EFFECT OF TEMPERATURE AND TIME OF PLATE INCUBATION ON THE ENUMERATION OF PASTEURIZATION-RESIS1ANT BACTERIA IN MILKl

This study was undertaken to determine the effect of temperature and time of plate incubation upon the count of thermoduric bacteria in milk. Specific types of thermoduric bacteria in pure culture, as well as those present in the mixed flora of commercial milk samples, were enumerated. Plate incubation at 28 C for 4 days was the temperature-time combination that produced the highest thermoduric bacterial count with laboratory-pasteurized milk. Incubation at 21, 32 or 35 C gave lower counts. Thermoduric bacteria subjected to pasteurization were more exacting in their growth temperature requirements than were unheated bacteria. Cultures of Arthrobacter sp. , Micrococcus varians and Streptococcus sp. grew over a much wider temperature range before laboratory pasteurization than after the heat treatment. The incubation temperature and time currently recommended for the standard plate c:ount, while presumably adequate for the enumeration of bacteria in raw milk, may not be equally satisfactory for the, determination of the maximum viable bacterial population of pasteurized mille. Recent changes in milk production and handling practices have necessitated reappraisal of certain bacteriological tests. Tests applied directly to raw milk are not always effective in detecting faulty production practices because the growth of bacteria in raw milk is greatly retarded by efficient cooling. Therefore, tests for specific groups of organisms that might serve as indices of contamination are now receiving considerable attention. The count of laboratory-pasteurized samples has been suggested as an index of unsanitary milk handling. The most common procedure for determining the viable bacterial population of milk is the agar plate method as outlined in Stomdard Methods for the Examination of Dairy Products (6). In this procedure, plates are incubated at 35 or 32 C for 48 3 hr for both the standard plate count of raw or commercially-pasteurized milk and the thermoduric bacterial count of laboratory-pasteurized milk. Incubation at 37 C for 2 days was originally employed to give pathogens that might be present in milk an opportunity to develop at their optimum growth temperature. This procedure had been fol'Journal Paper No. J-4666 of the Iowa Agricult~rral and Home Economics Experiment Station, Ames, Iowa. Project No. 1050. 'Present address: Division of Animal Science, University of Wyo~ing, Laramie. 'Present address: Department of Dairy Science, University of Arizona, Tucson. lowed for over 20 years before studies (2, 13, 16, 19) revealed that 32 C was nearer the optimum for many bacteria found in milk. The eighth edition of Standard Methods for the Examination of Dairy Products (3) recognized incubation at either 37 or 32 C for 48 hr for the agar plate method. Later reports (8, 24) concerning plate counts on raw and commercially pasteurized milk showed that counts after 48 hr of incubation of plates were somewhat higher at 35 than at 37 C and still slightly higher at 32 C than at 35 C. Plate incubation at 32 or 35 C for 48 hr was recognized in 1948 as standard procedure in this country (4). Although "Standard Methods" (6) continues to stipulate that plates be incubated at 35 or 32 C for 48 hr, some investigations (7, 18, 21) have suggested that longer incubation at these and other temperatures might be advantageous for the enumeration of bacteria in pasteurized milk. 'These investigations point out that the development of colonies on plates inoculated with pasteurized milk tended to be slower than on plates prepared from raw milk. Incubation for 3 ruiys is required for plate counts on dried milk (6). The present study was undertaken to determine the effect of temperature of plate incubation and length of the incubation period upon the enumeration of pasteurization-resistant (thermoduric) bacteria in milk. The identification of specific types of thermoduric bacteria was carried out, as well as their enumeration both in pure cultures and as the mixed flora of commercial milk samples. Exi•ERIMENTAL MErHODS Except for certain indicated modifications, the methods employed were those outlined in "Standard Methods" (6). To reduce the time required for preparing replicate plates, 1.0 ml and 10.0 ml pipettes graduated in tenths of a milliliter were ·used for delivery of 0.1 ml and 1.0 ml quantities. Twelve samples of manufacturing grade milk, co,oled in bulk tanks, and 20 can-cooled samples were examined. A standard plate count at 32 C was determined for each sample prior to laboratory pasteurization. A "complete immersion" laboratory p~-. teurization technique was employed. Ten ml. of ra"' D ow naded rom hp://m eridianenpress.com /jfp/article-pdf/26/11364/0022-2747-26_11_357. pdf by gest on 28 Feruary 2020 358 EFFECT OF TEMPERATURE AND TIME milk were pipetted aseptically into a sterile 125 x 15 mm test tube, using care to avoid contamination of the upper portion of the tube. The tube was closed with a sterile rubber stopper and was completely immersed in an electrically heated water bath. The pasteurization temperature was 62.5 + 0.1 C, and the pasteurization time was 30 min. Less than 5 min were required for the sample to reach pasteurization temperature. Immediately following pasteurization, the tube of milk was cooled in ice water. The tube was completely inverted 12 times; the upper part was thoroughly flamed before aliquots were removed for plating. Duplicate plates for each dilution were incubated at the following temperatures and times: Incubation temperature Incubation time 35 2 3 4 32 2 3 4 28 3 4 5 2I 4 5 7 10 7 14 21 28 At the end of each incubation period, colonies were counted with the aid of a Quebec colony counter, and the location of each colony was marked with ink on the bottom of the plate. Plates were protected from air-borne contamination by counting, unopened, in an inverted position. Various colored inks were used to designate colonies appearing after each respective incubation period. This system of colorcoding facilitated subsequent isolation and identification of bacteria according to their ability to form colonies during incubation at the various temperatures. Milk samples showing either a wide variation or no variation in thermoduric count, among the various incubation temperatures and times, were selected for study of the bacterial types encountered. Immediately after the colonies were counted, representative colonies from suitable plates were picked by a random method (15) and inoculated into tubes of sterile litmus milk. Following incubation at 32 C for 3 to 5 days, a loopful of milk from each tube was streaked onto a plate containing Plate Count Agar plus 0.25% non-fat milk solids. Surface colony characteristics were noted on the streaked plates after 72 hr at 32 C. To assure the isolation of pure cultures, a single colony picked from each streak plate was inoculated into a tube containing 5 ml of sterile litmus milk. The reaction was noted at intervals during a 14-day period at 32 C. Slants inoculated from the litmus milk tubes were mcubated at 32 C for 24 hr; smears were prepared from the slants, Gram stained and examined microscopically. A preliminary classification of the isolates. into genera was based upon cell morphology, Gram staining characteristics, reaction in litmus milk and colony characteristics. Additional cultural and biochemical testing of representative isolates verified classification into genera and, in some cases, into species. Four isolates, representative of the predominant genera of thermoduric bacteria found, were selected for study of the effect of temperature and time of plate incubation upon the growth of pure cultures before and after laboratory pasteurization. The selected isolates were classified as Microbacterium lacticum, Micrococcus varians, Streptococcus sp. and Arthrobacter sp. Stock cultures were prepared by inoculating sterile litmus milk with representative colonies from agar slants and then incubating the milk for 24 hr at 32 C. One ml of each culture was added to 100 ml of sterile reconstituted skim milk containing 10% non-fat milk solids. After thorough mixing, 10 ml of the inoculated skim milk were transferred to a sterile test tube for laboratory pasteurization. A second portion of the nonpasteurized, inoculated skim milk was refrigerated at 3.3 to 4.4 C for 24 hr before laboratory pasteurization. The pasteurized and nonpasteurized cultures were plated and incubated in the manner previously outlined for pasteurized samples of raw milk.