Microbiological Testing
of Foods, Beverages
and Pharmaceuticals
Introduction
The Membrane Filter Method
The consumer’s steadily growing requirements
for the quality and the longer shelf life of
foods and beverages must be met by the manufacturer. He cannot limit quality assurance to
inspection of the final product alone, such as
a bottled beverage or a prepared food product.
Instead, he continuously must inspect incoming raw materials and perform in-process
quality control tests throughout production
if he wants to avoid later losses and customer
complaints. Microbiological and aseptic
testing play a significant role in such quality
assurance.
Description
The Membrane Filter Method
A membrane filter of the appropriate pore
size is placed in a filter holder, and the sample
is filtered. In this process microorganisms
in the test sample are retained on the filter
surface by the screening action of the
membrane filter.
In the soft drink industry the microbiological
and hygienic quality including the biological
stability of the products are important criteria
for their assessment. The reason: just a few
microbes are often all it takes to spoil large
quantities of a beverage.
Although the explosive technological development has reduced the risk of contamination
by spoilage microbes, the issue of shelf life has
taken on new dimensions as a result of the
enormous production output now possible.
Quality control of bottling and filling, in terms
of chemical and, above all, biological stability,
must be adapted to this development by
state-of-the-art test methods.
The requirements for a practical microbiological test method are that it permit quantitative and reproducible detection of trace
contamination and that it can be performed
efficiently and economically under routine
conditions. These requirements are fullfilled
optimally by the membrane filter method.
The principle of this method is based on
the concentration of microorganisms from
relatively large samples on the surface of
the membrane filter, and on culturing these
microbes on a nutrient pad or an agar
culture medium.
For the Monitor MF-Methode the monitor is
ready to use due to a pre-asembled membrane
and pad inside.
Growth inhibitors can be removed by flushing
the holder with sterile water after filtration.
Afterwards, the membrane filter is placed on
a culture medium and incubated.
For the monitor method add the nutrient
media from the top and do a short vacuum
(< 1 sec.)
Nutrients and metabolites are exchanged
through the pore system of the membrane
filter. Colonies, which have developed on the
membrane filter surface during incubation,
are counted and related to the sample volume.
The advantages:
• Proofen accuracy
Compared with the direct method,
considerably larger sample volumes can be
tested. This concentration effect increases
the accuracy of microbial detection.
• Quantitative results
The visible colonies can be related directly
to the sample volume.
• Documentation
The membrane filter with colony growth
can be filed as a permanent record of
the test.
No inhibitors
Inhibitors, such as essential oils or disinfectants,
can be flushed from the membrane filter
after filtration.
GMP quality
Sartorius Membrane Filters are manufactured
under GMP conditions, ensuring consistent
quality and high reproducibility from batch to
batch and within each batch.
The Culture Media
Microorganisms can be detected by different
methods.
Methods involving culturing techniques and
the microscope are used to detect microbes,
whereas biochemical and serological techniques are commonly applied to differentiate
among such organisms.
For detecting microorganisms in cultures,
liquid and solid culture media are employed.
Microorganisms are concentrated by growth
in or on these culture media.
Quantitative detection is only possible with
solid culture media because the individually
developing colonies can be evaluated and
counted on the surface.
The following culture media can be used for
microbiological testing:
• Nutrient Pad Sets
Nutrient Pad Sets definitely optimize
the membrane filter method.
They standardize microbiological test
procedures, making them much
more efficient.
The simplify laboratory work. They
help to save time and money.
These sets are described on the following
pages and certainly offer the most convenient
way to use the membrane filter method.
• Absorbent pads to be wetted with culture
media
• Culture media with agar or gelatin as
the solidifying agent
Methods of CFU Determination
Methods of CFU Determination
Direct Method
Membrane Filter Method
The test sample is pipetted into
a petri dish …
The test sample is filtered through
a membrane filter
Standard MF method
The membrane filter is rinsed
and then placed on a culture
medium – a, b, or c –
and incubated.
Monitor MF method
… then mixed with the culture medium
and incubated
The nutrient media is given from
the top after filtration. Do a short
vacuum (<1 sec.). Close the monitor
with the plug at the bottom.
Remove the monitor and fit funnel
and base to a petri dish.
a) on a nutrient
pad wetted
with sterile
water
b) on an
c) on an agar
absorbent pad plate
wetted with
liquid culture
medium
Nutrient Pad Sets
User Benefits
Sartorius Nutrient Pad Sets have been used
successfully in the membrane filter method for
20 years. Practical and easy to handle, they
reduce labor and simplify many microbiological testing procedures.
Economical
Nutrient pads are sterile, dehydrated culture
media. Once they are moistened with 3.0–3.5 ml
of sterile and demineralized (or distilled) water
they are ready to use immediately.
