(370C.), its function as a solid medium is impaired and its usefulness

Transcription

1 GELATIN FOR BACTERIOLOGICAL USE Department of Bacteriology, Philadelphia College of Pharmacy & Science Received for publication September 21, 1940 The English word gelatin is derived (through the French gelatine and the Italian gelatina) from the Latin gelata and means that which is frozen, congealed or stiff. Gelatin is a protein of fairly uniform molecular constitution, is derived chiefly by the hydrolysis of collagen, and possesses well-defined physical and chemical characteristics. Collagens are a class of albuminoids, found abundantly in bones, skin, tendons, cartilage, and similar tissues of animals. The most important uses of pure gelatin are as a food, as a protective colloid in dietetics, to improve the texture and keeping qualities of ice-cream and certain food products, in photographic and pharmaceutical preparations, and as an ingredient of culture media. It was Koch who introduced gelatin into bacteriology, when he invented the gelatin tube method in 1875 and the plate method in This introduction of a solid culture method became the foundation on which all subsequent bacteriological investigation for the propagation of bacteria was established. Gelatin in concentrations of 1 per cent or more (this may be reduced to 0.6 per cent for a high grade gelatin) yields a very clear transparent solid medium which favors the development of a large variety of different species of microorganisms. Inasmuch as the melting point of such a solid preparation is below the optimum temperature required for the growth of most pathogens (370C.), its function as a solid medium is impaired and its usefulness for routine work is limited. Gelatin media when used to observe bacterial growth are employed for organisms which grow best at a temperature not exceeding 220C. Their chief use however is for studies of liquefaction by bacteria (producing 645

2 646 gelatinases). Gelatin also has been employed in media for the study of the fibrinolytic activity of hemolytic streptococci. In an examination of textbooks on Bacteriology and on Laboratory Methods, one finds that the gelatin concentration in Nutrient Gelatin varies from 10 to 15 per cent. Concentrations of from 20 to 25 per cent are recommended in hot climates. Sterilization is to be effected by the intermittent method (at 100'C.) or in the autoclave at a relatively low pressure (5 to 7 pounds). We are informed that if the temperature is too high there is interference with the setting property of gelatin, or that high temperatures interfere with its power of solidifying. Only in an occasional manual are there instructions given for sterilization by autoclaving for 20 minutes at 15 pounds pressure. GELATIN REQUIREMENTS AND STANDARDS There are no set standards for the requirements of gelatin to be employed in culture media other than those given in the A. P., H. A. "Standard Methods of Water Analysis" and the United States Pharmacopoeial requirements. The U. S. P. standards have a legal standing. The U. S. P. describes gelatin as follows: "A product obtained by the partial hydrolysis of collagen, derived from the skin, white connective tissue, and bones of animals. Description and physical propertie8-in sheets, flakes, shreds, or as a coarse or fine powder. It is colorless or yellowish, and has a very slight, characteristic odor and taste. When dry it is stable in the air, but when moist or in solution it is subject to bacterial decomposition. Gelatin is insoluble in cold water, but swells and softens when immersed in it, gradually absorbing from 5 to 10 times its own weight of water. It is soluble in hot water, in acetic acid, and in a hot mixture of glycerin and water. It is insoluble in alcohol, in chloroform, in ether, in benzene, in carbon disulfide, and in fixed or volatile oils." Other detailed requirements are presented. The "Standard Methods of Water Analysis", 7th Edition, prepared, approved and published jointly by the American Public Health Association and the American Water Works Association specifies the requirements for gelatin to be used in the prepara-

3 GELATIN FOR BACTERIOLOGICAL USE tion of Nutrient Gelatin. They are: "The gelatin used shall be of light color, shall not contain objectionable impurities and shall be free from preservatives. The melting point shall be such that a 10 per cent standard nutrient gelatin shall melt at 250C. or over." These requirements are even more general than those given in U. S. P. XI. The melting point requirement is one not commonly applied to gelatin when ordering the latter on the open market. Furthermore, these requirements concern preparing gelatin media for the analysis of water. The gelatin medium is used but infrequently today in water analyses and its greater use is for other purposes in bacteriological procedures. In any investigation dealing with the suitability of various gelatins for some specific purpose, a knowledge of certain details of manufacture is indispensable, since these greatly influence the properties and characteristics of the finished product. Edible gelatin (and gelatin for bacteriological use is in this class) is prepared chiefly from three carefully selected raw materials: clean bones, fresh frozen porkskins, and calfskins. Bone stock and calfskins are treated differently from porkskin for the production of gelatin. The physico-chemical properties of the gelatins produced are widely different and dependent upon the nature of the precursor. By far the most important factor in determining the nature of a given sample of gelatin is the preliminary treatment given its precursor, before actual hydrolysis of the collagen-yielding gelatin. For instance, due to the different methods of treatment, there will be noted a marked difference between the porkskin gelatin and the bone and calfskin gelatins in the region of their respective isoelectric points, as evidenced by the ph at which they exhibit maximum turbidity in a 12 to 2 per cent gel. Ossein (de-calcified bone) and calfskins are customarily treated with a lime suspension for a prolonged period of time, whereas porkskins are acid-plumped prior to extraction. Gelatin obtained from alkali-treated precursors has an isoelectric point at about ph 4.7 and is sold at a ph of from 5.2 to 6.6, whereas gelatin from the acid-treated precursor has a wide isoelectric zone of from ph 7.5 to 8.5 and is sold at a ph ranging from 3.5 to 4.6. The ash con- 647

