haunt-zhuo
BAM: Enumeration of Escherichia coli
and the Coliform Bacteria
September 2002
Bacteriological Analytical Manual
Chapter 4
Enumeration of
Escherichia coli
and the
Coliform Bacteria
Authors:
Peter Feng
, Stephen D.
Weagant, Michael A. Grant
Chapter
Contents
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Conventional Method for Determining
Coliforms and
E. coli
LST-MUG Method for
Detecting
E. coli
in Chilled
or Frozen Foods
Exclusive of Bivalve
Molluscan Shellfish
Bottled Water
Examination of Shellfish and Shellfish
Meats
Analysis
for
E. coli
in citrus
juices
Other
Methods for Enumerating Coliforms and
E. coli
References
Escherichia coli
, originally
known as
Bacterium coli
commune, was identified in
1885 by the
German pediatrician, Theodor Escherich
(
14
,
29
).
E.
coli
is widely
distributed
in the intestine of humans and warm-blooded
animals and is the
predominant
facultative anaerobe in the bowel and part of the
essential intestinal flora
that
maintains the physiology of the healthy host
(
9
,
29
).
E.
coli
is a member of the
family
Enterobacteriaceae
(
15
), which includes many
genera, including known
pathogens such
as
Salmonella
,
Shigella
, and
Yersinia
. Although most
strains of
E. coli
are not regarded as pathogens, they can
be opportunistic pathogens that cause
infections in immunocompromised hosts.
There are also pathogenic strains of
E.
coli
that when ingested,
causes gastrointestinal illness in healthy humans
(see Chap. 4A).
In 1892, Shardinger
proposed the use of
E. coli
as an indicator of fecal contamination.
This was based on the premise that
E. coli
is abundant in human
and animal feces and
not usually found
in other niches. Furthermore, since
E.
coli
could be easily detected
by its ability to ferment glucose
(later changed to lactose), it was easier to
isolate than
known gastrointestinal
pathogens. Hence, the presence of
E.
coli
in food or water
became
accepted as indicative of recent fecal
contamination and the possible presence
of frank pathogens. Although the
concept of using
E. coli
as
an indirect indicator of
health risk
was sound, it was complicated in practice, due to
the presence of other
enteric bacteria
like
Citrobacter
,
Klebsiella
and
Enterobacter
that can also
ferment
lactose and are similar to
E. coli
in phenotypic
characteristics, so that they are not
easily distinguished. As a result, the
term
group of enteric bacteria.
Coliform is not a taxonomic classification but
rather a
working definition used to
describe a group of Gram-negative, facultative
anaerobic
rod-shaped bacteria that
ferments lactose to produce acid and gas within 48
h at 35°
C.
In 1914, the U.S.
Public Health Service adopted the enumeration of
coliforms as a
more convenient standard
of sanitary significance.
Although
coliforms were easy to detect, their association
with fecal contamination
was
questionable because some coliforms are found
naturally in environmental
samples
(
6
). This led to the
introduction of the fecal coliforms as an
indicator of
contamination. Fecal
coliform, first defined based on the works of
Eijkman (
12
) is a
subset of total coliforms that grows
and ferments lactose at elevated incubation
temperature, hence also referred to as
thermotolerant coliforms. Fecal coliform
analyses are done at 45.5°
C
for food testing, except for water, shellfish and
shellfish
harvest water analyses, which
use 44.5°
C (
1
,
3
,
30
). The fecal coliform
group consists
mostly of
E.
coli
but some other enterics such as
Klebsiella
can also ferment
lactose at
these temperatures and
therefore, be considered as fecal coliforms. The
inclusion of
Klebsiella
spp
in the working definition of fecal coliforms
diminished the correlation
of this
group with fecal contamination. As a result,
E. coli
has reemerged as an
indicator, partly facilitated by the
introduction of newer methods that can rapidly
identify
E. coli.
Currently, all 3 groups are used as
indicators but in different applications.
