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
Raw milk typically has little bacterial contamination
as it leaves the udder of the animal; however, through a
variety of pathways, it can become contaminated with
bacteria originating from environmental sources, the
cow herself, and contact with contaminated equipment.
Although the types of bacteria found in raw milk are
very diverse, select groups are particularly important
from the perspective of finished product quality. In par-
ticular, psychrophilic and psychrotolerant bacteria that
grow quickly at low temperatures (e.g., species in the
genus Pseudomonas and the family Enterobacteriaceae)
and produce heat-stable enzymes, and sporeforming
bacteria that survive processing hurdles in spore form,
are the 2 primary groups of bacteria related to effects
on processed dairy products. Understanding factors
leading to the presence of these important bacterial
groups in raw milk is key to reducing their influence on
processed dairy product quality. Here we examine the
raw milk microbiological parameters used in the con-
temporary dairy industry for their utility in identifying
raw milk supplies that will perform well in processed
dairy products. We further recommend the use of a
single microbiological indicator of raw milk quality,
namely the total bacteria count, and call for the devel-
opment of a whole-farm approach to raw milk quality
that will use data-driven, risk-based tools integrated
across the continuum from production to processing
and shelf-life to ensure continuous improvement in
dairy product quality.
Key words: raw milk quality, microbiological testing,
bacterial contamination, farm practices
INTRODUCTION
The microbiological composition of raw bovine milk
has been extensively studied for over a century. Although
it is expected that raw milk will contain low levels of
Received June 16, 2022.
Accepted September 17, 2022.
*Corresponding author: nhw6@​cornell​.edu
1502
microbial contamination (here the focus will be on bac-
terial contamination), contemporary and historical at-
tention has emphasized the production of high-quality
raw milk as an indicator of overall hygienic production
practices at the farm. The term “high-quality,” from
a microbiological perspective, is often used to refer to
raw milk with low total bacteria counts; however, the
term may also be used to describe raw milk with low
levels of certain groups of bacteria (e.g., coliforms).
Indeed, tests for various groups of raw milk bacteria
have been used as indicators of raw milk quality beyond
the total bacteria count (TBC), most notably, prelimi-
nary incubation (PI) count for psychrophilic bacteria,
coliform count (CC), laboratory pasteurization count
(LPC) for thermoduric bacteria, and, less frequently,
spore count testing. Results of these tests may be used
to indicate specific deficiencies in hygienic practices;
for example, the presence of high levels of coliforms
(e.g., >100 cfu/mL) may indicate that cows are not
adequately cleaned before milking unit attachment
(Murphy and Boor, 2000).
Despite the common use of these tests to assess raw
milk quality, they are primarily used as indicators
of conditions on the farm and not as an indicator of
performance in finished products, an important consid-
eration when defining raw milk quality. An extensive
discussion of the effects of raw milk microbial contami-
nants on processed dairy product quality will not be
included here, as a recent review thoroughly examined
this topic (Murphy et al., 2016). However, it is notewor-
thy to our review that Murphy et al. (2016) identified
2 primary groups of raw milk microbial contaminants
that influence processed dairy product quality, includ-
ing (1) psychrophilic and psychrotolerant bacteria (e.g.,
Pseudomonas) and (2) sporeforming bacteria (e.g., Pae-
nibacillus and Clostridium). Briefly, high initial concen-
trations of psychrophilic and psychrotolerant bacteria,
or growth due to improper cooling or excessive storage
times, may lead to heat-stable enzyme development by
Pseudomonas and other related bacteria. Even though
the bacterial cells themselves do not survive process-
ing hurdles, the heat-stable enzymes produced when
cell concentrations reach 5 to 8 log cfu/mL continue
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
breaking down milk components even after processing,
leading to flavor, odor, and body defects in finished
products (Sørhaug and Stepaniak, 1997). For example,
cheese made from raw milk with elevated bacterial
numbers before processing (e.g., >1,000,000 cfu/mL)
leads to reduced yields and flavor defects during aging
(Banks et al., 1988). In UHT milk products, which are
stored at ambient temperatures, residual heat-stable
proteases and lipases produced by elevated raw milk
microbial loads cause gelation of the product over the
extended shelf-life, a defect termed “age-gelation,” along
with other textural defects such as sedimentation, and
off-odors and flavors (Murphy et al., 2016).
The second key group of bacteria in raw milk that
are relevant to finished product quality are sporeform-
ing bacteria, a diverse group belonging to the orders
Bacillales and Clostridiales. These bacteria produce
endospores, or spores, that originate from natural envi-
ronments on dairy farms, are transferred into raw milk,
survive processing hurdles, and subsequently grow,
causing spoilage in finished dairy products (Gopal et
al., 2015). For example, psychrotolerant sporeforming
bacteria grow at refrigeration temperatures, causing
fluid milk spoilage (Ranieri and Boor, 2009). Studies
have demonstrated that psychrotolerant sporeforming
bacteria are responsible for approximately 40 to 50%
of fluid milk in the United States reaching the Pasteur-
ized Milk Ordinance bacterial limit of 20,000 cfu/mL
during shelf-life (Ranieri and Boor, 2009; Alles et al.,
2018; Reichler et al., 2018) and, importantly, are the
primary bacterial agents limiting the extension of fluid
milk shelf-life. Another important group of sporeform-
ing bacteria, anaerobic butyric acid bacterial spores
(i.e., Clostridium spp.), cause late-blowing defect in
cheese (Klijn et al., 1995). Late blowing is of particular
concern in hard and semi-hard aged cheeses such as
Gouda and does not typically develop until 60 to 90 d
into aging, resulting in economic losses for cheesemak-
ers (Martin et al., 2021). A focus on these raw milk
microbial drivers of finished product quality is neces-
sary to prioritize efforts that will result in improved
finished product quality. This is critical to the entire
dairy continuum, as consumer dissatisfaction with
product quality influences their willingness and intent
to continue purchasing and consuming that product
(Quelch and Ash, 1980).
In this review we discuss common contemporary
microbiological parameters used to evaluate raw milk
quality, types of bacterial contaminants targeted by
these tests, sources of contaminants, and farm factors
associated with these parameters, as well as potential
shortcomings of these parameters for evaluating raw
milk quality from the perspective of finished product
quality. Further, we propose a new definition of raw
Journal of Dairy Science Vol. 106 No. 3, 2023
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milk quality that utilizes (1) an overall raw milk quality
indicator as a process control test to assess consistency
and compliance with hygienic farm practices, (2) a
set of tests that will aid in troubleshooting when the
overall raw milk quality indicator is at high levels, and
(3) tests that specifically assess the presence and levels
of groups of bacteria that influence finished product
quality. Finally, we discuss the potential for develop-
ment of predictive decision support tools that integrate
knowledge of raw milk microbial populations, as well
as farm management practices that influence micro-
bial presence and levels in raw milk and their effects
on finished product quality. Moving from the current
reactive, test-based system to a proactive and predic-
tive approach is a critical step in ensuring high-quality
dairy products across the continuum from production
to consumption.
