RAPID IDENTIFICATION OF CANDIDA ALBICANS1) by H. S. ANDLEICH2)

B. Sc., M.B.B.S., M.D., D.T.M., D. Bact. (London) (]3.II.1964) The rapid identification of a yeast isolated from a clinical material is essential for the early diagnosis of the infection and proper treatment. Routine identification of Candida albicans is usually dependent on the production, on certain media, of the characteristic chlamydospores. This m a y take any where from 2 to 3 days and a minimum of 24 hours. In recent years some techniques have been described where the criteria are less specific but which yield a diagnosis very much more quickly, rather within a few hours. REYNOLDS & BRAUDE (1956) observed the rapid formation of filaments by cells of Candida albicam when this yeast was incubated in blood, plasma, serum, cerebrospinal fluid, egg albumin or oleic acid albumin. BUCKLEY & VAN UDEN designated this as the R.B. effect after the name of the authors. TASCHDJIAN, BURCHALL KOZlNN (1960) reported that the rapid formation of germ tube-like filaments in human serum and serum substitutes is characteristic for Casdida albicans and the closely related Candida stdlatoidea. These authors pointed out that this phenomenon could be used for the identification of Candida albicass. Dog serum also proved satisfactory. MACKENZIE (1962) carried out extensive studies using the technique of TASCHDJIA~ et al. The test organism was inoculated into 0.5 ml of serum in a small tube or container and incubated at 37 ° C. MACKENZIE observed that rounded or short oval cell of Candida albicans rapidly gave rise to filamentous growth and within two to three hours numerous hand mirror forms were visible. These are characteristic of Candida albicans. They have been termed "Germ Tubes". He further observed that besides the human and dog serum, the serum of rabbit, guinea pig, horse and cattle also yielded equally good results. MACKENZIEcon1) Presented at the Sixth Indian Microbiological Congress (1963). 3) From the Department of Pathology and Bacteriology, S. P. Medical College, Bikaner. Mycopathol. et Mycol. Appl. X X I I I , 2.

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cluded that the serum tube test permitted a rapid presumptive diagnosis of Candida albica~s, although chlamydospore formation and biochemical characteristics remain the specific criteria for positive identification and differentiation from other species of candida. BUCKLEY & VAN UDEVT (1963) reported their observations of the studies on the rapid slide test. BUCKL~¥ & VAN UDEN at first followed the tube culture method of TASC~tDJIAN, BURCHALL & KOZINN (1960) substituting egg white for serum. Half ml of egg white were placed into clean, non-sterilised test tubes. There was no sterility problem since egg white is inhibitory for many organisms and also since the R. B. effect occurs, if at all, within a few hours in which time the microorganisms are unable to grow. The tubes were inoculated with a loopful of the yeast to be tested and incubated at 37 ° C. After every half hour intervals mounts were made from the tubes on slides and examined microscopically. The characteristic germ tubes appeared within 30 to 90 minutes. Buct~gEY & VAN UDEN then tried the slide culture method and have presented a simple slide culture method for which egg white is more readily adapted than serum. A drop of egg white is placed on a slide and mixed with a small amount of the yeast to be tested. The preparation is covered with a coverslip and incubated at 37 ° C. The slide is then observed after one hour or more for the R. B. effect. The preparations should be kept up to two hours at 37 ° C before calling the results negative. Unlike test with serum, slide preparations made with egg white need not be incubated in moist chambers to avoid drying out. BUCKLEY & VAN UI)EN found that this method gives satisfactory results. It was therefore considered worthwhile to find out as to which was the best and the most reliable method for a rapid identification of Caudida albicans species. The following four methods were tested. 1. Serum tube culture method. 2. Serum slide culture method. 3. Egg white tube culture method. 4. Egg white slide culture method. Twelve strains of Caudida albicans, six of which were isolated locally and six were received from Dr. HASENCLEVER of U.S.A. were used. The same test organism was subjected to all the four tests at one time. The technique for the tube test was as follows: About 0.5 mI of serum or egg white is taken in a small test tube (Kahn's or W. R. tube) and inoculated with a very small inoculum of the test culture by means of a straight wire. Inoculation with straight wire is essential because it was found, in preliminary experiments, that the proportion of yeast like cells forming filaments progressively decreased with increasing cell concentration. The tubes are then incubated at 37 ° C. As to the slide culture technique that employed by BVCKI.EY & VAN UDEX was followed. A drop of serum or egg white is placed on

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a slide and mixed with a small amount of the yeast to be tested. This preparation is covered with a coverslip and incubated at 37 ° C. The slide is then observed after one hour or more for the R. B. effect. The preparations are examined up to two hours before calling the test negative. Since the purpose of the investigations was to determine the most satisfactory method amongst those reported by various workers, only strains of Candida aIbicam were subjected to the test and the tests were carried out several times using the same strain. It was observed that the serum tube test suggested by MACKENZIE gave the best results. The beginning of formation of germ tubes was visible in this method within half an hour and within two hours definite and distinct germ tubes were seen. The number of germ tubes obtained by this method was much greater than by any of the other methods. The next best method proved to be the egg tube culture method. By this method also evidence of the starting of formation of germ tubes was present in about 30 minutes and fully formed germ tubes were visible by the end of two hours. The only difference that was seen between the serum tube and the egg white tube methods was that by the serum tube method the number of cells showing the growth of filamentous germ tubes was significantly more than by the egg white tube method. There was no significant difference in the time of appearance of the distinctly formed germ tube. As to the serum slide culture technique, filamentous germ tubes started appearing in tile culture in about 40-60 minutes and were present in much smaller numbers than either in the serum tube culture method or in the egg white tube culture method. In the egg white slide culture method tile number of fully formed filamentous germ tubes was again definitely less than in the serum slide culture preparation. The slide culture methods undoubtedly gave the poorest results. Not only the filamentous germ tubes appear later but also the number of positive results obtained by this method is only in the range of 50 to 60~o, while in the case of tube culture method it is 100°/o. It will thus be seen that statistically the serum tube culture method gives the best results. It may, however, be pointed out that as long as our aim is to decide whether a given culture of yeast like cells is Candida albicans or not, the egg white culture method yields as useful results as the serum tube culture method. Moreover, it has the advantage that egg white is much more easily procurable than the human serum or the serum of any other suitable animal such as rabbit or dog. I t is therefore concluded that the egg white tube culture technique is the method of choice for the rapid identification of Candida albicans. Cultures of Candida s~ellatoidea also give a positive R. B. effect, both by the serum tube culture method and the egg white tube culture method, Candida stellatoidea, however, can be differentiated from Candida albicans by its cultural characters and biochemical activities, subsequently, if necessary. 6*

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References l, BUCKLEY, H. R° (~ VAN UDEN, lX[. (1963) The identification of Candida albicans within two hours by the use of an egg white slide preparation. Sabouraudia 2: 205--208. 2. MACKENZIE, D. \~7. R. (1962) Serum tube identification of Candida albicans. J. clin. Path. 15: 563--568. 3. SILVA, M. S. (~ BUCKLEY (1962) Activity of egg white against fungi pathogenic to man - In "Therapy of fungous diseases" by DALLDORF G., Charles C. Thomas, Springfield, Ill. U.S.A. 4. TASCHDJIAN, C. L., BURCHALL, J. J. ~¢ KOZINN, P. J. (1960) Rapid identification of Candida albicans by filamentation on serum and serum substitutes. AMA J. Dis, Child. 99: 212--215, Nov.