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J. Dairy Sci.

106:1502–1517
https://doi.org/10.3168/jds.2022-22416
© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Invited review: Redefining raw milk quality—Evaluation of raw milk


microbiological parameters to ensure high-quality processed dairy products
N. H. Martin,* R. L. Evanowski, and M. Wiedmann
Milk Quality Improvement Program, Department of Food Science, Cornell University, Ithaca, NY 14853

ABSTRACT microbial contamination (here the focus will be on bac-


terial contamination), contemporary and historical at-
Raw milk typically has little bacterial contamination tention has emphasized the production of high-quality
as it leaves the udder of the animal; however, through a raw milk as an indicator of overall hygienic production
variety of pathways, it can become contaminated with practices at the farm. The term “high-quality,” from
bacteria originating from environmental sources, the a microbiological perspective, is often used to refer to
cow herself, and contact with contaminated equipment. raw milk with low total bacteria counts; however, the
Although the types of bacteria found in raw milk are term may also be used to describe raw milk with low
very diverse, select groups are particularly important levels of certain groups of bacteria (e.g., coliforms).
from the perspective of finished product quality. In par- Indeed, tests for various groups of raw milk bacteria
ticular, psychrophilic and psychrotolerant bacteria that have been used as indicators of raw milk quality beyond
grow quickly at low temperatures (e.g., species in the the total bacteria count (TBC), most notably, prelimi-
genus Pseudomonas and the family Enterobacteriaceae) nary incubation (PI) count for psychrophilic bacteria,
and produce heat-stable enzymes, and sporeforming coliform count (CC), laboratory pasteurization count
bacteria that survive processing hurdles in spore form, (LPC) for thermoduric bacteria, and, less frequently,
are the 2 primary groups of bacteria related to effects spore count testing. Results of these tests may be used
on processed dairy products. Understanding factors to indicate specific deficiencies in hygienic practices;
leading to the presence of these important bacterial for example, the presence of high levels of coliforms
groups in raw milk is key to reducing their influence on (e.g., >100 cfu/mL) may indicate that cows are not
processed dairy product quality. Here we examine the adequately cleaned before milking unit attachment
raw milk microbiological parameters used in the con- (Murphy and Boor, 2000).
temporary dairy industry for their utility in identifying Despite the common use of these tests to assess raw
raw milk supplies that will perform well in processed milk quality, they are primarily used as indicators
dairy products. We further recommend the use of a of conditions on the farm and not as an indicator of
single microbiological indicator of raw milk quality, performance in finished products, an important consid-
namely the total bacteria count, and call for the devel- eration when defining raw milk quality. An extensive
opment of a whole-farm approach to raw milk quality discussion of the effects of raw milk microbial contami-
that will use data-driven, risk-based tools integrated nants on processed dairy product quality will not be
across the continuum from production to processing included here, as a recent review thoroughly examined
and shelf-life to ensure continuous improvement in this topic (Murphy et al., 2016). However, it is notewor-
dairy product quality. thy to our review that Murphy et al. (2016) identified
Key words: raw milk quality, microbiological testing, 2 primary groups of raw milk microbial contaminants
bacterial contamination, farm practices that influence processed dairy product quality, includ-
ing (1) psychrophilic and psychrotolerant bacteria (e.g.,
INTRODUCTION Pseudomonas) and (2) sporeforming bacteria (e.g., Pae-
nibacillus and Clostridium). Briefly, high initial concen-
The microbiological composition of raw bovine milk trations of psychrophilic and psychrotolerant bacteria,
has been extensively studied for over a century. Although or growth due to improper cooling or excessive storage
it is expected that raw milk will contain low levels of times, may lead to heat-stable enzyme development by
Pseudomonas and other related bacteria. Even though
Received June 16, 2022.
the bacterial cells themselves do not survive process-
Accepted September 17, 2022. ing hurdles, the heat-stable enzymes produced when
*Corresponding author: nhw6@​cornell​.edu cell concentrations reach 5 to 8 log cfu/mL continue

1502
Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1503

breaking down milk components even after processing, milk quality that utilizes (1) an overall raw milk quality
leading to flavor, odor, and body defects in finished indicator as a process control test to assess consistency
products (Sørhaug and Stepaniak, 1997). For example, and compliance with hygienic farm practices, (2) a
cheese made from raw milk with elevated bacterial set of tests that will aid in troubleshooting when the
numbers before processing (e.g., >1,000,000 cfu/mL) overall raw milk quality indicator is at high levels, and
leads to reduced yields and flavor defects during aging (3) tests that specifically assess the presence and levels
(Banks et al., 1988). In UHT milk products, which are of groups of bacteria that influence finished product
stored at ambient temperatures, residual heat-stable quality. Finally, we discuss the potential for develop-
proteases and lipases produced by elevated raw milk ment of predictive decision support tools that integrate
microbial loads cause gelation of the product over the knowledge of raw milk microbial populations, as well
extended shelf-life, a defect termed “age-gelation,” along as farm management practices that influence micro-
with other textural defects such as sedimentation, and bial presence and levels in raw milk and their effects
off-odors and flavors (Murphy et al., 2016). on finished product quality. Moving from the current
The second key group of bacteria in raw milk that reactive, test-based system to a proactive and predic-
are relevant to finished product quality are sporeform- tive approach is a critical step in ensuring high-quality
ing bacteria, a diverse group belonging to the orders dairy products across the continuum from production
Bacillales and Clostridiales. These bacteria produce to consumption.
endospores, or spores, that originate from natural envi-
ronments on dairy farms, are transferred into raw milk, RAW MILK MICROBIOLOGICAL PARAMETERS
survive processing hurdles, and subsequently grow,
causing spoilage in finished dairy products (Gopal et Total Bacteria Count: An Overall Indicator
al., 2015). For example, psychrotolerant sporeforming of Raw Milk Microbiological Quality
bacteria grow at refrigeration temperatures, causing and Hygienic Production Practices
fluid milk spoilage (Ranieri and Boor, 2009). Studies
have demonstrated that psychrotolerant sporeforming The TBC is the most widely used measure of micro-
bacteria are responsible for approximately 40 to 50% bial quality of raw milk and is measured in the United
of fluid milk in the United States reaching the Pasteur- States using several approved methods including the
ized Milk Ordinance bacterial limit of 20,000 cfu/mL standard plate count (SPC), plate loop count (PLC),
during shelf-life (Ranieri and Boor, 2009; Alles et al., Petrifilm (3M) aerobic count, flow cytometry method-
2018; Reichler et al., 2018) and, importantly, are the ologies (e.g., Bactoscan, Foss Analytical), and others
primary bacterial agents limiting the extension of fluid (USPHS/FDA, 2019). In the US, the Pasteurized Milk
milk shelf-life. Another important group of sporeform- Ordinance limits the bacteria count in grade “A” raw
ing bacteria, anaerobic butyric acid bacterial spores milk to 100,000 cfu/mL for individual producers or
(i.e., Clostridium spp.), cause late-blowing defect in 300,000 cfu/mL for commingled raw milk (USPHS/
cheese (Klijn et al., 1995). Late blowing is of particular FDA, 2019). Outside of the US, similar limits on TBC
concern in hard and semi-hard aged cheeses such as are required. For example, the limit for total plate count
Gouda and does not typically develop until 60 to 90 d of raw milk intended for processing is 100,000 cfu/mL in
into aging, resulting in economic losses for cheesemak- Europe (European Commission, 2021), whereas in New
ers (Martin et al., 2021). A focus on these raw milk Zealand the limit is 300,000 cfu/mL (Ministry for Pri-
microbial drivers of finished product quality is neces- mary Industries, 2022), and Canadian standards limit
sary to prioritize efforts that will result in improved total aerobic mesophilic bacteria to 50,000 cfu/mL (Na-
finished product quality. This is critical to the entire tional Dairy Code, 1997). However, reported mean bulk
dairy continuum, as consumer dissatisfaction with tank TBC typically fall well below these regulatory lim-
product quality influences their willingness and intent its. For instance, Elmoslemany et al. (2009a) measured
to continue purchasing and consuming that product TBC and other microbiological quality parameters over
(Quelch and Ash, 1980). a 2-year period from approximately 11,100 bulk tank
In this review we discuss common contemporary raw milk samples collected from 235 dairy farms in
microbiological parameters used to evaluate raw milk Prince Edward Island, Canada. The authors report a
quality, types of bacterial contaminants targeted by TBC geometric mean of 5,300 cfu/mL with only 6% of
these tests, sources of contaminants, and farm factors samples above 50,000 cfu/mL. Similarly, Pantoja et al.
associated with these parameters, as well as potential (2009) measured TBC from 7,241 bulk tank raw milk
shortcomings of these parameters for evaluating raw samples from 16 farms in Wisconsin (USA) during a
milk quality from the perspective of finished product 1-year period, reporting a mean TBC of 3.1 log cfu/mL
quality. Further, we propose a new definition of raw (1,259 cfu/mL), with just 1.6% (n = 142) above the
Journal of Dairy Science Vol. 106 No. 3, 2023
Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1504

