John H. Hanks

Relation of Oxygen and Temperature in the Preservation of Tissues by Refrigeration

An inquiry has been made into conditions which may influence the viability of 1×1 cm areas of biopsied rabbit skin during refrigeration at 0° and 6-8°C. Since the availability of oxygen, as well as the nature of the storage medium, had an, important influence on the preservation of viability at these two temperatures, these relationships may be of interest to those wishing to store or ship tissues for surgical and other purposes. Tissues separated from the circulation rapidly become anoxemic and necrotic at room or body temperatures. This condition may be prevented in uterine or intestinal strips by oxygenation or by chilling. 1 , 2 The survival of ligated limbs 3 and of the cells in whole embryos 4 , 5 or organs 5 , 6 is optimal (among the widely spaced temperatures which have been studied) at 0°. At this temperature respiration is minimal, while oxygen solubility in water is twice that at 30°. Though Lambert 7 Carrel, 8 and Hetherington and Craig 5 found 0 to 7° favorable for preserving the small masses of crowded cells in embryonic tissue fragments, there is considerable evidence that the thin perimeter of migrating and dividing cells in established tissue cultures has maximal longevity around 30° 9 10 and are unable to re-establish growth after refrigeration for a few days. 9 , 11 , 13 Upon considering the fact that large tissues are killed more rapidly at higher temperatures and small groups of thinly spread cells at low temperatures, it seemed not unlikely that one of the common denominators might be a question of oxygen supply and demand. Methods. After shaving and scrubbing, the chosen area of rabbit skin was covered for 2 minutes with a wet pack of tincture of iodine diluted 1:2 in water, and again scrubbed with alcohol. The strips of biopsied skin, either full- or half-skin depth were cut into 1×1 cm pieces.

Determination of cell viability

Summary and conclusions Differentiation between life and death in unicellular beings should be based upon criteria which are more convenient and fundamental than measurements of capacity for growth. The principle of ion (eosin) exclusion has been substantiated as a simple, rapid tool for this purpose. The conditions for valid observations in cell and tissue cultures have been delineated in respect to: eosin, serum and electrolyte concentrations. These simplified methods have replaced cultivation procedures in studies on: the effects of exposure to pancreatin and to drying, and the exposure of sensitive cells to tuberculin; storage of stock suspensions of cells without renewal of medium; and use of such methods for investigating nutritional or metabolic requirements.

Limited Multiplication of Mycobacterium lepraemurium in Cell Cultures

Summary Quantitative determinations have revealed significant multiplication of M. lepraemurium in explanted spleen cells which had been infected in vivo; also in the L strain of mouse fibrocytes infected in vitro, provided the metabolism of the latter was altered by means of hydrocortisone. During periods of 8–10 days, the rate of multiplication closely approximated the maximal rates observed in susceptible animals.

Inorganic Aging of the Plasma Layer of Tissue Cultures

Summary The adsorption of calcium phosphate complexes to plasma substrates, and the conditions required for their removal, indicate that the plasma gel is the site of an ionic exchange in which preferentially adsorbed ions may greatly alter the desired ionic balance. This process is one of the factors which causes plasma to become growth inhibitory during prolonged maintenance of cells in this type of substrate.

Calcification of Cell Cultures in the Presence of Embryo Juice and Mammalian Sera

Summary and Conclusions 1. Chick embryo juice, in concentrations suitable for chick cell cultures, induces calcification of the cell colonies and plasma in mammalian cell cultures. 2. Fresh embryo pulp and extract are saturated with Ca and P. Ca and particularly P increase with age of the embryos, their ratios being 1:2.4 at 11 days. Upon incubation the P increases an additional 30%percnt;. 3. Pasteurization of embryo extracts inactivates the phosphatases and prevents calcification in cell cultures receiving high concentrations of embryo juice. 4. Calcification of cultures in the presence of mammalian sera is due to combining the Ca of serum and salt solution with the high levels of P in embryo juice, to the further liberation of P during incubation, and to the limited Ca X P products held in solution by mammalian sera. 5. The calcifying propensity of chicken serum is much less than that of 3 mammalian sera, which may be arranged as follows: human < bovine < rabbit.

