Media sterilization techniques

Using sterile techniques, transfer the appropriate bacterial cells from slant to a test tube or flask containing 10 mL of sterilized LB medium and 40 fiL of the ampicillin solution. Incubate the culture with vigorous shaking at 37°C for 12 to 15 hours or overnight. 

For aerobic fermentations, air needs to be supplied continuously. Typical aeration rates for aerobic fermentation are 0.5 - 1.0 vvm (air volume per liquid volume per minute). This requires an enormous amount of air. Therefore, not only the medium but also the air must be free of microbial contaminants. All of the sterilization techniques discussed for medium can also be employed for air. However, sterilization of air by means of heat is economically impractical and is also ineffective due to the low heat-transfer efficiency of air compared with those of liquids. The most effective technique for air sterilization is filtration using fibrous or membrane filters. 

Using sterile technique, add 10 ml of the inoculum from step 1 to the 1 liter of culture medium. Cultivate the bacteria with shaking (300 rpm) at 37 °C for 20 to 24 hr, whereupon the absorbance at 566 nm should be about 0.9. 

Transfer 5 to 10 ml of this culture to 100 ml of fresh SPI medium in a 500-ml sidearm flask so that the final cell suspension has an absorbance at 500 nm of about 0.1 when viewed against an SPI medium blank. (NOTE If you do not use a Klett colorimeter and a sidearm flask, remove 1-ml samples for absorbance readings in a spectrophotometer. Use sterile technique for all samplings. Do not return samples from the cuvettes to the cell suspension.) Incubate this culture with shaking at 37°C. 

After the 2-hr incubation at 37°C, use sterile technique to perform the following steps for dilution and plating, labeling all tubes and plates with the dilutions and medium used. 

Prepare the inoculation medium as follows Using sterile technique, withdraw 10 ml from the growth medium described above and place it in a sterile growth tube. Add 0.5 ml of sterile uracil from the previous paragraph to bring the inoculation medium to 30 p-g/ml. Add 150 fig ampicillin per ml of the medium by adding 30 gd of a 50-mg/ml solution of ampicillin in H20, which has been filter-sterilized. Grow and harvest the bacteria as described in the protocol for Experiment 9. 

To preserve cells and virus-infected cells, freezing medium containing 10% dimethyl sulfoxide (DMSO) is used, along with a slow freezing process. DMSO is light sensitive, hazardous to handle, and toxic to cells. Use appropriate protection, sterile technique, and speed. 

Sterile technique is used to ensure that the sterile medium is inoculated in such a way that only the desired micro-organism is introduced and encouraged to grow, with other micro-organisms - contaminants-being excluded. A medium inoculated and subsequently cultured in this way is said to be aseptic . 

Using sterile techniques, streak E. coli TGl strain cells from a glycerol stock onto a minimal medium plate. Grow at 37 °C for 24-36 h. ... 

It is necessary to estabUsh a criterion for microbial death when considering a sterilization process. With respect to the individual cell, the irreversible cessation of all vital functions such as growth, reproduction, and in the case of vimses, inabiUty to attach and infect, is a most suitable criterion. On a practical level, it is necessary to estabUsh test criteria that permit a conclusion without having to observe individual microbial cells. The failure to reproduce in a suitable medium after incubation at optimum conditions for some acceptable time period is traditionally accepted as satisfactory proof of microbial death and, consequentiy, stetihty. The appHcation of such a testing method is, for practical purposes, however, not considered possible. The cultured article caimot be retrieved for subsequent use and the size of many items totally precludes practical culturing techniques. In order to design acceptable test procedures, the kinetics and thermodynamics of the sterilization process must be understood. 

Another standard test, which is much simpler and more convenient, is the membrane filter technique. A suitable volume of sample is filtered through a sterile, 0.45-p.m membrane filter. The filter is placed in a petri dish containing a specific growth medium (M-Endo nutrient broth, M-Endo medium) and incubated for 24 h at 35°C. If after this time the colonies show the characteristic green sheen, this is taken as positive evidence for the presence of the coliform group (see Water, sewage). 

Sterile pharmaceutical preparations must be tested for the presence of fungal and bacterial contamination before use (see Chapters 18 and 23). If the preparation contains an antibiotic, it must be removed or inactivated. Membrane filtration is the usual recommended method. However, this technique has certain disadvantages. Accidental contamination is a problem, as is the retention of the antibiotic on the filter and its subsequent liberation into the nutrient medium. 

The build-up of waste products inhibits growth and it is necessary to change the culture medium at regular intervals, usually every few days. As with all cell culture techniques, asepsis is essential but very difficult to maintain. Antibiotics may therefore be incorporated into the culture media to assist in maintaining sterility. 

Soybean casein digest broth shall be used for normal media runs, but thioglycolate broth shall be used for the detection of anaerobic organisms, especially when a filtered nitrogen purge is used to ensure anaerobic conditions. Prepare the media according to manufacturing direction. Adjust pH the medium is sterilized by 0.2 p. Sterile filtration technique is used. 

Cooling Section For the cooling section, a quench cooler with adequate heat removal capacity is effective. Another technique is to inject the hot medium through an expansion valve into a vacuum chamber, which is known as flash cooling. Both of these take a very short time therefore, the sterilization during the cooling period can be assumed to be negligible. 

Employing aseptic techniques, the inocolum obtained is used to inoculate a 2 L Erlenmeyer flask containing a 500 ml portion of a sterilized vegetative culture medium having the following composition Soluble starch 10 g, Peptones 5 g, Beef extract 5 g, Sodium chloride 5 g, Yeast extract 2.5 g and tap water 1100 ml. 

In 1907, Ross Granville Harrison introduced tissue culture as a new technique for the study of nerve fibre outgrowth [24], At that time, it was hardly envisioned that cell culture would become the most widespread research tool in life sciences and an important method for preparing antibodies, vaccines and drugs. During the development of tissue culture, parameters such as sterility, temperature, gas mixture, medium composition and substrate features were found to be critical for... 

The in vitro technique for this species involves dissecting an infected fish aseptically and obtaining the plerocercoids in a sterile condition. This process can be facilitated by painting the surface of the fish with an aseptic solution (such as 1 % iodine in 90% ethanol), before dissection and rinsing of the removed larvae in saline and antibiotics and then culturing. Early experiments (786) established that larvae became sexually mature adult worms, when incubated in an appropriate medium at 40 °C (bird body temperature). 

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