Outline
Laboratory Support Equipment
Minnesota Department of Health
•
•
•
•
•
•
Labware
Sterilization
Equipment
Temperature Monitoring
Standards, Reagents, and Media
• Media Preparation
• Quality Control
• Water quality
Public Health Laboratory
•
June 18th, 2009
Laboratory Support Equipment
Quality Control Procedures (Part I)
• Sterility checks
• + / - controls
• Variability
Lab Support Equipment
• Labware
Labware
• Filtration equipment
• Funnels
• Bases
• Glassware
• Plasticware
Plasticware
• Serological pipets
• TC
• TD
Glassware
• Borosilicate
• Measurement
markings
1
Labware, Glassware, & Plasticware
Labware, Glassware, & Plasticware
Sterility QC
• Can be washed & reused
or single use items
• Quality Control testing
must be done before use
• Documentation of QC
testing must be recorded
Sterilization Equipment
• Autoclaves
Sterilization Equipment
• UV Sterilizer
• Steam sterilization
• Short wave ultraviolet light
Autoclaves & UV Sterilization
• Autoclaves must
be tested for
effectiveness
monthly
UV Sterilizer Maintenance
• Bulb replacement
• Scheduled
• UV Instruments
must be tested for
effectiveness
quarterly
cleaning
2
Autoclave Maintenance
• Performed annually
• Pressure check
• Temperature
calibration
• Record in maintenance
log
• Maintenance can be
performed internally or
by service contract
UV Sterilizer Records of Operation
• Must be documented for each use
• Wavelength
• Duration of exposure
Autoclave run log
Mechanical Timing Device
Calibration
• Must be checked
against a calibrated
timer/stopwatch
quarterly
• Records must be
maintained in
equipment log
Records of Autoclave Operation
• Must be documented for every cycle
•
•
•
•
•
•
•
Date
Contents
Max temperature
Pressure
Time in sterilization mode
Total run time
Operators initials
Incubators, Water baths, & Ovens
• Temperature
• Documentation
• Maintenance
3
Membrane Filters & Pads
Temperature Monitoring
• 47 mm diameter
• Thermometers
• Continuous
recording devices
• Verify calibration
& document
accuracy
• ≥ 70% pores
•
•
•
•
0.45 µm pore diameter
Sterility
QC
Documentation
Break time!
Standards, Reagents, & Media
• Commercially
prepared or
made in-house
• Storage
• Sterility
In-house Media
• Storage of
dehydrated media
• Preparation of
media
Media Logbook
• In-house
•
•
•
•
•
•
•
•
Date of preparation
Preparer’s initials
Manufacturer
Lot #
Type of media
Amount
Expiration date
pH
4
Media Logbook
Tracking Standards, Reagents, &
Media
• Commercially
prepared media
•
•
•
•
•
•
Tracking Log
B2008-77-….
Manufacturer
Lot #
Type of media
Amount
Date received
Expiration date
Media QC
• Sterility QC
• Growth of target
organism
• Inhibition of nontarget organisms
QC Record
Water Quality
• Distilled
• Deionized
• Reverse osmosis
• Quality monitoring
• Residual chlorine
• Heterotrophic bacteria
• Conductivity
5
ASTM Type I and II Reagent Water
Type
I
0.056
18.200
3.000
50.000
1.000
1.000
• Conductivity @ 25°C (microohms/cm)
• Resistivity @ 25°C (megaohms/cm)
• Total Silica (µg/L)
• Total Organic Carbon (µg/L)
• Chlorides (µg/L)
• Sodium (µg/L)
Test Variability
II
1.000
1.000
3.000
50.000
5.000
5.000
Quality Control Procedures (Part I)
• Documentation
• Acceptance criteria
• Types of QC procedures
• Sterility checks
• Sterility blanks
• +/- controls
Questions
• Duplicate
counts
• 10% variability
(2 analysts)