Detection
of coliforms is used as an
indicator of sanitary quality of water or as a
general
indicator of sanitary condition
in the food-processing environment. Fecal
coliforms
remain the standard indicator
of choice for shellfish and shellfish harvest
waters; and
E. coli
is used
to indicate recent fecal contamination or
unsanitary processing. Almost
all the
methods used to detect
E.
coli
, total coliforms or fecal
coliforms are
enumeration methods that
are based on lactose fermentation
(
4
). The Most Probable
Number (MPN) method is a statistical,
multi-step assay consisting of presumptive,
confirmed and completed phases. In the
assay, serial dilutions of a sample are
inoculated into broth media. Analysts
score the number of gas positive (fermentation
of lactose) tubes, from which the other
2 phases of the assay are performed and then
uses the combinations of positive
results to consult a statistical tables (Appendix
2), to
estimate the number of organisms
present. Typically only the first 2 phases are
performed in coliform and fecal
coliform analysis, while all 3 phases are done for
E.
coli
. The
3-tube MPN test is used for testing most foods.
The 5-tube MPN is used for
water,
shellfish and shellfish harvest water testing and
there is also a 10-tube MPN
method that
is used to test bottled water or samples that are
not expected to be highly
contaminated
(
3
).
There is
also a solid medium plating method for coliforms
that uses Violet Red Bile
Agar, which
contains neutral red pH indicator, so that lactose
fermentation results in
formation of
pink colonies. There are also membrane filtration
tests for coliform and
fecal coliform
that measure aldehyde formation due to
fermentation of lactose. This
chapter
also includes variations of above tests that use
fluorogenic substrates to detect
E.
coli
(
18
),
special tests for shellfish analysis, a brief
consideration of bottled water
testing
and a method for testing large volumes of citrus
juices for presence of
E.
coli
in conjunction with the
Juice HACCP rule.
I. Conventional
Method for coliforms, fecal coliforms and
E. coli
A.
Equipment and
materials
1.
Covered water bath, with circulating
system to maintain temperature of
45.5
±
0.2°
C. Water level should
be above the medium in immersed
tubes.
2.
Immersion-type thermometer,
1-55°
C, about 55 cm long, with
0.1°
C
subdivisions,
certified by National Institute of Standards and
Technology (NIST), or equivalent
3.
Incubator, 35 ±
1.0°
C
4.
Balance with
capacity of >2 kg and sensitivity of 0.1 g
5.
Blender and blender jar
(
see
Chapter 1)
6.
Sterile
graduated pipets, 1.0 and 10.0 mL
7.
Sterile
utensils for sample handling
(
see
Chapter 1)
8.
Dilution
bottles made of borosilicate glass, with
polyethylene screw
caps equipped with
Teflon liners. Commercially prepared dilution
bottles containing sterile
Butterfield's phosphate buffer can also be
used.
9.
Quebec colony
counter, or equivalent, with magnifying lens
10.
Longwave UV light [~365 nm], not to
exceed 6 W.
11.
pH meter
B.
Media
1
and
Reagents
2
Brilliant green lactose bile (BGLB)
broth, 2% (
M25
3
)
Lauryl tryptose (LST) broth
(
M76
4
)
EC broth
(
M49
5
)
Levine's eosin-methylene blue (L-EMB)
agar (
M80
6
)
Tryptone (tryptophane) broth
(
M164
7
)
MR-VP broth
(
M104
8
)
Koser's citrate broth
(
M72
9
)
Plate count agar (PCA) (standard
methods) (
M124
10
)
Butterfield's phosphate-buffered water
(
R11
11
) or
equivalent diluent (except
for
shellfish)
Kovacs' reagent
(
R38
12
)
Voges-Proskauer (VP) reagents
(
R89
13
)
Gram stain reagents
(
R32
14
)
Methyl red indicator
(
R44
15
)
Violet red bile agar (VRBA)
(
M174
16
)
VRBA-MUG agar
(
M175
17
)
EC-MUG medium
(
M50
18
)
Lauryl tryptose MUG (LST-MUG) broth
(
M77
19
)
Peptone Diluent, 0.1%
(
R56
20
)
C.
MPN -
Presumptive test for coliforms, fecal coliforms
and
E. coli
Weigh
50 g food into sterile high-speed blender jar.
