Bacteria
Bacteria
DOC316.53.01189
Prepared Agar Plates
Membrane Filtration
Scope and application: For low-turbidity water and wastewater.
Test preparation
Before starting
This method uses prepared agar plates for the analysis of different types of bacteria. Refer to Prepared agar plates
on page 2 to select a prepared agar plate for the test procedure.
Set the incubator to the specified temperature. Refer to Table 1 on page 3. Let the incubator temperature become stable,
then add the samples.
Wash hands thoroughly with soap and water.
Use a germicidal cloth, bactericidal spray, weak bleach solution or weak iodine solution to clean the work area.
Make sure that all of the materials that come in contact with samples are sterile.
During filtration, remove the vacuum as soon as the funnel is empty so that the membrane filter does not become dry.
As an alternative to the filter assembly with flask, use a sterile, disposable filter unit.
Items to collect
Description
Quantity
Prepared agar plate (refer to Consumables and replacement items on page 6)
1
Sterile buffered dilution water
1
Membrane filter, 0.45 micron
1
Filtration apparatus with aspirator or pump
1
Forceps, sterilized
1
Incubator
1
Microscope, low-power
1
Pipet(s) for dilution or for sample volumes less than 100 mL, if necessary
1
Refer to Consumables and replacement items on page 6 for order information.
1
Sample collection
•
•
•
•
•
•
•
Use a sterile glass or plastic container such as a Whirl-Pak bag that contains
sterilized sodium thiosulfate. The sodium thiosulfate is not necessary if the sample
does not contain a residual disinfectant.
Open the sample containers immediately before collection and close immediately
after collection. Do not put the lid or cap down. Do not touch the lip or inner surfaces
of the container. Do not rinse the containers before use.
To collect a potable water sample from a faucet, spigot, hydrant or pump, let the
water flow at a moderate rate for 2–3 minutes. Remove the screens or aerators. Do
not use faucets or spigots that have a bad seal or that show a leak between
components.
To collect a non-potable sample from a river, lake or reservoir, hold the container
below the water surface, then remove the cap. As an alternative, remove the cap and
push the container, mouth down, below the water surface to prevent the collection of
surface scum. Put the mouth of the container into the current. Fully fill the container
below the water surface.
Collect a minimum of 100 mL of sample. Keep a minimum of 2.5 cm (1 inch) of air
space in the container.
Write the sample information on the container and start the analysis as soon as
possible.
If immediate analysis is not possible, keep the sample at or below 10 °C (50 °F) for a
maximum of 8 hours. Do not let the sample freeze.
Sample volumes
Use a sample volume that is applicable to the sample type. For samples with a low level
of bacteria such as finished, potable water, use 100 mL of sample. Use less sample for
non-potable water or water that contains more bacteria.
When the approximate bacteria level is unknown, analyze three different sample
volumes. Use the results from the sample volume that shows approximately 20 to
200 colonies for each membrane filter.
When the sample volume is less than 20 mL (diluted or undiluted), add 10 mL of sterile
buffered dilution water to the filter funnel before the vacuum is applied. The additional
dilution water helps to apply the bacteria equally across the membrane filter.
Sample dilution
Dilute samples that contain a high level of bacteria so that approximately 20 to
200 bacteria colonies grow on the membrane filter. Use the steps that follow to make
serial dilutions of the sample.
1.
2.
3.
4.
5.
Wash hands thoroughly with soap and water.
Invert the sample container for 30 seconds (approximately 25 times).
Open a bottle of sterile buffered dilution water.
Use a sterile pipet to add 11 mL of sample into the dilution water bottle.
Put the cap on the dilution water bottle and invert for 30 seconds (25 times). This is a
10x dilution (sample is diluted by a factor of 10).
6. Add 11 mL of the 10-fold dilution to another dilution bottle (100x dilution). Mix well.
7. Add 11 mL of the 100-fold dilution to the third bottle (1000x dilution). Mix well.
8. If necessary, continue to dilute the sample.
Prepared agar plates
Table 1 shows the available prepared agar plates for the analysis of different types of
bacteria. The prepared agar plates include a certificate of analysis and expiration date.
Most of the plates have a shelf life of 1 year when stored at 2–8 °C (35–46 °F). The m-EI
2
Bacteria, MF, prepared agar plates
agar plates have a shelf life of 3 months. The sensitivity of all of the prepared agar plates
is 1 CFU/100 mL.
Table 1 Prepared agar plates
Bacteria
Prepared agar plate
Incubation time
Incubation temperature
Heterotrophic bacteria
m-HPC
24 hours
35 (± 0.5) °C
95 (± 0.9) °F
Total coliform bacteria
m-Endo1
24 hours
35 (± 0.5) °C
95 (± 0.9) °F
Fecal coliform bacteria
m-FC1
24 hours
44.5 (± 0.2) °C
112.1 (± 0.7) °F
Total coliform and E. coli
bacteria
m-ColiBlue24
24 hours
35 (± 0.5) °C
95 (± 0.9) °F
Enterococci bacteria
M-EI
24 hours
44.5 (± 0.5) °C
112.1 (± 0.9) °F
E. coli bacteria with
confirmation
Nutrient agar with
MUG
24 hours
35 (± 0.5) °C
95 (± 0.9) °F
E. coli bacteria
Modified m-TEC
2 hours at 35 (± 0.5) °C (95 (± 0.9) °F), then 22 hours at 44.5 °C
(112.1 °F)
Membrane filtration test procedure
1. Set up the membrane
filtration apparatus. Use a
sterile forceps to put a
membrane filter in the
assembly. Make sure that
the grid side is up.