The level of moisture is optimal when an
excess ring of water surrounding the pad
is visible.
All Nutrient Pad Set types are supplied with
the appropriate membrane filters, which are
also presterilized and individually packaged.
The membrane filters tailored to meet the
special requirements of microbial detection
are available with 47 mm or 50 mm diameters.
A standard package contains 100 sterile
nutrient pads each “preplated” in a petri dish
(each bag contains 10 petri dishes) and
100 individually sterile packed membrane
filters.
Eliminates time-consuming and laborintensive preparation of culture media
(sterilization and cleaning, among others).
• After wetting with 3,5 ml destilled water
NPS are ready to use: NPS and go
Simple to use
Nutrient Pad Sets can also be used in
laboratories which do not have extensive
microbiological equipment. Sterile water
for moistening the pads can be prepared
easily with a Sartorius Dosing Syringe and
an attached Syringe Filter Holder (0,2 µm).
• Everyone can use NPS
Consistent quality
During manufacture, each type of Nutrient
Pad Set is compared with the corresponding
agar medium with respect to their growthpromoting properties. This QA procedure
ensures consistent quality and reproducible
results.
• NPS are validated. In comparison of agar
which is done within different deviations
of amount and height NPS gives always
constant results
Trouble-free storage
Nutrient Pad Sets have a shelf life of
9 to at least 24 months at room temperature.
• No waste or overproduction
Highly versatile
Nutrient Pad Sets can be modified by additives
in the solution used to wet them; for example,
Wort or Orange Serum Nutrient Pads when
wetted with 5 % ethanol promote the growth
of acetic-acid bacteria.
• Advanced system
How to Handle Microorganisms
Microorganism cultures must always be
handled as carefully as if they contained
pathogens.
Before and after use, inoculating loops and
wires must be sterilized by flaming until they
glow red-hot.
General Directions
General Procedure.
To obtain reliable results for microbiological
tests, it is necessary to work under conditions
that rule out contamination by microorganisms which distort such results.
That is why you should work near the flame
of a Bunsen burner in a room protected from
drafts. Before beginning with the actual
procedure, spray or wash down your work area
with a disinfectant (e.g., 70% alcohol).
Before use, filter holders, forceps and scissors
should be sterilized by one of the standard
methods, such as flaming for routine tests.
Working with microorganisms is not
dangerous if the following safety rules
are observed:
Wash your hands thoroughly before and
after working in a laboratory.
Do not eat or drink in a laboratory.
Do not touch bacterial matter with your
hands.
Never pipet bacteria suspensions with
your mouth. Always use mechanical aids
for pipetting (e.g., Peleus ball).
All laboratory equipment which has come in
contact with bacteria must be sterilized.
To protect people and animals from contagious diseases or poisoning, living cultures
have to be destroyed before cleansing or
disposing of the containers. One method is
to coat them thoroughly with disinfectants
or to autoclave them in suitable containers.
How to Use Nutrient Pad Sets
You can see for yourself how easy it is to use Nutrient Pad Sets: NPS and go
Desinfect the working area
Cut open the packaging and remove the number
of nutrient pads needed
Wet the nutrient pads with 3,5 ml sterile and distilled
or demineralized water
Flame the stainless steel funnel
Flame the frit
Flame the lid and inside the funnel
Flame the forceps, shortly cool down
Take off the membrane
Place the filter on the frit of the filter holder,
discard the yellow paper (not shown here)
Filter the sample. Then rinse the inside of the filter holder
with sterile water or physiological saline solution
Place the filter on the pad without entrapping air bubbles
Incubate the nutrient pad in the petri dish with the lid
right side up
Typical Application Examples
Product
Detection|determination of
Nutrient pad type
Beer
Pediococci and lactobacilli
VLB-S7-S
Wild yeasts
Lysine
Yeasts and molds
Wort, Malt extract
Foods
Milk
Pharmaceuticals
and cosmetics
Soft drinks
Sugar
Water
Wine
Colony count
Standard TTC, Standard, Caso
Enterobacteria
Endo, Teepol, M-FC, Tergitol TTC, ECD,
MacConkey, Chromocult
Mesophilic bacteria and
thermophilic spore formers
Glucose-Tryptone
Pseudomonas aeruginosa
Cetrimide
Salmonellae
Bismuth-Sulfite
Staphylococci
Chapman
Streptococci
Azide
Yeasts and molds
Wort, Malt extract
E. coli and coliforms
Endo
Salmonellae
Bismuth-Sulfite
Streptococci
Azide
Candida albicans
Sabouraud
Colony count
Caso, R2A
Fecal streptococci
Azide
Enterobacteria
MacConkey
Pseudomonas aeruginosa
Cetrimide
Staphylococcus aureus
Chapman
Acid-tolerant microbes
Orange Serum
Colony count
Standard, Standart TTC
Lactic-acid bacteria
VLB-S7-S, Orange Serum
Slime-forming bacteria
(Leuconostoc)
Weman
Yeasts and molds
Wort, Schaufus-Pottinger, Malt extract
Mesophilic bacteria and
thermophilic spore formers
Glucose-Tryptone
Slime-forming bacteria
(Leucostonoc)
Weman
Yeasts and molds
Wort, Schaufus-Pottinger, Malt extract
Colony count
Standard TTC, Standard, R2A, Yeast extract
E. coli and coliforms
Endo, Tergitol TTC, Teepol, M-FC, ECD
Fecal streptococci
Azide
Pseudomonas aeruginosa
Cetrimide
Acetobacter
Wort, Orange serum, both wetted with 3%–5% ethanol
Lactic-acid bacteria
Orange Serum
Lactic-acid bacteria
(especially Leuconostoc oenos)
Tomato Juice “Jus de Tomate"
Yeasts and molds
Wort, Schaufus-Pottinger, Malt extract
Description and Growth Evaluation Examples
Standard TTC-NPS
Type 14055
Standard-NPS
Type 14064
Caso-NPS
Type 14063
Meat extract-peptone medium (dehydrated
nutrient broth) for determining the colony
count (total CFU count); formulated acc.
