Medium Inoculum Research Articles

Chemically Defined Medium for Penicillin Production

AbstractCorn steep liquor (CSL) is considered an essential constituent in the penicillin inoculum and production media. In the present work, the inoculum medium containing CSL followed by synthetic medium (production medium from which CSL was deleted) of initial pH value 7.3 gave the same penicillin yield as that of the inoculum and production media containing CSL. On the other hand this method for penicillin production gave a bright fermentation broth and the penicillin recovery may be easier.

Effets de la Contamination Simultanée ou Successive par Beauveria Tenella et par Entomopoxvirus Melolonthae des Larves de Melolontha Melolontha [Col. Scarabaeidae

SummaryP. Ferros & B. Hurpin: Effects of simultaneous or successive contamination by Beauveria tenella and by Entomopoxvirus melolonthae of Melolontha melolontha larvae [Col. Scarabaeidae].Following previous experiments which outlined the important part played by the physiological or health state of Melolontha melolontha L. on its susceptibility to the Beauveria tenella mycosis, the conditions of an experimental infection with Entomopoxvirus melolonthae resulting in the development of the mycosis have been investigated. The experiments have been realized with techniques previously described, at 20° ± 1° C on homogeneous batches of twenty second instar larvae, the pathologic state of the insects being checked every week during 8 months. The germs have been used simultaneously or separately. Refering to previous results the infecting doses have been settled to 5.104 (=B1), 5.105 (= B2), 5.106(= B3) Beauveria tenella conidiospores per gram of dried peat, and to one white grub reached by a virus infection respectively for 10, 5 and 1 liter of peat (VI, V2 and V3). After the contamination of the rearing peat with both germs, the development of a pure virus infection is not favoured but the number of mixed infections is fairly superior, especially after a primary infection by the virus followed one month later by a secondary infection by Beauveria. After cumulating the cases of mixed infection and of pure mycosis and relating those values to 100, the results are mentionned in table 4. These results show that, in the presence of virus infection the white grub susceptibility towards the mycosis increases, the number of dead insects due to mycosis being 1 to 5 times larger than observed in the control batch. This increased susceptibility is particularly obvious when using a low or medium inoculum of spores.

Effect of Novobiocin and Its Combination with Tetracycline, Chloramphenicol, Erythromycin, and Lincomycin on the Microbial Generation of Escherichia coli

Inhibition of the steady-state generation of Escherichia coli by the bacteriostatic antibiotic novobiocin is linearly related to drug concentration in the range of 0 to 30 mug/ml. Increased cell sizes result because the drug inhibits cell division. The generation rate dependence on drug concentration depends on the nonionized fraction of novobiocin and is invariant with inoculum size or medium composition. However, the antibacterial activity of novobiocin decreases as the concentration of nutrients and Mg(2+) increases, although the inhibitory action of novobiocin on generation rate remains unchanged for concentrations of Mg(2+) above 8.1 x 10(-4) M. Novobiocin is synergistic in combinations with tetracycline in broth, but not when the Mg(2+) was maintained at 4.05 x 10(-3) M. Combinations of novobiocin with the 50S ribosomal subunit inhibitors chloramphenicol, erythromycin, or lincomycin are antagonistic, and the degree of growth inhibition is determined only by that component of the binary combination that would have the greater potency if it were acting alone.

Formalin-inactivated Venezuelan equine encephalomyelitis (Trinidad strain) vaccine produced in rolling-bottle cultures of chicken embryo cells.

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus grown in rolling-bottle cultures of chicken embryo cells. Trinidad strain virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin (USP) and antibiotics. Manipulation of multiplicity of inoculum (0.06 to 0.00006) and maintenance medium volume (100 to 300 ml) resulted in high-titered virus yields and only moderate cell destruction when fluids from infected cultures were harvested at 18 to 24 hr. The virus was inactivated at 37 C by 0.05% Formalin within 8 to 10 hr and with 0.1% Formalin within 6 to 8 hr. Single dose, antigen extinction tests in mice performed with 30 small-scale vaccine lots showed excellent potency at either Formalin concentration with inactivation periods ranging from 24 to 96 hr.

Formalin-inactivated Venezuelan Equine Encephalomyelitis (Trinidad Strain) Vaccine Produced in Rolling-Bottle Cultures of Chicken Embryo Cells

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus grown in rolling-bottle cultures of chicken embryo cells. Trinidad strain virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin (USP) and antibiotics. Manipulation of multiplicity of inoculum (0.06 to 0.00006) and maintenance medium volume (100 to 300 ml) resulted in high-titered virus yields and only moderate cell destruction when fluids from infected cultures were harvested at 18 to 24 hr. The virus was inactivated at 37 C by 0.05% Formalin within 8 to 10 hr and with 0.1% Formalin within 6 to 8 hr. Single dose, antigen extinction tests in mice performed with 30 small-scale vaccine lots showed excellent potency at either Formalin concentration with inactivation periods ranging from 24 to 96 hr.

Observations on the maintenance of Trichomonas vaginalis and Trichomonas foetus; the effects of cortisone and agar on enhancement of severity of subcutaneous lesions in mice.

