How Alternative Differential t >g To IdentificationApproaches for ItI2 SImilar Baci i Commonly Studied in Microbiology Isaiah A. Benathen Two similarlyappearing, gram pos- reactionin this test while Bacillusmega- group D streptococci. After incuba- itive, aerobic, endospore forming terium shows a negative response. tion, a positive reaction on this me- Downloaded from http://online.ucpress.edu/abt/article-pdf/53/3/162/45120/4449251.pdf by guest on 30 September 2021 bacilli found in the soil which are This test poses some difficulties. The dium results in the hydrolysis of the studied in undergraduate microbiol- organic reagents alpha naphthol and glycoside esculin into glucose and es- ogy courses are Bacillus subtilis and potassium hydroxide-creatinemust be culetin. The esculetin produced reacts Bacillusmegaterium. These bacteriaare prepared. Alpha naphthol is light and with an iron salt in this media to form frequentlyused in exercises concerned temperature sensitive and should be a brown to black colored complex with the gram stain, the endospore stored in a darkened bottle in the (Power & McCuen 1988). stain and the series of exercises con- refrigerator.It is also a possible carcin- DNA medium is used to identify cerned with the differentialidentifica- ogen and must be handled with care pathogenic Staphylococcus aureus. tion of an unknown organism. (Claus 1989).These compounds have a DNase activity in Staphylococcusaureus The purpose of this paper is to strong irritating odor. Both reagents correlatesvery well with coagulase ac- present alternative simple procedures must be added to a 1 ml sample of the tivity in this organism (Oxoid Limited to the traditionalunknown tests which previously incubated glucose broth 1982).These two enzyme activitiesare permit a clear and unequivocal differ- culture of the organism. The tubes used to identify the pathogenic nature ential identification decision between must be thoroughly mixed to oxygen- of this organism (Difco Laboratories Bacillussubtilis and Bacillusmegaterium. ate the contents. A red-colored com- 1984). After incubation, cultures This paper shows that media used plex requiringoxygen for its formation which show DNase activity develop in the clinical microbiologylaboratory develops after 5 to 15 minutes of clear halos around the growth after for the identificationand characteriza- standing. This is a positive reaction. addition of 1N HCl to the plate. This tion of gram positive cocci such as Any red-brown color in the tube is halo is due to hydrolysis of the macro- Staphylococcusaureus and Enterococcus considered to be negative. The experi- molecule DNA to a mixture of single fecalis can be used to distinguish be- ence of the authorshows that students nucleotides and shorter polynucle- tween these two bacilli. These two have frequently misinterpreted this otides. These nucleotides are not pre- organisms are difficult to distinguish test and have been wrong in the dif- cipitated by reaction with 1N HCI. since they show the same gram reac- ferentialidentification of these two or- Where DNA was present, it forms a tion and identical responses on a wide ganisms. cloudy white precipitate in the agar range of test media used for purposes (Oxoid Limited 1982). of differentialidentification. Typically, both organisms show fermentation of The Alternative Solution dextrose to an Materials & Methods acids, absence of mixed This paper presents a simpler and acid fermentation, lack of indole and clearer alternative for distinguishing The hydrogen sulfide production, no cultures of Bacillussubtilis and between Bacillus subtilis and Bacillus growth on citrate and lack of urease Bacillusmegaterium are obtained from megaterium.The media used are plates activity. Both organisms are able to Presque Isle Cultures (P.O. Box 8191, of Phenylethyl Alcohol with hydrolyze macromolecules agar PresqueIsle, PA 16505).The trypticase such as Blood (PEAB), slants of Bile Esculin starch, proteins and lipids. soy broth without dextrose and bile agar and plates of DNA agar. These The results of the Voges Proskauer esculin medium are easily obtained media are used in the clinical test for the fermentation of glucose to microbi- from Difco Laboratories(Detroit, MI ology laboratory for the acetoin and the neutral product bu- differential 48232) or BBL (Cockeysville, MD identification of gram positive tanediol differs in these two organ- cocci. 