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Efficacy of 3M™ Petrifilm™ aerobic count plates for enumerating Bacillus sporothermodurans and Geobacillus stearothermophilus in UHT milk
International Dairy Journal 25 (2012) 147e149
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
Efficacy of 3MÔ PetrifilmÔ aerobic count plates for enumerating Bacillus sporothermodurans and Geobacillus stearothermophilus in UHT milk Rosa M. Casillas-Buenrostro a, Norma L. Heredia a, DeAnn L. Benesh b, Santos García a, * a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Apdo. Postal 124-F, Ciudad Universitaria, San Nicolás de los Garza, N.L. 66451, Mexico b 3M Company, 3M Center, St. Paul, MN 55144-1000, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2011 Received in revised form 10 January 2012 Accepted 10 January 2012
The effectiveness of the 3MÔ PetrifilmÔ aerobic count plate for determining the amount of Bacillus sporothermodurans and Geobacillus stearothermophilus in ultra high temperature milk was determined and its efficacy was compared with the pour-plate agar and one-streak nutrient agar methods. Tubes containing milk were inoculated with spore suspensions, heat shocked and incubated. Aliquots were collected after 0, 24, 48, and 72 h of incubation, inoculated using the three different plate methods, and incubated at 55 C. B. sporothermodurans was detected at 9 h on the Petrifilm plate, compared with 18 h for both the pour plate and one-streak methods. Depending on the strain, G. stearothermophilus was detected between 6 and 12 h by the Petrifilm plate method and between 9 and 12 h by the other methods. The Petrifilm plates gave reproducible results compared with the traditional methods and are compatible with industrial requirements for milk quality control. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Pasteurization of milk, during which it is heated at 61e66 C for 30 min or at 71.7 C for at least 15 s (usually 30e40 s), kills most vegetative bacterial cells, but spores can survive (Scheldeman, Herman, Foster, & Heyndrickx, 2006). Another procedure, ultra high temperature (UHT) processing, sterilizes foods at high temperatures for short periods of time, resulting in less chemical change compared with in-container commercial sterilization. In UHT processing a high temperature is applied for a short period of time (usually between 135 C and 150 C for 1e8 s) in a continuous flow (Scheldeman et al., 2006). After this treatment, virtually all microorganisms, including spores, are destroyed (Anonymous, 1992; Scheldeman et al., 2006). The European Union (EU) requires that UHT-treated milk have no deterioration after 15 days at a temperature of at least 30 C. UHT milk must meet the standard of <10 colony forming units (cfu) per 0.1 mL after incubation at 30 C for 15 days (Anonymous, 1992). Heat-resistant spore forming bacilli are widely distributed in nature and commonly found in the microbial flora of raw and pasteurized milk and dairy products. Spores surviving processing (filling, temperature, sterilization of packaging material, etc.) can
* Corresponding author. Tel./fax: þ52 818 376 3044. E-mail address: [email protected] (S. García). 0958-6946/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2012.01.004
germinate, grow, and proliferate in the product (Scheldeman et al., 2006). Geobacillus stearothermophilus, a thermophilic microorganism that produces high heat-resistant spores, is frequently isolated from milk and dairy products, and associated with flat sour spoilage of evaporated milk (Kalogridou-Vassiliadou, 1992). Due to its heat resistance, this microorganism is often used as a biological indicator for testing the efficacy of sterilization processes (Chopka & Mathijk, 1984; Phillips & Griffiths, 1986). Exceptionally high heat-resistant spores (HRS) can survive UHT processes in dairy plants or contaminate products after heat treatment (Hammer, Lembke, Suhren, & Heeschen, 1995; Pettersson, Lembke, Hammer, Stackebrant, & Priest, 1996). Some of these organisms belong to the species Bacillus sporothermodurans, first described by Pettersson et al. (1996). B. sporothermodurans is a non-pathogen with the ability to grow in milk and dairy products. Although no major sensory changes are produced during growth in milk, contamination levels particularly in UHT-treated milk frequently exceed the criterion of the EU regulations (Scheldeman et al., 2006), resulting in considerable economic losses. The dairy industry tests for the presence of microorganisms in UHT milk using the pour-plate count agar or the one-streak method on nutrient agar at 37 C (for total viable count) and 55 C (for thermophilic bacterial count). Although these methods are simple, they are time-consuming due to preparation of agar plates. The ready-to-use Petrifilm aerobic count (AC) plates (3M Company, St.