RAW MILK MICROBIOLOGICAL PARAMETERS
Total Bacteria Count: An Overall Indicator
of Raw Milk Microbiological Quality
and Hygienic Production Practices
The TBC is the most widely used measure of micro-
bial quality of raw milk and is measured in the United
States using several approved methods including the
standard plate count (SPC), plate loop count (PLC),
Petrifilm (3M) aerobic count, flow cytometry method-
ologies (e.g., Bactoscan, Foss Analytical), and others
(USPHS/FDA, 2019). In the US, the Pasteurized Milk
Ordinance limits the bacteria count in grade “A” raw
milk to 100,000 cfu/mL for individual producers or
300,000 cfu/mL for commingled raw milk (USPHS/
FDA, 2019). Outside of the US, similar limits on TBC
are required. For example, the limit for total plate count
of raw milk intended for processing is 100,000 cfu/mL in
Europe (European Commission, 2021), whereas in New
Zealand the limit is 300,000 cfu/mL (Ministry for Pri-
mary Industries, 2022), and Canadian standards limit
total aerobic mesophilic bacteria to 50,000 cfu/mL (Na-
tional Dairy Code, 1997). However, reported mean bulk
tank TBC typically fall well below these regulatory lim-
its. For instance, Elmoslemany et al. (2009a) measured
TBC and other microbiological quality parameters over
a 2-year period from approximately 11,100 bulk tank
raw milk samples collected from 235 dairy farms in
Prince Edward Island, Canada. The authors report a
TBC geometric mean of 5,300 cfu/mL with only 6% of
samples above 50,000 cfu/mL. Similarly, Pantoja et al.
(2009) measured TBC from 7,241 bulk tank raw milk
samples from 16 farms in Wisconsin (USA) during a
1-year period, reporting a mean TBC of 3.1 log cfu/mL
(1,259 cfu/mL), with just 1.6% (n = 142) above the
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
regulatory limit of 100,000 cfu/mL. In a more recent
study, Rodrigues et al. (2017) evaluated 472 raw bulk
tank milk samples from 19 farms in New York State
and reported mean TBC values by farm ranging from
3.01 log cfu/mL (1,023 cfu/mL) to 4.11 log cfu/mL
(12,882 cfu/mL). Although these reports indicate that
TBC values well below common regulatory limits in the
US and in other countries are commonplace, producers
are encouraged to pursue continuous improvement in
reducing bulk tank raw milk TBC, primarily through
the lever of premium payments from processors and
cooperative. Guidelines suggest that TBC targets for
good raw milk are in the range of <5,000 to <10,000
cfu/mL (Murphy and Boor, 2000; Jayarao and Wolf-
gang, 2003), yet these recommendations are somewhat
dated for contemporary producers. It should also be
noted that the regulatory limit for TBC (i.e., 100,000
cfu/mL) is not an appropriate cutoff for defining raw
milk quality, as raw milk with this level of microbial
contamination is at high risk of resulting in finished
product quality deterioration.
Farm sources and management practices associated
with elevated TBC in bulk tank raw milk have been
widely studied. Many possible pathways of bacterial
contamination throughout the production continuum
may lead to increased TBC in bulk tank milk, so com-
prehensive management is essential to limit contamina-
tion and subsequent growth of bacteria during storage.
The first potential source of bacterial contamination
is, of course, the udder itself, especially when an active
bacterial infection is present. For example, Hayes et al.
(2001) examined a variety of raw milk microbiological
quality parameters, including SPC, Petrifilm aerobic
count, presumptive gram-negative bacteria on Mac-
Conkey agar, and presumptive Streptococcus spp. on
Edwards medium, from bulk tank milk from 13 farms
over a 2-week period to characterize the causes of spikes
(i.e., sudden elevations above normal values) in TBC.
The authors found that 11 of the 20 observed spikes
were driven by increases in the mastitis pathogen Strep-
tococcus uberis (Hayes et al., 2001). Similarly, Zadoks
et al. (2004) found that streptococcal, staphylococcal,
and gram-negative counts were all significantly and
positively associated with bulk tank raw milk TBC,
with streptococcal counts accounting for 69% of TBC
variability in their study of mastitis-causing bacteria
in bulk tank raw milk on 48 New York farms. The
relationship between mastitis and TBC is further il-
lustrated by the relationship between SCC and TBC.
For example, in a study of the associations between
raw milk quality indicators in bulk tank raw milk on 16
farms, Pantoja et al. (2009) reported that the odds of
increased TBC increased by 2.4% for every 10,000 cell/
mL increase in SCC in the same milk load. A study of
Journal of Dairy Science Vol. 106 No. 3, 2023
1504
SCC and PLC, a measure of TBC, from monthly pro-
ducer data collected from 5 large milk processing facili-
ties in New York from March 1999 to December 2000,
indicated that farms with SCC below 200,000 cells/mL
had lower PLC levels compared with milk with higher
SCC (van Schaik et al., 2002). Additionally, Borneman
and Ingham (2014) reported a highly significant cor-
relation (P-value <2 × 10−16) between SPC and SCC in
2012 bulk tank raw milk samples; however, the authors
also report that the R2 value for this association was
quite small (0.02–0.03), suggesting that many other
factors also drive SPC outcomes. Indeed, the effects of
mastitis pathogens on bulk tank raw milk TBC depend
on multiple factors, including the causative bacterial
agent, the proportion of the herd infected, and the size
of the herd (Hayes et al., 2001).
In addition to the contribution of mastitis to bulk
tank TBC, other major factors that influence this
parameter include milking time hygiene (for example,
sufficient removal of soil from teat surfaces before milk-
ing) and equipment and raw milk handling factors (e.g.,
proper cleaning and sanitation of equipment and ad-
equate cooling of raw milk after harvest). Elmoslemany
et al. (2010) identified risk factors for elevated total
aerobic count, including the amount of dirt on teats be-
fore milking, use of water to wash teats during milking
preparation without subsequent drying, use of the same
towel for drying multiple cows, and manual cleaning of
the bulk tank. The authors also noted that the lowest
bulk tank bacterial counts were associated with pre-
dipping of teats during milking preparation followed
by drying with single-use towels, compared with other
methods of teat preparation. Similarly, Jayarao et al.
(2004) studied microbial quality parameters in bulk
tank raw milk from 126 dairy farms in Pennsylvania and
found that when cows were subjected to pre- and post-
dipping, SPC values were significantly lower, whereas
SPC values were significantly higher when cows were
sprayed with teat dip instead of using a cup for ap-
plication. According to a study by Doyle et al. (2016),
the microbial population of the teat surface was the
most significant source of contamination for raw milk,
which highlights the importance of pre-milking hygiene
to management of TBC in bulk tank raw milk. Further,
these studies also provide additional insight into the as-
sociations described earlier between TBC and SCC, as
effective milking preparation and hygiene are essential
to prevent mastitis from contagious and environmental
mastitis pathogens (Hogan, 2017; Middleton and Fox,
2017), which ultimately cause elevated SCC in bulk
tank milk. Finally, equipment hygiene and water qual-
ity factors also play an important role in management
of TBC in bulk tank raw milk. For example, Elmos-
lemany et al. (2009b) reported that high slug scores,
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
indicating sufficient physical cleaning of the pipeline,
along with use of water softener, were associated with
lower TBC in bulk tank raw milk and related. Hard
water and infrequent evaluation of milk house water for
bacterial contamination were associated with elevated
TBC in bulk tank raw milk. In a study specifically fo-
cused on the effects of cleaning and hygiene of milking
equipment on bacterial counts in raw milk, Bava et
al. (2011) categorized farms into a high SPC group (n
= 14 farms, mean SPC = 4.25 log cfu/mL) and a low
SPC group (n = 8, mean SPC = 3.68 log cfu/mL) and
examined equipment cleaning and sanitation factors
and equipment contamination between the groups. The
authors report that the high and low groups differed
significantly between bacterial contamination of liners
and milk receivers, as well as in water temperature
reached during the detergent phase of cleaning milking
equipment. Unfortunately, many of these studies report
associations, and few studies have assessed whether the
identified associations represent true “cause and effect”
relationships.
Importantly, raw milk handling, with particular at-
tention paid to achieving and maintaining proper cool-
ing (i.e., maintaining milk temperature below 40°F, or
4.4°C), is also critical to preventing increases in TBC
during storage, especially for raw milk supplies that
will be stored for longer than 24 h before processing.
