Microbiological efficacy of superheated steam. I. Communication: results with spores of Bacillus subtilis and Bacillus stearothermophilus and with spore earth.

For the spores of Bacillus subtilis and Bacillus stearothermophilus as well as for spore earth (acc. DIN 58,946 Part 4 of August 1982), the dependence of resistance on the superheating of the steam used to kill germs was determined. A material (glass fibre fleece) was used as the germ carrier which does not superheat on contact with steam. The temperature of the saturated steam was 100 degrees C (B. subtilis) and 120 degrees C (B. stearothermophilus and spore earth). The yardstick for the resistance of the spores or bioindicators was the exposure period of the saturated or superheated steam at which 50% of the treated test objects no longer showed any viable test germs. The spores of Bacillus subtilis were far more sensitive to superheating of steam and reacted far more than the spores of Bacillus stearothermophilus and the germs in the spore earth. When superheating by 4 Kelvin the spores of Bacillus subtilis were approximately 2.5 times more resistant than they were to saturated steam. The resistance of Bacillus stearothermophilus and spore earth was only slightly higher up to superheating by 10 Kelvin. The spores of Bacillus subtilis had the highest resistance during superheating by 29 Kelvin; they were 119 times more resistant than they were to saturated steam. The resistance maximum of the spores of Bacillus stearothermophilus was at an superheating by around 22 Kelvin. However, the spores were only 4.1 times more resistant than they were to saturated steam. When using steam to kill germs, we must expect superheated steam. This raises the question whether the spores of Bacillus stearothermophilus, with their weaker reaction to the superheating of steam, are suitable as test germs for sterilisation with steam in all cases.

[Effect on the microbicidal efficacy of formaldehyde, glutardialdehyde, peracetic acid, chloramine T (N-chloro-4-toluenesulfonamide), m-cresol, ethanol and benzyldimethyldodecacylammonium bromide by blood (model experiments for chemical disinfection of instruments)]. / Beeinflussung der mikrobiziden Wirksamkeit von Formaldehyd, Glutardialdehyd, Peressigsäure, Chloramin T (N-Chlor-4-toluolsulfonsäureamid), m-Kresol, Ethanol und Benzyldimethyldodecylammoniumbromid durch Blut (Modellversuche zur chemischen Instrumentendesinfektion).

In a preceding paper (Zbl. Hyg. 191 [1991] 457-477) we reported on the dependence of the microbicidal efficacy of active agents of the disinfection of instruments on the amount of coagulated blood adhering to the instruments. In the present investigation, we were interested in the dependence of the microbicidal effects on the amount of blood in the solutions of the active agents. Test areas of 2 cm2 were contaminated with 50 and 100 microliters coagulating blood, respectively, containing cells of Staphylococcus aureus as test germ. The solutions of the microbicidal agents were contaminated with heparinized blood up to a concentration of 4% immediately before starting the disinfection and 24 hours before, respectively. After a period of action lasting 1 hour at 20 degrees C, the relative number of test germs capable of multiplying (N/N0) was determined. The concentration of the microbicidal substances reducing the relative number of test germs capable to multiply to 10(-4) served for estimating the dependence of the microbicidal efficacy of the agents on the blood content of the solutions. The experimental results depended on the thickness of the layer of coagulated blood. The dependence of the efficacy of the microbicidal substances on the blood content of the solutions was the higher the thinner the blood layer was. At a thickness of the layer of the coagulated blood of 0.25 mm, a blood content of the solution of 4%, and applying it immediately after adding the blood, the concentration of glutardialdehyde had to be 1.6 times that without blood to reach the same microbicidal efficacy. When applying the solution 24 hours after adding the blood, the concentration of glutardialdehyde had to be 4.2 times that without blood. The quaternary ammonium compound reacted faster with the blood than did glutardialdehyde; the respective factors were 2.6 and 4.5. The concentration factors of chloramine T were 3.3 and 3.8. Under the conditions of the test, peracetic acid exhibited small concentration factors: 1.3 and 1.6. The microbicidal efficacy of ethanol, formaldehyde and m-cresol soap solution was not or only slightly altered by the amount of blood in the solution of the microbicidal agent.

Model tests for the efficacy of disinfectants on surfaces. IV. Communication: dependence of test results on the amount of contamination and the kind of active substance.

