Aeromicrobiology: an assessment of a new meat research complex.

Airborne microorganisms in food processing plants are extremely important because of the economic and health problems they may cause. Controlling the sources of microbial contamination in food processing plants can greatly reduce the chance of producing harmful food products (1).

Sneezing, talking, laughing, falling hair, using soiled laboratory coats, as well as shedding from hands and arms contributed to the microbial contamination of air in food processing plants (2). Immediate washing of floors containing materials that may support microbial growth has been observed to be critical in reducing the airborne counts in food processing plants (3).

The raw materials in processing plants contribute a major part of the microbial air contamination. From these raw materials, microbes may be deposited into the air during handling and processing of the raw materials. The high number of microorganisms in the first stages of food processing confirm the influence of raw materials on the microbial count in the air (4).

Monitoring the microbial population in the atmosphere surrounding food during processing is important in discovering possible emerging sources of contamination. These sources may include workers, incoming air and raw materials.

There are two main methods by which airborne microbes can be sampled: 1) collection into a liquid (buffer solution or liquid medium); and 2) collection onto solid and semi-liquid media or filters. Conditions such as particle size, wind speed and wind direction influence the efficiency of airborne particle collection. More information on air sampling can be obtained from the review papers of Al-Dagal and Fung (1) and Kang and Frank (5).

The main objectives of this study were 1) to monitor the microbiological quality of air in a new meat processing laboratory before and after occupancy; 2) to study the microbial profile of air in the new laboratory; 3) to monitor the effects of temperature, humidity, number of people and their activity on the microbial populations in the new meat processing complex; and 4) to study the microbiological profile before, during and after the slaughtering operation in the slaughtering room.

Materials and methods

This study was conducted in the new meat laboratory complex at Weber Hall of the Department of Animal Sciences and Industry, Kansas State University. The sampling operation began in March 1988 and finished in April 1989. The laboratory complex consists of three main rooms, three coolers, two freezers, a smoke house and a slaughtering room. The air was sampled at the designated sites, which included Sites 1, 2/1, 2/2, 2/3, 3 and 4. The microbial data from different spots (* and * *) were averaged and reported as the count of each site. In a pilot plant operation, slaughtering of animals (beef cattle, pigs and sheep) was usually conducted every Tuesday and further processing of meat for research or teaching took place during the rest of the week.

The instrument used to collect the microorganisms from air was the Surface Air Sampler (SAS, Pool Bioanalysis Italiana, Milano, Italy). The function of the SAS is to suck air at a fixed rate--3 liters/second--through a cover, which has been designed with small holes that direct the air flow onto a plate containing a suitable agar medium. The desired volume of air to be tested can be chosen by a 15-unit knob attached to the system. Practical use of this instrument in air microbiology was described by Ligugnana and Fung (6).

Temperature and humidity gauges (Scientific Device Laboratory, Inc., Glenview, Illinois) were used to monitor each site at the time of sampling. It took 15 minutes for the humidity gauge to equilibrate at each site.

Ethyl alcohol was applied as a sanitizer to kill the microorganisms charged to the SAS cover from the previous sampling operation.

Plate count agar (PCA, Difco) was the medium used in recovering air microbes. PCA was prepared and poured into 65 x 15 mm-Rodac plates (Replicate Organisms Detection and Count, Becton Dickinson and Company, Oxnard, California) three to five days before each sampling period. Violet Red Bile Agar (VRB) was used as a selective medium for recovery of gram-negative bacteria.

Sampling protocol

Air in the meat laboratory complex was sampled in two distinct time periods (14 weeks each); the first period was before the occupancy when no operation related to meat processing was occurring, and the second period was after the researchers and students had started processing meat. Dust, open rooms and construction materials were the predominant elements in the first period. Different conditions including continuous cleaning, controlled temperature and air flow, and increased number of people with different types of activity existed in the second period. The air of the different sites was sampled once a week. One SAS unit drawing 60 liters/20 seconds was chosen for each position in each site for the airborne plate count (ABPC). Using VRB agar, coliform bacteria were enumerated four weeks before occupancy and four weeks after occupancy. The air samples were taken (sucked downward) at a 4.5 to 5 foot height. Alcohol (95%) was applied after each sampling operation to sanitize the SAS cover. The temperature, humidity, number of people and their activities, and the presence of meat were always recorded.

