UNIVERSITY OF CALIFORNIA 
 
 AGRICULTURAL EXPERIMENT STATION 
 E. W. HILGARD, Director 
 
 PRESERVATION OF UNFERMENTED GRAPE-MUST 
 
 By F. T. Bioletti and A. M. dal Piaz 
 
 BULLETIN 130 
 
 BERKELEY 
 
 THE UNIVERSITY PRESS 
 August, 1900 
 
PRESERVATION OF UNFERMENTED GRAPE-MUST. 
 
 By Frederic T. Bioletti and A. M. dal Piaz. 
 
 The use of unfermented grape-juice or "must" as a beverage, both 
 in health and sickness, has been common in vine- growing countries 
 from time immemorial. It has, however, until lately been restricted 
 to the immediate vicinity of the vineyards and the season of ripe 
 grapes. This is owing to the great facility with which fruit juices of 
 all kinds spoil within a few days after being expressed from the fruit, 
 unless preserved artificially. The great progress made within the last 
 few decades in methods, both legitimate and illegitimate, of food 
 preservation, has made it possible to keep grape- juice for an indefinite 
 period, and to make use of it as a beverage at all seasons and in all 
 places. Accordingly the manufacture of grape-must has attained 
 notable proportions in some European countries, and in some parts of 
 the United States. Its use, however, has up to the present day been 
 almost exclusively medicinal, although it is one of the most wholesome 
 and agreeable beverages known, in health as well as disease. The 
 cause of this restricted use is twofold. In the first place, in order to 
 simplify and cheapen the processes of manufacture, injurious preserva- 
 tive agents have been made use of by the unscrupulous, and, in the 
 second place, the lack of the necessary special knowledge and technical 
 skill has resulted in many failures of attempts to preserve the must 
 in a legitimate manner, so that the price has been necessarily too high 
 for the regular consumer. 
 
 It is to remedy this lack of knowledge on the part of the manu- 
 facturer, to warn the consumer against the injurious effects of anti- 
 septics, and to call attention to the merits of this delicious beverage, 
 that this Bulletin is written. More stress is laid on general princi- 
 ples than on actual methods, as the methods will vary considerably 
 according to the scale on which the manufacture is conducted and 
 according to the facilities and appliances at the disposal of the indi- 
 vidual manufacturer. The business can be conducted profitably with 
 either small or large quantities, but must necessarily be commenced 
 on a modest scale by the inexperienced. The directions given here 
 should enable almost any grape- grower to commence operations, 
 and gradually, as he acquires confidence and skill, to engage more 
 largely in what should be an important industry in California. 
 
Composition of Grape- Must. — A consideration of the following- 
 table, showing the constituents of the normal juice of ripe grapes, will 
 make clear its value as a nourishing beverage in health, and also its 
 therapeutic efficacy in certain cases of disease: 
 
 Parts in 1,000. 
 
 Grape sugar (dextrose and levulose) 18U to 280 
 
 Free organic acids (tartaric, malic, tannic) 1 to 10 
 
 Salts of organic acids (cream of tartar, poo. sium malate, 
 
 calcium tartrate, calcium malate 4 to 8 
 
 Ash (containing potassium, sodium, calcium, magnesium, 
 
 ferric oxide, phosphoric and sulfuric acids).... 3 to 5 
 
 Nitrogenous matter (proteids, amido -compounds) 3 to 10 ■ 
 
 This table shows that some of the principal constituents of wine, 
 such as alcohol, glycerine, etc., are totally lacking in pure grape-juice; 
 and it is to be noted that it contains no unwholesome substance of 
 whatever kind. 
 
 Grape- juice should and can be delivered to the consumer so as to 
 contain no other substances than those shown above. If chemical 
 analysis shows any other ingredients, a fraud has been practiced; and 
 as all the additions usually made are in the nature of antiseptics or 
 preservatives, they are all more or less injurious. All the antiseptics 
 used are easily detected by more or less simple chemical tests, and if 
 an effective pure-food law were in operation it would be easy for the 
 consumer and the honest producer to protect themselves by occasional 
 chemical analyses of the various brands of grape-must on the market. 
 In the larger European countries, where such laws do exist, the use 
 of injurious adulterants is rendered dangerous, if not impossible. 
 