In the assessment of efficacy of surface disinfectants, many influencing factors have to be taken into account. One essential item is whether the surface to be disinfected is clean or soiled. Among the feasible soilings, the blood is of particular consequences because it ads impediments to many disinfecting agents. This paper shows to what extent the impairment of the efficacy of typical active agents depends on the blood burden of the surfaces. Therefore, test surfaces (varnished plywood) were contaminated with 0.01 to 0.08 ml of coagulating blood per test area (3 cm2). The blood contained cells of Staphylococcus aureus as test germs. The disinfection was effected by immersing the test objects in the disinfecting solution for 5 seconds and mingling the adhering disinfecting solution (about 0.02 ml) with the coagulated blood on the test surface with a glass spatula for about 20 seconds. Subsequently, the test objects remained in a horizontal position at room conditions for 4 hours and then the numbers of surviving test germs were determined. The graphical representation of the results shows that the efficacy curves of formaldehyde and phenol lie very closely together, i.e. their effect is hardly impaired by the different blood burdens of the test areas. The efficacy curves of glutaraldehyde, peracetic acid, chloramine T, and quaternary ammonium compounds lie very far apart from each other. To achieve the same microbicidal effect (log N/N0 = -5) when the contaminating amount is raised from 10 microliters/3 cm2 to 80 microliters/3 cm2, the concentration of chloramine T has to be raised by a factor of 5.4, peracetic acid by a factor of 9, glutaraldehyde by a factor of 24, quaternary ammonium compound even by a factor of 67. Ethanol and sodium hypochlorite showed a divergent behaviour. For ethanol, the efficacy diminution produced by increasing the contamination amount by a factor of 4 can be compensated by raising the concentration from 50% to about 70%. But again and again, there were test objects on which the number of germs able to reproduce had only been lowered by a factor of about 10(-3). At the highest contamination of 80 microliters/3 cm2, even 95% ethanol proved to be completely insufficient. With sodium hypochlorite even at the lowest contamination of 10 microliters/3 cm2, a microbicidal effect of only about 10(-5) was obtained. With increasing contamination, the highest achievable microbicidal effect clearly decreased. It is remarkable that the microbicidal effect of this active agent decreased with increasing concentrations. The results show how important it is in testing the efficacy of disinfecting agents to exactly lay down the amount of contaminating substances. To find out how safely an agent works under harder circumstances, the dependence of the microbicidal effect from the amount of contaminating substances per test area has to be determined.

[Suitability of Bacillus subtilis and Bacillus stearothermophilus spores as test organism bioindicators for detecting superheating of steam]. / Eignung der Sporen von Bacillus subtilis und Bacillus stearothermophilus als Testkeime von Bioindikatoren zum Nachweis einer Uberhitzung von Wasserdampf.

Biological indicators used to test sterilisation procedures for their efficacy consist of a so-called germ carrier to which the microorganisms used as test organisms adhere. In previous papers we demonstrated that carriers made of filter paper on contact with saturated steam show superheating while carriers made of glass fibre fleece as well as wetted filter paper do not. Using spores of Bacillus subtilis and Bacillus stearothermophilus as test organisms we have now investigated whether and to what extent carrier superheating affects the characteristic values (t50%) of these biological indicators. The indicators were exposed to saturated steam at 100 degrees C (B. subtilis) or 120 degrees C (B. stearothermophilus) under three different exposure conditions: 1. dry (i.e. conditioned to 45% relative humidity before introduction into the sterilising chamber), freely accessible; 2. dry with a substratum and a cover of filter card-board; 3. wet (moistened with twice distilled water before introduction into the sterilising chamber), freely accessible. For previously selected exposure periods, the incidence of indicators with surviving test organisms was determined. The reaction pattern of bioindicators with spores of B. stearothermophilus was different from that of bioindicators with spores of B. subtilis. For B. subtilis, the incidence of bioindicators exhibiting surviving test organisms depended on the nature of the carries as well as on the exposure conditions. On filter paper carriers, t50% increased in the order "wet, freely accessible", "dry, freely accessible", "dry, between filter card-board". On dry and wetted glass fibre fleece, resistance was approximately the same; when the indicators were sandwiched between layers of filter card-board, t50% increased. For B. stearothermophilus, t50% was largely dependent on the carrier material alone. The values obtained for filter paper were invariably much lower than those for glass fibre fleece. As the results show, using spores of B. subtilis it is possible to detect superheating, but the steam resistance of the spores is relatively low. Spores of B. stearothermophilus are of high steam resistance but they are practically unsuitable for detecting superheating. It is imperative to search for a test organism the resistance of which against steam is sufficiently high and which at the same time is capable of reacting to superheating (equivalent to reduced humidity) by a sufficiently large increase in resistance.

