9 Total and thermotolerant
coliforms and Escherichia coli
9.1 Introduction
Most of the guidelines contained in this chapter are
taken from the Chapter 8 of the 4th Edition of the Com-
pendium of Methods for the Microbiological Examination
of Foods (Kornacki and Johnson, 2001). When different
from or complementary to those of the Compendium,
they were completed with information and recommen-
dations from the 17th Edition of Standard Methods for
the Examination of Dairy Products (Wehr and Frank,
2004), specific to the microbiological examination of
dairy products, and section 9221 of the 21st Edition
of the Standard Methods for the Examination of Water
and Wastewater (Hunt and Rice, 2005), specific to the
microbiological examination of water.
9.1.1 Definition of total coliforms
The group of total coliforms is a subgroup of the Entero-
bacteriaceae family, which in the 2nd Edition of Bergey’s
Manual of Systematic Bacteriology (Brenner and Farmer
III, 2005) includes 44 genera and 176 species. The total
coliforms group comprises only Enterobacteriaceae capa-
ble of fermenting lactose with the production of gas, in
24 to 48 hours at 35°C. More than 20 species fit this
definition, among which one can find not only bacteria
that originate from the gastrointestinal tract of humans
and other hot-blooded animals (Escherichia coli), but
also non-enteric bacteria (Citrobacter, Enterobacter,
Klebsiella and Serratia species, among others).
The ability to ferment lactose can be verified by
the formation of gas and/or acid, in culture media
containing lactose. These characteristics are used in
the traditional methods to enumerate total coliforms.
The most modern methods directly detect the activ-
ity of the β-galactosidase enzyme, which is involved in
the fermentative metabolism of lactose, incorporating
7007TS-DASILVA-Book.indb 89
substrates for the enzyme in the culture medium.
One of these substrates is ONPG (ortho-nitrophenyl-
β-D-galactopyranoside) which, when degraded by
β-galactosidase, results in a yellow reaction product.
Other substrates are X-GAL (5-bromo-4-chloro-3-
indolyl-β-D-galactopyranoside), which results in an
intensely blue reaction and Salmon-Gal (6-chloro-
3-indolyl-β-D-galactopyranoside), which results in a
salmon-pink to red color.
9.1.2 Definition of thermotolerant
coliforms
The group of thermotolerant coliforms, commonly
called fecal coliforms, is a subgroup of the total colif-
orm group and includes only members that are capable
of fermenting lactose in 24 hours at 44.5–45.5°C, with
the production of gas. The objective of this definition
was, in principle, to select only Enterobacteriaceae origi-
nating from the gastrointestinal tract (E. coli), but it is
known that the group also includes members of non-fe-
cal origin (several strains of Klebsiella pneumoniae, Pan-
toea agglomerans, Enterobacter aerogenes, Enterobacter
cloacae and Citrobacter freundii). Because of this, the
term “fecal coliforms” has been gradually substituted by
“thermotolerant coliforms”
9.1.3 Escherichia coli
E. coli is included both in the group of total coliforms
as in that of thermotolerant coliforms. Its natural
habitat is the intestinal tract of hot-blooded animals,
although it may also be introduced into foods via non-
fecal sources. E. coli is traditionally distinguished from
the other thermotolerant coliforms by its growth char-
acteristics in L-EMB Agar (Levine’s Eosine Methylene
11/26/2012 11:15:23 AM
 90 Microbiological examination methods of food and water
Blue Agar) and the profile of the results when subjected
to the indole, methyl red, Voges Proskauer and citrate
(IMVC) tests. The most modern methods differentiate
E. coli by verifying the activity of the β-glucuronidase
enzyme, produced by 96% of the E. coli strains, includ-
ing the anaerogenic (i.e. non-gas producing) strains
(Feng and Hartman, 1982). One of the substrates uti-
lized to verify the activity of β-glucuronidase is MUG
(4-methylumbelliferyl-β-D-glucuronide), which, when
degraded by β-glucuronidase, results in 4-methyl-
umbelliferone, which is fluorescent under UV light.
Another substrate commonly used to this purpose is
BCIG (5-bromo-4-chloro-3-indolyl-β-D-glucuronide),
also called X-β-D-Glucuronide, which when degraded
by the enzyme, forms a blue reaction product.
9.1.4 Use as indicators
According Kornacki and Johnson (2001) E. coli was ini-
tially introduced as an indicator in 1892 in Australia
and in 1895 in the United States. It was used to indicate
the contamination of water by fecal material and, con-
sequently, to alert for the potential presence of enteric
pathogens (Salmonella, for example). The standard was
changed to total coliforms in 1915, by the U.S. Pub-
lic Health Service, based on the (questionable) premise
that all coliforms were of equal value as indicators of
fecal contamination. After their use as indicators of the
microbiological quality of water, they began to be uti-
lized for the same purpose for foods in general, without
any judicious and thorough evaluation of the validity
of their use to this purpose in different products. At
present, the premise that there is a direct correlation
between high numbers of E. coli, thermotolerant col-
iforms, total coliforms and Enterobacteriaceae in foods
with fecal contamination is no longer valid, for a series
of reasons: 1) E. coli, thermotolerant coliforms, total
coliforms or Enterobacteriaceae are not obligate inhabit-
ants of the intestinal tract of hot-blooded animals, and
can be found in a number of different environmental
reservoirs. 2) The presence of these microorganisms is
common in food processing environments, and may
even become part of the resident microbiota of the facil-
ity (especially when cleaning and sanitation conditions
are inadequate). 3) Several strains of E. coli, coliforms or
Enterobacteriaceae may grow in refrigerated foods.
Based on these facts, the Food and Agricultural
Organization and the World Health Organization
(FAO/WHO, 1979) concluded that it is not possible
7007TS-DASILVA-Book.indb 90
to assess the safety (innocuity) of foods as a function
of the levels of E. coli, thermotolerant coliforms, total
coliforms or Enterobacteriaceae. High levels of these
microorganisms may, under certain circumstances, be
related to or associated with a greater probability of the
presence of enteric pathogens, however, frequently this
is not the case. In the same way, its absence does not
necessarily mean that the products are free from enteric
pathogenic bacteria. Kornacki and Johnson (2001)
listed the following applications for these microorgan-
isms as indicators:
a) Enterobacteriaceae and coliforms – indicators of the
hygienic conditions of manufacturing processes,
since they are easily inactivated by sanitizing agents
and capable of colonizing several niches in process-
ing plants, when sanitation and cleaning procedures
are inappropriate or inadequately executed.
b) Coliforms – indicators of processing flaws or post-
