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Improvement of the soil bacteriological research methods on existence of anthrax spores
Statistical data on diseases of animals and humans anthrax in recent years show a gradual decrease of outbreaks in
Ukraine. However, the risk of new outbreaks of the disease is stored for decades. According to WHO data, the greatest inci-
dence of anthrax falls on the territory of Europe.
The aim of our study was to improve the selection method of dispute Bac. anthracis from soil. It is known that anthrax
the pathogen Bacillus anthracis is not fastidious to nutrient, grows well on ordinary nutrient media. After cultivation of sow-
ings it was found a slight difference in the number of units that form colonies (CFU) at different nutrient mediums: meat-
peptone agar (MPA) MPA blood, Hettinger’s agar, soda MPA, trypton-soy agar (TSA) and environment PLET.
According to the obtained of preliminary results of studies to extract Bacillus anthracis spores from the soil we have
used the mediums MPA and PLET. Due to the fact that the methods №2 and №3 of soil investigation on the presence of
spores as described in the guidelines "Laboratory diagnosis of anthrax in humans and extraction of anthrax from objects in
the environment" and "Laboratory diagnosis and detection of anthrax" are identical, so we have received identical results.
By results of researches while studying methods № 1 the number of spores introduced in 100 g soil was highest
(6969±9,46), compared to techniques according to № 2–5. However, using the methodology № 1, when using the nutrient
medium MPA, with concentration 6969 spores in the 100 g soil mass in average were extracted 200 CFU significantly lower
(p<0,001), that probably higher at 157 of cells compared with the method № 2, but in 100 cells decrease with method № 5
(р<0,001).
During the research of the samples with the method №1 and cultivation in medium PLET was showed insignificantly
greater amount of CFU (30 CFU) than in the medium of the cultivation MPA, which was significantly (p<0,001) higher,
number of CFU relative to the results obtained by the method № 2, at the same time, but significantly (p<0,01) lower relative
performance obtained by the methods № 5.
That is, with the method №1, only 200 CFU were extracted at the concentration of 6969 spores per 100 g of soil, which
corresponded to 2,87% of the total number of spores introduced into the soil. According to the results of research with the
method № 2, № 3 on the MPA was exacted the least amount of CFU – 43 comparing to the № 1 and № 5 methods – 200;
300, respectively. The amount of spores, introduced into the 100 g of soil was per 221 units more compared with the method
№ 4, and per 2729 more than in the soil with the method № 5.
The quantities of spores in 1 g of soil when determining with the method № 4 was 4182 CFU, so with this method is ex-
tracted CFU 4,09% that per 1,22% more, compared with the data obtained in the first method and per 3,12% more, compared
with indicators obtained with the method № 2 and № 3.
With applying the methodology № 5 it was extracted 300±0,22 CFU, while making 1673 spores per 100 g of soil. That
was on 5297 spores in 100 g soil less, compared to the number of spores made with the method № 1 and per 2729 – with
the method № 2, and per 2508 – with the method № 4.
It should be noted, that this method (№ 5) is more effective, compared with other methods. It detects spores at the their
slightest amount in 100 g of soil. However, with this method it is necessary to study only 2,5 g soil, whereas with the method
of № 1 – 60 g, with the method of № 2 – 95 g, № 4 – 10 g.
It should be mentioned, that the reliability of the results is much higher with the method № 5, while it does not require
large expenditures of time and reagents.
The results of the application of the methodology for the allocation of № 5 soil of spores is more effective compared to
methods № 1–4, it is confirmed more allocation CFU of 100 g soil on nutrient media and MPA and PLET. This technique
gives the opportunity to achieve results that will have a higher degree of reliability. In the future, further testing techniques
planned selection spores Bacillus anthracis is non-sterile soil.
Key words: anthrax, extraction, spores, soil, cultivation.
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