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Paul, MN, USA) are commonly used to enumerate aerobic mesophilic microorganisms. These plates include an indicator dye and built-in grid that aids in counting of colonies. Since there are no literature reports on the use of these plates for the enumeration of thermophilic microorganisms in milk, this study was undertaken to test the suitability of the Petrifilm AC plate for counting B. sporothermodurans and G. stearothermophilus in UHT milk. 2. Materials and methods 2.1. Microorganisms B. sporothermodurans DSM10599 and G. stearothermophilus strain ATCC 12980 were obtained from the National Collection of Microbial Strains, ENCB-IPN, Mexico City, Mexico. G. stearothermophilus strains NRRL B-4419 (9A2), NUB3621 (9A5), XL-56-6 (9A-19), and 10 (9A-21) were kindly provided by the Bacillus Genetic Stock Center, Columbus, OH, USA. These cultures were maintained in rehydrated skim milk at 81 C. 2.2. Spore production The inoculum was prepared by transferring 50 mL from the stock culture of B. sporothermodurans into 5 mL of brain heart infusion (BHI) broth (Bioxon, Becton Dickinson de Mexico, Mexico, D.F.) supplemented with vitamin B12 (1 mg L1). The spores were heat shock activated (80 C, 10 min) and then incubated at 37 C for 18 h. The strains of G. stearothermophilus were similarly heat activated (Flint & Brooks, 2001), except for strains ATCC 1298, 9A-19, and 9A21, which were inoculated into trypticase soy broth (TSB, Becton Dickinson Co. Sparks, MD, USA ) at 42 C, and strains 9A2 and (9A5) in nutrient broth (Difco, Becton Dickinson Co. Sparks MD, USA) at 55 C, as recommended by the suppliers. For spore production, 100 mL from the activated cultures were streaked onto various media. BHI agar with 1 mg L1 vitamin B12, MnSO4, and 1 g L1 CaCl2 was used for 7 mg L1 B. sporothermodurans, which were incubated at 37 C for 96 h. Trypticase soy agar (TSA, Becton Dickinson Co. Sparks MD, USA) was used for G. stearothermophilus 9A2, 9A5, 9A-19, and 9A-21, and TSA plus 50 mg L1 MnSO4, 100 mg L1 MgSO4, and 500 mg L1 CaCl2 was used for strain ATCC 1298. All the G. stearothermophilus cultures were incubated at 55 C for 60 h. Colonies with sporulated cells (observed by phase contrast microscopy) were removed from the agar and centrifuged at 10,000 g for 10 min. Spores were purified after repeated lowspeed centrifugation (500 g for 15 min) at 4 C and subsequent washing (usually 4 times) with ice-cold purified (Thermo Scientific Branstead II, Dubuque, IA, USA) sterile water (García-Alvarado, Labbé, & Rodríguez, 1992). Cleaned spores were suspended in water, adjusted to 106 cells mL1 (by counting cells in their respective nutrient agar), kept at 2 C, and used within 4 weeks. 2.3. Detection of B. sporothermodurans and G. stearothermophilus in UHT milk Tubes containing 9 mL UHT milk were inoculated with 1 mL of spore suspensions of B. sporothermodurans or G. stearothermophilus to give spore concentrations of 102 spores mL1. Samples were subjected to heat shock (80 C for 10 min), and the tubes were then incubated at 37 C. After 0, 24, 48, and 72 h of incubation, aliquots were inoculated onto Petrifilm AC plates (100 mL), pour-plate agar (100 mL), and one-streak nutrient agar plates (10 mL). For Petrifilm AC plates, samples were inoculated by dropping the inoculum onto the plate at a distance of no more than 5 cm. The drops were allowed to dry, and the plates were incubated at 55 C for the
G. stearothermophilus strains and at 37 C and 55 C for B. sporothermodurans in a humid chamber (plastic box with a piece of sterile water-wet cotton). Plates were examined every hour for microbial growth. The sterility of the milk matrix used was confirmed using standard plate count agar (ACS, Bioxon). 2.4. Determination of heat-resistant spores in UHT milk available for retail sale To determine the presence of HRS in commercially available UHT milk, samples from 40 different batches and expiration dates of a leading brand were collected. Sample size was determined using general inspection levels of the MIL STD 105E/D (Anonymous, 2010, Table II-A). Milk samples were analyzed using the three methods described above. 