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Volume 2.

3, 2010

Probiotics

Probiotics...........................p2

Genus Yersinia..................p5
Yersinia ..................p5

Marine Isolates
Lactobacillus bulgaricus added to dairy products
for bioleaching ................p7
in a milk protein matrix.
Probiotics
Jvo Siegrist, Product Manager Microbiology At the beginning of the 20th century, probiotics were thought to have a beneficial
ivo.siegrist@sial.com affect to the host by improving the intestinal microbial balance. At that time, it was
Probiotics take on a key role in observed that lactic-acid bacteria inhibit the growth of proteolytic bacteria due to low
pH. Through his studies and observations, the Russian biologist Metchnikoff noted
Microbiology

the functional food industry


that people from cultures which consumed large amounts of fermented milk, for
The name “Probiotics” is derived from example those in Bulgaria and the Russian steppes, generally had a higher life-span.
Latin (pro) and Greek (biotic) roots Therefore, he proposed that lactic-acid bacteria decrease the intestinal pH as a result
meaning “for life”. Probiotics are defined of fermentation, thereby suppressing the growth of proteolytic bacteria. Metchnikoff
as live bacteria with beneficial effects to propagated the consumption of sour milk fermented with the Lactobacillus bulgaricus
the health of the host organism. Today, [5] as a means of promoting intestinal balance and overall health.
primarily lactic acid bacteria (LAB) and
Today, diverse studies report the benefits of probiotics, such as inhibitory effects on
bifidobacteria are used as probiotics,
pathogens, aid in the management or prevention of chronic intestinal inflammatory
however certain yeasts and bacilli are
diseases or of atopic diseases, and support to the immune system. Potential beneficial
also known to have positive effects. In
applications abound, and the research around this topic continues to expand.
most cases, probiotics are produced
directly by fermentation in foods such Recently, scientists in Finland have discovered that cheese can help preserve and enhance
as yogurt, or are supplied through the immune system of the elderly by acting as a carrier for probiotic bacteria. The research
dietary supplements. Although the showed that daily consumption of probiotic cheese helps to bolster immunity in elderly
term “Prebiotics” is also used often in people. Cheese was confirmed to be an effective carrier for probiotics [1].
such cases, the two terms should not
be confused, since “prebiotics” actually Bifidobacteria
refers to non-digestible compounds that Bifidobacterium is one of the most important probiotic bacteria used in the dairy
stimulate the growth and/or activity industry. They are Gram-positive, non-motile, rod-shaped, and often branched anaerobic
of probiotics in the gut. Prebiotics are bacteria. They were first isolated from a breast-fed infant by Henry Tissier from the
frequently used in functional food, and Pasteur Institute. At that time, Tissier named the organism Bacillus bifidus communis [6].
typically consist of oligosaccharides. Bifidobacteria have a positive affect on the immune system and help to control intestinal
Prebiotics can be found in milk pH . In additional, bifidobacteria produce bacteriocins and bacteriocin-like inhibitory
(galactooligosaccharides, or GOS) and compounds which inhibit the growth of other bacteria.
plants with dietary fibers, however they
are often produced by fermentation. Bifidobacteria posesses many glycosylases able to degrade various plant- or milk-
derived oligosaccharides. Several such enzymes were identified on the Bifidobacterium
genome. Also diverse glycosyl hydrolase, ABC transporter and the fos gene cluster that
is involved in the processing of health-promoting fructooligosaccharides (prepiotics),
called bifidogenic factors, can be found on the genome [10]. Obviously Bifidobacteria
are able to utilize a broad range of substrates as energy sources, such as plant polymers,
glycoproteins and glycoconjugates, as well as having specialized proteins for the
catabolism of oligosaccharides.
Bifidobacteria also have a unique hexose metabolism called the bifid shunt. The key
enzyme, fructose-6-phosphate phosphoketolase is not found in any other Gram-positive
intestinal bacteria and therefore provides an ideal target for a diagnostic test.
It was found that live B. lactis bacteria can directly counteract the harmful effects of
coeliac-toxic gliadin and this may prove to be a future potential treatment of coeliac
disease [9].

