Nonstaining (KOH) method for determination of gram reactions of marine bacteria.

J D Buck Appl. Environ. Microbiol. 1982, 44(4):992.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1982, p. 992-993

Vol. 44, No. 4

0099-2240/82/100992-02$02.00/0 Copyright C 1982, American Society for Microbiology

Nonstaining (KOH) Method for Determination of Gram Reactions of Marine Bacteriat

JOHN D. BUCKS

Mote Marine Laboratory, Sarasota, Florida 33577

Received 6 May 1982/Accepted 15 June 1982

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A rapid nonstaining (KOH) method for the determination of the Gram reactions of bacteria is described, and its application to marine isolates is discussed. All gram-positive and gram-negative results obtained by Gram staining were confirmed by the KOH method. Gram-variable bacteria produced equivocal results. A century ago, Hans Christian Gram developed a staining procedure which enabled the separation of most commonly encountered bacteria into two groups: gram positive and gram negative. Today, Gram staining remains the most important differential technique applied to bacteria (2); in fact, for 8 of the 19 major groupings of bacteria (1), staining is required as a primary aid to identification. Although many biochemical features of diagnostic bacteriology have been adapted for increased time-cost efficiency, the Gram stain technique is essentially the same now as it was in 1884. Premixed stains can be purchased, but the procedure is still relatively time consuming, costly, and often messy, and reagents must be replaced periodically. Approximately 40 years ago, a simple and rapid nonstaining (KOH) method for the determination of the Gram reaction appeared in the Japanese literature (3). The method was described once again and used for 69 strains of bacteria encountered in veterinary microbiology clinics (3). The efficacy of the KOH method was confirmed further with 22 bacteria of importance in the brewing industry (6). Quality control for the traditional Gram stain procedure was achieved with 100 clinical isolates (4), and, to keep costs down, the KOH method has been suggested for routine use in hospitals (5). In this study, I extended the usefulness of the KOH technique to the characterization of a large collection of marine bacteria. I hope to remind the teaching and research community of or introduce it to the effectiveness of the method. To perform the test, place a drop of 3% aqueous KOH on a slide. If one prefers, 10 ,ul
t Contribution no. 149 from the University of Connecticut Marine Research Laboratory. t Present address: Department of Marine Sciences and Marine Sciences Institute, Marine Research Laboratory, The University of Connecticut, Noank, CT 06340.

from an automatic pipette is adequate. In practice, as many as 8 to 10 tests per slide can be done conveniently. Using a sterile loop, transfer a visible amount of bacterial growth from an agar culture to the drop of KOH. Mix the cells and KOH thoroughly on the slide, constantly stirring over an area about 1.5 cm in diameter. If the bacterium-KOH suspension becomes markedly viscid or gels within 5 to 60 s, the isolate is gram negative. If no gelling is observed, the isolate is gram positive. The best way to determine viscosity is to raise the loop about 1 cm from the slide. If an obvious stringiness is present, then the culture is gram negative. In this study, 400 isolates were tested by both the KOH method and conventional staining; for the latter, a commercial stain kit (Difco Laboratories) was used. Bacteria were isolated from seawater and a wide variety of fish. All cultures were streaked to ensure purity and maintained on slants of marine agar (Difco) or tryptic soy agar (Difco). Staining and the KOH procedure were performed at the same time with slant cultures ranging in age from several hours to several weeks. Using the stain procedure on 18- to 24-h cultures, I found 81% of the cultures to be gram negative, 11% to be gram positive, and 8% to be gram variable, i.e., having both purple and pink cells. All gram-positive and gram-negative results obtained by staining were confirmed by the KOH technique. Gram-variable bacteria gave equivocal results. In previous studies, only Fluharty and Packard (3) encountered a bacterium found to be gram variable by staining, and it was found to be gram negative by the KOH procedure. A total of 30 organisms were found to be gram variable by staining in the present study; when tested by the KOH method, 18 (60%) were found to be gram negative, and 12 (40%) were found to be gram positive. Six contaminated cultures contained initially both gram-positive and gram-negative bacteria and gelled with
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VOL. 44, 1982