O’Connell et al. (2016) showed that, when stored at
6°C, the bulk tank raw milk TBC increased signifi-
cantly from 3.43 log cfu/mL to 4.87 log cfu/mL after
96 h of storage, but storing raw milk at 2°C resulted in
no significant increase between 0 and 96 h of storage.
Driving increases in raw milk TBC during prolonged
storage, or storage at abusive temperatures (e.g.,
≥6°C), is the growth of Pseudomonas. De Jonghe et al.
(2011) demonstrated that under suboptimal simulated
storage conditions (i.e., 6°C storage with 7 brief spikes
to 10°C over 4 d, to simulate the addition of warm milk
to the bulk tank) not only was there a significant in-
crease in raw milk TBC compared with optimal storage
conditions (i.e., 3.5°C storage with 7 brief spikes to 6°C
over 4 d, to simulate the addition of warm milk to the
bulk tank), but Pseudomonas was the primary bacterial
agent causing the increase in TBC. This is of particular
importance, as noted earlier in this review, because a
variety of Pseudomonas species are known to produce
heat-stable enzymes that ultimately affect the quality
of processed dairy products.
As the name suggests, the TBC approximates the to-
tal levels of bacterial contaminants in raw milk, yet the
types of microbial contaminants in bulk tank raw milk
are diverse, and, importantly, not all of these contami-
nants are relevant to finished product quality, as will be
discussed. Advances in culture-independent molecular
Journal of Dairy Science Vol. 106 No. 3, 2023
1505
biological methodologies over the last 20 years have al-
lowed for more comprehensive evaluation of microbial
populations in raw milk using methods such as targeted
metagenomics. Although study design plays a large role
in the microbiome identified in any particular study
due to variations between farms and over time on the
same farm (Skeie et al., 2019), by stage of lactation
(McHugh et al., 2020), by geographic region (Skeie et
al., 2019), and under different meteorological condi-
tions (Kable et al., 2016), there are commonalities to
be drawn with regard to the raw milk microbiome from
these studies. In a recent thorough review of the raw
milk microbiome by Parente et al. (2020), the authors
evaluated results from 5 studies on the microbiome of
bulk tank raw milk from different geographic regions
to determine the most prevalent and abundant taxa
in raw bulk tank milk. These taxa included members
of the phyla Proteobacteria, Firmicutes, Bacteroidota
(formerly Bacteroidetes), Actinobacteria, and Mycoplas-
matota (formerly Tenericutes), with the most prevalent
and abundant genera including Pseudomonas, Strepto-
coccus, Lactococcus, Acinetobacter, and Staphylococcus
(Parente et al., 2020). In addition to the commonalities
in raw milk microbial content, it should be noted that,
overall, the microbiome of raw milk is tremendously
diverse, with many relevant bacterial taxa that are
important from the perspective of milk quality, human
safety, and animal health often present at very low lev-
els. For example, sporeforming bacteria typically are
found in raw milk at very low levels (Martin et al.,
2019). Psychrotolerant sporeforming bacteria, which
are the primary biological limiting factor for HTST
fluid milk shelf-life (Ranieri and Boor, 2009), have been
found to have a mean concentration of less than 1 spore
per milliliter of raw bulk tank milk [mean of −0.72 log10
most probable number (MPN)/mL; Buehler et al.,
2018a] in a study of 99 farms across New York State.
Further supporting that psychrotolerant sporeforming
bacteria are typically found at low levels, a recent study
of spores in bulk tank raw milk from 17 New York State
dairy farms reported that only 42% of the 34 bulk tank
raw milk samples tested had detectable levels of psy-
chrotolerant spores in the 1 mL of raw milk that had
been heat-treated and plated for spore enumeration
(Martin et al., 2019). In the aforementioned review by
Parente et al. (2020), Paenibacillus, the predominant
genus of psychrotolerant sporeforming bacteria causing
HTST fluid milk spoilage (Ranieri and Boor, 2009),
was not identified in the top 25 most prevalent and
abundant bacterial genera, yet it is a major contributor
to fluid milk spoilage and waste in the US (Martin et
al., 2021), highlighting the challenges associated with
detecting low-level bacterial contaminants of relevance
to raw milk quality.
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
A high TBC, whether gauged by the regulatory limit
(i.e., 100,000 cfu/mL) or by current examples of lim-
its defining high-quality raw milk (e.g., 5,000 cfu/mL;
Jayarao and Wolfgang, 2003) in and of itself is not a
definitive indication that dairy products manufactured
from that milk will have reduced quality. Indeed, as
discussed previously, the primary raw milk microbial
drivers of finished product quality include groups of
bacteria capable of producing heat-stable enzymes and
sporeforming bacteria. Other bacterial contaminants,
even when present at elevated levels, may have no dis-
cernable effect on finished product quality, although
they may indeed indicate a farm-level deficiency in
cleaning, sanitation, or other management factors.
However, evidence suggests that, when TBC counts
are high, the bacteria responsible for the overall in-
creases are of the groups that have been shown to
produce heat-stable enzymes. For example, a study of
the predominant microflora of low-quality bulk tank
milk (as defined by TBC >3.0 × 104) in Denmark re-
vealed that gram-negative, oxidase-positive bacteria,
including Pseudomonas, Chryseobacterium, Alcaligenes,
Comamonas, and Pasteurella, were present in 72% of
samples (Holm et al., 2004). Similarly, Rodrigues et al.
(2017), in a study of bulk tank raw milk microbiome
in high-SPC (>3.6 log cfu/mL) and low-SPC (≤3.6 log
cfu/mL) samples identified that high-SPC samples had
significantly higher abundances of Acinetobacter, En-
terobacteriaceae, Corynebacterium, and Streptococcus
(Rodrigues et al., 2017).
It is demonstrable that, as an indicator of raw milk
quality, the TBC has important utility from both farm
hygiene and management practice perspectives, as well
as potentially from a finished product perspective, as
the types of bacteria that typically drive high TBC
levels are those that are likely to produce heat-stable
enzymes; however, this parameter is not without limi-
tations. Studies using culture-independent methods to
evaluate microbial populations in raw milk (Doyle et al.,
2016; Rodrigues et al., 2017; Skeie et al., 2019; McHugh
et al., 2020) have provided a great deal of insight into
types and sources of bacterial contamination; however,
these insights have also raised questions about the limi-
tations of our current industry test standards to truly
assess the quality of raw milk. To illustrate this point,
many bacterial taxa found using culture-independent
methods in bulk tank raw milk are obligately anaero-
bic, including various members of the classes Clostridia
(e.g., Romboutsia, Ruminococcus, Mogibacterium) and
Bacteroidia (e.g., Bacteroides, Alistipes; Parente et al.,
2020). This is no surprise, as these obligate anaerobes
are associated with the gut microflora and are pres-
ent in high levels in manure (McGarvey et al., 2004).
However, culture-dependent methods currently used to
Journal of Dairy Science Vol. 106 No. 3, 2023
1506
evaluate the TBC (e.g., SPC, Petrifilm aerobic count)
are performed under aerobic conditions, a potential
limitation for the interpretation of these tests as they
relate to raw milk quality. Further limitations of these
standard culture-dependent TBC methods include the
use of mesophilic incubation temperatures (typically
30–35°C; Laird et al., 2004), which do not allow for the
growth of obligate psychrophiles or thermophiles, and
the common presence of bacteria that grow in clumps
or chains (e.g., gram-positive cocci such as Streptococ-
cus and Staphylococcus), resulting in multiple cells pre-
senting as single colonies on agar plates (Sutton, 2012).
Fortunately, the increased use of culture-independent
flow cytometric methods (e.g., Bactoscan) for estima-
tion of TBC in bulk tank raw milk has addressed many
of the major limitations associated with traditional
culture-dependent methods discussed here, although,
to ensure equivalency to the standard method (i.e.,
SPC), the output of individual bacteria count from the
flow cytometric methods is often converted to colony-
forming units, as the individual bacteria count is often
considerably higher due to the ability to enumerate
cells not detected by culture-based methods. This
conversion uses a linear regression equation that varies
by system and eliminates some of the benefits of this
method. Stakeholders may consider using individual
bacteria count values instead of converting to colony-
forming units, to maintain the benefits of this method
over culture-based methods.
