Cornell University Dairy Foods

Milk Quality Improvement Program

Department of Food Science
Stocking Hall, Ithaca, NY 14853
Phone: 607-255-2893
Science Notes

Provided with support from the NYS Dairy Promotion Advisory Board, dairy farmers dedicated to the production, manufacture & distribution of quality dairy products.
 Updated 09-28-08

- RAW MILK BACTERIA TESTS –

Standard Plate Count, Preliminary Incubation Count,
Lab Pasteurization Count and Coliform Bacteria Counts
&
SOURCES AND CAUSES OF HIGH BACTERIA COUNTS
– AN ABREVIATED REVIEW –
Dairy product quality assurance begins at the farm and ends in the hands of the consumer. In this regard,
raw milk quality is essential and is closely monitored. Regulations require that bacteria and somatic cell
counts of Grade “A” raw milk not exceed 100,000 Standard Plate Count (SPC) and 750,000 Somatic Cell
Count (SCC), respectively. Raw milk must also meet other quality standards; it should be free of drug
residues, free of added water and free of sediment, contaminants and other abnormalities. The overall
condition and cleanliness of the dairy farm, as determined by routine inspections, are also considered.
Although regulatory requirements have been instrumental in ensuring the quality of raw milk, most
segments of the dairy industry feel that more stringent standards (e.g. SPC < 10,000, SCC < 200,000)
would result in an even higher quality milk supply. Depending on the purchaser of the milk, dairy
farmers can also receive substantial monetary premiums by producing quality milk that meets standards
far more demanding than regulatory requirements.
The SPC, which measures the overall bacterial quality of a sample, is used extensively in both regulatory
and premium testing programs. In addition to the SPC, producer raw milk is often subjected to a number
of other bacteriological tests used as indicators of how that milk was produced. These tests may be
included in determining eligibility for premium payments or they may be used simply as an added quality
assurance tool. The procedures most often used in addition to the SPC are the Preliminary Incubation
Count (PIC), the Lab Pasteurization Count (LPC) and/or the Coliform Bacteria Count (Richardson,
1985). While the SPC gives an estimated count of the total bacteria in a sample that can be detected with
the method, the PIC, LPC and Coliform Count select for specific groups of bacteria that are often
associated with poor dairy practices. Results of these testing procedures are used to help identify
potential problems that may not be evident or detected with the SPC alone. This document provides an
overview of these bacteriological procedures and provides a general discussion on the causes of high
bacteria counts in raw milk.

Standard Plate Count

The Standard Plate Count (SPC) of a producer raw milk samples gives an indication of the total number of
aerobic bacteria present in the milk at the time of pickup. Milk samples are plated in a semi-solid nutrient
media and then incubated for 48 hours at 32°C (90°F) to encourage bacterial growth. Single bacteria or
tight clusters (e.g. chains or clumps) grow to become visible colonies that are then counted. All bacterial
plate counts are expressed as the number of colony forming units (cfu) per milliliter (ml).
Aseptically collected milk from clean, healthy cows generally has SPC values of less than 1,000. Higher
counts suggest that contaminating bacteria are entering the milk from a variety of possible sources.
Although it’s impossible to eliminate all sources of contamination, counts of less than 5,000 or even 1000
are possible; counts of 10,000 or less should be achievable by most farms. One of the most frequent
causes of high SPCs is poor cleaning of the milking system. Milk residues on equipment surfaces provide
nutrients for growth and multiplication of bacteria that can then contaminate the milk of subsequent
milkings. Other practices that might contribute to increased bulk-tank SPCs are milking soiled cows,
maintaining an unclean milking and housing environment, and failing to rapidly cool the milk to or
maintain it at less than 4.4°C (40°F). On rare occasions, mastitic cows that shed infectious bacteria can
also contribute to or cause high SPCs.