 An analysis of a pure grape-must made by a government chemist 
 in Austria, and one of must put up by Swett & Son, at Martinez, made 
 by Mr. G. E. Colby at this station, gave the following results: 
 
 Analyses of Grape -Must. 
 
 Austria. California. 
 
 Per Cent. Per Cent. 
 
 Solid contents by spindle (Balling) 21.62 20.60 
 
 Alcohol .none none 
 
 Total acid (as tartaric) .78 .53 
 
 Volatile acid 01 .03 
 
 Grape sugar 19.62 19.15 
 
 Cream of tartar .61 .59 
 
 Free tartaric acid 03 .07 
 
 Ash 37 .19 
 
 Phosphoric acid .02 .04 
 
 No cane sugar, starch sugar or antiseptics, were found in the Cali- 
 forniaftor the Austrian musts. Artificial (Anilin) coloring matter, 
 salicylic, benzoic and boracic acids, formalin and fluorides were tested 
 for in the California must but none were present. 
 
 This is approximately what should be shown by any pure grape- 
 juice. It is instructive to compare this with some partial analyses, 
 
made at this station, of some of the beverages offered to the consumer 
 in California under such titles as "Unfermented Wine," and "Pure 
 Grape Juice," recommended for invalids and for communion purposes: 
 
 SAMPLE 1 - PerCent. 
 
 Solid contents by spindle 22.00 
 
 Total acids (as tartaric) .59 
 
 Sulfurous acid (antiseptic) 06 
 
 SAMPLE 2 ' Per Cent. 
 
 Solid contents by spindle '. 20.80 
 
 ' Alcohol, by volume 2.00 
 
 Salicylic acid (antiseptic) 3.90 
 
 The first sample was sold as a "curative for throat and lung 
 troubles." The amount of sulfurous acid it contained was sufficient to 
 cause throat and lung as well as digestive troubles in a healthy person. 
 The second sample was sold as "pure unfermented grape-juice," but 
 besides containing a large amount of the injurious antiseptic salicylic 
 acid (more than twenty times as much as was necessary to preserve it) , 
 it contained two per cent, of alcohol. Even healthy persons, much 
 more invalids, would contract severe indigestion from the use of such 
 a product, which is a fraud upon the public. 
 
 Causes of spoiling. — In order to make clear the nature of the 
 problem which must be solved in order to preserve grape-juice indefi- 
 C\r\ r\ ***£& nitely, a short account 
 
 O ^r£==^3**k °*' ^ ne causes °f spoiling 
 
 ^\ 0c $ will be useful . When 
 
 Z3/c W^'& /I grapes ' or any fruits ' 
 
 q? W& c J J are gathered, the sur- 
 
 // faces in contact with 
 
 „ o^v. </\ f * JJ the air have the spores 
 
 rP *° /i of various fungi, yeasts, 
 
 and bacteria adhering 
 
 to them. All these 
 
 a, a', a". Various forms of a mold, (Mucor). 
 
 c, d, e. Various forms of yeasts, molds, and bacteria. Spores are miCrOSCOpic, 
 
 Fig. 1.-MICRO-ORGANISMS ON GRAPES. but ^ ^^ Qf theh , 
 
 appearance, when sufficiently enlarged by the microscope to be visible, 
 may be obtained by reference to Fig. 1, which shows various forms 
 of these organisms developed on the skin of a muscat grape. 
 
 When the grapes are crushed and the juice expressed, the latter 
 will be contaminated by these spores, washed off the skin. In the air 
 they are dry, and therefore inert; but very soon after they are 
 surrounded by the must, which is a very favorable medium for their 
 growth, they assume an active form and commence to multiply. If 
 the must is warm, this change to an active state occurs very soon and 
 
the consequent increase in numbers is proportionately rapid. If, on 
 the contrary, the grapes and therefore the must be cool, the increase 
 is much slower; but, eventually, if left alone the organisms increase 
 until the must ferments. This fermentation consists principally in 
 the changing of the grape sugar into alcohol and carbonic acid, and 
 is the essential part of the process which changes grape- juice into wine. 
 