Preliminary Incubation Count: Enrichment
of Psychrophilic Bacteria
The PI count has a long history of use in the dairy
industry, with Johns (1958) first suggesting the use of
this parameter in North America. As Johns describes
it, the adoption of bulk tank use on farms with proper
cooling may mask the sanitary conditions used to pro-
duce the milk, and therefore a novel test should be
used to identify milk produced under poor sanitary
conditions. The PI test conditions include incubation
of raw milk for 18 h at 12.8°C, thereby enriching for
psychrophilic bacteria, followed by enumeration using
the SPC method (Frank and Yousef, 2004). The US
currently has no regulatory requirements for PI count,
but guidelines typically suggest that PI count should
be either below a specific target (e.g., 50,000 cfu/mL)
or below a certain increase (e.g., 3- to 4-fold) above
the SPC value (Murphy and Boor, 2000a). Jayarao and
Wolfgang (2003) suggest that a good PI count should
fall below 10,000 cfu/mL. Reported PI counts vary
based on study, although few studies on contemporary
raw milk supplies have been published. Boor et al.
(1998) examined the microbial quality of raw bulk tank
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
milk from New York producers between 1993 and 1996,
reporting a mean PI count of 81,000 cfu/mL, with farm
means ranging from 13,000 cfu/mL to 216,000 cfu/mL
(Boor et al., 1998). Jayarao et al. (2004) reported a
mean PI count of 8,740 cfu/mL for 126 producers in
Pennsylvania over an 8-wk time period, with a range
of 500 to 139,750 cfu/mL. In a study of approximately
11,100 raw bulk tank milk samples collected from 235
dairy producers in Prince Edward Island, Elmoslemany
et al. (2009a) reported a geometric mean PI count of
12,000 cfu/mL.
Elevated PI counts have been tied to inadequate
cleaning and sanitation at the farm, in particular in
milking and milk storage equipment, but also potential-
ly of the cows themselves. Jayarao et al. (2004) suggest
that, when PI counts are elevated, investigation should
focus on equipment factors such as inadequate water
temperature used to clean equipment and water quality
issues (e.g., water hardness, pH), temperature of the
bulk tank 2 h after milking, and pre- and post-milking
hygiene procedures (e.g., use of pre- and post-dip, use
of forestripping). Similarly, Murphy and Boor (2000)
indicate that elevated PI counts are most likely the
result of dirty equipment, poor cooling, or residual soil
on teat surfaces at the time of milking. In a more re-
cent publication, Elmoslemany et al. (2009b) reported
that farm management practice risk factors for elevated
PI count included environment and cow hygiene fac-
tors, including cow stall hygiene, teat cleanliness, teat
wash, and pre-dip, as well as equipment hygiene fac-
tors, including temperature of bulk tank alkaline wash,
pipeline alkaline wash alkalinity, and milk house water
quality factors (Elmoslemany et al., 2009b).
The groups of bacteria detected by the PI method are
typically dominated by psychrophilic or psychrotolerant
taxa, as the procedure itself enriches the populations
that can grow at 12.8°C. The predominant populations
reportedly identified from PI counts include strepto-
cocci, coliforms, Pseudomonas, and at lower rates,
Staphylococcus and gram-positive rods (Gillespie et al.,
2012). In a dated study, Johns and Landerkin (1969)
reported that the primary organisms responsible for in-
creases in PI counts were gram-negative rods. Similarly,
a more recent report by Martin et al. (2011) indicated
a moderate correlation (R2 = 0.68) between PI counts
on SPC agar versus PI count on gram-negative selective
agar (i.e., crystal violet tetrazolium agar), indicating
that the predominant population were gram-negative
bacteria. However, it should be noted that the raw milk
samples tested in Martin et al. (2011) were collected
from storage tanks at processing facilities, not from
producer bulk tanks, which likely influenced the overall
bacterial populations. These limited studies indicate
that, although Pseudomonas and other gram-negative
Journal of Dairy Science Vol. 106 No. 3, 2023
1507
bacteria seem to predominate the population of bacte-
ria enriched during the PI method, the microbial driv-
ers of this parameter are more diverse than is typically
assumed.
The limitations of the PI count are driven by the
fact that this method is meant to predict the keeping
quality of raw milk under certain abusive conditions,
namely (1) storage at the farm or processing facility
for long periods of time (e.g., >2–3 d total), (2) storage
at higher-than-optimum temperature (i.e., >4°C) for
shorter periods of time, or (3) both conditions. Given
that these conditions do not necessarily occur for any
given load of raw milk, the PI count has no significant
correlation with finished product quality (Martin et al.,
2011) despite selecting for psychrophilic bacteria that
are often capable of producing heat-stable enzymes.
If storage and handling conditions at the farm and
the processing facility are adequately managed, most
bacteria detected by the PI count will not reach levels
where they produce heat-stable enzymes, and therefore
will not affect finished product quality. Importantly,
total bacteria levels were considerably higher in the
1950s and 1960s when the PI count was recommended
by Johns (1958), with the high-quality group in that
study having SPC values ranging from 5,000 to 29,000
cfu/mL (mean of ~14,600 cfu/mL, calculated here from
raw data presented in Johns), which would not be con-
sidered high quality under contemporary definitions.
Even so, some argue that, even in the contemporary
raw milk continuum, some milk supplies are held for
2 to 3 d at the farm and potentially another 2 to 3
d at the processing facility before pasteurization, and
therefore using the PI count is still an appropriate as-
sessment of the risk of reduced keeping quality. A more
direct and quantitative approach to consider when raw
milk with initial low TBC (i.e., <5,000 cfu/mL) is held
under abusive conditions, as defined here, is to evalu-
ate for TBC immediately before processing, to identify
total bacteria levels reaching concentrations that would
allow for production of heat-stable enzymes.
Coliform Count: Indicators of Fecal
and Environmental Contamination
Coliforms are a method-defined group of bacteria that
are aerobic or facultatively anaerobic gram-negative,
non-sporeforming rods capable of fermenting lactose to
produce gas and acid within 48h at 32 to 35°C (David-
son et al., 2004). Coliforms have been used for over a
century as indicators of fecal contamination in water,
dairy, and other food products (Martin et al., 2016),
although it is noteworthy that the coliform group in
general is not strictly associated with fecal contamina-
tion, and, indeed, fecal coliforms are a small subset of
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
the larger coliform group, many of which are associated
with environmental sources (e.g., water, vegetation).
The standard method for enumeration of CC is the
use of violet red bile agar, with confirmation testing of
typical colonies for gas and acid production in brilliant
green bile medium; however, the use of dehydrated film
media (e.g., 3M Coliform Petrifilm) is pervasive due to
the simplicity of the method (Davidson et al., 2004).
The Pasteurized Milk Ordinance does not set forth a
regulatory limit in the US for coliforms in raw milk;
however, California has established a regulatory limit
of 750 cfu/mL for raw milk (CDFA, 2022). Coliforms
are commonly evaluated in raw milk as indicators of
hygienic milking practices, specifically as indicators of
fecal contamination, and guidelines suggest that good-
quality raw milk should contain no more than 10 cfu/
mL (Jayarao et al., 2001), with levels between 100 and
1,000 cfu/mL indicating poor milking hygiene (Ruegg
and Reinemann, 2002). Pantoja et al. (2009) reported a
mean CC of 1.7 log cfu/mL (50 cfu/mL) from over 7,200
raw milk samples from 16 dairy farms in Wisconsin,
and Jayarao et al. (2004) reported a mean of 70 cfu/mL
from 126 dairy farms in Pennsylvania, both at or above
the guidelines for good- and acceptable-quality raw
milk, respectively. Jayarao and Wang (1999) reported a
considerably higher CC mean from 130 bulk tank raw
milk samples from South Dakota and Minnesota, of 3.4
log cfu/mL (2,500 cfu/mL).