2.5. Statistical analysis All the experiments were conducted three times each in duplicate. The results were evaluated with an analysis of variance (ANOVA) test using the SigmaStat program. 3. Results and discussion G. stearothermophilus is a thermophilic bacterium whose recommended temperature for growth is 55 C. On the other hand, B. sporothermodurans exhibits a wider range of temperature, growing well at 37 C. In this work, B. sporothermodurans was incubated at 37 and 55 C in the different methods. G. stearothermophilus and B. sporothermodurans were detected using each of the three methods. After inoculating 102 spores mL1 into UHT milk, viable cells were counted at 0, 24, 48, and 72 h. In all cases, Petrifilm AC plates revealed the microorganisms faster than, or in a similar time to, the pour-plate agar and one-streak methods (Table 1). No significant difference (P < 0.05) in detection of B. sporothermodurans was observed between incubations at 37 C or 55 C, except for B. sporothermodurans after 0 h of incubation, for which the microorganisms were detected faster at 37 C (9 h) than at 55 C (18 h). After the various incubation times in milk, B. sporothermodurans was always detected (when incubated at 37 C) by 9 h on Petrifilm AC plates compared with 18 h using the pour-plate agar and one-streak methods. When the plates were incubated at 55 C, the microorganism after (0 h) of incubation in milk, was detected at 18 h by all three methods, and after 24, 48, and 72 h in milk, the bacterium was detected within 9 h on Petrifilm AC plates and within 18 h using the pour-plate agar and onestreak methods. G. stearothermophilus strain 12980 was always detected at 6 h by the Petrifilm AC plate method and at 12 h by the pour-plate agar and one-streak methods. However, the results differed for other strains. When immediately inoculated (0 h) in milk, strain 9A2 was detected at 9 h by all three methods, but after 24 h in milk, this bacterium was detected at 9 h on Petrifilm AC plates and by 12 h using the pour-plate agar and one-streak methods. Interestingly, the organism was not detected after 48 h in milk by any of the methods tested. Similarly, strain 9A5 was detected within 12 h by the three methods, after 0 h of incubation in milk; however, the strain was not detected after 24, 48, or 72 h in milk by any method. Strain 9A19, was not detected after 0 h in milk, but after 24 h, it was detected within 9 h on Petrifilm AC plates and within 12 h by the pour-plate agar and one-streak methods. However, the strain was undetectable after 48 or 72 h in milk. Strain 9A21 was always detectable after the various times of incubation in milk, within 9 h on Petrifilm AC plates and within 12 h using the other methods.
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Table 1 Detection time (h) of G. stearothermophilus (G. st) and B. sporothermodurans (B. sp) by different methods in UHT milk. G. stearothermophilus was incubated for detection at 37 C and B. sporothermodurans at 37 C and 55 C.a Incubation time (h)
Detection method
Detection time in hours G. st 12980
G. st 9A2
G. st 9A5
G. st 9A19
G. st 9A21
B. sp 37 C
55 C
0
Petrifilm AC plate Pour plate One-streak
6 (9 1) 12 (7 1) 12 (þ)
9 (10 2) 9 (7 2) 9 (þ)
12 (26) 12 (13) 12 (þ)
ND ND ND
9 (>100) 12 (>100) 12 (þ)
9 (7 5) 18 (7 5) 18 (þ)
18 (4 2) 18 (4 2) 18 (þ)
24
Petrifilm AC plate Pour plate One-streak
6 (25 1) 12 (12 1) 12 (þ)
9 (>100) 12 (>100) 12 (þ)
ND ND ND
9 (>100) 12 (>100) 12 (þ)
9 (>100) 12 (>100) 12 (þ)
9 (18 5) 18 (22 2) 18 (þ)
9 (9 5) 18 (88 2) 18 (þ)
48
Petrifilm AC plate Pour plate One-streak
6 (>100) 12 (>100) 12 (þ)
ND ND ND
ND ND ND
ND ND ND
9 (>100) 12 (>100) 12 (þ)
9 (>100) 18 (>100) 18 (þ)
9 (>100) 18 (>100) 18 (þ)
72
Petrifilm AC plate Pour plate One-streak
6 (>100) 12 (>100) 12 (þ)
ND ND ND
ND ND ND
ND ND ND
9 (>100) 12 (>100) 12 (>100)
9 (>100) 18 (>100) 18 (þ)
9 (>100) 18 (>100) 18 (þ)
a
Values in parentheses are cfu per plate standard deviation, too numerous to count (>100) or positive growth (þ); ND, not detected.