 The main object, then, of the producer who wishes to place "pure 
 unfermented grape-juice" upon the market, is to permanently prevent 
 this fermentation. Besides this, the grape-juice must be quite clear, 
 in order to present an attractive appearance to the consumer. 
 
 To attain the first object there are two general groups of methods, 
 which may be called respectively chemical and physical . All the 
 chemical methods consist in the addition of germ poisons or antisep- 
 tics, which either kill the microscopic organisms of fermentation or 
 permanently prevent their growth and increase. Of these substances 
 the principal used are, besides salicylic and sulfurous acid already 
 mentioned, boracic acid, saccharin, and, of late, formalin. Many 
 patent preservatives are found on the market, but they nearly all 
 contain one or more of these substances as their active principle. 
 They are all injurious to digestion and in other ways; and it may be 
 said in general that any substance which prevents fermentation will 
 also interfere with digestion, and is therefore to be avoided. 
 
 The Physical methods work in one of two ways: they remove the 
 germs by some mechanical means, such as a filter, or a centrifugal 
 apparatus; or they destroy them by heat, cold, electricity, etc. The 
 methods which depend upon the removal of the germs are inapplicable, 
 as this cannot be done thoroughly except with very small quantities 
 of liquid, and the minute organisms with which we have to deal will 
 soon increase sufficiently to spoil the liquid, if a single one escapes the 
 filter. One yeast-cell, for instance, at ordinary temperatures will 
 increase to tens of millions in three or four days ; and if the tempera- 
 ture is warm the increase will be still more rapid. We are, then, 
 reduced to those physical methods which destroy the germs; and of 
 these the only one which has been found useful in this connection is 
 the use of high temperatures. This method depends on the fact that 
 when a liquid is heated up to a sufficiently high temperature all 
 organisms present are killed. This temperature is called the "death 
 point," and differs for each particular variety of organisms. The 
 death point will also differ according to the composition of the liquid 
 in which the organism is immersed. Yeast, for instance, is killed at 
 a lower temperature in must than in water, on account of the acidity 
 of the former. Time, also, is a factor in determining the death point. 
 An organism may not be killed if heated to a certain degree quickly 
 
and as quickly cooled ; while if it is kept at that same degree for some 
 time it will be killed. Some tests made at this station with a pure* 
 yeast isolated from a California wine illustrate these facts. The 
 yeast was placed in must which had previously been completely freed 
 from all germs, and w T as heated to various temperatures for various 
 lengths of time, with results as follows: The initial temperature of 
 the must was 20° C. (68°F.) and the yeast was killed by heating 
 it gradually up to 60° C. (140° F.) in fifteen minutes; that is to say, 
 the time taken to bring the temperature from 20° C. (68°F.) to 60° C. 
 (140° F.) was fifteen minutes, and at the end of this time the must 
 containing the yeast was allowed to cool in a room at 20° (68° F.). 
 This same yeast was not killed when heated in twenty-five minutes 
 from 20° C. (68° F. ) to 50° C. ( 122° F. ) , nor even when kept at the latter 
 temperature for five minutes longer. But when kept at this tempera- 
 ture for ten minutes longer, all the yeast cells were killed. Another 
 test with the same yeast showed that if heated from 20° C. (68° F.) to 
 45° C. (113° F. )in twenty minutes, and then kept at the latter tempera- 
 ture for twenty minutes, few or none of the yeast cells were killed, 
 though in thirty minutes most of them were rendered incapable of 
 growth. However, even in the last case some were left alive and 
 ultimately spoiled the must. We learn from these tests that heating 
 to 45° C. (113° F.), even for a somewhat prolonged time, cannot be 
 depended on to sterilize grape-must, and that even 50° C. (122° F.) 
 requires too much time to be practical. A heating to 60° C. ( 140° F. ) , 
 however, would probably be quite safe, provided that only this 
 particular variety of yeast were present in the must. In practice, 
 however, we have an unknown number of kinds of micro-organisms 
 present, and some of them may be able to withstand a somewhat 
 higher temperature than this. 
 
