Investigation and application of methods for enumerating heterotrophs and Escherichia coli in the air within piggery sheds

AIMS: To investigate methods for the recovery of airborne bacteria within pig sheds and to then use the appropriate methods to determine the levels of heterotrophs and Escherichia coli in the air within sheds. METHODS AND RESULTS: AGI-30 impingers and a six-stage Andersen multi-stage sampler (AMS) were used for the collection of aerosols. Betaine and catalase were added to impinger collection fluid and the agar plates used in the AMS. Suitable media for enumerating E. coli with the Andersen sampler were also evaluated. The addition of betaine and catalase gave no marked increase in the recovery of heterotrophs or E. coli. No marked differences were found in the media used for enumeration of E. coli. The levels of heterotrophs and E. coli in three piggeries, during normal pig activities, were 2.2 x 10(5) and 21 CFU m(-3) respectively. CONCLUSIONS: The failure of the additives to improve the recovery of either heterotrophs or E. coli suggests that these organisms are not stressed in the piggery environment. The levels of heterotrophs in the air inside the three Queensland piggeries investigated are consistent with those previously reported in other studies. Flushing with ponded effluent had no marked or consistent effect on the heterotroph or E. coli levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work suggests that levels of airborne heterotrophs and E. coli inside pig sheds have no strong link with effluent flushing. It would seem unlikely that any single management activity within a pig shed has a dominant influence on levels of airborne heterotrophs and E. coli.     

Comparison of dichloran rose bengal chloramphenicol and Sabouraud dextrose agar with cycloheximide and chloramphenicol for airborne mold sampling

The more the mold species isolated on a culture medium, the more the sampling environment is represented accurately. According to the sampling purpose, it is crucial to use the best culture medium for mold. However, no study is available regarding the comparison of dichloran rose bengal chloramphenicol (DRBC) and Sabouraud dextrose agar with cycloheximide and chloramphenicol (SDA-CHX-CHL) culture media in terms of their application for airborne sampling, isolation, and identification of fungi. Airborne mold samples were impacted onto both DRBC and SDA-CHX-CHL, simultaneously using single-stage Andersen sampler. The limit of detection (LOD) value for airborne mold count was 7 CFU m^?3 (1 colony growth on the Petri dish). The total mold counts (TMC) ranged between <7 and 504 CFU m^?3 (med 56 CFU m^?3) and <7 and 1218 CFU m^?3 (med 259 CFU m^?3), collected on SDA-CHX-CHL and DRBC, respectively. Significantly higher TMC were observed on DRBC than on SDA regardless of the sampling environment (i.e, indoor or outdoor) (p < 0.05). Among the most predominant mold genera, observation frequencies of Penicillium spp. and Aspergillus spp. on both culture media were found to be more than 70%. Observation frequencies of Cladosporium spp., Alternaria spp., and yeast were found to be higher in samples collected on DRBC than those on SDA-CHX-CHL. Finally, DRBC was found to be superior to SDA in terms of both number of colonies and number of genera isolated from the air.     

裂褶菌培养基形态学观察和DNA序列分析

目的通过观察裂褶菌在5种培养基上的生长状态、扫描电镜及DNA序列分析,了解该菌形态学及分子生物学等方面的特征。方法菌落转种于沙氏培养基(SDA),麦芽浸膏琼脂(MEA),马铃薯葡萄糖琼脂(PDA),玉米粉琼脂(CMA)和察氏琼脂(CZA)平皿培养基,27℃和37℃培养2周,观察菌落生长情况,进行扫描电镜检测及DNA序列分析。结果菌落在SDA,MEA和PDA上生长状态较好,呈蓬松白色羊毛状;尿素酶试验阳性,放线菌酮耐受试验阴性。光镜下见分支分隔菌丝、侧生的钉状突起及类水母体变异子实体。扫描电镜见菌丝分隔处闭锁联合、侧生钉状突起和泪滴状球形分泌物。经26S rDNAD1/D2区序列分析证实该菌株为裂褶菌。结论裂褶菌只有丝状型一种菌落形态;分支分隔菌丝及分隔处闭锁联合,侧生钉状突起和泪滴状球形分泌为其形态学特征;孢子由类水母状子实体产生。     