[Microbial resistance to formaldehyde. III> Dependence of the microbial effect on Staphylococcus aureus, Enterococcus faecium and spores of Bacillus stearothermophilus on temperature]. / Resistenz mikrobieller Keime gegenüber Formaldehyd III. Abhängigkeit des mikrobiziden Effektes von der Temperatur bei Staphylococcus aureus, Enterococcus faecium und Sporen von Bacillus stearothermophilus.

Temperature dependence of microbicidal efficacy of formaldehyde was examined with suspension tests (pH 7.0). Test germs were Staphylococcus aureus, Enterococcus faecium and spores of Bacillus stearothermophilus. The methodology was nearly the same as in previous investigations (5, 7). At given exposure periods and temperatures formaldehyde concentrations necessary to produce a microbicidal effect of log (N/N0) = -4.0 (concentrations of equal efficacy) were determined. N and N0 represent the numbers of colony-forming units in suspensions with and without formaldehyde, respectively. On rectangular graphic representation with the reciprocal value of absolute temperature on the abscissa and with the logarithm of the formaldehyde concentration on the ordinate, the formaldehyde concentrations of equal efficacy fitted straight lines. Lines referring to different exposure periods nearly paralleled each other. With increasing exposure periods the steepness of the lines decreased slightly. This effect was most pronounced with Staphylococcus aureus as a test germ. The ratio of formaldehyde concentrations of equal efficacy for exposure periods of 120 minutes at 20 degrees C and 30 degrees C, respectively, was 3.1:1 with Staphylococcus aureus, and 2.8:1 with Enterococcus faecium. The corresponding ratio obtained with spores of Bacillus stearothermophilus and referring to 60 degrees C and 70 degrees C, respectively, was 3.6:1. The logarithms of these ratios decreased with temperature in the same measure as the pertinent absolute temperatures increased. On the basis of the previously presented three-dimensional model of the relations between concentration, period of action and efficacy of microbicidal agents, it could be shown that deviations of the results from a linear and parallel course reflect an inconstant concentration exponent. When low formaldehyde concentration, long exposure period and "high" temperature coincide, the efficacy of formaldehyde is lower than calculated for a linear and parallel course of the relation.

[Further studies on the falsification of the steam resistance of bioindicators by superheating]. / Weitere Untersuchungen über die Verfälschung der Dampf-Resistenz von Bioindikatoren durch Uberhitzung.

As a supplement to a preceding paper (Zbl. Hyg., 194 (1993). 369-279) the resistance of bioindicators has been investigated which differed only in the material of which the carriers are made (filter paper and glass fibre fleece, respectively). The conditions of the tests were such that further evidence could be expected for the fact that the characteristic values of bioindicators can be falsified by superheating of the carrier and its envelope. As a test organism Enterococcus faecium has been used. To avoid side effects, the bacteria have been dried on to the carriers from a suspension in water distillators. The exposition basket of the resistometer has been loaded with 3 rows of 15 indicators (disc shaped; diameter: 6 mm), arranged parallel. At a temperature of 68 degrees C the dependency of (relative) frequency of indicators having surviving test organisms capable of multiplying on exposure time to saturated steam has been determined. Free exposed indicators made of filter paper required considerably longer exposure periods than indicators made of glass fibre fleece to change from "(nearly) all the indicators have test organisms capable of multiplying" to "(nearly) all the indicators are free from test organisms capable of multiplying". The exposure time to free 50% of carriers made of filter paper from test organisms capable of multiplying (t50%) amounted to 44.1 minutes. The frequency of indicators free from test organisms was higher at the ends than in the middle of the exposed rows. When such indicators had been wetted before exposure, they showed a t50% value of 2.3 minutes, and the frequency of indicators free from surviving test organisms was distributed over the exposed indicators evenly. Free exposed indicators made of glass fibre fleece showed a t50% value of 4.4 minutes. The frequency of indicators free from test organisms was evenly distributed. Wetting of the indicators before exposure changed t50% value only slightly. When indicators made of glass fibre fleece had been exposed between two layers of filter paper they showed a very high t50% value (47.5 minutes), and the frequency of indicators free from test organisms was unevenly distributed. Indicators being sterile could be found mainly at the ends of the exposed rows. In an envelope of parchment paper bioindicators made of glass fibre fleece showed considerably higher characteristic values than when free exposed. Superheating of the carriers amounted to about 3 Kelvin.(ABSTRACT TRUNCATED AT 400 WORDS)