Did you know… In adult intestines, only 3-6% of the fecal flora is composed of Bifidobacteria, while
in breast-fed infants Bifidobacteria can constitute up to 90%. With increasing age, the
that bacteria accounts number of Bifidobacteria decreases. It was observed that babies and adults with lower
numbers of Bifidobacteria have a higher risk for diarrhea and allergies. For this reason,
for about 7% of human Bifidobacteria are added as a probiotic supplement to infant formulas, drinks, yogurts
body weight? and a range of other products.
An adult person carries about 2.5 - Because of the wide use of bifidus, Fluka has developed a Bifidobacteria Selective Media
7.5 kg of living bacteria in the intestinal (BSM), available as an agar or a broth, as a standard for quality control. This medium
tract. There are about ten times as allows for easy and fast quality control of yogurt made with bifidus and can be used to
many bacterial cells as there are tissue control the count of bifidus bacteria.
cells in the entire body.

2 sigma-aldrich.com/microbiology
Organisms (ATCC) Growth Colony Appearance
Bifidobacterium longum (15707) +++ red-brown (maroon)
Bifidobacterium infantis (15697) +++ red-brown (maroon)
Streptococcus thermophilus (14486) - -
Lactobacillus acidophilus (314) - -

Microbiology
Lactobacillus bulgaricus (11842) - -
Table 1. Bifidobacterium sp. cultural characteristics on BSM Agar
Figure 1. Yogurt sample cultured on BSM Agar.
Bifidobacteria appears as purple-brown colonies.
Name Brand Cat. No. Pack Size
BSM Agar Fluka 88517 500 g Medium for Lactobacilli
BSM Broth Fluka 90273 500 g
Since lactobacilli prefer acidic conditions,
BSM Supplement Fluka 83055 5g
natural extracts and juices from tomatoes
Wilkins Chalgren Anaerobic Agar Fluka W1761 500 g and oranges, as well as other single
Mupirocin Sigma M7694 50 mg metabolic acids (e.g. malic acid), are often
100 mg used as media ingredients. Casein and
Lithium Mupirocin Sigma 07188 1g yeast extract provide rich amino acid
100 mg sources, and the maltose is used as a
Table 2. Selective medium and supplements for Bifidobacterium carbohydrate source for lactobacilli, which
cannot utilize glucose as fermentable
sugar. Fructose is the carbohydrate source
Bifidobacterium grow very well on this medium, while Lactobacillus and Streptococcus of Lactobacillus fructivorans. Polysorbate,
strains are inhibited. Bifidobacterium colonies grow within 24-48 hours (occasionally up sorbitan mono-oleate and other related
to three days because of the highly selective conditions). The Bifidobacterium colonies compounds act as a source of fatty acids
are purple-brown and therefore are easy to differentiate from other organisms. and stimulate the lactic acid bacteria.
In a Swiss governmental evaluation study for the enumeration of bifidobacteria in sour Today, it is standard practice to
milk products, the traditional method was compared to Wilkins-Chalgren Agar with differentiate lactobacilli based on their
100 mg/L mupirocine and BSM Agar. The traditional method produced statistically phenotype using selective media. Classical
significant differences, while Wilkins-Chalgren Agar and BSM Agar showed similar phenotypic tests for identification of
results without any significant variances. The study concluded, “On the BSM Agar, the lactobacilli are based on physiological
bifidobacteria forms purple-brown colonies which made the enumeration easy” [3]. characteristics, like motility, growth
temperature, respiratory type, and
Lactobacillus Species growth in sodium chloride, as well as
Lactobacilli are rod-shaped, Gram-positive, fermentative, facultative anaerobic or on diverse biochemical characteristics,
microaerophilic organotrophs. Normally they form straight rods, but under certain such as fermentation type, metabolism
conditions spiral or coccobacillary forms have been observed. In most cases, they form of carbohydrate substrates, production of
chains of varying length. Lactobacilli belong to the lactic acid bacteria and comprise lactic acid isomers, coagulation of milk,
the major part of this group. As their name implies, they produce lactic acid and and presence of specific enzymes like
derive energy from the fermentation of lactose, glucose and other sugars to lactate via arginine dihydrolase. In Bergey’s Manual,
homofermentative metabolism. About 85-90% of the sugar utilized in the fermentative Lactobacillus is described as a Gram-
process is converted to lactic acid. However, there are some heterofermentative positive rod, non-spore forming, acid
lactobacilli that produce alcohol in addition to lactic acid from sugars. This acid- fast negative and catalase negative. The
producing mechanism inhibits growth of other organisms and favors the growth of colony morphology on certain media is
lactobacilli that thrive in low pH environments. ATP is generated during the process by taken for the presumptive identification.
non-oxidative substrate-level phosphorylation.
Some strains of lactobacilli were shown to produce, like bifidobacteria, a bacteriocin-like
substance and are able to inhibit a broad range of pathogens [8]. Lactobacilli also produce
adhesins (proteins), which perform a vital role in recognizing specific host components
(extracellular matrix) important for the bacterial adhesion and colonization at host surfaces,
as well as in bacterial interaction with physiological and immunological processes [8].
In the last several years, several new Lactobacillus species have been introduced as
probiotics, including Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus
johnsonii. As of this time, probiotics have not been used in the pharmaceutical industry
due to the many open questions that remain to be answered [7].
Figure 2. 3D rendering of isolated Lactobacillus

