Bacteria incubation

Weinberg (26), who found that the bacterial death rate in synthetic media was accelerated by iron deprivation. The survival of serum-exposed bacteria incubated at 4°C makes it most unlikely that the iron-starvation per se is responsible for the killing of bacterial cells. Griffiths (5) found that the inhibition of E. coli by serum is accompanied by the appearance of abnormal bacterial phenylalanyl t-RNA. Such abnormal t-RNA was not found in iron-supplemented serum. It is possible, therefore, that permanent lesions develop in iron-starved bacteria which not only stop bacterial multiplication but eventually cause bacterial death. 

Opsonization of bacteria Incubate E. coli expressing GFP in 5% heat-inactivated FCS for 30 min at 37°C, immediately prior to use. 

Wash BMMs five times with DMEM to remove extracellular bacteria, incubate for 40 min in complete medium and then for 60 min in complete medium... 

Transform chemically competent Machl bacteria. For each reaction, transfer 2 pL of Dpwl-treated DNA to 50 pL of bacteria. Incubate on ice for 30 min. Heat shock the cells for 30 s at 42 °C and immediately place them on ice for 1 min. Add 250 pL of room temperature SOC medium and incubate at 250 rpm for 1 h at 37 °C. Plate 80—100 pL of each transformation on prewarmed LB plates containing the appropriate antibiotic (ampiciUin for pMSlOlC vector). Incubate overnight at 37 °C. 

Add 150 pL/weU of the bacteria culture or of the periplasmic extract. These solutions contain the soluble mutated protein which has been secreted by bacteria. Incubate for 1 h with gentle agitation (450 rpm). Wash the ELISA plate four times as described in step 6. 

As mentioned above, immobilized cells are studied mainly for practical reasons, since they show a number of economic advantages over the use of growing cells or cell suspensions. Production of organic acids is one of the prospective applications of immobilized cells. Another one is related to the release of nitrogenous bases and some nucleosides by immobilized cells. In nitrogen-starved immobilized cells the levels of all metabolites (first of all, nucleotides) are reduced (Leps and Ensign, 1979). It was shown (Ikonnikov et al., 1982) that immobilized cells of propionic acid bacteria, incubated periodically in nitrogen-free medium, released substances of protein and nucleic acid nature, whose quantity decreased with the time of incubation in... 

Use 10 pL of the final preparation to transform 100 pL of competent DH5a bacteria Incubate the freshly thawed bacteria with the DNA for 30 mm on ice, heat shock for 45 s at 42°C, add 900 pL of LB medium (no ampicillin), and allow the cells to recover at 37°C with vigorous shaking for 1 h Harvest the bacteria by centrifugation, resuspend in 200 pL of LB medium containing 100 pg/mL... 

Aliphatic-Garboxylics. There are only two herbicides present in this class, trichloroacetate [76-03-9] (TCA) and dalapon [75-99-0]. These are used primarily for the selective control of annual and perennial grass weeds in cropland and noncropland (2,299). Dalapon is also used as a selective aquatic herbicide (427). Dalapon and TCA are acidic in nature and are not strongly sorbed by sods. They are reported to be rapidly degraded in both sod and water by microbial processes (2,427). However, the breakdown of TCA occurs very slowly when incubated at 14—15°C in acidic sods (428). Timing not only accelerates this degradation but also increases the numbers of TCA-degrading bacteria. An HA has been issued for dalapon, but not TCA (269). 

The agar [9002-18-0] plate method consists of adding a known quantity of sample, usually 1.0 or 0.1 mL, depending on the concentration of bacteria, to a sterile petti plate and then mixing the sample with a sterile nutrient medium. After the agar medium solidifies, the petti plate is incubated at 32°C for 48 hours after which the bacterial colonies are counted and the number expressed ia terms of a 1 mL or 1 g sample. This procedure measures the number of viable organisms present and able to grow under test conditions, ie, 32°C. 

Tuberculocidal Test. The tubercle bacillus is resistant to disinfectants because the cells are protected with a waxy coating that is not readily penetrated. The tuberculocidal test is a use dilution practical type test that employs porcelain cylinders. The bacteria are different from those in the use dilution method (Table 10), the incubation time is longer, and the details of the procedure are different. For example, in the tuberculocidal test the test is divided into two parts, a presumptive test and a confirmatory test. The former employs Mycobacterium smegmatis and the latter employs Mycobacterium bovis (BCG). For the presumptive test the incubation time is 12 days, as against 48 hours for other bacteria used in the use-dilution method. For the confirmatory test the incubation time is 60 days, with an additional 30 days in case there is no growth. As shown in Table 10, the concentrations of the phenol standard are higher than used with other bacteria. 

A subsequent patent, U.S. Patent 2,828,246 described a commercial process for bacitracin production. A 1,230 gallon portion of a medium containing 10% soybean oil meal, 2.50% starch and 0.50% calcium carbonate having a pH of 7.0 was inoculated with a culture of bacitracin-producing bacteria of the Bacillus subtilis group and the inoculated medium incubated for a period of 24 hours with aeration such that the superficial air velocity was 12.1. An assay of the nutrient medium following the fermentation revealed a yield of bacitracin amounting to 323 units/ml. This was more than twice the yields previously obtained. 

The subsequent advance was rather fortuitous and rested more with serendipity than with scientific logic. A search was made for cheaper more effective replacements for casein hydrolysate. Amongst the tested materials was com steep liquor (CSL). CSL is a by-product of the manufacture of starch from maize kemals. Whole maize is incubated in warm water, at 50°C acidified with SO2. Thermophilic bacteria hydrolyse proteins and other components of the kemals, thereby loosening the starch granules. These are removed, leaving behind the steep liquor which is used to treat further maize kemals. Ultimately, the liquor is too viscous to re-use and the liquor is concentrated and used as cattle feed. It was this material that was used for penicillin fermentation. Surprisingly, the yield of penicillin increased by a further 5-10 fold giving yields of 50-100 ig ml. 

The optimum temperature varies widely from species to species but in general the common moulds will grow better at 22-25 °C than most human pathogenic and commensal bacteria. It is customary, therefore, to incubate mould cultures at lower temperatures than bacterial cultures. 

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