The Inoculum

Specific procedures exist for storing (16,17) and propagating microorganisms to obtain reproducible fermentations. The stock culture is stored frozen (<—80°C) or freeze-dried. To prepare the inoculum (seed) mixture, an aUquot is taken and grown in consecutive soHd or Hquid cultures of increasing volume. The volume of the last step, the seed fermentor, is typically 4—12% of the main fermentor volume. 

The experiment was carried out on the 1,000 gallon scale. Three impellers 1 8" diameter at 220 rpm were employed. The air rates were 0 to 5 hours, 40 cfm, 5 to 10 hours, 80 cfm and after 10 hours, 125 cfm. The inoculum rate was 10% v/v. It was prepared by the standard inoculum development technique on the following medium ... 

This was inoculated with a spore suspension of P. patu/um (1 liter containing 3-5 x 10 spores/ml) and grown at 25°C in 100 gallon tank. The inoculum is transferred at 40 hours or when the mycelial volume (after spinning 10 minutes at 3,000 rpm) exceeds 25%. The fermentation is conducted as near to the ideal pH curve as possible by addition of crude glucose, according to U.S. Patent 3,069,328. 

The broth culture so obtained is employed as an inoculum (1%). Into each of ten flasks containing 100 ml of sterile nutrient broth is added 1 ml of the inoculum. The flasks are agitated on a rotary shaker for 8 hours at 28°C at 240 strokes per minute. After this growth period, a solution of 25 mg of 16/3-methy(cortisone in 0.5 ml of methanol is aseptically added to each flask which in turn is reshaken and incubated for an additional 24 hours. The final pH is 7.8. 

To a 2-liter flask were added 300 g of finely divided corn. The flask and its contents were then sterilized and after sterilization 150 ml of sterile deionized water wereadded. To the mixture in the flask were then added 45 ml of the inoculum prepared by the process and the material was thoroughly mixed. The mixed material was then incubated for about 20 days at 25°C in a dark room in a water-saturated atmosphere. The following illustrates the recovery of the anabolic substance from the fermentation medium. 

The trays are charged with the hot medium and, on cooling to 30°C, the inoculum is added. Two days are normally required for germination and growth and towards the end of this period the pH drops. These first two days are crucial in a microbiological sense because it is here that problems of contamination are most likely to occur. 

Sterilised caldum carbonate slurry is added in increments to the sterilised medium to maintain pH. Addition of all die necessary caldum carbonate at the beginning of the process would irreversibly and adversely affect the inoculum. 

The medium used to produce the inoculum should be designed for rapid growth of the production organism without exopolysaccharide production. Production of the latter in the inoculum train can give rise to highly viscous cultures that are difficult to transfer from one vessel to another. 

The correct answer is seven. In practice, the number of inoculum steps does not usually exceed four to reduce the risk of contamination. This also reduces capital investment and production costs since fewer transfers require fewer vessels for development of the inoculum. 

Contamination of the production vessel leads to serious financial penalties and each step in the inoculum train is monitored for contamination. To reduce the risk of contamination during sampling it is usual to take a sample from the residue left in each vessel after its contents have been transferred to the next reactor. Since these contamination checks are retrospective, a heavy reliance is placed on the growth characteristics of the production organism. Kinetic variables such as growth rate and oxygen consumption rate are also used to assess the quality of the inoculum. 

Place the flask in a temperature-controlled shaker at 37 °C. The exponential growth phase will last from 2 to 24 hours after inoculation. The exact time and duration depend on the physiological condition of the inoculum. The data in Table 10.1 are plotted and a growth curve will be obtained for an exponentially growing culture. Figure 10.2 shows the typical growth curve obtained for a viable organism. 

In our screening program for virus inhibitors and stimulators we used cucumber as an assay host, in which starch lesions are formed in proportion to the concentration of virus in the inoculum. 

Example 12.7 Develop a model for the anaerobic batch fermentation of glucose to ethanol and coproduct CO2 using Saccharomyces cerevisiae. The starting mixture contains 10% glucose. The inoculum is 0.0005 w/w. Product inhibition stops cell growth at 14% ethanol. Assume ka = 0 but include the cannibalization of cellular material beginning when the substrate is completely consumed. 