to “Standard Methods for the Examination of
Water and Wastewater”, 1998, and modified
by the addition of TTC.
Meat extract-peptone medium (dehydrated
nutrient broth) for determining the colony
count (total CFU count); formulated acc. to
the “Standard Methods for the Examination
of Water and Wastewater,” 1998.
Soybean-Casein Digest Medium for isolating
microorganisms and for determining the total
CFU count acc. to the “USP 25, the german
drinking water law and ISO 9308-1.”
Type of membrane filter supplied
0.45 µm; green with green grid.
Type of membrane filter supplied
0.45 µm; green with green grid.
Type of membrane filter supplied
0.45 µm; green with green grid
Incubation conditions
48 hours at 30 °C. The incubation time and
temperature may be varied acc. to the type of
sample in compliance with the regulations.
Incubation conditions
48 hours at 30 °C. The incubation time and
temperature may be varied acc. to the type of
sample in compliance with the regulations.
Incubation conditions
24–72 hours at 30°C–37°C,
depending on the test sample
Evaluation
Evaluation
Bacteria predominantly grow on this medium. Bacteria predominantly grow on this
Their colonies are stained red by TTC reduction. medium. The morphology and color of their
colonies vary.
Evaluation
Depending on the microbes to be detected,
this medium can be converted into a selective
one by mixing the wetting liquid with
additives before moistening the pad. When
10% serum is added to the wetting liquid
a number of fastidious pathogenic bacteria
like the genuses Pneumococcus, Neisseria,
Streptococcus, Corynebacterium, Erysipelothrix
and Brucella are able to grow on the pad.
Total Count
Bacillus subtilis
Escherichia coli
Staphylococcus aureus
Mixed culture from well water
Mixed culture from drinking water
Mixed culture from process water
Description and Growth Evaluation Examples
Yeast extract-NPS
Type 14090
R2A-NPS
Type 14084
Chromocult-NPS
Type 14087
For the determination of CFU count in water
for the human use, water in container or
bottles for the human use and mineral water,
acc. to the German drinking water law and
ISO 6222.
For the detection of total colony count of
heterotrophic microorganisms and for the
subcultivation of bacteria in drinking water.
Acc. to the Standard Methods for the
Examination of Water and Waste Water and
EP 1998. The optimal growth for bacteria
which have adapted to the particular living
conditions of water low in nutrients.
For the determination of total coliforms
and E.Coli in water and food.
Type of membrane Filter supplied
0.45 µm; green with green grid
Type of membrane Filter supplied
0.45 µm; green with green grid
Type of membrane Filter supplied
0.45 µm; white with black grid
Incubation conclitions
44 ±4 h at 36 ±2°C; 68 ±4 h at 22 ±2°C
acc. to EN ISO 6222.
Incubation conditions
72 h at 35°C; 5 days at 20°C or 28°C
Incubation conclitions
24 h at 36 ±1°C; longer incubation time
disturbs the counting of the colonies.
Evaluation
Bacteria, yeasts and molds can grow.
Due to no dye most colonies, predominantly
bacteria, grow colorless.
Evaluation
Predominantly colonies of different size
and color, predominantly white or colorless
<1 mm diameter.
Evaluation
E. Coli develops dark-blue to violett
colonies. Coliforme red to salmone colonies.
Other gramnegative colonies are colorless,
few with ß-Glucuronidase activity are light
blue to turkese.
To confirm E. Coli give Kovacs reagent
to dark blue colonies. Then E. Coli stains
cherry-red.