(see Chapter 1 and current
FDA
compliance programs for instructions on sample
size and compositing)
Frozen samples
can be softened by storing it for <18 h at
2-5°
C, but do not
thaw. Add
450 mL of Butterfield's phosphate-buffered water
and blend for 2
min. If <50 g of sample
are available, weigh portion that is equivalent to
half
of the sample and add sufficient
volume of sterile diluent to make a 1:10
dilution. The total volume in the
blender jar should completely cover the
blades.
Prepare decimal
dilutions with sterile Butterfield's phosphate
diluent. Number
of dilutions to be
prepared depends on anticipated coliform density.
Shake all
suspensions 25 times in 30 cm
arc or vortex mix for 7 s. Do not use pipets to
deliver <10% of their total volume.
Transfer 1 mL portions to 3 LST tubes for
each dilution for at least 3
consecutive dilutions. Hold pipet at angle so that
its
lower edge rests against the tube.
Let pipet drain 2-3 s. Not more than 15 min
should elapse from time the sample is
blended until all dilutions are inoculated
in appropriate media.
NOTE:
Use 5-tube MPN for
analysis of shellfish and shellfish harvest
waters.
Incubate LST tubes at
35°
C. Examine tubes and record
reactions at 24 ±
2 h
for
gas, i.e., 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.
D.
MPN -
Confirmed test for coliforms
From each gassing LST tube, transfer a
loopful of suspension to a tube of
BGLB
broth, avoiding pellicle if present. Incubate BGLB
tubes at 35°
C and
examine
for gas production at 48 ±
2 h.
Calculate most probable number
(MPN)
(see Appendix 2) of coliforms based on proportion
of
confirmed
gassing LST tubes for 3 consecutive
dilutions.
E.
MPN
- Confirmed test for fecal coliforms and
E. coli
From each
gassing LST tube from the Presumptive test,
transfer a loopful of
each suspension
to a tube of EC broth (a sterile wooden applicator
stick may
also be used for these
transfers). Incubate EC tubes 24 ±
2 h
at 45.5 °
C and
examine for
gas production. If negative, reincubate and
examine again at 48 ±
2 h.
Use results of this test to calculate fecal
coliform MPN. To continue with
E.
coli
analysis, proceed to Section F
below. The EC broth MPN method may
be
used for seawater and shellfish since it conforms
to recommended
procedures
(
1
). (Caution: see Note
below).
NOTE:
Fecal coliform
analyses are done at 45.5±
0.2°
C for all foods, except
for water testing and in shellfish and
shellfish harvest water analysis, which
uses an incubation temperature of
44.5±
0.2°
C.
F.
MPN -
Completed test for
E.
coli
.
To perform
the Completed test for
E.
coli
, gently agitate each gassing EC
tube
and streak for isolation, a
loopful to a L-EMB agar plate and incubate for
18-24 h at 35°
C. Examine
plates for suspicious
E.
coli
colonies, i.e., dark
centered and flat, with or without
metallic sheen. Transfer up to
5
suspicious
colonies from each L-EMB plate to PCA
slants incubate for 18-24 h at 35°
C
and use for further testing.
NOTE:
Identification of any
1 of the 5 colonies as
E.
coli
is sufficient to
regard
that EC tube as positive; hence, not all 5
isolates may need to be tested.
Perform
Gram stain. All cultures appearing as Gram-
negative, short rods
should be tested
for the IMViC reactions below and also re-
inoculated back
into LST to confirm gas
production.
Indole production.
Inoculate tube of tryptone broth and incubate 24
±
2 h at
35°
C.
Test for indole by adding 0.2-0.3 mL of Kovacs'
reagent. Appearance of
distinct red
color in upper layer is positive test.
Voges-Proskauer (VP)-reactive
compounds. Inoculate tube of MR-VP broth
and incubate 48 ±
2 h at
35°
C. Transfer 1 mL to 13 x 100 mm
tube. Add 0.6
mL α
-naphthol
solution and 0.2 mL 40% KOH, and shake. Add a few
crystals
of creatine. Shake and let
stand 2 h. Test is positive if eosin pink color
develops.
