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The influence of Mannheimia haemolytica A1 seed culture inoculum cell density on the production of leukotoxin in submerged culture supernatant.

Mannheimia haemolytica leukotoxin is produced during the logarithmic growth phase in submerged culture in RPMI 1640 medium with and without the addition of foetal calf serum or albumin. In order to establish a pattern of optimal leukotoxin production in small volumes in submerged cultures and to define some parameters involved, two high leukotoxin producing Mannheimia haemolytica strains were grown in RPMI 1640 medium containing either FCS or BSA. The cell growth and leukotoxin production abilities of each strain were determined concomitantly every hour in RPMI 1640 medium containing each of the additives over a time period of 6 h. The growth performance of three dilutions of a standardized seed culture inoculum prepared with each of the cultures and additives were simultaneously compared with each other using the above parameters. The different seed culture inoculum dilutions had a definite effect on the time and quantity of leukotoxin production. Both strains demonstrated peak leukotoxin production after 4 h of active growth. The addition of albumin to both isolates gave slightly increased leukotoxin levels, and both showed that the peak leukotoxin was not associated with peak cell concentration. Obvious quantitative differences in the ability of different M. haemolytica strains to produce leukotoxin were noted. Strain 12296 produced optimal leukotoxin concentration from the medium (1/25) dilution of the seed culture inoculum after 4 h, whereas strain 1/10 produced the same concentration with the low (1/5) dilution seed culture inoculum, possibly reflecting the superior production ability of the first strain. However, each strain of M. haemolytica appeared to have its own specific logarithmic cell growth and leukotoxin production pattern. The peak cell density of M. haemolytica grown in submerged RPMI 1640 culture medium cannot be used as an indication of optimal leukotoxin levels.

Albumins↗

Influence of inoculum type and cultivation conditions on natamycin production by Streptomyces natalensis.

This study demonstrates the effect of cultivation conditions on the production of natamycin. Of these conditions, the effect of oxygen limitation and type of inoculum were extensively investigated. Increasing the shaking speed and decreasing the medium volume improved both the volumetric and specific natamycin production. Also, decreasing the dissolved oxygen level in the cultivation medium through the addition of soluble biopolymer (alginate) resulted in a significant decrease in the natamycin yield without effect on the cell growth. On the other hand, spore inoculum yielded higher concentration of natamycin compared to the vegetative cells by about 40%. The maximal cell productivity based on biomass [Yp/x] of about 0.4 [g/g] was obtained by using shake flask of 50 ml working volume agitated at 200 rpm and the inoculum was in the form of spore 2 x 10(8) spores/ml. These results showed that the production process of natamycin is highly dependent on oxygen level in the cultivation medium and type of inoculum as well.

Culture Media↗

[Effect of inoculum parameters on the specificity of secondary synthesis in cultures producing novobiocin and mycoheptin].

The impact of the vegetative inoculum parameters on specificity of the secondary synthesis in the cultures producing novobiocin and mycoheptin was studied. During the study the fermentation conditions were varied by using the vegetative inoculum differing in the respiration rate after its transfer to the fermentation medium. To show the decisive role of the inoculum parameters in regulation of the specificity of the secondary synthesis, the dynamics of accumulation of certain metabolites forming from glucose along with the main antibiotic and the activity of the key enzymes of the carbohydrate metabolism during the culture growth in the fermentation media were studied. It was found that the specificity of the secondary synthesis with respect to certain metabolites was defined by the intensity of carbohydrate metabolism, i. e. the ratio of the activity of enzymes of glycolysis and the pentosephosphate pathway. In this regard, the inoculum with the maximum respiration rate in an amount of 10 to 20 per cent promoted the highest productivity of the mycelium by the synthesis of novobiocin and mycoheptin while the rate of accumulation of fatty acids, carbohydrates and phenol compounds (for Streptomyces spheroides) and mycopentene (for Streptoverticillium mycoheptinicum) decreased.