regulatory limit of 100,000 cfu/mL. In a more recent SCC and PLC, a measure of TBC, from monthly pro-
study, Rodrigues et al. (2017) evaluated 472 raw bulk ducer data collected from 5 large milk processing facili-
tank milk samples from 19 farms in New York State ties in New York from March 1999 to December 2000,
and reported mean TBC values by farm ranging from indicated that farms with SCC below 200,000 cells/mL
3.01 log cfu/mL (1,023 cfu/mL) to 4.11 log cfu/mL had lower PLC levels compared with milk with higher
(12,882 cfu/mL). Although these reports indicate that SCC (van Schaik et al., 2002). Additionally, Borneman
TBC values well below common regulatory limits in the and Ingham (2014) reported a highly significant cor-
US and in other countries are commonplace, producers relation (P-value <2 × 10−16) between SPC and SCC in
are encouraged to pursue continuous improvement in 2012 bulk tank raw milk samples; however, the authors
reducing bulk tank raw milk TBC, primarily through also report that the R2 value for this association was
the lever of premium payments from processors and quite small (0.02–0.03), suggesting that many other
cooperative. Guidelines suggest that TBC targets for factors also drive SPC outcomes. Indeed, the effects of
good raw milk are in the range of <5,000 to <10,000 mastitis pathogens on bulk tank raw milk TBC depend
cfu/mL (Murphy and Boor, 2000; Jayarao and Wolf- on multiple factors, including the causative bacterial
gang, 2003), yet these recommendations are somewhat agent, the proportion of the herd infected, and the size
dated for contemporary producers. It should also be of the herd (Hayes et al., 2001).
noted that the regulatory limit for TBC (i.e., 100,000 In addition to the contribution of mastitis to bulk
cfu/mL) is not an appropriate cutoff for defining raw tank TBC, other major factors that influence this
milk quality, as raw milk with this level of microbial parameter include milking time hygiene (for example,
contamination is at high risk of resulting in finished sufficient removal of soil from teat surfaces before milk-
product quality deterioration. ing) and equipment and raw milk handling factors (e.g.,
Farm sources and management practices associated proper cleaning and sanitation of equipment and ad-
with elevated TBC in bulk tank raw milk have been equate cooling of raw milk after harvest). Elmoslemany
widely studied. Many possible pathways of bacterial et al. (2010) identified risk factors for elevated total
contamination throughout the production continuum aerobic count, including the amount of dirt on teats be-
may lead to increased TBC in bulk tank milk, so com- fore milking, use of water to wash teats during milking
prehensive management is essential to limit contamina- preparation without subsequent drying, use of the same
tion and subsequent growth of bacteria during storage. towel for drying multiple cows, and manual cleaning of
The first potential source of bacterial contamination the bulk tank. The authors also noted that the lowest
is, of course, the udder itself, especially when an active bulk tank bacterial counts were associated with pre-
bacterial infection is present. For example, Hayes et al. dipping of teats during milking preparation followed
(2001) examined a variety of raw milk microbiological by drying with single-use towels, compared with other
quality parameters, including SPC, Petrifilm aerobic methods of teat preparation. Similarly, Jayarao et al.
count, presumptive gram-negative bacteria on Mac- (2004) studied microbial quality parameters in bulk
Conkey agar, and presumptive Streptococcus spp. on tank raw milk from 126 dairy farms in Pennsylvania and
Edwards medium, from bulk tank milk from 13 farms found that when cows were subjected to pre- and post-
over a 2-week period to characterize the causes of spikes dipping, SPC values were significantly lower, whereas
(i.e., sudden elevations above normal values) in TBC. SPC values were significantly higher when cows were
The authors found that 11 of the 20 observed spikes sprayed with teat dip instead of using a cup for ap-
were driven by increases in the mastitis pathogen Strep- plication. According to a study by Doyle et al. (2016),
tococcus uberis (Hayes et al., 2001). Similarly, Zadoks the microbial population of the teat surface was the
et al. (2004) found that streptococcal, staphylococcal, most significant source of contamination for raw milk,
and gram-negative counts were all significantly and which highlights the importance of pre-milking hygiene
positively associated with bulk tank raw milk TBC, to management of TBC in bulk tank raw milk. Further,
with streptococcal counts accounting for 69% of TBC these studies also provide additional insight into the as-
variability in their study of mastitis-causing bacteria sociations described earlier between TBC and SCC, as
in bulk tank raw milk on 48 New York farms. The effective milking preparation and hygiene are essential
relationship between mastitis and TBC is further il- to prevent mastitis from contagious and environmental
lustrated by the relationship between SCC and TBC. mastitis pathogens (Hogan, 2017; Middleton and Fox,
For example, in a study of the associations between 2017), which ultimately cause elevated SCC in bulk
raw milk quality indicators in bulk tank raw milk on 16 tank milk. Finally, equipment hygiene and water qual-
farms, Pantoja et al. (2009) reported that the odds of ity factors also play an important role in management
increased TBC increased by 2.4% for every 10,000 cell/ of TBC in bulk tank raw milk. For example, Elmos-
mL increase in SCC in the same milk load. A study of lemany et al. (2009b) reported that high slug scores,
Journal of Dairy Science Vol. 106 No. 3, 2023
Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1505