Evaluation of media for recovery of aerosolized bacteria

Disease transmission by airborne bacteria is well known. Bacterial burden in indoor air is estimated by sampling the air and estimating Colony Forming Units (CFU) using a variety of media. In this study, the recovery of bacteria, after aerosolization in an aerosol chamber, and employing a variety of media, was compared to that achieved using Tryptic Soy Agar medium. The total number of cells present was determined by direct microscopy. All trials were conducted at approximately the same relative humidity (RH) and temperature using the same collection device. Twelve species of bacteria were tested and a total of 120 media or media combinations were evaluated. Recovery on 64 media formulations was significantly lower for all strains examined, and therefore, excluded from further consideration for the purposes of this study. Data for 56 of the media are presented. Three species (Bacillus subtilis, Staphylococcus aureus andSerratia marcescens) were selected as representative for reporting and testing recovery success. It is concluded that, for the media included in the study, there are large differences in recovery and successful recovery is related both to the effect of aerosolization and the type of medium employed for recovery. Brain Heart Infusion Agar (with horse serum), Tryptic Soy Agar and Mueller Hinton Agar yielded the best recoveries of aerosolized cultures. The most important finding was that only a small fraction of the airborne bacterial populations, enumerated by direct microscopy, could be recovered on any of the media tested, suggesting that culturable bacterial count is not a satisfactory means of estimating air microbial pollution.     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

Effect of Prehumidification on Sampling of Selected Airborne Viruses

Studies were undertaken to determine if a prewetting device (humidifier bulb) used in combination with an all glass impinger (AGI-30) would increase the recovery of airborne mengovirus-37A, vesicular stomatitis virus (VSV), and the S-13 coliphage. Suspensions of T3 coliphage with mengovirus-37A, VSV, or S-13 were aerosolized and collected by using the AGI-30-humidifier bulb combination to sample the aerosols before and after shifts in relative humidities (RH). These studies revealed the following. (i) At low RH values there was a 3 to 4 log increase in recovery of airborne T3 phage; (ii) concomitantly, the recovery of mengovirus-37A and VSV decreased; and (iii) only at the mid-range RH values was the recovery of S-13 enhanced. The prehumidification technique significantly increased the recovery of airborne T3 phage but decreased the recovery of the two animal viruses tested.     

Observations on the use of a medium detecting β-glucuronidase activity and lactose fermentation for the simultaneous detection of Escherichia coli and coliforms

Three hundred and forty clinical isolates of Candida species and Saccharomyces cerevisiae were tested in order to evaluate different methods for identification of Candida albicans using fluorogenic or chromogenic substrates. Detection of N -acetyl-β-D-galactosaminidase (NAGase) was performed with ready-to-use agars such as Fluoroplate Candida Agar (Merck, Germany), MUAG Candida Agar and MUAG Sabouraud Agar (Biolife, Italy) which contained 4-methylumbelliferyl- N -acetyl-β-D-galactosaminide (4-MUAG). MUAG Candi Kit and RAP-ID-ALBICANS (Biolife, Italy) and Albicans ID Agar (bioMérieux, France) were also used. The Vitek AMS System was used as a reference identification method for all isolates. NAGase activity could be detected for C albicans with Fluoroplate Candida Agar (98.8%), MUAG Sabouraud Agar (98.4%), Albicans ID (99.6%). MUAG Candi Kit (97.5%) and RAP-ID-ALBICANS (96.2%). Proline aminopeptidase examined with RAP-ID-ALBICANS was present in 98.7% of C. albicans. There was one false-positive result for C. tropicalis (9.1%) on Fluoroplate Candida Agar, one false-positive result for C. glabrata (2.2%) on Albicans ID Agar: five false-negative results for C. albicans (3.1%), but no false-positive results for the other tested species were observed with RAP-ID-ALBICANS.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: I. Enumeration1

Agar plating media containing solely activated sludge extracts yielded, in general, higher viable counts of activated sludge bacteria than any other culture medium tested. Activated sludge extracts made from different treatment plants varied in efficacy in evoking maximal viable counts. Frequently, homologous plating, i.e., plating inocula of activated sludges on extracts made from the same activated sludges, tended to yield lower counts than the heterologous platings tried in this investigation. The counts obtained by homologous plating of activated sludge were not significantly lower and sometimes were even significantly higher than the counts obtained on standard Nutrient Agar, which had been found by previous workers to be a good medium for counting activated sludge bacteria. The higher counts obtained with activated sludge extracts set objectives for formulating reproducible or defined culture media for the enumeration of activated sludge bacteria.     

