Efficiency of Selenite Cystine and TT Enrichment Broths for the Detection of Salmonella

Comparisons of selenite cystine (SC) and TT enrichment broths for detecting salmonellas were made with pure culture suspensions, with samples of naturally or artificially contaminated foods and with poultry feed. Selenite cystine recovered higher numbers of salmonellas from pure cultures and ground beef, while TT broth recovered higher numbers from pork sausage and poultry feed. Differences in the recovery of salmonellas from other food products appeared to be insignificant. The use of both SC and TT is thus recommended for maximal recovery of these organisms.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.     

Comparison of Media for the Enumeration of Clostridium perfringens

For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 10(4) organisms per g, but with 10(6) organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

Storage of cultures of Salmonella enteritidis PT4 at either 4 degrees or 8 degrees C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4-7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60 degrees rather than 55 degrees C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37 degrees C prior to heating enabled the organisms to recover heat resistance.     

Effect of Sodium Polyanetholesulfonate on Antimicrobial Systems in Blood

Sodium polyanetholesulfonate (SPS), an anticoagulant which inhibits the antimicrobial systems of blood, is used widely in blood culture media. The addition of SPS to experimental blood cultures inoculated with small numbers of a variety of organisms caused a striking increase in recovery of these organisms. Sodium fluoride also increased the incidence of positive blood cultures with some organisms. SPS completely inhibited serum antibacterial activity and serum-dependent phagocytosis (and killing) by isolated leukocytes at a concentration usually employed in blood culture media. SPS also stimulated both glucose C-1 oxidation in resting leukocytes and formate oxidation in both resting and phagocytosing leukocytes in serum-free systems. These in vitro studies support the concept that SPS is a useful additive to blood culture media and further elaborate on the mechanism of its inhibition of the microbicidal activity of blood.     

Comparison of four membrane filter methods for fecal coliform enumeration.

Four membrane filter methods fecal coliform enumeration were evaluated and compared in six laboratories based on determination of accuracy, specificity, upper counting limit, and recovery comparability. Recovery accuracy with pure cultures ranged from 89 to 100% for m-FC, mTEC (a procedure developed for thermotolerant Escherichia coli), and m-FC2 methods (the latter incorporating a 2-h, 35 degrees C resuscitation period), but was less than 60% for the MacConkey membrane broth method. These figures dropped by approximately 40 to 55% when the cultures were subjected to temperature (10 degrees C) stress. Close to 800 colonies were verified to determine specificity. False-positive colonies occurred most frequently with the m-FC2 method (18%), whereas false-negative colonies were most common on MacConkey membrane broth (26%). In counting range experiments using a variety of samples, the highest upper counting limit was 130 colonies per filter with the mTEC procedure. Recovery comparisons were based on over 130 samples including raw surface waters, raw sewage, and chlorinated and unchlorinated sewage effluents. In general, recoveries were significantly higher with the m-FC2 and mTEC methods; however, on m-FC2, growth of nontarget background organisms was also higher in most cases. Highest recoveries from chlorinated sewage effluents were obtained by the mTEC method, and the addition of a similar resuscitation period to the m-FC procedure (m-FC2) improved fecal coliform recovery from such samples. The best overall performance for fecal coliform enumeration was obtained with the mTEC method with high recovery and low levels of background colonies, good specificity and accuracy, and a high upper counting limit. This procedure also offers the advantage of enumerating E. coli within 24 h.     

Recovery efficiency of a hydrophilic ionic-liquid aqueous biphasic system for the primary purification of cytochrome c from simulated Saccharomyces cerevisiae fermentation broth

Ionic liquid-based aqueous biphasic system (ILABS) has emerged as an attractive green approach for the extraction and separation of various biomolecules. The growing market demands of cytochrome c (cyt c) due to its vast uses in medical applications have urged the search for cost-efficient approaches for the production and purification of cyt c. In this study, the feasibility of ILABS to recover cyt c from simulated Saccharomyces cerevisiae cell cultures was investigated by evaluating the effects of phase composition, pH, and additives concentration on the recovery efficiency of cyt c. The ILABS was developed using the hydrophilic ionic liquids, 1-hexyl-3-methylimidazolium bromide ((C6mim)Br) and potassium citrate. The optimal separation conditions for recovery of cyt c from simulated cells cultures were attained with ILABS of pH 9 comprising of 28% (w/w) of (C6mim)Br and 24% (w/w) of potassium citrate and the addition of 0.2% (w/w) NaCl at room temperature (25 °C). Cyt c was recovered in the IL-rich phase with partition coefficient (Kc) of 364.00 ± 1.09, recovery yield (Y) of 99.76% ± 0.03 and selectivity (S) of 223.31 ± 0.56. The results suggest that ILABS can efficiently recover cyt c from microbial fermentation broth with high recovery yields and separation efficiency.     