Total Count
E. coli and coliforms
Escherichia coli
Escherichia coli
Escherichia coli
Mixed culture from river water
Mixed culture from water
Mixed culture from water
Description and Growth Evaluation Examples
Endo-NPS
Type 14053
M-FC-NPS
Type 14068
Teepol-NPS
Type 14067
Selective medium for detecting E. coli and
coliform bacteria formulated according
to the “Standard Methods for the Examination of Water and Wastewater,” 1998.
For the detection of E. coli and fecal
coliform bacteria according to Geldreich
et al, recommended by the “Standard
Methods for the Examination of Water
and Wastewater,” 1998.
For the detection of E. coli and fecal coliform
bacteria according to Burman, N.P. (1967).
Type of membrane filter supplied
0.45 µm; white with green grid
Type of membrane Filter supplied
0.45 µm; white with green grid
Type of membrane filter supplied
0.45 µm; white with green grid
Incubation conclitions
24 hours at 37°C
Incubation conclitions
20 ±4 hours at 37°C in an incubator or
at 44,4°C in a water bath.
Incubation conditions
18–24 hours at 37°C
Evaluation
E. coli and coliforms bacteria develop sharply
contoured, as dark-red colonies. E. Coli has
a greenesh metallic sheen (Fuchsin sheen)
with a dark-red point on the underside
of the membrane.
Evaluation
E. coli and coliforms develop blue colonies
with diameters of 1–2 mm; colonies of
a different color are not evaluated.
Evaluation
E. coli and coliform bacteria form 1–2 mm
diameter yellow colonies surrounded
by a yellow zone. Non lactose-fermenting
bacteria develop red colonies of various
sizes.
Escherichia coli
Escherichia coli
Escherichia coli
E. coli and coliforms from river water
E. coli and coliforms from waste water
E. coli and coliforms from waste water
E. coli and coliforms
Description and Growth Evaluation Examples
ECD-NPS
Type 14082
MacConkey-NPS
Type 14097
Tergitol TTC-NPS
Type 14056
Selective culture medium for detecting
and identifying Escherichia coli, acc.
to ISO 9308-1.
For isolation and differentiation of enterobacteria. Complies with the specifications
of the DAB 10 (German Pharmacopoeia),
EP II and USP 25 and § 35 LMBG (German
Food and Drug Law).
For the detection of coliform bacteria
and E. coli according to Pollard; modified
acc. to Chapman, acc. to ISO 9308-1.
Type of membrane filter supplied
0,45 µm; white with green grid
Type of membrane filter supplied
0.45 µm; white with green grid
Type of membrane filter supplied
0.45 µm; white with green grid
Incubation conditions
18–24 hours at 37°C
Incubation conditions
18–24 at 37°C
Incubation conditions
20 ±4 hours at 37°C
Evaluation
Bile salt inhibits the accompanying flora
of microbes not living in the intestine.
Colonies with light blue fluorescence in
UV light indicate E. coli, confirm by
subsequent staining with Kovacs reagent.
Evaluation
Escherichia coli forms large red or reddish
colonies coliform microbes form large pinkcolored, sometimes slimy colonies lactosenegative enterobacteria form colorless
colonies. Gram-positive microbes are usually
inhibited or their growth is very small.
Evaluation
Coliform bacteria form red colonies.
E. coli and Enterobacter aerogenes colonies
are yellow to orange with a yellow zone.
The medium prevents Proteus colonies from
running.
Escherichia coli
Escherichia coli
Escherichia coli
E. coli colonies fluoresence in UV light
E. coli and coliforms from river water
E. coli and coliforms from waste water
E. coli and coliforms
Description and Growth Evaluation Examples
Wort-NPS
Type 14058
For the detection of yeasts and molds.
This culture medium is used in production
and quality control testing in the food,
pharmaceutical and cosmetics industries,
among other applications.
Schaufus-Pottinger-NPS
a) Type 14070
b) Type 14072
c) Type 14080
d) Type 14083
For detection and determination of the
total CFU count of yeasts and molds in
beverages and sugar according to Schaufus
and Pottinger.
Sabouraud-NPS
Type 14069
For culturing yeasts, molds, acid-tolerant and
acidophilic bacteria; also for detecting yeasts
and molds in beverages such as fruit juices,
sterility testing of pharmaceuticals and for
isolating of matopathogenic yeasts and fungi.
According to USP 25.
Type of membrane filter supplied
0.65 µm; grey with white grid
Type of membrane filter supplied
a) 0.65 µm; white with green grid
b) 1.2 µm; white with green grid
c) 0,8 µm; grey with white grid
d) 0,65 µm; grey with white grid
Type of membrane filter supplied
0.65 µm; grey with white grid
Incubation conditions
2–3 days at 25°C
Incubation conditions
2–3 days at 28–30°C
Incubation conditions
2–5 days at 25–30°C
Evaluation
Yeasts usually develop smooth white or
colored colonies. Molds generally form velvety
or fluffy cotton-like colonies in the early
growth phase and may take on various colors
after conidiospore production.
