Comparative growth of Bacteroides species in various anaerobic culture media

The growth of five species of Bacteroides in four anaerobic culture media was continuously monitored turbidimetrically. Interspecies differences were observed in the growth of Bacteroides spp. in the various media, but growth in Brain Heart Infusion broth supplemented with yeast extract, haemin and menadione, was consistently better than in Wilkins-Chalgren, Thioglycollate or Schaedler broths. Microscopy of cultures grown overnight in Brain Heart Infusion broth showed that the bacteria exhibited normal morphology but most species grown in the other media displayed filamentation or chain formation. Four of the five species grown in Schaedler broth also exhibited spheroplast formation. This morphological change occurred in the stationary phase of growth, was reduced by inclusion of NaCl in the medium and was abolished in Schaedler broth prepared at double the recommended strength.     

Nitrocellulose filter blots for species identification of Mobiluncus curtisii and Mobiluncus mulieris

Seventy strains of Mobiluncus, motile curved anaerobic bacteria associated with bacterial vaginosis, were correctly identified to species level by using bacteria fixed to nitrocellulose and hybridized with 32P-labeled DNA.     

Evaluation of liquid media for growth of Helicobacter pylori.

Helicobacter pylori has routinely been isolated and grown on solid media. Recently, we have succeeded in obtaining growth of this organism in several liquid media in large volumes, including tryptic soy broth, Mueller-Hinton broth, brucella broth, brain heart infusion broth, and Columbia broth. Growth was tested in the media with and without supplementation. Growth was obtained after incubation under microaerobic conditions and with CO2 enrichment. Growth in a stationary system versus that in an agitated system was evaluated. Results from these experiments show that H. pylori can be grown in any of the liquid media tested except buffered yeast extract-alpha-ketoglutarate if serum is added. No growth was observed on buffered yeast extract-alpha-ketoglutarate even with serum and other supplementation. Growth of H. pylori in most of the liquid media with supplements was improved if the culture was incubated in a CO2 atmosphere. The findings reported here may be useful in clinical, industrial, and research laboratories that require harvests of large quantities of H. pylori cells.     

Isolation of Mobiluncus species from clinical specimens by using cold enrichment and selective media.

New and selective Rlk and SA media, combined with cold enrichment at 4 to 5 degrees C, allowed isolation of Mobiluncus species from patients with bacterial vaginosis at higher rates than with conventional cultivation methods. Rlk medium consists of Columbia CNA agar supplemented with peptone, yeast extract, 5% laked rabbit or sheep blood, nalidixic acid, and tinidazole. SA medium consists of Columbia CNA agar supplemented with 2% rabbit serum, 1.6% laked rabbit or sheep blood, nalidixic acid, and tinidazole. Use of these selective media plus the cold enrichment technique permitted Mobiluncus species to propagate at rates similar to those of other anaerobic members of the vaginal flora.     

Production and characterization of monoclonal antibodies to Mobiluncus species.

Members of the genus Mobiluncus are anaerobic motile curved rods which are associated with bacterial vaginosis (BV). Murine monoclonal antibodies (MAbs) to the ATCC type strains of M. curtisii subsp. curtisii, M. curtisii subsp. holmesii, and M. mulieris were produced and characterized by enzyme-linked immunosorbent assay and indirect immunofluorescence assay. Four MAbs were subspecies specific and reacted with M. curtisii subsp. curtisii but not with M. curtisii subsp. holmesii; four were specific for M. mulieris. The remaining antibodies demonstrated some cross-reactivity: three were species specific and reacted with both subspecies of M. curtisii, and one defined an epitope shared by M. curtisii subsp. holmesii and M. mulieris but not by M. curtisii subsp. curtisii. None of the MAbs reacted with a panel of other bacteria commonly present in the vaginas of normal women or women with BV. Examination of the molecular specificities of the antibodies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed four antibodies which were specific for an 82,000-dalton molecule of M. curtisii subsp. curtisii and five antibodies which bound a major band of M. mulieris at 93,000 daltons. Selected MAbs reacted in the indirect immunofluorescence assay with 24 of 25 Mobiluncus spp. clinical isolates from local women with BV and could be used for direct detection of Mobiluncus spp. in vaginal fluid from a patient with BV.     

Application of impediometry to rapid assessment of liquid culture media

The impedance method provides as unique opportunity to determine microbial activity and kinetics. Since the metabolic processes depend on the nature and quality of the culture medium, impediometry allows the assessment of liquid culture media. Impedance microbiology represents an approach to quantitative microbiology. We investigated the influence of pH, composition and variation of the amounts of industrially made dry media, overheating during the dissolving or sterilisation processes, and qualitative differences between batches of the same culture medium. Using glucose broth as an example, we showed that impediometry allows quantitative, microbial assessment of culture media. Inaccurate preparation of the culture medium could be detected quickly by the use of impediometry. The method is very simple to perform, requires no sample preparation, allows rapid assessment of liquid culture media, and interprets results automatically with the aid of a microcomputer.     

