Colorful series (laboratory)

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The so-called colored series is a laboratory method for the determination (identification) of bacteria based on various characteristics. It checks for certain enzyme activities , the breakdown of substrates , the formation of metabolic products and other abilities (e.g. active movement ). The Bunte range consists of test tubes and , if necessary, Petri dishes , which are loaded with various nutrient media . The culture media contain reagents and indicators for the detection of certain properties that enable a determination of bacteria at the level of the genus or even the species . The tubes are inoculated with a pure culture of the bacterium to be determined. The results (positive or negative) are compared with a table and so you can assign the unknown pure culture to a species with a certain probability.

Small colorful series

The oldest and most compact version is the traditional, small Bunte series , which is primarily used to determine enterobacteria . It contains: Kligler agar , urea agar , MIO tubes and Simmons citrate agar . The culture media are inoculated differently depending on the type of medium. In most cases the most common enterobacteria can be identified quickly, but there are exceptions and irregularities. The examination is more precise using a system in which 20 to 30 biochemical examinations can be carried out within 24 hours and evaluated visually or electronically (for example Analytical Profile Index API-20E).

A MacConkey agar is streaked out to check the purity . The colonies must look like the ones used to inoculate the Bunte series and no other colonies must grow on the agar.

Kligler agar

This is a nutrient medium for identifying gram-negative intestinal bacteria according to Kligler (1917, 1918), for example Salmonella , Shigella , Escherichia , Enterobacter and Proteus . It is used to demonstrate the lactose breakdown by the enzyme β-galactosidase , the usability of the existing carbohydrates in a fermentation , recognizable by the formation of acids during the breakdown and possibly the formation of gas ( CO 2 ), as well as the formation of hydrogen sulfide (H 2 S) . Instead of the classic Kligler agar with two contained sugars, TSI agar ( triple sugar iron agar ) is often used, which provides comparable results. The formation of H 2 S can also be detected with the help of SIM agar ( hydrogen sulfide formation, indole formation, motility ). The formation of hydrogen sulfide is typical for some species in the genera Citrobacter , Proteus (or Cosenzaea ) and Salmonella .

Mode of action

Different results of TSI agar (from left to right): (a) Bacterium can use various carbohydrates, including lactose and has produced gas, (b) Bacterium can only use glucose , (c) Bacterium has produced H 2 S, (i.e. ) no reaction or nutrient medium not inoculated.

The nutrient medium is used as a slanting agar tube. During the inoculation, the agar is first pierced vertically and then streaked out on the inclined surface in a meandering line. The breakdown of glucose produces acid and at pH <7 the pH indicator phenol red turns yellow. Due to the formation of alkaline metabolic products from the breakdown of peptones and the oxidation of the acids on the inclined surface in the presence of air oxygen , the nutrient medium in the upper area is re-alkalized (the pH value rises above 7) and this part of the medium turns red. If, in addition to glucose, lactose is broken down (β-galactosidase activity positive), additional acid is generated, then the entire nutrient medium remains yellow due to the larger amount of acid. If this takes place in fermentation without the use of oxygen, CO 2 is formed , which becomes visible as small to large gas bubbles and cracks in the nutrient medium. H 2 S formed from the sulfur-containing inorganic compound sodium thiosulfate reacts with the iron salt it contains to form iron sulfide , which is precipitated as a black precipitate.

Typical composition

The nutrient medium usually consists of (data in grams per liter ):

Urea agar

With the help of the urea agar according to Christensen (1946), it is possible to differentiate between bacteria that have the enzyme urease and those that are urease-negative. Urease is an enzyme that can hydrolyze urea . Representatives of the genera Klebsiella , Proteus and Yersinia have urease, while, for example, Escherichia , Providencia , Salmonella , Serratia and Shigella are urease-negative. The genera Citrobacter and Enterobacter have both urease-positive and -negative species.

Mode of action

Serratia odorifera on urea agar according to Christensen, with negative result (the microorganism does not have the enzyme urease, which hydrolyzes urea to ammonia).
Cosenzaea myxofaciens ( synonym : Proteus myxofaciens ) on urea agar according to Christensen, with positive result (the microorganism has the enzyme urease, which hydrolyzes urea to ammonia).