3
Cat. No. Name Description

Media for Differentiation


17153 LS Differential Agar For the maximum growth and differentiation of lactobacilli and streptococci on the basis of
Antibiotic free skim milk powder (Fluka 70166) colonial morphology, T.T.C. reduction and casein reaction.
TTC Solution (Fluka 17779)

17158 Litmus Milk For maintenance of lactobacilli and for determining the action of bacteria on milk.
Microbiology

17222 WL Nutrient Agar For the examination of materials encountered in brewing and for industrial fermentations
containing mixed flora of yeasts and bacteria.
17215 WL Differential Agar For selective isolation and enumeration of bacteria encountered in breweries and
industrial fermentations.

Nonselective Media
Y3127 Yeast Malt Agar Used for the isolation and cultivation of yeasts, molds and other aciduric microorganisms.
17216 Tomato Juice Agar For the cultivation and enumeration of lactobacilli.
80957 Plate Count Skim Milk Agar For the enumeration of bacteria in milk and dairy products.
17218 Tomato Juice Broth For cultivation of yeasts and other aciduric microorganisms.
T2188 Tryptone Glucose Yeast Extract Agar Recommended for enumeration of bacteria in water, air, milk and dairy products.
17123 Elliker Broth For culturing streptococci and lactobacilli of importance in the dairy industry.

Selective Media (by low pH and selective agents)


R1148 Rogosa SL Agar Used as a selective medium for cultivation of lactobacilli.
85515 Sorbic Acid Agar (Base) For the isolation and differentiation of lactobacilli from food, faeces etc. according to Reuter.
17154 Lactobacillus Bulgaricus Agar (Base) Used with acetate buffer for isolation and identification of Lactobacillus bulgaricus.
69966 MRS Broth For the enrichment and isolation of all species of lactobacilli from all types of material.
41782 MRS Agar, Vegitone This MRS Agar is free of animal derived material. It is recommended for the isolation and
cultivation of Lactobacillus species.
69964 MRS Agar For the enrichment, cultivation and isolation of all species of Lactobacillus from all types of
material according to De Man, Rogosa and Sharpe.
38944 MRS Broth, modified, Vegitone This MRS Broth contains plant peptone instead of animal peptone. It is recommended for the
isolation and cultivation of Lactobacillus species.
30912 MRS Agar, original acc. For the enrichment, cultivation and isolation of all species of Lactobacillus from all types of
DeMan-Rogosa-Sharpe materials. Recommended by the “Schweizerisches Lebensmittelbuch” 5th ed., chapter 56A.
75405 Orange-serum Agar For the isolation, cultivation and enumeration of acid-tolerant spoilage microorganisms in fruit
juice and fruit juice concentrates, in particular from citrus fruit, according to Hays, Troy and Beisel.
17226 Universal Beer Agar For culturing microorganisms of significance in the brewing industry.
W2261 WL Nutrient Broth Recommended for the cultivation of bacteria encountered in breweries and industrial
fermentations.
02538 Raka Ray Agar A medium for selective isolation of lactic acid bacteria from beer and brewing processes.
Lactic Acid Supplement, modified (Fluka 14121)
Cycloheximide solution (Fluka 18079)

83920* Rogosa Agar Selective agar for the isolation and enumeration of lactobacilli belonging to the buccal and
intestinal flora, as well as from food such as milk and meat.
64198 NBB Agar Base, modified Selective medium used for the detection of contaminating/spoilage microorganisms in brewery.
50725 NBB Broth Base, modified Selective medium used for the detection of contaminating/spoilage microorganisms in brewery.