processing contamination of pasteurized foods,
since they are easily destroyed by heat and do not
survive heat treatment.
c) E. coli – indicator of fecal contamination in fresh
(“in natura”) foods (but not in processed foods).
9.1.5 Methods of analysis
Classical MPN method: The classical method to per-
form total coliforms, thermotolerant coliforms and
E. coli counts in water and foods is the Most Probable
Number (MPN) technique, which includes the follow-
ing steps: 1°) Presumptive test, in which three aliquots
of three dilutions of the sample are inoculated into a
series of three tubes containing Lauryl Sulphate Tryp-
tose (LST) broth per dilution. LST contains lactose and
the observation of growth accompanied by the produc-
tion of gas from lactose fermentation, after 24–48 h
incubation at 35°C, is considered indicative or suspect
(presumptive) of the presence of coliforms. 2°) For the
confirmation of total and thermotolerant coliforms, a
loopful of each suspected tube is transferred to tubes
with Brilliant Green Bile (BGB) Broth 2% and E. coli
Broth (EC), selective culture media containing lactose.
The observation of growth with the production of gas
in the BGB tubes, after 24–48 h incubation at 35°C, is
considered confirmative of total coliforms. Growth with
gas production in the EC tubes, after 24 h incubation at
45.5°C (or 44.5°C, in the case of water), is considered
confirmative of thermotolerant coliforms. 3°) The EC
11/26/2012 11:15:23 AM
 Total and thermotolerant coliforms and Escherichia coli
tubes testing positive for the presence of thermotolerant
coliforms are suspected of the presence of E. coli. For
confirmation, a loopful of each tube is streaked onto
Levine’s Eosine Methylene Blue (L-EMB) Agar, a differ-
ential selective medium to distinguish E. coli from other
thermotolerant coliforms. If any development of typi-
cal E. coli colonies is observed on L-EMB, two of these
colonies are to be isolated for the biochemical indole,
MR, VP and citrate (IMVC) assays. The cultures with
the + + − − (biotype 1) or − + − − (biotype 2) profiles
are considered confirmed.
In the classical MPN method, the last step of the
test is optional and many laboratories conclude the
analysis with the confirmation of thermotolerant colif-
orms. When the presence of E. coli is verified, the assay
is referred to as “complete test”. In water analysis, the
section 921 of the 21st Edition of the Standard Meth-
ods for the Examination of Water and Wastewater (Hunt
and Rice, 2005) recommends different procedures from
those used for the confirmation of E. coli. One of these
consists in transferring the suspected cultures obtained
in LST to tubes containing EC broth with MUG. After
incubation at 44.5ºC/24 h, the cultures showing blue
fluorescence under UV light are considered confirmed.
Another procedure is the transference from LST to Tryp-
tone (Tryptophane) Broth, incubation at 44.5ºC/24 h
and the indole test. The cultures that are positive in the
indole test are considered confirmed.
In the MPN method developed by ISO 7251:2005
for thermotolerant coliforms in foods, incubation in
EC broth is done at 44 ± 1ºC and the presumptive
confirmation of the presence of E. coli also utilizes the
indole test, after growth in Tryptone (Tryptophane)
Broth at 44 ± 1ºC. The main advantage of the ISO
method is that the acceptable temperature variation
is ±1ºC, which can be achieved in laboratory incu-
bators. In the Compendium, the maximum acceptable
variation is ±0.2°C, which requires a water bath for
incubation. The big problem with this requirement is
that water baths with this level of temperature stability
are rather expensive, as are the thermometers capable
of detecting this variation.
Chromogenic substrate method: For the determi-
nation of total coliforms and Escherichia coli in water,
an extremely simple and practical method is that of
the chromogenic and fluorogenic substrate (COLIL-
ERT®) AOAC 991.15 (Horwitz and Latimer, 2010), a
culturing technique based on the addition of a defined
or specific and at the same time differential culture
medium to the sample, in which the exact balance
7007TS-DASILVA-Book.indb 91
91
between all the components ensures the specificity of
the result. The medium contains two substrates for
enzymes: a) ortho-nitrophenyl-β-D-galactopyranoside
(ONPG), a substrate for the β-galactosidase enzyme
of coliforms, the reaction product of which is yellow.
b) 4-methylumbelliferyl-β-D-glucuronide (MUG), a
substrate for the β-glucuronidase enzyme of E. coli, the
reaction product of which is fluorescent under UV light.
The test can be conducted in two ways: a) Presence/
absence in 100 mL, by adding the culture medium to
100 ml of the sample (the sterile, dried medium is mar-
keted in ampoules containing the exact quantity neces-
sary for 100 ml samples). b) Most probable Number
(MPN) in 100 ml, by dividing the 100 ml into 10 aliq-
uots of 10 ml each.
Plate count method: For the enumeration of total
coliforms in foods, the Chapter 8 of the Compendium
(Kornacki and Johnson, 2001) and the Chapter 7 of the
Standard Methods for the Examination of Dairy Products
(Davidson et al., 2004) also recommend the direct plate
count method in plates containing Violet Red Bile Agar
(VRB). This method is based on the same counting
principle as that of the Enterobacteriaceae plate count,
described in a specific chapter, but uses lactose instead
of glucose, in the VRB Agar.
Petrifilm™ (3M Company). This is a modified ver-
sion of the Colony Formining Unit (CFU) plate count,
and consists of two sterile, rehydratable films impreg-
nated with culture medium and cold-water-soluble-
gelling agents. Inoculation is done directly onto the
bottom film, which, after inoculation, is covered with
the top film. The inoculum is evenly distributed over
the circular growth area of the bottom film by gentle
manual pressing with a plastic spreader and, after solidi-
fication of the gel, the plates are incubated for the devel-
opment of colonies.
The culture medium that forms the basis of the sys-
tem is Violet Red Bile (VRB), selective for enterobac-
teria, supplemented with triphenyltetrazolium chloride
(TTC), an indicator that, when reduced, colors colonies
red, thereby facilitating their visualization. The medium
contains lactose, which, fermented by coliforms or
E. coli, produces gas bubbles around the colonies. Typi-
cal colonies appear red surrounded by gas bubbles. In
the petrifilm version for coliforms + E. coli, the medium
also contains BCIG, a chromogenic substrate for the
β-glucuronidase enzyme, which allows to differenti-
ate E. coli by the formation of a blue precipitate sur-
rounding the colonies. The high-sensivity petrifilm
version (High-Sensitivity Coliform Count Plate) allows
11/26/2012 11:15:23 AM
 92 Microbiological examination methods of food and water
Table 9.1 Analytical kits adopted as AOAC Official Methods for coliforms and E. coli in foods (Horwitz and Latimer, 2010, AOAC Inter-