The urea agar can be used in test tubes as inclined agar tubes or in Petri dishes , a relatively large amount of the pure culture is spread out. The finished nutrient medium contains urea, if this is used by a bacterium that has the enzyme urease, carbon dioxide and ammonia are produced . The ammonia makes the medium alkaline (pH> 7) and the phenol red pH indicator it contains shows this by changing color from yellow to red-violet. If the bacterium does not have the enzyme urease, there is no breakdown of urea and the medium remains yellowish, due to the pH indicator it contains. Urease-negative bacteria can also grow on the medium as it contains glucose as a source of energy and carbon , but their growth does not change the color of the medium.

Typical composition

The nutrient medium usually consists of (data in grams per liter ):

MIO tubes

Positive indole test, recognizable by the red-violet discoloration of the reagent.

With the help of the MIO agar, bacteria from the order of the Enterobacterales (Enterobacteria) can be further differentiated from one another. The abbreviation MIO results from three characteristics that can be detected with this medium: (1) active range of motion ( M otilität, in English motility ). Active movement is caused by flagella in representatives of the Enterobacterales . (2) Formation of I ndol from tryptophan by the enzyme tryptophanase . (3) formation of putrescine from ornithine by the enzyme O rnithindecarboxylase (ODC). For example, Escherichia is positive for motility and indo formation, but negative for ODC, whereas Enterobacter is positive for motility and ODC, but negative for indo formation.

Mode of action

The MIO agar is used in test tubes as a high-layer tube and inoculated with the pure culture by puncturing an inoculation needle. The finished nutrient medium is a so-called soft agar , which contains agar-agar in a lower concentration than usual, namely only 1 - 4 g / L. In an agar gel with such a low agar concentration, flagellated bacteria can spread actively by swimming. This leads to a clouding of the medium by the bacteria outside of the branch duct, while in the case of bacteria without active movement only growth and thus clouding can be observed in the inoculation duct.

The medium also contains the amino acid L tryptophan as a substrate; some types of bacteria have the enzyme tryptophanase and can therefore break down tryptophan into indole, pyruvate and ammonia . The indole formed is detected in the indole test with Kovacs reagent . However, this test is only performed after incubation and evaluation of the other characteristics by adding the reagent. A cherry-red coloration of the solution indicates indole formation (indole-positive bacteria), if the solution remains colorless or turns yellowish, the bacterium is indole-negative.

The medium also contains the amino acid L - ornithine as substrate and the pH indicator bromocresol purple . If ornithine is used by a bacterium that has the enzyme ornithine decarboxylase (ODC), putrescine (butane-1,4-diamine) is produced through decarboxylation . This product makes the medium alkaline (pH> 7) and the included bromocresol purple pH indicator shows this by changing color from yellow to purple to violet, the ODC test is positive. If the bacterium does not have the enzyme ornithine decarboxylase, ornithine is not broken down and the medium appears yellow, due to the pH indicator it contains and the acids formed from glucose.

Simmons Citrate Agar

With the help of citrate agar according to Simmons (1926), bacteria can be identified that can grow with citrate as the only source of energy by generating energy through its breakdown. This applies to some representatives of the Enterobacteriaceae , as well as to some fungi. In the Enterobacteriaceae family, most members of the genera Citrobacter , Enterobacter and Klebsiella behave citrate-positive, the same applies to Serratia ( Yersiniaceae family ), while Escherichia and Shigella are citrate-negative. Species or serotypes of Salmonella behave differently.

Mode of action

Cosenzaea myxofaciens (synonym: Proteus myxofaciens ) on Simmons citrate agar, with negative result (the microorganism cannot utilize citrate).
Serratia odorifera on Simmons citrate agar, with positive result (the microorganism can utilize citrate).

The citrate agar can be used in test tubes as inclined agar tubes or in Petri dishes and is inoculated with a pure culture. The finished nutrient medium contains sodium citrate as the only energy source, the pH indicator bromothymol blue and only the inorganic compound ammonium dihydrogen phosphate as a nitrogen source . If a microorganism can utilize citrate (it then has the enzyme citrate lyase at its disposal), citrate is converted to acetyl-CoA and oxaloacetate . Oxaloacetate is further broken down into pyruvate and carbon dioxide (CO 2 ). Hydrogen carbonate (HCO 3 - ) is formed from the CO 2 with water, and ammonia (NH 3 ) is also released from the inorganic nitrogen source. These reactions lead to an alkalization (pH> 7) and the pH indicator bromothymol blue contained in the nutrient medium shows this by changing the color from green to blue. If the bacterium cannot utilize citrate, there is no pH increase and the medium remains green, due to the pH indicator it contains. In this case, however, there is no growth either, since it does not contain any other energy sources apart from citrate.

