SOP: Media for Thermal Death Time Studies

GIFSQ — Global Institute for Food Safety & Quality

Principle

Thermal death time studies require media for cultivating and sporulating the test organisms, for preparing spore suspensions, and for subculturing after heating. The book gives the composition and preparation of the standard media used in this work.

Apparatus required

• Tubes and bottles with cotton-wool plugs or caps
• Conical flask (for dissolving agar separately)
• Autoclave

Reagents

a. Basamin-Busch agar medium — Basamin-Busch 3 g, trypticase 2 g, dextrose 2 g, manganese sulphate 10 mg, dipotassium phosphate 3 g, monopotassium dihydrogen phosphate 1 g, agar 20 g, and 1000 ml of distilled water (note: “Basamin-Busch” is carried as printed; the name may be an OCR garble — check against the printed book).

b. Beef heart broth — see the SOP on preparation of spore suspension of Cl. sporogenes (PA 3679); for isolation of spores of other organisms, add 10 g of gelatin to the infusion, place residual meat particles to a height of 0.5 to 1 in. in 22 × 175 mm tubes, cover with broth to a depth of about an inch (about 15 ml), plug and sterilize.

c. Pea-peptone broth — grind 100 g of fresh peas and mix with 1,000 ml of water (if dried peas are used, simmer 100 g in 1,000 ml of distilled water for 1 hr); add 20 g of peptone and a few marble chips; distribute into tubes or flasks and autoclave for 25 min at 15 psig.

d. Potato dextrose agar — to 200 g of peeled white potatoes add 1 litre of water, steam for 1 hr, crush, and adjust the volume to 1,000 ml; to 200 ml of this infusion add 20 g of dextrose, 15 g of agar and 800 ml of water; sterilize by autoclaving at 15 psig for 15 min; cool to 120 °F and acidify to pH 3.5 with sterile 10% tartaric acid.

e. Crook’s No. 2 agar — mix peptone 10 g, glucose 20 g, magnesium sulphate 0.25 g, dipotassium phosphate 0.25 g, and agar 15 g with 1,000 ml of distilled water (adjustment of pH is not necessary); sterilize at 15 psig for 20 min.

f. Pork broth — boil 500 g of ground lean pork in 1,000 ml of distilled water slowly for about an hour; filter through cheese cloth and press the meat; cool overnight in a refrigerator; refilter through double folds of cheese cloth; to the infusion add 10 g of tryptone (bacto) and 1 g of dipotassium phosphate; adjust the pH to 7 (note: the printed “10 =” and “Adjust the H to7” are carried here as 10 g and pH 7 — unambiguous OCR garbles).

g. Pork infusion agar — prepare the pork infusion as for pork broth but do not adjust the pH; to 1,000 ml of infusion add peptone (bacto) 5 g, tryptone 1.5 g, dipotassium phosphate 1.25 g, and 1 g each of glucose and soluble starch; adjust the pH between 7.4 and 7.6; heat to boiling, add 15 g of agar and continue heating until it dissolves; sterilize at 15 psig for 30 min (the pH after sterilization should be 6.8 to 7.6).

Procedure — Basamin-Busch agar

Dissolve all the ingredients except the agar in 500 ml of distilled water; adjust the pH to 6.8–7

↓

Distribute into tubes (5 ml) or bottles; in a separate conical flask, dissolve the agar in the remaining 500 ml of water and distribute into tubes (5 ml) or bottles

↓

Plug the tubes with cotton wool or cap the bottles; sterilize at 15 psig for 20 min

↓

When required for use, melt the requisite number of agar tubes or bottles and transfer under aseptic conditions to the broth — to each tube or bottle of broth, use one tube or bottle of agar; cool and inoculate

Procedure — Pork broth for spore production

When required for spore production, distribute the residual meat particles into tubes or flasks (as in liver broth), add strips of iron and cover with the infusion; plug with cotton wool and sterilize by autoclaving at 15 psig for 20 min

Precautions

• The Basamin-Busch medium is useful for cultivation of B. stearothermophilus, particularly when good sporulation is required.
• Pea-peptone broth is an excellent medium for spore production of thermophilic anaerobes.
• Potato dextrose agar is good for the cultivation of yeasts and moulds; Crook’s No. 2 agar is useful for cultivation of a large number of yeasts and moulds for TDT studies, for inoculated packs, and for counting.
• Pork broth, like liver broth and beef heart broth, is suitable for preparing spores of putrefactive anaerobes (PA 3679); the spores produced are highly heat-resistant and not so suitable for inoculated pack studies, for which liver broth should be preferred.
• Pork infusion agar is suitable for culturing and spore counting of anaerobic bacteria in TDT studies; if required for counting of colonies, omit glucose in order to reduce the splitting of agar due to gas formation.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.