To study microbial loads before, during and after slaughtering operations, air of the slaughtering room and one cooler (2/3) was sampled for eight weeks. The samples were taken before the slaughtering operation, two times during the operation (two hours apart), and a final sample was taken two to three hours after the slaughtering room was cleaned. The sample from the cooler (site 2/3) usually was taken as soon as the carcasses were moved inside. Besides one unit used for testing ABPC, two units (120 liters/40 seconds) were selected for the coliform count (ABCC). After each sampling operation, the ABPC plates were incubated at 32 |degrees~ C for 48 hours and the VRB plates as 32 |degrees~ C for 24 hours before colonies were counted. Data from different spots of the same site were averaged. The airborne mold counts (ABMC), which were differentiated from bacteria by colony morphologies, were obtained along with the ABPC from PCA incubated at 32 |degrees~ C for 48 hours.

Calculation and statistical analysis of the ABPC

The ABPC of microbes obtained from 60 liters of air onto one plate containing PCA was converted to ABPC per cubic meter as follows:

# of microbes on one plate ------------------ x 1000 = CFU/|m.sup.3~ 60 liter x unit(s) (60L/20 Sec.)

Descriptive analysis was used for the total airborne counts obtained from sites other than slaughtering room. A multiple comparison analysis was used to compare the ABPC before, during and after the slaughtering operation (7).

Identification protocol

For identification of isolates, as many as eight colonies were randomly selected from rodac plates and transferred with sterilized toothpicks into microtiter wells containing PCA. Sterile microtiter plates (Dynatech Laboratories, Inc., Chantilly, Virginia) containing PCA were used to stock isolated colonies for identification. The plates were then incubated at room temperature for 24 hours for colonies to grow and then refrigerated for further steps in identification of unknowns. About 24 hours before starting the identification process, Brain Heart Infusion broth (two drops) was added to each microtiter well containing isolated cultures. The plates then were incubated at room temperature for 24 hours to activate the cultures. After the incubation period, cultures from this microtiter plate were transferred to another microtiter plate containing PCA and kept as a reference.

With a multi-inoculator (8), all 96 cultures were smeared onto four neighboring microscopic slides simultaneously for gram staining. This procedure provided information on isolates concerning purity and gram morphology (positive or negative, rod or coccus). Yeast cells could also be recognized. Biochemical test data were used only for pure cultures. Descriptive characterization of bacteria to genus level were made with the scheme described by Gailani (9), who obtained information from Buchanan and Gibbon (10).

A motility test (motility agar, Difco) and catalase test were also conducted in microtiter plates. From the original microtiter plate, cultures were stabbed into another microtiter plate containing motility agar (Difco) with a 96-pinpoint multi-inoculator. The motility results were read after incubation for 48 hours at 37 |degrees~ C with the help of a magnifying glass.

The catalase test was conducted by placing one drop of 3 % hydrogen peroxide (Fisher Scientific Co., St. Louis, Missouri) into each well containing reactivated isolates. Formation of bubbles indicates a positive catalase test.

Methyl red agar was used to test the anaerobic fermentation of glucose. This test was used to differentiate between Staphylococcus and Micrococcus. The nonmotile, gram positive and catalase positive microorganisms were individually stabbed into tubes containing methyl red agar (with added glucose). Mineral oil was added to each tube, and then the tubes incubated at 32 |degrees~ C for 48 hours. Staphylococcus provided a positive test (a yellow tube indicates fermentation) and Micrococcus gave a negative test (no color change indicates lack of fermentation). Mold counts (observation of mycelium and sporulations) were obtained along with the total microbial counts grown on PCA at 32 |degrees~ C for 48 hours.

