bacteriological incubators. Each anaerobic culture set consisted of two bottles containing brain heart infusion agar and CO₂. Gentamicin sulfate (50 μg/ml) was added to one of these to inhibit facultative enteric bacilli. Comparison of the anaerobic bottles with an identical aerobic bottle which was also routinely inoculated permitted early identification of anaerobic colonies. Representative species of most anaerobic genera of proven pathogenicity for man have been isolated from this system during 10 months of routine use.     

Detection of Lactobacillus acidophilus in Feces of Humans, Pigs, and Chickens

Lactobacilli in fecal material from humans, pigs, and chickens were enumerated on lactobacillus selective agar (LBS). In all samples, higher numbers of lactobacilli were detected when plates were incubated in a system flushed with CO₂ rather than in air. Much higher numbers of bacteria from human feces were detected when the LBS agar plates were incubated anaerobically in a hydrogen-carbon dioxide atmosphere (GasPak) than when incubated in CO₂. The bacteria from human feces isolated on LBS agar incubated anaerobically were predominately bifidobacteria. Cultures from all three sources isolated on LBS agar incubated under CO₂ were lactobacilli, including Lactobacillus acidophilus. Differences were observed in biochemical characteristics of some of the L. acidophilus isolated from all three sources. Guanine plus cytosine base ratios of deoxyribonucleic acid isolated from L. acidophilus cultures from humans were lower, in most cases, than those from pigs and chickens.     

Helicobacter canis bacteraemia in a 7-month-old child

On the basis of biochemical, phenotypic and 16S rRNA analyses, Helicobacter canis was isolated and identified from an otherwise healthy 7-month-old girl with intermittent fever. Blood cultures signalled bacterial growth after 5 days that was characterized as small gram-negative spiral rods. Subculturing on Colombia plates with 5% sheep blood, chocolate agar and brucella agar, aerobically and anaerobically as well as in a microaerophilic atmosphere, showed scanty growth after an additional 4 days. Secondarily seeded with fluid from the original bottle, the paediatric blood bottles repeatedly signalled growth after one night's incubation, whereas the conventially treated bottles did not support growth after 7 days' incubation. From the secondary seeded paediatric bottles a pure culture was isolated on chocolate agar plates, and identified as H. canis. This case indicates that blood culture systems should be compared and improved for their capacity to detect Helicobacter and related pathogenic bacteria species. Further studies are also needed to determine the importance of H. canis as a primary pathogen, and the role of cats in the possible zoonotic spread of H. canis to humans.     

The effect of store conditions on the rotting of apples, cv. Bramley's Seedling, by Nectria galligena

The log of the time interval between inoculation with Nectria galligena in October and the onset of rotting in apples held in air was proportional to the deficit between the temperature of incubation and 25°C, but temperature did not affect the rate of subsequent rot expansion. Rots expanded equally fast whether apples were held in dry or moist air. The quantity of rotted tissue obtained after incubating inoculated apples in atmospheres containing up to 12.5% CO₂ increased with increasing concentrations of CO₂ greater than 2.5%. The quantity of rotted tissue obtained in apples incubated in 10% CO₂ was three times as great as that obtained after incubation in air. The incidence of natural rots was lower in apples stored at 4% CO₂ than in those stored in air and rotting increased with increasing concentrations of CO₂ higher than 4%. Colonies of N. galligena grew faster on malt agar plates incubated in 5% CO₂ than in air, but growth was slower in 10% CO₂ than in air. The quantity of benzoic acid per mg hyphae accumulated in developing lesions was similarly related to the CO₂ concentrations up to 2.5% but decreased at higher concentrations, and the quantities found in apples stored in CO₂ concentrations >5.0% CO₂ were less than in those stored in air.     

A new MSPQC system for rapid detection of pathogens in clinical samples

A new multi-channel series piezoelectric quartz crystal (MSPQC) system for detection of pathogens in clinical sample was proposed. Some factors, which affect the detection of pathogens by using MSPQC, were all investigated. A total of 650 clinical samples were detected by MSPQC and compared with licensed BACTEC 9120 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA) simultaneously in the Third Xiangya Hospital of Central South University, China. When the incubation period was 5 days, two systems had similar detected results: the MSPQC system detected 123 growth of 650 (18.92%) bottles while the BACTEC 9120 detected 125 growth of 650 (19.23%) bottles. The MSPQC had 2 false-positive signals and 2 false-negative signals. However, BACTEC 9120 had 3 false-positive signals and 0 false-negative signals. Further identifications of bacteria were run by VITEK-2 (bioMérieux China Ltd.), 5% sheep blood trypticase soy agar (SBA) and chocolate agar (CA). Comparing with BACTEC 9120, MSPQC system possesses following advantages: shorter average detection time, less blood volume needed, less false-positive results and low cost. It can also provide information in real time. So MSPQC has a wonderful perspective in clinical application.     

