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The microbiological content of raw milk affects quality, shelf life, and safety of processed milk and other dairy products (3, 21, 24). There are several methods available for detection and enumeration of microorganisms in raw and processed milks. Culture techniques are the most common, but a major disadvantage of these is the time needed to produce results (27). Another significant disadvantage of traditional culture methods is their failure to isolate viable but nonculturable organisms (7). On the other hand, total microscopic count methods are relatively fast, but limitations of these techniques include operator fatigue from prolonged use of microscopes and inability to discriminate between living and dead bacteria. To alleviate problems associated with culture-based detection systems and direct microscopic methods, various other methods have been developed (11, 15, 26). These include colorimetric assay methods based on liberation of dyes from substrates by enzymes, dye reduction tests, and ATP bioluminescence (12, 18, 22, 30). Although biochemically and physiologically based assays are fast, they lack absolute specificity. It is therefore doubtful whether these nonculturing approaches provide complete quality and safety assurance.

Interest in rapid methods and automation in food microbiology has been growing in the past several decades (15). There have been some attempts to introduce direct, automated enumeration methods into dairy testing (6, 23, 26), and BactoScan was developed as an automated instrument for routine testing of the bacteriological quality of raw milk (6, 25). However, the current instrumentation is limited to measuring either total microbial or somatic cell counts, and it has not been developed for more diversified work such as microbial differentiation. By contrast, flow cytometers may suit the broad and specific needs of microbial analysis of milk and dairy products within one type of instrument (15, 23). Flow cytometry (FCM) is extremely sensitive, avoids the need for culturing or enrichment procedures, and can be both qualitative and quantitative (2, 23). Use of fluorescent stains or fluorogenic substrates in combination with FCM allows the detection and discrimination of viable culturable, viable nonculturable, and nonviable organisms (2, 7, 28). Furthermore, there is the possibility that numerous (or even rare) microbial cells could be detected against a background of other bacteria or nonbacterial particles by combining FCM and specific fluorescently-labeled antibodies or oligonucleotide probes (1, 10, 23, 29). As an early step towards demonstrating the potential application of flow cytometers in milk analyses, we have developed a rapid method for detecting total bacteria.

Milk-clearing treatments and detection of bacteria in milk by using FCM. Escherichia coli (XL1-Blue MRF) and Staphylococcus aureus (NCTC 4163) were chosen to represent gram-negative rods and gram-positive cocci that are potential contaminants of milk (13, 20). Bacteria were grown overnight in Trypticase soy broth (Oxoid, Sydney, Australia) at 28°C on a rotary shaker (180 rpm) for 16 h. Pure populations of E. coli and S. aureus were easily detected by FCM when they were suspended in phosphate-buffered saline (PBS) (Fig. 1A and B), but when they were inoculated into ultra-heat-treated (UHT) milk, no distinct separation appeared (Fig. 1C). This is due to the presence of proteins and lipid globules that can bind nonspecifically to fluorescent stains and interfere with staining and detection of bacteria. Treatment of milk by centrifugation to remove lipids without also treating samples with proteases was insufficient to allow definition of bacteria (Fig. 1D). Thus, the most critical barrier to FCM analysis of milk is the presence of protein globules. Therefore, we applied enzymatic treatment to remove or modify proteins and thereby enable distinction of bacteria by flow cytometry. We used 0.05 mg of proteinase K (EC 3.4.21.64; Sigma-Aldrich, Sydney, Australia) or 10 µl of savinase (EC 3.4.21.52; Novo Nordisk Bioindustrial Pty. Ltd., Sydney, Australia) to treat 100 µl of UHT milk samples and 50 µl of savinase plus 50 µl of 0.1% Triton X-100 to treat 100 µl of raw milk samples. Treated milk samples were incubated at 37°C for 30 to 45 min, after which 900 µl of 150 mM NaCl was added and mixed by inversion of tubes. Samples were then centrifuged at 14,000 × g for 10 min and lipids (top layer) and digested proteins of the milk were drawn off with a micropipette without disturbing the pelleted material, which contained bacteria. The pellet was resuspended in 100 µl of 150 mM NaCl. The samples were then stained and analyzed using FCM (see below for staining protocol and FCM analyses). After the application of proteases in concert with lipid removal, the bacteria in UHT milk appeared in exactly the same positions on bivariate dot plots as bacteria in PBS (Fig. 1A and E and 1B and F). Sorting of putative bacterial populations coupled with subsequent microscopic analysis and plating on selective agar confirmed that the sorted regions represented bacteria inoculated into the UHT milk samples (data not shown).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Detection of bacteria in UHT milk by FCM. (A) E. coli in phosphate-buffered saline; (B) S. aureus in phosphate-buffered saline; (C) untreated milk plus E. coli; (D) milk plus E. coli with fat removed; (E) milk plus E. coli with fat removed and with protease treatment; (F) milk plus S. aureus with fat removed and with protease treatment. Results shown are typical of two repeated experiments assayed in triplicate.