Results and discussion

The opportunity to test air quality before operation of a new meat processing laboratory provided an excellent chance to determine the background of microbial populations in the air of the facilities. The information, which includes the type, number of microorganisms, and the conditions influencing their presence, can be used for comparison with similar data after the facilities are used for teaching and research in meat processing.

In analyzing the obtained data, the total microbial count and mold count were plotted against time in weeks. A ranking system was designed to categorize the microbial counts in air before and after the occupancy. The designated ranges were:

* |is less than~100 CFU/|m.sup.3~, low count;

* |is greater than~100 CFU/|m.sup.3~, intermediate count; and

* |is greater than~300 CFU/|m.sup.3~, high count.

The three ranges were based on the descriptive analysis accompanying the sampling operation throughout the 28-week period. It was found that whenever the air sample was taken from a clean site, with low temperature, less human activity, ventilation and short stay of the meat, the total microbial counts were in the range of 100 CFU/|m.sup.3~ or lower. More activity (mostly meat processing) with higher numbers of people and less cleanliness resulted in moderate microbial count that ranged between 101 and 300 CFU/|m.sup.3~. Higher numbers than 300 CFU/|m.sup.3~ were associated with the presence of dust, high number of people (nine to 20), and live animals. These three ranges were applicable to the meat research complex at KSU or similar meat processing plants. Different ranges can be set in a similar way in meat processing plants with different conditions or other food processing plants.

Analysis of ABPC at Site 1 (teaching laboratory)

The microbial, temperature and humidity profiles of site 1 (teaching laboratory) are presented in Figure 2. In the first period of this study, the temperature range was between 23 and 26 |degrees~ C. The relative humidity (RH) was between 54 and 75 percent. The ventilation system was not in operation (at all sites) throughout the first period (except in the second week for equipment testing). More dust and construction personnel were in the facilities during the first 11 weeks of the first period, compared with weeks 12, 13 and 14, when preparations were started for occupancy. The ABPC at site 1 ranged from 37 CFU/|m.sup.3~ in the thirteenth week, when construction personnel started to move their tools and wash the floor, to 580 CFU/|m.sup.3~ in the first weeks of sampling. In the second period of this study (weeks 15 to 28) temperature at site 1 declined to 10 to 21 |degrees~ C, ventilation had begun to operate, continuous cleaning was initiated, and the airborne microbial counts dramatically declined. This site was also the least frequently used after occupancy.

Figure 3 shows the changes in the percent of samples that fell in each of the three microbial ranges before and after the occupancy of site 1. Before occupancy, the number of samples in each range was similar. After occupancy, however, a large percentage of samples fell in the low range and a few in the intermediate range, whereas none was found in the high range.

Sites 2/1, 2/2 and 2/3 (coolers) had the same conditions in the first period of air sampling. The temperature ranged from 22 to 26 |degrees~ C, and RH from 54 to 75 percent. Some dust, construction tools and working people (eight or fewer) existed at some sampling periods. The ABPC in these sites were in the range of 110 to 530 CFU/|m.sup.3~, which were relatively high.

In the second period (after occupancy) of sampling, the three coolers had different conditions that affected the microbial profiles.

Analysis of ABPC at site 2/1 (cooler) after occupancy

In site 2/1, the temperature was in the range between 0 and 6 |degrees~ C. The RH ranged from 58 to 72 percent. Higher microbial counts were obtained at this site because of higher frequency of the presence of meat carcasses during the sampling period, compared with coolers 2/2 and 2/3. The total airborne counts ranged between 33 and 340 CFU/|m.sup.3~, but 57.14 percent of the samples were higher than 100 CFU/|m.sup.3~. This result indicates that the presence of meat cuts for a relatively long time (three to five days) affected the microbial load in the air of the cooler. With the exception of the fifth and the sixth week, the mold count in the second period of site 2/1 was similar to results obtained in the first period (before occupancy), where more than 90 percent of the samples had lower than 5 CFU of mold/|m.sup.3~. Figure 4 shows the microbial, temperature and humidity profiles in both periods (before and after occupancy).

