The bismuth sulphite agar is a solid medium selective and differential culture, specially formulated for isolation of Salmonella enterica subspecies enterica serotype Typhi, among other species of Salmonella. The medium is known as BSA agar for its acronym in English Bismuth Sulfite Agar.
The original formula for bismuth sulfite agar was created in 1927 by Wilson and Blair (Glucose Bismuth Sulphite Iron Medium); it contained sodium sulfite, glucose, bismuth solution, ammonium citrate, ferrous sulfate, and agar-agar.
Growth of 48 hours of incubation of Salmonella sp on bismuth sulfite agar. Source: Pixnio.com Author: Dr. WR Erving, USCDCP
Today there is a modification of the original medium, composed of meat extract, meat and casein peptones, bismuth sulfite indicator, glucose, disodium phosphate, ferrous sulfate, bright green and agar-agar.
There are many means for the isolation of Salmonella species, but when it comes to recovering the Typhi serotype, bismuth sulfite agar has a notable advantage over them, since in most a very low or no recovery of this microorganism is obtained.
However, it is necessary to use more than one type of medium when trying to isolate enteropathogens, because bismuth sulfite agar is less effective for other species of Salmonella and for the genus Shigella, which are inhibited or develop very poorly. in this agar.
It should be noted that of all Salmonella species, the Typhi serotype is one of the most important enteropathogens in humans, this being its only reservoir. This serovar causes typhoid fever, gastroenteritis, bacteremia, and septicemia.
For this reason, it is relevant to include this agar when analyzing water, feces or food samples where its presence is suspected.
Basis
Like most culture media, Bismuth Sulfite Agar contains nutrients to promote bacterial growth, such as peptones and meat extract. Likewise, glucose functions as a source of energy and carbon.
However, not all bacteria will grow on this medium, as Bismuth Sulfite Agar is a selective medium. It contains compounds that inhibit the growth of Gram positive microorganisms and certain Gram negative bacteria. These compounds are: the indicator bismuth sulfite and bright green.
For its part, disodium phosphate maintains the osmolarity and pH of the medium.
Additionally, bismuth sulfite agar is a differential medium thanks to the presence of ferrous sulfate, which shows the formation of H 2 S. The H 2 S formed by bacteria reacts with ferrous sulfate and forms a clearly visible insoluble black precipitate.
Finally, the agar-agar provides the solid consistency to the medium.
Preparation
Weigh out 52.3 g of the dehydrated medium and dissolve in one liter of water. Heat the mixture to boil for 1 minute with frequent stirring, until completely dissolved. Do not overheat too much. This medium is not autoclavable, because extreme heat damages the culture medium.
Allow to cool to 45 ° C and shake before serving in sterile Petri dishes. It is recommended to make plates with good thickness. To do this, 25 ml must be poured into each plate. Let solidify. As it is a medium that is not sterilized, it is normal for its immediate use to be suggested.
However, a study carried out by D'aoust in 1977, showed that there is a better recovery of Salmonella typhimurium and Salmonella enteritidis as the bismuth sulfite agar medium ages, not affecting the performance for the serovars Typhi and Paratyphi B.
D'aoust recommends using the plates on day 4 of refrigeration, although he notes that as the medium ages, selectivity decreases, and Proteus vulgaris strains develop more easily.
For this reason, for highly contaminated samples, such as feces, it is preferable to use freshly prepared medium. Otherwise use on day 4 of its preparation. Other authors recommend using the plates the day after their preparation, stored in the refrigerator.
Chilled plates must be tempered before use. The pH of the medium should be 7.5 ± 0.2. The raw medium is beige and the prepared medium is greenish-gray opalescent.
Applications
Among the samples that can be planted in this medium are samples of faeces, drinking or waste water and food.
To improve the isolates, it is recommended to carry out a pre-enrichment treatment with lactose broth and after enrichment with tetrathionate broth or cystine selenite broth, before seeding on bismuth sulfite agar.
The plates are incubated at 35 ° C ± 0.2 for 24 to 48 hours, aerobically.
Characteristics of the colonies on bismuth sulfite agar
Salmonella Typhi colonies are typically seen on this agar within 24 hours with a black center and surrounded by a bright green halo. Whereas, in 48 hours they turn completely black due to the formation of hydrogen sulfide.
-This medium inhibits most species of the genus Shigella.
- S. Typhi and S. arizonae can give very similar colonies.
-The coliforms that produce H 2 S such as Proteus and Citrobacter produce colonies similar to those of Salmonella, so it is necessary to carry out biochemical identification tests.
-A good striation must be performed to obtain isolated colonies; It is the only way to observe the typical characteristics of colonies of the genus Salmonella.
QA
For the sterility control, an uninoculated plate is incubated at 37 ° C, it is expected that there will be no growth or color change.
For quality control, known strains such as:
Escherichia coli ATCC 25922, Salmonella enteritidis ATCC 13076, Salmonella Typhi ATCC 19430, Shigella flexneri ATCC 12022, Enterococcus faecalis ATCC 29212.
Escherichia coli and Shigella flexneri are expected to be partially inhibited by developing greenish-brown and brown colonies respectively. Whereas, both salmonellas must have an excellent development with black colonies with metallic luster, and finally Enterococcus faecalis must be totally inhibited.
References
- Wilson, W., & EM McV. Blair. Use of a Glucose Bismuth Sulphite Iron Medium for the Isolation of B. typhosus and B. proteus. The Journal of Hygiene, 1927; 26 (4), 374-391. Retrieved from.jstor.org
- D'aoust JY. Effect of storage conditions of the performance of bismuth sulfite agar. J Clin Microbiol. 1977; 5 (2): 122–124. Available in: ncbi.nlm.nih.gov
- IVD Laboratories. Bismuth-sulfite agar according to WILSON-BLAIR. 2009.Available at: BismuthSulfitagar_span_Jan_2009% 20 (2).pdf
- Himedia Laboratories. Bismuth Sulphite Agar. 2017.Available at: himedialabs.com
- Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
- Morales R, de la Cruz D, Leyva G and Ybarra M. Bacteriological Quality of Raw Goat Milk Produced in Miravalles, Puebla. Rev Mex de Ing Quím 2012; 11 (1): 45-54