Fluorescent staining and FMC. Bacterial cells in treated milk samples were stained with SYTO BC (Molecular Probes Inc., Bioscientific Pty. Ltd., Sydney, Australia). SYTO BC (excitation and emission maxima of 480 and 500 nm) is a high-affinity nucleic acid stain that penetrates both gram-positive and gram-negative bacteria, giving a bright green fluorescent signal (Molecular Probes [Eugene, Ore.] handbook). SYTO BC, diluted 1:20 in dimethylsulfoxide, was mixed 1:50 with resuspended bacteria and incubated in the dark at 37°C for 5 to 10 min. The viabilities of bacterial cultures were determined by dual staining of the subsamples with propidium iodide (PI) and SYTO BC. A solution of PI (1 mg ml¹) was prepared in dimethyl sulfoxide, and 1 µl of this was added per ml of bacterial suspension. PI is generally excluded by intact plasma membranes; thus, uptake of PI (orange/red fluorescence) indicates cell death (23; Molecular Probes handbook).

Microscopic and plate count methods. A Carl Zeiss (Sydney, Australia) Axioskop 2 epifluorescence microscope fitted with 10× eyepieces and 40× and 100× (oil immersion) objectives was used to confirm and count cells. Excitation of SYTO BC- or PI-stained cells was by a 100-W Hg vapor arc lamp with an appropriate filter block giving excitation at 450 to 490 nm and examination at 520 nm. Direct microscopic counts of bacterial suspensions were carried out using bright-field microscopy and Thoma counting chamber procedures (5).

Comparison between FCM, total microscopic count, and plate count methods. Dual staining of pure bacterial cultures with PI and SYTO BC showed that inocula (16-h bacteria cultures) were >99% viable. UHT milk was inoculated with E. coli and S. aureus cells at different cell concentrations between 10³ and 10⁸ ml¹ and analyzed using FCM and traditional techniques such as plate count and direct microscopy. Strong correlations (r = 0.98) between FCM and plate counts or direct microscopic counts were obtained for both E. coli and S. aureus (data not shown). E. coli counts measured by FCM in the range of 10⁴ to 10⁸ ml¹ were not significantly (P < 0.05) different from those given by the Chromocult coliform agar plate count method, although FCM estimated a significantly (P < 0.05) greater number of cells at the level of 10³ bacteria ml¹ (Fig. 2A). For S. aureus, the numbers measured by FCM were not significantly (P < 0.05) different from those obtained by the Baird-Parker agar plate count methods in the range of 10³ to 10⁸ bacteria ml¹ (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Comparison of bacterial counts given by FCM with plate count methods. Bacteria were inoculated into UHT milk at levels between 103 and 108 ml1. Data (three replicates) were analyzed using Student's t test (an asterisk denotes significant difference at a P value of <0.05). , FCM counts; , plate counts. (A) E. coli; (B) S. aureus.

Bacterial counts in raw milk. Raw milk was obtained refrigerated fresh from the dairy plant and tested the same day or, where indicated, after 48 h of cold (4°C) storage. Unlike in UHT milk, when proteinase K or savinase was used separately on raw milk, no regions of distinct particle populations could be observed with FCM (data not shown). It is likely that heat-treated proteins that are already partly or fully denatured are more sensitive to proteolysis than native proteins in raw milk. However, following the modified proteolytic treatment (protease and a detergent), a bacterial population could be separated from other milk particles based on light-scattering characteristics (Fig. 3A). Statistical treatment of data showed there was a significant correlation (P < 0.01) and good agreement (r = 0.91) between FCM and the standard plate count method (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   (A) Detection of bacteria in raw milk using FCM. The boxed region defines the bacterial population. (B) Correlation between total bacterial counts for raw milk samples obtained by FCM and standard plate count methods. , Bacterial counts of fresh raw milk samples (n = 38); , bacterial counts after cold storage (4°C) for 48 h (n = 21). Assays of individual samples were performed in triplicate.

Potential of FCM in milk microbiology. This study demonstrates the ability of FCM to determine total bacterial numbers after clearing of milk and staining of bacteria with a readily available fluorescent stain. The sensitivity of the FCM procedure was 10⁴ total bacteria ml of milk¹. A limit of 10⁴ total bacteria ml of raw milk¹ is below the level of detection required to satisfy legislation in many countries and states (14). The assay takes 45 to 60 min depending on whether processed or raw milk is being analyzed. The total FCM analysis time is between 30 s and 2 min, depending on numbers of bacteria in milk. This time frame compares favorably with current culturing methods, which take 72 h (8).