2. Invert the sample or the
diluted sample for
30 seconds (25 times) to
make sure that the sample
is mixed well.
3. Pour or use a pipet to
add the sample into the
funnel. If the volume is less
than 20 mL, add 10 mL of
sterile buffered dilution
water to the funnel.
4. Apply the vacuum until
the funnel is empty. Stop the
vacuum.
5. Rinse the funnel with 20
to 30‑mL of sterile buffered
dilution water. Apply the
vacuum. Rinse the funnel
two more times.
6. Stop the vacuum when
the funnel is empty. Remove
the funnel from the filter
assembly. Use sterile
forceps to lift the membrane
filter.
7. Put the membrane filter
on the prepared agar plate.
Let the membrane filter
bend and fall equally across
the agar to make sure that
air bubbles are not caught
below the filter.
8. Put the lid on the petri
dish and invert the petri
dish.
1
Additional media is necessary for confirmation.
Bacteria, MF, prepared agar plates
3
9. Put the inverted petri
dish in a plastic bag and
seal the bag.
10. Incubate the inverted
petri dish at the specified
temperature for the
specified time. Refer to
Table 1 on page 3.
11. Remove the petri dish
from the incubator. Use a 10
to 15x microscope to count
the number of bacteria
colonies on the membrane
filter.
Interpret and report the coliform results
Report the coliform density as the number of colonies in 100 mL of sample. For total
coliforms, use a sample volume that gives 20–80 coliform colonies on the membrane
filter. For fecal coliforms, use a sample volume that gives 20–60 fecal coliform colonies
on the membrane filter.
If there are more than 200 colonies, dilute the sample and use the diluted sample in the
test procedure. Use the sample volume before dilution in the coliform density
determination.
1. Use the microscope to look at the colonies on the membrane filter. Count the number
of isolated coliform colonies.
2. Determine the coliform density as follows:
Membrane filter(s)
Coliform density determination
One membrane
filter
Coliform colonies in 100 mL = Coliform colonies counted ÷ mL sample
× 100
Example: 50 coliform colonies were counted. The sample volume was
20 mL. The coliform density is 50 ÷ 20 mL × 100 = 250 coliforms in
100 mL of sample.
Multiple filters,
dilutions or
duplicates for
each sample
Average coliform colonies in 100 mL = Sum of coliform colonies in all
samples ÷ sum of mL sample × 100
Example: Two 50-mL samples gave 5 colonies on one filter and
9 colonies on another filter. The coliform density is (5 + 9) ÷ (50 + 50) ×
100 = 14 coliforms in 100 mL of sample.
3. If colonies are not isolated or if there are more than 200 colonies of all types:
a. Report the results as “Confluent growth with or without coliforms” when the
bacteria grows together across some or all of the membrane filter.
b. Do the test procedure again with half the sample volume. If the total number of
colonies (coliforms plus non-coliforms) is more than 200 for each membrane or
the colonies are not isolated, report the results as “Too numerous to count”
(TNTC).
c. Do the test procedure again with a dilution that gives approximately 50 coliform
colonies and not more than 200 colonies of all types.
4
Bacteria, MF, prepared agar plates
Interpret and report the HPC results
Use the steps that follow to determine the colony-forming units for each mL of sample
(CFU/mL). A colony density of 20 to 200 colonies on the membrane filter is recommended
for best results. If there are more than 200 colonies, dilute the sample and use the diluted
sample in the test procedure. Use the sample volume before dilution in the CFU/mL
determination.
1. Use the microscope to look at the colonies on the membrane filter. Estimate the
number of colonies in each square of the membrane filter.
Note: Make estimated counts only when there are isolated colonies without spreaders.
2. Determine the CFU/mL of sample for the estimated colony count as follows:
Estimated count
CFU/mL determination
1 to 2 colonies in
each square
1.
2.
Count all of the colonies on the filter.
Divide the total number of colonies by the sample volume.
Example: 122 total colonies were counted. The sample volume was
100 mL. 122 colonies/100 mL = 1.2 CFU/mL
3 to 10 colonies in 1.
each square
2.
3.
4.
Count the colonies in 10 representative squares.
Divide by 10 to get the average number of colonies in each square.
Multiply the average number in each square by 100.
Divide by the sample volume.
Example: The average number of colonies was 8 colonies in each
square. The sample volume was 10 mL. 8 colonies x 100/10 mL =
80 CFU/mL
10 to 20 colonies
in each square
1.
2.
3.
4.
Count the colonies in 5 representative squares
Divide by 5 to get the average number of colonies in each square.
Multiply the average number in each square by 100.
Divide by the sample volume.