Farm sources and management practices associated
with high CC may include poor milking hygiene (e.g.,
not adequately removing residual soil from teats be-
fore unit attachment), or environmental contamination
through insufficiently cleaned equipment or, occasion-
ally, from presence of coliform mastitis in the milking
herd (Murphy and Boor, 2000). Jayarao and Wolfgang
(2003) suggest that, when bulk tank raw milk CC
levels are high, pre-milking hygiene should be evalu-
ated, including whether udders are wet during milking,
whether the milking unit falls into manure during milk-
ing, whether rubber hoses and gaskets show wear, and
whether clinical coliform mastitis occurs in the herd. A
study by Elmoslemany et al. (2009b) indicated that cow
hygiene factors, including leg hygiene score and teat
cleanliness score, are significantly associated with bulk
tank raw milk CC; in addition, several equipment fac-
tors, including temperature of multiple equipment wash
steps, cleaning and sanitation chemistry factors, and
milk house water quality factors, are also significantly
associated with bulk tank raw milk CC. Pantoja et al.
(2011) also report that handling of milking unit clusters,
specifically reducing rate of unit fall-offs and increasing
cluster washes, are associated with in-line CC and that
milking machine wash failures (e.g., lower-than-normal
wash temperature reached, failure to dispense preset
Journal of Dairy Science Vol. 106 No. 3, 2023
1508
amount of detergent) are strongly associated with in-
line raw milk CC. The authors further found a positive
association between SCC and CC, indicating that milk-
ing cows with coliform mastitis may have contributed
to CC levels in their study (Pantoja et al., 2011).
Coliforms are a diverse group of bacteria, falling
primarily within the Enterobacteriaceae family but
also including members of the Aeromonadaceae fam-
ily (Abbott et al., 2003). Genera commonly isolated
from raw milk include Citrobacter, Enterobacter, Esch-
erichia, and Klebsiella (Jayarao and Wang, 1999).
Kagkli et al. (2007) further report the presence of
Hafnia and Serratia in bulk tank raw milk. Of these
genera, only Escherichia coli is predominantly associ-
ated with fecal contamination, whereas the remaining
coliform contaminants are often associated with, and
persist in, environmental contamination, as from soil,
water, and vegetation (Martin et al., 2016), which is
consistent with the reports that bulk tank raw milk
CC is significantly associated with equipment clean-
ing and sanitation factors. Indeed, even members of
the coliform group traditionally thought to be superior
indicators of fecal contamination, Klebsiella and Esch-
erichia coli, have been shown to persist and grow in
natural environments such as water and soil (Ferguson
and Signoretto, 2011). Many genera of environmental
coliforms, including Enterobacter, Citrobacter, and Ser-
ratia, have also been shown to grow substantially at
low temperatures (Masiello et al., 2016), meaning that,
over prolonged cold storage of raw milk, these organ-
isms may contribute to high overall TBC in bulk tank
raw milk. Finally, coliforms that cause environmental
mastitis include Escherichia, Klebsiella, Enterobacter,
and Serratia (Hogan and Smith, 2003), which may also
contribute to CC in bulk tank raw milk. Hayes et al.
(2001) found that, of 20 bulk tank raw milk TBC spikes
evaluated over a 2-wk period from 13 farms, 4 were as-
sociated with high levels of Escherichia coli, potentially
due to milking of mastitic cows; however, the authors
suggested that these spikes were more likely due to in-
sufficient cleaning and sanitation of equipment.
As discussed previously, elevated raw milk CC
(>100–1,000 cfu/mL) is an indicator of inadequate cow
hygiene, milking time hygiene, or equipment hygiene.
However, similarly to the other hygiene indicators
discussed here, the ability of CC to influence finished
product quality is almost entirely dependent on the
conditions allowing these organisms to reach levels
where they may begin to produce heat-stable enzymes.
Coliforms, as detailed for other gram-negative bacte-
ria (e.g., Pseudomonas), have been shown to produce
a variety of enzymes when cell concentrations reach
high enough levels (Trmčić et al., 2015), some of which
are heat stable (Vithanage et al., 2016). Due to the
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
limited ability of CC to indicate fecal contamination,
this parameter does not provide considerable additional
information beyond that of other indicator organisms
from the perspective of finished product quality.
Laboratory Pasteurization Count: A Measure
of Thermoduric Bacteria
The use of the LPC method dates back to the early
20th century, when early dairy bacteriologists studied
the presence of thermoduric bacteria in raw milk and
their ability to survive pasteurization treatments, in
particular low-temperature, long-time pasteurization
(Hileman, 1940). This method consists of heat treating
raw milk to 62.8°C for 30 min to eliminate heat-sensi-
tive bacteria, followed by enumeration using the SPC
method (Frank and Yousef, 2004), although variations
on this method will be discussed further herein. Similar
to the PI and CC parameters, there are no regulatory
limits for LPC in raw milk in the US, but this param-
eter is often used as a component of producer quality
programs. It is generally recommended that LPC be
below 100 cfu/mL as an indication of high-quality milk
and that levels above 200 cfu/mL are an indication
of poor equipment hygiene (Murphy and Boor, 2000;
Jayarao and Wolfgang, 2003). Several publications have
reported LPC values for bulk tank raw milk, including
a mean of 1.9 log cfu/mL (79 cfu/mL) from a study of
7,220 bulk tank raw milk samples collected from Wis-
consin producers (Pantoja et al., 2009), a mean LPC
of 129 cfu/mL from 855 bulk tank raw milk samples in
New York (Boor et al., 1998), and a mean of 43 cfu/mL
from 1,141 bulk tank samples collected from Tennessee
dairy farms (Gillespie et al., 2012).
It is widely accepted that LPC values exceeding
guidelines as described above (i.e., >200 cfu/mL) are
associated with poor equipment cleaning and sanita-
tion. These associations date back to the earliest pub-
lications on the use of LPC to identify populations of
bacteria capable of surviving pasteurization. A review
of the thermoduric bacteria in pasteurized milk, pub-
lished in the Journal of Dairy Science by Hileman in
1940, describes improperly sterilized milk contact sur-
faces, dirty milk cans, and improper caring for equip-
ment and utensils as sources of thermoduric bacteria in
raw milk (Hileman, 1940). It should be noted that, in
this 1940 review, a low LPC is designated as 5,000 cfu/
mL, nearly 2 orders of magnitude higher than current
recommendations for LPC limits for good-quality milk.
Certainly, in the last century, cleaning and sanitation
practices of milking and milk handling equipment have
advanced dramatically. Current research, however, cor-
roborates these early reports that equipment hygiene
is of particular importance to LPC levels in bulk tank
Journal of Dairy Science Vol. 106 No. 3, 2023
1509
raw milk. Elmoslemany et al. (2009b) evaluated the
association between LPC in bulk tank raw milk and
farm risk factors, reporting that higher temperatures
used during equipment cleaning and sanitation, high
chlorine concentration, high slug score (indicating suf-
ficient physical cleaning of the pipeline), and the use
of water softener (to eliminate hard water) are all pro-
tective against high LPC (Elmoslemany et al., 2009b).
Meanwhile, Bava et al. (2011) found that LPC of liner
swabs before milking (indicating residual thermoduric
bacteria in cleaned and sanitized milking liners) is sig-
nificantly and positively associated with bulk tank raw
milk SPC levels, further supporting the importance of
equipment cleaning and sanitation on bacterial con-
tamination of the bulk tank milk.