Table 3. Media for lactobacilli detection, differentiation, isolation, enumeration and cultivation

*Available in Europe only.

4 sigma-aldrich.com/microbiology
Modern Methods to Detect Lactobacilli
As a modern alternative, molecular biology-based methods, like the need for PCR amplification. The sandwich hybridization test,
PCR, can be consulted. However, they are often quite expensive. called HybriScan, is performed on a microtiter plate. The range of
Under our Fluka Brand, Sigma-Aldrich provides a revolutionary lactobacilli detected by HybriScan tests is listed in Table 4.
molecular biology method that is rapid, easy and cost effective. More information about the test and the technical principles
Based on the detection of rRNA, this method completely avoids

Microbiology
behind it are found on our web site sigma-aldrich.com/hybriscan

Cat. No. Brand Description Specifity Reactions


62533 Fluka HybriScan®D Beer Detects all beer spoiling bacteria of the genera Lactobacillus, Pediococcus, 96
Pectinatus and Megasphaera
68301 Fluka HybriScan®D Drinks Detects yeast of the genera Saccharomyces, Zygosacchromyces, Brettanomyces, 96
Torulaspora, Pichia, Candida and bacteria of the genera Lactobacillus,
Acetobacteraceae and Alicyclobacillus
59744 Fluka HybriScan®D Lactobac Detects bacteria of the genera Lactobacillus and Pediococcus in fruit juices and 96
non-alcoholic beverage.
75724 Fluka HybriScan®I Identification of Lactobacillus brevis 48
Lactobacillus brevis
80065 Fluka HybriScan®I Identification of Lactobacillus buchneri 48
Lactobacillus buchneri
86827 Fluka HybriScan®I Identification of Lactobacillus lindneri 48
Lactobacillus lindneri

Table 4. HybriScan products (HybriScan®D = detection kit; HybriScan®I = identification kit)

References
1. F. Ibrahim, S. Ruvio, L. Granlund, S. Salminen, M. Viitanen, A.C. Ouwehand, 6. H. Tissier, Recherchers sur la flora intestinale normale et pathologique du
Probiotics and immunosenescence: cheese as a carrier, FEMS Immunology & nourisson, Thesis, University of Paris (1900)
Medical Microbiology, 59, 1, p53-59 (2010) 7. G.W. Tannock, Probiotics: time for a dose of realism, Curr. Issues Intest.
2. Rada V. and Koc J.: The use of mupirocin for selective enumeration of Microbiol., 4(2), 33-42 (2003)
bifidobacteria in fermented milk products. Milchwissenschaft. 55, 65–67 (2000) 8. Å. Ljungh, T. Wadström, Lactobacillus Molecular Biology: From Genomics to
3. Bundesamt für Gesundheit: Schweizerisches Lebensmittelbuch (SLMB): Probiotics, Caister Academic Press (2009)
Kapitel 56, «Mikrobiologie» Neuausgabe 2000, Stand 2004 9. K. Lindfors, etal., Live probiotic Bifidobacterium lactis bacteria inhibit the
4. J. Nowroozi, M. Mirzaii, M. Norouzi, Study of Lactobacillus as Probiotic toxic effects induced by wheat gliadin in epithelial cell culture, Clin. Exp.
Bacteria, Iranian J. Publ. Health, Vol. 33, No. 2, pp.1 -7 (2004) Immunol., 152(3), 552-8 (2008)
5. R.B. Vaughan, The romantic rationalist: A study of Elie Metchnikoff, Medical 10. J.F. Kim, et al., Genome sequence of the probiotic bacterium Bifidobacterium
History 9: 201–15 (1965) animalis subsp. lactis AD011, J. Bacteriol., 191(2), 678-9 (2009)