indicating sufficient physical cleaning of the pipeline, biological methodologies over the last 20 years have al-
along with use of water softener, were associated with lowed for more comprehensive evaluation of microbial
lower TBC in bulk tank raw milk and related. Hard populations in raw milk using methods such as targeted
water and infrequent evaluation of milk house water for metagenomics. Although study design plays a large role
bacterial contamination were associated with elevated in the microbiome identified in any particular study
TBC in bulk tank raw milk. In a study specifically fo- due to variations between farms and over time on the
cused on the effects of cleaning and hygiene of milking same farm (Skeie et al., 2019), by stage of lactation
equipment on bacterial counts in raw milk, Bava et (McHugh et al., 2020), by geographic region (Skeie et
al. (2011) categorized farms into a high SPC group (n al., 2019), and under different meteorological condi-
= 14 farms, mean SPC = 4.25 log cfu/mL) and a low tions (Kable et al., 2016), there are commonalities to
SPC group (n = 8, mean SPC = 3.68 log cfu/mL) and be drawn with regard to the raw milk microbiome from
examined equipment cleaning and sanitation factors these studies. In a recent thorough review of the raw
and equipment contamination between the groups. The milk microbiome by Parente et al. (2020), the authors
authors report that the high and low groups differed evaluated results from 5 studies on the microbiome of
significantly between bacterial contamination of liners bulk tank raw milk from different geographic regions
and milk receivers, as well as in water temperature to determine the most prevalent and abundant taxa
reached during the detergent phase of cleaning milking in raw bulk tank milk. These taxa included members
equipment. Unfortunately, many of these studies report of the phyla Proteobacteria, Firmicutes, Bacteroidota
associations, and few studies have assessed whether the (formerly Bacteroidetes), Actinobacteria, and Mycoplas-
identified associations represent true “cause and effect” matota (formerly Tenericutes), with the most prevalent
relationships. and abundant genera including Pseudomonas, Strepto-
Importantly, raw milk handling, with particular at- coccus, Lactococcus, Acinetobacter, and Staphylococcus
tention paid to achieving and maintaining proper cool- (Parente et al., 2020). In addition to the commonalities
ing (i.e., maintaining milk temperature below 40°F, or in raw milk microbial content, it should be noted that,
4.4°C), is also critical to preventing increases in TBC overall, the microbiome of raw milk is tremendously
during storage, especially for raw milk supplies that diverse, with many relevant bacterial taxa that are
will be stored for longer than 24 h before processing. important from the perspective of milk quality, human
O’Connell et al. (2016) showed that, when stored at safety, and animal health often present at very low lev-
6°C, the bulk tank raw milk TBC increased signifi- els. For example, sporeforming bacteria typically are
cantly from 3.43 log cfu/mL to 4.87 log cfu/mL after found in raw milk at very low levels (Martin et al.,
96 h of storage, but storing raw milk at 2°C resulted in 2019). Psychrotolerant sporeforming bacteria, which
no significant increase between 0 and 96 h of storage. are the primary biological limiting factor for HTST
Driving increases in raw milk TBC during prolonged fluid milk shelf-life (Ranieri and Boor, 2009), have been
storage, or storage at abusive temperatures (e.g., found to have a mean concentration of less than 1 spore
≥6°C), is the growth of Pseudomonas. De Jonghe et al. per milliliter of raw bulk tank milk [mean of −0.72 log10
(2011) demonstrated that under suboptimal simulated most probable number (MPN)/mL; Buehler et al.,
storage conditions (i.e., 6°C storage with 7 brief spikes 2018a] in a study of 99 farms across New York State.
to 10°C over 4 d, to simulate the addition of warm milk Further supporting that psychrotolerant sporeforming
to the bulk tank) not only was there a significant in- bacteria are typically found at low levels, a recent study
crease in raw milk TBC compared with optimal storage of spores in bulk tank raw milk from 17 New York State
conditions (i.e., 3.5°C storage with 7 brief spikes to 6°C dairy farms reported that only 42% of the 34 bulk tank
over 4 d, to simulate the addition of warm milk to the raw milk samples tested had detectable levels of psy-
bulk tank), but Pseudomonas was the primary bacterial chrotolerant spores in the 1 mL of raw milk that had
agent causing the increase in TBC. This is of particular been heat-treated and plated for spore enumeration
importance, as noted earlier in this review, because a (Martin et al., 2019). In the aforementioned review by
variety of Pseudomonas species are known to produce Parente et al. (2020), Paenibacillus, the predominant
heat-stable enzymes that ultimately affect the quality genus of psychrotolerant sporeforming bacteria causing
of processed dairy products. HTST fluid milk spoilage (Ranieri and Boor, 2009),
As the name suggests, the TBC approximates the to- was not identified in the top 25 most prevalent and
tal levels of bacterial contaminants in raw milk, yet the abundant bacterial genera, yet it is a major contributor
types of microbial contaminants in bulk tank raw milk to fluid milk spoilage and waste in the US (Martin et
are diverse, and, importantly, not all of these contami- al., 2021), highlighting the challenges associated with
nants are relevant to finished product quality, as will be detecting low-level bacterial contaminants of relevance
discussed. Advances in culture-independent molecular to raw milk quality.
Journal of Dairy Science Vol. 106 No. 3, 2023
Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1506

A high TBC, whether gauged by the regulatory limit evaluate the TBC (e.g., SPC, Petrifilm aerobic count)
(i.e., 100,000 cfu/mL) or by current examples of lim- are performed under aerobic conditions, a potential
its defining high-quality raw milk (e.g., 5,000 cfu/mL; limitation for the interpretation of these tests as they
Jayarao and Wolfgang, 2003) in and of itself is not a relate to raw milk quality. Further limitations of these
definitive indication that dairy products manufactured standard culture-dependent TBC methods include the
from that milk will have reduced quality. Indeed, as use of mesophilic incubation temperatures (typically
discussed previously, the primary raw milk microbial 30–35°C; Laird et al., 2004), which do not allow for the
drivers of finished product quality include groups of growth of obligate psychrophiles or thermophiles, and
bacteria capable of producing heat-stable enzymes and the common presence of bacteria that grow in clumps
sporeforming bacteria. Other bacterial contaminants, or chains (e.g., gram-positive cocci such as Streptococ-
even when present at elevated levels, may have no dis- cus and Staphylococcus), resulting in multiple cells pre-
cernable effect on finished product quality, although senting as single colonies on agar plates (Sutton, 2012).
they may indeed indicate a farm-level deficiency in Fortunately, the increased use of culture-independent
cleaning, sanitation, or other management factors. flow cytometric methods (e.g., Bactoscan) for estima-
However, evidence suggests that, when TBC counts tion of TBC in bulk tank raw milk has addressed many
are high, the bacteria responsible for the overall in- of the major limitations associated with traditional
creases are of the groups that have been shown to culture-dependent methods discussed here, although,
produce heat-stable enzymes. For example, a study of to ensure equivalency to the standard method (i.e.,
the predominant microflora of low-quality bulk tank SPC), the output of individual bacteria count from the
milk (as defined by TBC >3.0 × 104) in Denmark re- flow cytometric methods is often converted to colony-
vealed that gram-negative, oxidase-positive bacteria, forming units, as the individual bacteria count is often
including Pseudomonas, Chryseobacterium, Alcaligenes, considerably higher due to the ability to enumerate
Comamonas, and Pasteurella, were present in 72% of cells not detected by culture-based methods. This
samples (Holm et al., 2004). Similarly, Rodrigues et al. conversion uses a linear regression equation that varies
(2017), in a study of bulk tank raw milk microbiome by system and eliminates some of the benefits of this
in high-SPC (>3.6 log cfu/mL) and low-SPC (≤3.6 log method. Stakeholders may consider using individual
cfu/mL) samples identified that high-SPC samples had bacteria count values instead of converting to colony-
significantly higher abundances of Acinetobacter, En- forming units, to maintain the benefits of this method
terobacteriaceae, Corynebacterium, and Streptococcus over culture-based methods.
(Rodrigues et al., 2017).
It is demonstrable that, as an indicator of raw milk Preliminary Incubation Count: Enrichment
quality, the TBC has important utility from both farm of Psychrophilic Bacteria
hygiene and management practice perspectives, as well
as potentially from a finished product perspective, as The PI count has a long history of use in the dairy
the types of bacteria that typically drive high TBC industry, with Johns (1958) first suggesting the use of
levels are those that are likely to produce heat-stable this parameter in North America. As Johns describes
enzymes; however, this parameter is not without limi- it, the adoption of bulk tank use on farms with proper
tations. Studies using culture-independent methods to cooling may mask the sanitary conditions used to pro-
evaluate microbial populations in raw milk (Doyle et al., duce the milk, and therefore a novel test should be
2016; Rodrigues et al., 2017; Skeie et al., 2019; McHugh used to identify milk produced under poor sanitary
et al., 2020) have provided a great deal of insight into conditions. The PI test conditions include incubation
types and sources of bacterial contamination; however, of raw milk for 18 h at 12.8°C, thereby enriching for
these insights have also raised questions about the limi- psychrophilic bacteria, followed by enumeration using
tations of our current industry test standards to truly the SPC method (Frank and Yousef, 2004). The US
assess the quality of raw milk. To illustrate this point, currently has no regulatory requirements for PI count,
many bacterial taxa found using culture-independent but guidelines typically suggest that PI count should
methods in bulk tank raw milk are obligately anaero- be either below a specific target (e.g., 50,000 cfu/mL)
bic, including various members of the classes Clostridia or below a certain increase (e.g., 3- to 4-fold) above
(e.g., Romboutsia, Ruminococcus, Mogibacterium) and the SPC value (Murphy and Boor, 2000a). Jayarao and
Bacteroidia (e.g., Bacteroides, Alistipes; Parente et al., Wolfgang (2003) suggest that a good PI count should
2020). This is no surprise, as these obligate anaerobes fall below 10,000 cfu/mL. Reported PI counts vary
are associated with the gut microflora and are pres- based on study, although few studies on contemporary
ent in high levels in manure (McGarvey et al., 2004). raw milk supplies have been published. Boor et al.
However, culture-dependent methods currently used to (1998) examined the microbial quality of raw bulk tank
Journal of Dairy Science Vol. 106 No. 3, 2023
Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1507