Methyl red-
reactive compounds. After VP test, incubate MR-VP
tube
additional 48 ±
2 h at
35°
C. Add 5 drops of methyl red
solution to each tube.
Distinct red
color is positive test. Yellow is negative
reaction.
Citrate. Lightly inoculate
tube of Koser's citrate broth; avoid detectable
turbidity. Incubate for 96 h at
35°
C. Development of distinct turbidity
is
positive reaction.
Gas
from lactose. Inoculate a tube of LST and incubate
48 ±
2 h at 35°
C. Gas
production (displacement of medium from
inner vial) or effervescence after
gentle agitation is positive reaction.
Interpretation:
All cultures
that (a) ferment lactose with gas production
within 48 h at 35°
C, (b)
appear as Gram-negative nonsporeforming rods and
(c)
give IMViC patterns of ++--
(biotype 1) or -+-- (biotype 2) are considered to
be
E. coli
.
Calculate MPN (see Appendix 2) of
E.
coli
based on proportion of
EC tubes in 3 successive dilutions that
contain
E. coli
.
NOTE:
Alternatively, instead
of performing the IMViC test, use API20E or
the automated VITEK biochemical assay
to identify the organism as
E.
coli
.
Use growth from the
PCA slants and perform these assays as described
by the
manufacturer.
G.
Solid medium
method - Coliforms
Prepare
violet red bile agar (VRBA) according to
manufacturer's instructions.
Cool to
48°
C before use. Prepare, homogenize,
and decimally dilute sample as
described in section I. C above so that
isolated colonies will be obtained when
plated. Transfer two 1 mL aliquots of
each dilution to petri dishes, and use
either of the following two pour
plating methods, depending on whether
injured or stressed cells are suspected
to be present (
1
).
Pour 10 mL VRBA tempered to
48°
C into plates, swirl plates to mix,
and let
solidify. To prevent surface
growth and spreading of colonies, overlay with 5
mL VRBA, and let solidify. If
resuscitation is necessary, pour a basal layer of
8-10 mL of tryptic soy agar tempered to
48°
C. Swirl plates to mix, and
incubate at room temperature for 2
±
0.5 h. Then overlay with 8-10 mL of
melted, cooled VRBA and let solidify.
Invert solidified plates and incubate
18-24 h at 35°
C. Incubate dairy
products
at 32°
C
(
2
). Examine plates under
magnifying lens and with illumination.
Count purple-red colonies that are 0.5
mm or larger in diameter and
surrounded
by zone of precipitated bile acids. Plates should
have 25-250
colonies. To confirm that
the colonies are coliforms, pick at least 10
representative colonies and transfer
each to a tube of BGLB broth. Incubate
tubes at 35°
C. Examine at 24
and 48 h for gas production.
NOTE:
If gas-positive BGLB
tube shows a pellicle, perform Gram stain to
ensure that gas production was not due
to Gram-positive, lactose-fermenting
bacilli.
Determine the
number of coliforms per gram by multiplying the
number of
suspect colonies by percent
confirmed in BGLB by dilution factor.
Alternatively,
E.
coli
colonies can be distinguished
among the coliform
colonies on VRBA by
adding 100 ?
g of
4-methyl-
umbelliferyl-
β
-D-glucuronide
(MUG) per mL in the VRBA overlay.
After
incubation, observe for bluish fluorescence around
colonies under
longwave UV light. (see
LST-MUG section II for theory and applicability.)
H.
Membrane
Filtration (MF) Method - coliforms: see Section
III. Bottled
Water.
NOTE:
Food homogenates will
easily clog filters, hence MF are most suitable
for analysis of water samples; however,
MF may be used in the analysis of
liquid foods that do not contain high
levels of particulate matter.