New Type of Colony-forming Cell located in the Pleural Cavity

A semi-solid agar culture system has been developed which supports the clonal growth of granulocytic and/or macrophage colonies from their specific progenitor cells^1,2. In the course of studies on the level of these progenitor cells in the peripheral blood of mice, whole blood was cultured in agar-medium. Cultures were prepared in 35 mm plastic Petri dishes and each culture contained 1 ml. of 0.3% agar in modified Eagle's medium. The media and general technique of agar culture have been described elsewhere³. In these experiments, 0.2 ml. of whole, unheparinized blood was taken directly from the axilla of anaesthetized 3 months old C57BL mice and added to 5 ml. of agar-medium, held liquid at 37° C. One ml. portions of this mixture were pipetted into four replicate culture dishes, allowed to gel and incubated for 7 days at 37° C in a fully humidified atmosphere of 10% CO₂ in air. Each culture contained 0.1 ml. of a 1:6 dilution of pooled sera from C57BL mice injected 3 h previously with 5 µg endotoxin to provide an adequate concentration of the specific colony-stimulating factor (CSF), required for granulocytic and macrophage colony formation.     

Enrichment and isolation of Bacillus beveridgei sp. nov., a facultative anaerobic haloalkaliphile from Mono Lake, California, that respires oxyanions of tellurium, selenium, and arsenic

Mono Lake sediment slurries incubated with lactate and tellurite [Te(IV)] turned progressively black with time because of the precipitation of elemental tellurium [Te(0)]. An enrichment culture was established from these slurries that demonstrated Te(IV)-dependent growth. The enrichment was purified by picking isolated black colonies from lactate/Te(IV) agar plates, followed by repeated streaking and picking. The isolate, strain MLTeJB, grew in aqueous Te(IV)-medium if provided with a small amount of sterile solid phase material (e.g., agar plug; glass beads). Strain MLTeJB grew at high concentrations of Te(IV) (~8 mM) by oxidizing lactate to acetate plus formate, while reducing Te(IV) to Te(0). Other electron acceptors that were found to sustain growth were tellurate, selenate, selenite, arsenate, nitrate, nitrite, fumarate and oxygen. Notably, growth on arsenate, nitrate, nitrite and fumarate did not result in the accumulation of formate, implying that in these cases lactate was oxidized to acetate plus CO₂. Strain MLTeJB is a low G + C Gram positive motile rod with pH, sodium, and temperature growth optima at 8.5–9.0, 0.5–1.5 M, and 40°C, respectively. The epithet Bacillus beveridgei strain MLTeJB^T is proposed.     

Conditions for strict autotrophic culture of tobacco callus

Organic gelling agents such as agar and agarose provide a heterotrophic substrate for growth of illuminated tobacco callus. When green cells are incubated in CO₂-free air on a medium lacking sucrose but solidified with 1% agar, an increase in relative dry weight is sustained through two passages. Similar results with different inoculum sources, and with three brands of agar and two forms of agarose, suggest this is a general phenomenon. A fully autotrophic culture system was developed employing polyurethane pads to support cells in a liquid medium lacking sucrose. Growth was negligible in two passages in CO₂-free air, and increased with each added increment in CO₂ concentration.     

New Strategies for Cultivation and Detection of Previously Uncultured Microbes

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O₂ [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO₂ (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO₂. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.     

Growth Conditions Influence Expression of Cell Surface Hydrophobicity of Staphylococci and Other Wound Infection Pathogens