milk from New York producers between 1993 and 1996, bacteria seem to predominate the population of bacte-
reporting a mean PI count of 81,000 cfu/mL, with farm ria enriched during the PI method, the microbial driv-
means ranging from 13,000 cfu/mL to 216,000 cfu/mL ers of this parameter are more diverse than is typically
(Boor et al., 1998). Jayarao et al. (2004) reported a assumed.
mean PI count of 8,740 cfu/mL for 126 producers in The limitations of the PI count are driven by the
Pennsylvania over an 8-wk time period, with a range fact that this method is meant to predict the keeping
of 500 to 139,750 cfu/mL. In a study of approximately quality of raw milk under certain abusive conditions,
11,100 raw bulk tank milk samples collected from 235 namely (1) storage at the farm or processing facility
dairy producers in Prince Edward Island, Elmoslemany for long periods of time (e.g., >2–3 d total), (2) storage
et al. (2009a) reported a geometric mean PI count of at higher-than-optimum temperature (i.e., >4°C) for
12,000 cfu/mL. shorter periods of time, or (3) both conditions. Given
Elevated PI counts have been tied to inadequate that these conditions do not necessarily occur for any
cleaning and sanitation at the farm, in particular in given load of raw milk, the PI count has no significant
milking and milk storage equipment, but also potential- correlation with finished product quality (Martin et al.,
ly of the cows themselves. Jayarao et al. (2004) suggest 2011) despite selecting for psychrophilic bacteria that
that, when PI counts are elevated, investigation should are often capable of producing heat-stable enzymes.
focus on equipment factors such as inadequate water If storage and handling conditions at the farm and
temperature used to clean equipment and water quality the processing facility are adequately managed, most
issues (e.g., water hardness, pH), temperature of the bacteria detected by the PI count will not reach levels
bulk tank 2 h after milking, and pre- and post-milking where they produce heat-stable enzymes, and therefore
hygiene procedures (e.g., use of pre- and post-dip, use will not affect finished product quality. Importantly,
of forestripping). Similarly, Murphy and Boor (2000) total bacteria levels were considerably higher in the
indicate that elevated PI counts are most likely the 1950s and 1960s when the PI count was recommended
result of dirty equipment, poor cooling, or residual soil by Johns (1958), with the high-quality group in that
on teat surfaces at the time of milking. In a more re- study having SPC values ranging from 5,000 to 29,000
cent publication, Elmoslemany et al. (2009b) reported cfu/mL (mean of ~14,600 cfu/mL, calculated here from
that farm management practice risk factors for elevated raw data presented in Johns), which would not be con-
PI count included environment and cow hygiene fac- sidered high quality under contemporary definitions.
tors, including cow stall hygiene, teat cleanliness, teat Even so, some argue that, even in the contemporary
wash, and pre-dip, as well as equipment hygiene fac- raw milk continuum, some milk supplies are held for
tors, including temperature of bulk tank alkaline wash, 2 to 3 d at the farm and potentially another 2 to 3
pipeline alkaline wash alkalinity, and milk house water d at the processing facility before pasteurization, and
quality factors (Elmoslemany et al., 2009b). therefore using the PI count is still an appropriate as-
The groups of bacteria detected by the PI method are sessment of the risk of reduced keeping quality. A more
typically dominated by psychrophilic or psychrotolerant direct and quantitative approach to consider when raw
taxa, as the procedure itself enriches the populations milk with initial low TBC (i.e., <5,000 cfu/mL) is held
that can grow at 12.8°C. The predominant populations under abusive conditions, as defined here, is to evalu-
reportedly identified from PI counts include strepto- ate for TBC immediately before processing, to identify
cocci, coliforms, Pseudomonas, and at lower rates, total bacteria levels reaching concentrations that would
Staphylococcus and gram-positive rods (Gillespie et al., allow for production of heat-stable enzymes.
2012). In a dated study, Johns and Landerkin (1969)
reported that the primary organisms responsible for in- Coliform Count: Indicators of Fecal
creases in PI counts were gram-negative rods. Similarly, and Environmental Contamination
a more recent report by Martin et al. (2011) indicated
a moderate correlation (R2 = 0.68) between PI counts Coliforms are a method-defined group of bacteria that
on SPC agar versus PI count on gram-negative selective are aerobic or facultatively anaerobic gram-negative,
agar (i.e., crystal violet tetrazolium agar), indicating non-sporeforming rods capable of fermenting lactose to
that the predominant population were gram-negative produce gas and acid within 48h at 32 to 35°C (David-
bacteria. However, it should be noted that the raw milk son et al., 2004). Coliforms have been used for over a
samples tested in Martin et al. (2011) were collected century as indicators of fecal contamination in water,
from storage tanks at processing facilities, not from dairy, and other food products (Martin et al., 2016),
producer bulk tanks, which likely influenced the overall although it is noteworthy that the coliform group in
bacterial populations. These limited studies indicate general is not strictly associated with fecal contamina-
that, although Pseudomonas and other gram-negative tion, and, indeed, fecal coliforms are a small subset of
Journal of Dairy Science Vol. 106 No. 3, 2023
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the larger coliform group, many of which are associated amount of detergent) are strongly associated with in-
with environmental sources (e.g., water, vegetation). line raw milk CC. The authors further found a positive
The standard method for enumeration of CC is the association between SCC and CC, indicating that milk-
use of violet red bile agar, with confirmation testing of ing cows with coliform mastitis may have contributed
typical colonies for gas and acid production in brilliant to CC levels in their study (Pantoja et al., 2011).
green bile medium; however, the use of dehydrated film Coliforms are a diverse group of bacteria, falling
media (e.g., 3M Coliform Petrifilm) is pervasive due to primarily within the Enterobacteriaceae family but
the simplicity of the method (Davidson et al., 2004). also including members of the Aeromonadaceae fam-
The Pasteurized Milk Ordinance does not set forth a ily (Abbott et al., 2003). Genera commonly isolated
regulatory limit in the US for coliforms in raw milk; from raw milk include Citrobacter, Enterobacter, Esch-
however, California has established a regulatory limit erichia, and Klebsiella (Jayarao and Wang, 1999).
of 750 cfu/mL for raw milk (CDFA, 2022). Coliforms Kagkli et al. (2007) further report the presence of
are commonly evaluated in raw milk as indicators of Hafnia and Serratia in bulk tank raw milk. Of these
hygienic milking practices, specifically as indicators of genera, only Escherichia coli is predominantly associ-
fecal contamination, and guidelines suggest that good- ated with fecal contamination, whereas the remaining
quality raw milk should contain no more than 10 cfu/ coliform contaminants are often associated with, and
mL (Jayarao et al., 2001), with levels between 100 and persist in, environmental contamination, as from soil,
1,000 cfu/mL indicating poor milking hygiene (Ruegg water, and vegetation (Martin et al., 2016), which is
and Reinemann, 2002). Pantoja et al. (2009) reported a consistent with the reports that bulk tank raw milk
mean CC of 1.7 log cfu/mL (50 cfu/mL) from over 7,200 CC is significantly associated with equipment clean-
raw milk samples from 16 dairy farms in Wisconsin, ing and sanitation factors. Indeed, even members of
and Jayarao et al. (2004) reported a mean of 70 cfu/mL the coliform group traditionally thought to be superior
from 126 dairy farms in Pennsylvania, both at or above indicators of fecal contamination, Klebsiella and Esch-
the guidelines for good- and acceptable-quality raw erichia coli, have been shown to persist and grow in
milk, respectively. Jayarao and Wang (1999) reported a natural environments such as water and soil (Ferguson
considerably higher CC mean from 130 bulk tank raw and Signoretto, 2011). Many genera of environmental
milk samples from South Dakota and Minnesota, of 3.4 coliforms, including Enterobacter, Citrobacter, and Ser-
log cfu/mL (2,500 cfu/mL). ratia, have also been shown to grow substantially at