2
Preliminary Incubation Count
The Preliminary Incubation Count (PIC) reflects milk production practices. This procedure involves
holding the milk at 12.8°C (55°F) for 18 hours prior to plating. This step encourages the growth of groups
of bacteria that grow well at cool temperatures. Bacteria in the incubated sample are counted with the SPC
procedure and compared to the SPC of the un-incubated sample to determine if a significant increase has
occurred. PICs are generally higher than SPCs, although in some cases no growth occurs or counts may
even be lower. Counts 3-4 fold higher are often considered significant, but this depends on the initial
SPC. Some consider counts of > 50,000 to be of concern regardless of the SPC, though in some cases the
counts may be equal and in rare cases the PIC may be lower. It should be noted that contaminating
bacteria from the same source can vary in their growth rates in this procedure, which can result in very
different PICs at the same level of contamination.
High PICs are most often associated with a failure to thoroughly clean and sanitize either the milking
system or, in some cases, the cows. Bacteria considered to be natural flora of the cow, including those
that cause mastitis, are not thought to grow significantly at the PI temperature, although there may be a
few exceptions. If a PIC is approximately equal to or slightly higher, or even lower, than a high SPC
(e.g., > 50,000), it may suggest that the high SPC is possibly due to mastitis. Marginal cooling (e.g., milk
is held over 40°F) or prolonged storage times may also result in unacceptable PIC levels by allowing
organisms that grow at refrigeration temperatures to multiply. Bacteria that grow well at refrigeration
temperatures (psychrotrophic bacteria), are most frequently associated with high PICs. Although the
same types of bacteria that cause high PICs can also cause defects in pasteurized milk products, the PIC
of a raw milk supply does not indicate the potential quality or shelf-life of a pasteurized product made
from that milk. These types of bacteria are mostly destroyed by pasteurization, but can occur as post-
pasteurization contaminants in pasteurized milk.
Lab Pasteurized Count
Although most bacteria are destroyed by pasteurization, there are certain types that are not. The Lab
Pasteurized Count (LPC) estimates the number of bacteria in a sample that can survive the pasteurization
process. Milk samples are heated to 62.8°C (145°F) for 30 minutes, which simulates batch pasteurization.
Bacteria that survive the heat treatment (thermoduric bacteria) are enumerated using the SPC procedure.
LPCs are generally much lower than SPCs, with counts > 200-300 deemed high. The natural bacterial
flora of the cow, as well as those associated with mastitis, are generally not thermoduric, although there
may be a few exceptions. High LPCs are most often associated with a chronic or persistent cleaning
failure in some area of the system or significant levels of contamination from soiled cows. Other common
causes of high LPCs are leaky pumps, old pipe-line gaskets, inflations and other rubber parts, and
milkstone deposits.
Coliform Count
The Coliform Count procedure selects for bacteria that are most commonly associated with manure or
environmental contamination. Milk samples are plated on a selective bacterial media that encourages the
growth of coliform bacteria, while preventing the growth of others. Though coliforms are often used as
indicators of fecal contamination, there are strains that commonly exist in the environment. Coliforms
may enter the milk supply as a consequence of milking soiled cows or dropping the milking claw into
manure during milking. Generally, counts >50 would indicate poor milking hygiene or other sources of
contamination. Higher coliform counts more often result from dirty equipment and in rare cases result
from milking cows with environmental coliform mastitis.

3
Quality Standards - SPC, LPC, PIC and Coliform Count
Suggested standards used for quality premiums or for trouble-shooting purposes are listed in table 1. The
tests included in a quality program, as well as the limits used, will vary depending on the philosophy and
requirements of the processor or cooperative. Generally, standards used to determine premium eligibility
are based on values established for well managed farms. Although some of the test methods are not
considered “official,” it is important that they be carried out in a professional manner using standardized
procedures. It is not recommended that these tests be used to penalize dairy farms.
Table 1. Suggested Bacterial Test Standards for Quality Premiums
Testing Procedure Suggested Standard 1 Regulatory Standard 1

Standard Plate Count < 10,000 < 100,000

Laboratory Pasteurization Count < 200 - 300 none

Preliminary Incubation Count 50,000 – 100,000 none

and/or Significantly > SPC
Coliform Bacteria Count < 50 California
(< 750)
1
All counts expressed as colony forming units per ml.

