Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006–2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO₂, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.     

Micro-organisms isolated from cadaveric samples of allograft musculoskeletal tissue

Allograft musculoskeletal tissue is commonly used in orthopaedic surgical procedures. Cadaveric donors of musculoskeletal tissue supply multiple allografts such as tendons, ligaments and bone. The microbiology laboratory of the South Eastern Area Laboratory Services (SEALS, Australia) has cultured cadaveric allograft musculoskeletal tissue samples for bacterial and fungal isolates since 2006. This study will retrospectively review the micro-organisms isolated over a 6-year period, 2006–2011. Swab and tissue samples were received for bioburden testing and were inoculated onto agar and/or broth culture media. Growth was obtained from 25.1 % of cadaveric allograft musculoskeletal tissue samples received. The predominant organisms isolated were coagulase-negative staphylococci and coliforms, with the heaviest bioburden recovered from the hemipelvis. The rate of bacterial and fungal isolates from cadaveric allograft musculoskeletal tissue samples is higher than that from living donors. The type of organism isolated may influence the suitability of the allograft for transplant.     

The visual assessment of broth cultures for tissue bank samples

The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5–5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.     

Fungal culture of musculoskeletal tissue: what’s the point?

There have not been any studies that review the prevalence of fungal isolates using selective media from samples of banked musculoskeletal tissue retrieved from living and cadaveric donors. A total of 2,036 swab and 2,621 biopsy samples of musculoskeletal tissue from tissue banks were received from the 1st August 2008 till 31st December 2010. Routine culture for fungi using selective media with a prolonged incubation period failed to demonstrate a greater prevalence of fungal isolates than by using non-selective culture media alone. Using selective culture fungi were recovered from only two Sabouraud agar plates (0.1%) but not from non-selective media. During the same period fungi were isolated from three graft samples cultured in non-selective broth media only (0.1%). There was no correlation of fungal isolates from selective or non-selective media inoculated at the same time nor from multiple graft samples collected from the same donor supporting the possibility of an exogenous source for fungal isolates rather than an endogenous source.     

Swab or biopsy samples for bioburden testing of allograft musculoskeletal tissue?

Swab and biopsy samples of allograft musculoskeletal tissue are most commonly collected by tissue banks for bacterial and fungal bioburden testing. An in vitro study was performed using the National Committee for Clinical Laboratory Standards standard ‘Quality control of microbiological transport systems’ (2003) to validate and evaluate the recovery of six challenge organisms from swab and biopsy samples of allograft musculoskeletal tissue. On average, 8.4 to >100 and 7.2 to >100 % of the inoculum was recovered from swab and biopsy samples respectively. A retrospective review of donor episodes was also performed, consisting of paired swab and biopsy samples received in this laboratory during the period 2001–2012. Samples of allograft femoral heads were collected from living donors during hip operations. From the 3,859 donor episodes received, 21 paired swab and biopsy samples each recovered an isolate, 247 swab samples only and 79 biopsy samples only were culture positive. Low numbers of challenge organisms were recovered from inoculated swab and biopsy samples in the in vitro study and validated their use for bioburden testing of allograft musculoskeletal tissue. Skin commensals were the most common group of organisms isolated during a 12-year retrospective review of paired swab and biopsy samples from living donor allograft femoral heads. Paired swab and biopsy samples are a suitable representative sample of allograft musculoskeletal tissue for bioburden testing.     

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched in Bolton broth and Preston broth, followed by plating on Skirrow, mCCDA, and blood agar (a membrane filter on its surface) media, respectively. Thirty-nine of 99 samples were positive and 71 Campylobacter were isolated by one or more methods. More than one species of Campylobacter were obtained in 8 (20.5 %) of 39 positive samples and two genotypes were yielded on the same medium (11 samples, 28.2 %) by pulsed-field gel electrophoresis (PFGE) genotyping. Enrichment by Preston broth was significantly better than by Bolton broth (P?<?0.05). Moreover, the latter failed to detect Campylobacter jejuni strains. Skirrow medium was significantly less efficient than mCCDA medium and membrane filtration method (P?<?0.05). Overall, the combination of PC (primary enrichment in Preston broth, followed by selective enrichment on mCCDA agar), PF (primary enrichment in Preston broth, followed by membrane filtration culture onto blood agar), and BF (primary enrichment in Bolton broth, followed by membrane filtration culture onto blood agar) methods provided the optimum isolation rate of Campylobacter spp.     

