Bacteriological techniques for dairy purposes.

  • Great Britain. Ministry of Agriculture, Fisheries and Food.
Date:
1968
Catalogue details

Licence: Public Domain Mark

Credit: Bacteriological techniques for dairy purposes. Source: Wellcome Collection.

    53/192 (page 45)
    cubated to check sterility. Examine larger amounts of the sample where it is suspected that the number of organisms is below 2 per 100 mil. Immediately after the distribution in 6 x 3 in. tubes cover with grease- proof paper to protect the plugs. Place the tubes in a warm steamer or autoclave, and bring to steaming within 15 min; maintain in flowing steam _ for a further 30 min. Cool to incubation temperature. (c) Incubation (i) Total Spores. mesophiles, 3 days at 30-+-1° or 2 days at 37-+1°; thermo- philes, 2-3 days at 55 +3°. (ii) Resistant Spores. mesophiles, 21 days at 30+1° or 14 days at 37-+1°; thermophiles, 3 days at 55 + 3°. It is advisable to place plates and tubes incubated at 55° in a cannister containing moist cotton-wool, or in plastic bags. Alternatively, a large dish of water should be placed in the incubator. This prevents drying out. REFERENCE I. FRANKLIN, J. G., WILLIAMS, D. J. and CLEGG, L. F. L. (1956). ¥. appl. Bact., 19, 46. 25. COUNT OF ANAEROBIC SPORES (DILUTION COUNT TECHNIQUE) 1. Medium The medium recommended is a semi-solid broth suitable for the enumera- tion of anaerobic spores by a dilution technique (see Ref. 1) Reinforced Clostridial Medium (R.C.M,) to which is added aqueous neutral red solution to give a final indicator concentration of 1/10,000 (see Technique No. 11). 2. Dilution Counts Dilution counts on solutions containing a small number of organisms usually necessitate working with turbid solutions, and consequently there may be difficulty in reading positive growth tubes. The addition of a suitable indicator, which gives a colour change when anaerobic growth has taken place, facilitates the identification of positive tubes. Neutral red gives a yellow green fluorescence at an Eh of—o-34 V. at pH 7-0, which is in the range reached by anaerobic growth but lower than the level normally produced by aerobic growth. Neutral red is, therefore, used in detecting the presence of anaerobes. (Streptococci and Lactobacilli may also give fluorescence but as these are not sporing organisms, they should not confuse the result). The medium is dispensed in 9 ml quantities into 6 x 3} in. test-tubes and sterilized by autoclaving at 121° for 15 min. If they are not used at once the tubes of medium should be boiled for 30 min immediately before use. The number of anaerobic spores may be determined by making dilutions in R.C.M. broth and subsequent inoculation of these dilutions in 1 ml quantities into replicate tubes containing 9 ml of R.C.M. broth. When the expected count per ml is low, it is advisable to use twofold dilutions with at least four tubes at each level and at least eight levels; where tenfold dilutions are made, five tubes should be put up at each level.