Rapid slide coagglutination test for identifying and typing group B streptococci.

A Cowan I strain of Staphylococcus aureus was labeled with either group B streptococcal grouping or typing antiserum. These antibody-labeled reagent cells (ARC) were used in a slide coagglutination test to identify and type group B streptococci from blood agar plates. All streptococci were also identified by the standard Lancefield capillary precipitin test. In a blind study, all 141 group B streptococci were correctly identified by the coagglutination grouping test. None of the 148 non-group B streptococci caused agglutination of ARC. The coagglutination grouping test required an acid extract prepared from only four colonies and could be completed less than 30 min after colonies were removed from plates. The coagglutination typing test correctly identified 98.6% of the types of the 141 group B streptococcal strains tested. At least 88.6% of these streptococci could be typed directly from blood agar plates within 5 min by the coagglutination typing test. The remaining 11.4% of the group B streptococci were acid extracted (less than a 30-min procedure), and the extract was used for coagglutination typing. Coagglutination typing can be performed with only four colonies. The coagglutination grouping and typing tests are inexpensive, rapid, reliable, and easy to perform.     

Comparison of Meritec-Strep with Streptex for direct colony grouping of beta-hemolytic streptococci from primary isolation and subculture plates.

Meritec-Strep (Meridian Diagnostics, Inc., Cincinnati, Ohio) a coagglutination method, was compared with Streptex (Wellcome Diagnostics, Research Triangle Park, N.C.), a latex agglutination method, for the identification of beta-hemolytic streptococcal groups A, B, C, F, and G by the direct colony method. A total of 124 beta-hemolytic streptococcal isolates were tested, which included 77 from group A, 15 from group B, 10 from group C, 1 from group F, and 21 from group G. All were tested from subculture, and 74 (60%) were also tested from primary isolation plates. For Meritec-Strep, usually one colony was directly applied to the reaction card for testing each grouping reagent, while for Streptex, five colonies were tested after a 1-h extraction process. Complete agreement was obtained for all isolates tested from subculture with the kits. From primary isolation plates, Meritec-Strep correctly identified 97.3% of the isolates compared with 94.6% correctly identified for Streptex. Meritec-Strep produced a false-negative for one group A isolate and positive reactions for group A and F reagents with another group A isolate. A diphtheroid contaminant caused the positive group F reaction. Streptex produced false-negative results for one group A and three group C isolates. Most positive reactions were strong and rapid (less than 30 s) for both kits. The negative test control provided in the individual group A and B kits was nonreactive for all isolates. Meritec-Strep accurately identified isolated colonies of beta-hemolytic streptococci on primary isolation and subculture plates. It provided faster results than Streptex by eliminating the time and manipulation of antigen extraction and needed fewer colonies when individual group A or B reagents were used.     

Two Cases of Chromobacterium violaceum Infection after Injury in a Subtropical Region

Chromobacterium violaceum is a gram-negative rod and is isolated from soil and water in tropical and subtropical regions. The species have pigmented and nonpigmented colony types. Infections caused by nonpigmented strains are rare. We report on two cases of infection caused by both pigmented and nonpigmented strains of C. violaceum. Two 24-year-old Korea Airline stewardesses were admitted to Inha University Hospital, Inchon, South Korea, on 9 August 1997, 3 days after an airplane accident in Guam. Both had multiple lacerations on exposed parts of their bodies. There was swelling, tenderness, and pus discharge. The wounds contained many small fragments of stones and weeds. A pigmented strain was isolated from the left hand and a nonpigmented strain was isolated from the left knee of one patient. For the other patient only a nonpigmented strain was isolated from a foot wound. The nonpigmented colonies from the left-knee and the left-foot wounds did not produce any pigment even after an extended period of incubation. The biochemical characteristics were the same for each strain except for oxidase and indole reactions. The pigmented strain was oxidase negative and indole positive, whereas the nonpigmented strains were oxidase positive and indole negative. The patients were successfully treated by debridement and with appropriate antibiotics.     

Properties and type antigen patterns of group B streptococcal isolates from pigs and nutrias.