When higher concentrations of spores were inoculated in milk, the detection time was similar (not shown). The inability to detect G. stearothermophilus after incubation in milk by the methods studied could be due to the ability of milk components to inhibit the growth of G. stearothermophilus. Spore germination and vegetative growth of G. stearothermophilus are inhibited by components of raw and mildly heated milks (Ashton & Busta, 1968; Ashton, Busta, & Warren, 1968; Busta, 1966). It has been suggested that milk proteins deplete essential cations from the medium, which results in the inhibition of spore germination or vegetative growth (Ashton & Busta, 1968; Ashton et al., 1968; Busta, 1966). Depletion of available cations or a decrease in pH by initial microbial growth could be the cause of subsequent growth inhibition (Ashton & Busta, 1968; Kalogridou-Vassiliadou, 1992). In the current study, this behaviour was strain dependant, since three of six strains (B. sporothermodurans DSM10599 and G. stearothermophilus 12980 and 9A21) were not inhibited during the incubation time analyzed. Although the presence of mesophilic bacteria is commonly detected using rapid methods, such as Petrifilm AC plates, thermophilic bacteria are usually detected by more traditional methods. In this study, the efficiency with which Petrifilm AC plates can be used to enumerate B. sporothermodurans and G. stearothermophilus in UHT milk was clearly demonstrated. No HRS were detected in the samples from 40 different batches and expiration dates of UHT milk available for retail sale by the three methods described above. Advantages of rapid methods like Petrifilm AC plates include pre-prepared and quality controlled culture media, ease of use and training, and reduced laboratory space and disposal requirements. Reduction of the 2,3,5-triphenyltetrazolium chloride (TTC, indicator dye) in the media by the growing spore formers produces a red colour (formazan), which facilitates colony counting. In the current study, six 100-mL samples were screened on one plate, allowing six samples to be evaluated on a single Petrifilm AC plate. In conclusion, the three methods studied in this work were able to detect levels of organisms as low as 10 cfu 0.1 mL1, complying with the EU standards; however, the Petrifilm AC plates detected the organisms in less time than the other methods. The Petrifilm AC plates gave reproducible results compared with the traditional
methods and are compatible with the demanded rapid industrial requirements for milk quality control.
Acknowledgements We are thankful to Consejo Nacional de Ciencia y Tecnología de México (CONACYT) for the scholarship granted to Rosa M. CasillasBuenrostro.