 It must be kept in mind also that fungi, including yeasts, exist in 
 two states, the vegetative or growing state, and the spore or resting 
 state. The latter is more resistant than the former; and it has been 
 found that yeast spores, for instance, to be killed must be heated 
 about 5° C. (9° F.) higher than the same yeast in the growing state. 
 The above tests were made with yeast containing no spores; but, as 
 in practice, spores may be and undoubtedly usually are present, a 
 temperature 5° C.(9° F.) higher than indicated would be necessary. 
 Practical experiments made at this station indicate that must can be 
 safely sterilized at a temperature of 75° C.(167°F.) or 80° C.(176°F.) 
 if all the precautions indicated below are observed. At this tempera- 
 ture the flavor of the grape-juice is hardly changed, though at between 
 90° C (194° F.) and 100° C. (212° F.) it is slightly affected. 
 
 * Pure in this case means a yeast consisting of only one variety of micro-organism. 
 
Another property of fungi and their spores, which is of importance 
 in this connection, is their great resistance to heat when dry. Yeast 
 can be heated in a dry state to a temperature above that of boiling 
 water without being killed; the spores of some fungi (e.g., common 
 mold) are even more resistant. The bearing of this upon the preser- 
 vation of must is that during the final sterilization, which takes place 
 in glass bottles or similar vessels, portions of the inner surface of the 
 cork and of the bottle above the liquid are comparatively dry; and if 
 any spores should be adhering to these parts there is danger that they 
 will not be killed, and that afterwards, when they come in contact 
 with the must, they will grow and cause fermentation or mold. For 
 this reason both the bottles and the cork must be thoroughly sterilized 
 before being used. This can be accomplished for the bottles by boiling 
 them for at least half an hour after thorough washing, and then 
 allowing them to drain in a place where they are exposed to no 
 draughts or dust. This boiling should be done as short a time as 
 possible before filling the bottles, and they should be handled care- 
 fully, taking care not to touch their mouths, for with the greatest care 
 the hands cannot be kept free from mold spores. To sterilize the 
 corks this method is not always sufficient, as spores that may be in 
 cracks are liable to escape. For sterilizing the corks, some closed 
 receptacle should be used which will safely withstand considerable 
 pressure. The corks when placed in this receptacle can best be steri- 
 lized by steam under pressure, which is allowed to flow in until the 
 pressure, as shown by a gauge, is at least ten pounds. This pressure 
 indicates a temperature of about 115° C. (239° F.), and should 
 continue for at least twenty minutes. 
 
 Apparatus required. — The apparatus necessary for preserving grape- 
 must on any but the very smallest scale consists of: 1, a continuous 
 pasteurizer; 2, a pressure filter; 3, a pressure sterilizer for corks 
 (this may be dispensed with) ; 4, a bottle pasteurizer; 5, a boiler for 
 pressure steam. Certain other utensils are, of course, necessary, but 
 they are such as are found in almost every wine-cellar. 
 
 The manner of operating. — The method of proceeding is as follows: 
 Sound, clean grapes, preferably those having high natural acidity, are 
 picked carefully, while cool, into clean boxes. They should not be 
 too ripe or the must will be too sweet and difficult to clear. They 
 should be crushed as soon as possible after picking, and the juice run 
 into perfectly clean puncheons or other receptacles which have been 
 previously steamed. If the must is cold — 15° C. (59° F.) or under — 
 it may be safely left to settle for twenty-four hours or more. This 
 settling is an advantage, as it rids the juice of most of the floating 
 solid matter, and facilitates the subsequent filtering. During this 
 
settling the must should be closely watched, in order to anticipate 
 even a commencement of fermentation. After this settling, when the 
 must has become almost clear, it is run through a continuous pasteur- 
 izer, of one form of which Fig. 2 gives an idea. 
 