• 5% variability
(1 analyst)
6
Dilutions
ƒ Typical Coliform Dilutions
ƒ Based on 100 ml Sample
Dilutions and Calculations
Minnesota Department of Health
Public Health Laboratory
June 18th, 2009
Sample
Dilution
Preparation
Sample
Diluent
Volume (mL)
100
50
10
1
0.1
0.01
0.001
0.0001
Factor
1x
2x
10x
100x
1000x
10,000x
100,000x
1,000,000x
Vol. (mL)
100
50
10
1
10 (100x)
1 (100x)
10 (1,000x)
1 (1,000x)
(mL)
0
50
90
99
90
99
90
99
Dilutions
Calculations
Estimation of Bacterial Density
ƒ Typical Heterotrophic Dilutions
ƒ Based on 1 ml Sample
ƒ MPN five 20-mL Portions
Sample
Dilution
Preparation
Sample
Diluent
Volume
Volume (mL)
1
0.5
0.1
0.01
0.001
0.0001
Factor
1x
2x
10x
100x
1000x
10,000x
Vol. (mL)
1
0.5
0.1
1
1
1 (100x)
on Dish (mL)
1.0
0.5
0.1
1.0
0.1
0.1
(mL)
0
0
0
99
99
99
Calculations
Estimation of Bacterial Density
ƒ MPN ten 10-mL Portions
#
Tubes Positive
0
1
2
3
4
5
6
7
8
9
10
MPN Index
100 mL
<1.1
1.1
2.2
3.6
5.1
6.9
9.2
12.0
16.1
23.0
>23.0
#
Tubes Positive
0
1
2
3
4
5
MPN Index
100 mL
<1.1
1.1
2.6
4.6
8.0
> 8.0
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive 10mL
(Five Tubes)
#
Tubes Positive
0-0-0
0-0-1
0-1-0
0-2-0
1-0-0
1-0-1
1-1-0
1-1-1
1-2-0
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
MPN Index
100 mL
<2
2
2
4
2
4
4
6
6
4
7
7
9
9
12
8
11
11
14
14
17
13
17
17
21
26
#
Tubes Positive
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1
5-3-2
5-3-3
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
100 mL
22
26
27
33
34
23
30
40
30
50
60
50
70
90
80
110
140
170
130
170
220
280
350
240
300
500
900
1600
>=1600
1
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive (Five Tubes)
10
1
5
5
#
Tubes Positive
0-0-0
0-0-1
0-1-0
0-2-0
1-0-0
1-0-1
1-1-0
1-1-1
1-2-0
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
0.1
2
5-5-2
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive 10 mL
(Five Tubes)
1
5
0.1
0.01
5
2
0.001
1
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive 10 mL
(Five Tubes)
5
0.1
0.01
3
1
5-3-2
MPN Index
100 mL
<2
2
2
4
2
4
4
6
6
4
7
7
9
9
12
8
11
11
14
14
17
13
17
17
21
26
#
Tubes Positive
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1
5-3-2
5-3-3
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
100 mL
22
26
27
33
34
23
30
40
30
50
60
50
70
90
80
110
140
170
130
170
220
280
350
240
300
500
900
1600
>=1600
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive (Five Tubes)
5-5-2
1
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive 10 mL
(Five Tubes)
#
Tubes Positive
0-0-0
0-0-1
0-1-0
0-2-0
1-0-0
1-0-1
1-1-0
1-1-1
1-2-0
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
MPN Index
100 mL
<2
2
2
4
2
4
4
6
6
4
7
7
9
9
12
8
11
11
14
14
17
13
17
17
21
26
#
Tubes Positive
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1
5-3-2
5-3-3
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
100 mL
22
26
27
33
34
23
30
40
30
50
60
50
70
100X70=70,00
90
80
110
140
170
130
170
220
280
350
240
300
500
900
1600
>=1600
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive (Five Tubes)
0.001
1
#
Tubes Positive
0-0-0
0-0-1
0-1-0
0-2-0
1-0-0
1-0-1
1-1-0
1-1-1
1-2-0
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
MPN Index
100 mL
<2
2
2
4
2
4
4
6
6
4
7
7
9
9
12
8
11
11
14
14
17
13
17
17
21
26
#
Tubes Positive
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1
5-3-2
5-3-3