References Anonymous. (1992). Council directive 92/46/EEC of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heattreated milk and milk-based products. Official J European Union, L268, 0001e0032. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri¼CELEX: 31992L0046:EN:HTML Accessed 29.10.10. Anonymous. (2010). Sampling by attributes e military standard 105E tables. SQC online. http://www.sqconline.com/download/ Accessed 28.10.10. Ashton, D. H., & Busta, F. F. (1968). Milk components inhibitory to Bacillus stearothermophilus. Journal of Dairy Science, 51, 842e847. Ashton, D. H., Busta, F. F., & Warren, J. A. (1968). Relief of casein inhibition of Bacillus stearothermophilus by iron, calcium, and magnesium. Applied Microbiology, 16, 628e635. Busta, F. F. (1966). Milk component(s) inhibitory to Bacillus stearothermophilus. Journal of Dairy Science, 49, 751e756. Chopka, A. K., & Mathijk, D. K. (1984). Isolation, screening and characterization of thermophilic Bacillus species isolated from dairy products. Journal of Applied Bacteriology, 57, 263e271. Flint, S. H., & Brooks, J. D. (2001). Rapid detection of Bacillus stearothermophilus using impedance-splitting. Journal of Microbiological Methods, 44, 205e208. García-Alvarado, J. S., Labbé, R. G., & Rodríguez, M. A. (1992). Sporulation and enterotoxin production of Clostridium perfringens type A at 37 and 43 C. Applied and Environmental Microbiology, 58, 1411e1414. Hammer, P., Lembke, F., Suhren, G., & Heeschen, W. (1995). Characterization of a heat resistant mesophilic Bacillus species affecting quality of UHT milk e a preliminary report. Kieler Milchwirtschaftliche Forschungsberichte, 47, 303e311. Kalogridou-Vassiliadou, D. (1992). Biochemical activities of Bacillus species isolated from flat sour evaporated milk. Journal of Dairy Science, 75, 2681e2686. Pettersson, B., Lembke, F., Hammer, P., Stackebrandt, E., & Priest, F. G. (1996). Bacillus sporothermodurans, a new species producing highly heat-resistant endospores. International Journal of Systematic Bacteriology, 46, 759e764. Phillips, J. D., & Griffiths, M. W. (1986). Factors contributing to the seasonal variation of Bacillus spp. in pasteurized dairy products. Journal of Applied Bacteriology, 61, 275e285. Scheldeman, P., Herman, L., Foster, S., & Heyndrickx, M. (2006). Bacillus sporothermodurans and other highly heat-resistant spore formers in milk. Journal of Applied Microbiology, 101, 542e555.
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Short communication
Efficacy of 3MÔ PetrifilmÔ aerobic count plates for enumerating Bacillus sporothermodurans and Geobacillus stearothermophilus in UHT milk Rosa M. Casillas-Buenrostro a, Norma L. Heredia a, DeAnn L. Benesh b, Santos García a, * a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Apdo. Postal 124-F, Ciudad Universitaria, San Nicolás de los Garza, N.L. 66451, Mexico b 3M Company, 3M Center, St. Paul, MN 55144-1000, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2011 Received in revised form 10 January 2012 Accepted 10 January 2012
The effectiveness of the 3MÔ PetrifilmÔ aerobic count plate for determining the amount of Bacillus sporothermodurans and Geobacillus stearothermophilus in ultra high temperature milk was determined and its efficacy was compared with the pour-plate agar and one-streak nutrient agar methods. Tubes containing milk were inoculated with spore suspensions, heat shocked and incubated. Aliquots were collected after 0, 24, 48, and 72 h of incubation, inoculated using the three different plate methods, and incubated at 55 C. B. sporothermodurans was detected at 9 h on the Petrifilm plate, compared with 18 h for both the pour plate and one-streak methods. Depending on the strain, G. stearothermophilus was detected between 6 and 12 h by the Petrifilm plate method and between 9 and 12 h by the other methods. The Petrifilm plates gave reproducible results compared with the traditional methods and are compatible with industrial requirements for milk quality control. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Pasteurization of milk, during which it is heated at 61e66 C for 30 min or at 71.7 C for at least 15 s (usually 30e40 s), kills most vegetative bacterial cells, but spores can survive (Scheldeman, Herman, Foster, & Heyndrickx, 2006). Another procedure, ultra high temperature (UHT) processing, sterilizes foods at high temperatures for short periods of time, resulting in less chemical change compared with in-container commercial sterilization. In UHT processing a high temperature is applied for a short period of time (usually between 135 C and 150 C for 1e8 s) in a continuous flow (Scheldeman et al., 2006). After this treatment, virtually all microorganisms, including spores, are destroyed (Anonymous, 1992; Scheldeman et al., 2006). The European Union (EU) requires that UHT-treated milk have no deterioration after 15 days at a temperature of at least 30 C. UHT milk must meet the standard of <10 colony forming units (cfu) per 0.1 mL after incubation at 30 C for 15 days (Anonymous, 1992). Heat-resistant spore forming bacilli are widely distributed in nature and commonly found in the microbial flora of raw and pasteurized milk and dairy products. Spores surviving processing (filling, temperature, sterilization of packaging material, etc.) can
