Mycobacterium tuberculosis grows on blood agar
The causative agent of tuberculosis Mycobacterium tuberculosis is a slow-growing microorganism.
M. tuberculosis was first isolated by R. Koch from pulmonary tuberculosis after 10 days of incubation in a medium containing coagulated lamb and bovine serum. The facts of isolating mycobacteria are also described using blood agar, but this is enriched egg agar, which since 1920 has become the standard for the isolation of M. tuberculosisbecause of the convenience of its sterilization. Subsequently, according to the information available to the authors of this communication, no comparative study of the effectiveness of blood agar and egg media has been carried out, and even the ability of blood agar to support the growth of M. tuberculosis has been forgotten.
Dense agar or egg culture media containing malachite green, as well as broth or dense Middlebrook medium, is recommended as a "gold standard" for isolation, culture and differential diagnosis of M. tuberculosis.
Despite a literature report on the isolation of M. tuberculosis from blood agar (M. Arvand, ME Mielke, T. Weinke, T. Regnath, H. Hahn, Lettr, Infection 26: 254, 1998), for decades it was believed that the isolation of these microorganisms required a special egg environment, such as the Lowenstein-Jensen medium.
An article published in April 2003 in the Journal of Clinical Microbiology reports the random allocation of M. tuberculosis on blood agar.
A group of French researchers, after a prolonged incubation of the ganglionic tissue on blood agar to isolate Bartonella henselae, was surprised to receive M. tuberculosis instead.
The purpose of the study following this case was to determine whether blood agar during prolonged incubation can assure the growth of strains of M. tuberculosis isolated from various samples.
As a result of the study, it was found that modern blood media, widely used in almost all cases for the primary isolation of bacteria from clinical material, are at least as effective as the widely recommended Levenshtein-Jensen medium.
The study included 23 positive samples with Tsil-Nielson staining (15 samples of material from the respiratory tract and 8 aspirations from the lymph nodes). All the samples were introduced simultaneously into tubes with Coletsos egg medium (enriched in Levenshtein-Jensen medium, bio-Merieux, France) and 5% blood agar (with sheep blood) (Bio Technologie, France ). Sputum for homogenization and decontamination was pretreated with dithiothreitol and 2% NaOH. The inoculated tubes were incubated at 37 ° C in a normal atmosphere (without CO2) for 30 days and examined every 2 days for the presence of colonies. Identification of the isolates was confirmed by acid-resistant staining and probe hybridization to a 16S rRNA target (GenProbe, San Diego).
Contamination of crops has not been observed; M. tuberculosis were isolated from all samples on two types of media after 30 days of incubation. On blood agar, the colonies had a typical morphology: small, wrinkled, non-pigmented. The differences in time of onset and size of colonies on blood agar and on egg agar were not statistically significant (p = 0.11).
Among the samples containing, under microscopy, more than 50 acid-resistant rods in the field of vision, M. tuberculosis were isolated after 1 week since the start of incubation, from samples containing less than 1 acid-resistant rods in the field of vision, after 2 weeks of incubation.
In subsequent experiments, the researchers compared the growth of 38 strains of M. tuberculosis isolated from sputum and lymph nodes on two media. 10 µl of a suspension containing 106-mycobacteria / ml of each isolate was applied to egg agar and 5% blood agar in test tubes, incubated for 6 days at 37 ° C. After 6 days, 21 of the 38 isolates developed on egg agar and 27 out of 38 on blood agar. The mean number of colonies on blood agar (1,049 ± 2,867) was significantly higher than on egg agar (621 ± 2,256) (P less than 0.001).
The materials in this report demonstrate that the primary isolation of M. tuberculosis from clinical material can be achieved within 10-15 days of incubation, transplanting to the same medium after 6 days of incubation. Thus, provided the medium does not dry out (using test tubes instead of plates), blood agar is a suitable alternative for the primary isolation of M. tuberculosis and may be even more appropriate for transplanting these bacteria. Using blood agar can also detect other "finicky" slow-growing microorganisms that may be present in clinical material with M. tuberculosis (for example, Bartonella spp.). However, since blood agar is a non-selective medium, it may be more suitable for testing uncontaminated samples. If it is impossible to urgently deliver clinical material to the reference laboratory in special environments, it must be immediately applied to blood agar.
When bacteriologically examining the tissue of enlarged lymph nodes, the use of blood agar and prolonged incubation is recommended. The data obtained suggest that, with Bartonella spp. and other demanding pathogens, the laboratory can meet M. . tuberculosis. Therefore, the high infectious properties of M. tuberculosis must be combated by appropriate microbiological safety measures.