The vitamin requirements of Staphylococcus cohnii

In heat-resistance studies with spores of Clostridium sporogenes BC-2, an improved recovery medium was needed for severely heat-damaged spores as the used previously--Wynne medium in Miller-Prickett tubes--did not allow accurate counts of spores because of gas formation and disruption of agar. Initial test with pour plates of Viande-Leyure medium containing egg-yolk gave much increased counts for spores previously heated for 50 min at 112 degrees C; this increase was attributed to the presence of egg-yolk. Addition of egg-yolk to Reinforced Clostridial Agar, All-Culture Medium and Trypticase Soy Agar showed that Trypticase Soy Agar with egg-yolk was the best recovery medium. For the final formulation, the value of supplementation with cysteine-HCl and methylene blue was also shown. The resultant Egg-yolk Trypticase Soy Agar is conveniently prepared from BBL Trypticase Soy Agar (40 g) with the addition of 0.4 g/l cysteine-HCl, 4 mg/l of methylene blue and 2% Oxoid egg-yolk emulsion aseptically to the melted basal medium. For optimal spore counts, pour plates are incubated anaerobically for 5--7 d at 30 degrees C.     

Control of microbial contamination of Franz diffusion cell receptor phase in the development of transcutaneous breast cancer therapeutics

Aims:  To determine the micro-organism contamination of excised porcine (pig) ear, and evaluate the use of Cyclopore track-etched membranes (CTEM) for preventing ingress into Franz-type diffusion cells.
Methods:  Swabs were taken from four locations and used to inoculate Tryptone Soya Agar (TSA) and Sabouraud Dextrose Agar (SDA) plates. Diffusion cells were assembled to include porcine skin with and without CTEM, and the receptor phase sampled periodically and spread onto plates.
Results:  Five distinct colony types were isolated after incubation of all swabs on TSA plates at 37°C; on SDA plates, one fungal colony was found at 30°C and one at 37°C. The SDA agar plate incubated at 30°C resulted in the growth of a large diffused white fungal colony. No regional differences were observed. Without the CTEM, the receptor phase became contaminated within 6 h. With the CTEM present, microbial ingress was substantially retarded with visible presumptive fungal growth occurring at 24 h and detectable contamination on both microbiological media at 48 h.
Conclusions:  As expected, the native porcine ears were considerably contaminated. The ingress of contamination into the diffusion cell receptor phases can be largely, but not entirely, eliminated using CTEM. The addition of antimicrobial agents was necessary to eliminate micro-organisms that were observed at later time points.
Significance and Impact of the Study:  This article, while highlighting the presence of a high number of micro-organisms on native porcine skin, presents a practical means to reduce the risk of microbial contamination in transdermal/transcutaneous permeation studies, particularly in the study of cell cultures grown within Franz diffusion cell receptor compartments.     

Yeast communities associated with sugarcane in Campos, Rio de Janeiro, Brazil.

Yeast communities associated with sugarcane leaves, stems and rhizosphere during different phases of plant development were studied near Campos, in Rio de Janeiro, Brazil. Atmospheric temperature, soil granulometry and pH, and sugar cane juice degree Brix and pH were determined. Yeast communities associated with sugarcane were obtained after cellular extraction by shaking, blending and shaking plus sonication, and cultured on Yeast Nitrogen Base Agar plus glucose (0.5%) and Yeast Extract-Malt Extract Agar. No significant differences in yeast counts were found among the cellular extraction treatments and culture media. 230 yeast cultures were identified according to standard methods, and distinct yeast communities were found for each substrate studied. The prevalent species isolated from sugarcane were Cryptococcus laurentii, Cryptococcus albidus, Rhodotorula mucilaginosa and Debaryomyces hansenii.     

红色毛癣菌临床分离株的菌落形态和镜下结构特征再分析

目的探讨红色毛癣菌的菌落形态和镜下结构特征以及与感染部位的相关性。方法采用传统培养方法对192株红色毛癣菌进行表型分型,选取其中39株在28℃、30℃、35℃3种温度孵育6d、10d、14d时观察菌落形态和生长速度。结果192株红色毛癣菌共分离出3种表型：绒毛型、沟纹型、粉末型（或颗粒型）。在相同培养基上,28℃、30℃时菌落生长速度无显著差异（P〈0.05）,均快于35℃（P〈0.05）。在相同温度时,菌落在SDA上的生长速度快于PDA培养基。在28℃、30℃时菌落形态比较稳定,在35℃时变异较大。菌落在PDA培养基上产孢丰富,镜下显示有较多的大、小分生孢子,而在SDA培养基上只有少量的小分生孢子,几乎见不到大分生孢子。各种浅部感染均以绒毛型为主,绒毛型在手足癣中占比例最高,沟纹型在体股癣中所占比例较高,粉末型在甲癣中占的比例较高。结论红色毛癣菌的菌落形态与培养基和培养温度有关,其表型和镜下结构与感染部位均有一定的相关性。     

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.     

Comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples.

