3. Fd Technol. (1976) 11, 427-430

Technical note: Aseptic technique for obtaining sterile beef tissue

J. BUCKLEY, P. A. MORRISSEY A N D M I C H E L E DALY

The changes which take place in meats during refrigerated storage may be caused by external bacterial sources as well as by naturally occurring enzymatic and chemical reactions. To obtain an understanding of these changes and of their relative importance in meat spoilage, it is necessary to differentiate between the bacterial-induced changes and those due to muscle autolysis. The success of such a study depends upon the method used for obtaining sterile tissue. The size of the animal from which the muscle tissue is to be taken determines to a great extent the procedure and precautions to be used. The larger the animal the more difficult it is to obtain sterile tissue and the more expensive the study becomes.

TABLE 1. Techniques for obtaining sterile muscle tissue

Method

1 Surgical room technique

2 Alcohol flame 3 Alcoholic dye-dip 4 Chlortetra-cycline solution 5 Gnotobiotic 6 Surgical isolator

Animal

Rabbit and lamb

Rabbit Rabbit Chicken Small animals Beef

Author

Zender et al. (1958) ;

Sharp (1963) Sharp (1963) Van den Berg et al. (1964) Ockerman et al. (1964) Ockerman & Cahill (1967)

Radouco-Thomas et al. (1959)

Methods of obtaining sterile muscle tissue have been reviewed by Ockerman et al. (1969) and are classified in Table 1. Sharp (1963) reported that the alcoholic-dye-dip gave sterile tissue approximately 50% of the time and he recommended the use of an alcoholic-flame procedure which gave about 70% success. This is an extremely useful method when a large number of sterile samples are required from the same muscle. The chlortetracycline procedure used by Van den Berg, Lentz & Khan (1963) and by Khan & Van den Berg (1964) can only be used in autolytic studies while the gnotobiotic method of Ockerman et al. (1964) is a very expensive procedure. Of the procedures listed above the surgical isolator procedure outlined by Ockerman (1966) and Ockerman & Cahill (1967) is perhaps the most suitable for larger animals, such as the bovine and

Authors’ address : Departments of Dairy and Food Technology and Chemistry, University College, Cork.

427

428 J . Buckley, P . A . Morrissty and Michelle Duly

porcine species. Hascgawa et al. (1970) used essentially this procedure to obtain porcine muscle, but without the surgical isolator. The procedure is time consuming and the success of the method depends entirely on having complete control over the slaughtering operation.

This note reports on an alternative and relatively inexpensive method for obtaining sterile muscle tissue from a beef carcass. The method was developed with the co-operation of a local abbatoir.

Materials and methods

Knives, calipers, grinder and grinder plates were sterilized for 15 min at 121°C prior to use. Sterile disposable gloves were worn by the operators during the entire sampling procedure.

Slaughtering and evisceration of animals were carried out in the usual manner on an automated line in a local abbatoir. The total time from stunning to entry into chill room was approximately 45 min. Carcasses were held overnight at 2" to 5°C and then boned out at 10°C. The boner wore sterile gloves and used a sterile knife to remove the strip loin or lumbar region of the longissirnus dorsi muscle (about 5-6 kg) from the carcass which was then placed in a sterile covered container and transported to the laboratory.

In this study, the samples were prepared using aseptic technique in a laminar air flow unit. Plate 1 shows the design of the laminar flow assembly. Air was filter-sterilized and blown over the table and grinder towards the operator, by an air sterilizing unit supplied by Microfilm Ltd, Fleet, Hants., England. The air flow was directed by a Perspex hood. As an added precaution the table and hood were washed with a hypochlorite solution prior to use.

The strip-loin with skin-side down, was placed on sterile aluminium foil and an incision made longitudinally along the exposed surface with a sterile knife. A second sterile knife was used to strip off a 2-3 cm thick slice longitudinally along the strip loin while a second operator held the slice with a sterile calipers. This was performed on both sides of the incision. Slices about 2 cm thick were then removed aseptically from the exposed interior and transferred to a sterile container. It is possible to obtain 2 kg sterile tissue from a 5 kg striploin using this procedure. The excised slices were ground through a sterile grinder having a 4 mm grinding plate and collected in a sterile container. Samples were then prepared for each sampling period by transferring approximately 5 g minced tissue to sterile 50 ml screw-cap bottles with caps loosely screwed on so as to maintain aerobic conditions. Using one bottle at each sampling period the danger of contamination during the storage period was minimized. All samples were stored at 7°C until required.

