Pseudomonas Media and Tests

Jvo Siegrist

Analytix Volume 2007 Article 5

ivo.siegrist@sial.com

Detection, Identification, Differentiation, and Cultivation of Pseudomonas Species

Figure 1. HiFluoro Pseudomonas Agar under UV light

Pseudomonas 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 wounds.

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 as well as 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 industry.

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 (Figure 2). Another feature associated with Pseudomonas is the secretion of pyoverdin (fluorescein, a siderophore), a fluorescent yellow-green pigment under iron-limiting conditions. Certain Pseudomonas species may also produce additional pigments, such as pyocyanin (blue pigment, a siderophore) by P. aeruginosa, quinolobactin (yellow, dark green in presence of iron, a siderophore) by P. fluorescens, a reddish pigment called pyorubrin and pyomelanin (brown pigment). On blood agar a hemolytic reaction can be observed.

Figure 2. Oxidase test

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

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. We supply 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 and diagnostic tests.

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

Table 1 Media for Pseudomonas

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

References

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2. Mead GC, Adams BW. 1977. A selective medium for the rapid isolation of pseudomonads associated with poultry meat spoilage. British Poultry Science. 18(6):661-670. https://doi.org/10.1080/00071667708416418

3. Meyer J, Geoffroy VA, Baida N, Gardan L, Izard D, Lemanceau P, Achouak W, Palleroni NJ. 2002. Siderophore Typing, a Powerful Tool for the Identification of Fluorescent and Nonfluorescent Pseudomonads. AEM. 68(6):2745-2753. https://doi.org/10.1128/aem.68.6.2745-2753.2002

4. Lau GW, Hassett DJ, Ran H, Kong F. 2004. The role of pyocyanin in Pseudomonas aeruginosa infection. Trends in Molecular Medicine. 10(12):599-606. https://doi.org/10.1016/j.molmed.2004.10.002

5. Matthijs S, Tehrani KA, Laus G, Jackson RW, Cooper RM, Cornelis P. 2007. Thioquinolobactin, a Pseudomonas siderophore with antifungal and anti-Pythium activity. Environ Microbiol. 9(2):425-434. https://doi.org/10.1111/j.1462-2920.2006.01154.x