[Model tests for effectiveness assay of disinfectants on surfaces. III. Dependence of test results on the type of substances and the test microbes (Staphylococcus aureus, Mycobacterium terrae)]. / Wirksamkeitsprüfung von Desinfektionsmitteln an Oberflächen in Modellversuchen. III. Mitteilung: Abhängigkeit der Versuchsergebnisse von der Art des Wirkstoffes und des Testkeimes (Staphylococcus aureus, Mycobacterium terrae).

A new test method for surface disinfectants was applied to investigate the efficacy of the most important active components of disinfectants to Staphylococcus aureus and Mycobacterium terrae. The test germs were embedded in coagulated blood. Frosted glass served as test surface. The disinfection was performed by applying a fixed amount of the disinfectant and mixing it with the contamination by rubbing. The number of surviving germs was determined quantitatively. Generally, the two test germs showed a distinctly different behaviour towards the active substances applied. Mycobacteria proved to be clearly more resistant than staphylococci, except with formaldehyde and the cresol-soap solution. The formaldehyde, the mycobacteria were only a little more resistant, while to cresol-soap solution they were even a little more sensitive than were staphylococci. Compounds containing active chloride showed a sufficient effect on mycobacteria only if the consumption of the active component by blood was nearly excluded. The quaternary ammonium compound and glyoxal, even the high concentrations, showed a totally insufficient efficacy to mycobacteria. The results shall provide the basis for a new guideline to be established by the Federal Health Office concerning the efficacy testing of surface disinfectants effective against mycobacteria, especially tuberculosis bacteria.

[Superheating of germ carriers falsifies the steam resistance of bioindicators]. / Uberhitzung der Keimträger verfälscht die Dampf-Resistenz von Bioindikatoren.

In the course of experiments on the resistance of bioindicators in saturated steam the authors noticed that carriers made of filter paper showed superheating by hygroscopic condensation. The differences in temperature between the bioindicators and the saturated steam amounted to about 5 Kelvin. Even after 20 minutes temperature equilibrium has not been reached. As a result of overheating and reduced water activity the frequency of bioindicators with surviving test organisms was increased. Beyond that, the microbiologic results depended on storage conditions of the bioindicators before putting them into the resistometer. The lower (higher) the-relative humidity, the higher (lower) the superheating, and the higher (smaller) the frequency of indicators with test organisms having survived. Carriers made of glass fibre fleece did not show any superheating and related effects. But carriers onto which small amounts of a suspension of test organisms in blood have been dried on, gave a superheating of about 1.7 Kelvin. Under identical conditions t50% values of bioindicators made of filter paper and bioindicators made of glass fibre fleece differed considerably. Experiments with Enterococcus faecium as test organism at 75 degrees C resulted in 14.4 and 1.7 minutes, respectively. Therefore, the high resistance of test organisms dried onto filter paper is an artefact caused by superheating. The experimental results should have consequences on the manufacture and use of bioindicators as well as chemoindicators and on the evaluation of results got with them.

[Sterilization--the microbiology between claim and reality]. / Sterilisation--Die Mikrobiologie zwischen Anspruch und Wirklichkeit.