A number of spoilage type microorganisms which were isolated from spoiled beef, were purified and reinoculated into sterile mince. The reinoculated mince was then distributed in 5 g amounts into sterile 50 ml screw-cap bottles as for the sterile mince

Sterile beef tissue

(Facing p. 428)

Sterile beef tissue 429

TABLE 2. Typical example for microbial numbers and pH values for sterile control and reinoculated samples

Enterobacter Non-pigmented Control aerogenes pseudomonas Day

S.P.C. PH S.P.C. PH S.P.C. PH

0 4 6

1 1 13 15

20 25

a

ia

None None None None None None None None None None

5.64 5.68 5.72 5.62 5.72 5.66 5.75 5.63 5.70 5.70

1 .ox 10%

4.0 x 10' 1 - o x 108 1 *ox 10'0 2.0 x 10'0 3.0 x 10'0 1.0 x 10'0 1 a 0 x 10'0 1.0 x 10'0

2 . 0 ~ 104 5.70 5 -69 5.65 5.60 6.64 6.50 6.86 6.83 6-94 7.0

7.0 x 104 3.0 x 106 1 -4 x 10'0 2 . 1 x 10'0 3.0 x 10'0 I * o x 10'0 1 . o x 10'0 2.5 x 1010 1 * 9 x 1010 1.6 x 1010

- 5.79 6.56 7.42 7.66 7.96 7.98 7.90 7.94 7-85 7.90

and stored at 7°C. Every other day one each of the control and inoculated samples were analysed for total counts using the standard plate count at 20°C for 72 hr, during a storage period of twenty-seven days. This procedure has been used on six occasions up to this time. On each occasion approximately 200 x 5 g samples have been prepared, half of these being used as sterile controls. Only two bottles out of a total of 600 controls have been found to contain any bacteria. I t is critical to use aseptic precautions at all stages particularly, during the transfer into the sterile 50 ml bottles.

Table 2 summarizes and is a typical example of the microbial numbers and pH values obtained for the sterile control plus two species of bacteria which had been purified and reinoculated.

Acknowledgments

The authors wish to thank Internation Meat Packers, Midleton, Co. Cork for their valued co-operation and assistance during the course of this work. We are grateful to the National Science Council, Dublin, for a grant to finance this project.

References HASEGAWA, T., PEARSON, A.M., PRICE, J.F. & LECHOWICH, R.V. [ 1970) ApPl. Microbiol. 20, 117. KHAN, A.W. & VAN DEN BERG, L. (1964) 3. Fd Sci. 29, 49. OCKERMAN, H.W. (1966) 3. Enu. Hlth, 29, 243. OCKERMAN, H.W. & CAHILL, V.R. (1967) The National Provisioner, 156 (lo), 35. OCKERMAN, H.W., CAHILL, V.R., DAVIS, K.E. & DAVIS, C.E. (1964) 3. Anim. Sci. 23, 142.

430 J . Bucklty, P . A . Morrissey and Michelle Dab

OCKERMAN, H.W., CAHILL, V.R., WEISER, H.H., DAVIS, C.E. & SIEFKER, J.R. (1969) 3. Fd Sci. 34,93. RADOUCO-THOMAS, C., LATASTE DOROLLE, C., ZENDER, R., BUSSET, R., MEYER, H.M. & MOUTON, R.F.

SHARP, J.G. (1963) 3. Sci. Fd Agric. 14,468. VAN DEN BERG, L. LENTZ, C.P. & KHAN, A.W. (1968) Fd Technol., Champaign, 18, 135. ZENDER, R., LATASTE-DOROLLE, C., COLLET, R.A., ROWINSKI, P. & MOUTON, R.F. ( 1958) Fd Res. 23,305.

(1959) Fd Res. 24,453.

(Received 8 February 1976)