Early studies indicated that predominant populations
detected by the LPC consisted primarily of streptococci,
micrococci, coryneform bacteria, aerobic sporeforming
rods, and occasionally gram-negative rods (Thomas et
al., 1967). More modern evaluations of thermoduric
populations in raw milk revealed similar outcomes, with
Kikuchi et al. (1996) reporting the presence of Bacillus
(30.7% of total isolates), Microbacterium (26.9% of to-
tal isolates), Micrococcaceae (23.4% of isolates), coryne-
form bacteria (7.9% of isolates), Streptococcus (6.7% of
isolates), and others at a lower rate in 400 laboratory
pasteurized raw milk samples from Japan. Delgado et
al. (2013) reported that nearly 60% of isolates from 22
raw milk samples from Spain subjected to laboratory
pasteurization were identified as Streptococcus ther-
mophilus (56%), with other major contributors identi-
fied as Lactobacillus (18%), Enterococcus (13%), and
others at below 1% of the isolates identified, including
aerobic bacilli (Delgado et al., 2013). Finally, Ribeiro
Júnior et al. (2018) reported 8 genera of thermoduric
bacteria isolated from 20 Brazilian raw milk samples,
including Bacillus, Brachybacterium, Enterococcus,
Streptococcus, Micrococcus, Kocuria, Paenibacillus, and
Macrococcus, with Bacillus representing approximately
50% of the isolates identified. Although the overall
types of thermoduric bacteria found in raw milk over
time appear to be consistent across studies, the pro-
portions vary considerably by study and presumably
by raw milk supply, although this cannot be confirmed
through current literature. It should also be noted that
only one (Delgado et al., 2013) of the above-cited stud-
ies indicates what criteria were used to select bacterial
isolates (e.g., all colonies selected, selected based on
colony morphology, other), so appropriate caution must
be used when interpreting the specific bacterial popula-
tion proportions reported here.
Limitations of the LPC method for identifying high-
quality raw milk include methodological issues and
interpretation issues. First, regarding methodological
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
limitations of the LPC test, the recommendation is to
pour plate using SPC agar, as many of the expected ther-
moduric bacteria selected by the laboratory pasteuriza-
tion treatment are micro-aerophilic and therefore do
not form robust colonies on the surface of agar, making
enumeration difficult. Many contemporary laboratories
have moved away from using the pour plating method,
as it is more time consuming and requires the use of an
autoclave. In its place, dehydrated film plates (e.g., 3M
Petrifilm) are increasingly used, because they require
no specialized equipment beyond a pipette and an incu-
bator. A study by Byrne and Bishop (1991) evaluated
3M Aerobic Count Petrifilm as a suitable alternative
for LPC enumeration compared with SPC pour plates.
The authors reported that, for 45 samples of naturally
contaminated raw milk, there was no significant differ-
ence between LPC conducted using SPC pour plates
and AC Petrifilm. However, when evaluating raw milk
experimentally contaminated with various thermoduric
bacteria, the authors found that recovery of 6 of the 7
Micrococcus species tested was significantly lower on
AC Petrifilm than on SPC pour plates. The authors
further noted that, when raw milk samples were allowed
to sit at room temperature for 2 to 4 h before plating,
this difference diminished. It should therefore be noted
that, although overall differences between these 2 plat-
ing methods for naturally contaminated milk appear to
be small based on this one study, high proportions of
Micrococcus in the thermoduric population of any given
raw milk supply would likely result in far lower LPC
with the use of AC Petrifilm.
Beyond the LPC methodological issues discussed,
the issue of interpretation represents a much larger
limitation to the contemporary dairy industry. The first
misinterpretation of the LPC test is that it represents
populations of thermoduric bacteria capable of sur-
viving pasteurization. Although that may be true for
low-temperature, long-time pasteurization, also called
batch or vat pasteurization, which is conducted at 63°C
for 30 min, it is not true for HTST pasteurization (72°C
for 15 s), which is the heat treatment used for the vast
majority of fluid milk in the US. Previous studies of
HTST fluid milk populations indicate that the pre-
dominant bacteria recovered are Bacillus spp., whereas,
at the end of shelf-life, the predominant bacteria are
the psychrotolerant sporeforming bacteria Paenibacillus
(Huck et al., 2008; Ranieri and Boor, 2009). Of course,
Bacillus and other aerobic sporeforming bacteria are
among the bacterial taxa selected for by the LPC
method, as previously seen. However, given the large
variation in the proportions of these organisms within
the overall thermoduric population found in raw milk
supplies, LPC is a poor predictor of the microbiological
performance of pasteurized fluid milk, as demonstrated
Journal of Dairy Science Vol. 106 No. 3, 2023
1510
by Martin et al. (2011). Another frequent misconcep-
tion is that the LPC is a proxy for spore populations
in raw milk. As described earlier, aerobic sporeformers
may represent a range of proportions of the thermodu-
ric populations in raw milk and are typically present in
very low levels. For these reasons, if a raw milk spore
count is desired, the method used should be specific
for spores, as will be described in the next section.
Finally, it should be acknowledged that, although non-
sporeforming thermoduric bacteria are not drivers of
processed dairy product quality in most cases, these
organisms can represent important non-starter lactic
acid bacteria, especially in raw milk cheeses.
Sporeforming Bacteria: Low-Level Contaminants
Contributing to Dairy Product Quality
The final raw milk microbiological parameter that
will be discussed in this review is the spore count.
Overall, this parameter is used less frequently to moni-
tor raw milk quality than the others discussed here,
and, indeed, to call it one parameter is a misnomer;
as sporeforming bacteria are diverse and have varied
effects on finished product quality, several different
methods are used to quantify different types of spore-
formers. The primary groups of sporeforming bacteria
monitored in raw milk supplies are (1) psychrotolerant
sporeformers (e.g., Paenibacillus spp.), which cause
spoilage of fluid milk; (2) mesophilic and thermophilic
sporeformers (e.g., Bacillus licheniformis), which con-
tribute to levels of spores in dairy powders; and (3)
anerobic butyric acid bacteria (e.g., Clostridium tyro-
butyricum), which cause late-blowing defect in certain
styles of cheese. The methods used to detect each of
these groups include application of an initial heat treat-
ment to eliminate all vegetative cells in the sample,
including other thermodurics (e.g., Micrococcus) and
sporeforming bacteria that are not in spore form, fol-
lowed by selective enumeration methods that allow for
growth of the group of interest. For psychrotolerant,
mesophilic, and thermophilic spore count, raw milk is
heat treated to 80°C for 12 min, followed by plating on
standard methods agar, which is then incubated at 6°C
for 10 d, 32°C for 48 h, and 55°C for 48 h before enu-
meration, respectively (Martin et al., 2019). Anaerobic
butyric acid bacteria (BAB) are enumerated using a
MPN method due to their presence in low levels in
raw milk, which is performed by distributing raw milk
into tubes of Bryant and Burkey medium in successive
dilution series (e.g., 5 tubes with 9 mL of medium and 1
mL of raw milk; 5 tubes with 9.9 mL of medium and 0.1
mL of raw milk), which are then capped with melted
paraffin wax to maintain an anaerobic environment and
heat treated at 75°C for 15 min, followed by incubation
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
at 35°C for 6 d. Tubes are scored for gas production (as
indicated by the movement of the wax plug upward in
the tube), and final MPN is calculated (Martin et al.,
2019).
The US currently has no regulatory spore count lim-
its for raw milk, and spore count monitoring is primar-
ily driven by processors that want to control finished
product quality or spoilage originating from spores in
raw milk. Unlike other bacterial parameters described
herein, there are no generally accepted guidelines for
limits of spores in bulk tank milk, with the exception of
a limit of 3.0 log spores/L (1,000 spores/L or 1 spore/
mL) for BAB spores suggested by Vissers et al. (2006)
to prevent late-blowing defect in cheese. Spore levels
in raw milk are reportedly very low, which contributes
to the limitations of this method, as we will discuss.