Sterilization means to free an object from all living and viable germs. To fulfill this claim, the microbiology is confronted with some fundamental problems. Under the action of a microbicidal agent the microorganisms do not die at the same time, even if there is a homogeneous population. The destruction of microorganisms follows a special destruction order. The number of dying organisms is always proportional to the number of viable organisms present. On theoretical grounds it is, therefore, impossible to free an object from all living and viable germs; there will ever be left a certain proportion of viable germs, even if this proportion may be very small. The aim of sterilization must be to keep that proportion very small. To reach this goal in a practical time without damaging the objects to be sterilized, microorganisms of high resistance should be excluded, i.e. the objects to be sterilized should not be contaminated by microorganisms of high resistance to the sterilizing agent(s). The assurance level that an object is free from all living and viable germs must be so high that it is practically impossible to proof this by a test for sterility. On the other hand, the absence of microorganisms of high resistance in the objects to be sterilized makes it feasible to use microorganisms of high resistance as test organisms for the manufacture of microbiologic indicators. Such indicators enable to record as an integral the action of all microbicidal parameters of the noxious agent(s) far beyond the region for which the application of a test for sterility is sensible. Nevertheless, bioindicators have a similar status as the physical and physico-chemical controlling instruments. Microbiological indicators make it possible to guarantee the application of a certain (minimal) efficacy of the microbicidal agent(s). Whether this efficacy is sufficient to reach the wanted sterility assurance level depends, last not least, on the microbiological contamination (bioburden) of the object to be sterilized. Each sterilization process has its own prerequisites.

[A simple apparatus for the determination of the resistance of bioindicators to saturated steam at temperatures less than 100 degrees C., tested with Enterococcus faecium as test microbe]. / Eine einfache Apparatur zur Bestimmung der Resistenz von Bioindikatoren gegen gesättigten Wasserdampf bei Temperaturen von weniger als 100 degrees C, erprobt mit Enterococcus faecium als Testkeim.

An apparatus is described by means of which the resistance of microbiological indicators to water vapor at temperatures below 100 degrees C can be determined. The apparatus can be assembled from parts generally available in laboratories. The principle of the apparatus consists in the production of water vapor of the desired temperature under conditions of reduced pressure and its recondensation to water after having passed a special chamber. Accordingly, the device consists of a heated round-bottom flask serving as steam generator, an exposure chamber (B), and a condenser (D) attached to a receiver (E). The bioindicators are exposed to the water vapor in the exposure chamber. A bypass located between the steam generator and the condenser allows for continuous operation even when the exposure chamber is opened. The reduced pressure was achieved by means of a waterjet pump and adjusted by two tandem-joined pressure-regulating valves as needed. The apparatus was tested using water vapor of 73, 75 and 77 degrees C, respectively, and bioindicators containing Enterococcus faecium as test organism. In the range of exposure periods in which bioindicators change from the status "all indicators having surviving test organisms" to the status "all indicators free from surviving test organisms" the bioindicators showed D values of 5.7, 4.4 and 2.9 min, respectively. For the temperature dependence of resistance a z value of 12.5 Kelvin resulted.

[The activity of formaldehyde, glutardialdehyde, peracetic acid, chloramine T (N-chlor-4-toluolsulfonamide), m-cresol, ethanol and benzyldimethyldodecylammonium bromide against bacteria which are found in coagulated blood. (Model studies for chemical disinfection of instruments]. / Wirksamkeit von Formaldehyd, Glutardialdehyd, Peressigsäure, Chloramin T (N-Chlor-4-toluolsulfonsäureamid), m-Kresol, Ethanol und Benzyldimethyldodecylammoniumbromid gegen Bakterien, die sich in geronnenem Blut befinden (Modellversuche zur chemischen Instrumenten-Desinfektion).