Farm sources and management practices associated low temperatures (Masiello et al., 2016), meaning that,
with high CC may include poor milking hygiene (e.g., over prolonged cold storage of raw milk, these organ-
not adequately removing residual soil from teats be- isms may contribute to high overall TBC in bulk tank
fore unit attachment), or environmental contamination raw milk. Finally, coliforms that cause environmental
through insufficiently cleaned equipment or, occasion- mastitis include Escherichia, Klebsiella, Enterobacter,
ally, from presence of coliform mastitis in the milking and Serratia (Hogan and Smith, 2003), which may also
herd (Murphy and Boor, 2000). Jayarao and Wolfgang contribute to CC in bulk tank raw milk. Hayes et al.
(2003) suggest that, when bulk tank raw milk CC (2001) found that, of 20 bulk tank raw milk TBC spikes
levels are high, pre-milking hygiene should be evalu- evaluated over a 2-wk period from 13 farms, 4 were as-
ated, including whether udders are wet during milking, sociated with high levels of Escherichia coli, potentially
whether the milking unit falls into manure during milk- due to milking of mastitic cows; however, the authors
ing, whether rubber hoses and gaskets show wear, and suggested that these spikes were more likely due to in-
whether clinical coliform mastitis occurs in the herd. A sufficient cleaning and sanitation of equipment.
study by Elmoslemany et al. (2009b) indicated that cow As discussed previously, elevated raw milk CC
hygiene factors, including leg hygiene score and teat (>100–1,000 cfu/mL) is an indicator of inadequate cow
cleanliness score, are significantly associated with bulk hygiene, milking time hygiene, or equipment hygiene.
tank raw milk CC; in addition, several equipment fac- However, similarly to the other hygiene indicators
tors, including temperature of multiple equipment wash discussed here, the ability of CC to influence finished
steps, cleaning and sanitation chemistry factors, and product quality is almost entirely dependent on the
milk house water quality factors, are also significantly conditions allowing these organisms to reach levels
associated with bulk tank raw milk CC. Pantoja et al. where they may begin to produce heat-stable enzymes.
(2011) also report that handling of milking unit clusters, Coliforms, as detailed for other gram-negative bacte-
specifically reducing rate of unit fall-offs and increasing ria (e.g., Pseudomonas), have been shown to produce
cluster washes, are associated with in-line CC and that a variety of enzymes when cell concentrations reach
milking machine wash failures (e.g., lower-than-normal high enough levels (Trmčić et al., 2015), some of which
wash temperature reached, failure to dispense preset are heat stable (Vithanage et al., 2016). Due to the
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limited ability of CC to indicate fecal contamination, raw milk. Elmoslemany et al. (2009b) evaluated the
this parameter does not provide considerable additional association between LPC in bulk tank raw milk and
information beyond that of other indicator organisms farm risk factors, reporting that higher temperatures
from the perspective of finished product quality. used during equipment cleaning and sanitation, high
chlorine concentration, high slug score (indicating suf-
Laboratory Pasteurization Count: A Measure ficient physical cleaning of the pipeline), and the use
of Thermoduric Bacteria of water softener (to eliminate hard water) are all pro-
tective against high LPC (Elmoslemany et al., 2009b).
The use of the LPC method dates back to the early Meanwhile, Bava et al. (2011) found that LPC of liner
20th century, when early dairy bacteriologists studied swabs before milking (indicating residual thermoduric
the presence of thermoduric bacteria in raw milk and bacteria in cleaned and sanitized milking liners) is sig-
their ability to survive pasteurization treatments, in nificantly and positively associated with bulk tank raw
particular low-temperature, long-time pasteurization milk SPC levels, further supporting the importance of
(Hileman, 1940). This method consists of heat treating equipment cleaning and sanitation on bacterial con-
raw milk to 62.8°C for 30 min to eliminate heat-sensi- tamination of the bulk tank milk.
tive bacteria, followed by enumeration using the SPC Early studies indicated that predominant populations
method (Frank and Yousef, 2004), although variations detected by the LPC consisted primarily of streptococci,
on this method will be discussed further herein. Similar micrococci, coryneform bacteria, aerobic sporeforming
to the PI and CC parameters, there are no regulatory rods, and occasionally gram-negative rods (Thomas et
limits for LPC in raw milk in the US, but this param- al., 1967). More modern evaluations of thermoduric
eter is often used as a component of producer quality populations in raw milk revealed similar outcomes, with
programs. It is generally recommended that LPC be Kikuchi et al. (1996) reporting the presence of Bacillus
below 100 cfu/mL as an indication of high-quality milk (30.7% of total isolates), Microbacterium (26.9% of to-
and that levels above 200 cfu/mL are an indication tal isolates), Micrococcaceae (23.4% of isolates), coryne-
of poor equipment hygiene (Murphy and Boor, 2000; form bacteria (7.9% of isolates), Streptococcus (6.7% of
Jayarao and Wolfgang, 2003). Several publications have isolates), and others at a lower rate in 400 laboratory
reported LPC values for bulk tank raw milk, including pasteurized raw milk samples from Japan. Delgado et
a mean of 1.9 log cfu/mL (79 cfu/mL) from a study of al. (2013) reported that nearly 60% of isolates from 22
7,220 bulk tank raw milk samples collected from Wis- raw milk samples from Spain subjected to laboratory
consin producers (Pantoja et al., 2009), a mean LPC pasteurization were identified as Streptococcus ther-
of 129 cfu/mL from 855 bulk tank raw milk samples in mophilus (56%), with other major contributors identi-
New York (Boor et al., 1998), and a mean of 43 cfu/mL fied as Lactobacillus (18%), Enterococcus (13%), and
from 1,141 bulk tank samples collected from Tennessee others at below 1% of the isolates identified, including
dairy farms (Gillespie et al., 2012). aerobic bacilli (Delgado et al., 2013). Finally, Ribeiro
It is widely accepted that LPC values exceeding Júnior et al. (2018) reported 8 genera of thermoduric
guidelines as described above (i.e., >200 cfu/mL) are bacteria isolated from 20 Brazilian raw milk samples,
associated with poor equipment cleaning and sanita- including Bacillus, Brachybacterium, Enterococcus,
tion. These associations date back to the earliest pub- Streptococcus, Micrococcus, Kocuria, Paenibacillus, and
lications on the use of LPC to identify populations of Macrococcus, with Bacillus representing approximately
bacteria capable of surviving pasteurization. A review 50% of the isolates identified. Although the overall
of the thermoduric bacteria in pasteurized milk, pub- types of thermoduric bacteria found in raw milk over
lished in the Journal of Dairy Science by Hileman in time appear to be consistent across studies, the pro-
1940, describes improperly sterilized milk contact sur- portions vary considerably by study and presumably
faces, dirty milk cans, and improper caring for equip- by raw milk supply, although this cannot be confirmed
ment and utensils as sources of thermoduric bacteria in through current literature. It should also be noted that
raw milk (Hileman, 1940). It should be noted that, in only one (Delgado et al., 2013) of the above-cited stud-
this 1940 review, a low LPC is designated as 5,000 cfu/ ies indicates what criteria were used to select bacterial
mL, nearly 2 orders of magnitude higher than current isolates (e.g., all colonies selected, selected based on
recommendations for LPC limits for good-quality milk. colony morphology, other), so appropriate caution must
Certainly, in the last century, cleaning and sanitation be used when interpreting the specific bacterial popula-
practices of milking and milk handling equipment have tion proportions reported here.
advanced dramatically. Current research, however, cor- Limitations of the LPC method for identifying high-
roborates these early reports that equipment hygiene quality raw milk include methodological issues and
is of particular importance to LPC levels in bulk tank interpretation issues. First, regarding methodological
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limitations of the LPC test, the recommendation is to by Martin et al. (2011). Another frequent misconcep-