Evaluation
Molds develop velvety or fluffy whitish or
greenish colonies which can take on various
colors after conidiospore production. Yeast
and bacteria colonies have smooth surfaces.
Acid forming sugar fermenters are whitish
to yellow non-acid formers are, by contrast,
greenish to blue-green.
Evaluation
Yeasts usually develop smooth white or
colored colonies. Molds generally form
velvety or fluffy, cotton-like colonies in the
early growth phase and may take on various
colors after conidiospore production.
Saccharomyces cerevisiae
Torula lipolytica
Alternaria humicola
Yeasts and molds from spoiled beer
Mixed culture from a soft drink
Yeasts and molds from cough syrup
Yeasts and molds
Description and Growth Evaluation Examples
Malt extract-NPS
Type 14086
Azide-NPS
Type 14051
Bismuth-Sulfite-NPS
Type 14057
For the determination of yeasts and molds,
recommended by AOAC and APHA. Especially
for use in drinks and food.
For the detection of enterococci according
to Slanetz and Bartley. Enterococci are
considered indicator organisms of fecal
contamination. They are less sensitive to
chemical effects than are E. coli organisms
and are therefore longer detectable, for
instance, in waste water and in chlorinated
water.
Selective culture medium for detecting salmonellae in water, food, animal feed, acc. to Wilson
and Blair and USP 25. If a very slight contamination by salmonellae is suspected, prepare an
enrichment culture with selenite or potassium
tetrathionate broth and to subsequentIy plate
(streak with an inoculating loop) the sample on
the membrane filter of the nutrient pad.
Type of membrane filter supplied
0.8 µm; grey with white grid
Type of membrane filter supplied
0.45 µm; green with green grid
Type of membrane filter supplied
0.45 µm; green with green grid
Incubation conditions
2–3 days at 25–30°C,
or modifies acc. to the determination
Incubation conditions
24–48 hours at 37°C
Incubation conditions
18–48 hours at 37°C
Evaluation
Yeasts usually develop smooth white,
random colored. Molds generally form velvety
or fluffy, cotton-like colonies in the early
growth phase and may take on various colors
after conidiospore production. No growth
of bacteria due to low pH-factor.
Evaluation
Enterococci form small red to reddish
brown colonies (approx. 1 mm d) with
smooth peripheries.
Evaluation
Most salmonellae form light-colored colonies
with brown to black centers, surrounded by
a black zone with a metallic sheen (“fish eye”).
Some salmonella species develop uniformly
dark brown to black colonies which may lack
the typical zone.
Yeasts and molds
Fecal bacteria
Saccaromyces cerevisiae
Streptococcus faecalis
Salmonella typhosa, streak
Mixed culture from Saccaromyces and Rhodutorula
Streptococci from waste water
Salmonellae from waste water
Description and Growth Evaluation Examples
VLB-S7-S-NPS
Type 14059
For the detection of pediococci and lactobacilli in routine microbiological quality control
and testing in breweries according to Emeis;
modified acc. to Rinck and Wackerbauer.
Orange Serum-NPS
a) Type 14062
b) Type 14096 (pH 3.2)
For the detection of acid-tolerant microbes
accordina to the “Recommended Methods
for the Microbiological Examination of Foods”
(1966) of the APHA.
Lysine-NPS
Type 14061
Selective medium for detecting “wild yeasts”
in breweries according to Morris and Eddy.
Type of membrane filter supplied
0.45 µm; white with green grid
Type of membrane filter supplied
0.45 µm; green with green grid
Type of membrane filter supplied
0.65 µm; grey with green grid
Incubation conditions
Anaerobic (microaerophilic),
2–3 days at 25–28°C for quick microscopic
examination (microcolonies) 5–7 days
until macroscopically visible colonies are
formed (trace detection).
Incubation conditions
Aerobic or anaerobic (microaerophilic),
2–3 days at 25–28°C
Incubation conditions
Aerobic, 2–5 days at 25–28°C
Evaluation
Pediococci (“Sarcina”) develop round pale
green colonies with smooth peripheries and
an approx. 1 mm d. Lactobacilli grow as
slightly rounded, irregularly lobed approx.
2 mm d colonies which are initially light
green and later dark green.
Evaluation
Only acid-tolerant microbes, such as microflora in fruit juices, can grow on this medium.
Such microorganisms are predominantly
Lactobacillus Leuconostoc, Bacillus, yeasts
and molds.
Evaluation
Only “wild yeasts” (not belonging to the
genus Saccharomyces), which break down
Iysine, can develop on this medium.