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
100 mL
22
26
27
33
34
23
30
40
30
50
60
50
70
90
80
110
140 140*10=1,400
170
130
170
220
280
350
240
300
500
900
1600
>=1600
2
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive 10 mL
(Five Tubes)
1
0
0.1
2
0.01
0
Calculations
Estimation of Bacterial Density with
Various Combinations of Positive 10 mL
(Five Tubes)
0.001
0
0-2-0
#
Tubes Positive
0-0-0
0-0-1
0-1-0
0-2-0
1-0-0
1-0-1
1-1-0
1-1-1
1-2-0
2-0-0
2-0-1
2-1-0
2-1-1
2-2-0
2-3-0
3-0-0
3-0-1
3-1-0
3-1-1
3-2-0
3-2-1
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
MPN Index
100 mL
<2
2
2 2*10=20
4
2
4
4
6
6
4
7
7
9
9
12
8
11
11
14
14
17
13
17
17
21
26
#
Tubes Positive
4-2-0
4-2-1
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
5-2-0
5-2-1
5-2-2
5-3-0
5-3-1
5-3-2
5-3-3
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
MPN Index
100 mL
22
26
27
33
34
23
30
40
30
50
60
50
70
90
80
110
140
170
130
170
220
280
350
240
300
500
900
1600
>=1600
Calculations of Coliform Density
Calculations of Coliform Density
ƒ Count Plates with 20-80 colonies
ƒ Report as Colonies/100 mL
ƒ No filter within ideal range, total all
ƒ Colonies/100 mL = colonies counted x 100
mL sample Filtered
ƒ Drinking water count any colonies
counts on all filters
ƒ 50 mL, 10mL, 1mL
ƒ 19 counts, 4 counts, <1
ƒ [(19+4+0)x100] = 37.7 /100 mL
(50+10+1)
Calculations of Coliform Density
ƒ One filter within ideal range, total only
counts on filter within range
ƒ 50 mL, 10mL, 1mL
ƒ 33 counts, 4 counts, <1
ƒ [(33x100] = 66 /100 mL
50
If total bacteria count was >200 colonies
then result is reported >= 66 / 100 mL
3
Questions
4
Coliform Microbiology Methods
Presenters:
Jeffrey Brenner
Lindsey Mattila
Public Health Laboratory
Minnesota Department of Health
June 18th, 2009
Determine If Water Contains
Pathogens
ƒ Which pathogens?
ƒ Bacteria
ƒ Viruses
ƒ Parasites
ƒ Methods not available
or unreliable
ƒ Organisms present in
low numbers
Determine If Water Contains
Pathogens
Pathogen Detection vs. Indicator
System
ƒ Testing for all possible pathogens is:
ƒ Coliform Bacteria are the commonly-used bacterial
indicator for water quality
ƒ Coliforms are abundant in the environment
ƒ Complex
ƒ Time consuming
ƒ Expensive
ƒ Aquatic
ƒ Soil
ƒ Vegetation
ƒ Feces of all warm-blooded animals and humans
ƒ Most unlikely to cause illness
ƒ Relatively easy and inexpensive to test for coliform
bacteria
Pathogen Detection vs. Indicator
System
What are Coliform Bacteria?
ƒ Presence indicates disease-causing
organisms could be in the water system
ƒ Most pathogens that contaminate water
supplies come from the feces of humans or
animals and may include
ƒ There are three different classifications of
coliform bacteria:
ƒ Total coliform
ƒ Fecal coliform
ƒ E. coli
ƒ Bacteria
ƒ Viruses
ƒ Parasites
Heterophic
1
What are Total Coliform Bacteria?
What are Total Coliform Bacteria?
ƒ Total Coliform Bacteria
ƒ Defined as:
ƒ Large collection of bacteria
ƒ Citrobacter
ƒ Enterobacter
ƒ Escherichia
ƒ Hafnia
ƒ Klebsiella
ƒ Serratia
ƒ Yersinia
ƒ
ƒ
ƒ
ƒ
ƒ
Facultative anaerobic
Rod-shaped
Nonsporulating
Gram-negative organisms
Ferment lactose with the production of
acid and gas at 35-37oC
What are Total Coliform Bacteria?
What are Fecal Coliform Bacteria?
ƒ If only Total Coliform Bacteria detected
ƒ Fecal Coliform are a sub-group of total coliform
bacteria.
