Evaluation of Four Blood Culture Systems Using Parallel Culture Methods

Commercially prepared Vacutainer thioglycolate broth with SPS (TBSPS), the B-D Vacutainer culture tube with SPS (VCT), the Pfizer E-Vac hypertonic brucella broth with SPS (BBSPS), and our own Trypticase-soy hypertonic broth with SPS (TSBH) were studied by parallel culture methods for their ability to allow the survival of a wide spectrum of organisms, their ability to allow recovery of organisms, and the time required to obtain a recovery from clinical blood cultures. Organisms considered to be clinically significant were recovered from 176 blood cultures. The TSBH system produced all 176 recoveries, the BBSPS system allowed for 170 recoveries, the TBSPS recovered 131, and the VCT accounted for 106 recoveries. The time required to recover any given organism from both the BBSPS and the TSBH systems did not exceed 48 h, whereas 86 of the 131 recoveries from the TBSPS system required more than 72 h and 74 of the 106 VCT recoveries also required more than 72 h. A wide spectrum of organisms were recovered from both the BBSPS and the TSBH systems. The TBSPS system failed to allow the recovery of many streptococci and Pseudomonas spp. and the VCT system failed to recover many organisms.     

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.     

Rapid Diagnosis of Bacteremia

Early appropriate treatment of bacteremia is important in minimizing morbidity and mortality. Standard blood culture methods are not optimal since several days are often required for recovery and identification of organisms which may be present in the blood. The use of a membrane filter technique allows one to grow any organisms present in blood much more rapidly than by broth or pour plate culture. Furthermore, growth is in the form of typical colonies on the surface of solid media, and a series of rapid diagnostic tests may be used to provide speedy identification. Use of membrane filters also facilitates removal by washing of normal antibacterial factors and antimicrobial drugs which may be present in blood. Although the filter technique yielded the most rapid growth, broth culture and whole blood pour plates yielded more positive cultures and use of all three systems was necessary for maximal recovery of organisms in blood cultures. Data on quantitative aspects of bacteremia in the antimicrobial era are also presented. The number of low level bacteremias (10 colonies/ml or less) is surprisingly high. This is particularly true for gram-negative bacilli; antimicrobial therapy at the time of culture undoubtedly influenced these results greatly. Finally, suggestions are given for a much simpler and more efficient membrance filter blood culture technique.     

Rapid Diagnosis of Bacteremia

Early appropriate treatment of bacteremia is important in minimizing morbidity and mortality. Standard blood culture methods are not optimal since several days are often required for recovery and identification of organisms which may be present in the blood. The use of a membrane filter technique allows one to grow any organisms present in blood much more rapidly than by broth or pour plate culture. Furthermore, growth is in the form of typical colonies on the surface of solid media, and a series of rapid diagnostic tests may be used to provide speedy identification. Use of membrane filters also facilitates removal by washing of normal antibacterial factors and antimicrobial drugs which may be present in blood. Although the filter technique yielded the most rapid growth, broth culture and whole blood pour plates yielded more positive cultures and use of all three systems was necessary for maximal recovery of organisms in blood cultures. Data on quantitative aspects of bacteremia in the antimicrobial era are also presented. The number of low level bacteremias (10 colonies/ml or less) is surprisingly high. This is particularly true for gram-negative bacilli; antimicrobial therapy at the time of culture undoubtedly influenced these results greatly. Finally, suggestions are given for a much simpler and more efficient membrance filter blood culture technique.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

Detection of Bacteremia in Children with Sodium Polyanethol Sulfonate: a Prospective Clinical Study

A prospective study was made of 1,000 consecutive duplicate blood cultures obtained from sick children to evaluate the usefulness of sodium polyanethol sulfonate (SPS). With the small volumes of blood (1 to 5 ml) usually obtained for blood cultures in children, SPS did not increase the frequency of recovery of organisms judged to be associated with clinical infections, with the possible exception of Diplococcus pneumoniae. However, the use of SPS was associated with an increased frequency of recovery of organisms judged to be contaminants, such as Staphylococcus epidermidis and propionibacteria, possibly because SPS enhanced the recovery of a very small inoculum of skin bacteria.     