Genus Yersinia
Jvo Siegrist, Product Manager Microbiology Pigs, rodents, rabbits, sheep, cattle, horses, dogs, and cats are
ivo.siegrist@sial.com the natural sources of Yersinia. At the moment, most human
Detection, identification, differentiation and illness cases caused by Yersinia originate from Y. enterocolitica.
This organism is the cause of yersiniosis, an infectious disease
cultivation of Yersinia species
with symptoms like fever, abdominal pain, and diarrhea. Other
Yersinia is a rod shaped and facultative anaerobic Gram-negative clinically important species of this genus are Y. pseudotuberculosis
bacteria belonging to the family of Enterobacteriaceae. It has (symptoms similar to Y. enterocolitica except in most cases
a fermentative metabolism, is oxidase-negative, mannitol- no diarrhea is seen) and Y. pestis (organism responsible for
positive, glucose-positive and lactose negative. On the Bismuth the bubonic plague). Most infections are acquired through
sulfite Agar (Fluka 95388) Yersinia can be differentiated from contaminated food, like raw or undercooked pork products,
Salmonella because it is not able to produce hydrogen sulphide. seafood, vegetables, unpasteurized milk or untreated water.
It is a psychrophilic organism, surviving and proliferating at low However, infections may also occur after contact with infected
temperatures of 0-4 °C (e.g., on food products in a refrigerator). animals or faeces, or through transmission by fleas.
Some Yersinia species are also relatively highly heat resistant.
The bacteria received its name from A. E. J. Yersin, a Swiss micro-
However, they can be quite easily inactivated by oxidizing agents
biologist, who discovered the Yersinia pestis bacterium in 1894 in
such as hydrogen peroxide and potassium permanganate.
Hong Kong.

5
Species Differentiation Properties
Y. enterocolitica catalase-positive, most strains are ornithine-positive, motile at 22-26 °C,
non-motile at 37 °C, urea-positive, sorbitol- and cellobiose- positive, most
strains are sucrose-positive
Y. pseudotuberculosis motile at 22-26 °C, non-motile at 37 °C, urea-positive, rhamnose-positive,
esculin-positive, ornithine decarboxylase negative, indole negative
Microbiology

Y. pestis produces two antiphagocytic components (antiphagocytic slime), catalase


positive non-motile at 22-26 °C and 37 °C, esculin-positive, indole negative,
ornithine decarboxylase negative Figure 2. Bacteriophage øR1-37 attached to
Y. enterocolitica YeO3-R1 cell (length of cell = 1,7 μm;
Table 1. Some differentiation of the important Yersinia species C Source Mikael Skurnik, University of Turku, Finland)

Cat. No. Description Cat. No. Supplements


Nonselective Broths 38266 Ferrioxamine E
53286 Brain Heart Broth 75258 Yersinia selective Supplement
40893 Peptone Water, phosphate-buffered, Vegitone 17778 Ticarcillin Supplement
77187 Peptone Water, phosphate-buffered 17777 Potassium chlorate Supplement
Table 3. Selective and growth supplement for Yersinia
Selective Enrichment Broths
17156 ITC Broth (Base)
69965 Mossel Broth Cat. No. Test for Yersinia Diagnostics
17192 Peptone Sorbitol Bile Broth 80507 Bile Esculin Disks
Basal Media for Carbohydrates Utilization 88597 Catalase Test
A0715 Andrade Peptone Water 56481 Cellobiose Disks
28943 Andrade Peptone Water, Vegitone 63367 Dextrose (Glucose) Disks
05686 DMACA Indole Disks
Medium for Autoagglutination
49825 DMACA Reagent
39484 Methyl Red Voges Proskauer Broth
67309 Kovac’s Reagent for Indoles
Nonselective Agars for Differentiation & Confirmation 78719 Kovac’s Reagent Strips
27048* Christensen’s Urea Agar 28816 Lactose Disks
60787 Kligler Agar 94438 Mannitol Disks
M1428 Motility Indole Ornithine Medium 08714 Methyl Red Solution
75315 OF Test Nutrient Agar 07345 Oxidase Reagent acc.
O5386 Ornithine Decarboxylase Broth Gaby-Hadley A
51463 Urea Broth 07817 Oxidase Reagent acc.
Gaby-Hadley B
Selective Agars for Detection and Isolation 18502 Oxidase Reagent acc.
95760* Yersinia Selective Agar (CIN Agar) Gordon-McLeod
40560 Oxidase Strips
Selective Agars with Differential System for Differentiation, Detection and Isolation 70439 Oxidase Test
95388 Bismuth sulfite Agar 93999 Rhamnose Disks
70135 DCLS Agar 92971 Salicin Disks
90035 DCLS Agar No. 2 93998 Sorbitol Disks
17213 Violet Red Bile Glucose Agar without Lactose 94309 Sucrose Disks
53605 Violet Red Bile Glucose Agar without Lactose, Vegitone
Table 4. Test for identification and differentiation
95273 VRB MUG Agar of Yersinia
Table 2. Media for Yersinia

*Available in Europe only.