ƒ Source is probably environmental
ƒ Fecal contamination not likely
ƒ However, if environmental
contamination can enter the system,
there may be a way for pathogens to
enter the system
ƒ
ƒ
ƒ
ƒ
Citrobacter
Enterobacter
Escherichia
Klebsiella
ƒ Appear in greater quantities in the intestines and
feces of warm blooded animals and humans
ƒ Indicate fecal contamination and possible
contamination by pathogens.
What are Fecal Coliform Bacteria?
What are E. coli Bacteria?
ƒ Defined as:
ƒ E. coli is a sub-group of the fecal
ƒ
ƒ
ƒ
ƒ
Rod-shaped
Gram-negative organisms
Facultative-anaerobic
Ferment lactose with the production of acid and
gas at 35-37oC and within 48 hours at 44oC
ƒ Capable of growth in the presence of bile salts
ƒ Oxidase negative
coliform group.
ƒ Most E. coli are harmless
ƒ Found in great quantities in the
intestines of warm-blooded animals
and humans
2
What are E. coli Bacteria?
Total Coliform Methods
Fermentation
Technique
ƒ Presence of E. coli almost always
Membrane
Filtration
Substrate
Technology
indicates recent fecal contamination.
ƒ Some strains cause illness. Most
outbreaks have been caused by a
specific strain O157:H7
ƒ When drinking water sample is
reported as “E. coli present” usually not
O157:H7
Multi-Tube Fermentation Technique
“Equivalent” But Not The Same
Multi-Tube Fermentation Technique
ƒ
ƒ
ƒ
ƒ
ƒ
Standard Coliform Fermentation
Estimation of Bacteria Density
Presence-Absence Coliform Test
Fecal Coliform Test
Advantages
ƒ
ƒ
ƒ
ƒ
Disadvantages
ƒ
ƒ
Requires a lot of prep support,
supplies, and equipment
Presumptive Phase
ƒ
Confirmed Phase
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
Standard Coliform Fermentation
ƒ Three Phase Test
ƒ Presumptive
ƒ lauryl tryptose broth
ƒ Confirmed
ƒ Brilliant green lactose bile broth
Suitable for a wide variety of water
P/A or MPN
Better recovery of injured
organisms
lauryl tryptose broth
brilliant green lactose bile broth
False (+) & false (-) can occur
(mixed culture)
Test takes 48-96 hours
MPN is labor intensive
High non-coliform population may
mask coliform detection
Standard Coliform Fermentation
Presumptive Phase
ƒ Lauryl tryptose broth (LTB)
ƒ Sample portion to medium will not reduce ingredient
concentration below standard
ƒ LTB (g/L) = (35.6* FV)/ M
LTB = Dehydrated LTB g/L
FV = Volume of Medium + Sample
M = Volume of Medium
ƒ Series of 1, 5, 10 tubes per dilution
3
Standard Coliform Fermentation
Presumptive Phase
ƒ Preparation of Lauryl Tryptose Broth
Standard Coliform Fermentation
Presumptive Phase
ƒ Incubate at 35 +/- 0.5C for
24 +/- 2 hours
ƒ Sample + Broth = Ingredient Concentration
ƒ LTB (g/L) = (35.6* FV)/ M
Sample (mL)
Broth (mL)
Final
Volume
(mL)
Dehydrated
LTB
Required
(g/L)
5
10
15
53.4
1.5
10
10
20
71.2
2.0
20
10
30
106.8
3.0
50
50
100
71.2
2.0
100
50
150
106.8
3.0
100
20
120
213.6
6.0
Factor
ƒ Examine for growth, gas, and
acidic reaction (shades of
yellow color)
ƒ Reincubate and reexamine at
48 +/- 3 hours
ƒ Production of an acidic
reaction or gas constitutes a
positive presumptive
Standard Coliform Fermentation
Presumptive Phase
Standard Coliform Fermentation
Confirmation Phase
ƒ Absence of acidic reaction or gas at
ƒ If all presumptive tubes are positive in two
or more consecutive dilutions, within 24 h
then:
the end of 48+/- 3 hours constitutes
negative test.