正常人和肝病患者血中“致密体”及其与纳米细菌的关系

目的研究正常和肝病患者血液中圆球体样微生物的生物学特性,初步确定纳米细菌与该种可滤过物的关系。方法将正常人和肝病患者培养阴性的血培养物进行细菌L型、穿菌和厌氧菌培养。同时取沉淀用透射电镜观察。L型培养液用0．45μm和0．22μm滤器过滤,接种RPMI 1640培养基,细胞培养条件下培养45d,用鼠抗纳米细菌单克隆抗体8D10免疫组化和钙盐染色进行纳米细菌鉴定。血浆纳米细菌培养阳性者12000×g离心后,进行普通和L型细菌培养。结果36／39患者和60／60健康对照血培养液中呈现类似于L型的巨型体、圆球体、原生小体的不明微生物。电镜观察圆球体内为电子致密样物质,周围未见细胞壁结构。电镜和光镜下可见其粘附在红细胞上或存在于红细胞内。滤过后培养物钙盐染色有5／39阳性,但纳米细菌免疫组化染色均阴性。纳米细菌阳性的培养物转种,未见一般细菌、细菌L型和上述血中“致密体”。结论血培养中致密体样微生物与纳米细菌无关,可能在维持机体正常免疫功能上具有一定意义。     

Comparison of a Blood Culture System Containing Liquoid and Sucrose with Systems Containing Either Reagent Alone

By utilizing parallel culture methods on multiple blood specimens, three blood culture systems were evaluated for their ability to allow rapid recovery of organisms. The same base medium was used for all three systems. System or flask 1 contained base medium with Liquoid added, flask 2 contained base medium with sucrose added, and flask 3 contained both Liquoid and sucrose. During the study period, there were 259 patients considered to have positive blood cultures. Organism recovery was accomplished from flask 1 in 214 of these patients, from flask 2 in 180 cases, and from flask 3 in 257 instances. The use of anticoagulants and osmotic stabilizers in blood culture systems is discussed in detail.     

Experimental studies on bacterial product cantastim derived from Pseudomonas aeruginosa. VII. Activation of immune cells of healthy controls and cancer patients

CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa with beneficial effects related to enhancing the immune responses in conditions such as chronic viral and bacterial infections, immunodeficiencies and cancer immunosuppression. The aim of this study was to determine the capacity of this biological product to stimulate in vitro human leukocytes in whole blood. Blood samples from healthy donors and cancer patients were incubated with CS for 24 h and leukocytes were assessed for induction of inflammatory cytokines (TNF-alpha and IL-1beta) by ELISA and expression of early activation marker CD69 by flow-cytometry. For both groups of investigated subjects, the levels of TNF-alpha and IL-1beta in the supernatants of whole blood culture stimulated with CS were significantly higher than in unstimulated cultures, although lower than in LPS-stimulated samples. Stimulation of whole blood cultures with CS increased both the frequency and the expression of CD69 on the surface of T lymphocytes and NK cells. Importantly, this was noticed not only for healthy controls, but also for cancer patients. These data demonstrate the capacity of bacterial immunomodulator CS to activate human leukocytes of healthy subjects and cancer patients.     

Growth of cultured blood cells in the presence of the cryoprotector polyethylene oxide 400

The effect of polyethylene oxide 400 (PEO-400) cryoprotector on the mitotic activity of human peripheral blood leukocytes stimulated with PHA was studied in a culture. The cytologic peculiarities of the blood cell growth on addition of PEO-400 prior to cultivation are manifested in the preservation of the transformation capacity and mitosis despite a significant reduction of the mitotic activity in comparison with control cultures.     

Cometabolism of polychlorinated biphenyls: enhanced transformation of Aroclor 1254 by growing bacterial cells

Acinetobacter sp. strain P6 and a soil isolate, Arthrobacter sp. strain B1B, were tested for their ability to transform Aroclor 1254 as washed resting cells and as growing cells with biphenyl as the substrate. Growing cells were far superior to resting-cell suspensions in terms of total polychlorinated biphenyl (PCB) transformation, transformation of specific PCB congeners, and diversity of congeners that were attacked. Growing cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B transformed 32 and 23% of the [14C]Aroclor 1254, respectively, whereas resting cells of the same respective cultures transformed only 17 and 8%. Transformation was significantly greater with resting cells in only 2 of 39 cases in which congeners were transformed by both growing and resting cells of both cultures. The components of 19 and 12 capillary gas-chromatographic peaks of Aroclor 1254 were transformed by biphenyl-grown resting cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B, respectively, whereas the components of an additional 6 and 7 peaks were attacked by growing cells of the same respective cultures. Biphenyl oxidation by resting cells of both cultures decreased with time to less than 8% in 28 h. In addition to the normal 2,3-dioxygenase attack on PCBs, Acinetobacter sp. strain P6 also attacked congeners lacking an open 2,3-position. The ability of Acinetobacter sp. strain P6 to transform the components of 25 of the 40 largest peaks of Aroclor 1254 makes it one of the most versatile PCB-transforming organisms yet reported.     