Equation (12.17) postulates that spontaneous deaths occur throughout the batch cycle. This means that dXjdt is initially negative. Is it possible to lose the inoculum completely if the induction period is too long Long induction periods correspond to small values of ot in the lag phase model of Problem 12.6. Find the critical value for ot at which the inoculum is lost. 

In phase B it is assumed that the inoculum has adapted itself to the new environment and growth then proceeds, each cell dividing into two. Cell division by binary fission may take place every 15-20 minutes and the increase in numbers is exponential or logarithmic, hence the name log phase. Phase C, the stationary phase, is thought to occur as a result of the exhaustion of essential nutrients and possibly the accumulation... 

The inoculum stage media are designed to provide the organism with all the nutrients that it requires. Adequate oxygen is provided in the form of sterile air and the temperature is controlled at the desired level. Principal criteria for transfer to the next stage in the progression are fieedom from contamination and growth to a pre-determined cell density. 

During the disinfection process a change of pH can, at one and the same time, affect 1 the rate of growth of the inoculum ... 

In all experiments the inoculum size should be controlled and clearly stated in any account of the experiment. 

Graded doses ofthe test substance are incorporated into broth dispensed in McCartney boules and the bottles inoculated with the test organism and incubated. The point at which no growth occurs is taken as the bacteriostatic concentration (minimum inhibitory concentration, MIC). It is essential when performing these tests to determine the size of the inoculum as the position of the end-point varies considerably with inoculum size, which should always be defined in any description of result. 

A medium of Linsmayer Skoog (8) supplemented with 0.2 mg/L 2.4-dichlorophenoxyacetic acid as a growth regulator was used for the growth of cell suspension The cultivation was carried out in Erlenmayer flasks with 1/5 net volume on a shaker (11.6 rad/s) in the dark, at 26 - 28°C. In compliance with the nature of the experiment, flasks of different size were used 100 - 1000 cm, and the duration of the cultivation was 5 days for growing the inoculum and 12 days for studying the time course of growth. 

H.annuus 1805 cell suspension was cultivated on a shaker (11.6 rad/s) at 26-28 °C in the dark in 1/5 net volume flasks. Duration of growth was different from 5 days for obtaining the inoculum to 10 days for studying the course of growth, the course of changes in the cell walls and the courses of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase as well. For inoculation 20 % (v/v) five-day cell suspension, containing 9 g/L dry cell biomass, was used. 

Laboratory experiments using natural consortia under defined conditions have particular value from several points of view. They are of direct environmental relevance, and their use minimizes the ambiguities in extrapolation from the results of studies with pure cultures. They provide valuable verification of the results of studies with pure cultures and make it possible to evaluate the extent to which the results of such studies may justifiably be extended to the natural environment. It should be appreciated, however, that in some cases the habitats from which the inoculum was taken might already have been exposed to xenobiotics so that natural enrichment (preexposure) could already have taken place. This has been discussed in Chapter 4. 

Laboratory studies on the rate of degradation of phenanthrene using samples from a contaminated site showed that this depended critically on the source of the inoculum within the site (Sandoli et al. 1996). 

Biopsy or blood specimens may be cultured for Leishmania spp. or T. cruzi with Novy-MacNeal-Nicolle (NNN) medium. Biopsy specimens are ground in a small amount of saline. Biopsies from skin lesions or other tissues which may contain bacteria may have penicillin (0.1 ml of 1,000 U/ml) added to the medium with the inoculum. The inoculum is 1 drop of ground tissue or blood. Incubate the culture at room temperature (22°C), and at days 3 and 7, examine a direct mount of liquid from the bottom of the slant at x400 magnification. These cultured organisms are potentially infective for humans. 

When temperature studies are conducted, it is important to concurrently incubate an additional tube of medium inoculated with the fungus at 25 °C to ensure viability of the inoculum. For an isolate to be considered dimorphic, only a few cells of its typical tissue form need... 

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