Selective plating underestimates abundance and shows differential recovery of bifidobacterial species from human feces

The aim of the present work was to compare the efficacies and levels of selectivity of different culture-dependent and -independent methods for analyzing bifidobacteria in human stool samples. The three different culture media used here significantly differed from each other, particularly with regard to the recovery of Bifidobacterium adolescentis. Bifidobacterium medium failed to recover B. adolescentis; Beerens medium recovered some B. adolescentis organisms (17% of total bifidobacteria), whereas tomato-Eugon medium recovered mainly B. adolescentis organisms (58% of total bifidobacteria). A culture-independent method that combines GC fractionation of bacterial community DNA and 16S rRNA sequencing indicated that B. adolescentis organisms accounted for 85% of all bifidobacteria. Methodological biases, such as those described in this paper, should be taken into account in interpreting earlier studies and designing future experiments.     

Culture of Ethiopian Strains of Borrelia recurrentis

A number of standard bacteriological media with supplements were tested for their ability to support in vitro growth of Ethiopian strains of Borrelia recurrentis. Propagation of 18 out of 21 strains occurred in Trypticase soy yeast broth to which bovine albumin (fraction V), N-acetyl glucosamine, and sodium pyruvate had been added. This medium supported a population of 10(7) organisms per ml and yielded a harvest of four to five times the original inoculum during the logarithmic phase of growth. Maximal yield varied from 1.4 x 10(7) to 3.4 x 10(7) organisms per ml. Generation time in optimal media was 11.3 h. Lesser multiplication of organisms occurred in other media tested. Strains from primary cultures were infective for the green monkey. Recovery of viable organisms from subculture has not been successful.     

Development of a two-step enrichment procedure for recovery of Yersinia enterocolitica from food.

Two new enrichment media were formulated for the recovery of Yersinia enterocolitica from foods: (i) yeast extract-rose bengal broth for preenrichment at 4 or 10 degrees C; and (ii) bile-oxalate-sorbose broth, a selective enrichment incubated at 22 degrees C. Comparison of these media in a two-step enrichment procedure against cold enrichment and modified Rappaport broth showed improved and more rapid recovery of human strains of Y. enterocolitica from inoculated foods. The use of bile-oxalate-sorbose broth as a selective enrichment also improved the performance of cold enrichment with phosphate-buffered saline. Determination of the best enrichment system for recovery of Y. enterocolitica from samples of retail pork and fresh pork tongues depended on whether the criterion was the number of positive samples, the variety of different serotypes recovered, or the ability to recover the important human serotype O:3. A single enrichment system with the widest selectivity would include preenrichment at 4 degrees C with either phosphate-buffered saline for 14 days or yeast extract-rose bengal broth for 9 days followed by selective enrichment with bile-oxalate-sorbose broth at 22 degrees C for 5 days.     

Extraction and recovery of organochlorine pesticides from fungal mycelia

An extraction method was developed to recover organochlorine pesticides (OCPs) associated with live and mercuric chloride-treated fungal mycelia. A Cladosporium sp. was inoculated into potato dextrose broth, DDT (90 mg/l) added and incubated for seven days. The combination of a microextraction procedure for aqueous-phase-associated DDT and a sonication extraction for mycelia-bound DDT, using dichloromethane as the extracting solvent, resulted in the recovery of 31-51% of added DDT. DDT recovery was increased to 62-65% by grinding the fungal mycelia before sonication. Alkali and nitric acid pretreatments were tested to increase the recovery of DDT associated with fungal mycelia, however, these treatments resulted in the production of unidentified DDT transformation products. Pretreating mycelia containing DDT in concentrated hydrochloric acid at 60 degrees C for 2, 4 and 6 h resulted in DDT recoveries of 90-91%, 99% and 101-102% respectively without the production of transformation products. When an OCP mixture (DDT, DDD and DDE) was added to fungal mycelia, between 89% and 96% of DDT, DDD and DDE were recovered from live cultures compared to 85-91% in mercuric chloride-treated cultures using the microextraction/hydrochloric acid pretreatment (60 degrees C/6 h) sonication extraction method.     