Comparison of microbiological culture methods in screening allograft tissue. Swab versus nutrient broth

In order to reduce the risk of transmission of infectious diseases through transplantation of tissue allografts, one should examine tissues for the presence of microorganisms. However, there are no detailed tissue banking guidelines describing the culture method or incubation time to be used. Therefore, we compared two culture methods--blood agar plate versus Wilkins Chalgren broth--and three incubation times--2, 7 or 14 days for their performance. The ultimate aim is to use the optimal setting as standard operating procedure (SOP) for tissue allograft cultures. From 70 consecutive donors, 919 tissue samples were taken. All 919 tissue samples were incubated on blood agar as well as in Wilkins Chalgren broth for 7 days. 567 of these 919 tissue samples were left to incubate up to 14 days. Wilkins Chalgren broth yielded 24.5% (139/567) positive cultures after 14 days of incubation. This was slightly more than the growth on blood agar after 14 days (22.9%--130/567) (p=n.s.) and significantly more than the growth in Wilkins Chalgren broth after 7 days of incubation (21.9%--124/567) (p<0.05). Based on these results, Wilkins Chalgren broth has been implemented as the SOP. Since the yield of positive cultures increased from 2 to 7 days of incubation in broth (1.8 times) and the variability of species cultured from 7 to 14 days of incubation shifted towards mostly microorganisms known to be common contaminants, we established the cut-off at 7 days of incubation in Wilkins Chalgren broth.     

Guar gum: a cheap substitute for agar in microbial culture media

AIMS: To determine the possibility of using guar gum, a colloidal polysaccharide, as a cheap alternative to agar for gelling microbial culture media. METHODS AND RESULTS: As illustrative examples, 12 fungi and 11 bacteria were cultured on media solidified with either guar gum or agar. All fungi and bacteria exhibited normal growth and differentiation on the media gelled with guar gum. Microscopic examination of the fungi and bacteria grown on agar or guar gum gelled media did not reveal any structural differences. However, growth of most of the fungi was better on guar gum media than agar, and correspondingly, sporulation was also more advanced on the former. Bacterial enumeration studies carried out for Serratia sp. and Pseudomonas sp. by serial dilution and pour-plate method yielded similar counts on both agar and guar gum. Likewise, a selective medium, succinate medium used for growth of Pseudomonas sp. did not support growth of Bacillus sp. when inoculated along with Pseudomonas on both agar or guar gum supplemented medium. CONCLUSIONS: Guar gum, a galactomannan, which is 50 times cheaper than Difco-bacto agar, can be used as a gelling agent in place of agar in microbial culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: As the media gelled with guar gum do not melt at temperature as high as 70 degrees C, these can be used for isolation and maintenance of thermophiles.     

Improved method for detection of Vibrio parahaemolyticus in seafood.

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Unique advantages of using low temperature scanning electron microscopy to observe bacteria

Summary Electron microscopy (EM) has greatly helped to elucidate our understanding of bacterial structure and function. However, several recent studies have cautioned investigators about artifacts that result from the use of conventional EM preparation procedures. To avoid these problems, the use of low temperature scanning electron microscopy (LTSEM) was evaluated for examining frozen, fully hydrated specimens. Spinach leaves (Spinacia oleracea L. cv. New Jersey), which were naturally infected or inoculated with bacteria, were used as the experimental material. 1 cm segments of the infected leaves were plunge frozen in liquid nitrogen, transferred to a cryochamber for sputter coating and then moved onto a cryostage in an SEM. After observation, some of the frozen, hydrated leaf segments were transferred onto agar medium to determine whether preparation for LTSEM was nondestructive to the bacteria. The other tissue segments were chemically fixed by freeze-substitution. The results indicated that after cryopreparation and observation in the LTSEM: (i) viable bacteria, which were recovered from the leaf sample, could be cultured on agar medium for subsequent study, and (ii) the frozen samples could be freeze substituted and embedded so that transmission electron microscopic (TEM) observations could be carried out on the same specimen. In conclusion, frozen, hydrated leaf tissue infected with bacteria can be observed using LTSEM and then can be either processed for TEM observation to obtain further structural details or recovered to culture the pathogenic bacteria for supplementary studies.Abbreviations EPS extracellular polysaccharide - EM electron microscopy - LTSEM low temperature scanning electron microscopy - SEM scanning electron microscopy - TEM transmission electron microscopy - TSA tryptic soy agar - TSB tryptic soy broth Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.     

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.     

Effect of a culturing method and growth phase on composition of lipopolysaccharides in Yersinia pseudotuberculosis

The effects of the culturing method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lypopolysaccharide (LPS) composition of Yersinia pseudotuberculosis (O:Ib serovar, strain KS 3058) grown in cold (5 degrees C) were studied. The amount the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and achieved maximum in the stationary growth phase for both media. The bacteria culturing on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultured in the liquid medium. It was proposed that the culturing of Yersinia pseudotuberculosis in cold as colonies on the agar surface causes an increase in the bacterial virulence.     

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.     