pour plate using SPC agar, as many of the expected ther- tion is that the LPC is a proxy for spore populations
moduric bacteria selected by the laboratory pasteuriza- in raw milk. As described earlier, aerobic sporeformers
tion treatment are micro-aerophilic and therefore do may represent a range of proportions of the thermodu-
not form robust colonies on the surface of agar, making ric populations in raw milk and are typically present in
enumeration difficult. Many contemporary laboratories very low levels. For these reasons, if a raw milk spore
have moved away from using the pour plating method, count is desired, the method used should be specific
as it is more time consuming and requires the use of an for spores, as will be described in the next section.
autoclave. In its place, dehydrated film plates (e.g., 3M Finally, it should be acknowledged that, although non-
Petrifilm) are increasingly used, because they require sporeforming thermoduric bacteria are not drivers of
no specialized equipment beyond a pipette and an incu- processed dairy product quality in most cases, these
bator. A study by Byrne and Bishop (1991) evaluated organisms can represent important non-starter lactic
3M Aerobic Count Petrifilm as a suitable alternative acid bacteria, especially in raw milk cheeses.
for LPC enumeration compared with SPC pour plates.
The authors reported that, for 45 samples of naturally Sporeforming Bacteria: Low-Level Contaminants
contaminated raw milk, there was no significant differ- Contributing to Dairy Product Quality
ence between LPC conducted using SPC pour plates
and AC Petrifilm. However, when evaluating raw milk The final raw milk microbiological parameter that
experimentally contaminated with various thermoduric will be discussed in this review is the spore count.
bacteria, the authors found that recovery of 6 of the 7 Overall, this parameter is used less frequently to moni-
Micrococcus species tested was significantly lower on tor raw milk quality than the others discussed here,
AC Petrifilm than on SPC pour plates. The authors and, indeed, to call it one parameter is a misnomer;
further noted that, when raw milk samples were allowed as sporeforming bacteria are diverse and have varied
to sit at room temperature for 2 to 4 h before plating, effects on finished product quality, several different
this difference diminished. It should therefore be noted methods are used to quantify different types of spore-
that, although overall differences between these 2 plat- formers. The primary groups of sporeforming bacteria
ing methods for naturally contaminated milk appear to monitored in raw milk supplies are (1) psychrotolerant
be small based on this one study, high proportions of sporeformers (e.g., Paenibacillus spp.), which cause
Micrococcus in the thermoduric population of any given spoilage of fluid milk; (2) mesophilic and thermophilic
raw milk supply would likely result in far lower LPC sporeformers (e.g., Bacillus licheniformis), which con-
with the use of AC Petrifilm. tribute to levels of spores in dairy powders; and (3)
Beyond the LPC methodological issues discussed, anerobic butyric acid bacteria (e.g., Clostridium tyro-
the issue of interpretation represents a much larger butyricum), which cause late-blowing defect in certain
limitation to the contemporary dairy industry. The first styles of cheese. The methods used to detect each of
misinterpretation of the LPC test is that it represents these groups include application of an initial heat treat-
populations of thermoduric bacteria capable of sur- ment to eliminate all vegetative cells in the sample,
viving pasteurization. Although that may be true for including other thermodurics (e.g., Micrococcus) and
low-temperature, long-time pasteurization, also called sporeforming bacteria that are not in spore form, fol-
batch or vat pasteurization, which is conducted at 63°C lowed by selective enumeration methods that allow for
for 30 min, it is not true for HTST pasteurization (72°C growth of the group of interest. For psychrotolerant,
for 15 s), which is the heat treatment used for the vast mesophilic, and thermophilic spore count, raw milk is
majority of fluid milk in the US. Previous studies of heat treated to 80°C for 12 min, followed by plating on
HTST fluid milk populations indicate that the pre- standard methods agar, which is then incubated at 6°C
dominant bacteria recovered are Bacillus spp., whereas, for 10 d, 32°C for 48 h, and 55°C for 48 h before enu-
at the end of shelf-life, the predominant bacteria are meration, respectively (Martin et al., 2019). Anaerobic
the psychrotolerant sporeforming bacteria Paenibacillus butyric acid bacteria (BAB) are enumerated using a
(Huck et al., 2008; Ranieri and Boor, 2009). Of course, MPN method due to their presence in low levels in
Bacillus and other aerobic sporeforming bacteria are raw milk, which is performed by distributing raw milk
among the bacterial taxa selected for by the LPC into tubes of Bryant and Burkey medium in successive
method, as previously seen. However, given the large dilution series (e.g., 5 tubes with 9 mL of medium and 1
variation in the proportions of these organisms within mL of raw milk; 5 tubes with 9.9 mL of medium and 0.1
the overall thermoduric population found in raw milk mL of raw milk), which are then capped with melted
supplies, LPC is a poor predictor of the microbiological paraffin wax to maintain an anaerobic environment and
performance of pasteurized fluid milk, as demonstrated heat treated at 75°C for 15 min, followed by incubation
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at 35°C for 6 d. Tubes are scored for gas production (as raw milk. These can broadly be categorized as (1)
indicated by the movement of the wax plug upward in housing area and bedding practices, (2) milking routine
the tube), and final MPN is calculated (Martin et al., practices, (3) cow-level factors, and (4) feed factors.
2019). For example, Murphy et al. (2019) demonstrated that
The US currently has no regulatory spore count lim- bedding material is associated with spores in bulk tank
its for raw milk, and spore count monitoring is primar- raw milk, with level of spores in the bedding material
ily driven by processors that want to control finished directly associated with MSC and TSC in raw milk.
product quality or spoilage originating from spores in Magnusson et al. (2007) reported the same finding when
raw milk. Unlike other bacterial parameters described specifically investigating the levels of raw milk contami-
herein, there are no generally accepted guidelines for nation with Bacillus cereus spores. Miller et al. (2015a)
limits of spores in bulk tank milk, with the exception of also reported that the use of sand or sawdust bedding
a limit of 3.0 log spores/L (1,000 spores/L or 1 spore/ for lactating cows was associated with lower MSC in
mL) for BAB spores suggested by Vissers et al. (2006) bulk tank raw milk, and the use of straw bedding was
to prevent late-blowing defect in cheese. Spore levels associated with lower TSC in bulk tank raw milk. Mur-
in raw milk are reportedly very low, which contributes phy et al. (2019) and Martin et al. (2019) both reported
to the limitations of this method, as we will discuss. that the increased frequency of topping up or chang-
Martin et al. (2019) reported mean psychrotolerant ing bedding was associated with reduced bulk tank
spore count of −0.24 log cfu/mL (0.57 cfu/mL), meso- raw milk spore levels. Studies have also suggested that
philic spore count (MSC) of 0.50 log cfu/mL (3 cfu/ milking routine practices may affect spore levels in bulk
mL), and thermophilic spore count (TSC) of 0.36 log tank raw milk; this may be driven by the effectiveness
cfu/mL (2.3 cfu/mL) in 34 samples of bulk tank raw of different milking practices with regard to removal
milk collected from 17 New York dairy farms. Similarly, of residual soil (e.g., manure, bedding material) from
Buehler et al. (2018a) reported a mean psychrotoler- teat ends. Relatedly, the amount of soil on teat and
ant spore count of −0.72 log MPN/mL (0.19 MPN/ udder surfaces has also been shown to be significantly
mL) based on data collected by Masiello et al. (2014), associated with bulk tank spore levels (Vissers et al.,
also demonstrating low-level psychrotolerant spore 2006; Masiello et al., 2014; Martin et al., 2019; Murphy
contamination in raw milk. In a study of bulk tank et al., 2019). Indeed, Evanowski et al. (2020) reported
raw milk from farms in 18 US states, Murphy et al. that significant reductions in bulk tank MSC and TSC