Mobiluncus species in bacterial vaginosis: aspects of pathogenesis

Mobiluncus is an anaerobic motile rod associated with bacterial vaginosis. In this work, luminol-enhanced chemiluminescence was used to study the ability of Mobiluncus spp. from the vaginas of women with bacterial vaginosis to induce, in the presence of normal adult serum, oxidative metabolism of polymorphonuclear leukocytes, which is an indirect measure of phagocytic activity. M. curtisii induced a significantly (p less than 0.05) lower response than M. mulieris, which indicates that M. curtisii escapes phagocytosis more easily. Indirect immunofluorescence assays showed IgG antibodies to M. curtisii at significantly (p less than 0.01) higher titres than to M. mulieris in women with bacterial vaginosis. The titres were higher in patients with bacterial vaginosis than in women without vaginosis and healthy men. No antibodies to Mobiluncus spp. of secretory IgA type were found in vaginal washings. These results indicate that M. curtisii is a more virulent species than M. mulieris, and agree with reports of M. curtisii found in postoperative and extragenital infections.     

Novel chemically modified liquid medium that will support the growth of seven bartonella species

Bacteria of the genus Bartonella, a member of the Alphaproteobacteria, are fastidious, gram-negative, aerobic bacilli that comprise numerous species, subspecies, and subtypes. In human and veterinary medicine, species isolation remains a vital component of the diagnostic and therapeutic management of Bartonella infection. We describe a novel, chemically modified, insect-based liquid culture medium that supports the growth of at least seven Bartonella species. This medium will also support cocultures consisting of different Bartonella species, and it facilitated the primary isolation of Bartonella henselae from blood and aqueous fluid of naturally infected cats. This liquid growth medium may provide an advantage over conventional direct blood agar plating for the diagnostic confirmation of bartonellosis.     

Demonstration of heterogeneity among the antigenic proteins of Mobiluncus species.

The protein and antigenic profiles of the American Type Culture Collection type strains of Mobiluncus species and those of 114 clinical isolates were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and immunoblotting with homologous polyvalent antisera. The majority of isolates (82%) possessed characteristic protein profiles and could be identified to the species level by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The major protein bands were also antigenic, and some antigenic cross-reactivity was noted between the two Mobiluncus species. All of the isolates were examined for reactivity with a panel of 12 monoclonal antibodies previously prepared against the type strains. While 56 of 60 clinical isolates of Mobiluncus curtisii (93%) reacted with one or more of the monoclonal antibodies, only 23 of 54 clinical isolates which were identified as Mobiluncus mulieris by biochemical methods (48%) reacted with one or more of the monoclonal antibodies. One of the 4 M. curtisii isolates (25%) and 11 of the 31 M. mulieris isolates (35%) which did not react with the monoclonal antibodies also had atypical protein profiles. These results demonstrate a high degree of heterogeneity in the protein and antigenic profiles of Mobiluncus isolates and suggest that further taxonomic division may be appropriate.     

Evaluation of ten anaerobic blood culture media.

Selection of an anaerobic blood culture based upon clinical findings that have compared the isolation rates of bacteremic agents from different blood culture media. No agreement has been reached as to which of the commercially available blood culture media is optimal for detection of bacteremia. The purpose of this study was to determine the rates of recovery of anaerobic microorganisms from various anaerobic blood culture media. The blood culture media were inoculated with a small inoculum of microorganisms in the presence or absence of an erythrocyte-serum mixture. The results demonstrated that the type of medium and the erythrocyte-serum mixture influenced the ability of blood culture media to support the growth of microorganisms. The majority of the media failed to support the growth of 87% or more of the microorganisms within four days after inoculation. Pre-reduced brain-heart infusion broth supported the growth of a larger proportion of microorganisms than the other types of blood culture media.     

Evaluation of vented blood culture media with sorbitol.

Two consecutive studies comparing the recovery of microorganisms from transiently vented blood cultures in tryptic soy or brain heart infusion broth with added sorbitol and that from tryptic soy broth without sorbitol failed to demonstrate any significant advantage of hypertonic media. A significantly greater number of organisms was isolated in both studies from the medium without sorbitol.     

Comparison of liquid growth media for Legionella pneumophila.

Ten liquid media were compared under standard conditions for their ability to support the growth of Legionella pneumophila. Modified gonococcal-ferric cysteine broth (without sodium chloride) supplemented with 1% yeast extract yielded the best overall growth of the one strain of L. pneumophila examined. Growth rates were independent of pH changes which occurred during incubation. The growth rates of 10 different strains of L.pneumophila were compared in this medium. There appeared to be little difference in the growth rates of strains passaged frequently or infrequently, or between environmental and clinical isolates. Moderate aeration resulted in a faster growth rate and in approximately a 1 log10 higher final cell concentration as compared to a static broth culture. These experiments demonstrate that there are moderate to marked differences among the various media described in the literature and that no liquid medium yet developed supports rapid growth of L. pneumophila incubated without shaking.     