national, 2010).
Method Kit Name and Manufacturer
986.33 Petrifilm™ Coliform Count Plate,
3M Microbiology Products
989.10 Petrifilm™ Coliform Count Plate,
3M Microbiology Products
996.02 Petrifilm™ HSCC (High Sensitivity
Coliform Count Plate),
3M Microbiology Products
991.14 Petrifilm™ E.coli/ Coliform Count Plate,
Petrifilm™ Coliform Count Plate,
3M Microbiology Products
2000.15 Petrifilm™ Rapid Coliform Count Plate,
3M Microbiology Products
2005.03 SimPlate Coliforms and
E. coli Color Indicator,
BioControl Systems Inc.
991.15 Colilert, Idexx Laboratories Inc.
992.30 ColiComplete®,
BioControl Systems Inc.
983.25 ISO-GRID, Neogen Corp.
990.11 ISO-GRID, Neogen Corp.
2009.02 TEMPO® System, bioMérieux Inc.
MPN = Most probable number, MUG = 4-methylumbelliferyl-β-D-glucoronide, ONPG = o-nitrophenyl-β-D-galactopyranoside, X-GAL =
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
the inoculation of volumes of 5 ml while the petrifilm
version for rapid counts (Rapid Coliform Count Plate)
allows to obtain the result within 6 to 14 h.
Other methods that already have been officially rec-
ognized by the AOAC International are the microbio-
logical test kits described in Table 9.1.
9.2 Most probable number (MPN)
method APHA 2001 for total
coliforms, thermotolerant
coliforms and E. coli in foods
Method of the American Public Health Association
(APHA), as described in Chapter 8 of the 4th Edition
7007TS-DASILVA-Book.indb 92
Principle Matrices
Cultural, plate count in dry Milk.
rehydratable film
Cultural, plate count in dry Dairy products.
rehydratable film
Cultural, plate count in dry Dairy products.
rehydratable film
Cultural, plate count in dry Foods.
rehydratable film
Cultural, plate count in dry Foods (not applicable to hash
rehydratable film brown potatoes).
Cultural (MPN), sample distributed Foods.
into a device with multiple wells
containing substrates for β-galactosidase
and β-glucuronidase
ONPG and MUG substrates for Water.
β-galactosidase and β-glucuronidase
X-GAL and MUG substrates for All foods.
β-galactosidase and β-glucuronidase
Cultural, membrane filtration MPN Foods.
Cultural, membrane filtration MPN Foods.
Cultural (MPN) automated, sample Raw ground beef, bagged lettuce,
distributed into a device with multiple cooked chicken, pasteurized
wells containing substrates for crabmeat, frozen green beans,
β- glucuronidase and pasteurized whole milk.
of the Compendium of Methods for the Microbiological
Examination of Foods (Kornacki and Johnson, 2001).
Included also recommendations from Chapter 7 of the
Standard Methods for the Examination of Dairy Products
(Davidson et al., 2004), specific to the examination of
dairy products. In the Compendium is also applied to
the examination of bottled water.
9.2.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
11/26/2012 11:15:23 AM
 Total and thermotolerant coliforms and Escherichia coli
• Dilution tubes containing 9 ml 0.1% Peptone Water
(PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Total and thermotolerant coliforms count
• Lauryl Sulfate Tryptose (LST) Broth tubes (10 ml)
• Brilliant Green Bile (BGB) Broth 2% with Durham
tubes
• E. coli (EC) Broth with Durham tubes
• Pure culture E. coli
• Pure culture E. aerogenes
• Laboratory incubator set to 35 ± 0.5°C
• Water bath set to 45.5 ± 0.2°C
• Water bath set to 44.5 ± 0.2°C
E. coli count (traditional method for foods)
(optional)
• Levine’s Eosin Methylene Blue (L-EMB) Agar
• Plate Count Agar (PCA) slants
• Koser’s Citrate Broth or Simmons Citrate Agar slants
• Tryptone (Tryptophane) Broth
• MR-VP Broth
• Indole Kovacs Reagent
• Methyl Red Solution
• Voges-Proskauer (VP) Test Reagents (5% α-naphthol
alcoholic solution, 40% potassium hydroxide aque-
ous solution, creatine phosphate crystals)
• Laboratory incubator set to 35 ± 0.5°C
9.2.2 Procedure
b) Incubation for presumptive test: Incubate LST
A general flowchart for the detection of total coliforms,
thermotolerant coliforms and E. coli in foods by the
MPN Method APHA 2001 is shown in Figure 9.1.
Before starting activities, carefully read the guide-
lines in Chapter 4, which deals with all details and care
required for performing MPN tests. The procedure
described below does not present these details, as they
are supposed to be known to the analyst.
a) Preparation of the samples and inoculation:
Following the procedures described in Chapter 2,
homogenize 25 g or 25 ml of sample with 225 ml
of 0.1% Peptone Water (PW) or Butterfield’s Phos-
phate Buffer (10−1 dilution). If the food is acid
7007TS-DASILVA-Book.indb 93
93
check the pH of the 10−1 dilution and adjust to 6.8
if necessary (with sterile NaOH).
Prepare subsequent decimal dilutions of the sam-
ple. Select three appropriate dilutions and inoculate
three 1 ml portions of each dilution onto three tubes
with 10 ml of Lauryl Sulfate Tryptose Broth (LST).
Follow the orientation of the Table 4.1 (Chapter 4)
to select the dilutions according the expected level
of sample contamination by coliforms
Note a.1) Use 5-tube MPN series for analysis of shellfish and
shellfish harvest waters.
If the expected count is low, inoculate three
10 ml aliquots of the 10−1 dilution onto three tubes
with 10 ml of LST Broth double strength, three
1 ml aliquots of the 10−1 dilution onto three tubes
with 10 ml of LST Broth single strength, and three
1 ml aliquots of the 10−2 dilution onto three tubes
with 10 ml of LST Broth single strength.
For liquid samples with low count it is possible to
start the series without dilution: 3 × 10 ml of sam-
ple without dilution onto 3 × 10 ml of LST Broth
double strength, 3 × 1 ml of sample without dilu-
tion onto 3 × 10 ml of LST Broth single strength
and 3 × 10 ml of 10−1 dilution onto 3 × 10 ml of
LST Broth single strength. If the food is acid adjust
the pH of the three double strength LST tubes to
6.8 after the inoculation (with sterile NaOH).
For liquid samples with low count it is also possi-
ble to apply a MPN single dilution test, inoculating
5 × 10 ml aliquots of the sample without dilution
onto 5 × 10 ml of LST Broth double strength. If the
food is acid adjust the pH of the five double strength
LST tubes to 6.8 after the inoculation (with sterile
NaOH).
tubes at 35 ± 0.5°C/24 ± 2 h. Examine tubes and
record reactions at 24 ± 2 h for gas (displacement of
medium in fermentation vial or effervescence when
tubes are gently agitated). Re-incubate gas-negative
tubes for an additional 24 h and examine and record
reactions again at 48 ± 2 h. Perform confirmed test
on all presumptive positive (gas) tubes.
Note b.1) The accepted temperature variation in the Standard
Methods for the Examination of Dairy Products (Dav-
idson et al., 2004) is ± 1°C. In the Compendium of
Methods for the Microbiological Examination of Foods
(Kornacki and Johnson, 2001) is ± 0.5°C to har-
monize with the Standard Methods for the Exami-
nation of Water and Wastewater (Hunt and Rice,
2005).
11/26/2012 11:15:23 AM
 -1 -2 -3
10 10 10
Homogenization 1ml 1ml