Growth of Small Numbers of Acid-fast Bacteria in Blood and in Serum.∗

The survival or growth of tubercle bacilli in blood is an important question from several points of view: the immunological action of serum and leucocytes, the investigation of tuberculous bacillemia, and the suitability of blood as a culture medium. Some investigators have emphasized the unfavorable action of blood on the growth of tubercle bacilli on solid media, while others have reported the growth of these organisms in blood. These points of view were reconciled when it was learned that the influence of blood in the two situations is quite different. This note indicates that, although blood interferes with the growth of tubercle bacilli on a solid substrate, it is actually an excellent medium. Saline, defibrinated rabbit blood, or serum, .45 cc, were mixed with .05 cc volumes of 4 decimal dilutions of “clump-free” suspensions of human, bovine, avian, and timothy bacilli in 10 × 75 mm Pyrex tubes. Of these mixtures .05 cc was spread evenly on each of two slants of Corpers egg-yolk medium (in screw-capped tubes) which were free of surface moisture. The small tubes containing the mixtures were sealed with paraffined corks; both solid and liquid mediums were incubated with their surfaces horizontal. From the results in Table I, several conclusions may be drawn: (a) Rabbit blood and serum were as favorable as the solid medium, with two exceptions: the irregular growth of bovine bacilli in blood, and the delayed growth of timothy bacilli in serum. (b) There was no evidence of the growth-inhibitory effects hitherto ascribed to hemoglobin or to “autolytic products” of blood. (c) The failure of tubercle bacilli to grow suitably on solid medium inoculated with blood was again confirmed. Positive results cannot be obtained with small numbers of bacilli.

Analysis of Present Methods of Collecting Tubercle Bacilli from Urine.

In a study on the concentration of tubercle bacilli from urine 1 two standard procedures were used as controls: Petroffs tannic-acid method† and direct centrifugation for one hour. Since the detailed study of newer methods will not appear for some time, and since the results of one of the present technics are definitely improved by simplification, a brief analysis of this procedure is desirable. Using Breeds counting method, the 2 procedures were analyzed quantitatively for their ability to collect human tubercle bacilli which had been added from “clump-free” suspensions to clear normal urine in predetermined numbers. The results were expressed as “concentration-factors” which indicated the increase in the average number of bacilli per microscopic field following concentration. The concentration-factors were determined for direct centrifugation, for each of the steps outlined below for the Petroff procedure, and for a procedure which duplicated Petroff s, except for the omission of the tannic acid. Two methods—milk and HCl—for “fixing” the sediments to the glass slides were compared. The results shown in Fig. 1 are average values from 4 experiments in which different concentrations of bacilli were added to the urines. There were no exceptions to the general ratings as illustrated. The results in the chart illustrate several useful facts: (a) The Petroff method appears to depend on the spontaneous precipitation of urates from acidified, chilled urine rather than on the use of tannic acid. It could be designated more properly as a “urate” concentration-method. (b) The first “urate” (or tannic acid) sediment produces a higher concentration-factor than is obtained by direct centrifugation. (c) The remainder of the Petroff procedure results in a loss of the majority of the bacilli which were collected in the first step by precipitation, (d) Slide losses are reduced by the use of milk as a fixative.

Sterilization of Skin

It is frequently stated that in the strict bacteriological sense it is impossible to sterilize skin. This concept apparently arises from the use of methods which do not insure that an appropriate disinfectant is applied in active form for an adequate interval of time. Since the rich and; complex nutrients used for cell cultivation provide an excellent pabulum for a variety of microorganisms, long experience in sterilization of leprous and normal skin prior to biopsy for tissue culture induces us to present evidence that skin sterilization is not a difficult problem. Early attempts to sterilize well scrubbed skin by painting with tincture of iodine were at times successful and at times failures. The inconstancy of the results suggested the desirability of controlling the action of iodine with respect to concentration, and time of action. By covering the area to be biopsied with a heavy patch of fabric, dripping iodine solution (tincture of iodine 1:2 in 70%percnt; alcohol by weight) on this patch until it was saturated, and then removing the patch and recleaning with alcohol after an interval of 5 minutes, the occurrence of scattered contamination was terminated. During the next several years it was noted that epithelial cells rarely if ever migrated from fragments ex-planted from the papillary or uppermost layer of the skin, while abundant epithelial cells appeared around fragments explanted from the deeper or reticular layer of the skin. Upon the first occasion when a biopsy was taken after painting the skin with 2%percnt; Mercurochrome in Duponol as a wetting agent, good epithelial growth around papillary ex-plants appeared for the first time in our experience. Nevertheless, one after another of the culture tubes gave evidence of contamination by staphylococci until 40%percnt; of the cultures had been lost. The period of applying the iodine (1:2) patch was next shortened to 2 minutes, without trouble from contamination and with the result that the superficial layer of skin produced moderate to excellent epithelial migration.