* Corresponding author. Tel./fax: þ52 818 376 3044. E-mail address: [email protected] (S. García). 0958-6946/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2012.01.004
germinate, grow, and proliferate in the product (Scheldeman et al., 2006). Geobacillus stearothermophilus, a thermophilic microorganism that produces high heat-resistant spores, is frequently isolated from milk and dairy products, and associated with flat sour spoilage of evaporated milk (Kalogridou-Vassiliadou, 1992). Due to its heat resistance, this microorganism is often used as a biological indicator for testing the efficacy of sterilization processes (Chopka & Mathijk, 1984; Phillips & Griffiths, 1986). Exceptionally high heat-resistant spores (HRS) can survive UHT processes in dairy plants or contaminate products after heat treatment (Hammer, Lembke, Suhren, & Heeschen, 1995; Pettersson, Lembke, Hammer, Stackebrant, & Priest, 1996). Some of these organisms belong to the species Bacillus sporothermodurans, first described by Pettersson et al. (1996). B. sporothermodurans is a non-pathogen with the ability to grow in milk and dairy products. Although no major sensory changes are produced during growth in milk, contamination levels particularly in UHT-treated milk frequently exceed the criterion of the EU regulations (Scheldeman et al., 2006), resulting in considerable economic losses. The dairy industry tests for the presence of microorganisms in UHT milk using the pour-plate count agar or the one-streak method on nutrient agar at 37 C (for total viable count) and 55 C (for thermophilic bacterial count). Although these methods are simple, they are time-consuming due to preparation of agar plates. The ready-to-use Petrifilm aerobic count (AC) plates (3M Company, St.
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Paul, MN, USA) are commonly used to enumerate aerobic mesophilic microorganisms. These plates include an indicator dye and built-in grid that aids in counting of colonies. Since there are no literature reports on the use of these plates for the enumeration of thermophilic microorganisms in milk, this study was undertaken to test the suitability of the Petrifilm AC plate for counting B. sporothermodurans and G. stearothermophilus in UHT milk. 2. Materials and methods 2.1. Microorganisms B. sporothermodurans DSM10599 and G. stearothermophilus strain ATCC 12980 were obtained from the National Collection of Microbial Strains, ENCB-IPN, Mexico City, Mexico. G. stearothermophilus strains NRRL B-4419 (9A2), NUB3621 (9A5), XL-56-6 (9A-19), and 10 (9A-21) were kindly provided by the Bacillus Genetic Stock Center, Columbus, OH, USA. These cultures were maintained in rehydrated skim milk at 81 C. 2.2. Spore production The inoculum was prepared by transferring 50 mL from the stock culture of B. sporothermodurans into 5 mL of brain heart infusion (BHI) broth (Bioxon, Becton Dickinson de Mexico, Mexico, D.F.) supplemented with vitamin B12 (1 mg L1). The spores were heat shock activated (80 C, 10 min) and then incubated at 37 C for 18 h. The strains of G. stearothermophilus were similarly heat activated (Flint & Brooks, 2001), except for strains ATCC 1298, 9A-19, and 9A21, which were inoculated into trypticase soy broth (TSB, Becton Dickinson Co. Sparks, MD, USA ) at 42 C, and strains 9A2 and (9A5) in nutrient broth (Difco, Becton Dickinson Co. Sparks MD, USA) at 55 C, as recommended by the suppliers. For spore production, 100 mL from the activated cultures were streaked onto various media. BHI agar with 1 mg L1 vitamin B12, MnSO4, and 1 g L1 CaCl2 was used for 7 mg L1 B. sporothermodurans, which were incubated at 37 C for 96 h. Trypticase soy agar (TSA, Becton Dickinson Co. Sparks MD, USA) was used for G. stearothermophilus 9A2, 9A5, 9A-19, and 9A-21, and TSA plus 50 mg L1 MnSO4, 100 mg L1 MgSO4, and 500 mg L1 CaCl2 was used for strain ATCC 1298. All the G. stearothermophilus cultures were incubated at 55 C for 60 h. Colonies with sporulated cells (observed by phase contrast microscopy) were removed from the agar and centrifuged at 10,000 g for 10 min. Spores were purified after repeated lowspeed centrifugation (500 g for 15 min) at 4 C and subsequent washing (usually 4 times) with ice-cold purified (Thermo Scientific Branstead II, Dubuque, IA, USA) sterile water (García-Alvarado, Labbé, & Rodríguez, 1992). Cleaned spores were suspended in water, adjusted to 106 cells mL1 (by counting cells in their respective nutrient agar), kept at 2 C, and used within 4 weeks. 