Factors affecting growth of Legionella pneumophila in liquid media

The growth in liquid media of Legionella pneumophila serogroups 1-6 was monitored turbidimetrically and factors affecting growth rate were studied. The presence of inhibitors, use of detoxifying agents and the method of broth preparation each had significant effects on cultivation. Cysteine was essential for growth; the optimal concentration was 100 micrograms/ml, but supplemental iron had no demonstrable effect.     

Evaluation of liquid and lyophilized preservatives for urine culture

Culture results of urine specimens transported conventionally (sterile cup) and in a commercial liquid or an investigational lyophilized preservative were compared in a hospital that experiences substantial delays in specimen transport to the laboratory (greater than 40% of specimens received after a delay of greater than or equal to 2 h). At the time of initial plating in the laboratory, 106 of 111 (95.5%) specimens that were positive (greater than or equal to 10(5) CFU of a single organism per ml in pure culture) after conventional transport were also positive in liquid preservative. After a 24-h holding period (cup refrigerated, preserved urine at room temperature), agreement was 91.4% (96 of 105). At the time of initial plating, agreement between results obtained by the conventional method and those obtained by using lyophilized preservative was 96.9% (63 of 65); after 24 h, agreement was 92.4% (61 of 67). Complete inhibition of growth of three Klebsiella pneumoniae isolates was observed in liquid preservative; however, urine processed in the lyophilized preservative did not show inhibition. The proportion of urine cultures showing no change in quantitative growth between the time of initial plating and repeat plating at 24 h was virtually identical for all three processing methods (83.6 +/- 0.9%). After the 24-h holding period, specimens processed in lyophilized preservative were less likely to show diminished quantitative growth than were specimens processed conventionally or in liquid preservative but were more likely to show an increase in growth of greater than or equal to 1 log. Nonetheless, the apparent lack of toxicity of lyophilized preservative may make it preferable to the currently available liquid preservative.     

Performance of six culture media for isolation ofSalmonella species from stool samples

The relative performance of five plating media [Rambach agar; salmonella-shigella (SS) agar, novobiocin-brilliant green-glycerol-lactose (NBGL), modified semisolid Rappaport-Vassiliadis medium (MSRV), andSalmonella Detection and Identification-2 (SM2)] and selenite broth (SB) subcultured in SS agar in the recovery ofSalmonella spp. from 500 human stool specimens was evaluated. On Rambach agar and SS agar, the C₈-esterase test was also used for selection of suspicious colonies. Eighty-one samples were positive for salmonellae on at least one of the six media. Sensitivities and specificities of MSRV, SB, NBGL, SS, Rambach agar, and SM2 were 95.1 and 98.1%, 87.6 and 99.8%, 79 and 91.9%, 69.1 and 99.3%, 56.8 and 96.9%, and 54.3 and 92.4%, respectively. There were statistically significant differences between MSRV and NBGL, Rambach agar, SS agar, and SM2 (p < 0.005), between SB and SS agar, Rambach agar, and SM2 (p < 0.05), and between NBGL and SM2 and Rambach agar (p < 0.005). The greatest number of isolates was recovered with MSRV, whose performance surpassed that of enrichment in selenite broth, probably because the subcultures were not repeated on MSRV. This hypothesis is now under investigation. The specificity of each of the five solid media was greater than 90%.     

Detection and identification of Mobiluncus species by direct filter hybridization with an oligonucleotide probe complementary to rRNA.

A hybridization assay for direct detection and identification of Mobiluncus species has been developed and tested. A [32P]-labelled synthetic oligonucleotide probe, complementary to a nucleotide sequence in the variable region V8 of Mobiluncus 16S ribosomal RNA, was utilized. One of the advantages of using rRNA as target molecule for the hybridization assays is the copy number of rRNA, which can be as high as 10(4), and that additionally three to six sites on the minus strand of the DNA gene can be utilized. This probe was found to be sensitive and to react with 62 of 68 tested typical or atypical Mobiluncus isolates. It was also specific, and was shown not to react with 96 tested unrelated bacterial species and isolates, including taxonomically closely related species like Actinomyces or Bifidobacterium spp., or with bacteria isolated from the vagina of both healthy persons with an undisturbed flora, as well as from patients suffering from the bacterial vaginosis syndrome (BV).     

Evaluation of agar media for growth of Campylobacter pylori

The supporting ability for the growth of Campylobacter pylori was tested on various agar plates. C. pylori grew on media supplemented with blood, serum or egg-yolk, and it showed good growth on Mueller Hinton agar with horse blood, and on brain-heart infusion agar with yeast extract and horse blood. The organism grew moderately on Mueller Hinton agar, but could not grow on the other unsupplemented media. Mueller Hinton medium may be of use as a proper base of blood agar for growth of C. pylori. Egg-yolk may be substituted as a low-priced enrichment material for blood.