Sources and Causes of High Bacteria Counts in Raw Milk: An Abbreviated Review
Milk is synthesized in specialized cells of the mammary gland and is virtually sterile when secreted into
the alveoli of the udder (Tolle, 1980). Beyond this stage of milk production, microbial contamination can
generally occur from three main sources (Bramley & McKinnon, 1990); from within the udder, from the
exterior of the udder and from the surface of milk handling and storage equipment. The health and
hygiene of the cow, the environment in which the cow is housed and milked, and the procedures used in
cleaning and sanitizing the milking and storage equipment are all key factors in influencing the level of
microbial contamination of raw milk. Equally important are the temperature and length of time of storage,
which allow microbial contaminants to multiply and increase in numbers. All these factors will influence
the total bacteria count (SPC) and the types of bacteria present in bulk raw milk.
Microbial Contamination from within the Udder: Raw milk as it leaves the udder of healthy cows
normally contains very low numbers of microorganisms and generally will contain less than 1000 total
bacteria per ml (Kurweil, 1973). In healthy cows, the teat cistern, teat canal, and the teat apex may be
colonized by a variety of microorganisms though microbial contamination from within the udder of
healthy animals is not considered to contribute significantly to the total numbers of microorganisms in the
bulk milk, nor to the potential increase in bacterial numbers during refrigerated storage. Natural flora of
the cow generally will not influence LPCs, PICs or Coliform Counts.
While the healthy udder should contribute very little to the total bacteria count of bulk milk, a cow with
mastitis has the potential to shed large numbers of microorganisms into the milk supply. The influence of
mastitis on the total bacteria count of bulk milk depends on the strain of infecting microorganism(s), the
stage of infection and the percentage of the herd infected. Infected cows have the potential to shed in
excess of 10 7 bacteria per ml. If the milk from one cow with 10 7 bacteria per ml comprises 1% of the bulk
tank milk, the total bulk tank count, disregarding other sources, would be 10 5 per ml (Bramley &
McKinnon, 1990).
Mastitis organisms found to most often influence the total bulk milk count are Streptococcus spp., most
notably S. agalactiae and S. uberis (Bramley & McKinnon, 1990; Bramley et al., 1984; Gonzalez et al.,
1986; Jeffrey and Wilson, 1987) though other mastitis pathogens have the potential to influence the bulk

4
tank count as well. Staphylococcus aureus is not thought to be a frequent contributor to total bulk tank
counts though counts as high as 60,000/ml have been documented (Gonzalez et al., 1986). Detection of
implied pathogens does not necessarily indicate that they originated from cows with mastitis. Potential
environmental mastitis pathogens and/or similar organisms can occur in milk as a result of other
contributing factors such as dirty cows, poor equipment cleaning and/or poor cooling. An increase in
SCC can sometimes serve as supportive evidence that a mastitis bacterium may have caused an increase in
the bulk milk bacteria count. This seems to hold true more for Streptococcus spp. than for S. aureus,
which appears to be shed into the milk in lower numbers (Fenlon et al., 1995). Correlations of somatic
cell responses and environmental mastitis organisms, including coliform bacteria, streptococci, and
certain coagulase-negative Staphylococcus spp., were found to be poor as well. These organisms are by
nature associated with the cow’s environment and may influence bulk milk bacteria counts through other
means (Bramley, 1982; Zehner et al., 1986). S. agalactiae and S. aureus are not thought to grow
significantly on soiled milking equipment or under conditions of marginal or poor cooling. Their
presence in bulk tank milks is considered strong evidence that they originated from infected cows
(Gonzalez et al., 1986; Bramley and McKinnon, 1990). In general, mastitis organisms will not influence
LPCs or PICs though in some cases of coliform mastitis, coliform counts may be elevated.
Microbial Contamination from the Exterior of the Udder: The exterior of the cows’ udder and teats
can contribute microorganisms that are naturally associated with the skin of the animal as well as
microorganisms that are derived from the environment in which the cow is housed and milked. In
general, the direct influence of natural inhabitants as contaminants in the total bulk milk count is
considered to be small and most of these organisms do not grow competitively in milk. Of more
importance is the contribution of microorganisms from teats soiled with manure, mud, feeds or bedding.
Teats and udders of cows inevitably become soiled while they are lying in stalls or when allowed in
muddy barnyards. Used bedding has been shown to harbor large numbers of microorganisms. Total
counts often exceed 10 8-1010 per gram (Bramley, 1982; Bramley & McKinnon, 1990; Hogan et al., 1989;
Zehner et al., 1986). Organisms associated with bedding materials that contaminate the surface of teats
and udders include streptococci, staphylococci, spore-formers, coliforms and other Gram-negative
bacteria. Both thermoduric and psychrotrophic strains of bacteria are commonly found on teat surfaces
(Bramley & McKinnon, 1990) indicating that contamination from the exterior of the udder can influence
LPCs, PICs and coliform counts.
The influence of dirty cows on total bacteria counts depends on the extent of soiling of the teat surface
and the wash procedures used immediately before milking. For example, if one gram of teat soil
containing 108 bacteria is allowed into the milk of one cow giving approximately 30 lb. (~13,400 gm) of
milk, the total bacteria count for that cow’s milk, excluding other sources, would be in excess of 7,000
cfu/ml. Milking heavily soiled cows could potentially result in bulk milk counts exceeding 10 4 cfu/ml.
Several studies have investigated pre-milking udder hygiene techniques in relation to the bacteria count of
milk (Bramley &McKinnon, 1990; Galton et al 1984; McKinnon et al 1990, Pankey, 1989). Generally,
thorough cleaning of the teat with a sanitizing solution (spray, wet towel or dip) followed by thorough
drying with a clean towel is effective in reducing the numbers of microorganisms in milk contributed
from soiled teats. Counts of coliform bacteria, though highly associated with manure, barnyard mud, and
used bedding, were relatively low in these studies, even for the untreated cows suggesting that higher
coliform counts in bulk milk are more likely to occur due to other factors (e.g., equipment, mastitis).
Influence of Equipment Cleaning and Sanitizing Procedures: The degree of cleanliness of the
milking system probably influences the total bulk milk bacteria count as much, if not more than any other
factor (Olson and Mocquat, 1980). Milk residues left on equipment contact surfaces support the growth
of a variety of microorganisms. Organisms considered as natural inhabitants of the teat canal, apex and
skin are not thought to grow significantly on soiled milk contact surfaces or during refrigerated storage of
milk. This generally holds true for a number of organisms associated with contagious mastitis (e.g., S.