Martin et al. (2019) reported mean psychrotolerant
spore count of −0.24 log cfu/mL (0.57 cfu/mL), meso-
philic spore count (MSC) of 0.50 log cfu/mL (3 cfu/
mL), and thermophilic spore count (TSC) of 0.36 log
cfu/mL (2.3 cfu/mL) in 34 samples of bulk tank raw
milk collected from 17 New York dairy farms. Similarly,
Buehler et al. (2018a) reported a mean psychrotoler-
ant spore count of −0.72 log MPN/mL (0.19 MPN/
mL) based on data collected by Masiello et al. (2014),
also demonstrating low-level psychrotolerant spore
contamination in raw milk. In a study of bulk tank
raw milk from farms in 18 US states, Murphy et al.
(2019) reported mean MSC and TSC of 0.26 log cfu/
mL (1.8 cfu/mL). Finally, BAB spores are also pres-
ent in raw milk at low levels. For example, Vissers et
al. (2007) reported a mean BAB from 24 farms in the
Netherlands of 2.70 log spores/L (501 spores/L or 0.5
spores/mL; Vissers et al., 2007). Despite these low lev-
els of contamination, spores present in raw milk survive
processing hurdles and go on to cause quality deterio-
ration of finished dairy products, making them espe-
cially important parameters of raw milk quality. With
no generally accepted limits on aerobic spore levels in
raw milk to prevent finished product quality issues, an
alternative approach for quantifying and reducing the
effects of these organisms is to use predictive modeling
tools such as those discussed later in this review. Brief-
ly, stakeholders use established distributions of target
sporeforming populations (e.g., psychrotolerant spores)
in raw milk, or develop distributions based on their own
supply chain, to predict the impact on finished product
outcomes. These models can then be used to determine
the incremental improvement on finished product out-
comes (e.g., spoilage) when spore concentrations are
reduced in the supply.
Numerous publications have demonstrated the role
that farm management practices and factors play in
the levels of sporeforming bacteria found in bulk tank
Journal of Dairy Science Vol. 106 No. 3, 2023
1511
raw milk. These can broadly be categorized as (1)
housing area and bedding practices, (2) milking routine
practices, (3) cow-level factors, and (4) feed factors.
For example, Murphy et al. (2019) demonstrated that
bedding material is associated with spores in bulk tank
raw milk, with level of spores in the bedding material
directly associated with MSC and TSC in raw milk.
Magnusson et al. (2007) reported the same finding when
specifically investigating the levels of raw milk contami-
nation with Bacillus cereus spores. Miller et al. (2015a)
also reported that the use of sand or sawdust bedding
for lactating cows was associated with lower MSC in
bulk tank raw milk, and the use of straw bedding was
associated with lower TSC in bulk tank raw milk. Mur-
phy et al. (2019) and Martin et al. (2019) both reported
that the increased frequency of topping up or chang-
ing bedding was associated with reduced bulk tank
raw milk spore levels. Studies have also suggested that
milking routine practices may affect spore levels in bulk
tank raw milk; this may be driven by the effectiveness
of different milking practices with regard to removal
of residual soil (e.g., manure, bedding material) from
teat ends. Relatedly, the amount of soil on teat and
udder surfaces has also been shown to be significantly
associated with bulk tank spore levels (Vissers et al.,
2006; Masiello et al., 2014; Martin et al., 2019; Murphy
et al., 2019). Indeed, Evanowski et al. (2020) reported
that significant reductions in bulk tank MSC and TSC
were observed after milking employees were trained on
enhanced teat end cleaning procedures and laundered
towels used for milking preparation were prepared by
washing with detergent, sanitizing with bleach, and
thoroughly drying, demonstrating the importance of
milking preparation procedures on transmission of
spores into bulk tank raw milk. Finally, feed factors
influence BAB spore levels in bulk tank raw milk. For
example, Vissers et al. (2007) found that the driving
factor for BAB spore levels in bulk tank raw milk was
the concentration of BAB spores in mixed silage fed
to the lactating herd. The pathway of contamination
from feed to milk includes concentration of spores in
the manure and subsequent contamination of the udder
and teat surfaces. These studies all demonstrate the
importance of comprehensive management in control of
the transmission of spores from environmental sources
into bulk tank raw milk.
Concerning the characterization of sporeforming
bacteria found in bulk tank raw milk, major genera
reported include Bacillus, Paenibacillus, Clostridium,
Brevibacillus, Lysinibacillus, Sporosarcina, Ureibacillus,
Viridibacillus, Oceanobacillus, and others (Coorevits et
al., 2008; Miller et al., 2015a). Multiple reports suggest
that the most prevalent sporeforming bacterium pres-
ent in raw milk is Bacillus licheniformis (Crielly et al.,
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
1994; Miller et al., 2015b), a facultative anaerobe that
has a broad range of growth temperatures. Bacillus
cereus group members, which includes both pathogens
(e.g., Bacillus cereus sensu stricto) and dairy spoilage
organisms (e.g., Bacillus weihenstephanensis; Ivy et al.,
2012), are also common contaminants in bulk tank raw
milk (Lin et al., 1998; te Giffel et al., 2002), representing
spoilage and potential foodborne disease risks in down-
stream dairy products. Other Bacillus species identified
in raw milk include Bacillus clausii, Bacillus pumilus,
Bacillus subtilis, and others (Miller et al., 2015b). Pae-
nibacillus dominates the psychrotolerant sporeforming
taxa found in raw milk (Ivy et al., 2012), but other
genera found in raw milk have also been reported to
grow at low temperatures, including Bacillus weihenste-
phanensis and Viridibacillus spp. (Trmčić et al., 2015).
Similarly, strictly anerobic sporeforming bacteria are
predominantly within the genus Clostridium (Doyle et
al., 2015), with the notable member being Clostridium
tyrobutyricum as the primary causative agent of late-
blowing defect in certain styles of cheese (Klijn et al.,
1995). Finally, raw milk is occasionally demonstrated
to be a source of sporeforming extremophiles that sur-
vive very harsh conditions that are destructive to most
other sporeforming organisms. For example, Schelde-
man et al. (2005) found a surprising diversity of highly
heat-resistant spores (recovered after heat treatment of
100°C for 30 min) in raw milk supplies from 17 farms
in Belgium, including Bacillus sporothermodurans and
Geobacillus spp. These extremophiles have particular
importance to the quality of dairy powders and UHT
fluid milks (Miller et al., 2015b; Alonso et al., 2021).
Overall, the diversity of sporeforming bacteria in raw
milk supplies contributes to their ability to cause qual-
ity deterioration in finished dairy products and repre-
sents a challenge for detecting and enumerating specific
subgroups, as we will describe subsequently.
The primary limitations of monitoring raw milk
supplies for sporeforming bacteria are related to their
presence in very low concentrations, often below 1 cfu/
mL, which is the detection limit of most traditional
microbiological methods. In response, methods that
estimate low-level concentrations of spores (i.e., MPN)
are used for BAB and have been described for psy-
chrotolerant spores (Masiello et al., 2014); however,
these methods are time consuming and tedious, and
require a great deal of space and laboratory ware (e.g.,
test tubes), which are barriers to routine use. Due to
the low levels of contamination and the associated
methodological challenges with enumerating low-level
contaminants—as the number of recovered colonies are
often at such low levels (i.e., <20–25 cfu/mL) that reli-
able assessment of spore loads is not assured—the as-
sociation of raw milk spore count with finished product
Journal of Dairy Science Vol. 106 No. 3, 2023
1512
quality is low. For example, Martin et al. (2011) found
no significant association between spore count and
fluid milk SPC at 17 d of refrigerated storage, despite
Paenibacillus being the predominant genus detected at
that point in shelf-life. The authors acknowledged that
the limitation of the enumeration method, specifically
that many samples tested had spore counts below the
limit of detection, likely influenced the outcome of
their study. It is further noteworthy that, in addition to
recommendations for BAB limits in raw milk intended
for use in cheesemaking, as described earlier, no well-
established guidelines for aerobic spore limits in raw
milk currently exist, limiting the ease of interpretation
of this parameter to dairy industry stakeholders.