2.3. Detection of B. sporothermodurans and G. stearothermophilus in UHT milk Tubes containing 9 mL UHT milk were inoculated with 1 mL of spore suspensions of B. sporothermodurans or G. stearothermophilus to give spore concentrations of 102 spores mL1. Samples were subjected to heat shock (80 C for 10 min), and the tubes were then incubated at 37 C. After 0, 24, 48, and 72 h of incubation, aliquots were inoculated onto Petrifilm AC plates (100 mL), pour-plate agar (100 mL), and one-streak nutrient agar plates (10 mL). For Petrifilm AC plates, samples were inoculated by dropping the inoculum onto the plate at a distance of no more than 5 cm. The drops were allowed to dry, and the plates were incubated at 55 C for the
G. stearothermophilus strains and at 37 C and 55 C for B. sporothermodurans in a humid chamber (plastic box with a piece of sterile water-wet cotton). Plates were examined every hour for microbial growth. The sterility of the milk matrix used was confirmed using standard plate count agar (ACS, Bioxon). 2.4. Determination of heat-resistant spores in UHT milk available for retail sale To determine the presence of HRS in commercially available UHT milk, samples from 40 different batches and expiration dates of a leading brand were collected. Sample size was determined using general inspection levels of the MIL STD 105E/D (Anonymous, 2010, Table II-A). Milk samples were analyzed using the three methods described above. 2.5. Statistical analysis All the experiments were conducted three times each in duplicate. The results were evaluated with an analysis of variance (ANOVA) test using the SigmaStat program. 3. Results and discussion G. stearothermophilus is a thermophilic bacterium whose recommended temperature for growth is 55 C. On the other hand, B. sporothermodurans exhibits a wider range of temperature, growing well at 37 C. In this work, B. sporothermodurans was incubated at 37 and 55 C in the different methods. G. stearothermophilus and B. sporothermodurans were detected using each of the three methods. After inoculating 102 spores mL1 into UHT milk, viable cells were counted at 0, 24, 48, and 72 h. In all cases, Petrifilm AC plates revealed the microorganisms faster than, or in a similar time to, the pour-plate agar and one-streak methods (Table 1). No significant difference (P < 0.05) in detection of B. sporothermodurans was observed between incubations at 37 C or 55 C, except for B. sporothermodurans after 0 h of incubation, for which the microorganisms were detected faster at 37 C (9 h) than at 55 C (18 h). After the various incubation times in milk, B. sporothermodurans was always detected (when incubated at 37 C) by 9 h on Petrifilm AC plates compared with 18 h using the pour-plate agar and one-streak methods. When the plates were incubated at 55 C, the microorganism after (0 h) of incubation in milk, was detected at 18 h by all three methods, and after 24, 48, and 72 h in milk, the bacterium was detected within 9 h on Petrifilm AC plates and within 18 h using the pour-plate agar and onestreak methods. G. stearothermophilus strain 12980 was always detected at 6 h by the Petrifilm AC plate method and at 12 h by the pour-plate agar and one-streak methods. However, the results differed for other strains. When immediately inoculated (0 h) in milk, strain 9A2 was detected at 9 h by all three methods, but after 24 h in milk, this bacterium was detected at 9 h on Petrifilm AC plates and by 12 h using the pour-plate agar and one-streak methods. Interestingly, the organism was not detected after 48 h in milk by any of the methods tested. Similarly, strain 9A5 was detected within 12 h by the three methods, after 0 h of incubation in milk; however, the strain was not detected after 24, 48, or 72 h in milk by any method. Strain 9A19, was not detected after 0 h in milk, but after 24 h, it was detected within 9 h on Petrifilm AC plates and within 12 h by the pour-plate agar and one-streak methods. However, the strain was undetectable after 48 or 72 h in milk. Strain 9A21 was always detectable after the various times of incubation in milk, within 9 h on Petrifilm AC plates and within 12 h using the other methods.