5
agalactiae), although there may be exceptions. Certain strains associated with environmental mastitis
(e.g., coliforms) may be able to grow to significant numbers on milk contact surfaces. In general,
environmental contaminants (e.g., from bedding, manure, feeds) are more likely to grow on soiled
equipment surfaces. Water used on the farm might also be a source of microorganisms, especially
psychrotrophs that might seed soiled equipment and/or the milk (Bramley & McKinnon, 1990).
Cleaning and sanitizing procedures can influence the degree and type of microbial growth on milk contact
surfaces by leaving behind milk residues that support growth, as well as by setting up conditions that
might select for specific microbial groups. More resistant and/or thermoduric bacteria may endure in low
numbers on equipment surfaces that are considered to be efficiently cleaned with hot water. If milk
residue is left behind (e.g., milk stone) growth of these types of organisms, though slow, may persist. Old
cracked rubber parts are also associated with higher levels of thermoduric bacteria. Significant build-up
of these organisms to a point where they influence the total bulk tank count may take several days to
weeks (Thomas et al., 1966), although increases could be detected in the LPC procedure.
Less efficient cleaning, using lower temperatures and/or the absence of sanitizers tends to select for the
faster growing, less resistant organisms, principally Gram-negative rods (coliforms and Pseudomonads)
and lactic streptococci. This will result in potential high PICs and in some case elevated LPCs. Effective
use of chlorine or iodine sanitizers has been associated with reduced levels of psychrotrophic bacteria that
cause high PICs (Jackson & Clegg, 1965). Psychrotrophic bacteria tend to be present in higher count milk
and are often associated with occasional neglect of proper cleaning or sanitizing procedures (Olson &
Mocquat, 1980; Thomas et al., 1966) and/or poorly cleaned refrigerated bulk tanks (MacKenzie, 1973;
Thomas, 1974).
Milk Storage Temperature and Time: Refrigeration storage, while preventing the growth of non-
psychrotrophic bacteria, will select for psychrotrophic microorganisms that enter the milk from soiled
cows, dirty equipment and the environment. Minimizing the level of milk contamination from these
sources will help prevent psychrotrophs from growing to significant levels in the bulk tank during the
storage period on the farm or at the dairy plant. In general these organisms are mostly not thermoduric
and will not survive pasteurization. The longer raw milk is held before processing (potential up to 5 days;
2 days on the farm, 3 at the plant), the greater the chance that psychrotrophs will increase in numbers.
Holding milk near the legal limit of 7.2°C (45°F) allows much quicker growth than milk held below 4.4°C
(40°F). Although milk produced under ideal conditions may have an initial psychrotroph population of
less than 10% of the total bulk tank count, psychrotrophic bacteria can become the dominant microflora
after 2-3 days at 4.4°C (40°F) (Gehringer, 1980). This could result in a significant influence on PICs.
Colder temperatures of 1-2°C (34-36°F) will delay this shift, although not indefinitely.
Under conditions of poor cooling with temperatures greater than 7.2°C (45°F), bacteria other than
psychrotrophs are able to grow rapidly and can become predominant in raw milk. Though incidents of
poor cooling still occur, this defect is not as common as when milk was held and transported in cans.
Streptococci have historically been associated with poor cooling of milk, appearing as pairs or chains of
cocci (spherical bacteria) on microscopic examination of milk smears (Atherton & Dodge, 1970). These
bacteria will potentially increase the acidity of milk. Certain strains are also responsible for a “malty
defect” that is easily detected by its distinct odor. Storage temperatures greater than 15°C (60°F) tend to
select for these types of contaminants (Gehringer, 1980). Although poor cooling conditions allow growth
of bacteria that normally will not grow in properly refrigerated milk, it will not prevent typical
psychrotrophic strains from growing. The types of bacteria that grow and become significant will depend
on the initial microflora of the milk (Bramley & McKinnon, 1990).