All 59 group B streptococcal cultures isolated from pigs and nutrias reacted with group B-specific antiserum and gave a positive CAMP reaction in the zone of staphylococcal beta-lysin. Most of the cultures were pigmented; all cultures hydrolyzed Na hippurate and utilized salicin, maltose, and saccharose but not esculin, mannitol, or inulin. Fifty-three percent of the group B streptococci from pigs and none of those from nutrias were lactose positive. Serotyping revealed that most of the group B streptococci from pigs were of serotype III and most of those from nutrias were of type Ia/c. Protein c was present as c beta antigen. All group B streptococci were susceptible to penicillin and bacitracin (10 U), and most of the porcine cultures were resistant to tetracycline. According to these results, group B streptococci from pigs and nutrias differ from bovine and human group B streptococci and seem to play no role in cross-infections between animals or between animals and humans.     

The isolation and identification of Bacteroides ssp. from the normal human vaginal flora

The occurrence of gram-negative anaerobic bacilli in the normal vaginal flora was studied in 20 normal healthy women attending a family-planning clinic. A swab was taken from the cervix and posterior fornix and Bacteroides spp. were isolated on a selective medium from 13 (65%) subjects. A heavy growth of Bacteroides was obtained from 11 specimens but only a few colonies were isolated from two specimens. Where possible, 10 representative colonies from each subject were studied and 113 isolates were identified by conventional bacteriological tests. Most isolates (78%) belonged to the B. melaninogenicus/oralis group. The commonest species identified in this group were the B. bivius/disiens complex (42%), B. melaninogenicus ss. intermedius (22%) and ss. melaninogenicus (16%). Asaccharolytic strains were isolated in smaller numbers from 54% of subjects, but only five strains of the B. fragilis group were isolated from two subjects; fusobacteria were not detected.     

Carbohydrate fingerprints of streptococcal cells.

The carbohydrates of whole cells of group A, B, C, D, F, and G streptococci were analyzed with a highly sensitive gas chromatographic procedure. Characteristic chromatographic fingerprints were obtained for each group of streptococci grown in broth cultures or as single colonies on a blood agar plate. Rhamnose, glucose, and N-acetylglucosamine were major components of all the groups. Groups A, C, and F contained very little galactose, and groups A and B showed almost a complete absence of N-acetylgalactosamine. Chromatograms obtained for group B streptococci were distinguished by the presence of 1,4-anhydroglucitol.     

Evaluation of the Spot-CAMP test for the rapid presumptive identification of group B streptococci

A rapid Spot-CAMP test was evaluated for its ability to accurately identify colonies of Streptococcus agalactiae (Lancefield Group B) growing on primary sheep blood agar plates. The test uses a beta-lysin-containing filtrate, which is prepared from a broth culture of Staphylococcus aureus. A drop of beta-lysin filtrate is applied adjacent to a suspected group B Streptococcus (GBS) colony and the plate is incubated and then examined for a zone of synergistic hemolysis. The Spot-CAMP test demonstrated 100% correlation with both a Standard CAMP procedure and Lancefield serogrouping. The rapid Spot-CAMP test was easy to perform and inexpensive, and could presumptively identify within 30 minutes colonies of GBS growing on primary isolation plates.     

Feedback suppression of B cell colony formation in healthy individuals

Proliferation and differentiation of B cells has been extensively studied and the study of feedback suppression of B cell proliferation has been limited to humoral factors. However, very little is known about feedback suppression of B cell proliferation by cellular influences. We have previously reported on the role of T cells and their subsets on B cell proliferation in that we did not observe suppression of B cell colony growth by T cells. We now report on the role of B cells in limiting B cell proliferation. B cell colonies were grown in methyl cellulose for either 3 days or 5-6 days utilizing 2 x 10(5) T cells irradiated with 9,000 rads, and 2 x 10(5) B cells. The B cells were then obtained from these colonies and increasing numbers of cells were added to fresh autologous B cells that were further cultured for 5 days to form new B cell colonies. At the end of this period, B cell colony numbers were determined. Our data show that addition of CD19- and CD20- positive B cells recovered from mature colonies after 5 days to fresh B cells suppressed further B cell colony growth in all cases tested, whereas addition of CD19-positive B cells recovered from immature colonies after 3 days of culture did not suppress further B cell colony growth. Elimination of CD 19- or CD20-positive cells with monoclonal antibody to CD19 and complement or by the technique of panning enhanced colony growth. Supernatants obtained from B cell colonies did not suppress B cell colony formation. Our data suggest that there is feedback suppression of normal progenitor B cell proliferation by constituent B cells and that this effect develops during maturation of colonies during the growth phase.     