ƒ Submit drinking water samples that
demonstrate growth to the confirmation
phase
Standard Coliform Fermentation
Confirmation Phase
ƒ If all presumptive tubes are positive in two
or more consecutive dilutions, within 24 h
then:
ƒ Submit only tubes of the highest dilution in
which all are positive
ƒ Submit tubes in higher dilutions
10
1
0.1
ƒ Submit only tubes of the highest dilution in
which all are positive
ƒ Submit tubes in higher dilutions
10
1
0.1
Standard Coliform Fermentation
Confirmation Phase
ƒ Gently shake or rotate
presumptive tubes or bottles.
ƒ Inoculate brilliant green
lactose bile broth
ƒ Incubate at 35 +/- 0.5C
ƒ Positive confirmation
ƒ Gas production any time within
48 hours
4
Standard Coliform Fermentation
Completed Phase
ƒ Inoculate EC broth (Fecal)
or
ƒ EC-MUG broth (E. coli)
Estimation of Bacteria
ƒ To be covered in the dilutions and
calculation section of the presentation
ƒ Negative EC or EC-MUG indicate
positive nonfecal coliforms
Presence-Absence Coliform Test
Presence-Absence Coliform Test
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ Yellow color indicates acid conditions
Similar to multiple-tube procedure
Use 100 mL test sample
Mostly for use with drinking water
Use P-A broth
Incubate at 35 +/- 0.5C
Inspect at 24 hours and 48 hours
Presence-Absence Coliform Test
Confirmation
(lactose fermentation)
ƒ Gently shake for determination of gas
production
ƒ Foaming indicates gas production
Fecal Coliform Test
ƒ Gently shake or rotate
ƒ Test to distinguish fecal coliform
presumptive bottles.
ƒ Inoculate brilliant green
lactose bile broth
ƒ Incubate at 35 +/- 0.5C
ƒ Positive confirmation
ƒ Elevated temperature
ƒ Two Medium
ƒ EC broth (requires presumptive phase)
ƒ A-1 broth (direct isolation)
ƒ Gas production any time within
48 hours
5
Fecal Coliform Test
Fecal Coliform Test
ƒ EC broth (requires presumptive
phase)
ƒ Inoculate all presumptive tubes or
bottles to EC broth
ƒ Incubate at 44.5 +/- 0.2C for 24
hour +/- 2 hours
ƒ Positive Fecal
Fecal Coliform Test
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
A1 Broth
Direct isolation
No presumptive medium
Inoculate as total coliform
Incubate 3 hours at 35 +/- 0.5C
Transfer to water bath at 44.5 +/- 0.2C
Gas production within 24 hours indicates
positive fecal
Membrane Filtration Technique
ƒ Gas production
ƒ Growth within 24 +/- 2 hours
ƒ Multiple tubes (MPN)
ƒ Single tube (P/A)
Membrane Filtration Technique
ƒ Advantages
ƒ Enumeration is easy
ƒ Minimal support needed/most
supplies disposable
ƒ More precise than MPN methods
ƒ Disadvantages
ƒ Presence of iron or particulates
ƒ Overgrowth of non-coliforms
ƒ Typical (sheen) colonies &
atypical (dark green or red)
colonies must be verified
ƒ Stressed organisms may not
grow
ƒ False (+) and false (-) can occur
Standard Total Coliform Membrane Filter
ƒ Agar or liquid medium
ƒ Standard Total Coliform Membrane Filter
ƒ Delayed-Incubation Total Coliform
ƒ Fecal Coliform Membrane Filter
ƒ M-Endo
ƒ LES Endo
ƒ Determine ideal sample volume
ƒ Yields 20- 80 coliforms
ƒ Not more than 200 colonies of all types
ƒ
ƒ
ƒ
ƒ
Filter ideal volumes
Place prepared filter on pad
Invert dish
Incubate for 22-24 hours at 35 +/- 0.5C
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Standard Total Coliform Membrane Filter
Standard Total Coliform Membrane Filter
ƒ Incubation >24 hours may make it
ƒ Typical Coliform Colonies
difficult to differentiate colonies
ƒ Use low magnifications to count
colonies
ƒ Two types of coliform colonies
ƒ Pink to dark-red with metallic surface
sheen
ƒ Typical
ƒ Atypical
Standard Total Coliform Membrane Filter
Standard Total Coliform Membrane Filter
ƒ Atypical Coliform Colonies
ƒ Coliform verification
ƒ Dark red or nucleated without sheen
ƒ Colonies that lack sheen may be pink,
red, white or colorless on Endo-type
medium
ƒ Typical sheen colonies from noncoliforms
ƒ Atypical colonies occasionally may be coliforms.