Anti-Bacterial Agents↗

[In vitro simulation of rabbit cecal fermentation in a semi- continuous flow fermentor. II. Effect of inoculum type].

Two Rusitec fermentors were operated under identical conditions. One was seeded with an inoculum of rabbit caecal contents, and the other with bovine rumen contents. The fermentation substrate was rabbit feed that had been digested with amylase and pepsin. The substrate constituents (organic matter, OM and NDF) were lost in 48 h at a significantly higher rate in the presence of rumen inoculum (OM: +10%, NDF: +15%). The pHs of the 2 fermentors were similar at pH 6.6. The fermentors produced similar amounts of protein nitrogen per 24 h, after 6 d of adaptation. Volatile fatty acid production was slightly higher in the presence of rumen inoculum. The fermentor inoculated with rumen contents produced a higher percentage of propionic acid (25%) than of butyric acid (7%), while fermentation with rabbit caecal contents gave the opposite ratio (C3/C4 = 0.81). Consequently, only the rabbit caecal inoculum provided the fermentation profile characteristic of the species.

Amylases↗

[Influence of the inoculum effect on minimal inhibitory concentrations of 3 derivatives of 5-nitro-imidazole and 11 other antibiotics on Bacteroides fragilis].

Three nitro-imidazole derivatives and eleven other antibiotics were studied for both their antimicrobial activity and the degree to which they produced inoculum effect on twenty strains of Bacteroides fragilis group. Five inocula were studied ranging from 10(5) to 10(9) CFU/ml. Statistical analysis show the significant influence of inoculum and those of strains. Nitro-imidazole derivatives exhibited a large inoculum effect while other antibiotics exhibited only a minimal to-no-inoculum effect.

Anti-Bacterial Agents↗

Active immunization against the malaria parasite Plasmodium berghei in mice. The immunizing inoculum.

In the immunization procedure of Swiss and C3H/StZ mice against P. berghei the inoculum plays an important role. Only viable parasites are able to induce immunity when multiple inoculations (10(5) P.E. per mouse) or a single inoculation (1-4 X 10(7) P.E. per mouse) are administered. The inoculated parasitized erythrocytes should enter the vascular system. The subcutaneous route is inappropriate, since subsequent immune reactions are notably absent. In combination with a given suppressive regimen successful immunization depends on an optimum number of viable P.E. in the inoculum. All conditions that affect the proportion of viable parasites in the inoculum (route, storage, medium, temperature, donor) should be recognized and controlled. The actual immunizing capacity of the inoculum also depends on the magnitude and time of initiation of sulfathiazole treatment after inoculation. Suppressive treatment (300 mg/L) starting 2 days after inoculation was found optimal in order to render the procedure less sensitive to small differences in the number of P.E. inoculated. Conditions which lead to antimalarial immunity are apparently strain-specific.

Animals↗

Synergistic enhancement of in vitro antimicrobial activity of cefmetazole and cefotiam, cefamandole or cefoperazone in combination against methicillin-sensitive and -resistant Staphylococcus aureus. II. Effect of inoculum size.

The inoculum effect was studied on the activity of cefmetazole and cefotiam, cefamandole or cefoperazone alone and in combination against 9 strains of methicillin-sensitive Staphylococcus aureus (MSSA) and 20 strains of methicillin-resistant Staphylococcus aureus (MRSA) by means of the checkerboard titration method with Mueller-Hinton agar plate using 10(6) and 10(8) CFU/ml. The antimicrobial activity against MSSA and MRSA was potentiated synergistically in combination of cefmetazole and these cephalosporins either with inoculum size 10(6) or 10(8) CFU/ml. At a concentration of cephalosporins < or = 6.25 micrograms/ml, the combination effect of cefmetazole and cefotiam or cefamandole against MRSA was more potent than that of cefmetazole and cefoperazone. With a higher inoculum size the effect was reduced. Under these conditions with a low dose level of drugs, the enhancement of the activity of cefmetazole by cefotiam was least influenced by inoculum size among cephalosporins studied.