Figure 5 indicates the changes in the microbial ranges before and after occupancy at site 2/1. Before occupancy, most samples were in the intermediate to high microbial ranges, whereas, after occupancy, more samples fell in the low microbial range.

Analysis of ABPC at site 2/2 (cooler) after occupancy

In site 2/2 (cooler), the temperature readings were below 4.5 |degrees~ C for all samples in the second period. The RH was in the range between 62 and 75 percent. This cooler was used least for meat chilling. At this site, 80 percent of the samples had lower than 100 CFU/|m.sup.3~. The mold counts were less than 17 CFU/|m.sup.3~ in 95 percent of the samples. Figure 6 shows the microbial, temperature and RH profiles at site 2/2. Figure 7 indicates that changes in the microbial ranges occurred before and after occupancy at site 2/2. Before occupancy, all samples were higher than 100 CFU/|m.sup.3~, whereas, after occupancy, most of the samples were in the low microbial range.

Analysis of ABPC at site 2/3 (cooler) after occupancy

The human activity and total microbial count at site 2/3 (cooler) were between those observed and collected at sites 2/1 and 2/2. With the exception of the 24th week, the temperature readings were 4.5 |degrees~ C or less. The RH was in the range between 65 and 75 percent. This cooler was always used to receive the carcasses after slaughtering operations. Figure 8 shows the microbial, temperature and RH profiles in site 2/3.

Figure 9 indicates the changes in the microbial ranges occurred before and at occupancy at site 2/3. Before occupancy the microbial counts were all above 100 CFU/|m.sup.3~, but after occupancy most samples (78.57 percent) fell in the low microbial range.

Analysis of ABPC at site 3 (meat fabrication laboratory)

The conditions at site 3 (meat fabrication laboratory) presented in the first period were similar to those in the other sites. The temperature was in the range between 22 and 26 |degrees~ C, and the RH between 54 and 73 percent. Dust and number of people were similar to those in site 1, except in the 12th week, when more people and equipment were present in site 3. The total airborne counts in the first period range between 107 and 833 CFU/|m.sup.3~ in the first 12 weeks, but were lower (73 and 87 CFU/|m.sup.3~) in the 13th and 14th weeks, as a result of clean-up operations. In the second period of air sampling, the temperature at site 3 declined and remained at the level between 6 and 13 |degrees~ C. The RH ranged from 55 to 78 percent. During most of the sampling periods, from three to seven people were found working with meat cuts. About 60 percent of the samples had total microbial counts less than 100 CFU/|m.sup.3~, and the rest of the samples were in the intermediate range (100 to 300 CFU/|m.sup.3~). The mold count was similar to those in the first period, except in the sixth and seventh weeks. Figure 10 shows the microbial, temperature and RH profiles in site 3.

Figure 11 indicates the changes in the microbial ranges before and after occupancy of site 3. Before occupancy all samples were above 100 CFU/|m.sup.3~, whereas, after occupancy, most of the microbial counts were in the low and intermediate ranges.

Analysis of ABPC at site 4 (slaughtering room)