ƒ Verify both typical and atypical
ƒ Simultaneous inoculation of
ƒ Lactose fermentation (lauryl tryptose broth)
ƒ Brilliant green lactose broth
ƒ Rapid test
ƒ Reaction with cytochrom oxidase and B-galactosidase
within 4 hours
ƒ Commercial multi-test systems
Fecal Coliform Membrane Filter
Fecal Coliform Membrane Filter
ƒ Chlorinated effluents should first demonstrate
comparable results obtained in Multiple-tube
ƒ M-FC medium Agar
ƒ Determine ideal sample volume
ƒ Use low magnifications to count colonies
ƒ Two types of fecal colonies
ƒ Yields 20- 60 coliforms
ƒ Not more than 200 colonies of all types
ƒ
ƒ
ƒ
ƒ
Filter ideal volumes
Place prepared filter on pad
Invert dish
Incubate for 24 +/- 2 hours at 44 +/- 0.2C
ƒ Incubation temperature is critical
ƒ Seal in waterproof plastic bags and submerge
ƒ Typical fecal colonies
ƒ Various shades of blue
ƒ Non fecal colonies
ƒ Gray to cream-colored
ƒ Elevating the temperature to 45.0 +/- 0.2C
eliminates environmental Klebsiella strains
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E. coli Coliform Membrane Filter
E. coli Coliform Membrane Filter
ƒ M-TEC Agar and Modified m-TEC
ƒ Determine ideal sample volume
ƒ M-TEC Agar
ƒ Yields 20-80 coliforms
ƒ Not more than 200 colonies of all types
ƒ
ƒ
ƒ
ƒ
Filter ideal volumes
Place prepared filter on pad
Invert dish
Incubate for 22 +/-2 hours at 44.5 +/- 0.5C
ƒ After incubation remove filter and place on pad
ƒ Saturate pad with 2 mL Urease
ƒ Positive yellow to yellow-brown colonies after 15
minutes
ƒ Modified m-TEC agar
ƒ Red – magenta-colored colonies
Chromogenic Substrate Technology
Chromogenic Substrate Technology
ƒ Nutrient indicator (substrate) is targeted to
organism-specific enzyme
ƒ Nutrient indicator is the major source of
carbon
ƒ Nutrient indicator changes color or
fluoresces when metabolized
ƒ Non-targets are both starved and
suppressed
ƒ Advantages
Chromogenic Substrate Technology
Chromogenic Substrate Technology
ƒ Advantages (cont.)
ƒ Disadvantages
ƒ Recovers stressed
coliforms
ƒ Detects a single
viable organism
(1CFU/100 ml)
ƒ Minimal equipment
needed
ƒ No media
preparation
ƒ P/A or MPN formats
ƒ Cost effective (large
sample numbers)
ƒ Generally more sensitive than other
methods
ƒ Easy to perform, use, and train
ƒ No confirmation step required
ƒ Simultaneous detection of E. coli
ƒ Rapid
ƒ Minimal interferences: non-coliforms
suppressed
ƒ Too sensitive?
ƒ Media costlier than other coliform methods
ƒ Limited suppliers
ƒ Colilert®
ƒ ColitagTM
ƒ Readycult®
ƒ E*Colite®
ƒ M-ColiBlue24®
ƒ Water with color makes reading more difficult.