Example: The average number of colonies was 17 colonies in each
square. The sample volume was 10 mL. 17 colonies x 100/10 mL =
170 CFU/mL
More than
20 colonies in
each square
1.
2.
Divide 2000 by the volume of the original (undiluted) sample.
Report results as more than the result of step 1.
Example: The original sample volume was 0.1 mL. 2000/0.1 mL =
> 20,000 CFU/mL
3. Report the test results as colony forming units for each mL (CFU/mL). Report
averaged counts as estimated CFU/mL. Include in the report the method used, the
incubation temperature, time and the nutritional medium. Example: 98 CFU/mL,
membrane filter method, 35 °C, 24 hours, m-TGE Broth.
Summary of method
The membrane filtration procedure is used for samples that are low in turbidity and have
low bacteria counts. The sample is poured through a membrane filter. The bacteria in the
sample stays on the membrane filter. The membrane filter is moved to a petri dish that
contains a nutritional broth or agar. During incubation, the bacteria grow and form
colonies on the membrane filter. After incubation, the filter is examined with a microscope
for bacteria colonies.
Bacteria, MF, prepared agar plates
5
Consumables and replacement items
Required reagents
Description
Quantity/test
Unit
Item no.
m-ColiBlue24® prepared agar plates
1
15/pkg
2805215
m-EI prepared agar plates
1
15/pkg
2811715
m-Endo prepared agar plates
1
15/pkg
2811615
m-FC prepared agar plates
1
15/pkg
2811515
m-HPC prepared agar plates
1
15/pkg
2811415
m-TEC, modified, prepared agar plates
1
15/pkg
2811815
Nutrient agar with MUG prepared agar plates
1
15/pkg
2812115
Dilution water, buffered, 99 mL, sterile2
1
25/pkg
1430598
Description
Unit
Item no.
Membrane filter holder, magnetic, 300-mL funnel
each
1352900
Filter pump, aspirator
each
213100
Flask, filtering, glass, 1000 mL
each
54653
Forceps, stainless steel
each
2141100
Membrane filter, 0.45 micron, 47 mm diameter, sterile
200/pkg
1353001
Membrane filter, 0.45 micron, 47 mm diameter, sterile EO (ethylene oxide)
150/pkg
2936100
each
2947050
25/pkg
209798
each
1465100
each
1970010
Pipet tips, TenSette, 1.0–10.0 mL, sterile, individually wrapped
50/pkg
2558996
Stopper, rubber, size 8, for filtration assembly
6/pkg
211908
3.66 m (12 ft)
56019
Description
Unit
Item no.
Laboratory incubator, culture, 110 VAC
each
2619200
Laboratory incubator, culture, 230 VAC
each
2619202
Portable incubator with 12 VDC power socket
each
2569900
AC power supply for portable incubator, 110–240 VAC
each
2968100
Battery pack, rechargeable, for portable incubator 12 VDC
each
2580300
Portable incubator rack, general purpose/petri dish
each
2580502
Required apparatus
Microscope, compound
Pipet, serological, 10–11 mL, sterile, disposable
Pipet filler, safety bulb
®
Pipet, TenSette , 1.0–10.0 mL
Tubing, rubber, 7.9 mm (5/16-in.) inside diameter
Incubators
2
6
Buffered dilution water is prepared with magnesium chloride and potassium dihydrogen phosphate.
Bacteria, MF, prepared agar plates
Sample collection
Description
Unit
Item no.
Sampling bags, Whirl-Pak® with dechlorinating reagent, 177 mL
100/pkg
2075333
Sampling bags, Whirl-Pak without dechlorinating reagent, 207 mL
100/pkg
2233199
Sampling bottles, sterilized, with dechlorinating agent, 100-mL sample
100/pkg
8888006
Sampling bottles, sterilized, without dechlorinating reagent, 100-mL sample
12/pkg
2495012
Sampling bottles, sterilized, without dechlorinating reagent, 100-mL sample
50/pkg
2495050
each
2568700
Unit
Item no.
Disposable filter funnels with membrane filters, sterile
150/pkg
2586300
Pipet, serological, 10–11 mL, sterile, disposable
25/pkg
209798
Pipet, serological, 2 mL, sterile, glass
35/pkg
2093136
Pipet filler, safety bulb
each
1465100
Support base for disposable filter funnels
each
2586201
Vacuum pump, hand-operated
each
1428300
Sample transport kit, includes 100 sample bags with dechlorinating agent, refrigerant
pack, rack and 9-L cooler
Optional reagents and apparatus
Description
Bacteria, MF, prepared agar plates
7
FOR TECHNICAL ASSISTANCE, PRICE INFORMATION AND ORDERING:
In the U.S.A. – Call toll-free 800-227-4224
Outside the U.S.A. – Contact the HACH office or distributor serving you.
On the Worldwide Web – www.hach.com; E-mail – [email protected]
©
Hach Company/Hach Lange GmbH, 2007–2017. All rights reserved.
HACH COMPANY
WORLD HEADQUARTERS
Telephone: (970) 669-3050
FAX: (970) 669-2932
04/2017, Edition 9
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