The experiments were performed using frosted glass as carrier with its surface being contaminated with whole blood containing Staphylococcus aureus as test organism. At the time of sampling, a heparin preparation was added to the blood to prevent premature coagulation. After addition of the staphylococci, coagulation was initiated by means of a heparin antagonist. 10, 25, 50, 100, and 150 microliters, respectively, of the blood were homogeneously spread on rectangular test areas of 10 x 20 mm. After the blood had coagulated, each of the test objects was placed in 15 ml of the solution (20 degrees C) containing the active ingredient tested for 60 min. After that, the test objects were removed from the disinfectant and, in order to inactivate any adhering active components, treated with a neutralizing solution of suitable composition. The number of viable germs (colony-forming units) was determined quantitatively. The blood samples were ground together with quartz sand. Aliquots of the diluted suspensions were mixed with molten agar medium. The plates then were incubated at 37 degrees C over a period of 14 days. The relative number of viable germs (N/No) per test object was calculated from the number of colonies. Plotting of the microbicidal effects obtained (log N/No] versus the concentration of the active substance (see Figs. 1-3) yielded curves differing in some characteristics as e.g. curvature, slope of the lower curve section (log N/No). less than -3), concentration range according to the layer thickness of the contamination. To visualize the reduction of the efficacy of the respective disinfectants caused by blood, the concentrations of active components were determined which are necessary to achieve a microbicidal effect of log (N/No) = -4. These concentrations were plotted versus the amounts of blood per test area (Fig. 4). The resulting curve for formaldehyde was slightly U-shaped. With a raising amount of blood, the concentration required slightly decreased in the beginning and increased again from an amount of ca. 100 microliter blood per test area. For all other active substances, the required concentration of these substances increased with the amount of blood used. The curve obtained for ethanol exhibited the lowest slope. The slope of the curves increased in the following order: ethanol, m-cresol, peracetic acid, chloramine T, glutardialdehyde, benzyldimethyldodecylammoniumbromide. The curves for chloramine T and glutardialdehyde nearly paralleled each other.(ABSTRACT TRUNCATED AT 400 WORDS)

[The significance of heat activation for the testing of bioindicators on surviving microorganisms exposed to formaldehyde]. / Bedeutung der Hitzeaktivierung für die Prüfung von Bioindikatoren auf überlebende Keime, die Formaldehyd ausgesetzt waren.

Heat activation is a special phenomenon: After an additional heat treatment, a larger share of the bacterial spores which had been exposed to formaldehyde proves to be viable than without such heat activation. Model studies have been performed to test the effects of heat activation on the examination of bioindicators and test objects for surviving organisms. Test objects (cotton threads of 1 cm length) contaminated with spores of Bacillus stearothermophilus were used for these trials. The test objects were exposed to a 2% formaldehyde solution at 60 degrees C. After periods of action of 30, 45, 60 ... and 105 min, formaldehyde adhering to the test objects was neutralized. For testing these objects for surviving organisms, they were placed into a nutrient medium and incubated for 40 days at 56 degrees C. The investigation consisted of 2 parallel test series which only differed in one single point. In one series, the test objects were incubated at 56 degrees C as soon as they had been placed into the nutrient solution. In the other series, the test objects were exposed to a temperature of 95 degrees C for 1 h (heat activation) before starting incubation. The culture tubes were checked daily to see whether signs of growth (turbidity and deposits) could be observed. The frequencies of test objects with surviving organisms depending on the period of action of formaldehyde and the period of incubation determined in this way are based on the examination of 72 test objects each. Without heat activation, the share of test objects on which surviving test organisms could be detected, increased slowly with the period of incubation. Only after 30 days the counts did not increase any more when continuing the incubation (cf. Fig. 1). In the test series in which the spores had been subjected to heat activation before the incubation period, useful results were obtained already after 3 days. They only changed slightly when incubation was continued. Moreover, the frequency of test objects on which surviving organisms could be detected was always considerably higher than without heat activation. When the frequency of test objects with surviving organisms was plotted against the period of action of formaldehyde (cf. Fig. 2A), S-shaped curves resulted.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological indicators and monitoring systems for validation and cycle control of sterilization processes.

The article is divided in four parts. The first part deals with terms and definitions. After that there is given a description of the most common types of biological indicators. These two parts furnish material and introduce to the third part, a survey of the components of monitors, their functions and the criteria one has to take into consideration when designing them. The fourth and last part deals with the most essential feature of biological indicators, the resistance, its calibration, description and adjustment.

[Dependence of microbiologic test results of formaldehyde gas sterilization methods on the nature of the test material]. / Abhängigkeit der mikrobiologischen Prüfungsergebnisse von Formaldehyd-Gassterilisationsverfahren von der Materialbeschaffenheit des Testkörpers.