ƒ Colisure ® eliminates some of those problems
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Chromogenic Substrate Technology
Chromogenic Substrate Technology
ƒ Select appropriate procedure
ƒ Coliforms grow in substrate technology
they use β-galactosidase to metabolize
ONPG and change it from colorless to
yellow
ƒ Presence-absence
ƒ Most Probable Number (MPN)
ƒ Determine ideal sample volume
ƒ Add appropriate medium
ƒ Incubate at 35 +/- 0.5C
Chromogenic Substrate Technology
Chromogenic Substrate Technology
ƒ Negative total coliform
ƒ Positive total coliform
ƒ Colilert®-18 hr
ƒ Colilert®
ƒ Colisure® (Magenta)
ƒ Positive total coliform
ƒ Colilert®
ƒ Colilert®-18 hr
Chromogenic Substrate Technology
Chromogenic Substrate Technology
ƒ E. coli use β-glucuronidase to metabolize MUG
and create fluorescence
ƒ Positive E. coli
ƒ Fluorescence
9
Reasons Why the MTF, MF and
Substrate Technology Results Don’t
Always Agree
Reasons Why the MTF, MF and
Substrate Technology Results Don’t
Always Agree
ƒ Problems associated with sampling:
ƒ Method Definition of “coliform group”
ƒ Samples not collected simultaneously
(i.e. not true splits)
ƒ Uneven distribution of bacteria
ƒ Statistical variability
ƒ Low numbers of organisms
ƒ Contamination at collection
ƒ MF total coliform is based on the ability to
produce acid from lactose fermentation
ƒ Substrate technology total coliform has
the enzyme B-D galactosidase
ƒ Therefore, the substrate technology coliform
group is inherently broader
Reasons Why the MTF, MF and
Substrate Technology Results Don’t
Always Agree
Reasons Why the MTF, MF and
Substrate Technology Results Don’t
Always Agree
ƒ Performance of the tests:
ƒ No test method utilizing biochemical
reactions of organisms is perfect:
ƒ Were both sets of samples transported
under the same conditions to the labs?
ƒ Were the analyses performed at
approximately the same time?
ƒ Were both methods performed correctly?
ƒ Individual organisms and groups of organisms
are always changing through mutations,
exchanges of genetic material, etc.
ƒ Strains of species don’t always exhibit typical
reactions.
ƒ Environmental stressors may affect the
organisms metabolic activity.
Reasons Why the MTF, MF and
Substrate Technology Results Don’t
Always Agree
Summary
ƒ Difference in test sensitivity
ƒ Testing water for all possible pathogens is:
ƒ Generally accepted that substrate
technology is more sensitive test than
the MF method.
ƒ Complex
ƒ Time consuming
ƒ Expensive
ƒ Coliform Bacteria are the commonly-used
bacterial indicator for water quality.
ƒ Presence in water indicates that diseasecausing (pathogens) organisms could be in
the water system.
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Summary (cont.)
Coliform Microbiology Methods
ƒ Total Coliform Methods are
“Equivalent” But Not The Same
ƒ Substrate technology coliform group is
inherently broader
ƒ Generally accepted that substrate
technology is more sensitive test than
the MF method
Heterotrophic Plate Count
Heterotrophic Plate Count
ƒ Estimates live heterotrophic bacteria
ƒ Compare data using same procedure
ƒ Pour Plate Method
and medium
ƒ Information about water quality
ƒ Significance of coliform test
ƒ HPC useful in judging efficiency of
treatment processes
ƒ Spread Plate Method
Heterotrophic Plate Count
ƒ Membrane Filter Method
ƒ SimPlate
Heterotrophic Plate Count
ƒ Pour Plate
ƒ Agar at 44 - 46°C
ƒ Sample volume 0.1 – 2.0 mL
ƒ Serial dilutions using 0.1 % peptone
ƒ Surrounded by liquid agar for short
water
ƒ 30 - 300 colonies
ƒ Approximately 15 mL of agar
medium
period, organisms intolerant to
elevated temperature may be killed
ƒ Obligate aerobes may not be able to
develop
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Heterotrophic Plate Count
ƒ Invert solidified plates and incubate
ƒ NWRA agar (HPCA)
ƒ 35°C ± 0.5°C for 48 - 72 ± 2 hours
ƒ R2A Agar
Heterotrophic Plate Count
ƒ Count all colonies
ƒ Report as “colony-forming units”
ƒ 360 CFU/mL, pour plate method,
350C/48 hr,R2A agar
ƒ Used for compromised organisms
ƒ 35°C ± 0.5°C for 48 - 72 ± 2 hours
Heterotrophic Plate Count
SimPlate
Heterotrophic Methods
ƒ Substrate Technology
ƒ 10 ml sample / medium
ƒ Results reported MPN/mL
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