The initial adhesion of microbes to tissue and solid surfaces can be mediated by hydrophobic interaction. Expression of microbial cell surface hydrophobicity (CSH) is influenced by growth conditions, and often best expressed after growth under nutrient-poor conditions, or “starvation.” In the present study, the CSH of 133 strains of Enterobacteriaceae, Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, group A streptococcus, Pseudomonas aeruginosa, Clostridium perfringens, Bacteroides fragilis, Peptococcus magnus, and of 8 Candida albicans strains was measured by the salt aggregation test after growth on hematin agar in a 5% CO₂ atmosphere, or under anaerobiosis. Cells of all but 8 strains expressed pronounced or moderate CSH, i.e., they aggregated in 0.01-2 M ammonium sulfate. When the agar surface was covered by human serum (diluted 1:5) to mimic growth conditions in a wound, 94 strains expressed higher CSH, and 44 strains the same CSH as after growth without serum. The CSH of 12 strains of different species was measured after growth on blood, hematin and PDM agar, with or without serum, and in an aerobic or a 5% CO₂ atmosphere. The highest CSH was expressed after growth in 5% CO₂ with serum, and the lowest growth after on blood agar in aerobic atmosphere. Identical results were obtained with native and heat-inactivated (56 C, 20 min) serum. The reduced surface tension obtained in 5% CO₂, as well as yet unidentified serum factors, promotes expression of CSH.     

Effects of Long Term Exposure to High-CO2 During Storage at 0°C on Biology and Infectivity of Botrytis cinerea in Red Chicory

The effects on Botrytis cinerea of prolonged exposures to CO₂‐enriched atmospheres were studied in vitro and in vivo at 0°C. Mycelial growth on potato dextrose agar decreased linearly with increasing CO₂ concentrations from 5, 10, 15 and 20% CO₂. The growth reduction was greater after 30–40 days of incubation. A reduced production of sclerotia in air by the colonies formerly exposed to various CO₂ concentrations was also detected. Conidial germination was delayed and the amount of germinated conidia decreased with increased CO₂ and at 20% CO₂ it was inhibited. Germ tube elongation was affected in the same way. In artificially inoculated red chicory, lesion area caused by B. cinerea decreased with increasing concentrations of CO₂ up to 60 days storage, later only 10 and 15% CO₂ were really effective, while in the final inspection after 120 days all the concentration tested showed a low efficacy. Similar results were obtained in naturally infected chicory where the severity of the disease decreased by increasing CO₂ from 5 to 10%, higher values did not improve the suppressive effect or determined, after 150 days of storage, an increased vulnerability of the tissues to disease due to the phytotoxic effects of the gas. An atmosphere enriched with 10% CO₂ is advised to suppress Botrytis rot during storage at 0°C of red chicory.     

CO2 enhances effects of hypoxia on mortality,development, and gene expression in cowpea bruchid, Callosobruchus maculatus

Modified atmosphere based on lack of O₂ offers a safe, residue-free alternative to chemical fumigants for pest control in stored grains. In this study, we intended to determine whether elevated CO₂ (at a biologically achievable level) has an enhanced suppressive effect over low O₂ atmosphere alone on the cowpea bruchid (Callosobruchus maculatus), a storage pest of cowpea and other legumes. Experiments were performed under two modified atmospheric conditions, (1) 2% O₂ + 18% CO₂ + 80% N₂ and (2) 2% O₂ + 98% N₂. Both hypoxic environments significantly affected the development and survival of all insect developmental stages. Eggs were most vulnerable to hypoxia, particularly at the early stage (4–6 h old), surviving only up to a maximum of 2 days in both treatments. These were followed by adults, pupae and larvae, in order of decreasing susceptibility. The 3rd and 4th instar larvae were most resilient to hypoxia and could survive up to 20 days of low O₂. The presence of 18% CO₂ significantly increased the mortality of adults, the later stage of eggs, as well as 1st and 4th instar larvae caused by hypoxia. However, the surviving insects exhibited faster development, evidenced by their earlier emergence from cowpea seeds compared to those without CO₂. One interesting observation was the frequent, premature opening of the emergence windows in the 4th instar larvae when CO₂ was involved. This phenomenon was not observed at all in insects stressed by low O₂ alone. Differential expression profiling of metabolic genes and proteolytic activity of midgut digestive enzymes suggested that the rate of metabolic activity could contribute in part to the difference in insect development and survival under hypoxia in the presence and absence of CO₂.     

Validation of the BacT/ALERT?3D automated culture system for the detection of microbial contamination of epithelial cell culture medium

Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(?)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(?)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(?)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(?)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(?)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.     