Demonstration of the capsular antigens of bovine group B streptococci by the serum-soft agar method.

The elaboration of type-specific capsular antigens by group B streptococci can be demonstrated by the serum-soft agar technique. Group B streptococci isolated from bovine mastitis, namely, strains 9F, 14Mi, 8Mo, 44B, and 4S, were shown to form diffuse and compact types of colony morphology in serum-soft agar. Immunochemical and chemical analyses of antigens isolated from diffuse and compact colonies of strain 9F indicated that the diffuse-type growth of this strain was due to the elaboration of a galactose-rich surface antigen, whereas the compact 9F strain was devoid of this antigen. Specific 9F antiserum was effective in converting the diffuse 9F colonies of the compact type, indicating the presence of capsular material. Preliminary evidence suggests that the serum-soft agar technique could also be used to determine the antigenic diversity of the surface antigens of group B streptococci, thus providing an effective means of typing those organisms.     

成年大鼠眼睫状体缘色素上皮组织的神经前体细胞的培养和鉴定

目的　探讨成年大鼠眼睫状体缘色素上皮产生神经前体细胞的潜力及其生物学特征。方法　取成年SD大鼠睫状体缘处的色素上皮组织块 ,置于含bFGF和B2 7的DMEM/F12 无血清培养液中进行神经前体细胞培养 ,免疫组化反应染色鉴定细胞的表型。结果　培养 5～ 8d ,组织块长出由众多无色素和色素细胞构成的细胞集落 ,集落的大多数细胞处于增殖状态(BrdU反应阳性 ) ,并表达神经前体细胞的标志物 (nestin)。撤掉bFGF和B2 7,加入胎牛血清培养 3～ 5d ,集落的细胞发生分化 ,分别表达神经元和星形胶质细胞的标志物 (NSE、GFAP) ,但不表达少突胶质细胞的标志物 (O4 )。结论　由成年大鼠睫状体缘色素上皮长出的细胞集落不仅含有增殖能力和多分化潜能的神经前体细胞 ,而且尚含有色素细胞 ,该部位可能是视网膜神经感觉层和色素上皮共同保守的细胞生发带     