4 648 stituents are likewise quite different. Ossein and calfskin gelatins contain primarily calcium salts existing usually as phosphates, sulfates, or chlorides, since after liming and washing the ph is lowered by the use of phosphoric acid, sulfuric or hydrochloric acid. Porkskin gelatin has an ash which is almost entirely sodium chloride with traces of aluminum. Other than the raw material and its treatment, the important variables in gelatin as a product are its jelly strength, viscosity, ph, clarity, bacterial count, and content of oxidizing or reducing substances. Since the gelatin industry manufactures only gelatin of such quality as to be acceptable for food use, these last three characteristics are rigidly maintained at the prescribed level. Gelatin is found in commerce in different forms. After concentration and clarification, the gelatin congealed in the form of a ribbon is fed automatically onto wire net frames (aluminum, nickle wire or allegheny metal wire) and passed through carefully regulated dryers. Sheet gelatin is thus produced. Shred gelatin is made by cutting sheet gelatin into very narrow shreds by a shearing machine. Gelatin also is dried by feeding the solutions onto large hot wheels, and the dried gelatin is obtained in the form of flakes. Granulated gelatin, the variety most desirable from the standpoint of use and ease of handling is prepared by grinding the broken sheets or flakes into granules. The jelly strength of gelatin is specified commercially by the expression, "Bloom Rating," the determination of which is made by the Bloom gelometer, a very ingenious device developed and used by the industry for this purpose. The higher the Bloom rating, the greater is the power of gel formation. Edible gelatin of average grade available on the market has a Bloom rating of 100 to 225. Higher grade gelatins are available in commerce today with Bloom ratings of 250 to 300. The high Bloom rating gelatins are most desirable for bacteriological use. Media prepared from such gelatins can withstand such high temperatures (autoclaving) for long periods of time, as are required during sterilization for the greater assurance of obtaining a sterile product.

5 GELATIN FOR BACTERIOLOGICAL USE 649 PURPOSE OF INVESTIGATION Due to inconstancy in results in the jellying properties of gelatin media after sterilization at high temperatures as experienced by different workers, we carried out a series of tests on various kinds of marketed gelatin obtained from bone and skin tissues and compared the properties of gelatin media prepared from these gelatins as to jellying after autoclaving, employing varying time limits. In selecting gelatins for this study samples were collected representing a cross section of commercial grades of food gelatin on the market. A history of each sample was noted as to raw material and the exact treatment received in processing. This proved to be of great importance as will be noted in our report. PROCEDURE Nine different samples of gelatin were used. Samples 1, 2, 3, 7, and 8 consisted of gelatin derived from bone tissue. Sample 4 was a high grade porkskin gelatin and sample 6 was a low grade porkskin gelatin. Samples 5 and 9 were samples available on the market and recommended specifically for the preparation of culture media. The following table presents additional information concerning these samples. Throughout the tests the media were prepared by adding gelatin in the designated concentrations to Nutrient Broth (made with 10 grams of peptone, 5 grams of sodium chloride, and 3 grams of beef extract, with water sufficient to make 1000 ml.). The hydrogen-ion concentration of the finished preparations was adjusted to ph 7. The sterile solid media were inoculated separately with Proteus vulgaris, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Observations were made every 24 hours during incubation which was carried out for 96 hours at 370C. and at room temperature. Gelatin media were prepared separately on three different occasions from the nine samples in 10, 15, and 20 per cent concentrations, placed in tubes and flasks and autoclaved at 15 pounds

