Blood agar

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Alpha hemolysis on cooked blood agar plate

Blood agar is used in microbiology

  • as a breeding ground for microorganisms that need components of the blood of mammals for growth,
  • to study the effects of microorganisms on red blood cells (erythrocytes), for example the dissolution of erythrocytes.

The culture medium consists of 5 to 10% defibrinated blood (usually sheep or horse blood, sometimes rabbit blood, rarely human blood; pig blood only for special cases) and enables the detection of certain classes of pathogens and the assessment of the haemolytic properties of the cultured bacteria ( e .g . Streptococci ). The blood agar can also be used with an addition of antibiotics .

Types of hemolysis

Beta hemolysis on blood agar plate
  • Alpha -hemolysis (alpha-hemolysis): The bacteria do not produce any hemolysins , they cause a greenish zone on blood agar, which is not due to real hemolysis , but to discoloration and a loss of potassium in the red blood cells. The reduction of the red blood pigment hemoglobin to biliverdin causes the green coloration, the alpha hemolysis is therefore also known as greening. There are still intact erythrocytes .
  • β-hemolysis (beta-hemolysis): The bacteria produce streptolysin O or S and are surrounded by a clear zone of hemolysis. The hemoglobin is completely broken down, this is a real hemolysis. In this area all erythrocytes are completely hemolyzed.
  • γ-hemolysis (gamma-hemolysis): These bacteria show no hemolytic behavior.

Types of blood agar used

Base for blood agar

For the quick preparation of ready-to-use Petri dishes with the blood agar nutrient medium , a ready-made mixture of the dried components - with the exception of the blood - is often used. Such granulates or powders contain, for example (data in g / l):

The figures indicate the mass concentration of the constituents (in grams per liter ) in the nutrient medium mixed with water. The pH should be 7.3. Either blood is then added or the nutrient medium can also be used without this addition for the cultivation of demanding microorganisms , but then no hemolysis can be detected.

Manufacture of blood agar

After the basic nutrient medium (see above) has cooled to a temperature below 50 ° C., a certain volume of defibrinated blood is mixed in and the nutrient medium is filled into Petri dishes. The recommended amount of blood is usually 50–80 ml (based on one liter of blood agar) or a proportion of 5–10% in the finished medium. Usually sheep blood or rabbit blood is used. After the addition of blood, the pH value should be 6.8 so that the erythrocytes are retained. The filled, solidified blood agar can be used for up to three months if stored in a cool place.

Instead of the basic nutrient medium with heart extract and a nutrient medium, on the basis of soybean - Tryptone be used. A different production method is recommended for the cultivation of some bacteria. Thus, the isolation of succeeds Mycobacterium tuberculosis and other pathogens of the tuberculosis from sputum with a blood agar, 3.0% of human blood, 0.1% glycerol and penicillin contains.

Cooked blood agar

Colonies of Neisseria gonorrhoeae on cooked blood agar

The chocolate agar (because of its color also erroneously chocolate agar called) is a variant of blood agar, in which by brief heating of the blended with blood medium at 80 ° C, a lysis of the erythrocytes is obtained. The lysis releases hemin (“factor X”) and NAD + (“factor V”) into the nutrient medium, which causes the agar to turn medium brown in color. The released growth factors can be metabolized there by bacteria that are not hemolytic themselves (e.g. Haemophilus influenzae ). Cooking blood plates are sometimes incubated in an atmosphere with an increased CO 2 concentration, especially capnophile germs, i.e. CO 2 -preferring bacteria, as well as all Neisseria reproduce much better.

Columbia blood agar

In 1966, the development of a nutrient medium with a new blood agar composition called Columbia agar or Columbia blood agar was reported. It is often used as Columbia agar with 5% sheep blood (English: Columbia agar with 5% sheep blood ) and has the advantage that the colonies of bacteria on this blood agar grow faster and are larger in comparison to other blood agar culture media. The improved growth of the bacteria is attributed to the use of two different peptones and yeast extract as a source of B vitamins .

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

  • Pancreatically degraded casein 12.0
  • Peptically degraded animal tissue 5.0
  • Yeast extract 3.0
  • Beef extract 3.0
  • Corn starch 1.0
  • Sodium chloride 5.0
  • Agar-agar 13.5
  • defibrinated sheep blood 5%
Colonies of Staphylococcus aureus on Columbia horse blood agar

Instead of sheep blood, horse blood can also be used; the nutrient medium thus obtained is z. B. as Columbia agar with 5% horse blood (English: Columbia agar with 5% horse blood or simply Columbia horse blood agar ). Columbia blood agar is recommended in the microbiological-infectious quality standards (MiQ) of the German Society for Hygiene and Microbiology (DGHM) as a nutrient medium for the initial isolation of numerous clinical samples. The nutrient media inoculated with the sample material are then incubated at a temperature of 35 ± 2 ° C. in an aerobic atmosphere enriched with carbon dioxide (CO 2 ) .

Numerous microorganisms which are of interest as pathogens in medical microbiology grow on this medium, for example representatives of the Enterobacteriaceae family , Pseudomonas species and other aerobic gram-negative rods. In the case of gram-positive bacteria, the nutrient medium is u. a. suitable for streptococci , enterococci , staphylococci and corynebacteria . Representatives of the yeast genus Candida can also be cultivated with it.

Blood agar with antibiotics

To isolate some bacteria as selectively as possible, a blood agar with the addition of a certain antibiotic or several antibiotics can be used to inhibit the rest of the accompanying flora.