Anticomplementary, Anticoagulatory, and Serum-Protein Precipitating Activity of Sodium Polyanetholsulfonate

Sodium polyanetholsulfonate (SPS) at 7.8 mug/ml completely abolished complement-mediated hemolysis of 1:10 diluted fresh guinea pig and human serum; at least twice as much SPS was required to reduce complement activity in 1:2 diluted human serum. The coagulation of 90 and 20% human blood was inhibited by 250 and 125 mug of SPS per ml, respectively. When added to fresh human serum, SPS precipitated beta 1C-globulin (C3), C4, beta lipoproteins, immunoglobulin IgG, IgM, and IgA, though incompletely.     

Cometabolism of polychlorinated biphenyls: enhanced transformation of Aroclor 1254 by growing bacterial cells.

Acinetobacter sp. strain P6 and a soil isolate, Arthrobacter sp. strain B1B, were tested for their ability to transform Aroclor 1254 as washed resting cells and as growing cells with biphenyl as the substrate. Growing cells were far superior to resting-cell suspensions in terms of total polychlorinated biphenyl (PCB) transformation, transformation of specific PCB congeners, and diversity of congeners that were attacked. Growing cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B transformed 32 and 23% of the [14C]Aroclor 1254, respectively, whereas resting cells of the same respective cultures transformed only 17 and 8%. Transformation was significantly greater with resting cells in only 2 of 39 cases in which congeners were transformed by both growing and resting cells of both cultures. The components of 19 and 12 capillary gas-chromatographic peaks of Aroclor 1254 were transformed by biphenyl-grown resting cells of Acinetobacter sp. strain P6 and Arthrobacter sp. strain B1B, respectively, whereas the components of an additional 6 and 7 peaks were attacked by growing cells of the same respective cultures. Biphenyl oxidation by resting cells of both cultures decreased with time to less than 8% in 28 h. In addition to the normal 2,3-dioxygenase attack on PCBs, Acinetobacter sp. strain P6 also attacked congeners lacking an open 2,3-position. The ability of Acinetobacter sp. strain P6 to transform the components of 25 of the 40 largest peaks of Aroclor 1254 makes it one of the most versatile PCB-transforming organisms yet reported.     

Effect of Phenylbutazone on Phagocytosis and Intracellular Killing by Guinea Pig Polymorphonuclear Leukocytes

The anti-inflammatory drug phenylbutazone has been found to inhibit both engulfment and intracellular killing of E. coli by guinea pig peritoneal polymorphonuclear (PMN) leukocytes. The bactericidal activity of leukocytic homogenates was also inhibited by the drug. Addition of the drug at various time intervals to a phagocytic reacting system caused an almost immediate cessation of bactericidal activity. Metabolic studies showed that the drug sharply curtailed glucose-l-(14)C and (14)C-formate oxidation of both resting and phagocytizing PMN leukocytes. These data indicated an effect upon the hexose monophosphate shunt and H(2)O(2) formation. Further investigation showed that the sites of inhibition were on glucose-6-phosphate and 6-phosphogluconate dehydrogenase. These inhibitions resulted in decreased H(2)O(2) production. It is suggested that H(2)O(2) activates lysosomes and subsequently complexes with the lysosomal enzyme, myeloperoxidase. This complex is a potent bactericidal agent in the phagocyte.     

Primary cultures of rat hepatocytes in hollow fiber chambers

Summary Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O₂-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P≤0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O₂-enriched conditions to maintain long-term P450 activity of rat hepatocytes.     