The last part of this study involved an analysis of airborne microorganisms in the slaughtering room, as well as in site 2/3 (cooler), which was the receiving room for the carcasses. In the slaughtering room air quality was monitored only after occupancy of the complex. The temperature before, during and after the slaughtering operation ranged from 12 to 21 |degrees~ C, and the RH from 30 to 86 percent. Higher RH readings were recorded during the slaughtering operation because of the use of water and boiling water. The site was always watered and soap-washed after slaughtering. During operation, animals including cattle (one to three), pigs (three to six) and/or sheep (two to four), as well as from nine to 20 students and personnel, were in the slaughtering area. A multiple comparison procedure was used to ascertain if there were significant differences in the total airborne counts before (31 CFR/|m.sup.3~); hour 0 (796 CFU/|m.sup.3~, at the beginning of operation); hour 2 (564 CFU/|m.sup.3~, two hours after operation); and after (56 CFU/|m.sup.3~) the slaughtering operation. There was no significant difference (P|is less than~0.05) in the total airborne counts before and after slaughtering (31 CFU/|m.sup.3~ vs. 56 CFU/|m.sup.3~) and at hour 0 and hour 2 (796 CFU/|m.sup.3~ vs. 564 CFU/|m.sup.3~). A significant difference (P|is greater than~0.05), however, was found between the total counts during slaughtering (hour 0, 796 CFU/|m.sup.3~; and hour 2,564 CFU/|m.sup.3~) versus before (31 CFU/|m.sup.3~) or after (56 CFU/|m.sup.3~) slaughtering.

From Table 1, we concluded that the total airborne counts were higher during slaughtering than before or after slaughtering. Airborne mold counts were similar in all sampling periods. The airborne coliform bacteria were only detectable during slaughtering. These findings indicate that live animals entering the facilities were responsible for the presence of coliform bacteria, as well as the high airborne microbes during the slaughtering operation.

The determination of the total airborne count in site 2/3 (cooler) after receiving the carcasses from the slaughtering room showed similar results (87.5 percent of the samples had lower than 100 CFU/|m.sup.3~) to the samples (87.57 percent of the samples had lower than 100 CFU/|m.sup.3~) taken at the beginning of this investigation (the second 14 weeks of the initial sampling of this site). This result indicates that after the slaughtering operation, effective water-jet cleaning of the carcasses before sending the meat to the cooler (2/3) will reduce microbial contamination of the air in the cooler. About 25 percent of the samples (eight samples) had coliform bacteria on VRB medium. The mold count did not exceed 17 CFU/|m.sup.3~. Table 2 shows the total airborne, mold and coliform counts after receiving the carcasses in site 2/3.

In general, the conditions (temperature, RH, type and intensity of activities and cleanness) of all sites studied were similar in the first period (during construction). A decline of the microbial load was observed at the last few weeks of the first period because of the reduced construction activity and more clean-up operations.

The conditions were dramatically changed in the second period of this study. The temperature declined in the coolers to less than 6 |degrees~ C, in the fabrication room to the range of 8 to 13 |degrees~ C, and in the teaching laboratory to the range of 10 to 21 |degrees~ C. The TABULAR DATA OMITTED relative humidity increased, especially in the coolers, because of the continuous use of water and the presence of meat. The simultaneous presence of desirable factors, including low temperature, frequent cleaning, ventilation and good handling of the meat cuts, resulted in an acceptable level (|is less than~ 100 ABPC/|m.sup.3~) of the total airborne count. Although low temperature in a cooler usually results in lower microbial counts in the air, the results of this study indicate that site 2/1, which had similar conditions (including low temperature) to site 2/2, had higher microbial counts (153 ABPC/|m.sup.3~ vs. 52 ABPC/|m.sup.3~). This was because of the presence of carcasses in site 2/1 versus absence or short stay of carcasses in site 2/2.

Table 2.
Total airborne, mold and coliform counts after receiving
carcasses in site 2/3 (cooler)
Week        ABPC(1)          ABMC             ABCC
        CFU/|m.sup.3~    CFU/|m.sup.3~    CFU/|m.sup.3~
1             50               ND                4
2             33               ND               ND
3            125               ND                4
4             92               17               ND
5             42               ND               ND
6             17               ND               ND
7             92                8               ND
8             75               ND               ND
ABPC = airborne plate count
ABMC = airborne mold count
ABCC = airborne coliform count
ND = not detectable
1 Total numbers per |m.sup.3~

In the slaughtering room (site 4), most of the total airborne counts, as well as the coliform counts, were attributed to dirt, hair and activities of live animals.

The pattern (decline of ABPC as the carcasses are further processed) has not been found only in meat processing plants, but also in poultry processing plants (11).