The interest in functional foods, probiotics and prebiotics requires a proper method to determine specific bacterial groups in the intestinal flora, especially bifidobacteria and lactobacilli. Three media for lactobacilli (MRS, Rogosa, LAMVAB), three media for bifidobacteria (RB, NPNL, Beerens medium) and nine media for total anaerobes have been tested for selectivity and recovery. For total anaerobes Faecal Reinforced Clostridial Agar (FRCA) showed the highest cfu/g, followed by Columbia Blood Agar and BHI Blood agar. There were no significant differences between the media tested. Reduced physiological salt solution was found to be the best dilution medium. For bifidobacteria and lactobacilli samples of human faeces, cat faeces and pig ileal contents were used. Bifidobacteria could reliably be determined on all three media tested in human faeces, but not on pig ileal contents or cat faeces. Absolute counts were highest in human samples. No lactobacilli could be isolated on MRS in either sample, none of the colonies in the countable plates were lactobacilli. For Rogosa over 90% of the colony types observed in human samples were not lactobacilli. For cat faeces this was 58%, but no false positives were observed in the pig ileal samples. For LAMVAB the percentages of false positive colony types were 9, 14 and 0% for human, cat and pig samples. It can be concluded that for bifidobacteria RB and Beerens medium show comparable results, and can be used to quantify bifidobacteria in human faeces, but none of the media tested is suitable for reliably counting bifidobacteria from pig and cat samples. For lactobacilli LAMVAB shows the highest sensitivity.     

Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water

Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner’s 2nd Agar or Broth (R2A or R2AB), Brain–Heart Infusion Broth (BHIB), Mueller–Hinton Broth (MHB), and Ashdown’s (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.     

Effects of medium composition on the recovery of bacteria from sea water

Water samples were collected from offshore and inshore localities at various depths off the Connecticut coast over a two-year period. Spread plates for bacterial counts at 20 °C were made on a variety of complex solid media. Counts on Difco-Marine Agar were controls in all cases with counts on test media related to these in ratio form. Initially, nine media were used and represented some from the literature as well as personal formulations. Differences between inshore and offshore samples were greatest with media containing the highest peptone concentrations. Two media containing the peptones Gelysate and Trypticase showed the highest overall counts. A second phase concerned a comparative study of these peptones varying in concentration from 0.1 to 10.0 g/l in a constant basal medium. None of the media invariably gave counts greater than the control, but peptone concentrations of 10.0 and 5.0 g/l resulted in the lowest comparative counts. Considering all samples, peptone levels of 0.1 and 1.0 g/l showed the highest counts. Counts for both inshore and offshore water samples decreased as peptone concentration increased. Qualitatively, high peptone media showed large, mucoid, confluent colonies which made the counting of smaller ones difficult. Pigmented colonies were more frequent on low peptone media. Bacteria were isolated from all media and from all stations; the percentage of various groups varied with peptone concentration and source of sample.Media containing three fish peptones in varying concentrations have also been investigated. None produced overall counts greater than Difco-Marine Agar and counts decreased with increasing peptone levels: there was a trend towards higher counts in offshore waters with fish extracts. Quantitative and qualitative aspects of the work are discussed.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

不同消毒和培养基条件对兰花菌根真菌分离的影响

研究了不同的消毒时间和培养基类型对兰花菌根真菌分离效果的影响,以期获得兰花菌根真菌的最佳分离方法.采用了次氯酸钠和酒精作为消毒试剂,发现依次浸没于75 %酒精60 s、2 %次氯酸钠60 和75 %酒精30 s的组合消毒条件下分离效果最佳;选择了3种培养基（PDA、CZA和MEA）,结果表明MEA培养基的分离效果最为稳定.通过组织分离法分离兰花菌根真菌,筛选得到最佳的分离培养条件,旨在为兰花菌根真菌的多样性以及共生效应研究提供研究基础.     

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.     

A comparison of different culture media for the membrane filter quantification of Aeromonas in water

A comparative assessment of culture media for the membrane filter enumeration of Aeromonas spp. in water was performed, testing the effects of different incubation conditions (aerobic and anaerobic), temperatures (30 and 37 degrees C) and times (24 and 48 h). Different water samples seeded with test suspensions of Aeromonas spp., fecal material or raw sewage were examined. Results indicate clearly that plates should be incubated aerobically at 30 degrees C for 24 h. If the bacterial contamination is likely to be low, the use of most sensitive culture media, such as SAA, mA, ADA or PADE Agar, is recommended. By contrast, samples with an expected high level of background microbial flora should be analysed through more selective media, such as MIX Agar. However, the low selectivity of all media tested and the high likelihood of false negatives based upon the macroscopic examination of colonies means that further research directed to the development of more efficient media is needed.