CNA blood agar

The CNA blood agar , or CNA agar for short, is a further development of the Columbia blood agar . The name of the CNA agar refers to the two antibiotics contained in the culture medium, colistin (polymyxin E) and nalidixic acid . They are usually contained in a mass concentration of 10 mg / l each, the further composition corresponds to that of Columbia blood agar with 5% sheep blood. The two additionally contained active ingredients suppress the growth of gram-negative bacteria, so that CNA agar is used as a selective medium for the isolation of gram-positive bacteria (mainly streptococci and staphylococci) from numerous clinical and non-clinical samples.

Yeasts such as Candida are not inhibited in growth and Gram-negative bacteria to the antibiotics used resistant , are able to grow on CNA agar. There are slightly modified variants which, in addition to a lower concentration of nalidixic acid, also contain a third antibiotic - aztreonam - which is supposed to inhibit these gram-negative bacteria.

Blood agar with other antibiotics

Another example of a blood agar with the addition of an antibiotic is the gentamicin blood agar (GBA), which is based on the Columbia blood agar (with 10% sheep blood) and additionally contains 5.5 mg / l gentamicin . This nutrient medium enables the selective cultivation of Streptococcus pneumoniae and other streptococci, as well as species from the genera Bacteroides , Clostridium and various yeasts.

In order to selectively isolate Aeromonas species from human and animal faecal samples , the addition of ampicillin is recommended. Tests of various selective media for Aeromonas have shown that a Columbia blood agar (with 5% sheep blood) with an addition of 30 mg / l ampicillin achieves the highest recovery rate (98%). Such a medium is also referred to as ASBA 30 (ampicillin sheep blood agar with 30 mg / l ampicillin).

The American Public Health Association (the Food and Drug Administration in the US) recommends in its guidelines for microbiological analysis of food, among others, the addition of the following antibiotics: ampicillin, cycloheximide , moxalactam , novobiocin , oxytetracycline and polymyxin B .

See also

Individual evidence

  1. ↑ a b 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 , p. 562.
  2. ↑ a b c d e f Technical information blood agar (base). In: Website of Carl Roth GmbH & Co. KG. Retrieved December 26, 2013 .
  3. ↑ a b c d e f data sheet blood agar (PDF) from Merck , accessed on February 11, 2013.
  4. ↑ a b Bacteriological Analytical Manual, Media 20: Blood Agar. In: Website of the Food and Drug Administration (FDA). April 10, 2013, accessed December 26, 2013 .
  5. ↑ MS Tarshis et al .: Further experience with a new blood medium for the cultivation of Mycobacterium tuberculosis . In: American Journal of Public Health and the Nation's Health . tape 45 , no. 9 , September 1955, p. 1157–1161 , doi : 10.2105 / ajph.45.9.1157 , PMID 13249005 , PMC 1623456 (free full text).
  6. ↑ a b Herbert Hof and Rüdiger Dörries: Medical Microbiology . 5th edition. Thieme, Stuttgart 2014, ISBN 978-3-13-152965-7 , pp. 428 .
  7. ↑ a b c d e f QC / PI Manual: Columbia Agar with 5% Sheep Blood. In: Becton, Dickinson and Company (BD) website . Retrieved December 26, 2013 . ( German-language instructions for use, as of April 2013; PDF; 36 kB).
  8. ^ PD Ellner et al .: A new culture medium for medical bacteriology . In: American Journal of Clinical Pathology . tape 45 , no. 4 , April 1966, p. 502-504 , doi : 10.1093 / ajcp / 45.4_ts.502 , PMID 5325709 .
  9. ↑ Columbia agar + 5% horse blood. In: website of bioMérieux Deutschland GmbH. Retrieved December 26, 2013 .
  10. ↑ Issues 3, 6 and 7 . In: German Society for Hygiene and Microbiology [DGHM]. A. Podbielski, M. Herrmann, E. Kniehl, H. Mauch (Eds.): MiQ: Quality standards in microbiological-infectious diagnostics. MiQ basic work, issue 1–25 . 1st edition. Urban & Fischer Verlag in Elsevier GmbH, Munich 1997, ISBN 3-437-41569-7 .
  11. ↑ a b c FP Downes, K. Ito (Ed.): Compendium of Methods for the Microbiological Examination of Foods . 4th edition. American Public Health Association, Washington, DC 2001, ISBN 978-0-87553-175-5 , pp. 47, 210 .
  12. ↑ a b c QC / PI Manual: Columbia CNA Agar with 5% Sheep Blood. In: Becton, Dickinson and Company (BD) website . Retrieved December 26, 2013 . ( German-language instructions for use, as of April 2013; PDF; 36 kB).
  13. ↑ Columbia CNA Agar with 5% Sheep Blood, Improved II (German-language instructions for use, as of September 2011). (PDF; 48 kB) In: Website from Becton, Dickinson and Company (BD) . Retrieved December 26, 2013 .
  14. ^ WA Black, F. Van Buskirk: Gentamicin blood agar used as a general-purpose selective medium. In: Applied microbiology. Volume 25, Number 6, June 1973, pp. 905-907, PMID 4352009 . PMC 380938 (free full text).
  15. ^ S. Mishra et al .: Comparison of selective media for primary isolation of Aeromonas species from human and animal feces. In: Journal of Clinical Microbiology . tape 25 , no. November 11 , 1987, pp. 2040-2043 , PMID 3693536 .

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