6
Summary
Microbial contamination of raw milk can occur from a variety of microorganisms from a variety of
sources. Because of this, determining the cause of bacterial defects is not always straight-forward. High
bacteria counts can result from a combination of factors (e.g., dirty equipment coupled with marginal
cooling). Other than the SPC, a number of testing procedures may be used to evaluate the quality of raw
milk, including the LPC, PIC and the coliform count. These tests generally select for bacteria that occur
as contaminants that are not considered to be the natural flora of the cow. In some cases, though not
covered in this paper, more extensive procedures (e.g., bacterial culturing for mastitis bacteria) may be
useful. The likelihood of bacteria from different sources being responsible for high counts in the
discussed procedures is summarized in table 2.

Table 2. Sources Of Microbial Contamination As Detected By Bacteriological Procedures.

Procedure Natural Mastitis Dirty Cows Dirty Equip. Poor Cooling

Flora
SPC >10,000 Not likely Possible Possible Possible Possible

SPC >100,000 Not likely Possible Not likely Possible Possible

(rare) * *
LPC >200-300 Not likely Not likely Possible Possible Not likely
*
PIC High vs. SPC Not likely Not likely Possible Possible Possible
* *
SPC High/ no Not likely Possible Possible but Possible but Not likely
increase in PIC not likely not likely but possible
Coliform Count Not likely Possible Possible Possible Not likely
High (rare) But Possible
* A more likely possible source.

References
Atherton, H.V. and W.A. Dodge. 1970. Milk Under the Microscope. Vermont Extension Service, Univ. of Vt.
Bramley, A.J. 1982. Sources of Streptococcus uberis in the dairy herd I. Isolation from bovine feces and from
straw bedding of cattle. J. Dairy Res. 49:369.
Bramley, A.J., C.H. McKinnon, R.T. Staker and D.L. Simpkin. 1984. The effect of udder infection on the
bacterial flora of the bulk milk of ten dairy herds. J. Appl. Bacteriol. 57:317.
Bramley, A.J. and C.H. McKinnon. 1990. The microbiology of raw milk. pp. 163-208 in Dairy Microbiology,
Vol. 1. Robinson, R.K. (ed.) Elsevier Science Publishers, London.
Fenlon, D.R., D.N. Logue, J. Gunn, and J. Wilson. 1995. A study of mastitis bacteria and herd management
practices to identify their relationship to high somatic cell counts in bulk tank milk. Brit. Vet. J. 151:17.
Galton, D.M., R.W. L.G. Petersson, W.G. Merrill, D.K. Bandler, and D.E. Shuster. 1984. Effects of
premilking udder preparation on bacterial counts, sediment and iodine residue in milk. J. Dairy Sci. 67:2580.
Gehringer, G. 1980. Multiplication of bacteria during farm storage. In Factors influencing the bacteriological
quality of raw milk. International Dairy Federation Bulletin, Document 120.
Gonzalez, R.N., D.E. Jasper, R.B. Busnell, and T.B. Farber. 1986. Relationship between mastitis pathogen
numbers in bulk tank milk and bovine udder infections. J. Amer. Vet. Med. Assoc. 189:442.