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KOH (i.e., responded as gram negative). This pattern has been noted earlier (3, 6). In one study (3), the results for young (16- to 20-h) and older (2- to 10-month) cultures obtained by staining and the KOH method were found to be identical. In the current study, 4week-old cultures of recently obtained Bacillus, Staphylococcus, and Streptococcus isolates held at room temperature never showed any gelling. These strains remained gram positive by the KOH method, whereas staining revealed mixtures of pink and purple cells after a few days. The advantages of time, ease, and cost of the KOH technique are obvious. In a study in progress on the bacteria used in these experiments that were isolated from sharks and other fish, it was necessary to routinely separate gram-negative, oxidase-negative rods from the other bacteria for subsequent identification by rapid diagnostic tests for members of the Enterobacteriaceae. The KOH technique, together with a wet-mount preparation for observing morphology and cytochrome oxidase test strips, provided an efficient and rapid screening procedure. The gel reaction is apparent; generally there is no confusion regarding stringiness, although some cultures are slow to react, and the reaction is not obvious if too few cells are used. Holding the slide at an appropriate angle against a dark background aids observation. The KOH technique does have some disadvantages. It does not by itself enable any notation of cell morphology. However, additional characteristics necessary for eventual identification (e.g., motility) would furnish this. The culture sample necessary for the KOH test is bigger than that needed for Gram staining, although a large colony and a small drop of KOH will function if necessary. The KOH procedure does not enable the detection of gram-variable bacteria: as shown above, these bacteria are seen as gram positive or gram negative. The gram-variable cultures used in this study were members of the coryneform group (Corynebacterium and Arthrobacter). Several isolates of both genera were found to be gram positive by the KOH method, whereas some

gelled weakly (gram-negative reaction). A larger collection of gram-variable bacteria should be studied to clarify this anomaly further. It is clear that pure cultures are required for reliable observations; the KOH method will not enable the detection of contamination in unknown cultures. The present study did not include a detailed parallel comparison of staining results with KOH method results for cultures over time; however, no discrepancies among cultures of different ages were noted. There seems to be no evidence in this report or elsewhere (3) suggesting that the KOH technique cannot be used for cultures up to at least several weeks old. The above results show that the KOH method enabled the accurate determination of the Gram reactions of 92% of a large collection of marine bacteria. The procedure can be used to efficiently and rapidly characterize a wide variety of isolates encountered in both clinical and nonmarine environmental material. Early instruction should continue to stress the staining procedure as it relates to cell chemistry and should introduce the KOH technique as an appropriate example of modern, economically practical microbiology.
This work was supported by the Mote Marine Laboratory, Sarasota, Fla. I thank William H. Taft, Director, Mote Marine Laboratory, for providing space and facilities during a sabbatical leave. I am grateful to Mary Parks for tracing several literature citations.
LITERATURE CITED 1. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams

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& Wilkins Co., Baltimore. 2. Doetsch, R. N. 1981. Determinative methods of light microscopy, p. 21-33. In P. Gerhardt (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 3. Fluharty, D. M., and W. L. Packard. 1967. Differentiation of Gram-positive and Gram-negative bacteria without staining. Am. J. Vet. Clin. Pathol. 1:31-35. 4. Kohn, F. S., and S. A. Henneman. 1977. Novel quality assurance procedure for the Gram stain. J. Am. Med. Technol. 39:20-21. 5. Kuhn, P. J. 1981. Practical ideas for cost containment in microbiology. Med. Lab. Observ. 13:73-86. 6. Lin, Y. 1980. Use of potassium hydroxide technique for the differentiation of Gram-positive and Gram-negative bacteria. Brew. Dig. 55:36-37.