Microbiological Testing of Foods, Beverages and Pharmaceuticals

Introduction

The Membrane Filter Method

The consumers 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. 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.

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. 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
The test sample is pipetted into a petri dish

Membrane Filter Method

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. then mixed with the culture medium and incubated

Monitor MF method

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. Nutrient pads are sterile, dehydrated culture media. Once they are moistened with 3.03.5 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.

Economical Eliminates time-consuming and laborintensive preparation of culture media (sterilization and cleaning, among others). 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 m). 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. Trouble-free storage Nutrient Pad Sets have a shelf life of 9 to at least 24 months at room temperature. 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 No waste or overproduction NPS are validated. In comparison of agar which is done within different deviations of amount and height NPS gives always constant results Everyone can use NPS After wetting with 3,5 ml destilled water NPS are ready to use: NPS and go

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.

How to Handle Microorganisms Microorganism cultures must always be handled as carefully as if they contained pathogens. 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).

Before and after use, inoculating loops and wires must be sterilized by flaming until they glow red-hot. 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 Beer Detection|determination of Pediococci and lactobacilli Wild yeasts Yeasts and molds Foods Colony count Enterobacteria Mesophilic bacteria and thermophilic spore formers Pseudomonas aeruginosa Salmonellae Staphylococci Streptococci Yeasts and molds Milk E. coli and coliforms Salmonellae Streptococci Pharmaceuticals and cosmetics Candida albicans Colony count Fecal streptococci Enterobacteria Pseudomonas aeruginosa Staphylococcus aureus Soft drinks Acid-tolerant microbes Colony count Lactic-acid bacteria Slime-forming bacteria (Leuconostoc) Yeasts and molds Sugar Mesophilic bacteria and thermophilic spore formers Slime-forming bacteria (Leucostonoc) Yeasts and molds Water Colony count E. coli and coliforms Fecal streptococci Pseudomonas aeruginosa Wine Acetobacter Lactic-acid bacteria Lactic-acid bacteria (especially Leuconostoc oenos) Yeasts and molds Nutrient pad type VLB-S7-S Lysine Wort, Malt extract Standard TTC, Standard, Caso Endo, Teepol, M-FC, Tergitol TTC, ECD, MacConkey, Chromocult Glucose-Tryptone Cetrimide Bismuth-Sulfite Chapman Azide Wort, Malt extract Endo Bismuth-Sulfite Azide Sabouraud Caso, R2A Azide MacConkey Cetrimide Chapman Orange Serum Standard, Standart TTC VLB-S7-S, Orange Serum Weman Wort, Schaufus-Pottinger, Malt extract Glucose-Tryptone Weman Wort, Schaufus-Pottinger, Malt extract Standard TTC, Standard, R2A, Yeast extract Endo, Tergitol TTC, Teepol, M-FC, ECD Azide Cetrimide Wort, Orange serum, both wetted with 3%5% ethanol Orange Serum Tomato Juice Jus de Tomate" Wort, Schaufus-Pottinger, Malt extract

Description and Growth Evaluation Examples

Standard TTC-NPS Type 14055 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.

Standard-NPS Type 14064 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.

Caso-NPS Type 14063 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 m; 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.

Type of membrane filter supplied 0.45 m; 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.

Type of membrane filter supplied 0.45 m; green with green grid Incubation conditions 2472 hours at 30C37C, depending on the test sample 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.

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.

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 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.

R2A-NPS Type 14084 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. Type of membrane Filter supplied 0.45 m; green with green grid Incubation conditions 72 h at 35C; 5 days at 20C or 28C

Chromocult-NPS Type 14087 For the determination of total coliforms and E.Coli in water and food.

Type of membrane Filter supplied 0.45 m; green with green grid Incubation conclitions 44 4 h at 36 2C; 68 4 h at 22 2C acc. to EN ISO 6222. Evaluation Bacteria, yeasts and molds can grow. Due to no dye most colonies, predominantly bacteria, grow colorless.

Type of membrane Filter supplied 0.45 m; white with black grid Incubation conclitions 24 h at 36 1C; longer incubation time disturbs the counting of the colonies. Evaluation E. Coli develops dark-blue to violett colonies. Coliforme red to salmone colonies. Other gramnegative colonies are colorless, few with -Glucuronidase activity are light blue to turkese. To confirm E. Coli give Kovacs reagent to dark blue colonies. Then E. Coli stains cherry-red.

Evaluation Predominantly colonies of different size and color, predominantly white or colorless <1 mm diameter.

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 Selective medium for detecting E. coli and coliform bacteria formulated according to the Standard Methods for the Examination of Water and Wastewater, 1998.

M-FC-NPS Type 14068 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.

Teepol-NPS Type 14067 For the detection of E. coli and fecal coliform bacteria according to Burman, N.P. (1967).

Type of membrane filter supplied 0.45 m; white with green grid Incubation conclitions 24 hours at 37C

Type of membrane Filter supplied 0.45 m; white with green grid Incubation conclitions 20 4 hours at 37C in an incubator or at 44,4C in a water bath. Evaluation E. coli and coliforms develop blue colonies with diameters of 12 mm; colonies of a different color are not evaluated.

Type of membrane filter supplied 0.45 m; white with green grid Incubation conditions 1824 hours at 37C

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 coliform bacteria form 12 mm diameter yellow colonies surrounded by a yellow zone. Non lactose-fermenting bacteria develop red colonies of various sizes.

E. coli and coliforms

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

Description and Growth Evaluation Examples

ECD-NPS Type 14082 Selective culture medium for detecting and identifying Escherichia coli, acc. to ISO 9308-1.

MacConkey-NPS Type 14097 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).