References
1. K.J. Ryan, C.G. Ray (editors), Sherris Medical Microbiology, 4th ed., McGraw Hill, 368–70 (2004)
2. F.M. Collins, Pasteurella, Yersinia, and Francisella. In: Barron’s Medical Microbiology (S. Barron et al, eds.), 4th ed., University of Texas Medical Branch (1996)
3. F. Bichai, P. Payment, B. Barbeau, Protection of waterborne pathogens by higher organisms in drinking water: a review, Can. J. Microbiol. 54 (7), 509-524 (2008)
4. Bergey’s Manual of Systematic Bacteriology, 1994, 9th ed., Williams and Wilkins, Baltimore

More details about the media or tests can be found on our website sigma-aldrich.com/microbiology.

6 sigma-aldrich.com/microbiology
Native Marine Isolates from Indian Ocean Nodules
Abhilash, R. Venkat Raman, K.D. Mehta and B.D. Pandey, MEF Division, National Metallurgical
7-BIOSN
Laboratory (CSIR), Jamshedpur-831007, India
biometnml@gmail.com; www.nmlindia.org

Microbiology
The marine isolates from sea nodules can be worthy catalysts to explore the possibilities
of faster bioleaching of low index ocean nodules mined from Indian Ocean. The native
microbes were isolated from Indian Ocean Nodules in Artificial Sea water Nutrient
Broth (ASWNB) at pH 7.0. Two new and pure species were isolated after 12 rounds of
culture purification on ASWNB Agar. They were termed as 7-BIOSN and A-BIOSN. Their
microbiological and biochemical features are summarized below.

7-BIOSN A-BIOSN
Microscopic
Long chains of rod shaped cells Short rod shaped cells
Cells entangled in dense network Cells clubbed in pairs
Gram Positive Gram Positive
Non Acid Fast Staining Non Acid Fast Staining
Non-Endospore forming Endospore forming
Mesophilic Mesophilic
Biochemical A-BIOSN
Indole Test + +
Methyl Red Test + +
Voges Proskauer Test - +
Citrate Utilization Test + +
TSI Agar Test Glucose fermenting Glucose, lactose fermenting
Starch Hydrolysis Test + +
Gelatin Liquefication Test + -
DNase Test + +
Catalase Test - -
Oxidase Test + +
Salt Tolerance Test Could not grow in Could not grow in
>5% NaCl conc. >7% NaCl conc.
Antibiotic Resistant to Penicillin Resistant to Penicillin
Susceptibility Test Resistant to Ampicillin, etc. Sensitive to Ampicillin etc.
Generation time 1.3 h 0.7 h
Figures 1 & 2. Two isolates from Indian Ocean
Table 1. Microbiological and Biochemical Features
nodules capable of metal oxidation from its
sulphides and oxides

The pure isolates would be molecularly characterized for phylogenetic homology and (Under Oil Immersion 1000X Magnification in Phase
Contrast Microscope)
subsequently tested for toxicity on metal ions (Cu, Ni, Co, Fe, Mn) for applications in
bioleaching.
The Isolated species from sea nodules were tested for metal toxicity evaluation on various
concentrations of synthetic salts of copper, nickel and cobalt. It was found that both species
exhibited good resistance towards 1000 ppm Ni (II) concentration, whereas resistance
towards Cu(II) and Co(II) could only be seen at a metal concentration of 100 ppm.

Metal ion Cu Ni Co
Concentration [ppm] A-BIOSN 7-BIOSN A-BIOSN 7-BIOSN A-BIOSN 7-BIOSN
10 R R R R R R
50 R R R R R R
100 R R R R R R These values can be improvised on subsequent
sub-culturing for definite applications in
500 S S R R S S bioleaching of low-index sea nodules and pose a
1000 s S R S S S promising alternative technology for the future.
Table 2. results of sensitivity testing; R = resistance, S = sensitive

7
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