II. LST-MUG Method for Detecting
E. coli
in Chilled or Frozen
Foods Exclusive
of Bivalve Molluscan
Shellfish
The LST-
MUG assay
is based on the enzymatic activity of
β
-glucuronidase (GUD),
which
cleaves the substrate
4-
methylumbelliferyl
β
-D-glucuronide (MUG), to release
4-methylumbelliferone (MU). When
exposed to longwave (365 nm) UV light, MU
exhibits a bluish fluorescence that is
easily visualized in the medium or around the
colonies. Over 95% of
E.
coli
produces GUD, including
anaerogenic
(non-gas-producing)
strains. One exception is enterohemorrhagic
E. coli
(EHEC) of
serotype O157:H7, which is consistently
GUD negative (
11
,
17
). The lack of GUD
phenotype in O157:H7 is often used to
differentiate this serotype from other
E. coli
,
although
GUD positive variants of O157:H7 do exist
(
24
,
26
). The production of
GUD by other members of the family
Enterobacteriaceae
is rare,
except for some
shigellae (44 -58%) and
salmonellae (20-29%) (
18
,
27
). However, the
inadvertent
detection of these
pathogens by GUD-based assays is not considered a
drawback from
a public health
perspective. Expression of GUD activity is
affected by catabolite
repression
(
8
) so on occasion, some
E. coli
are GUD-negative,
even though they carry
the
uid
A gene
(
gus
A) that encodes for the
enzyme (
19
). In most
analyses however,
about 96% of
E. coli
isolates tested are
GUD-positive without the need for enzyme
induction (
27
).
MUG can be incorporated into almost any
medium for use in detecting
E.
coli
. But
some media such as
EMB, which contain fluorescent components, are not
suitable, as
they will mask the
fluorescence of MU. When MUG is incorporated into
LST
medium, coliforms can be enumerated
on the basis of gas production from lactose and
E. coli
are presumptively
identified by fluorescence in the medium under
longwave
UV light, thus it is capable
of providing a presumptive identification of
E. coli
within
24
h (
18
,
28
). The LST-MUG method
described below has been adopted as Official
Final Action by the AOAC for testing
for
E. coli
in chilled or
frozen foods, exclusive
of shellfish
(
28
). For information on MUG
assay contact,
Dr. Peter
Feng
FDA,
CFSAN, College
Park, MD, 20740; 301-436-1650.
CAUTION:
To observe for
fluorescence, examine inoculated LST-MUG tubes
under
longwave (365 nm) UV light in the
dark. A 6-watt hand-held UV lamp is adequate
and safe. When using a more powerful UV
source, such as a 15-watt fluorescent lamp,
wear protective glasses or goggles.
Also, prior to use in MUG assays, examine all
glass tubes for auto fluorescence.
Cerium oxide, which is sometimes added to glass as
a quality control measure, will
fluoresce under UV light and interfere with the
MUG
test (
25
).
The use of positive and negative control strains
for MUG reaction is
essential.
1.
Equipment and
material:
see section I.A above and in
addition,
New, disposable borosilicate
glass tubes (100 x 16 mm)
New,
disposable borosilicate glass Durham vials (50 x 9
mm) for gas
collection.
Longwave UV lamp, 6-watt or equivalent
2.
Media and reagents:
see
section I.B above
3.
Presumptive
LST-MUG test for
E. coli.
Prepare food samples and
perform the MPN Presumptive test as described in
section
I.C. above, except use LST-MUG
tubes instead of LST. Be sure to inoculate one
tube
of LST-MUG with a known GUD-
positive
E. coli
isolate as
positive control (ATCC
25922). In
addition, inoculate another tube with a culture of
Enterobacter aerogenes
(ATCC 13048) as negative control, to
facilitate differentiation of sample tubes that
show only growth from those showing
both growth and fluorescence. Incubate tubes
for 24 to 48 ±
2 h at
35°
C. Examine each tube for growth
(turbidity, gas) then
examine tubes in
the dark under longwave UV lamp (365 nm). A bluish
fluorescence
is a positive presumptive
test for
E. coli
. Studies by
Moberg et al. (
28
) show that
a 24
h fluorescence reading is an
accurate predictor of
E.
coli
and can identify 83-95% of
the
E.
coli
-positive tubes. After 48 h of
incubation, 96-100% of
E.
coli
-positive tubes
can be
identified (
28
). Perform a
confirmed test on all presumptive positive tubes
by
streaking a loopful of suspension
from each fluorescing tube to L-EMB agar and
incubate 24 ±
2 h at
35°
C. Follow protocols outlined in I.
F, above, for Completed test