(2019) reported mean MSC and TSC of 0.26 log cfu/ were observed after milking employees were trained on
mL (1.8 cfu/mL). Finally, BAB spores are also pres- enhanced teat end cleaning procedures and laundered
ent in raw milk at low levels. For example, Vissers et towels used for milking preparation were prepared by
al. (2007) reported a mean BAB from 24 farms in the washing with detergent, sanitizing with bleach, and
Netherlands of 2.70 log spores/L (501 spores/L or 0.5 thoroughly drying, demonstrating the importance of
spores/mL; Vissers et al., 2007). Despite these low lev- milking preparation procedures on transmission of
els of contamination, spores present in raw milk survive spores into bulk tank raw milk. Finally, feed factors
processing hurdles and go on to cause quality deterio- influence BAB spore levels in bulk tank raw milk. For
ration of finished dairy products, making them espe- example, Vissers et al. (2007) found that the driving
cially important parameters of raw milk quality. With factor for BAB spore levels in bulk tank raw milk was
no generally accepted limits on aerobic spore levels in the concentration of BAB spores in mixed silage fed
raw milk to prevent finished product quality issues, an to the lactating herd. The pathway of contamination
alternative approach for quantifying and reducing the from feed to milk includes concentration of spores in
effects of these organisms is to use predictive modeling the manure and subsequent contamination of the udder
tools such as those discussed later in this review. Brief- and teat surfaces. These studies all demonstrate the
ly, stakeholders use established distributions of target importance of comprehensive management in control of
sporeforming populations (e.g., psychrotolerant spores) the transmission of spores from environmental sources
in raw milk, or develop distributions based on their own into bulk tank raw milk.
supply chain, to predict the impact on finished product Concerning the characterization of sporeforming
outcomes. These models can then be used to determine bacteria found in bulk tank raw milk, major genera
the incremental improvement on finished product out- reported include Bacillus, Paenibacillus, Clostridium,
comes (e.g., spoilage) when spore concentrations are Brevibacillus, Lysinibacillus, Sporosarcina, Ureibacillus,
reduced in the supply. Viridibacillus, Oceanobacillus, and others (Coorevits et
Numerous publications have demonstrated the role al., 2008; Miller et al., 2015a). Multiple reports suggest
that farm management practices and factors play in that the most prevalent sporeforming bacterium pres-
the levels of sporeforming bacteria found in bulk tank ent in raw milk is Bacillus licheniformis (Crielly et al.,
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1994; Miller et al., 2015b), a facultative anaerobe that quality is low. For example, Martin et al. (2011) found
has a broad range of growth temperatures. Bacillus no significant association between spore count and
cereus group members, which includes both pathogens fluid milk SPC at 17 d of refrigerated storage, despite
(e.g., Bacillus cereus sensu stricto) and dairy spoilage Paenibacillus being the predominant genus detected at
organisms (e.g., Bacillus weihenstephanensis; Ivy et al., that point in shelf-life. The authors acknowledged that
2012), are also common contaminants in bulk tank raw the limitation of the enumeration method, specifically
milk (Lin et al., 1998; te Giffel et al., 2002), representing that many samples tested had spore counts below the
spoilage and potential foodborne disease risks in down- limit of detection, likely influenced the outcome of
stream dairy products. Other Bacillus species identified their study. It is further noteworthy that, in addition to
in raw milk include Bacillus clausii, Bacillus pumilus, recommendations for BAB limits in raw milk intended
Bacillus subtilis, and others (Miller et al., 2015b). Pae- for use in cheesemaking, as described earlier, no well-
nibacillus dominates the psychrotolerant sporeforming established guidelines for aerobic spore limits in raw
taxa found in raw milk (Ivy et al., 2012), but other milk currently exist, limiting the ease of interpretation
genera found in raw milk have also been reported to of this parameter to dairy industry stakeholders.
grow at low temperatures, including Bacillus weihenste-
phanensis and Viridibacillus spp. (Trmčić et al., 2015). Defining Raw Milk Microbiological Quality from
Similarly, strictly anerobic sporeforming bacteria are the Perspective of Finished Product Quality:
predominantly within the genus Clostridium (Doyle et A 3-Tiered Approach
al., 2015), with the notable member being Clostridium
tyrobutyricum as the primary causative agent of late- Each of the microbiological parameters discussed
blowing defect in certain styles of cheese (Klijn et al., herein has benefits and limitations for identifying and
1995). Finally, raw milk is occasionally demonstrated defining raw milk quality, but, importantly, it has been
to be a source of sporeforming extremophiles that sur- demonstrated that these various parameters are often
vive very harsh conditions that are destructive to most significantly associated with each other and with other
other sporeforming organisms. For example, Schelde- non-microbiological measures of quality, specifically
man et al. (2005) found a surprising diversity of highly bulk tank SCC. For example, Jayarao et al. (2004) re-
heat-resistant spores (recovered after heat treatment of ported that bulk tank raw milk with low SPC (<5,000
100°C for 30 min) in raw milk supplies from 17 farms cfu/mL) also had a significantly lower SCC (<200,000
in Belgium, including Bacillus sporothermodurans and cells/mL), PI (<10,000 cfu/mL), LPC (<100 cfu/mL),
Geobacillus spp. These extremophiles have particular and other bacterial measures of quality. Similarly, Pan-
importance to the quality of dairy powders and UHT toja et al. (2009) found that an increased TBC was 6.3
fluid milks (Miller et al., 2015b; Alonso et al., 2021). times more likely for bulk tank raw milk with elevated
Overall, the diversity of sporeforming bacteria in raw CC, and 1.3 times more likely for bulk tank milk with
milk supplies contributes to their ability to cause qual- elevated LPC. The authors further note that the odds
ity deterioration in finished dairy products and repre- of increased TBC increased by 2.4% for every 10,000
sents a challenge for detecting and enumerating specific cell/mL increase in bulk tank raw milk SCC (Pan-
subgroups, as we will describe subsequently. toja et al., 2009). Schukken et al. (1992) reported that
The primary limitations of monitoring raw milk herds that produced milk with higher PLC, a measure
supplies for sporeforming bacteria are related to their of TBC, had higher SCC, and Gillespie et al. (2012)
presence in very low concentrations, often below 1 cfu/ identified a significant association between SPC and
mL, which is the detection limit of most traditional PI. Finally, Masiello et al. (2017) reports a significant
microbiological methods. In response, methods that association between bulk tank SCC and TBC, PI, and
estimate low-level concentrations of spores (i.e., MPN) psychrotolerant spore count. A key takeaway from the
are used for BAB and have been described for psy- associations between raw milk microbiological param-
chrotolerant spores (Masiello et al., 2014); however, eters for dairy industry stakeholders is that monitoring
these methods are time consuming and tedious, and raw milk quality, especially as it pertains to identifying
require a great deal of space and laboratory ware (e.g., raw milk supplies likely to perform well throughout the
test tubes), which are barriers to routine use. Due to continuum from production to processing and shelf-life,
the low levels of contamination and the associated should be streamlined to focus on an overall microbio-
methodological challenges with enumerating low-level logical quality indicator, which we propose should be
contaminants—as the number of recovered colonies are TBC. As noted by Hutchison et al. (2005), although
often at such low levels (i.e., <20–25 cfu/mL) that reli- TBC is not without its own shortcomings, no better
able assessment of spore loads is not assured—the as- bacterial marker for overall raw milk hygiene currently
sociation of raw milk spore count with finished product exists, especially given its ease and rapidity of measure-
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Table 1. Suggested 3-tiered approach to using microbiological testing to define raw milk quality