Influence of gradual adaptation of cattle to Leucaena leucocephala leaf meal on biodegradation of mimosine and 3- hydroxy-4(1H)-pyridone (3,4 DHP) in rumen,their levels in blood,fate and influence of absorbed DHP on thyroid hormones and liver enzymes

Three rumen fistulated Karan Fries crossbred (Holstein X Tharparkar) calves were fed increasing dry matter (DM) levels of 25%, 50%, 75% and 100% through leucaena leaf meal (LLM) starting at week 1, 2, 3 and 4, respectively. The mimosine, 3,4 DHP and 2,3 DHP levels were determined in strained rumen liquor (SRL) and serum at 0, 1, 2, 4, 8, 12 and 24 h postfeeding on days 1, 8, 15, 22, 29 and 42. LLM was incubated for 24 h with SRL in vitro on days 0, 7, 14, 21, 28 and 41 to study mimosine and dihydroxypyridone (DHP) biodegradation. On day 43, 1–1.5 l of rumen liquor was transferred to another set of three unadapted calves which were fed 50% LLM after transfer of inoculum. DM intake was 1.78%, 2.13%, 2.27%, 1.66%, 1.54% and 1.35% of live weight during the 1st through 5th week, respectively. Both in vitro and in vivo studies showed extensive degradation of mimosine to 3,4 DHP and 2,3 DHP from first day of LLM feeding. The overall in vitro DHP degradation was nil, 28.6%, 43.3% and 40.1% on day upto 15, 21, 28 and 42 of LLM feeding. No mimosine was found in serum on any day of sampling. The 3,4 DHP detected (56.94±31.65 μg ml⁻¹ serum) one hour post feeding on day 1 exhibited a decline from day 22 onwards. The serum also contained 2,3 DHP on days 8, 15, 22, 42. The faecal and urinary excretion of 3,4 DHP and 2,3 DHP as percent of mimosine intake declined from first week (76.3±2.8) to 4th week (42.1±4.1). The feeding of LLM resulted in reduced level of T₃ and T₄ within a week of LLM feeding. The level of T₃ improved to normal by 6th week while that of T₄ remained low. The SGOT and SGPT activities were within normal range. The gradual adaptation to LLM feeding caused Karan Fries calves to acquire DHP degrading ability to nontoxic compounds and this ability was transferred through transfer of rumen liquor from such calves to other unadapted calves at as early as 9th day of LLM feeding. The results revealed the possibility of two types of microbes degrading mimosine and 3,4 DHP to 2,3 DHP. One type of 2,3 DHP degrading microbes may be inhibited in the presence of 3,4 DHP whereas the other type may be active.     

Enrichment and isolation of Acetitomaculum ruminis,gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen

Five strains of acetogenic bacteria were isolated by selective enrichment from the rumen of a mature Hereford crossbred steer fed a typical high forage diet. Suspensions of rumen bacteria, prepared from contents collected 7 h postfeeding, blended and strained through cheesecloth, were incubated in a minimal medium containing 10% clarified rumen fluid under either H₂:CO₂ (80:20) or N₂:CO₂ (80:20) headspace atmosphere. The selection criterion was an increment of acetate in the enrichments incubated under H₂:CO₂. Periodically, the enrichment broths were plated onto agar media and presumed acetogenic bacteria subsequently were screened for acetate production. Selected acetogenic bacteria utilized a pressurized atmosphere of H₂:CO₂ to form acetate in quantities 2 to 8-fold higher than when grown under N₂:CO₂. All presumptive acetogenic isolates were derived from either the 10^-7 or 10^-8 dilutions of rumen contents. All 5 strains were Gram-positive rods, and all utilized formate, glucose and CO. One strain required, and all were stimulated by, rumen fluid. No spores were observed with phase-contast microscopy and two strains were motile. No methane was detected in the headspace of pure cultures grown under either gas phase. The isolation of these bacteria indicates that acetogenic bacteria are inhabitants of the rumen of the bovine fed a typical diet and suggests that they may be participants in the utilization of hydrogen in the rumen ecosystem. Strain 139B (= ATCC 43876) is named Acetitomaculum ruminis gen. nov., sp. nov. and is the type strain of this new species. Portions of this work were presented previously (Greening RC, Leedle JAZ (1987) Abstr Annu Meet Am Soc Microbiol I 131, pp 194)     

Life cycle of Rhodnius brethesi Matta, 1919 (Hemiptera, Reduviidae, Triatominae), a potential vector of Chagas disease in the Amazon region

R. brethesi is a sylvatic species from the Amazon region; it has been incriminated as responsible for the transmission of Chagas disease in collectors of piacaba in this region. The aim of present study was to investigate the efficiency of these insects as potential vectors of Trypanosoma cruzi. Aspects related with feeding and defecation patterns, life time, and mortality had been observed in each instar of R. brethesi. We use 5th instar nymphs to get adults virgins, after the moulting 3 groups with 6 females and 2 males each were created to obtain eggs. After hatching, 1st instar nymphs had been weighed and kept in bottles until the next moult. Insects were fed once a week in mice. Results showed that the average period of incubation was 17 days, the number of blood meal was increasing from the 1st to the 5th instar nymph with 7 (average) to become adult, a significative numbers of the defecations occurring immediately after the bloodmeals. The total percentual of mortality was 16%. This results suggests that this species presents a good exploitation of blood meals and a brief nymphal development in laboratory conditions reflecting its behavior in sylvatic environments.     