National hospital survey of anaerobic culture and susceptibility methods: III

To assess the current status of anaerobic bacteriology in the United States, we surveyed, by means of a questionnaire, 150 hospitals selected at random with bed capacities of 200-1000 and we received responses from 98 (65%). Ninety-eight percent processed anaerobic culture specimens with 21% sending them to reference laboratories. Almost all these hospitals processed blood and wound cultures for anaerobes and all used selective media for identification, including BBE (52%), LKV (77%), and PEA (53%) agars. All hospital laboratories attempted identification of blood culture isolates including 80% that attempted speciation. Wound cultures for anaerobic bacteria and sterile site cultures were also processed for anaerobes by almost all labs. Identification of B. fragilis group species to species level was performed only in 56% of labs always and 37% sometimes. Preformed enzyme kits were used by 66% of labs and 30% used special potency disks for identification. Susceptibility testing was performed in-house by 21% of hospital labs and sent out to reference labs an additional 20%. Susceptibility testing was attempted for all blood culture isolates by both hospital (21% of total labs) and reference laboratories, but only performed by 17% for sterile body site and 14% of the time for wound isolates. Etest was used most often followed by broth microdilution. No labs used the agar dilution or disk elution methods. The antimicrobials most often tested in hospital labs, predicated on the commercial panel used, were penicillin/ampicillin and clindamycin (15/18; 83%; 15% of total labs), metronidazole (16/18; 89%; 16% of total labs) and cefotetan and ampicillin/sulbactam (12/18; 67%; 12% of total labs), piperacillin/tazobactam (7/18; 39%; 7% of total labs), cefoxitin (9/18; 50%), imipenem (8/18; 44%), and chloramphenicol (6/18; 33%). Our current survey suggests that while many labs are processing anaerobic cultures, especially blood cultures, the identification of isolates and the performance of antimicrobial susceptibility testing of isolates are in disarray and in dire need of improvement.     

Influence of culture conditions on cell surface hydrophobicity of rods of genus Serratia

The cell surface hydrophobicity (CSH) is a non-specific adhesion factor that is important in the proliferation of microorganisms on solid surfaces. Serratia spp. is a bacterium that has been increasingly implicated as a primary pathogen in numerous human infections, particularly in urinary tract infections. CSH of 60 Serratia spp. strains isolated from clinical materials was evaluated using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed), enrichment agar with 5% human blood and medium composed of agar granulated (Becton Dickinson), neopeptone (Difco) and 1% (v/v) glycerol. CSH was estimated most frequently when the analyzed strains in enrichment broth were cultured. When grown in enrichment broth cells of Serratia spp. at room temperature were more hydrophobic (43% after 24 h and 47% after 48 h) than those at 37 degrees C (30% after 24 h and 33% after 48 h). CSH of the examined Serratia spp. strains were depended on the temperature, time of the culture of bacteria and the kind of media. The influence of the culture conditions on the changes in CSH of the analyzed bacteria may suggest significance of these properties in the pathogenesis of Serratia spp.     

Selection of Media for the Isolation of Common Bacterial L-Phase Organisms from a Clinical Specimen

The L-phase of 13 bacteria commonly associated with disease were induced by penicillin and inoculated into various solid and broth media; their growth was recorded for a period of 14 days. Plates containing highly purified agar and sucrose as the stabilizing agent and those incubated under aerobic conditions gave the best results. Magnesium seems to be necessary for growth in broth media on primary isolation, although it may not be necessary on multiple transfers after a more stable state has been reached. Growth in broth media is much more difficult to achieve. Reversion is aided by using a higher concentration of agar in plates, by decreasing the sucrose concentration, and by omitting the antibiotics and horse serum. A procedure has been outlined for the routine culture and identification of L-phase organisms from a clinical specimen.     

Improved Method for Detection of Vibrio parahaemolyticus in Seafood

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

United States National Hospital Survey of anaerobic culture and susceptibility methods, II

To assess the status of clinical anaerobic bacteriology in the United States, we surveyed (by means of a questionnaire) 120 hospitals selected at random with bed capacities of 200-1000, and we received responses from 78 (65%), all of which performed some degree of clinical anaerobic microbiology. Separate anaerobic blood culture bottles were used by 73 labs (94%) (median, 450 specimens/mo): 56% used Bactec 7, 27 or 37; 15% used 'BacT-Alert'; 11% used Columbia broth; 5% used thioglycolate and 'lytic'; 3% each used, Dupont Isolator, Supplemented peptone or other media. Selective media was used for primary anaerobe isolation by 89% labs which included: LKV, 76%; PEA, 53%; BBE, 31%; CNA, 28%; 'CDC', 12%. Sixty labs (78%) stored anaerobes after isolation (median 7 days), most using blood agar plates (31%), chopped meat (26%) or thioglycolate broth (27%) either for further identification (30 out of 78) or susceptibility testing (33 out of 78), if clinically indicated. Only 23% performed routine anaerobic susceptibility testing of clinical isolates. Of the 77% that do not perform susceptibility studies, 59% would not even perform them upon physician request; 30% relied on published surveys; 68% did not publish results of anaerobic susceptibility in annual summaries. When susceptibility testing was performed, the test agents selected were related to availability on a commercial system (21), NCCLS recommendation (20), hospital formulary (15) or hospital committee input (20). Nine of 78 labs (12%) had discussed stopping or decreasing the performance of both anaerobic bacteriology and susceptibility testing. Despite educational and published guidelines, clinical anaerobic bacteriology is not uniformly practiced and could be improved. In addition, an educational effort must be made in order to stress the relevance and increase performance of anaerobic bacteriology.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.