Tergitol TTC-NPS Type 14056 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 m; white with green grid

Type of membrane filter supplied 0.45 m; white with green grid

Type of membrane filter supplied 0.45 m; white with green grid

Incubation conditions 1824 hours at 37C 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.

Incubation conditions 1824 at 37C 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.

Incubation conditions 20 4 hours at 37C 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.

E. coli and coliforms

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

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. Type of membrane filter supplied a) 0.65 m; white with green grid b) 1.2 m; white with green grid c) 0,8 m; grey with white grid d) 0,65 m; grey with white grid Incubation conditions 23 days at 2830C 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.

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 m; grey with white grid

Type of membrane filter supplied 0.65 m; grey with white grid

Incubation conditions 23 days at 25C 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.

Incubation conditions 25 days at 2530C 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.

Yeasts and molds

Saccharomyces cerevisiae

Torula lipolytica

Alternaria humicola

Yeasts and molds from spoiled beer

Mixed culture from a soft drink

Yeasts and molds from cough syrup

Description and Growth Evaluation Examples

Malt extract-NPS Type 14086 For the determination of yeasts and molds, recommended by AOAC and APHA. Especially for use in drinks and food.

Azide-NPS Type 14051 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. Type of membrane filter supplied 0.45 m; green with green grid Incubation conditions 2448 hours at 37C

Bismuth-Sulfite-NPS Type 14057 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 0.45 m; green with green grid Incubation conditions 1848 hours at 37C

Type of membrane filter supplied 0.8 m; grey with white grid Incubation conditions 23 days at 2530C, or modifies acc. to the determination

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 m; white with green grid Incubation conditions Anaerobic (microaerophilic), 23 days at 2528C for quick microscopic examination (microcolonies) 57 days until macroscopically visible colonies are formed (trace detection). 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.

Type of membrane filter supplied 0.45 m; green with green grid Incubation conditions Aerobic or anaerobic (microaerophilic), 23 days at 2528C

Type of membrane filter supplied 0.65 m; grey with green grid Incubation conditions Aerobic, 25 days at 2528C

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.

Product Spoiling bacteria

Lactobacillus pastorianus

Rhodotorula spec.

Torulopsis spec.

Lactobacilli and pediococci from sediment, streak

Mixed culture from a soft drink

Wild yeasts from lager beer

Description and Growth Evaluation Examples

Weman-NPS Type 14065 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)

Tomato Juice Jus de Tomate-NPS Type 14079 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.

Glucose-Tryptone-NPS Type 14066 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 m; white with green grid Incubation conditions Mesophilic microbes: 23 days/2830C; Thermophilic spore formers: 12 days at 55C 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 25 mm, are round smooth-edged, yellowish-green and surrounded by a yellow zone.

Type of membrane filter supplied 0.45 m; green with green grid Incubation conditions 23 days at 2830C

Type of membrane filter supplied 0.45 m; green with green grid Incubation conditions 46 days at 2530C (it is necessary to check for growth after 10 days to detect bacteria which develop slowly) Evaluation Lactobacilli form compact, whitish to slightly yellowish colonies with 13 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 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.

Product Spoiling bacteria

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

Description and Growth Evaluation Examples

Chapman-NPS Type 14074 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.

Cetrimide-NPS Type 14075 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 m; white with green grid Incubation conclitions 48 hours at 37C

Type of membrane filter supplied 0.45 m; white with green grid Incubation conditions 48 hours at 37C

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 12 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

Growth comparison

Troubleshooting Guide

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. Growth of Pseudomonas aeruginosa on Cetrimide-NPS

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

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 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.

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.

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 m 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 0,45 m 0,45 m 0,45 m 0,65 m 0,8 m 8 m

Membrane color white green grey grey grey white

Grid color green dark green white white white

Order no. of individually sterile packed membranes* Box of 100 pcs. Box of 1,000 pcs. 13906-050 ACN** 13806-050 ACN** 13006-050 ACN** 13005-050 ACN** 13004-050 ACN** 11301-050 ACN** 13906-050 ACR** 13806-050 ACR** 13006-050 ACR** 13005-050 ACR -

* 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 Woulffs 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 3.03.5 ml of sterile and demineralized water they are ready to use immediately. Target of detection 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

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). 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

NPS Type Azide Bismuth-Sulfit Caso Cetrimide Chapman Chromocult ECD Endo Glucose-Tryptone Jus de Tomate Lysine Mac Conkey Malt Extract M-FC Orange Serum Orange Serum ph 3.2 R2A Sabouraud Schaufus Pottinger

Order no. 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 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

Included membrane pore size, filter/grid 0.45 m, green/green 0.45 m, green/green 0.45 m, green/green 0.45 m, green/green 0.45 m, white/green 0.45 m, white/black 0.45 m, white/green 0.45 m, white/green 0.45 m, white/green 0.45 m green/green 0.65 m, green/green 0.45 m, white/green 0.8 m, grey/white 0.45 m, white/green 0.45 m, green/green 0.45 m, green/green 0.45 m, green/green 0.65 m, grey/white 0.65 m, white/green 1.2 m, white/green 0.8 m, grey/white 0.65 m, grey/white 0.45 m, green/green 0.45 m green/green 0.45 m, white/green 0.45 m, white/green 0.45 m, green/green 0.45 m, green/green 0.65 m, grey/white 0.45 m, green/green

*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

Standard Standard TTC Teepol Tergitol TTC VLB-S7-S Weman Wort Yeast Extract

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 fr 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 Bedarfsgegenstndegesetz der Bundesrepublik Deutschland, Bundesgesundheitsamt 16 = MEBAK Mitteleuropische 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 ber Trinkwasser und ber Wasser fr Lebensmittelbetriebe

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