地中海贫血患儿血清特异性PRA影响脐血造血干细胞增殖、分化能力的观察

目的： 探讨反复输血地中海贫血患儿血清中特异性群体反应性抗体（PRA）对脐血造血干/祖细胞增殖、分化能力的影响。 方法： 采用1×105脐血单个核细胞（MNC）与实验血清（健康儿童AB血清50 μL、PRA血清 0 μL、50 μL、100 μL）、补体联合孵育后半固体集落培养，倒置显微镜下观察并计数第7 d、第14 d总集落数和各种集落数。结果： 脐血造血干/祖细胞受PRA血清作用后，在半固体集落培养第7 d，总集落数、CFU-GM分别为A组88.20±9.41、79.00±11.39和B组88.60±9.12、79.20±10.44，显著高于C组20.60±7.39、15.20±4.66和D组4.00±2.05、1.40±0.51，P<0.01；其余各组的各种集落数两两比较，差异无显著（P>0.05）。A组与E组比较：两组的各种集落数比较均无显著差异（P>0.05）。在半固体集落培养第14 d，总集落数、CFU-GM分别为A组216.00±31.10、117.40±24.80和B组213.20±31.06、116.00±19.75，显著高于C组97.80±14.43、32.80±8.10和D组31.40±13.41、8.40±4.30，P<0.01;第14 d CFU-GEMM分别为A组45.60±8.51和B组42.60±7.03，显著高于C组20.80±6.96和D组7.80±6.06，P<0.05；第14 d BFU-MK分别为A组12.80±4.42、B组11.00±2.74，显著高于D组1.00±0.55，P<0.05；B组第 14 d CFU-E17.20±4.03显著高于D组5.60±2.87，P<0.05。A组与E组比较：两组的各种集落数比较差异均无显著（P>0.05）。PRA血清量与各种集落数目之间的Kendall相关分析：PRA血清量与第7 d的总集落数及CFU-GM、第14 d的总集落数、CFU-GM、CFU-GEMM、BFU-E、BFU-MK呈负相关（tau-b分别为-0.793、-0.849、-0.808、-0.804、-0.645、-0.674、-0.624，P<0.01；与第14 d CFU-MK呈负相关（tau-b为 -0.466，P<0.05）。结论： 特异性PRA对脐血造血干/祖细胞的增殖、分化能力具有抑制作用；在一定浓度下其抑制作用与PRA剂量有相关性：PRA剂量越大，抑制作用越强。     

Differences in blood group B-specific mucinase activity between virulent and avirulent Shigella flexneri 2a strains

Based on our previous findings we postulate that the production of blood group B-degrading mucinase by Shigella flexneri 2a is related to virulence. The virulent S. flexneri 2a strain M4243 produced a blood group B mucinase which decreased the blood group B reactivity of germ-free mouse mucins by a factor of 16 and the B reactivity of human saliva by a factor of 32. Avirulent S. flexneri 2a B-1, serologically similar, but not genetically identical to the M4243 strain, failed to degrade the blood group reactivity. The mucin-degrading ability of S. flexneri 2a M4243 harboring a large virulence-conferring 140 MDa plasmid was then compared with a genetically similar large plasmid-free avirulent S. flexneri 2a M4243A1. Virulent S. flexneri M4243 grew in human salivary mucins while the genetically identical avirulent M4243A1 did not. Supernatant of virulent M4243 culture decreased the blood B reactivity of salivary mucins by a factor of 32 while the avirulent M4243a1 had no effect. Eleven of 12 colonies of the transconjugant hybrid Escherichia coli K12 (7300-1-5) containing the shigella PWR 110 plasmid and chromosomal markers decreased the blood group B reactivity by a factor of 4-32, and two of four colonies of the E. coli strain 7262 containing only the plasmid reduced the B reactivity by a factor of 4-16. These findings suggest that blood group B-specific mucinase production may be related to S. flexneri virulence.     

Neural differentiation in the OTT-6050 mouse teratoma: Enzymatic and immunofluorescence characterization of a tumor fraction showing melanogenesis in neuroepithelial cells

Summary A pigmented tumor fraction, designated IB-9, obtained following cellular dissociation and elutriation procedures applied to the solid transplants of the OTT-6050 mouse teratoma cell line, was characterized enzymatically and by immunofluorescence for the presence of tyrosinase and tyrosine hydroxylase (TH). Enzymatic assays of the pigmented tumors were compared with those obtained on non-pigmented teratoma-derived tumors, on pigmented tumors obtained from the mouse melanoma B16 line as a control for tyrosinase activity, and on whole brains of adult 129/J mice as a control for TH activity.All the teratoma-derived tumors, including the IB-9 fraction, showed a predominance of TH over tyrosinase activity. The levels of TH activity appeared independent of the presence or the extent of melanin pigment. All pigmented teratoma-derived tumors showed low levels of tyrosinase activity.On the basis of the enzymatic assays, the IB-9 tumors were divided into two groups: group I, which showed low enzyme activity, almost certainly entirely tyrosinase; and group II, in which the enzyme activity appeared largely due to TH, with presumably a very low background of tyrosinase activity. Immunofluorescence demonstrated the localization of TH activity to non-pigmented cells of the IB-9 fraction, whereas the pigmented cells showed absence of TH activity.These findings, taken in conjunction with the presence by electron microscopy of premelanosomes and melanosomes, indicate that pigment formation associated with melanosomal differentiation in the neural cells of IB-9 with the histologic patterns of primitive CNS neuroepithelium results from tyrosinase activity only and is therefore unrelated to the metabolic pathways involved in catecholamine synthesis and degradation. It is suggested that, at this stage of differentiation and in this system, the expression of catecholamine synthesis via tyrosine hydroxylase in neuroepithelial cells, and of melanin pigment via tyrosinase, are probably mutually exclusive.Supported by Research Grant CA 11689 of the National Cancer Institute; MH 23861 of the National Institute of Mental Health; and Neuropathology Training Grant NS 5 T32 NS 7111 of the National Institute of Neurological and Communicative Diseases and Stroke, USPHS     