High-throughput metal susceptibility testing of microbial biofilms

Background  

Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxiCity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated.     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

Detection of Bacteremia with Liquid Media Containing Sodium Polyanetholsulfonate

Two liquid blood culture media, Tryptic soy broth (TSB) and Thiol broth, containing sodium polyanetholsulfonate were compared in 8,654 cultures. Pseudomonas and Corynebacterium (including Propionibacterium) were isolated significantly more frequently (P < 0.001) from TSB than from Thiol. Escherichia coli, Haemophilus, and Bacteroidaceae were isolated more frequently in TSB; however, the differences were not statistically significant. In no instance was Thiol superior to TSB in detecting bacteremia. In an additional 2,977 cultures, aerobic and anaerobic Vacutainer culture tubes with supplemented peptone broth were inoculated in parallel with TSB and Thiol. Significantly greater rates of detection (P < 0.01) in TSB or Thiol were noted with Pseudomonas, E. coli, Enterobacter, viridans, and group A streptococci, Bacteroidaceae, and staphylococci.     

Identification of coliform genera recovered from water using different technologies

Aims:  Methods for the detection of coliforms in water have changed significantly in recent years with procedures incorporating substrates for the detection of β- d -galactosidase becoming more widely used. This study was undertaken to determine the range of coliform genera detected with methods that rely on lactose fermentation and compare them to those recovered using methods based upon β- d -galactosidase.
Methods and Results:  Coliform isolates were recovered from sewage-polluted water using m-endo, membrane lauryl sulfate broth, tergitol TTC agar, Colilert-18^®, ChromoCult^® and ColiScan^® for primary isolation. Organisms were grouped according to whether they had been isolated based upon lactose fermentation or β- d -galactosidase production.
Conclusions:  A wide range of coliform genera were detected using both types of methods. There was considerable overlap between the two groups, and whilst differences were seen between the genera isolated with the two method types, no clear pattern emerged. Substantial numbers of 'new' coliforms (e.g. Raoutella spp.) were recovered using both types of methods.
Significance and Impact of the Study:  The results presented here confirm that both methods based on lactose fermentation or detection of β- d -galactosidase activity recover a range of coliform organisms. Any suggestion that only methods which are based upon fermentation of lactose recover organisms of public health or regulatory significance cannot be substantiated. Furthermore, the higher recovery of coliform organisms from sewage-polluted water using methods utilizing β- d -galactosidase-based methods does not appear to be because of the recovery of substantially more 'new' coliforms.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Comparison of the BACTEC System with Blind Subculture for the Detection of Bacteremia

One thousand blood specimens were cultured in BACTEC vials containing modified Columbia broth in aerobic, anaerobic, and hypertonic formulations. Radiometric readings and subcultures were performed on aerobic and hypertonic vials at 24 h and 7 days, and on anaerobic vials at 48 h and 7 days. Significant numbers of false-positive BACTEC readings were obtained. Although all positive cultures were eventually detected by the BACTEC, approximately 20% of blood specimens yielding positive subcultures at 24 h did not give positive BACTEC readings until 48 h.     

Differential Effects of Lithium Chloride on In Vitro Growth of Clavibacter michiganense subsp. nebraskense Depending upon Inoculum Source

The bacterium Clavibacter michiganense subsp. nebraskense (Corynebacterium michiganense subsp. nebraskense) was grown in broth cultures and inoculated into corn plants. The plating efficiency of cells from broth cultures was essentially the same on nutrient broth-yeast extract and the semiselective medium for this bacterium, CNS. However, when cells were isolated from Goss bacterial wilt- and blight-infected corn, very few were recovered on CNS compared with the amount recovered on nutrient broth-yeast extract agar. When lithium chloride was omitted from the CNS, recoveries from infected corn were nearly the same as on nutrient broth-yeast extract agar. No other ingredient of CNS was inhibitory, nor did substitution of other salts for lithium chloride cause equal inhibition. The amount of inhibition was proportional to lithium chloride concentration. The inhibition by lithium chloride occurred with several strains of the bacterium isolated from one corn cultivar and with one of the strains recovered from three different cultivars of infected corn.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.