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Table 1 Detection time (h) of G. stearothermophilus (G. st) and B. sporothermodurans (B. sp) by different methods in UHT milk. G. stearothermophilus was incubated for detection at 37 C and B. sporothermodurans at 37 C and 55 C.a Incubation time (h)
Detection method
Detection time in hours G. st 12980
G. st 9A2
G. st 9A5
G. st 9A19
G. st 9A21
B. sp 37 C
55 C
0
Petrifilm AC plate Pour plate One-streak
6 (9 1) 12 (7 1) 12 (þ)
9 (10 2) 9 (7 2) 9 (þ)
12 (26) 12 (13) 12 (þ)
ND ND ND
9 (>100) 12 (>100) 12 (þ)
9 (7 5) 18 (7 5) 18 (þ)
18 (4 2) 18 (4 2) 18 (þ)
24
Petrifilm AC plate Pour plate One-streak
6 (25 1) 12 (12 1) 12 (þ)
9 (>100) 12 (>100) 12 (þ)
ND ND ND
9 (>100) 12 (>100) 12 (þ)
9 (>100) 12 (>100) 12 (þ)
9 (18 5) 18 (22 2) 18 (þ)
9 (9 5) 18 (88 2) 18 (þ)
48
Petrifilm AC plate Pour plate One-streak
6 (>100) 12 (>100) 12 (þ)
ND ND ND
ND ND ND
ND ND ND
9 (>100) 12 (>100) 12 (þ)
9 (>100) 18 (>100) 18 (þ)
9 (>100) 18 (>100) 18 (þ)
72
Petrifilm AC plate Pour plate One-streak
6 (>100) 12 (>100) 12 (þ)
ND ND ND
ND ND ND
ND ND ND
9 (>100) 12 (>100) 12 (>100)
9 (>100) 18 (>100) 18 (þ)
9 (>100) 18 (>100) 18 (þ)
a
Values in parentheses are cfu per plate standard deviation, too numerous to count (>100) or positive growth (þ); ND, not detected.
When higher concentrations of spores were inoculated in milk, the detection time was similar (not shown). The inability to detect G. stearothermophilus after incubation in milk by the methods studied could be due to the ability of milk components to inhibit the growth of G. stearothermophilus. Spore germination and vegetative growth of G. stearothermophilus are inhibited by components of raw and mildly heated milks (Ashton & Busta, 1968; Ashton, Busta, & Warren, 1968; Busta, 1966). It has been suggested that milk proteins deplete essential cations from the medium, which results in the inhibition of spore germination or vegetative growth (Ashton & Busta, 1968; Ashton et al., 1968; Busta, 1966). Depletion of available cations or a decrease in pH by initial microbial growth could be the cause of subsequent growth inhibition (Ashton & Busta, 1968; Kalogridou-Vassiliadou, 1992). In the current study, this behaviour was strain dependant, since three of six strains (B. sporothermodurans DSM10599 and G. stearothermophilus 12980 and 9A21) were not inhibited during the incubation time analyzed. Although the presence of mesophilic bacteria is commonly detected using rapid methods, such as Petrifilm AC plates, thermophilic bacteria are usually detected by more traditional methods. In this study, the efficiency with which Petrifilm AC plates can be used to enumerate B. sporothermodurans and G. stearothermophilus in UHT milk was clearly demonstrated. No HRS were detected in the samples from 40 different batches and expiration dates of UHT milk available for retail sale by the three methods described above. Advantages of rapid methods like Petrifilm AC plates include pre-prepared and quality controlled culture media, ease of use and training, and reduced laboratory space and disposal requirements. Reduction of the 2,3,5-triphenyltetrazolium chloride (TTC, indicator dye) in the media by the growing spore formers produces a red colour (formazan), which facilitates colony counting. In the current study, six 100-mL samples were screened on one plate, allowing six samples to be evaluated on a single Petrifilm AC plate. In conclusion, the three methods studied in this work were able to detect levels of organisms as low as 10 cfu 0.1 mL1, complying with the EU standards; however, the Petrifilm AC plates detected the organisms in less time than the other methods. The Petrifilm AC plates gave reproducible results compared with the traditional
methods and are compatible with the demanded rapid industrial requirements for milk quality control.
Acknowledgements We are thankful to Consejo Nacional de Ciencia y Tecnología de México (CONACYT) for the scholarship granted to Rosa M. CasillasBuenrostro.
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