ANTIGEN BINDING TO LYMPHOID CELLS OF UNIMMUNIZED MICE

In order to determine the cell type responsible for the antigen-binding reaction in the bone marrow and spleen of mice, cells derived from pure in vitro derived colonies of neutrophils, eosinophils, macrophage-megakaryocytes and B lymphocytes were tested for their ability to bind fluorescent protein antigens. Only B lymphocytes bound antigen. An unexpectedly high percentage of bone marrow B lymphocytes (20%) bound a given antigen. This frequency was considerably higher than that found for spleen cells. As might be expected from such high binding frequencies, some cells bound two fluorchromated antigens when these are added together. As a direct test of the clonality of antigen binding to bone marrow B lymphocytes, whole colonies of B cells were tested for antigen binding of two non-cross-reacting protein antigens. The frequency of antigen-binding clones, including double antigen-binding clones, reflects exactly the frequencies observed for dispersed colony B cells and for in vivo derived Ig-bearing bone marrow B cells. The frequency of double antigen-binding colonies was equal to the product of the frequencies of the colonies binding each of the two antigens alone. No ‘mixed’ colonies containing single binding cells for each antigen were found. Thus, the ability to bind any two given antigens is a clonally distributed property of the bone marrow B lymphocyte population. Heterogenous receptors for multiple antigen binding on each cell are either randomly distributed among the B cell population, or homogenous antigen-binding receptors on each cell have a random chance of cross-reaction with the two antigens tested.     

Characterisation of Exiguobacterium aurantiacum isolates from blood cultures of six patients

Exiguobacterium spp. are alkaliphilic, halotolerant, non-spore-forming Gram-positive bacilli, hitherto uncharacterised from human infections. Six isolates of Exiguobacterium aurantiacum were obtained from patients with bacteraemia, three of whom had myeloma. All isolates formed orange–yellow pigmented colonies on blood agar, were catalase- and DNase-positive, and grew on nutrient agar at pH 10 and in the presence of NaCl 6% w/v. The six isolates were susceptible to all antimicrobial agents tested and were uniform in their fatty acid and mass spectrum profiles.     

Rapid detection of Bordetella pertussis by a monoclonal antibody-based colony blot assay.

Monoclonal antibodies to Bordetella pertussis filamentous hemagglutinin (FHA) and lipopolysaccharide (LPS) were used in a colony blot enzyme-linked immunosorbent assay designed for rapid detection of B. pertussis. Bacterial colonies from Bordet-Gengou agar plates were blotted onto nitrocellulose filter disks, lysed by immersion in chloroform, and reacted with monoclonal antibodies. Following reaction with peroxidase-conjugated rabbit anti-mouse immunoglobulin antisera and 4-chloro-1-naphthol, blue dots representing single colonies appeared on the filters. Blotting of single B. pertussis colonies could be performed after incubation for 40 h, i.e., before the colonies were visible by eye on the agar surface. Ten of ten B. pertussis strains showed positive blotting reactions with antibodies specific for B. pertussis FHA and LPS. Fourteen of fourteen B. parapertussis strains reacted with two of the FHA-specific antibodies but not with two of the LPS-specific antibodies. Strains of B. bronchiseptica showed a variable reaction pattern. No cross-reactions were observed with strains of Streptococcus mitis, S. pyogenes, S. pneumoniae, Staphylococcus aureus, Branhamella catarrhalis, or Klebsiella pneumoniae. This assay may be useful for identification of B. pertussis and B. parapertussis in suspected cases of whooping cough.     