Microbial Manganese Reduction by Enrichment Cultures from Coastal Marine Sediments

Manganese reduction was catalyzed by enrichment cultures of anaerobic bacteria obtained from coastal marine sediments. In the absence of oxygen, these enrichment cultures reduced manganates when grown on either lactate, succinate, or acetate in both sulfate-free and sulfate-containing artificial seawaters. Sodium azide as well as oxygen completely inhibited microbial manganese reduction by these enrichment cultures, whereas molybdate had no effect on them. The addition of nitrate to the medium slightly decreased the rate of Mn²⁺ production by these enrichment cultures. These findings are consistent with the hypothesis that the manganese-reducing organisms in these enrichment cultures use manganates as terminal electron acceptors and couple manganese reduction in some way to the oxidation of organic matter.     

Thyrotropin regulation of plasminogen activator activity in primary cultures of ovine thyroid cells

In primary cultures of ovine thyroid cells, a high level of plasminogen activator (PA) activity was detected in the culture media. This level is much higher than in primary cultures of Sertoli cells, granulosa cells, and pituitary cells. PA activity increased with time in culture and was regulated by TSH and insulin. Activity gel analysis of the culture media revealed a major band of 43,000 daltons and a minor one of 70,000 daltons, suggesting the presence of both of the urokinase-type and the tissue-type PA in the media.     

Chemokines: extracellular messengers for all occasions?

Movement of leukocytes from peripheral blood into tissues, also called leukocyte extravasation, is absolutely essential for immunity in higher organisms. Over the past decade, our understanding of the molecular mechanisms involved in white blood cell extravasation during both normal immune surveillance and the generation of protective immune responses has taken a great leap forward with the discovery of the chemokine gene superfamily. Chemokines are low-molecular-weight cytokines whose major collective biological activity appears to be that of chemotaxis of both specific and overlapping subsets of leukocytes. They are therefore likely to play a critical role in the directed movement of leukocytes from the bloodstream into tissue. These molecules are almost exclusively secreted and act as extracellular messengers for the immune system. However, emerging data also show that various members of the chemokine gene superfamily exert other biological effects outside the immune system. All nucleated cells and all tissues examined to date are capable of expressing at least some chemokines, and it seems likely therefore that by the time all the chemokines are identified, and all their biological functions elucidated, we will find that, as a family, these molecules perform an extracellular messenger role in all tissues and systems of the body.     

Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs

The effect of growth in different mineral media on subsequent oxidation of trichloroethylene (TCE) by type I and type II aquifer methanotrophs was evaluated. Mixed culture MM1, containing a type II methanotroph, and a type I pure culture tentatively identified as aMethylomonas sp., were enriched and isolated from an uncontaminated groundwater aquifer. The second-order rate coefficients (k/K_s) for TCE oxidation by these cultures varied by more than an order of magnitude when the cultures were grown in different mineral media. The presence of a chelator (NaEDTA) in one of these media, termed Whittenbury, significantly enhanced rates of TCE oxidation by all the cultures tested. When pregrown in this mineral medium, the resting cells of the pure cultureMethylomonas sp. MM2 exhibited second-order TCE oxidation rates as great as 0.78 liter/mg·day, whereas when pregrown in Whittenbury lacking the chelator, the rates did not exceed 0.018 liter/mg·day. The rate of TCE oxidation byMethylomonas sp. MM2 pregrown in another mineral medium formulation, devoid of chelators (termed Fogel), was intermediate in value (0.26 liter/mg·day), and adding EDTA to this medium did not affect the rate. Adding 1.6 μM copper to both Whittenbury and Fogel mineral media reduced the TCE oxidation rates about an order of magnitude; subsequent addition of 84 μM EDTA partially alleviated this effect. The maximal rate coefficients (k) for TCE oxidation byMethylomonas sp. MM2 were significantly higher, and the half saturation coefficients (K_s) for TCE significantly lower, following growth in the presence of EDTA. Stationary phase TCE oxidation rates as great as 2.3 liter/mg·day were achieved whenMethylomonas sp. MM2, grown in Whittenbury medium was provided formate as a source of reducing power. Omitting EDTA from Whittenbury medium also significantly reduced the methane oxidation rate and the growth yield. Copper addition did not significantly affect the methane oxidation rate or growth yield. The internal membrane structures ofMethylomonas sp. MM2 evaluated by transmission electron microscopy showed the presence of internal membranes, the ultrastructure of which was the same regardless of growth medium or TCE oxidation rate. The methane monooxygenase responsible for TCE oxidation inMethylomonas sp. MM2 under the conditions of this study appears to be associated with the particulate fraction.