Sterilization of Plastic Petri Dishes

By ALBERTSCHATZ, GEORGE G. BRANDON,and JAMESD. WEBBER Downloaded from http://online.ucpress.edu/abt/article-pdf/32/5/294/26987/4443055.pdf by guest on 26 September 2021

Microbiology is receiving increasing attention in cated that this concentration range should be inves- secondary school science curricula. This is also tigated.) true in some primary grades. In the first publication in elementary devoted specifically to microbiology Table 1. Concentrations of NaOCI tested for schools (Commonwealth of Pennsylvania, 1963) antifungal activity. some of the activities involved enrichment cultures is not neces- Original bleach solution NaOCIContent or mixed cultures for which sterility (contained 5.25%NaOCI) sary. But where organisms are isolated and grown as pure cultures, sterile containers are required. % by volume % by weight ppm For this kind of work, many schools cannot afford Dilution ml/liter (ml/100 ml) (gm/100 ml) glass petri dishes and an autoclave or hot-air oven. 1:500 2.0 0.2 0.01 100 Disposable plastic petri dishes are considerably less 1:200 5.0 0.5 0.025 250 than glass, but they cannot be sterilized 1:100 10.0 1.0 0.05 500 expensive 1:50 20.0 2.0 0.10 1,000 by heat. Also, where many students are involved, the cost of the large number of plastic dishes required exceeds the budget of many schools. This Plastic petri dishes were heavily contaminated report describes a simple, safe, and inexpensive with soil microflora. For this purpose, 250 grams of procedure for the sterilization of plastic and glass a lawn soil were shaken for 10 minutes in one liter petri dishes, test-tubes, vials, bottles, jars, and other of tap water and allowed to settle for about two equipment that must be rendered germ-free. hours. The petri dishes were then immersed for 10 minutes in the supernatant solution, which had Materials and Methods been decanted. A plate count showed that this con- The sterilizing agent is a commercial liquid- tained some 120,000 fungi per milliliter. The dishes chlorine bleaching solution containing 5.25% hypo- were then shaken free of the solution and air-dried chlorite (NaOCl) as the active principle. In exper- overnight at room temperature. iments leading to the adoption of this agent, tap- Four petri dishes were immersed for 10 minutes water dilutions were prepared to contain the four in each dilution of NaOCl; with no air bubbles en- concentrations of NaOCl shown in table 1. (Pre- trapped, the entire plastic surface was in contact vious experiments, not reported here, had indi- with the sterilizing solution. All dishes were again air-dried overnight at room temperature. For this, they were inverted and inclined with one edge Authors' addresses: raised about a centimeter, to facilitate drainage, on Albert Schatz-College of Education, Temple University, several layers of clean but nonsterile Philadelphia, Pa. 19122. newspaper George G. Brandon and James D. Webber-Webster Col- in a cabinet with the doors closed to minimize air lege, Webster Groves, Mo. 63119 currents.