9ml 9ml
PW PW

25g of samle +
225ml of Peptoned Water (PW)

1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml

PRESUMPTIVE
TEST
Lauryl Sulfate Tryptose
(LST) broth (10ml)

o
35±0.5 C/24±2h - 48±2h

Tubes of LST
with growth and gas production

1 loopful 1 loopful

THERMOTOLERANT E. coli TOTAL COLIFORMS

COLIFORMS CONFIRMATIVE TEST
E. aerogenes
CONFIRMATIVE TEST

o
E.coli (EC) broth 44.5±0.2 C/24±2h Brilliant Green Bile Broth (BGB) 2%
45.5±0.2oC/24±2h (shellfish) 35±0.5oC/24±2h - 48±2h
Water bath
BGB tubes with growth and gas production
EC tubes with growth and gas production

TOTAL COLIFORMS
THERMOTOLERANT (MPN/g)
COLIFORMS
(MPN/g)

Reincubate more 24±2h

E.coli confirmation

Figure 9.1 Continued.

7007TS-DASILVA-Book.indb 94 11/26/2012 11:15:24 AM

 Streak a loopful from EC positive tubes

Levine’s Eosin Methylene Blue (L-EMB) Agar

35±1oC/24±2h

Typical colonies

Plate Count Agar (PCA)

35±1oC/24±2h

BIOCHEMICAL SERIE

Koser’s Citrate Broth or

Simmons Citrate Agar MR-VP Broth Tryptone (Tryptophane) Broth

Gram staining
35±1oC/48±2h (VP)
35±1oC/96±2h 35±1oC/96±2h (MR) 35±1oC/24±2h

Gram negative rods

CITRATE TEST (-) VP(-) MR (+) TESTS INDOLE TEST (+ or -)

Confirmed tubes

E.coli
MPN/g

Figure 9.1 Scheme of analysis for the enumeration of total and thermotolerant coliforms and E. coli in foods using the MPN Method APHA
2001 (Kornacki and Johnson, 2001).

