Tryptose phosphate

In the procedure for the surface test (313), the vims is grown in a monolayer of baby hamster kidney cells and incubated in Eagles medium supplemented with tryptose phosphate broth and calf semm. After separation of the vims from the cells by sonification and centrifugation, amounts of the suspension containing 3 x 10 plaque-forrning units are dried on coversHps. The inoculated coversHps are placed in 5 ml of the disinfectant for 1, 5, or 10 min, then rinsed, sonicated, and assayed. 

The samples of medium taken for sterility checks may require the presence of protein or peptides to allow growth of potential contaminants Thus, about 2 mL of sterile serum or tryptose phosphate broth (TPB) are added before leaving the samples at 37°C for as long as possible. Because the rate of contamination may be very low or the presence of antibiotics retard growth, 100-mL samples may not be sufficient to detect this. Therefore, the last bottle of a batch of medium should be left at 37°C and monitored. 

In the early work, embryo and tissue extracts were commonly used to encourage growth of cells but now these are only occasionally added to medium and then only for cultivation of certain primary cells (e.g. see 2.2 and 2.3). Today s media, however, as well as containing amino acids, vitamins, salts and glucose normally include serum at about 10% and sometimes also other additives such as Bactopeptone or tryptose phosphate (Appendix 1). 

There are a number of media available which are not based on a detailed investigation of growth requirements, but rather include crude mixtures of nutrients added to promote cell growth. These include lactalbumin hydrolysate (Appendix 1 Table 9) or yeast extract (Appendix 4) to provide an inexpensive source of amino acids or vitamins. Thus Melnick s monkey kidney media A and B (Melnick, 1955) contain lactalbumin hydrolysate and calf serum in Hanks and Earle s BSS, respectively. Chick embryo extract and tryptose phosphate broth (Appendix 1, Tables 11 and 12) are also used occasionally and their use is referred to where appropriate throughout the book. Mitsuhashi and Maramorosch mosquito cell medium contains lactalbumin hydrolysate, yeast extract and foetal calf serum in a specially developed saline (Mitsuhashi and Maramorosch, 1964 Singh, 1967). 

Resuspend the cells in 20 ml warm growth medium, e.g. medium 199 containing tryptose phosphate (2%) and calf serum (2%). Allow the large clumps to settle and transfer cell suspension to a universal container. 

Add 20 ml calf or foetal calf serum and 20 ml tryptose phosphate broth (Appendix 1) to 80 ml double strength medium (e.g. Glasgow MEM Appendix 1) and warm the mixture to 44°C. 

Various other broths for detecting bacterial and fungal contamination include brain heart infusion broth, tryptose phosphate broth and trypticase soy broth. These should be made up as per manufacturers (Oxoid Ltd. or Difco Labs. see Appendix 3) instructions, but some procedures are given in Appendix 4. 

Suspend BHK21/C13 cells at 106/10 ml Eagle s Glasgow modification supplemented with 10% tryptose phosphate, 0.05 mM L-serine, 0.1 mM L-ornithine, 0.1 mM hypoxanthine and 0.25% calf serum and plate into 5 cm dishes. With this parent cell line very little DNA synthesis or cell growth occurs, but with the polyoma transformed PYY cells two doublings occur in low serum. 

Seed 5 X 10s BHK21/C13 cells into 100 mm dishes in Dulbecco s modification of Eagle s medium supplemented with 10% calf serum, 10% tryptose phosphate and bromodeoxyuridine at 3.3fig/ml. This kills most of the cells and after 10 days only about 20 cells have survived to form colonies. 

Seed 12 roller bottles each with 2 X 107 BHK C13 cells in ETC10 (Eagle s medium containing 10% tryptose phosphate broth plus 10% calf serum) and grow at 37°C for 3 days. 

After 1 h add 5 ml Eagle s medium +10% tryptose phosphate broth +10% calf serum (pseudorabies) or 5% human serum (herpes) and return to the incubator. 

Tryptose phosphate (Difco bacto or Oxoid - Appendix 3) Distilled water to... 

Tryptose phosphate broth (Difco) Thallous acetate (1.25%)... 

Add the horse serum, yeast extract, tryptose phosphate broth, thallous acetate, and penicillin to the agar. 

EFC EGF EMS ESG ETC Eagle s MEM supplemented with foetal calf serum epidermal growth factor ethyl methane sulphonate Ewing sarcoma growth factor Eagle s MEM supplemented with tryptose phosphate (10%) and calf serum (10%)... 

Bacterial counts on the inoculated sewage and the treated sewage were determined by appropriate dilution and plating on tryptose phosphate dextrose agar. The plates were counted after 24 to 48 hours incubation at 37° C. In addition, in several experi-... 

Consideration may have to be given to using a supplemented medium during the initial stages of culture to aid cell attachment and to offset the effects of low cell density, which will be more critical in microcarrier than stationary culture. The supplementation may be simple, e.g. non-essential amino acids, pyruvate (0.1 mg mH), adenine (10 jxg mH), hypoxanthine (3 xg ml" ) and thymidine (10 xg mH). Other supplements include tryptose phosphate broth (1 mg mH), HEPES (5 mM), transferrin (10 mg T ) and fibronectin (2 xg mH). Serum, unless serum factors are added, may have to be used at 5-10% initially before being reduced after 1-2 days of culture. 

---