Bacteriological Techniques↗

Effect of leukocyte concentration and inoculum volume on the laboratory identification of cytomegalovirus in peripheral blood by the centrifugation culture-antigen detection methodology.

OBJECTIVE: To investigate the rate of Cytomegalovirus (CMV) detection in peripheral blood by shell vial assay-indirect immunofluorescent assay technology using two leukocyte inocula concentrations and different inoculum volumes containing equivalent cell concentrations. DESIGN: Leukocyte inocula concentrations of 2 x 10(5) and 4 x 10(5) cells per 0.2 mL were assayed for the presence of CMV by shell vial assay-indirect immunofluorescent assay. The effect of different inoculum volumes (0.2 and 0.4 mL) containing an equivalent cell concentration of 4 x 10(5) was evaluated as well. The data were compared to conventional MRC-5 tube cultures, including blind passage. PATIENTS: Ninety-five patients (101 specimens) were tested sequentially. The test population consisted primarily of patients suffering from the acquired immunodeficiency syndrome. SETTING: The diagnostic virology laboratory, acquired immunodeficiency syndrome clinics, and hospital wards. RESULTS: Among the 101 specimens tested by shell vial assay-indirect immunofluorescent assay, the rate of CMV sensitivity increased by 36% using the higher leukocyte inoculum concentration of 4 x 10(5) cells per 0.2 mL (P = .002; Cochran's Q test). No significant difference in CMV yield was identified using equivalent cell concentrations with inocula volumes of 0.2 or 0.4 mL. The CMV sensitivity rate using the higher leukocyte inoculum surpassed that obtained by conventional tube culture-blind passage. CONCLUSION: These data denote the importance of leukocyte concentration on the rate of CMV detection in peripheral blood by the shell vial assay-indirect immunofluorescent assay. The data also point out the need to establish a standardized blood preparation protocol to achieve optimal clinical relevance of this widely used laboratory test.

AIDS-Related Opportunistic Infections↗

Implementation of conditions of the inoculum stage for Streptosporangium cultures.

Different factors concerning the inoculum stage of cultures classified within the genus Streptosporangium have been studied. These factors include a comparison among several inoculum media and sources, the influence of aeration and volume of medium in the growth of the cultures, and the effect of the inoculum size on the course of the production of two antibacterial substances. The results suggest that a slight variation in the status of the seed culture (age, biomass concentration, etc.) affect the course of the batch culture during its initial exponential phase (3-5 days), whereas, at longer times, the behavior of the culture does not depend greatly on the characteristics of the inoculum. The results obtained have allowed to define a set of conditions producing the maximum increase of biomass in the minimum time.

Actinomycetales↗

Methods for storage of antimicrobial effectiveness test inoculum suspensions below freezing.

Studies were conducted to determine the suitability of storage below freezing of some antimicrobial effectiveness test inoculum organisms: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Several solutions used for protecting microorganisms subjected to storage below freezing were compared. Comparison of 10% dextrose with other solutions (distilled water; 0.07 M phosphate buffer, H 7.0, with 15% glycerol; and 0.07 M phosphate buffer, pH 7.0, with 7.5% dimethyl sufoxide) demonstrated that inoculum suspensions were most stable when prepared with 10% dextrose. After storage for 6 months at-50 degrees in a freezer, inoculum suspensions prepared with 10% dextrose retained viability and demonstrated suitability for use in antimicrobial effectiveness tests of dosage forms containg preservatives.

Candida albicans↗

Effect of inoculum level of xylitol production from rice straw hemicellulose hydrolysate by Candida guilliermondii.

The effect of inoculum level on xylitol production by Candida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L-1 to 2.41 g L-1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g-1) and volumetric productivity (0.56 g L-1 h-1) were reached with an inoculum level of 0.9 g L-1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeast C. guilliermondii with efficiency values as high as 77% of the theoretical maximum.

