Overnight broth culture

If larger culture volumes are required then 2.5-L Ehrlenmeyer flasks containing a maximum of one liter of Mueller-Hinton broth are prepared and autoclaved. Once cooled, this broth may be seeded directly with colonies from the agar plate however, this often results in poor yields and slow growth. It is better to seed this volume of culture with a small amount of overnight broth culture. 

Sterile graduated pipettes of 10, 5, 2 and 1 ml, sterile capped 7.5 x 1.3 cm tubes/ small screw-capped bottles, Pasteur pipettes, an overnight broth culture of the test and control organisms (same as for disc diffusion tests), the required antibiotic in powder form, the required solvent for the antibiotic, sterile distilled water - 500 ml and suitable nutrient broth medium. A suitable rack to hold 22 tubes in two rows, i.e., 11 tubes in each row. 

A modification of the Szybalski method was used for the in vitro tests (Szybalski, 1958). Molten 0.6% agar (2 ml) was added to 0.1 ml of an overnight broth culture of Salmonella and the mixture was poured over the surface of a minimal agar base plate. After incubation, a positive result is indicated by a ring of mutant colonies around the sample of test compound. 

Inoculate five 45-ml sporulation agar slopes (in 100-ml medical flats) with approximately 1.0 ml of the 24-hour broth culture and incubate at 32 2°C for 5 days. Concurrently, plate the 24-hour broth culture onto TSA to check for purity. Incubate at 32 2°C overnight. The next day, if it is pure, discard otherwise purify it. 

A culture of Bacillus polymyxa in a tube with Trypticase soybean broth was incubated overnight at 25°C. 5 ml of this culture was transferred to 100 ml of the tank medium in a 500 ml Erlenmeyer flask which was incubated for 48 hours at room temperature. This 100 ml culture served as inoculum for one tank. During the course of fermentation the medium was aerated at the rate of 0.3 volume of air per volume of mash per minute. The temperature was maintained at about 27 C. Samples of mash were taken every 8 hours in order to determine pH and the presence of contaminants and spores. After 88 hours of fermentation the pH was about 6.3 and an assay using Escherichia coll showed the presence of 1,200 units of polymyxin per cubic centimeter. The polymyxin was extracted and purified by removing the mycelia, adsorbing the active principle on charcoal and eluting with acidic methanol. 

The culture broth was recovered after 72 h of fermentation, the biomass removed and the total protein content measured. Broth aliquots with a protein content of 1 mg were collected and their pH regulated at different values ranging from 3.5 to 8.0. To each broth fraction, 50 mg of the microspheres sample, previously equilibrated at the corresponding pH, was added and the suspension left under stirring overnight. Then, the microspheres were removed by centrifugation and the protein content and the PG activity were assayed on the resulting supernatant. 

Appropriate Xenorhabdus or Photorhabdus bacterial cells are aseptically transferred to 5 ml of nutrient broth in a test tube and kept overnight on a shaker. The flasks containing autoclaved material are inoculated with the bacterial culture by pouring the contents of one culture tube. The flask is shaken well and stored for 2-3 days at 25°C to allow multiplication of the bacteria. 

Inoculate 2-L baffle flasks containing 500 mL LB broth and 100 pg/mL ampicil-lin with 1 mL of the washed overnight culture, and grow at 30°C in an orbital incubator until the absorbance at 600 nm is approx 0.6... 

Add 0.1 mL of an overnight culture of E. coli strain C600, grown m nutrient broth containing 0.2% maltose, to 10 mL of fresh broth. Incubate the cells at 37°C, with shaking until they reach an absorbance of between 1 4—1 8 at 450 nm (A450 0.64 = 2x 10 cells/mL). This takes approx 2—3 h. 

In the morning, BHI (brains-heart infusion) nutrient broth was inoculated with bacteria, from an overnight culture or agar, to be used within 4 h. Within that incubation time, Mueller-Hinton (MH) plates were allowed to reach room temperature. 

It is useful to spread the cells remaining after amplification (Subheading 3.2.9.) to obtain single colonies for screening. Add 2 mL of LB broth to the pellet of overnight culture. Make serial dilution (10 , 10-5, 10 should be fine) in LB medium. Use 200 pL to spread on a plate. 

To prepare a seeding culture, fill a 25 pL universal with approximately 10 pL of sterile Mueller-Hinton broth and seed these with 1-2 colonies from the blood agar plate. This is then incubated at 37°C overnight with vigorous shaking at approximately 200 rpm. 

Grow 50 ml of an overnight culture (—18 hr) in Luria broth, NZYM, or Circle Grow medium (BIO 101 Labs, La Jolla, CA) with shaking (225 rpm). Harvest by centrifuging at 7000 g for 5 min. 

The night before the experiment, grow 5-ml overnight cultures of each of the following E. coli strains in YT broth with shaking at 37°C. 

Prepare a 50-ml overnight culture of E. coli (strain TGI) containing pGEX-2T in YT broth with 100 /tg/ml ampicillin. Incubate with shaking at 37°C overnight (—20 hr). 

The next morning, inoculate 2 liters of fresh YT broth containing 100 /tg/ml ampicillin with the 50 ml overnight culture. Incubate at 37°C with shaking for 1 hr. 

Dilute culture into 250-500 mL of Luria broth with 100 pg/mL of ampicillin, and grow overnight. 

Add 2 mL of the overnight culture to 200 mL of L-broth. Continue incubation at 37°C with vigorous shaking (300 cycles/min in a rotary shaker) until the culture has reached the logarithmic phase of growth. This normally takes 3 h. 

For the long-term storage of bacteria inoculate the colony in 1-2 ml of LB broth and ampicillin (100 mg/ml), place in culture overnight at 37 °C until saturation. Mix gently 85 pi culture medium with 150 pi of glycerol and store at -80 °C. The bacteria in this way can be preserved for long periods of time and can be possibly reseeded in liquid medium to perform the purification of the plasmids with commercial kits. 

Obtain suspensions of microorganisms by overnight growth at 37 C of stock cultures in nutrient broth (S. enteritidis or coryneform broth L. monocytogenes. ... 

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