How A Sim ple Alternative to the To Do IMViC7 Test in Microbiology Isaiah A. Benathen

Two bacteria commonly studied in The Problem ative results for the last two, while E. microbiology courses are Escherichia aerogenesyields negative results for the coliand Enterobacteraerogenes. Both are This paper presents a simple, single first two tests and positive results for described as coliforms, meaning that alternativeto the four tests used in the the last two (Benathen 1992). These they are gram-negative, non-spore IMViC test to distinguish between tests are still widely used by the food forming short bacilli that ferment lac- these two organisms in the classroom processing industry to detect post- tose broth to produce gas within 48 and laboratory.This is desirable since treatment contamination of foods Downloaded from http://online.ucpress.edu/abt/article-pdf/54/2/112/45539/4449419.pdf by guest on 28 September 2021 hours at 35 C. Both organisms are culturesof these two organismsdo not (Post 1988) and in water purification readily isolated on MacConkeyagar, a always give the typical expected re- for differentiationbetween fecal and differential and selective medium for sults for these four tests. nonfecal pollution in water. coliforms and enteric bacteria. Gram- In the IMViCtest, the "I" stands for positive bacteria are inhibited by the the indole test, which indicates pro- The Alternative crystal violet in this medium, while duction of indole after hydrolysis of gram-negatives that ferment lactose tryptophan. The "M" is the methyl- This paper uses bile-esculin agar as produce pink to red colonies. Gram- red test, which indicates fermentation an additional alternativeto the IMViC of negative lactose nonfermenters pro- glucose to mixed acids and a pH of test to distinguish between these two 4.4 or less. The "V" is the duce colorless colonies on this me- Voges- coliforms. Bile-esculin agar slants are Proskauer test, which indicates fer- dium. Typically, E. coli produces red commonly used to differentiate the mentation of glucose to the neutral colonies surrounded by a zone of pre- group D Enterococcusfaecalis from other end product acetoin. (The letter "i" is cipitated bile salts and E. aerogenes streptococci.This enterococcushydro- inserted for pronunciation.) The "C" lyses the esculin compound to escule- produces pink mucoid colonies on this stands for the citrate test, which indi- tin and glucose. The esculetin reacts medium. cates growth on medium containing with iron salts to form a black to E. coli is the microbial standard for citrate salts as the sole carbon source. brown colored complex in the agar fecal pollution of drinking water, The first three tests in this sequence (Koneman et al. 1983). This paper while E. aerogenesis a possible total require that special reagents be pre- demonstrates that only one of these coliform contaminant of water. Com- pared and added before the results can two coliforms hydrolyses the esculin monly, E. coli is found after fecal pol- be determined. They are Kovac's re- compound and blackens the medium. lution of water by humans and warm agent, Methyl Red pH indicator solu- blooded animals, while E. aerogenesis tion and Barritt'sA and B reagents, found growing in waters contam- respectively. Barritt'sA reagent is 5 Materials & Methods inated with industrialwastes or in raw percent alpha naphthol solution in ab- One possible source of cultures of or treated municipal water supplies solute alcohol. Barritt'sB reagent is 40 Escherichiacoli and Enterobacteraero- (Clesceri, Greenberg & Trussell 1989). percent potassium hydroxide in dis- genesused for these studies is Presque Their presence in water may indicate tilled water with 0.3g of creatine. The Isle Cultures (P.O. Box 8191, Presque the presence of enteric pathogens following changes are produced in Isle, PA 16505). The MacConkeyagar which produce disease symptoms positive tests after the appropriatere- and the bile-esculin agar medium are such as diarrhea,cramps, nausea and agents are added to these media. A easily obtained from Difco Laborato- jaundice (Environmental Protection red color is observed at the surface of ries (Detroit,MI 48232), BBL (Cockeys- Agency 1989). E. coli is the most com- the broth in an indole test; a red color ville, MD 21030) or Unipath Co. - mon cause of uncomplicated urinary is observed in the broth in a methyl- Oxoid Division (Ogdensburg, NY tractinfection in women (Stamm1988) red and Voges-Proskauer test. The 13669). and also causes gram-negative noso- Voges-Proskauertest reactionrequires Single pure cultures of E. coliand E. comial pneumonia (Bradley & Norris shaking to promote the reaction be- aerogenesor a mixed culture of these 1989). E. aerogenescauses urinarytract tween oxygen, acetoin, alpha naph- bacteria is streaked for single colony infections (awetz et al. 1989). thol and potassium hydroxide to form isolation to MacConkey agar for pre- diacetyl, which produces the red color sumptive identification. These streak on reaction with creatine. plates are incubated at 35 C for 24 Negative results for these three tests hours and examined for the different fail to show these changes in color. types of Isaiah A. Benathen is an associate pro- colonies present. Colonies fessor in the department of biological Growthon citrateagar slants produces that appear distinctively different are sciences at KingsboroughCommunity an alkaline reaction and a change in picked and streaked to separate bile- College, 2001 OrientalBlvd., Brooklyn, NY color of the pH indicatorfrom green to esculin agar slants. These slants are 11235. blue. E. coli usually yields positive incubated at 35 C for 24 hours and results for the first two tests and neg- evaluated.