7
Hogan, J.S., K.L. Smith, K.H. Hoblet, D.A. Todhunter, P.S. Schoenberger, W.D. Hueston, D.E. Pritchard,
G.L. Bowman, L.E. Heider, B.L. Brockett and H.R. Conrad. 1989. Bacterial counts in bedding materials used
on nine commercial dairies. J. Dairy Sci. 72:250.
Jackson, H. and F.L. Clegg. 1965. Effect of preliminary incubation on the microflora of raw bulk tank milk
with observation of the microflora of milking equipment. J. Dairy Sci. 48:407.
Jeffrey, D.C. and J. Wilson. 1987. Effect of mastitis-related bacteria on the total bacteria counts of bulk milk
supplies. J. Soc. Dairy Technol. 40(2):23.
Kurweil, R., & M. Busse. 1973. Total count and microflora of freshly drawn milk. Milchwissenschaft 28:427.
Richardson, G.H. 1985. Standard Methods for the Examination of Dairy Products, 15th ed. American Public
Health Association. Washington D.C.
MacKenzie, E. 1973. Thermoduric and psychrotrophic organisms on poorly cleaned milking plants and farm
bulk tanks. J. Appl. Bacteriol. 36:457.
McKinnon, C.H., G.J. Rowlands, & A.J. Bramley. 1990. The effect of udder preparation before milking &
contamination from milking plant on bacterial numbers in bulk milk of eight dairy herds. J. Dairy Res. 57:307.
Olson, J.C. Jr., and G. Mocquat. 1980. Milk and Milk Products. P.470. In Microbial Ecology of Foods. Vol. II.
J.H. Silliker, R.P. Elliott. A.C. Baird-Parker, F.L. Bryan, J.H. Christion, D.S. Clark, J.C. Olson, and T.A.
Roberts (eds.). Academic Press, New York, NY.
Palmer, J. 1980. Contamination of milk from the milking environment. In Factors Influencing the
Bacteriological Quality of Raw Milk. International Dairy Federation Bulletin, Doc. 120.
Pankey, J.W. 1989. Premilking udder hygiene. J. Dairy Sci. 72:1308.
Pasteurized Milk Ordinance. 1995. U.S. Department of Health and Human Services. Food and Drug
Administration. Washington D.C.
Thomas, S.B. 1974. The microflora of bulk collected milk-part 1. Dairy Ind. Int. 39:237.
Thomas S.B., R.G. Druce and K.P. King. 1966. The microflora of poorly cleansed farm dairy equipment. J.
Appl. Bacteriol. 29:409.
Tolle, A. 1980. The microflora of the udder. p 4. In Factors Influencing the Bacteriological Quality of Raw
Milk. International Dairy Federation Bulletin, Document 120.
Zehner, M.M., R.J. Farnsworth, R.D. Appleman, K. Larntz, and J.A. Springer. 1986. Growth of environmental
mastitis pathogens in various bedding materials. J. Dairy Sci. 69:1932.

Prepared by Steven C. Murphy (scm4@cornell.edu )

This paper is an update of:
Murphy, S. 1997. Raw milk bacteria tests: SPC, PIC, LPC and coliform count - what do they mean for your farm?
pp. 34-42 in Proceedings for the National Mastitis Council 1997 Regional Meeting. Syracuse, NY.
A modified version of this was published as:
Murphy, S. C. and K. J. Boor. 2000. Trouble-shooting sources and causes of high bacteria counts in raw milk.
Dairy, Food and Environmental Sanitation 20:606-611.