Candida↗

Effect of inoculum size on antimicrobial susceptibility of Helicobacter pylori.

An agar dilution assay was used to assess the effect of inoculum size and culture period on the susceptibility of 15 clinical isolates of Helicobacter pylori to ampicillin, erythromycin, tetracycline, chloramphenicol, metronidazole and tinidazole. The mean MIC of the isolates increased 2.2- to 21.2-fold as the inoculum size progressed from 10(3) to 10(7) cfu/spot. Identical results were noted when isolates were maintained for two or four days prior to testing. Inoculum size should be carefully controlled when assessing the in vitro susceptibility of Helicobacter pylori.

Helicobacter pylori↗

Effects of pneumoperitoneum on tumor implantation with decreasing tumor inoculum.

INTRODUCTION: The aim of this study was to determine the effect of pneumoperitoneum on the rate of trocar-site implantation with decreasing inoculum of cancer cells. METHODS: A total of 0.5 ml of GW-39 human colon cancer cell suspensions at 1 percent (approximately 3.2 x 10(5) cells) and at 0.5 percent (approximately 1.6 x 10(5) cells; v/v) were injected into the abdomen of hamsters through a midline incision. Animals in each group were randomized to receive either pneumoperitoneum (1 percent = 33; 0.5 percent = 43) or not (1 percent = 32; 0.5 percent = 39). Gross and microscopic tumor implants were documented seven weeks later at four trocar sites. RESULTS: In the 1 percent group, pneumoperitoneum significantly increased trocar-site tumor implants from 50 to 71 percent (P < 0.001). Pneumoperitoneum also resulted in the following: 1) more frequent involvement of all four concurrent sites (38 vs. 10 percent; P < 0.02); 2) more frequent palpable tumors (13 vs. 5 percent; P < 0.01); 3) larger tumor mass (2.1 +/- 0.6 g vs. 0.2 +/- 0.1 g; P < 0.02). In the 0.5 percent group, pneumoperitoneum did not significantly increase trocar-site tumor implants, and it did not result in a larger tumor mass. The percent increase in trocar-site implants owing to pneumoperitoneum was influenced by the amount of tumor inoculum (21 percent in the 1 percent group; 10 percent in the 0.5 percent group). The mass of palpable tumor implants after pneumoperitoneum decreased with decreased inoculum: 1 percent = 2.1 +/- 0.6 g; 0.5 percent = 0.3 +/- 0.1 g (P < 0.0001). CONCLUSIONS: Pneumoperitoneum significantly increased both tumor implantation rate and mass when approximately 3.2 x 10(5) colon cancer cells were injected into the peritoneal cavity. These effects of pneumoperitoneum diminished with one-half as many tumor cells injected in the peritoneal cavity.

Animals↗

Effect of age, amount of inoculum and inoculation medium composition on lactic acid production from glucose by Lactobacillus casei subsp. rhamnosus.

Effect of composition of the medium used for the inoculum cultivation, of the age and amount of the inoculum was investigated using a 3-L glass fermentor with a working volume of 1 L. The highest productivity of the culture was obtained when using a 20% (V/V) 1-d inoculum grown in the MRS medium. Yields of lactic acid were 88-97%, while the L(+)-isomer represented about 80% of the total product.

Bacteriological Techniques↗

Inoculum production of the ectomycorrhizal fungus Pisolithus microcarpus in an airlift bioreactor.