Typical composition

The nutrient medium usually consists of (data in grams per liter ):

Expanded Colorful Series

Since the small colored series does not always provide sufficient identification of the unknown pure culture, further tests can be carried out according to the same principle. To distinguish the enterobacteria, in particular which are imvic reactions carried out here is the above-mentioned I ndol-test, the M ethyl red sample to acid formation, the V Oge-Proskauer test on acetoin formation and also already described detection the C Itrat utilization .

In addition, the representatives of the Enterobacteriaceae, for the further differentiation and the SIM agar used, can be examined with the three characteristics simultaneously: the formation of S chwefelwasserstoff (H 2 S), the I ndol formation and the active movement ( M otilität ) of bacteria.

In addition, there are numerous other tests that are often arranged as a combination of many miniaturized reaction vessels in one system in order to be able to carry out a quick test, such as the test system Analytical Profile Index API-20E. Some of the test reactions used there or in comparable systems are also prepared and investigated on a conventional scale, that is to say in test tubes or microreaction vessels , and will be described using a few examples.

Test for nitrate reduction

Escherichia coli in the test for nitrate reduction, with a positive result.

With this test, it is investigated whether a bacterium nitrate to nitrite , or further to elemental nitrogen (dinitrogen N 2 ) reduce can. The reduction of nitrate to dinitrogen is known as denitrification and is a step in the nitrogen cycle . In the bacteria of the Enterobacteriaceae family, nitrate is only reduced to the level of nitrite.

Mode of action

In order to prove the nitrate reduction, a liquid complete medium (nutrient broth) is inoculated which, in addition to the components that enable growth such as glucose and peptone, also contains 1 g / L potassium nitrate (KNO 3 ). After the incubation, sulfanilic acid solution and 1-naphthylamine solution are added. With these reagents, formed nitrite is detected by a red color. If this test is positive, the bacterium has reduced nitrate to nitrite.

If no discoloration can be observed, a small amount of zinc dust is added. If the bacterium could not utilize nitrate, it is still present in the medium and is now reduced to nitrite, which can be recognized by the red color. In this case the bacterium is to be assessed as negative with regard to the nitrate reduction. If no color change can be observed after adding the zinc, the nitrate has been reduced to elemental nitrogen, which can be collected as a gas in a fermentation tube if necessary . The bacterium is therefore to be assessed as positive in terms of nitrate reduction.

Methyl red sample

Methyl red sample: Escherichia coli (left) with a positive result and Enterobacter cloacae (right) with a negative result.

With this test a further differentiation of the representatives of the enterobacteria is possible, it is examined whether a bacterium produces large amounts of acid in the fermentative breakdown of glucose . The gram-negative intestinal bacteria can be divided into two groups with regard to their fermentation products. On the one hand, there is the Escherichia coli type, which mainly forms various organic acids from glucose, in mixed acid fermentation and, on the other hand, the Enterobacter type, which is made from glucose in 2,3-butanediol fermentation , mainly butanediol (and acetoin as an intermediate product ) and gas (CO 2 and H 2 ) forms, but only a little acid. The formation of acid is detected with the methyl red test (also called MR test), while the formation of acetoin as a precursor of 2,3-butanediol can be demonstrated in the Voges-Proskauer test (also abbreviated as VP test).

Mode of action

For this detection, a liquid medium (nutrient broth) is inoculated, which only contains the components glucose and peptone necessary for growth, as well as potassium dihydrogen phosphate as a buffer substance . After the incubation, the medium is divided up, the Voges-Proskauer test is carried out with one part and the methyl red test with the other. For this purpose, an ethanolic solution of the pH indicator methyl red is added. Large amounts of acid formed lower the pH value significantly, the indicator shows this by changing color to red, the pH value is then below pH 4.5. The test is positive (MR-positive) and the enterobacterium belongs to the group of mixed acid fermenters . If the bacterium has produced little or no acid, the indicator shows a yellow color (pH> 4.5), the test result is MR-negative and the intestinal bacterium belongs to the group of butanediol fermenters , which is confirmed by the Voges-Proskauer test can be. An orange discoloration of the indicator (pH value in the range 5 to 6) is to be interpreted as a negative result, but must be confirmed with a positive VP test.