They mostly form white or cream-colored
colonies.
Lactobacillus pastorianus
Rhodotorula spec.
Torulopsis spec.
Lactobacilli and pediococci from sediment, streak
Mixed culture from a soft drink
“Wild yeasts” from lager beer
Product Spoiling bacteria
Description and Growth Evaluation Examples
Weman-NPS
Type 14065
Tomato Juice “Jus de Tomate”-NPS
Type 14079
Glucose-Tryptone-NPS
Type 14066
For detection and determination of the
total CFU count of slime-forming, mesophilic
bacteria according to Weman modified
(Lorenz, S., 1961, source: “Zucker,” page 14)
Tight-fitting, special petri dishes for microaerophilic incubation. For the detection of
spoilage bacteria (Lactobacillus, Leuconostoc,
Pediococcus, etc.) in wine and fruit juices
acc. to Dubois, Bindan and Lafon Lafourcade.
In tight-fitting petri dishes for detection
and determination of the total CFU count of
mesophilic and thermophilic spore formers
in foods according to Williams. This medium is
recommended by the NCA (National Canners
Association, USA 1956) and the ICUMSA
(lnternational Commission for Uniform
Methods of Sugar Analysis, 1974).
Type of membrane filter supplied
0.45 µm; green with green grid
Type of membrane filter supplied
0.45 µm; green with green grid
Type of membrane filter supplied
0.45 µm; white with green grid
Incubation conditions
2–3 days at 28–30°C
Incubation conditions
4–6 days at 25–30°C (it is necessary to
check for growth after 10 days to detect
bacteria which develop slowly)
Incubation conditions
Mesophilic microbes: 2–3 days/28–30°C;
Thermophilic spore formers: 1–2 days at 55°C
Evaluation
Some of the colonies of slime-forming,
mesophilic bacteria have a diameter greater
than 5 mm, are smooth, round, usually
colorless, and transparent or translucent.
Evaluation
Lactobacilli form compact, whitish to slightly
yellowish colonies with 1–3 mm diameters.
Pediococci usually develop somewhat smaller,
approx. 1 mm diameter colonies that later
take on a whitish to a slightly brownish
color. Leuconostoc oenus grows as colorless
to whitish colonies with diameters smaller
than 1 mm.
Evaluation
Microorganisms that ferment glucose
and produce acid grow as yellowish green
colonies. Typical “flat-sour” colonies (such
as Bacillus coagulans, Bacillus stearothermophilus) have a diameter of 2–5 mm,
are round smooth-edged, yellowish-green
and surrounded by a yellow zone.
Leuconostoc mesenteroides
Lactic-acid bacteria, streak
Bacillus coagulans, the “flat sour” colony
Mixed culture from sugar syrup
Leuconostoc oenos from wine
Mixed culture from canned vegetables
Product Spoiling bacteria
Description and Growth Evaluation Examples
Chapman-NPS
Type 14074
Cetrimide-NPS
Type 14075
Mannitol-sodium chloride-phenol red
medium for detecting pathogenic
staphylococci in foods and other materials
according to Chapman (1945) modified.
Recommended by the USP and APHA.
For detection and determination of the CFU
count of Pseudomonas aeruginosa according
to Lowbury (1951). This culture medium
conforms to the USP and APHA recommendations.
Type of membrane filter supplied
0.45 µm; white with green grid
Type of membrane filter supplied
0.45 µm; white with green grid
Incubation conclitions
48 hours at 37°C
Incubation conditions
48 hours at 37°C
Evaluation
Staphylococcus aureus develops golden
yellow to orange-colored colonies with
a yellow zone (mannitol-positive).
Staphylococcus epidermis forms whitish
colonies without changing color.
Evaluation
Pseudomonas aeruginosa forms blue colonies
with 1–2 mm diameters and blue zones.
Occasionally the colonies can also be bluishgreen, yellowish-green or colorless. Other
pseudomonas develop whitish colonies.
Non-fecal bacteria
Staphylococcus aureus
Pseudomonas aeruginosa
Mixed culture of staphylococci
Mixed culture of pseudomonas
Troubleshooting Guide
Growth comparison
Due to the variance in allocation of the pores,
all membranes do not guarantee sufficient
nutrient supply.
The pore size alone ist not a meaningful
criteria. A comparison of a cellulose nitrate
membrane with a mixed esters membrane
reveals significant differences in growth.
Failure to follow the directions may lead
to unsatisfactory results listed below:
1. Inhibited growth, dwarf colonies
pad too dry: not enough water used
Growth of E. coli on Endo-NPS
Growth of Pseudomonas aeruginosa
on Cetrimide-NPS
2. Colonies run
pad too wet, water film on the membrane
filter: too much water used.