Defining Raw Milk Microbiological Quality from
the Perspective of Finished Product Quality:
A 3-Tiered Approach
Each of the microbiological parameters discussed
herein has benefits and limitations for identifying and
defining raw milk quality, but, importantly, it has been
demonstrated that these various parameters are often
significantly associated with each other and with other
non-microbiological measures of quality, specifically
bulk tank SCC. For example, Jayarao et al. (2004) re-
ported that bulk tank raw milk with low SPC (<5,000
cfu/mL) also had a significantly lower SCC (<200,000
cells/mL), PI (<10,000 cfu/mL), LPC (<100 cfu/mL),
and other bacterial measures of quality. Similarly, Pan-
toja et al. (2009) found that an increased TBC was 6.3
times more likely for bulk tank raw milk with elevated
CC, and 1.3 times more likely for bulk tank milk with
elevated LPC. The authors further note that the odds
of increased TBC increased by 2.4% for every 10,000
cell/mL increase in bulk tank raw milk SCC (Pan-
toja et al., 2009). Schukken et al. (1992) reported that
herds that produced milk with higher PLC, a measure
of TBC, had higher SCC, and Gillespie et al. (2012)
identified a significant association between SPC and
PI. Finally, Masiello et al. (2017) reports a significant
association between bulk tank SCC and TBC, PI, and
psychrotolerant spore count. A key takeaway from the
associations between raw milk microbiological param-
eters for dairy industry stakeholders is that monitoring
raw milk quality, especially as it pertains to identifying
raw milk supplies likely to perform well throughout the
continuum from production to processing and shelf-life,
should be streamlined to focus on an overall microbio-
logical quality indicator, which we propose should be
TBC. As noted by Hutchison et al. (2005), although
TBC is not without its own shortcomings, no better
bacterial marker for overall raw milk hygiene currently
exists, especially given its ease and rapidity of measure-
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY 1513
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)
Monitoring parameters that Spore counts including butyric acid bacteria
directly influence finished count and psychrotolerant spore count,
product quality lactic acid bacteria
Troubleshooting Preliminary incubation count, coliform count,
and laboratory pasteurization count
High frequency, every load of milk
High/moderate frequency initially to develop a baseline
distribution in the supply, then subsequently lower
frequency to identify deviations from the baseline
Low frequency, only when needed to troubleshoot high
TBC, paired with on-farm risk assessment

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

Journal of Dairy Science Vol. 106 No. 3, 2023

 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
The development of decision support tools at the
production and processing levels has demonstrated how
relevant data can be used to predict outcomes of inter-
est to dairy industry stakeholders. For example, Ca-
brera (2018) outlines the development and application
of over 20 computerized dairy farm decision support
tools to assist producers with farm management related
to nutrition, reproduction, calf and heifer management,
replacement, price risk, and environment (Cabrera,
2018). Other similar tools have also been described
for dairy production (Rose et al., 2016; Balhara et al.,
2021), and numerous commercial products exist on the
marketplace (e.g., iDDEN.org, MyDairyDashboard.
com). Similarly, decision support tools have been de-
veloped to assist processors with dairy product quality
improvements. For example, Monte Carlo simulations
have been used to predict the shelf-life of fluid milk
based on the presence of psychrotolerant spores in
raw milk supplies (Buehler et al., 2018a), to predict
consumer exposure to spoiled yogurt (Buehler et al.,
2018b), and to predict the occurrence of late-blowing
defect in cheese (Qian et al., 2022).
Despite the demonstrated utility of data-driven, risk-
based decision support tools, they are not without their
limitations. In particular, dairy producers face several
barriers to the adoption of these tools. A recent com-
mentary on improving the adoption rates of integrated
decision support systems identified major barriers in-
cluding (1) perceived limited value by producers, (2) is-
sues of ease of use or interpretation, (3) lack of practical
and direct application, (4) data collection, quality, and
standardization challenges, (5) lack of data integration,
(6) limited data sharing and farm infrastructure, and
(7) limited multidisciplinary collaboration between sci-
entists developing new knowledge and those developing
decision support tools (Baldin et al., 2021). Similarly,
Rose et al. (2016) outlined a similar set of factors af-
fecting adoption of decision support tools, including
what the authors describe as core factors (e.g., ease of
use, peer recommendation, cost), enabling factor (i.e.,
facilitating conditions such as internet and phone con-
nection), driving factors (e.g., level of marketing and
legislative compliance), and modifying factors (e.g.,
producer age, scale of business; Rose et al., 2016).
Importantly, these barriers are likely to have the larg-
est influence on dairy producers who are most in need
from the perspective of quality improvements (e.g.,
those with limited resources, remote farm locations).
However, despite these limitations, development of
data-driven, risk-based decision support tools applied
to the microbiological quality of raw milk, integrated
throughout the production to processing chain, has the
potential to revolutionize how dairy industry stakehold-
Journal of Dairy Science Vol. 106 No. 3, 2023
1514
ers define raw milk quality. Further cross-disciplinary
efforts should be made, to ensure broad adoption and
implementation of these tools in the future. For ex-
ample, co-ops and processors could use these models to
set target levels for measures that are directly linked
to finished product quality and conversion efficiency
(e.g., BAB counts, psychrotolerant spore counts), with
associated premiums. This approach would allow for
strategic targeting of specific on-farm practices to
ensure consistent production of raw milk that meets
specifications, and subsequent verification of this ap-
proach through periodic, low-frequency monitoring of
relevant parameters (e.g., BAB counts) paired with
frequent process control monitoring (i.e., TBC testing)
on every load of milk.
CONCLUSIONS
Bacterial populations in raw milk play an important
role in the quality of processed dairy products. How-
ever, as discussed in this review, parameters currently
used to assess microbiological raw milk quality have
varying degrees of capacity to inform stakeholders
on their potential implications for finished product
quality. The future of raw milk quality determina-
tion should focus on a whole-farm approach that ac-
knowledges the microbiological risk factors at various
stages of the production process and transitions from
the reactive test-based system of our current dairy
industry to a predictive, risk-based system. This re-
quires standard use of an appropriate microbiological
indicator, namely TBC, periodic monitoring of micro-
bial parameters that directly affect finished product
quality, use of troubleshooting tests when the process
control test is out of compliance, and implementation
of integrated decision support tools that will provide
producers with the necessary guidance to produce
high-quality raw milk for processing into high-quality
finished dairy products. This approach will require
stakeholder collaboration throughout the dairy con-
tinuum from production, processing, regulatory agen-
cies, and academic institutions.
ACKNOWLEDGMENTS
The authors acknowledge the role that the New York
State Dairy Promotion Advisory Board (Albany, NY)
has played in their continued support of research aimed
at improving the quality and safety of dairy products
in New York and beyond. The authors have not stated
any conflicts of interest. The authors acknowledge that
N. Martin is a section editor for the Journal of Dairy
Science.
 Martin et al.: INVITED REVIEW: RAW MILK QUALITY
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ORCIDS
N. H. Martin https:​/​/​orcid​.org/​0000​-0003​-1704​-0634
R. L. Evanowski https:​/​/​orcid​.org/​0000​-0002​-2692​-3140
M. Wiedmann https:​/​/​orcid​.org/​0000​-0002​-4168​-5662