Summary of further tests

Other tests used within a Bunte series are tests for the utilization of various carbohydrates with acid formation in the OF test (oxidation-fermentation test, also known as the Hugh Leifson test) and for the formation of exoenzymes . This includes the gelatin liquefaction test, in which proteolytic enzymes can hydrolytically break down the protein gelatin ; the starch hydrolysis test , in which exo- amylases of the microorganisms are detected; the cellulose hydrolysis test, which detects exo- cellulases , or the DNase test (also called DNAse test), which detects the exoenzyme deoxyribonuclease, which microorganisms can use to break down DNA .

Further tests are based on the detection of endoenzymes, i.e. enzymes that are formed within the cell and catalyze metabolic processes there. In addition to the ornithine decarboxylase (ODC) already mentioned in the description of the MIO tube , there is evidence of lysine decarboxylase (LDC), here the bacteria are able to decarboxylate the amino acid L - lysine , whereby carcassine ( 1,5-diaminopentane ) arises. When testing for arginine dihydrolase (ADH), several reactions take place through bacterial enzymes that break down the amino acid L - arginine into ornithine, ammonia and carbon dioxide. The detection of ADH, LDC and ODC helps to determine the enterobacteria.

For the determination of the representatives of the Morganellaceae family , the detection of the enzyme phenylalanine deaminase (PAD) is also important. The representatives of the genera Morganella , Providencia and Proteus have this enzyme. The detection of tryptophan deaminase (TDA) is also common among them. The enzymes catalyze the deamination of the amino acids L - phenylalanine or L- tryptophan, the degradation products are detected by adding iron (III) chloride solution, and a dark green (PAD) or red-brown (TDA) color appears.

Individual evidence

  1. a b c d e f g h i j k Roland Süßmuth, Jürgen Eberspächer, Rainer Haag, Wolfgang Springer: Biochemical-microbiological internship . 1st edition. Thieme Verlag, Stuttgart / New York 1987, ISBN 3-13-685901-4 .
  2. a b Technical information Kligler agar for identifying gram-negative intestinal bacteria (iron two-sugar agar according to Kligler ). In: Merck Millipore website . Accessed January 1, 2020 .
  3. Technical information Triple Sugar Iron Agar (iron three-sugar agar). In: Merck Millipore website . Accessed January 1, 2020 .
  4. a b c d e f g h i j Michael T. Madigan, John M. Martinko, Jack Parker: Brock Mikrobiologie. German translation edited by Werner Goebel, 1st edition. Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin 2000, ISBN 978-3-8274-0566-1 .
  5. a b c d Technical information urea agar (base) according to Christensen (according to ISO 6579, ISO 10273, ISO 19250, ISO 21567). In: Merck Millipore website . Accessed January 1, 2020 .
  6. Eckhard Bast: Microbiological Methods: An Introduction to Basic Working Techniques . 2nd Edition. Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin 2001, ISBN 978-3-8274-1072-6 .
  7. a b c Technical information Simmons Citrate Agar. In: Merck Millipore website . Accessed January 1, 2020 .
  8. ^ Betty A. Forbes, Daniel F. Sahm, Alice S. Weissfeld: BAILEY & SCOTT'S Diagnostic Microbiology , 10th edition, Don Ladig 1998 ,, ISBN 0-8151-2535-6 .
  9. ^ A b Hans G. Schlegel, Christiane Zaborosch: General microbiology . 7th edition. Thieme Verlag, Stuttgart / New York 1992, ISBN 3-13-444607-3 .
  10. BW Senior: Media and tests to simplify the recognition and identification of members of the Proteeae . In: Journal of Medical Microbiology . tape 46 , no. 1 , January 1997, p. 39-44 , doi : 10.1099 / 00222615-46-1-39 , PMID 9003744 .