Detection of nuclear polyhedrosis virus infection in Heliothis spp. by agar gel double diffusion

Nuclear Polyhedrosis virus infection was detected in single Heliothis zea larvae by the agar gel double diffusion technique using antiserum to alkali-solubilized polyhedra. Virus bands were observed in 2nd-5th stadium larvae following homogenization in 0.1 M Na₂CO₃-0.05 M NaCl, pH 11.0, in approximately one-half the time necessary for mortality to occur. In a field test, virus infection was detected by this method as early as 3 days after virus treatment.     

Relation of feeding,growth, and metabolism to age in the larval,female house cricket

The maximum growth rate occurs in the first half of the 7th and 8th larval instars of the house cricket, at which time food and water consumption is maximal. Growth ceases in the last 2 to 3 days of each instar when food consumption is almost nil. The metabolic rate is twice, and the locomotory activity is four times, higher in the first 2 to 3 days than in the last 2 to 3 days of each instar. The %-gain in dry wt is 120% for the 7th and 139% for the 8th instar. The average digestive coefficient (AD) for the 8th instar is 67% and the average efficiency of food conversion to tissue (dry wt ECI) is 27%. The average daily food consumption for 8th instar larvae is 31.4 mg, and, therefore, we calculate that an average of 12 mg food is burnt per day for energy which is confirmed by the observed average V_O2 of 1.2 ml O₂/g-cricket/hr. Total lipids as %-total wt (239% mg-gain) increases in the first 4 days then remains constant in spite of cessation of feeding in the last 2 days. The RQ confirms the conversion of carbohydrates to lipids in the first half of each instar. In the moulting cycle carbohydrates are used for maintenance (when not feeding), but lipids are used at apolysis.     

Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a <Emphasis Type="Italic">Desulfovibrio</Emphasis> species under anaerobic conditions

Past handling practices associated with the manufacturing and processing of the high explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has resulted in extensive environmental contamination. In-situ biodegradation is a promising technology for remediating RDX contaminated sites but often relies on the addition of a cosubstrate. A sulfate-reducing bacterium isolated from an RDX-degrading enrichment culture was studied for its ability to grow on RDX as a sole source of carbon and nitrogen and for its ability to mineralize RDX in the absence of a cosubstrate. The results showed the isolate degraded 140 μM RDX in 63 days when grown on RDX as a carbon source. Biomass within the carbon limited culture increased 9-fold compared to the RDX unamended controls. When the isolate was incubated with RDX as sole source of nitrogen it degraded 160 μM RDX in 41 days and exhibited a 4-fold increase in biomass compared to RDX unamended controls. Radiolabeled studies under carbon limiting conditions with ¹⁴C-hexahydro-1,3,5-trinitro-1,3,5-triazine confirmed mineralization of the cyclic nitramine. After 60 days incubation 26% of the radiolabel was recovered as ¹⁴CO₂, while in the control bottles less than 1% of the radiolabel was recovered as ¹⁴CO₂. Additionally, ~2% of the radiolabeled carbon was found to be associated with the biomass. The 16S rDNA gene was sequenced and identified the isolate as a novel species of Desulfovibrio, having a 95.1% sequence similarity to Desulfovibrio desulfuricans. This is the first known anaerobic bacterium capable of mineralizing RDX when using it as a carbon and energy source for growth.     

Agarose Medium for Germination of Oospores of Phytophthora cactorum

When oospores of Phytophthora caetorum from 30-day-old culture were treated with 0.25% KMnO₄ for 20 min and incubated at 24°C under light for 10 days, 65–75% germinated on water agar and water agarose but only 1–21% germinated on V-8 agar and S+L agar. Water agarose was selected because germinated oospores formed restrieted colonies on this medium that could be isolated easily. KMnO₄ treatment killed sporangia, chlamydospores and mycelial fragments present in oospore suspensions. Under the above conditions, approximately 44% of oospores from 10-day-old culture germinated and the optimum germination rate of about 75% was obtained when oospores reached about 20 days old.