294 THE AMERICANBIOLOGY TEACHER, MAY 1970 The next day, 10 to 12 milliliters of a sucrose- hydrolysis of the gelatin protein. Autoclaving is gelatin medium at pH 3.8 to 4.0 were added to therefore not required. The low pH has other ad- each dish. The plates were then incubated at room vantages: at pH 3.8 to 4.0, only filamentous fungi temperature and colony counts made after three and an occasional yeast develop; and bacteria and days and six days. antinomycetes generally cannot tolerate this degree of acidity. Results Table 2 shows the number of fungi that grew on the NaOCl-treated plates. Control plates, not exposed to NaOCl, were completely overgrown in Table 2. Growth of soil fungi on plastic petri dishes treated with NaOCI.Each datum representsthe number of colonies three days. The data indicate that a two-hour ex- on a petri dish.C indicatessuch heavy growthover the entire posure to 0.01% NaOCl provided adequate steriliza- surfacethat a colony count was not possible. tion. However, it was found more convenient, from the point of view of a Colonies Period of exposure to NaOCI, hours teaching schedule-especially NaOCl per plate, when students in a particular class did the steriliz- % after days 0.5 1 2 24 ing-to keep the dishes immersed overnight. Control tests with previously unused plastic pet- 0.01 3 6,7,8,11 8,10,11,12 7,9,9,12 9,10,10,11 6 C,C,C,C C,C,C,C C,C,C,C C,C,C,C Downloaded from http://online.ucpress.edu/abt/article-pdf/32/5/294/26987/4443055.pdf by guest on 26 September 2021 ri dishes, sterilized by the manufacturer, showed 0.025 3 6,6,8,9 2,2,2,2 1,1,1,3 1,2,3,4 that the few colonies that developed on plates ex- 6 C,C,C,C 2,2,C,C, 2,2,2,C 3,3,6,C posed to 0.01% NaOCl for two hours and for 24 0.05 3 1,1,3,3 1,2,2,3 1,3,4,4 1,4,4,6 hours were air contaminants. When the medium 6 C,C,C,C 2,4,C,C 1,C,C,C 1,C,C,C 0.1 3 0,0,0,0 0,0,0,1 0,0,0,1 0,0,1,1 was poured into these plates under normal class- 6 0,1,1,3 0,1,C,C 0,0,2,2 0,0,1,1, room conditions with no special precautions to prevent contamination, one or two colonies usually developed on each dish. This low level of contamination did not interfere with a wide range of micro- Molds lend themselves particularly well to class- biologic experiments carried out with this medium room use for the following reasons: (i) A wide in NaOCl-sterilized glass and plastic containers. variety of fungi is readily available from soil and To determine whether residual traces of NaOCl air throughout the year and at no expense; conse- interfered with mold growth, 24 species of fungi, quently, there is no problem in obtaining organisms of 18 genera, were grown in pure culture in plastic to work with. (ii) Molds grow fairly rapidly at petri dishes that were previously unused (that is, room temperature. (iii) Their colonies have char- sterilized by the manufacturer) and exposed over- acteristics (size, shape, color) that are easily dis- night to a 0.01% solution of NaOCl. The results tinguishable with the naked eye, so that staining showed no inhibition by residual traces of NaOCl. and microscopic examination are not necessary; in Each organism grew at the same rate in both petri other words, schools do not need microscopes for dishes. a number of experiments that can be done with fungi. (iv) Many biologic principles observed with Discussion and Conclusions molds are applicable to other groups of microbiota. Hypochlorous acid in the form of NaOCl or Ca (OCl)2 has been used to kill pathogenic fungi * REFERENCES on babies' bottles in hospitals, to treat seeds, to COMMONWEALTH OF PENNSYLVANIA, Department of Public In- struction. 1963. Microbiology for elementary school chil- prevent mold growth on fruit and vegetables, to dren. (Curriculum Development Series No. 9.) sanitize food-processing plants, and in other situa- EDUCATIONAL SERVICES, INC. 1965. Illustrated handbook of some tions where molds are unwanted (see references). common molds. The effectiveness of hypochlorite in these areas GREENFIELD, J. R., and R. R. DUFFY. 1968. Evaluation of sensi- suggested the use of NaOCl to sterilize plastic tizers. In Proceedings of The American Society of Brewing Chemists, p. 46-51. petri dishes and thereby make it possible for them LINCOLN, P. A., G. D. BREACH, and J. G. DAVIS. 1965. The ac- to be reused in schools under normal classroom tivity of sodium hypochlorite against Candida albicans in conditions. This allows schools with limited budgets milk suspensions and films of dried milk. Medical Officer to include more microbiologic activities, involving 114: 169-172. MCCLUNG, L. S. 1960. Microbiology teaching aids. American pure-culture techniques, in their science programs. Biology Teacher 22: 352-385. The simple sucrose-gelatin medium is also ad- SEGALL, R. H. 1968. Fungicidal effectiveness of chlorine as in- vantageous for school use. This medium and a solu- fluenced by concentration, temperature, pH, and spore ex- tion containing a predetermined amount of citric posure time. Phytopathology 58: 1412-1414. acid, which lowers the pH to 3.8 to 4.0, are sterilized SLEETH, B. 1946. The effect of fungicidal seed treatments on guayule seedling emergence. Phytopathology 36: 999-1010. separately by boiling for 10 minutes and then SUSSMAN, A. S. 1963. Microbes: their growth, nutrition, and mixed. This separate sterilization minimizes acid interaction.