T lymphocyte colonies are the progeny of single cells.

T lymphocyte colonies, arising from phytohemagglutinin (PHA) stimulated mononuclear cells cultured in a semi-solid agar matrix, could be the progeny of single cells (monoclonal) or of multiple cells (polyclonal). We have conducted several studies to determine if these colonies are monoclonal or polyclonal in origin. Normal human peripheral blood mononuclear cells from male-female, HLA-A and B disparate donor pairs were incubated for 18 h in RPMI 1640 containing PHA and fetal calf serum (FCS) and then cultured in a two-layer semi-solid agar system. After 5 days of incubation, the clonality of the colonies was assessed by in situ Y chromatin analysis, and by analysis of HLA-A and B locus antigens. Overlayers were stained with quinicrine dihydrochloride and the number of cells in the T cell colonies with Y chromatin enumerated using fluorescence microscopy. In other studies, colonies were picked from the agar with a capillary pipette and expanded in culture media. After 17 days of culture, cells were harvested and HLA-A and B phenotypes were determined. The results indicate that 87% of the T cell colonies had cells of either male or female origin. In addition, 90% of the colonies possessed HLA-phenotypes of only one donor. We conclude that Y chromatin and HLA analysis of individual colonies from cocultures suggest the monoclonality of T lymphocyte colonies.     

Induction of B lymphocyte colony growth in vitro by thymus-derived stimulating factor.

Murine lymphoid cells which were stimulated in liquid culture containing thymus culture fluid (Thy-CF) and seeded in a soft agar culture system, proliferated and developed into B cell colonies. Two types of colonies were formed: large colonies within the upper layer and small flat colonies on the surface of the upper layer. Thy-CF prepared from cells of normal hydrocortisone-treated mice had a higher cloning potential than Thy-CF prepared from untreated mice. At concentrations of Thy-CF in culture medium greater than 35%, Thy-CF prepared from normal mice had an inhibitory effect on colony formation. Cells of nude mice were also able to form B cell colonies if thymocytes of normal mice were mixed with lymphoid cells in the culture medium. Thymocytes elaborate a B lymphocyte colony-stimulating factor which, with the help of T cells, triggers a B cell population into colony formation and immunoglobulin production.     

Recognition of group B streptococci in dip-slide cultures of urine.

One hundred strains of group B streptococci isolated from human infections were tested for growth on dip-slides available for the culture of urine. All grew on CLED agar, and none grew on MacConkey agar. The colonies were barely or not at all visible to the naked eye after overnight incubation (diameter, around 0.1 mm). The colony size increased eith prolonged incubation, but not if the inoculum density exceeded 10(6)/ml. Differences were found between lots of dip-slides. Poor growth on dip-slides may explain why group B streptococci have received little attention as pathogens of the urinary tract. The dip-slide screening personnel of one laboratory were informed of the experimental findings, and they started the practice of frequent subculture and prolonged incubation. The proportion of group B streptococci in significant bacteriuria increased from 0 to about 2% of positive cultures, whereas there was no conmitant increase of group B streptococci in dip-slides screened in several other laboratories serving as controls.     

Human B cell colony growth from pre-B cells in vitro.

We describe a simple one-step technique for the growth of human B cell colonies in semi-solid agar in vitro. This method used conditioned medium from the human plasmacytoma cell line LICR-LON-H My 2 as a source of stimulating activity. A linear relationship exists between the number of B cells seeded and the number of colonies formed (r = 0.95). Most colony forming cells, approximately 1 in 500 of B cells seeded, lack surface immunoglobulin, possess Fc receptors and mark with the Leu 12 monoclonal antibody. Cells within developing colonies are found to have cytoplasmic IgM, IgA and IgG depending on the length of time in culture.