Methods are described for the freezing of trichomonads, suspended in a liverinfusion broth, to – 20 °C and – 70 °C, and storage at these temperatures. After 9 months’ storage at – 70 °C, with dimethyl sulphoxide, six strains of T. vaginalis and three strains of T. foetus were alive; three trichomonas strains frozen earlier were also alive after 13 months. Four of the T. vaginalis strains survived equally well for 6 months at – 70 °C, in the presence of either dimethyl sulphoxide or glycerol.Although trichomonads may be maintained by subculture in a modified Diamond medium, storage at – 70 °C is more economical and less tedious; – 20 °C is not a satisfactory storage temperature for T. vaginalis or T. foetus, even in the presence of dimethyl sulphoxide or glycerol.The severity of subcutaneous lesions in mice produced by six freshly isolated strains of T. vaginalis, and two out of three stock culture strains of T. foetus, was enhanced by either the addition of agar to the suspending medium or administration of cortisone to the mice. One strain of T. foetus produced lesions only in the presence of agar, cortisone, on the dose schedule used, being ineffective.After storage at – 70 °C or cultivation in agar medium for 6 months, the virulence of trichomonads of either species was little affected, and lesions in mice were enhanced by agar in a similar degree to that found originally.For enhancing the severity of subcutaneous lesions in mice the agar method, involving only one injection, is simpler than the repeated administration of cortisone to infected mice; agar was shown to be more effective than cortisone with small infecting doses of one strain of T. vaginalis quantitatively tested.The subcutaneous injection of mice with trichomonads suspended in a liver-infusion broth containing 0·06% agar regularly produces lesions of the degree of severity required for the evaluation of compounds for therapeutic activity.We express our gratitude to Mr S. A. Price for constructive criticism and helpful suggestions during the preparation of the manuscript. Dr S. R. M. Bushby kindly provided details of the dimethyl sulphoxide method and apparatus in use in his laboratory for the low-temperature preservation of tumour cells and trichomonads. For supplying the trichomonas strains we are grateful to Dr L. P. Joyner, Miss M. Moore-Tucker and Mr C. S. Ledwich. We thank Mr W. A. Freeman for advice on the composition of the inoculum medium.

Utilization of carbohydrates and polyhydric alcohols by Mycobacterium tuberculosis.

Sweeney, Edward E. (University of California, Los Angeles) and Gregory J. Jann. Utilization of carbohydrates and polyhydric alcohols by Mycobacterium tuberculosis. J. Bacteriol. 84:459-465. 1962.-A new procedure, using a massive inoculum and nongrowth basal medium, was employed for testing carbohydrate and polyhydric alcohol utilization by human tubercle bacilli. A positive reaction was represented by acidification of the test medium rather than by growth, which was the criterion for carbohydrate utilization in studies by earlier workers. The new procedure was both more sensitive and more rapid than growth techniques; results were obtained within days, compared to weeks or months required for growth testing. The massive-inoculum technique may be applied to compounds other than carbohydrates and polyhydric alcohols, and is a sensitive means of detecting changes wrought by various chemical and physical agents upon the metabolism of tubercle bacilli. Three H(37)Rv strains and six strains of human tubercle bacilli freshly isolated from patients were tested with 21 carbohydrates and polyhydric alcohols. All nine strains gave strong positive reactions for glucose and glycerol, and usually weak positive reactions for ribose and sorbose. Five of the nine strains were trehalose positive, and six (all fresh patient isolates) of the nine were mannose positive.

Propagation of strain L (Earle) cells in agitated fluid suspension cultures.

SummaryThe agitated culture has been used in studying the growth cycle of strain L (Earle) mouse fibroblasts. From data available, it is seen that mammalian cells growing in vitro display a growth pattern quite similar to that of other microbiological populations. Some points which have been studied and can now be evaluated are the following: 1) the lag period for these cells appeared to be eliminated completely when log-phase cells were used as inoculum in media equilibrated to pH 7,2) growth rate appeared to be independent of the inoculum size and also independent of serum concentration, 3) generation time for these connective tissue cells. under conditions studied, was approximately 40 hours: 2 generations could usually be observed in these cultures and saturation concentration in some instances reached 2,000.000 cells per ml, 4) steady-state cultivation of tissue cells has been approached. The results indicated that populations could be maintained in such a manner, and this problem will be investigated.

The nutritional requirements of Haemophilus pertussis.

SUMMARY: Cohen & Wheeler medium prepared from casein hydrolysate previously treated with charcoal supported the growth of small inocula of Haemophilus pertussis (1–10 organisms/ml.). Almost as good growth was obtained from small inocula in medium in which the casein hydrolysate was replaced by a balanced mixture of 17 amino acids and which contained a mixture of growth factors. Medium containing either cystine and glutamic acid or cystine and aspartic acid as the only amino acids also supported the growth of small inocula; the rate of growth, however, was low. Nicotinic acid proved to be an essential growth factor for all strains tested. Guanine, hypoxanthine and xanthine enhanced growth although they were not essential. Biotin and haemin, also not essential, enhanced growth during the early phase; this was especially evident with freshly isolated strains.

PRODUCTION AND PROPERTIES OF 2,3-BUTANEDIOL: XX. INFLUENCE OF THE INOCULUM ON THE AEROBACILLUS POLYMYXA FERMENTATION

Whole wheat fermentations were started with known numbers of Aerobacillus polymyxa in the range from 50 thousand to 4000 million per ml. of mash. Increasing the initial numbers of the bacteria decreased the lag phase and increased the maximum numbers obtained. Although the initial rate of fermentation was increased, all the fermentations were finished at the same time and gave similar yields of 2,3-butanediol.The effects of added nutrients, incubation temperature, aeration, and reduced pressure on bacterial growth in the inoculum media were investigated but none of the treatments used had an appreciable effect on the subsequent fermentation. The use of a backstocking technique appears to be a practical method of inoculation.