Pseudomonas Media and Tests

By: Jvo Siegrist, Product Manager Microbiology,, Analytix Volume 2007 Article 5

Detection, identification, differentiation and cultivation of Pseudomonas species

HiFluoro Pseudomonas Agar under UV light

Figure 1. HiFluoro Pseudomonas Agar under UV light

are motile (one or more polar flagella), rod shaped and aerobe Gram-negative bacteria. They are found almost everywhere, in soil, water, plants and animals. In most cases it is not pathogenic and in fact can be beneficial. For example, P. putida is used as a bio-scrubber to aid in the biodegradation of diverse organic compounds in polluted air and waste water. However, P. aeruginosa is an infamous opportunistic human pathogen most commonly affecting immuno-compromised patients. Along with P. maltophilia, it accounts for the majority of human infections. Pathogenic Pseudomonas are found throughout the body, most commonly in the urinary tract, respiratory tract, blood and wounds1.

Rugged and opportunistic, Pseudomonas use a wide range of nutritional sources, even very simple nutritional environments without any organic compounds. They can remain viable for long periods of time in many different habitats and under very adverse conditions. They are also widespread, being found in water, saline solutions, utensils and even in cosmetics, pharmaceuticals and disinfectants, and many natural and manufactured foods. Psychrotrophic (cold-tolerant) Pseudomonas species are a significant food spoilage problem in refrigerated meat, fish, shell fish and dairy products. Because Pseudomonas thrive in water systems, they can be the source of contamination in the food and beverage industry2.

Pseudomonas are not generally fastidious microorganisms. They can grow on very simple media like Kind Agar, for example, which contains a protein hydrolysate, magnesium chloride, potassium sulphate and agar. Analytical microbiology leverages a microbe’s unique biochemistry to aid in its identification. For example, selective Pseudomonas media use cetrimide, nalidixic acid, cephaloridine, penicillin G, pimaricin, malachite green and other inhibitory agents. The proteolytic activity, lipolytic activity, fluorescent pigment formation, nitrate utilisation, glutamate utilisation, hemolytic reaction and other biochemical reactions are used in the media for the identification and differentiation of Pseudomonas species.

Pseudomonas gives negative Voges Proskauer, indole and methyl red tests, but a positive catalase test. While some species show a negative reaction in the oxidase test, most species, including P. fluorescens, give a positive result (see Figure 2). Another feature associated with Pseudomonas is the secretion of pyoverdin (fluorescein, a siderophore), a fluorescent yellow-green pigment under iron-limiting conditions3. Certain Pseudomonas species may also produce additional pigments, such as pyocyanin (blue pigment, a siderophore) by P. aeruginosa4, quinolobactin (yellow, dark green in presence of iron, a siderophore) by P. fluorescens5, a reddish pigment called pyorubrin and pyomelanin (brown pigment). On blood agar a hemolytic reaction can be observed.

Oxidase test

Figure 2. Oxidase test

Scientific classification of Pseudomonas:

Kingdom: Bacteria Order: Pseudomonadales
Phylum: Proteobacteria Family: Pseudomonadaceae
Class: Gamma Proteobacteria Genus: Pseudomonas

utilizes sugars as an energy source by using the Entner-Doudoroff pathway with pyruvate as the end product (dissimilation). The reaction utilizes a different set of enzymes from those used in glycolysis and the pentose phosphate pathway. Fermentation catabolism is not observed in Pseudomonas, but some species, like P. aeruginosa, P. stutzeri and P. denitrificans, are able to use nitrate as an electron acceptor instead of oxygen. Growth can also occur under anaerobic conditions when the denitrification pathway is used. Sigma-Aldrich supplies a wide array of products for the detection, identification, differentiation, enumeration and cultivation of Pseudomonas, using its biochemical characteristics, including Gram staining kit, and many types of selective growth media (Table 1) and diagnostic tests (Table 2). Additional information on media and tests for Pseudomonas and a wide range of other microbes can be found on our web site:

Table 1 Media for Pseudomonas

Cat. No. Brand Nonselective Broths
A0465 Sigma Alternative Thioglycollate Medium
53286 Sigma-Aldrich Brain Heart Broth
D3435 Sigma-Aldrich Dey-Engley Neutralizing Broth
63649 Sigma-Aldrich Membrane filter Rinse Fluid (USP)
70149 Sigma-Aldrich Nutrient Broth No 3
03856 Sigma-Aldrich Nutrient Broth No. 4
70179 Sigma-Aldrich Peptone Water
77187 Sigma-Aldrich Peptone Water, phosphate-buffered
40893 Sigma-Aldrich Peptone Water, phosphate-buffered,Vegitone
70157 Sigma-Aldrich Thioglycollate Broth (USP Alternative)
41960 Sigma-Aldrich Vegitone Infusion Broth


Cat. No. Brand Selective Enrichment Broths & Biochemical Identification Broths
00185 Sigma-Aldrich Acetamide Nutrient Broth
17129 Sigma-Aldrich Asparagine Proline Broth
78886 Sigma-Aldrich Cetrimide Broth
63163 Sigma-Aldrich Malachite Green Broth
39484 Sigma-Aldrich Methyl Red Voges Proskauer Broth
14305 Sigma-Aldrich Motility Nitrate Medium
72548 Sigma-Aldrich Nitrate Broth