112 THEAMERICAN BIOLOGY TEACHER, VOLUME 54, NO. 2, FEBRUARY1992 Table 1. Results observed on Conclusions References bile-esculin agar. This investigationdemonstrates that Benathen, I. (in press). Microbiologywith a medium designed to identify the healthcare applications. Belmont, CA: Star Microbe ObservedResults gram-positive Group D enterococcus Publishing Co. Bradley, 0. & Norris, J. (1989). Bacterial E. coli Growth and no can also be a convenient way to distin- nosocomial pneumonia. PhysicianAssis- blackening of agar guish between two common gram- tant, 13(6), 35-52. E. aerogenes Growth and blackening negative coliform bacilli. And, it Clesceri, L., Greenberg,A. & Trussell, R. of agar shows that a medium designed for one (Eds.). (1989). Standardmethods for the purpose can have other diagnostic examinationof waterand wastewater(17th value. The use of bile-esculin offers a ed.). Washington, DC: American Public Health Association. single medium to replace the three EnvironmentalProtection Agency. (1989). differentmedia used in the IMViCtest 40 CFRparts 141 and 142. Drinking wa- and offers a simpler, more convenient ter; national primarydrinking water reg- Results alternative that does not require re- ulations; total coliforms (including fecal agents for interpretationand coliform coliforms and E. coli); final rule. Federal E. coli forms red colonies sur- differentiation.This test can be used as Register,54(124), 27543-27568.Washing- rounded by a zone of precipitatedbile an additional differentialconfirmatory ton, DC: U.S. Government Printing Of- on MacConkey agar. E. aerogenes fice. Downloaded from http://online.ucpress.edu/abt/article-pdf/54/2/112/45539/4449419.pdf by guest on 28 September 2021 option after the IMViC test or when Jawetz, E., Melnick, J., Adelberg, E., forms pink glistening mucoid colonies the IMViC test results are atypical. Brooks, G., Butel, J. & Ornstein, L. on this medium. Both of these bacteria Students can also use the bile-esculin (1989). Medical microbiology(18th ed.). tolerate the oxgall bile salts and grow test in unknown studies in the labora- Norwalk, CT:Appleton and Lange. on the bile-esculin medium. However, tory to distinguish between these two Koneman, E.W., Allen, S.D., Dowell, only E. aerogenescan hydrolyse esculin coliforms. D.W. Jr. & Sommers, H.M. (1983). Color atlasand textbookof diagnosticmicrobiology to esculetin and blacken this slant. (2nd ed.). Philadelphia:J.B. Lippincott This positive result is diagnostic for Co. the coliform E. aerogenesand shows Post, F. (1988). A laboratorymanual for food that a microbiologicalmedium differ- microbiologyand biotechnology.Belmont, entially designed to identify Group D Acknowledgment CA: Star Publishing Co. Stamm, W. (1988).Protocol for diagnosis of enterococcican have another selective The authorthanks Marion Saccardi for urinarytract infection: Reconsidering the value. The results are summarized in thepreparation of allmicrobiological media criterionfor significant bacteriuria.Sup- Table 1. andcultures for this investigation. plementto Urology,32(2), 6-10.

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