6 650 pressure for 30 minutes. Batches of similar strengths were prepared and autoclaved for 60 minutes. All samples solidified upon cooling to room temperature. However, media prepared from sample 1 revealed upon autoclaving a heavy sediment in the containers here and in all other instances throughout this investigation. Furthermore, media prepared from this sample (no. 1) and from samples 2, 3, 7, and 8 were too cloudy to be employed in bacteriological procedures. Tubes of media thus sterilized and inoculated separately with the bacteria mentioned above showed liquefaction in all cases, except in those containers where Escherichia coli was used for inoculation. TABLE 1 UABUILD ORIGIN BLOOM ph 2 Ossein Ossein Porkskin* Marketed as Bacteriological Gelatint $ 6 Porkskin Ossein prepared with HOl Ossein Marketed as Bacteriological Gelatint T 1 Ossein * Available on the market as Pharmagel A. t Analyses revealed a porkskin or at least a gelatin from an acid-treated precursor. t The ph here was higher than that observed for pure marketed porkskin gelatin. In reality these samples were processed, containing admixed chemicals which accounts for the higher ph. Attention is directed here to sample 7. This was a special ossein gelatin in which hydrochloric acid was used to wash and adjust the ph of the ossein after living, instead of phosphoric acid which is generally used. Thus in this gelatin, the calcium salts existed chiefly as chlorides and were not precipitated in very large amounts when the media were adjusted to ph 7 as is the case in ordinary ossein gelatins, wherein the calcium salts exist chiefly as phosphates. It also is likely that what small amount of precipitation was observed in sample 7 would have been absent if this gelatin had been extracted at a lower ph, as this would have resulted in a lower calcium content.

7 GELATIN FOR BACTERIOLOGICAL USE Other batches of gelatin media were prepared separately on three different occasions from the nine samples in 10, 15, and 20 per cent gelatin concentrations, placed in tubes and flasks, and autoclaved at 15 pounds pressure for 90 (instead of 60) minutes. All media of 10 per cent gelatin concentration, sterilized for 90 minutes, failed to solidify upon cooling to room temperature with the exception of media prepared with sample 3. Media of 15 per cent gelatin concentration prepared with samples 2, 3, 4, 7, and 8 solidified and prepared with samples 1, 5, 6, and 9 did not solidify upon cooling. Media of 20 per cent gelatin concentration prepared with samples 2, 3, 4, 7, and 8 solidified and those prepared with samples 1, 5, 6, and 9 did not solidify upon cooling after they were sterilized at 15 pounds pressure for 90 minutes. All samples which solidified were inoculated separately with Proteus vulgaris, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. All organisms except the latter liquefied the gelatin media (in 10, 15, and 20 per cent concentrations), which remained solid after sterilization for 90 minutes. The sixty and ninety minute periods for autoclaving were used as being a test for determining the jellying properties of gelatin medium more rigid than the 20 or 30 minutes commonly employed in practice. DISCUSSION AND CONCLUSION The literature does not yield definite information concerning specifications for gelatin to be employed for bacteriological use. The requirements of the U. S. P. and those of the A. P. H. A. "Standard Methods" are too general and certainly not sufficiently detailed for a grade of gelatin required in the preparation of Nutrient Gelatin. From private sources, data are available which reveal that bacteriological gelatins as now sold vary in ph from 4.4 to 6.4, the Bloom rating from 102 to 260, and the viscosity from 28.7 to 56. In this investigation, the following observations were noted as of sufficient importance to form the basis for specifications for gelatin for use in preparing Nutrient Gelatin. Gelatins originating from alkali-treated precursors are not satisfactory because they produce Nutrient Gelatin media which 651

8 652 are too cloudy. One sample of ossein gelatin prepared with hydrochloric acid gave indications that it probably could be made satisfactory by changes in the manufacturing process. Porkskin gelatin is undoubtedly the gelatin of choice for bacteriological use. A high Bloom rating gelatin should be used because it withstands autoclaving for prolonged periods of time. This rating should not be less than 200 and preferably higher (250 to 300). We recommend a porkskin gelatin with a Bloom rating of 250 or higher as a product which should be used for preparing Nutrient Gelatin, the latter to be sterilized in the autoclave at 15 pounds pressure for 20 or 30 minutes (or even longer if desired). Such finished preparations are always sterile. They are solid at room temperature, liquefy at body temperature, and react with known species of bacteria in the usual prescribed manner. SUMMARY Nine different samples of marketed gelatin were obtained. Four of these samples were of bone origin manufactured as commonly practiced, one was of bone origin prepared with HCl, two originated from porkskin, and two samples recommended specifically for the preparation of culture media were of unknown origin. The latter two samples, however, upon analyses revealed that they were either porkskin gelatins or at least gelatins from an acid-treated precursor. The ph and Bloom rating of each sample were noted. Nutrient Gelatin in concentrations of 10, 15 and 20 per cent were prepared and individual batches of each (in tubes and flasks) were sterilized separately in the autoclave at 15 pounds pressure for 30, 60, and 90 minutes respectively. Samples which solidified upon cooling after sterilization were inoculated separately with three different species of bacteria which usually liquefy and one species which does not liquefy gelatin. The transparency and jellying properties on all samples which were sterilized and the effects of known species of bacteria upon samples which solidified were noted. ACKNOWLEDGMENT We wish to thank Mr. Melvin C. Firman for his assistance in some of the technical work during this investigation.