Many important tree species in reforestation programs are dependent on ectomycorrhizal symbiosis in order to survive and grow, mainly in poor soils. The exploitation of this symbiosis to increase plant productivity demands the establishment of inoculum production methods. This study aims to propose an inoculum production method of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) using liquid fermentation in an airlift bioreactor with external circulation. The fungus grew as dark dense pellets during a batch fermentation at 25.5 degrees C and air inlet of 0.26-0.43 vvm. The maximum biomass (dry weight) achieved in the airlift bioreactor was approximately 5 g.l(-1) after 10-11 days. The specific growth rate (micro(x)) in the exponential phase was 0.576 day(-1), the yield factor (Y(X/S)) 0.418, and the productivity (P(X)) 0.480 g.l(-1).day(-1). This specific growth rate was higher than that observed by other authors during fermentation processes with other Pisolithus isolates. The method seems to be very suitable for biomass production of this fungus. However, new studies on the fungus growth morphology in this system, as well as on the efficiency of the process for the cultivation of other ectomycorrhizal fungi, are necessary. It is also necessary to test the infectivity and efficiency of the inoculum towards the hosts.

Biomass↗

Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum.

Arbuscular mycorrhizal fungi (AMF) form a number of different infective propagules that are used to form new mycorrhizal associations. These are spores, extraradical hyphae and infected roots. However, not all fungi are equally capable of colonizing roots with all of the above-mentioned propagules and there is conflicting evidence of major differences in colonization strategy between members of the Glomineae and Gigasporineae. In this study, we tested the abilities of eight fungal species from four different genera to colonize roots using three different types of inoculum. Glomus and Acaulospora isolates colonized from all inoculum types, whereas Gigaspora and Scutellospora isolates colonized mainly from spores and to a limited degree from root fragments. Extraradical hyphae were not suitable propagules for the species of Gigaspora and Scutellospora tested. This indicates that AMF have different colonization strategies and that this is largely differentiated at the suborder level. It is unclear why there is such a difference among the fungi in inoculum types. Future research should examine differences in the anatomy and physiology to discern a mechanism for such differences in life-history strategies.

Ascomycota↗

Mycorrhizal inoculum potentials of pure reclamation materials and revegetated tailing sands from the Canadian oil sand industry.

Recent improvements in the management of oil sand tailings used by the Canadian oil sand industry have resulted in the production of composite tailing sands (CT): a new challenging material for reclamation work. Jack pine (Pinus banksiana Lamb.), hybrid poplar (Populus deltoides Bartr. ex Marsh. xPopulus nigra L.) and red clover (Trifolium pratense L.) plants were used in an 8-week greenhouse bioassay to evaluate the mycorrhizal inoculum potential of CT. This inoculum potential was compared with that of three other reclamation materials [common tailing sands (TS), deep overburden (OB) and muskeg peat (MK)], and with three sites reclaimed in 1982 (R82), 1988 (R88) and 1999 (R99). CT was devoid of active mycorrhizal propagules while all other materials showed some level of inoculum potential. Arbuscular mycorrhizal fungi were observed on roots of clover or poplar grown in TS, OB, and all substrates containing peat (MK, R82, R88 and R99). Pine roots were also colonized by vesicle-forming hyphae of an unidentified fine endophyte and by dark septate fungi. Ectomycorrhizas (ECM) were observed on pine and poplar grown in OB, MK, and in soils from the two older reclaimed sites (R82 and R88). Using morpho- and molecular typing, six ECM fungi were identified to the genus or species level: Laccaria sp., Thelephora americana, Wilcoxina sp. (E-strain), Tuber sp. (I-type), a Sebacinoid, and a Pezizales species. Laccaria sp. and Wilcoxina sp. were the most frequently observed ECM species.

Biodegradation, Environmental↗

The influence of the size of the initial inoculum on the efficacy of isometamidium (samorin) on a stock of Trypanosoma congolense.

The effect of the number of trypanosomes in the initial inoculum on the minimum curative dose, was determined for an experimental infection of Trypanosoma congolense in mice treated with isometamidium. Mice were infected by the intravenous route and were then treated three hours later by intraperitoneal injection. The minimum curative dose was shown to be dependent on the size of the initial inoculum, with a difference of a factor of 7.5 as the initial inoculum was increased from 10(3) to 10(6) trypanosomes per mouse. It is concluded that this may be a significant variable for in vivo drug sensitivity test, and may also have implications for treatment of infections in the field.

Animals↗