Colonies of motile microbes (such as Bacillus
or Proteus) tend to run even though the
water dosage is correct. To prevent this,
add NaCI or a similar emulsifier.
3. Contamination from underneath
inhibited colony growth, excess ring
of liquid cloudy, often including
discoloration of the pad:
a) membrane placed with grid facedown
on pad instead of faceup
Sartorius cellulose nitrate membrane
Sartorius cellulose nitrate membrane
b) contamination of the water used for
rehydration
c) contamination during preparation
(by airborne microbes or by contact)
d) microbes rinsed off the membrane filter
by incomplete vacuum filtration of
the sample or rinse liquid or by tilting
the prepared petri dish
e) contaminated filter support
f) contaminated forceps
4. Growth on one side only
petri dish slanted in the incubator
Mixed esters membrane
Mixed esters membrane
E. coli shows no metallic sheen on a mixed
esters membrane. In this case it is very
difficult to differenciate between E. coli and
coliforms without any further test. The red
color of the mixed esters membrane stands
for running colonies. A quantitative statement is difficult.
Pseudomonas grow as blue colonies with
a blue zone, which is clear shown on the
Sartorius cellulose nitrate membrane.
On the mixed esters membrane grow less
colonies and without the blue zone. Due to
the variance in the allocation of the pores,
here the mixed esters membrane did not
guarantee a sufficient nutrient supply. This
may cause in false negative results.
5. Too profuse or too sparse growth
(optimum microbial number between
20 and 200 per filter)
wrong dilution selected or sample
inadequately mixed with the diluent.
6. Non-uniform growth
sample volume less than 5 ml filtered
without adding sterile water as a diluent
or sample volume inadequately mixed
with the diluent.
Membrane Filters for Use on Agar Plates or on Absorbent Pads
If agar plates or absorbent pads to be wetted
with liquid culture medium are used instead
of Nutrient Pad Sets, we recommend the
following types of membrane filters.
Naturally, the membrane filters must be free
of microbes.
For this purpose, they can be boiled or
autoclaved. However, it is more convenient
to order presterilized and individually sterile
packed membrane filters (see table below).
Cellulose acetate prefilters
11301, a white membrane filter with a pore
size of 8 µm is used as a prefilter in a special
prefilter attachment (16807) for bacteriological analyses. It retains coarse suspended
particles, whereas it allows microorganisms
to pass through. These microbes are trapped
on the surface of the underlying bacteriaretentive membrane filter.
For detection of bacteria in dyed media.
They provide optimal contrast to lightcolored or transparent bacteria colonies
during counting.
For detection of yeasts and molds.
Pore size
Membrane color
Grid color
Order no. of individually sterile packed membranes*
Box of 100 pcs.
Box of 1,000 pcs.
0,45 µm
white
green
13906-050 ACN**
13906-050 ACR**
0,45 µm
green
dark green
13806-050 ACN**
13806-050 ACR**
0,45 µm
grey
white
13006-050 ACN**
13006-050 ACR**
0,65 µm
grey
white
13005-050 ACN**
13005-050 ACR
0,8 µm
grey
white
13004-050 ACN**
-
8 µm
white
–
11301-050 ACN**
-
* Also available as a non-sterile version.
To order boxes with 100 pcs. replace ACN with N and for boxes of 1,000 pcs. replace ACR with R.
** Also available in 47 mm diameter.
Accessories
Order no.
16201 Filter holder
16692 Vacuum pump
Order no.
16612 Vacuum pump
16610 Woulff’s bottle
Order no.
16807 Stainless steel funnel with
prefilter attachment
Order no.
16842 3-branch Manifold
16401-47-06-K BioSart 100 Monitor
Order no.
16842 3-branch Manifold
16407--25-ALK BioSart 250 Funnel
Order no.
16843 6-branch Manifold
Order no.
16685 Dosing syringe
Order no.
17649 Colony counter
Order no.
15410-47-ALR Adsorbent Pads
Order no.
16757 AirPort MD8 air sampler
Order no.
16746 MD8 Airscan air sampler
Order no.
16671 Anaerobic container
Order no.
A standard package contains 100 sterile
nutrient pads, each preplated in a petri dish
(each bag contains 10 petri dishes) and
100 individually sterile packed membranes.
Nutrient pad sets are sterile dehydrated
culture media and have a diameter of 50 mm.
Once they are moistened with 3.0–3.5 ml of
sterile and demineralized water they are
ready to use immediately.
NPS Type
Order no.