7007TS-DASILVA-Book.indb 95 11/26/2012 11:15:24 AM

 96 Microbiological examination methods of food and water
c) Confirmed test for total coliforms: From each gas-
sing LST tube, transfer a loopful of suspension to a
tube of Brilliant Green Bile Broth (BGB). Incubate
BGB tubes at 35 ± 0.5°C/24 ± 2 h and examine
for gas production, indicative of a positive result.
Re-incubate gas-negative tubes for an additional
24 h and examine and record reactions again at
48 ± 2 h. Calculate most probable number (MPN)
following the instructions described in Chapter 4,
using a MPN table.
d) Confirmed test for thermotolerant (fecal) colif-
orms: From each gassing LST tube, transfer a loop-
ful of suspension to a tube of E. coli Broth (EC).
Incubate EC tubes at 45.5 ± 0.2°C/24 ± 2 h for
most foods and 44.5 ± 0.2°C/24 ± 2 h for waters
and shellfish, preferably in a circulating water bath.
If a variety of food types are to be examined, a single
incubation temperature of 45.0 ± 0.2°C/24 ± 2 h
should be suffice. Examine tubes for gas produc-
tion, indicative of a positive result. Calculate the
thermotolerant coliforms most probable number
(MPN) following the instructions described in
Chapter 4, using a MPN table.
Note d.1) Place all the EC tubes in the water bath within
30 min after inoculation. Maintain a sufficient
water depth in the water bath incubator to immerse
the EC tubes to upper level of the medium. Incu-
bate an EC tube of E. coli and an EC tube of E.
aerogenes in the same water bath as positive and
negative control.
Note d.2) If the analysis will continue (E. coli count com-
pleted test below), re-incubate negative EC tubes
for an additional 24 h.
e) Completed test for E. coli in foods (optional):
Procedure described by the Compendium (Kornacki
and Johnson, 2001) and by the Standard Methods
for the Examination of Dairy Products (Davidson
et al., 2004).
From each gassing 48 h EC tube, streak (for iso-
lation) a loopful to a Levine’s Eosin Methylene Blue
Agar (L-EMB) plate. Incubate at 35 ± 1°C/24 ± 2 h
and examine plates for suspicious E. coli colonies
(dark centered and flat, with or without metallic
sheen).
Transfer two suspicious colonies from each
L-EMB plate to Plate Count Agar (PCA) slants,
incubate at 35 ± 1°C/24 ± 2 h and use for further
testing.
From PCA slants prepare a smear for Gram stain
and inoculate cultures into the following broths,
7007TS-DASILVA-Book.indb 96
for confirmation by IMVC tests (indole, methyl
red, Voges-Proskauer and citrate).
e.1) Citrate test: Lightly inoculate a tube of
Koser’s Citrate Broth and incubate at
35 ± 1°C/96 ± 2 h. Development of dis-
tinct turbidity (growth) is positive reaction.
Alternatively, Simmons Citrate Agar (slants)
may be used. Inoculate by streaking slant and
stabbing butt. Incubate at 35°C/96 h. Posi-
tive test is indicated by presence of growth,
usually accompanied by color change from
green to blue. Negative test is indicated by
no growth or very little growth and no color
change.
e.2) Indole test: Inoculate tube of Tryptone
(Tryptophane) Broth and incubate at
35 ± 1°C/24 ± 2 h. Test for indole by add-
ing 0.2–0.3 ml of Indole Kovacs Reagent.
Appearance of distinct red color in upper
layer is positive test and lack of red color is
negative test).
e.3) Methyl red (MR) and Voges-Proskauer
(VP) tests: Lightly inoculate tube of MR-VP
Broth and incubate at 35 ± 1°C/48 ± 2.
Perform Voges-Proskauer (VP) test at room
temperature as follows: Transfer 1 ml 48 h
culture to test tube and incubate remain-
der of MR-VP broth an additional 48 h at
35 ± 1°C. Add Voges-Proskauer (VP) Test
Reagents as follows: 0.6 ml α-naphthol,
shake well, 0.2 ml of 40% KOH solution and
shake. To intensify and speed reaction, add
a few crystals of creatine. Read results after
4 h. Development of pink-to-ruby red color
throughout medium is positive test and lack
of red color is negative test. Perform methyl
red test as follows: To 5 ml of 96 h MR-VP
broth, add 5–6 drops of methyl red indicator.
Read results immediately. Most E. coli cul-
tures give positive test, indicated by diffuse
red color in medium. A distinct yellow color
is negative test.
Consider as E. coli the cultures of Gram
negative rods, indole negative or positive, MR
positive, VP negative and citrate negative.
Calculate most probable number (MPN)
following the instructions described in
Chapter 4, using a MPN table.
11/26/2012 11:15:24 AM
 Total and thermotolerant coliforms and Escherichia coli
9.3 Most probable number (MPN)
methods ISO 4831:2006 and
ISO 7251:2005 for total coliforms
and presumptive E. coli in foods
These methods of the International Organization for
Standardization are applicable to products intended for
human consumption or for the feeding of animals, and
to environmental samples in the area of food produc-
tion and food handling.
9.3.1 Material required for analysis
Preparation of the sample and serial
dilutions
• Diluent: Saline Peptone Water (SPW) or Buffered
Peptone Water (BPW)
• Dilution tubes containing 9 ml Saline Peptone
Water (SPW) or Buffered Peptone Water (BPW)
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Total coliforms and presumptive
E. coli count
• Lauryl Sulfate Tryptose (LST) Broth tubes (10 ml)
• Brilliant Green Bile (BGB) Broth 2% with Durham
tubes
• E. coli (EC) Broth with Durham tubes
• Tryptone (Tryptophane) Broth
• Indole Kovacs Reagent
• Laboratory incubator set to 37 ± 1°C
• Water bath or laboratory incubator set to 44 ± 1°C
• Laboratory incubator set to 30 ± 1°C (optional for
total coliforms count only)
9.3.2 Procedure
A general flowchart for the enumeration of total colif-
orms and presumptive E. coli in foods using the Most
Probable Number (MPN) methods ISO 4831:2006
and ISO 7251:2005 is shown in Figure 9.2.
Before starting activities, carefully read the guide-
lines in Chapter 4, which deals with all details and care
required for performing MPN tests. The procedure
described below does not present these details, as they
are supposed to be known to the analyst.
7007TS-DASILVA-Book.indb 97
97
a) Preparation of the samples and inoculation:
Following the procedures described in Chapter 2,
homogenize m grams of the test sample with 9 m
milliliters of Saline Peptone Water (SPW) or Buff-
ered Peptone Water (BPW) (10−1 dilution). If the
food is acid check the pH of the 10−1 dilution and
adjust to 6.8 if necessary (with sterile NaOH).
Note a.1) ISO 4831:2006 and ISO 7251:2005 do not establish
the sample quantity in the analytical unity. A com-
monly weight used is 25 g of the sample in 225 ml of
the diluent.
Prepare subsequent decimal dilutions of the sam-
ple. Select three appropriate dilutions and inoculate
three 1 ml portions of each dilution onto three tubes
with 10 ml of Lauryl Sulfate Tryptose Broth (LST).
Follow the orientation of the Table 4.1 (Chapter 4)
to select the dilutions according the expected level
of sample contamination by coliforms.
Note a.2) For some products and/or each time that results of
greater accuracy are required, it may be necessary to
inoculate series consisting of more then three tubes
(e.g. five tubes).
If the expected count is low, inoculate three
10 ml aliquots of the 10−1 dilution onto three tubes
with 10 ml of LST Broth double strength, three
1 ml aliquots of the 10−1 dilution onto three tubes
with 10 ml of LST Broth single strength, and three
1 ml aliquots of the 10−2 dilution onto three tubes
with 10 ml of LST Broth single strength.
For liquid samples with low count it is possible to
start the series without dilution: 3 × 10 ml of sam-
ple without dilution onto 3 × 10 ml of LST Broth
double strength, 3 × 1 ml of sample without dilu-
tion onto 3 × 10 ml of LST Broth single strength
and 3 × 10 ml of 10−1 dilution onto 3 × 10 ml of
LST Broth single strength. If the food is acid adjust
the pH of the three double strength LST tubes to
6.8 after the inoculation (with sterile NaOH).
For liquid samples with low count it is also possi-
ble to apply a MPN single dilution test, inoculating
5 × 10 ml aliquots of the sample without dilution
onto 5 × 10 ml of LST Broth double strength. If
the food is acid adjust the pH of the five double
strength LST tubes to 6.8 after the inoculation
(with sterile NaOH).
b) Incubation for presumptive test (ISO 4831:2006
and 7251:2005): Incubate LST tubes at
37 ± 1°C/24 ± 2 h. Examine tubes and record reac-
tions at 24 ± 2 h for gas (displacement of medium
in fermentation vial or effervescence when tubes are
11/26/2012 11:15:24 AM
 -1 -2 -3
10 10 10
Homogenization 1ml 1ml

9ml 9ml
SPW SPW

25g of sample +
225ml of Saline Peptone Water (SPW)