Identification of airborne microbial isolates

Another aspect of this investigation was to determine the kinds of microorganisms in the air before and after occupancy of the facilities. The morphological, physical and biochemical analysis of 728 microbial isolates from the first period (before occupancy) of the study showed that 38.9 percent was gram-positive cocci (most of which were Micrococcus); 56.6 percent was gram-positive rods (most of which were Bacillus); and 4.5 percent was mixed cultures. After occupancy, microbial identification of 444 isolates indicated that 25 percent was gram-positive cocci (most of which were Micrococcus); 46.4 percent was gram-positive rods (most of which were Bacillus); 18.5 percent was yeasts; 3.6 percent was mixed cultures; and 6.3 percent was dead cultures (no growth at the time of identification). Identification of mold was not made in this survey study, although mold counts were made.

It is noteworthy that gram-negative cells were not isolated from non-selective medium (plate count agar), indicating that the number of gram-negative cells were much lower than gram-positive cells in the air before and after occupancy of the facilities. ABCC data during the two periods also showed negative growth of gram-negative bacteria on VRB agar. Other investigators (4) also presented similar data. The appearance of large numbers of yeast (18.5 percent) after occupancy compared with non-detectable yeast isolates before occupancy indicates that yeast in the air was associated with meat coming into the facilities. The role of yeast in meat microbiology is not entirely clear and may deserve further investigation. With the use of selective medium (VRB), coliform bacteria were found in the slaughtering room and in the samples taken from site 2/3 after receiving the carcasses. However, the numbers were low (5 and 4 ABCC/|m.sup.3~). Molds were found before and after occupancy of the facilities at low levels (|is greater than~99 percent of the samples were |is less than~100 ABMC/|m.sup.3~).

References

1. Al-Dagal, M. and D.Y.C. Fung (1990), Aeromicrobiology: a review. Critical Review in Food Science and Nutrition, Academic Press, 29(5):333-340.

2. York, G.K. (1973), Airborne microorganisms in meat plants. Proceeding of Meat Processing Conference, University of California-Davis, March 28-29, 1973. Pp. 42-45.

3. Heldman, D.R. and T.I. Hedrick (1971), Airborne contamination control in food processing plants, Mich. State Ag. Exp. Station Res. Bull. 33, pp. 1-78.

4. Kotula, A.W. and B.S. Emswiler-Rose (1988), Airborne microorganisms in a pork processing establishment, J. Food Prot. 51:935-937.

5. Kang, Y. and J.F. Frank (l989), Biological aerosols: a review of airborne contamination and its measurement in dairy processing plants, J. Food Prot. 52(7):512-524.

6. Ligugnana, R. and D.Y.C. Fung (1990), Training of food and dairy staff for microbiological air and surface hygiene, Food and Enviro. Sanit. 10(3): 130-135.

7. Snedecor, G.L. and W.G. Cochran (1976), Statistical Methods, 6th Ed., Iowa State University Press, Ames, Iowa.

8. Fung, D.Y.C. and P.A. Hartman (1975), Miniaturized microbiological techniques for rapid characterization of bacteria. In: Heden, G.C. and T. Illeni (eds.), New Approaches to Identification of Microorganisms, John Wiley and Sons, New York, NY.

9. Gailani, M.B. (1985), Water activity in relation to microbiology during processing and storage of Sudanese dried beef (Sharmoot), a Ph.D. dissertation, Dept. of Animal Sciences and Industry, Kansas State University, KS.

10. Buchanan, P.S. and N.E. Gibbon (1974), Bergey's Manual of Determinative Bacteriology (8th ed.), The Williams and Wilkins Co., Baltimore, MD.

11. Kotula, A.W. and J.A. Kinner (1964), Airborne microorganisms in broiler processing plants, Appl. Microbiol. 12: 179-184.

Daniel Y.C. Fung, Ph.D., Kansas State University, Dept. of Animal Sciences and Industry, Call Hall, Manhattan, KS 66506-1600.