21030). The DNA agar medium for PEAB is strongly selective for gram isms. Bacillussubtilis shows a positive these studies is obtained from Oxoid, positive cocci. It can be used to isolate U.S.A. (Columbia, MD 21045). It is enterococci which cause urinary tract also availablefrom Difco Laboratories. infections from urine cultures. This Bacillussubtilis and Bacillus Isaiah A. Benathen, Ph.D., is an associ- megate- ate professor of biological sciences at medium strongly inhibits gram nega- riumare inoculatedinto separatetubes Kingsborough Community College of tive bacteria(Power & McCuen 1988). of trypticase soy broth and incubated the City Universityof New York,Brooklyn, Bile Esculin agar is used for the for 18 hours at 30?C. Both organisms NY 11235. differentiationof the group D strepto- show good growth at this tempera- coccus Enterococcusfecalis from non ture. After incubation each organism 162 THEAMERICAN BIOLOGY TEACHER, VOLUME 53, NO. 3, MARCH1991 is separately streaked on plates of PEAB agar to isolate pure colonies. Table 1. Results observed on specialized media. are Separateslants of Bile Esculin agar Bile Esculin DNA Agar streaked and separate plates of DNA Microbe PEAB agar are streaked. The plates and S. aureus XXXXXX XXXXXXXX clear halo around slants are incubated for 72 hours at (control) growth 30?C. After incubation, 1N HCI is E. faecalis XXXXXXX black color XXXXXX added to the DNA agar plates. The (control) plates and slants are then evaluated. If B. subtilis gray colony with black color to clear halo around desired, a trypticase soy broth mixed white centers growth growth culture of these two bacteria can be B. megaterium white colony no color no halo prepared and incubated for five hours change at 30?C. This culture is then streaked onto PEAB agar to isolate the two bacilli. After overnight incubation, esculin hydrolysis and DNase activity students to the uses of readily avail- samples of the different colored colo- in B. subtilisare diagnostic. These pos- able selective media which make the nies can be separately streaked to Bile itive results for Bacillussubtilis permit identification of very similar bacilli Esculin agar slants and to DNA agar the differential identification of this clear and simple to perform and eval- plates. After incubation, the plates bacillus from B. megaterium.The re- uate. and slants are evaluated. sults are indicated in Table 1. Downloaded from http://online.ucpress.edu/abt/article-pdf/53/3/162/45120/4449251.pdf by guest on 30 September 2021 Results Conclusions References Bacillussubtilis forms gray colonies These investigations demonstrate Benathen, I.A. (1990). Isolation of pure with white centers on PEAB agar, that media used in the clinical micro- cultures from mixed cultures: A modern the identifica- approach. The AmericanBiology Teacher, while B. megateriumforms white colo- biology laboratory for 52(1), 46-47. nies on this medium. Growth of B. tion of gram positive cocci can be used Claus, G.W. (1989). Understandingmicrobes: subtilis on PEAB agar was demon- in a classroom setting to safely, easily A laboratoryworkbookfor microbiology. New strated earlier by this author (Bena- and conveniently distinguish between York:W.H. Freemanand Co. then 1990).The Bile Esculinagar slants two very similar gram positive bacilli Difco Laboratories(1984). Difcomanual: De- show unusual distinctive results. B. that are commonly studied in microbi- hydratedculture media and reagentsfor mi- subtilisproduces a black color in this ology classes. This paper also shows crobiology(10th ed.). Detroit:Difco Labo- medium, while B. megateriumhas no that these microorganismscan be con- ratories. effect on the color of this medium. The veniently identified without the neces- Oxoid Limited (1982). The Oxoidmanual of and culturemedia, ingredients, and otherlabora- observationof esculin hydrolysis by B. sity for preparing, refrigerating tory services(5th ed.). Hampshire, U.K.: subtilis, a bacillus, is highly unusual. using irritating,strong smelling chem- Oxoid Ltd. In addition, B. subtilis shows DNase icals. This alternativeapproach avoids Power, D.A. & McCuen, P.J. (1988). Man- activity while B. megateriumlacks this the potential risk of exposure of stu- ual of BBL productsand laboratoryproce- enzyme activity. The observations of dents and faculty to a possible carcin- dures(6th ed.). Cockeysville, MD: Becton different growth responses on PEAB, ogen. This paper also introduces the Dickinson MicrobiologySystems. JOIN NABT IN Teaching is NASHVILLE! easier with NOVEMBER6-10,1991 V ideodiscs STOUFFERHOTEL/ ! I e I NASHVILLE CONVENTION __I___ CENTER Call today for Form& Function: | Award-winningscience videodiscs your FREEcatalog How Should They Be Taught? 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