1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml

PRESUMPTIVE
TEST
Lauryl Sulfate Tryptose
(LST) broth (10ml)

o
37±1 C/24±2h - 48±2h

Tubes with growth and gas production

1 loopful 1 loopful

PRESUMPTIVE E. coli TOTAL COLIFORMS TEST

E. coli TEST
E. aerogenes

E.coli (EC) Broth Brilliant Green Bile Broth (BGB) 2%

44±1oC/24±2h 37±1oC/24±2h - 48±2h
Water bath
BGB tubes with growth and gas production
1 loopful
Tubes with growth and gas
TOTAL COLIFORMS
MPN/g

Tryptone (Tryptophane) Broth

o
44±1 C/48±2h

INDOLE TEST

Tubes with positive results

PRESUMPTIVE E.coli
MPN/g

Figure 9.2 Scheme of analysis for the enumeration of total coliforms and presumptive E. coli in foods using the Most Probable Number
(MPN) methods ISO 4831:2006 and ISO 7251:2005.

7007TS-DASILVA-Book.indb 98 11/26/2012 11:15:24 AM

 Total and thermotolerant coliforms and Escherichia coli
gently agitated). Re-incubate gas-negative tubes for
an additional 24 h and examine and record reac-
tions again at 48 ± 2 h. Perform confirmed test on
all presumptive positive (gas) tubes.
Note b.1) When performing only the total coliform
count (ISO 4831:2006) it is possible to choose
between two incubation temperatures, 30 ± 1°C
or 37 ± 1°C (30 ± 1°C is indicated for milk
and milk products). The temperature is subject
of agreement between parties concerned. The
incubation time for double strength LST tubes
is 24 ± 2 h without re-incubation of negative
tubes. For single strength LST tubes the nega-
tive tubes are re-incubated for an additional
24 ± 2 h.
c) Confirmed test for total coliforms (ISO
4831:2006): From each double strength LST tube
showing gas after 24 ± 2 h of incubation, transfer
a loopful of the suspension to a tube of Brilliant
Green Bile Broth (BGB).
From each single strength LST tube showing gas
after 24 ± 2 h or 48 ± 2 h of incubation, transfer
a loopful of the suspension to a tube of Brilliant
Green Bile Broth (BGB).
Incubate the BGB tubes at 37 ± 1°C/24 ± 2 h and
examine for gas production, indicative of a posi-
tive result. Re-incubate gas-negative tubes for an
additional 24 h and examine and record reactions
again at 48 ± 2 h. Calculate most probable number
(MPN) following the instructions described in
Chapter 4, using a MPN table.
Note c.1) If the LST tubes were incubated at 30 ± 1°C in the
previous step, use the same temperature to incubate
the BGB tubes.
d) Test for presumptive E. coli (ISO 7251:2005)
d.1) Test for growth and gas production in EC
Broth at 44°C: From each LST tube show-
ing gas, transfer a loopful of the suspension
to a tube of E. coli Broth (EC). Incubate the
EC tubes at 44 ± 1°C/24 ± 2 h (water bath
or incubator). Examine the tubes for gas
production and re-incubate the gas-negative
tubes for an additional 24 h ± 2 h.
Note d.1) For some milk products (e.g. casein), the Dur-
ham tube may stick to the bottom of the LST
tubes. If, after 48 h incubation period, growth
is observed but no gas production, inoculate
this LST culture into EC broth.
Note d.2) For double strength LST tubes, the opacity or
cloudiness may difficult the gas observation. In this
case inoculate this LST culture into EC broth.
Note d.3) For live shellfish, an incubation time of not
more than 24 ± 2 h may be used.
7007TS-DASILVA-Book.indb 99
99
d.2) Indole test at 44°C: From each EC tube show-
ing gas (in 24 or 48 h of incubation) inocu-
late a tube of Tryptone (Tryptophane) Broth.
Incubate the tubes at 44 ± 1°C/48 ± 2 h and
test for indole production: Add 0.5 ml of
the Indole Kovacs Reagent to each tube of
Tryptone (Tryptophane) Broth, mix well and
examine after 1 min. A red color in the alco-
holic phase (surface of the liquid) indicates
indole production.
Consider as presumptive E. coli the cultures
showing gas in EC Broth at 44°C and indole
production in Tryptone (Tryptophane) Broth
at 44°C. Calculate most probable number
(MPN) following the instructions described
in Chapter 4, using a MPN table.
9.4 Most probable number (MPN)
method APHA/AWWA/WEF 2005
for total and thermotolerant
coliforms and E. coli in water
Method of the American Public Health Associa-
tion (APHA), American Water Works Association
(AWWA) & Water Environment Federation (WEF),
as described in Section 9221 of the Standard Methods
for the Examination of Water and Wastewater (Hunt and
Rice, 2005).
9.4.1 Material required for analysis
Preparation of the sample and serial
dilutions
• Magnesium Chloride Phosphate Buffer (Dilution
Water)
• Dilution tubes containing 9 ml Magnesium Chlo-
ride Phosphate Buffer (Dilution Water)
Total and thermotolerant coliforms count
• Lauryl Sulfate Tryptose (LST) Broth tubes (10 ml)
• Brilliant Green Bile (BGB) Broth 2% with Durham
tubes
• E. coli (EC) Broth with Durham tubes
• Pure culture E. coli
• Pure culture E. aerogenes
• Laboratory incubator set to 35 ± 0.5°C
• Water bath set to 44.5 ± 0.2°C
11/26/2012 11:15:24 AM
 Figure 9.3 Scheme of analysis for the enumeration total and thermotolerant coliforms and E. coli in water using the Most Probable Number
(MPN) method APHA/AWWA/WEF 2005 (Hunt and Rice, 2005).