Cat. No. Brand Nonselective Agars for Cultivation, Enumeration and Isolation
70147 Sigma-Aldrich Milk Agar
70148 Sigma-Aldrich Nutrient Agar
44776 Sigma-Aldrich Nutrient Agar Plates (Diameter 55 mm)
80957 Sigma-Aldrich Plate Count Skim Milk Agar
17209 Sigma-Aldrich R-2A Agar
17175 Sigma-Aldrich Skim Milk Agar, modified
51414 Sigma-Aldrich Skim Milk Agar, modified
70159 Sigma-Aldrich Tryptone Glucose Extract Agar
T2188 Sigma-Aldrich Tryptone Glucose Yeast Extract Agar
01497 Sigma-Aldrich Yeast Extract Agar


Cat. No. Brand Nonselective Agars for Differentiation
70133 Sigma-Aldrich Blood Agar (Base)
21065 Sigma-Aldrich Calcium caseinate Agar
55420 Sigma-Aldrich CLED Agar
16636 Sigma-Aldrich HiCrome(™) UTI Agar, modified
60788 Sigma-Aldrich King Agar A
60786 Sigma-Aldrich King Agar B
75315 Sigma-Aldrich OF Test Nutrient Agar
P1852 Sigma-Aldrich Pseudomonas Agar (for Fluorescein)
91015 Sigma-Aldrich Tributyrin Agar


Cat. No. Brand Selective Agars for Detection and Isolation
11012 Sigma-Aldrich Cetrimide Nalidixic acid Agar
22470 Sigma-Aldrich Cetrimide Agar
70887 Sigma-Aldrich Cetrimide Agar
P2102 Sigma-Aldrich Pseudomonas Agar Base
14521 Sigma-Aldrich Cetrimide Agar Plates (Diameter 55 mm)
17168 Sigma-Aldrich Milk Agar, modified according to Brown & Scott
17208 Sigma-Aldrich Pseudomonas Isolation Agar


Cat. No. Brand Selective Agars with Differential System for
Differentiation, Detection and Isolation
50875 Sigma-Aldrich GSP Agar
78996 Sigma-Aldrich HiFluoro (™) Pseudomonas Agar Base


Table 2 Test for identification and differentiation of Pseudomonas

Cat. No. Brand Diagnostic Tests for Pseudomonas
88597 Sigma-Aldrich Catalase Test
05686 Sigma-Aldrich DMACA Indole Disks
49825 Sigma-Aldrich DMACA Reagent
78719 Sigma-Aldrich Kovac‘s Reagent Strips
60983 Sigma-Aldrich Kovac‘s Reagent for indoles
67309 Sigma-Aldrich Kovac‘s Reagent for indoles
08714 Sigma-Aldrich Methyl Red Solution
70439 Sigma-Aldrich Oxidase Test
40560 Sigma-Aldrich Oxidase Strips
07345 Sigma-Aldrich Oxidase Reagent acc. Gaby-Hadley A
07817 Sigma-Aldrich Oxidase Reagent acc. Gaby-Hadley B
18502 Sigma-Aldrich Oxidase Reagent acc. Gordon-McLeod

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1. Collins, F.M. Pasteurella, Yersinia, and Francisella. In Barron‘s Medical Microbiology, 4th ed., S. Barron, et al., eds., University of Texas Medical Branch at Galveston: Galveston, TX, 1996; Chapter 27.
G C Mead, B W Adams
British Poultry Science 1977-11-01
1. A new selective medium (CFC) has been developed for the rapid isolation of pigmented and non-pigmented pseudomonads associated with the spoilage of poultry meat held under chill conditions. It comprises Difco Heart Infusion Agar supplemented with 50 microgram cephaloridine, 10 microgram fucidin and 10 microgram cetrimide/ml. ...Read More
Jean-Marie Meyer, Valérie A Geoffroy, Nader Baida, Louis Gardan, Daniel Izard, Philippe Lemanceau, Wafa Achouak, Norberto J Palleroni
Applied and Environmental Microbiology 2002-06-01
A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specifici...Read More
Gee W Lau, Daniel J Hassett, Huimin Ran, Fansheng Kong
Trends in Molecular Medicine 2004-12-01
Pyocyanin (PCN) is a blue redox-active secondary metabolite that is produced by Pseudomonas aeruginosa. PCN is readily recovered in large quantities in sputum from patients with cystic fibrosis who are infected by P. aeruginosa. Despite in vitro studies demonstrating that PCN interferes with multiple cellular functions, its impo...Read More
Sandra Matthijs, Kourosch Abbaspour Tehrani, George Laus, Robert W Jackson, Richard M Cooper, Pierre Cornelis
Environmental Microbiology 2007-02-01
Under conditions of iron limitation Pseudomonas fluorescens ATCC 17400 produces two siderophores, pyoverdine, and a second siderophore quinolobactin, which itself results from the hydrolysis of the unstable molecule 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Pseudomonas fluorescens ATCC 17400 also d...Read More
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