The efficiency of a formaldehyde gas sterilization procedure was evaluated with the aid of test pieces consisting of various materials. Both rigid and flexible tubes served as test pieces. The tubes were 75 cm long with an inner diameter of 1 mm and were sealed at one end. The bioindicators were placed inside the tubes close to the sealed end. Dried spores of Bacillus stearothermophilus adhering to linen threads served as test organisms. The test results varied according to the material of the test pieces and the thickness of their walls (see Table 1). In flexible tubes made of silicon rubber, all bioindicators became sterile, in tubes of stainless steel, all bioindicators exhibited test organisms that had survived. The findings for materials such as polyvinyl chloride, polyethylene, polyamide and polytetrafluorethylene ranged between these two extremes; the frequencies of bioindicators containing viable germs were 10, 55, 68 and 85%, respectively. Rigid and flexible tubes which had been sealed at both ends served to demonstrate that silicon rubber and polyvinyl chloride were highly permeable for formaldehyde and water vapour. Also the other plastic materials tested were permeable for formaldehyde and water vapour but longer exposure periods were needed to create conditions in the interior of the tubes that would result in a killing of the test organisms (see Fig 2). In this respect, polyamide exhibited a peculiar behaviour. The number of viable spores remained at the initial level for a long period before a decline took place. From the results of testing, it is concluded that test pieces must conform to the objects to be sterilized not only in their dimensions (length, inner diameter) but also in the characteristics of their material. The walls of the test pieces should not have a higher permeability for formaldehyde and water vapour than the material to be sterilized. The highest demands on the efficiency of formaldehyde gas sterilization procedures are those created by mental tubes and thick-walled flexible polytetrafluorethylene. Instruments and devices to be sterilized by a formaldehyde gas procedure should be preferentially made of materials which are sufficiently permeable for formaldehyde and water vapour as e.g. silicon rubber. Such gas-permeable components may considerably facilitate the sterilization of cavities which have a small lumen and are difficult to reach.

[Method for contaminating test objects with coagulated blood]. / Eine Methode zur Kontamination von Testobjekten mit gerinnendem Blut.

A special method to contaminate test objects with blood is described. It is characterized by the use of coagulable blood and its coagulation when adhering to the test objects. Above all, the method is suitable for the microbiological evaluation of the efficacy of agents and methods for the disinfection of surfaces to which coagulated blood will adhere (e.g. instruments). Immediately upon sampling, the blood is heparinized (Liquemin 500 from Hoffmann-La Roche AG at a ratio of 0.1 ml per 100 ml blood). Until use, the coagulable blood is stored at 0 degrees C. For the contamination of test objects, the test organisms are added to the blood (at 0 degrees C), either by mixing the blood with a small amount of a suspension of the test organism or by centrifuging this suspension and dispersing the sedimented organisms in the blood. To start coagulation, Protamin 1000 (Hoffmann-La Roche) is added at a ratio of 0.15 ml per 10 ml blood. Immediately afterwards, the test objects are contaminated with the coagulating blood. Until complete coagulation of the blood, taking ca. 15 min, the test objects are stored in a humid chamber at ca. 20 degrees C.

[An expedient semi-automatic procedure for the preparation of large quantities of bioindicators especially for use in gas sterilization processes]. / Ein rationelles halbautomatisches Verfahren zur Herstellung grosser Mengen Bioindikatoren, insbesondere für Gas-Sterilisationsverfahren.

Bioindicators serve to test the efficacy of disinfection and sterilization procedures. Such indicators mostly consist of a support (filter paper, as a rule) to which micro-organisms have been fixed by drying. The authors have used a thread as support and a special apparatus for semi-automatic preparation of the bioindicators. The components of the device are either commercially available or may be prepared from commercially available material without difficulty. The principle of the method is as follows: The thread serving as the support is drawn slowly, at constant speed, through the suspension of test organisms and dried in an air stream immediately afterwards. The apparatus consists of a cylindrical glass tube of a few centimeters in diameter, an electric motor slowly rotating the cylinder, a fan, a magnetic stirrer, and an ice-water bath. A small vial containing the germ suspension is immersed in the ice-water bath. The vial is sealed by a screw cap with two glass tubes of about 3 mm inner diameter passing through it. One of the glass tubes being bent in its upper part reaches far down into the vial to leave just enough play for free rotation of a magnetic stirring rod. This tube serves to introduce the thread into the germ suspension. The second straight tube does not reach as far down as the first one. Its lower opening should not be immersed in the germ suspension. This tube serves as a guide for the returning thread. Preparation begins by winding the thread to be soaked with the suspension around the cylinder.(ABSTRACT TRUNCATED AT 250 WORDS)