7007TS-DASILVA-Book.indb 100 11/26/2012 11:15:24 AM

 Total and thermotolerant coliforms and Escherichia coli
Thermotolerant coliforms and E. coli count
(EC-MUG method for water) (optional)
• E. coli Broth with 4-methylumbelliferyl-β-D-
glucuronide (EC-MUG)
• Water bath set to 44.5 ± 0.2°C
E. coli count (indole method for water)
(optional)
• Tryptone (Tryptophane) Broth
• Indole Kovacs Reagent
• Water bath set to 44.5 ± 0.2°C
9.4.2 Procedure
A general flowchart for the enumeration of total and
thermotolerant coliforms and E. coli in water using
the Most Probable Number (MPN) method APHA/
AWWA/WEF 2005 is shown in Figure 9.3.
Before starting activities, carefully read the guide-
lines in Chapter 4, which deals with all details and care
required for performing MPN tests. The procedure
described below does not present these details, as they
are supposed to be known to the analyst.
a) Preparation of the samples and inoculation:
Homogenize the sample by shaking vigorously
about 25 times. For potable water apply a MPN
single dilution test, inoculating 5 × 20 ml aliq-
uots of the sample onto 5 × 10 ml of Lauryl Tryp-
tose Broth (LST) triple strength (or 5 × 20 ml of
LST double strength LST). It is also possible to
inoculate 10 × 10 ml aliquots of the sample onto
10 × 10 ml of Lauryl Tryptose Broth (LST) double
strength.
For non-potable water select three appropriate
dilutions and apply a MPN multiple dilution test,
inoculating five tubes of LST per dilution. Fol-
low the orientation of the Table 4.1 (Chapter 4) to
select the dilutions according the expected level of
sample contamination by coliforms.
b) Incubation: Incubate LST tubes at
35 ± 0.5°C/24 ± 2 h. Examine tubes and record
reactions at 24 ± 2 h for gas (displacement of
medium in fermentation vial or effervescence when
tubes are gently agitated). Re-incubate gas-negative
tubes for an additional 24 h and examine and record
reactions again at 48 ± 2 h. Perform confirmed test
on all presumptive positive (gas) tubes.
7007TS-DASILVA-Book.indb 101
101
c) Confirmed test for total coliforms: From each
gassing LST tube, transfer a loopful of suspen-
sion to a tube of Brilliant Green Bile Broth (BGB).
Incubate BGB tubes at 35 ± 0.5°C/24 ± 2 h and
examine for gas production, indicative of a posi-
tive result. Re-incubate gas-negative tubes for an
additional 24 h and examine and record reactions
again at 48 ± 2 h. Calculate most probable number
(MPN) following the instructions described in
Chapter 4, using a MPN table.
Note c.1) Alternative procedure only for polluted water
known to produce positive results consistently:
If all LST tubes are positive in two or more con-
secutive dilutions within 24 h, submit to the
confirmed test for coliforms only the tubes of
the highest dilution (smallest sample aliquots)
in which all tubes are positive and the positive
tubes of the subsequent dilution. For LST tubes
in which the positive result is produced only after
48 h, all should be submitted to the confirmed test
for coliforms.
d) Confirmed test for thermotolerant (fecal) col-
iforms (EC method): From each gassing LST
tube, transfer a loopful of the suspension to a
tube of E. coli (EC) Broth. Incubate EC tubes at
44.5 ± 0.2°C/24 ± 2 h in a water bath. Examine
tubes for growth and gas production, indicative of
a positive result. Calculate most probable number
(MPN) following the instructions described in
Chapter 4, using a MPN table.
Note d.1) Place all the EC tubes in the water bath within
30 min after inoculation. Maintain a sufficient
water depth in the water bath incubator to immerse
the EC tubes to upper level of the medium. Incu-
bate an EC tube of E. coli and an EC tube of E.
aerogenes in the same water bath as positive and
negative control.
e) Completed test for thermotolerant coliforms
and E. coli (EC-MUG method): From each gassing
LST tube, transfer a loopful of the suspension to
a tube of E. coli Broth with 4-methylumbelliferyl-
β-D-glucuronide (EC-MUG). Incubate EC-
MUG tubes at 44.5 ± 0.2°C/24 ± 2 h in a water
bath.
Note e.1) Place all the EC-MUG tubes in the water bath
within 30 min after inoculation. Maintain a suf-
ficient water depth in the water bath incubator
to immerse the EC tubes to upper level of the
medium. Incubate an EC-MUG tube of a MUG
positive strain of E. coli, an EC-MUG tube of
Klebsiella pneumoniae (glucuronidase negative)
and an EC-MUG tube of E. aerogenes (do not
11/26/2012 11:15:25 AM
 102 Microbiological examination methods of food and water
growth at 44.5°C) in the same water bath as
controls.
Thermotolerant coliforms: Examine EC-MUG
tubes for growth and gas production, indicative of
a positive result for thermotolerant coliforms. Cal-
culate most probable number (MPN) following the
instructions described in Chapter 4, using a MPN
table.
E. coli: Examine al EC-MUG tubes exhibiting
growth for fluorescence using a long-wavelength
UV lamp (366 nm, preferably six watts). The
presence of bright blue fluorescence is consid-
ered a positive result for E. coli. Calculate most
probable number (MPN) following the instruc-
tions described in Chapter 4, using a MPN
table.
f ) Completed test for E. coli (indole method): From
each gassing LST tube, transfer a loopful of the sus-
pension to a 5 ml tube of Tryptone (Tryptophane)
Broth. Incubate tubes at 44.5 ± 0.2°C/24 ± 2 h in
a water bath.
Note f.1) Place all the tubes in the water bath within
30 min after inoculation. Maintain a sufficient
water depth in the water bath incubator to
immerse the tubes to upper level of the medium.
Incubate a tube of an indole positive strain of
E. coli, a tube of E. cloacae (indole negative) and
an uninoculated tube in the same water bath as
controls.
After incubation add 0.2 to 0.3 ml of the
Indole Kovacs Reagent to each 5 ml tube of
Tryptone (Tryptophane) Broth and examine for
appearance of a deep red color in the upper layer
(indole test positive). The presence of red color is
considered a confirmative result for E. coli. Calcu-
late most probable number (MPN) following the
instructions described in Chapter 4, using a MPN
table.
9.5 Plate count method APHA 2001
for total coliforms in foods
Method of the American Public Health Association
(APHA), as described in Chapter 8 of the 4th Edition
of the Compendium of Methods for the Microbiologi-
cal Examination of Foods (Kornacki and Johnson,
2001) and Chapter 7 of the Standard Methods for
the Examination of Dairy Products (Davidson et al.,
2004).
7007TS-DASILVA-Book.indb 102
9.5.1 Material required for analysis
Preparation of the sample and serial
dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
• Dilution tubes containing 9 ml 0,1% Peptone Water
(PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Coliform count
• Sterile, empty 20 × 100 mm Petri dishes
• Violet Red Bile (VRB) Agar
• Brilliant Green Bile Broth (BGB)
• Laboratory incubator set to 35 ± 1°C (32 ± 1°C for
milk and dairy products)
9.5.2 Procedure
A flowchart for the enumeration of total coliforms in
foods using the plate count method APHA 2001 is
shown in Figure 9.4.
Before starting activities, carefully read the guide-
lines in Chapter 3, which deals with all details and care
required for performing plate counts of microorganisms,
from dilution selection to calculating the results. The
procedure described below does not present these details,
as they are supposed to be known to the analyst.
a) Preparation of the samples and inoculation.
For preparation of the samples and serial dilutions
(10−1, 10−2 and serial subsequent) follow the proce-
dures described in Chapter 2. Select three appropri-
ate dilutions of the sample and inoculate in Violet
Red Bile (VRB) Agar. Use the pour plate technique
and, after complete solidification of the medium,
cover the surface with a 5–8 ml thick layer of the
same medium.
For processed foods expected to contain sub-
lethally damaged or stressed coliforms (unable to
growth and form typical colonies on VRB), include
a recovery step: pour plate in a non-selective agar
(e.g. Trypticase Soy Agar, TSA), allow 2 ± 0.5 h of
recovery at room temperature, and then overlay the
TSA layer with VRB.
11/26/2012 11:15:25 AM
 Total and thermotolerant coliforms and Escherichia coli 103