Target of detection
Azide
Bismuth-Sulfit
Caso
Cetrimide
Chapman
Chromocult
ECD
Endo
Glucose-Tryptone
14051-47 N
14057-47 N
14063-47 N
14075-47 N
14074-47 N
14087-47 N
14082-47 N
14053-47 N
14066-47 N
Jus de Tomate
Lysine
Mac Conkey
Malt Extract
M-FC
Orange Serum
Orange Serum ph 3.2
R2A
Sabouraud
Schaufus Pottinger
14079-47 N
14061-47 N
14097-47 N
14086-47 N
14068-47 N
14062-47 N
14096-47 N
14084-47 N
14069-47 N
14070-47 N
14072-47 N
14080-50 N
14083-47 N
14064-47 N
14055-47 N
14067-47 N
14056-47 N
14059-47 N
14065-47 N
14058-47 N
14790-47 N
Enterococci
Salmonellae
Total CFU count
Pseudomonas aeruginosa
Staphylococcus aureus
E.Coli and coliform bacteria
Escherichia coli
Escherichia coli and coliforms
Mesophilic bacteria and
thermophilic spore forming bacteria
Spoiling bacteria
Wild yeasts
Enterobacteria
Yeats and molds
E.Coli and coliform bacteria
Acid-tolerant microorganisms
Acid-tolerant microorganisms
Total CFU count
Yeats and molds
Yeats and molds
Yeats and molds
Yeats and molds
Yeats and molds
Total CFU count
Total CFU count
E.Coli and coliform bacteria
E.Coli and coliform bacteria
Lactobacilli and Pediococci
Slime forming bacteria
Yeats and molds
Total CFU count
Standard
Standard TTC
Teepol
Tergitol TTC
VLB-S7-S
Weman
Wort
Yeast Extract
The membrane filters tailored to meet the
special requirements of microbial detection
are available with 50 mm (see table below)
or 47 mm diameter (order no. like 50 mm –
but replace 50N by 47N).
Included membrane
pore size, filter/grid
0.45 µm, green/green
0.45 µm, green/green
0.45 µm, green/green
0.45 µm, green/green
0.45 µm, white/green
0.45 µm, white/black
0.45 µm, white/green
0.45 µm, white/green
0.45 µm, white/green
0.45 µm green/green
0.65 µm, green/green
0.45 µm, white/green
0.8 µm, grey/white
0.45 µm, white/green
0.45 µm, green/green
0.45 µm, green/green
0.45 µm, green/green
0.65 µm, grey/white
0.65 µm, white/green
1.2 µm, white/green
0.8 µm, grey/white
0.65 µm, grey/white
0.45 µm, green/green
0.45 µm green/green
0.45 µm, white/green
0.45 µm, white/green
0.45 µm, green/green
0.45 µm, green/green
0.65 µm, grey/white
0.45 µm, green/green
References
*3, 20
*1, 2, 3, 6, 10, 12, 14, 18, 19, 20
*2, 3, 5, 9, 10, 12, 18, 19, 20
*5, 6, 9, 10, 19, 20
*3, 10, 19
*3, 6, 15, 18
*3, 14, 18
*2, 11, 13
*2, 3, 5, 9, 15, 18, 19
*2, 3, 13
*3, 10, 14, 18
*3, 13, 17
*3, 9
*3, 9, 19, 20
*19
*19
*19
*19
*3
*3
*1, 2, 4, 8, 10, 14, 18, 19
*14, 20
*7, 16
*11
*3
References NPS
* 1 = AFNOR
Association Franchaise de Normalisation
2 = AOAC
Associacion of Official Analytical Chemists
3 = APHA
American Public Health Association
4 = BS
British Standards
5 = DAB
Deutsches Arzneibuch
6 = DIN
Deutsches Institut für Normung
7 = EBC
European Brewery Communitiy
8 = EPA
Environmental Protection Agency
9 = EP
European Pharmacopeia
10 = FDA
Federal Drug Administration
11 = ICUMSA
International Commission for Uniform
Methods of Sugar Analysis
12 = IDF
International Dairy Federation
13 = IFU
14 = ISO
International Standards Organisation
15 = LMBG
Lebensmittel- und Bedarfsgegenständegesetz der Bundesrepublik Deutschland,
Bundesgesundheitsamt
16 = MEBAK
Mitteleuropäische Brauereitechnische
Analysenkommission
17 = SMWW
Standard Methods for the Examination of
Water and Wastewater
18 = USDA
US Department of Agriculture
19 = USP
US Pharmacopeia
20 = TVO
Verordnung über Trinkwasser und über
Wasser für Lebensmittelbetriebe
Sartorius AG
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Phone +33.1.69192100
Fax +33.1.69200922
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50011 Antella (FI), Italy
Phone +39.055.634041
Fax +39.055.6340526
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Tokyo 168-0074, Japan
Phone +81.3.33295533
Fax +81.3.33295543
Sartorius S.A.
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28050 Madrid, Spain
Phone +34.91.3586100
Fax +34.91.3588804
Specifications subject to change
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W/sart-119a · G
Publication No.: SM-4017-e02067
Order No.: 85030-503-99