Goal of testing Recommended microbiological test(s) Testing frequency


Process control Total bacteria count (TBC) High frequency, every load of milk
Monitoring parameters that Spore counts including butyric acid bacteria High/moderate frequency initially to develop a baseline
directly influence finished count and psychrotolerant spore count, distribution in the supply, then subsequently lower
product quality lactic acid bacteria frequency to identify deviations from the baseline
Troubleshooting Preliminary incubation count, coliform count, Low frequency, only when needed to troubleshoot high
and laboratory pasteurization count TBC, paired with on-farm risk assessment

ment, widespread use, and interpretability. Publica- that discussed herein. Once a baseline is established,
tions reviewed here and industry guidance suggest that monitoring tests may be performed less frequently
high-quality contemporary bulk tank raw milk should (e.g., monthly) unless a deviation from the baseline is
have no more than 5,000 cfu/mL TBC, which typically detected. Outcomes of monitoring tests may also be
would be an indicator of implementation of compre- used to reward producers who consistently produce
hensive farm hygiene and management. Conceptually, raw milk with low spore concentrations through pre-
we suggest that TBC is a test that should be used to mium payments. The second method, troubleshoot-
assess “process control,” which would mean that this ing tests, should be used only when TBC exceeds the
test should be used frequently (ideally for every bulk acceptable quality specification and includes the PI,
tank or load of milk) to verify processes and parameters CC, and LPC tests. In conjunction with evaluation of
on the farm (Table 1). Although an acceptable quality on-farm risk factors (e.g., pre-milking routine, equip-
specification limit of 5,000 cfu/mL for bulk tank TBC ment cleaning, and sanitation), troubleshooting tests
may be a good starting point for many operations, these should be used on a case-by-case basis to investigate
limits ultimately should be defined by each stakeholder, causes of increased TBC.
and more stringent limits may be set over time as part Overall, this 3-tiered approach to identifying high-
of a continuous improvement process. Some operations quality raw milk through the strategic use of raw milk
may even want to apply statistical process control pro- microbiological test parameters will facilitate a more
cedures using TBC data, as has been described for bulk efficient system with better outcomes for stakeholders
tank SCC (Lukas et al., 2005). across the dairy industry and can be used as the basis
Beyond the use of TBC as an overall process control of establishing a quality control program for raw milk.
test, we suggest that other microbiological tests dis-
cussed in this review should be utilized in 1 of 2 ways: Applying Data-Driven, Risk-Based Tools to Ensuring
(1) monitoring parameters that directly influence the Raw Milk Microbiological Quality for Optimum
finished product, or (2) troubleshooting causes of high Finished Product Performance
TBC (Table 1). The first, monitoring tests, should be
used periodically to assess parameters that directly In addition to the raw milk microbiological testing
affect finished product quality, including various types approach outlined here, future dairy industry stake-
of spore tests, especially BAB count and psychrotol- holders should adopt predictive tools that allow for
erant spore count. Additional microbial monitoring the integration of data across the dairy continuum to
parameters should be considered based on the specific support risk-based decision making at the farm. The
dairy product manufactured from the raw milk sup- research discussed in this review reveals considerable
ply. For example, although not covered in detail here overlap in on-farm risk factors for different raw milk
because their presence in raw milk is not routinely microbiological parameters. For example, adequate
tested for, certain raw milk lactic acid bacteria (lacto- equipment hygiene factors are associated with elevated
bacilli, streptococci, and others) may affect the qual- levels of TBC, CC, PI, and LPC (Elmoslemany et
ity of artisanal raw milk cheeses, in the development al., 2009b; Bava et al., 2011). Leveraging the research
of both desirable and undesirable attributes, making on these common factors will be essential to transi-
this group an appropriate target for raw milk micro- tion from a solely reactive, test-based system that is
bial monitoring in some cases. Initially, stakeholders predominantly used in the dairy industry today to a
should monitor these parameters frequently (e.g., whole-farm, risk-based system where farm management
weekly) to develop a baseline distribution of the spore practices and testing data are paired to proactively
concentrations (or alternative monitoring target) in reduce the likelihood of adverse events occurring, as
a particular supply and to ensure that this baseline relating to raw milk microbiological quality and result-
falls within expected ranges based on research such as ing finished product quality.

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Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1514

The development of decision support tools at the ers define raw milk quality. Further cross-disciplinary
production and processing levels has demonstrated how efforts should be made, to ensure broad adoption and
relevant data can be used to predict outcomes of inter- implementation of these tools in the future. For ex-
est to dairy industry stakeholders. For example, Ca- ample, co-ops and processors could use these models to
brera (2018) outlines the development and application set target levels for measures that are directly linked
of over 20 computerized dairy farm decision support to finished product quality and conversion efficiency
tools to assist producers with farm management related (e.g., BAB counts, psychrotolerant spore counts), with
to nutrition, reproduction, calf and heifer management, associated premiums. This approach would allow for
replacement, price risk, and environment (Cabrera, strategic targeting of specific on-farm practices to
2018). Other similar tools have also been described ensure consistent production of raw milk that meets
for dairy production (Rose et al., 2016; Balhara et al., specifications, and subsequent verification of this ap-
2021), and numerous commercial products exist on the proach through periodic, low-frequency monitoring of
marketplace (e.g., iDDEN.org, MyDairyDashboard. relevant parameters (e.g., BAB counts) paired with
com). Similarly, decision support tools have been de- frequent process control monitoring (i.e., TBC testing)
veloped to assist processors with dairy product quality on every load of milk.
improvements. For example, Monte Carlo simulations
have been used to predict the shelf-life of fluid milk CONCLUSIONS
based on the presence of psychrotolerant spores in
raw milk supplies (Buehler et al., 2018a), to predict Bacterial populations in raw milk play an important
consumer exposure to spoiled yogurt (Buehler et al., role in the quality of processed dairy products. How-
2018b), and to predict the occurrence of late-blowing ever, as discussed in this review, parameters currently
defect in cheese (Qian et al., 2022). used to assess microbiological raw milk quality have
Despite the demonstrated utility of data-driven, risk- varying degrees of capacity to inform stakeholders
based decision support tools, they are not without their on their potential implications for finished product
limitations. In particular, dairy producers face several quality. The future of raw milk quality determina-
barriers to the adoption of these tools. A recent com- tion should focus on a whole-farm approach that ac-
mentary on improving the adoption rates of integrated knowledges the microbiological risk factors at various
decision support systems identified major barriers in- stages of the production process and transitions from
cluding (1) perceived limited value by producers, (2) is- the reactive test-based system of our current dairy
sues of ease of use or interpretation, (3) lack of practical industry to a predictive, risk-based system. This re-
and direct application, (4) data collection, quality, and quires standard use of an appropriate microbiological
standardization challenges, (5) lack of data integration, indicator, namely TBC, periodic monitoring of micro-
(6) limited data sharing and farm infrastructure, and bial parameters that directly affect finished product
(7) limited multidisciplinary collaboration between sci- quality, use of troubleshooting tests when the process
entists developing new knowledge and those developing control test is out of compliance, and implementation
decision support tools (Baldin et al., 2021). Similarly, of integrated decision support tools that will provide
Rose et al. (2016) outlined a similar set of factors af- producers with the necessary guidance to produce
fecting adoption of decision support tools, including high-quality raw milk for processing into high-quality
what the authors describe as core factors (e.g., ease of finished dairy products. This approach will require
use, peer recommendation, cost), enabling factor (i.e., stakeholder collaboration throughout the dairy con-
facilitating conditions such as internet and phone con- tinuum from production, processing, regulatory agen-
nection), driving factors (e.g., level of marketing and cies, and academic institutions.
legislative compliance), and modifying factors (e.g.,
producer age, scale of business; Rose et al., 2016). ACKNOWLEDGMENTS
Importantly, these barriers are likely to have the larg-
est influence on dairy producers who are most in need The authors acknowledge the role that the New York
from the perspective of quality improvements (e.g., State Dairy Promotion Advisory Board (Albany, NY)
those with limited resources, remote farm locations). has played in their continued support of research aimed
However, despite these limitations, development of at improving the quality and safety of dairy products
data-driven, risk-based decision support tools applied in New York and beyond. The authors have not stated
to the microbiological quality of raw milk, integrated any conflicts of interest. The authors acknowledge that
throughout the production to processing chain, has the N. Martin is a section editor for the Journal of Dairy
potential to revolutionize how dairy industry stakehold- Science.

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Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1515
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Journal of Dairy Science Vol. 106 No. 3, 2023

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