-1 -2 -3
10 10 10
Homogenization 1ml 1ml

9ml 9ml
PW PW

25g of sample +
225ml of Peptone Water (PW)
1ml 1ml 1ml

VRB Violet Red Bile (VRB) Agar VRB

(pour plate with overlay)

o
35±1 C/18-24h
or
32±1o C/24±2h
(dairy products)

TOTAL COLIFORMS
CFU/g

Figure 9.4 Scheme of analysis for the enumeration of total coliforms in foods using the plate count method APHA 2001 (Kornacki and
Johnson, 2001).

Note a.1) The Chapter 7 of the Standard Methods for the
Examination of Dairy Products (Davidson et al.,
2004) recommends to overlay the TSA layer with
fortified VRB (VRB containing double strength of
bile salts, neutral red and crystal violet).
b) Incubation and colony counting. Incubate the
plates in an inverted position at 35 ± 1°C/18–24 h
(32 ± 1°C/24 ± 2 h for milk and dairy products).
Select plates with 15–150 colonies and count only
the typical coliforms colonies on the VRB medium:
red-purple, 0.5 mm or greater in diameter, sur-
rounded by a reddish halo characteristic of the pre-
cipitation of the bile salts.
c) Confirmation: Select five suspicious colonies
and inoculate each colony into a tube of Brilliant
Green Bile Broth (BGB). Incubate BGB tubes at
35 ± 0.5°C/24 ± 2 h (32 ± 1°C for milk and dairy
products) and examine for gas production. Consider
confirmed cultures exhibiting gas without surface
pellicle. If gas and pellicle are observed, perform
Gram stain and oxidase test. Consider confirmed the
cultures of Gram negative rods, oxidase negative.
d) Calculation of the results. Calculate number of
total coliforms/g of sample based on percentage
of colonies tested that are confirmed as total col-
iforms. Example: The presumptive count obtained
with 10−4 dilution of sample was 65 and four of five
colonies tested were confirmed (80%). The number
of coliforms cells/g of food is 65 × 0.8 × 104 =
520,000 CFU/g = 5.2 × 105 CFU/g.
9.6 References
AOAC International (2010) Rapid Methods Adopted as AOAC
Official MethodsSM. [Online] Available from: http://www.aoac.
org/vmeth/oma_testkits.pdf [Accessed 26th April 2011).
Brenner, D.J. & Farmer III, J.J. (2005) Family I. Enterobacteriaceae.
In: Brenner, D.J., Krieg, N.R. & Staley, J.T. (eds). Bergey’s Manual
of Systematic Bacteriology. Volume 2. 2nd edition. New York,
Springer Science+Business Media Inc. pp. 587–607.
Davidson, P.M., Roth, L.A. & Gambrel-Lenarz, S.A. (2004)
Coliform and other indicator bacteria. In: Wehr, H.M. & Frank,
J.F (eds). Standard Methods for the Examination of Dairy Products.
17th edition. Washington, American Public Health Association.
Chapter 7, pp. 187–226.

7007TS-DASILVA-Book.indb 103 11/26/2012 11:15:25 AM

 104 Microbiological examination methods of food and water
FAO/WHO (1979) Report of a joint FAO/WHO Working Group on
Microbiological Criteria for Foods. Food and Agricultural Organi-
zation and the World Health Organization. Report number:
Document WG/Microbiol./79/1.
Feng, P.C.S. & Hartman, P.A. (1982) Fluorogenic assay for immedi-
ate confirmation of Eschericihia coli. Applied and Environmental
Microbiology, 43, 1320–1329.
Horwitz, W. & Latimer, G.W. (eds) (2010) Official Methods of Anal-
ysis of AOAC International. 18th edition., revision 3. Gaithersburg,
Maryland, AOAC International.
Hunt, M.E. & Rice, E.W. (2005) Microbiological examination. In:
Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds).
Standard Methods for the Examination of Water & Wastewater.
21st edition. Washington, American Public Health Association
(APHA), American Water Works Association (AWWA) & Water
Environment Federation (WEF). Part 9000, pp. 9.1–9.169.
7007TS-DASILVA-Book.indb 104
International Organization for Standardization (2005) ISO
7251:2005. Microbiology of food and animal stuffs – Horizontal
method for the detection and enumeration of presumptive Escherichia
coli – Most probable number technique. Geneva, ISO.
International Organization for Standardization (2006) ISO
4831:2006. Microbiology of food and animal feeding stuffs – Hori-
zontal method for the detection and enumeration of coliforms – Most
probable number technique. Geneva, ISO.
Kornacki, J.L. & Johnson, J.L. (2001) Enterobacteriaceae, coliforms,
and Escherichia coli as quality and safety indicators. In: Downes,
F.P. & Ito, K. (eds). Compendium of Methods for the Microbio-
logical Examination of Foods. 4th edition. Washington, American
Public Health Association. Chapter 8, pp. 69–82.
Wehr, H.M. & Frank, J.F (eds) (2004) Standard Methods for the
Examination of Dairy Products. 17th edition. Washington, Ameri-
can Public Health Association.
11/26/2012 11:15:25 AM