Assessment of the Use of Gentian Violet and Malachite Green as Selective Agents in the Isolation of Brucella

The aim of the work is to evaluate ability of gentian violet and malachite green to inhibit 2 Brucella test strains and 8 strains of unwanted organisms and the influence of autoclaving on their activity. The study also aims to evaluate variability of inhibition in different batches of colorants and necessity in adjustment of their concentration.

Materials and methods. The study included 3 imported lots of gentian violet and malachite green. Inhibitors were put into nutrient medium containing pancreatic hydrolysates of gelatin and fsh meal, yeast extract, sodium chloride, glucose, sodium pyrosulfate, and agar. The same medium without colorants was used as a control medium. Effect of the colorants was studied with the help of test strains of Brucella abortus 19 ВА, B. melitensis Rev I, Enterococcus faecium VKM B-602, Escherichia coli ATCC 25922, Proteus vulgaris HX 19 222, Pseudomonas aeruginosa 22/99, Serracia plymuthica 1, Shigella flexneri 1a 8516, Staphylococcus aureus ATCC 25923, Yersinia enterocolitica C-187.

Results and discussion. We have determined biological parameters of nutrient media containing gentian violet (5,0; 2,5 1,25 mg/L) and malachite green (2.0; 1.0; 0.5 mg/L) when cultivating brucella. Gentian violet is able to inhibit Brucella greatly at concentration of 5 mg/L. S. aureus ATCC 25923 did not show any growth on the media containing malachite green or gential violet (5.0 and 2.5 mg/L). Malachite green inhibited the growth of enterococcus more actively than gentian violet. However, it inhibited swarming growth of Proteus less successfully than gentian violet. Other unwanted organisms were not sensitive to such concentrations of colorants at all. We have demonstrated the necessity to combine colorants with other selective agents, as well as advisability to put gentian violet into sterile media, while malachite green – into the media before its autoclaving. Adjustments to the concentration of gentian violet should be made when producing nutrient medium, as variations in inhibiting activity in different batches of this colorant were revealed.

1. Shepelin A.P., Dyatlov I.A. [Nutrient Media for Enterobacteria]. Moscow: Publishing house “Dynasty”; 2017. 76 p.

2. Podpletennya I.M., Mishchenko D.N. [Modifed Kessler medium for the detection of E. coli bacteria]. RF patent No. 2202618, publ. 20.04.2003. Bul. No. 11.

3. Vasil’ev D.A., Shcherbakov A.A., Karpunina L.V., Zolotukhin S.N. [Special Bacteriology Methods. Study Guide]. Ulyanovsk: Ulyanovsk State Agricultural Academy; 2004. 222 p.

4. [Nutrient media for the cultivation of brucellosis]. (Cited 17 Nov 2020). [Internet]. Available from: https://virus-infekciya.ru/bakterialnye/pitatelnye-sredy-dlya-kultivirovaniya-brucelleza.html.

5. Jones L.M., Morgan W.J.B. A preliminary report on a selective medium for the culture of Brucella, including fastidious types. Bull. World Health Organ. 1958; 19(1):200–3.

6. Keness J., Friedrich I., Banai M., Schonfeld S. Brucella melitensis growth on Loewenstein-Jensen egg medium from a case of Brucella meningitis. Diagn. Microbiol. Infect. Dis. 1993; 17(4):271–3. DOI: 10.1016/0732-8893(93)90035-6.

7. Friedrich I., Schonfeld S., Keness Y. The ability of Brucella melitensis to grow on Loewenstein-Jensen egg medium: presentation of a case with Brucella meningitis. Isr. J. Med. Sci. 1992; 28(11):806–7.

8. Hornsby R.L. Selective media for isolation of Brucella abortus strain RB51. Iowa, Ames: Iowa State University; 1998. (Cited 22 Aug 2019) [Internet]. Available from: https://lib.dr.iastate.edu/rtd/17852.

9. Ponomarenko D.G., Rusanova D.V., Khachaturova A.A., Skudareva O.N., Logvinenko O.V., Rakitina E.I., Kostyuchenko M.V., Semenko O.V., Maletskaya O.V., Кulichenko A.N. [Analysis of the epidemic and epizootic situation on brucellosis around the world in 2019 and the forecast for the Russian Federation for 2020]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2020; (2):48–56. DOI: 10.21055/0370-1069-2020-2-48-56.

10. Karagul M.S., Ikiz S. The evaluation of Brucella spp. isolation rates in ruminant abortion cases by using different selective media. Mac. Vet. Rev. 2018; 41(2):177–86. DOI: 10.2478/macvetrev-2018-0024.

11. Vicente A.F., Antunes J.M.А.Р., Lara G.H.В., Mioni M.S.R., Allendorf S.D., Peres M.G., Appolinário C.M., Listoni F.J.P., Ribeiro M.G., Megid J. Evaluation of three formulations of culture media for isolation of Brucella spp. regarding their ability to inhibit the growth of contaminating organisms. BioMed. Res. Int. 2014; 2014:702072. DOI: 10.1155/2014/702072.

12. Kovtun Yu.S., Kurilova A.A., Katunina L.S., Vasilenko E.I. [Comparative evaluation of protein hydrolysates in the process of constructing based on them nutrient medium for brucella cultivating]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2016; (4):93–7. DOI: 10.21055/0370-1069-2016-4-93-97.