 It is heated in this to 80° C. (175° F.) and should come out cool, 
 not warmer than 25° C. (77° F.) and should pass into fresh settling 
 receptacles. For this purpose puncheons or other casks may be used, 
 if they have been thoroughly sterilized by steam, though the best 
 receptacles would doubtless be casks or vats of metal lined with 
 enamel, such as are now made. The greatest care must be taken to 
 avoid contamination of the must as it flows from the pasteurizer. It 
 
 ST, Steam pipe. H, Outlet for hot pasteurized must. U, Inlet for unpasteurized must. 
 W, Water bath. P, Outlet for cooled pasteurized nmst. T, T' , Thermometers. 
 
 Fig. 2. — Continuous Pasteurizer. 
 should pass directly, by means of a block-tin pipe, from the pasteurizer 
 into the receiving casks. The end of this pipe should be thoroughly 
 sterilized by plunging into boiling water, and should never be allowed 
 to touch the hands or any exposed surface. When a receiving cask is 
 full it should be closed immediately with a wooden bung, sterilized 
 preferably in the way already described for corks. If all these opera- 
 tions have been conducted with the requisite care, and the casks kept 
 in a cool cellar, the must will remain without fermenting for many days 
 or even weeks. During this time it deposits more or less sediment 
 which has been formed in heating. It is then ready for filtering. 
 
 Filtration. — This filtration is best accomplished by means of a 
 filter so constructed that the must passes upward through the filtering 
 medium, under pressure. Such a filter, made by the International 
 Filter Co. of Chicago, is shown in Fig. 3. 
 
9 
 
 This filter consists essentially of two shallow bowls clamped together mouth to 
 mouth with the filtering medium between them. The unfiltered must enters the 
 lower bowl through the pipe on the right of the figure, passes through the filtering, 
 medium into the upper bowl and makes its exit when clear through the faucet a little 
 to the left of the middle of the figure. The small faucet at the bottom of the lower 
 bowl is for the purpose of cleaning the filter. Occasionally, when filtration 
 becomes slow, this faucet is opened for a few moments. This allows the sediment 
 accumulated at the bottom to escape and at the same time the entering must takes 
 a rotary course in the lower bowl, thus cleaning off the surface of the filtering 
 medium; so that when the cleaning faucet is closed filtration proceeds as before. 
 On a large scale, a filter press, such as is used in large wineries and in beet sugar 
 factories, might conveniently be used. 
 
 It is impossible to prevent a certain amount of contamination by 
 
 fungous spores during 
 nitration; but it should 
 be minimized as much as 
 possible by the greatest 
 cleanliness, and attention 
 to sterilizing everything 
 with which the must 
 comes in contact. In 
 this regard it should 
 be kept constantly in 
 mind that in an ordinary 
 room or cellar, where 
 there is little dust, there 
 is comparatively little 
 danger of contamination 
 from the air, the main 
 danger being from the 
 solid surfaces with which 
 the must comes in con- 
 tact. The must may be 
 bottled directly as it 
 flows from the filter, or 
 it may pass into a steri- 
 lized temporary recep- 
 tacle, from which it is 
 bottled. It should, how- 
 ever be placed in its final 
 receptacles (bottles, etc.) 
 the same day on which 
 it is filtered, corked im- 
 mediately, and sterilized as soon as possible, preferably within 24 hours. 
 
 Fig. 3.— Filter for Clarifying Must. 
 
10 
 
 Final sterilization. — On account of the recontamination during 
 nitration, a final sterilization must be made after the bottles are corked. 
 This is accomplished by means of a bottle-sterilizer, which the pro- 
 ducer can construct himself. A simple and efficient form is shown 
 in Fig. 4. 
 
 It consists of a wooden box or trough provided with a wooden 
 grating placed about two inches from the bottom. The bottles, after 
 being filled with the filtered must and corked, are placed in perforated, 
 or wire baskets, which rest upon the grating. The trough should 
 contain enough water to completely submerge the bottles. The water 
 should be kept at a constant temperature of about 85°C. (185°F.) by 
 means of a steam coil placed beneath the grating. The bottles should 
 be left in this pasteurizer for exactly fifteen minutes if they are one- 
 quart champagne bottles. For other sizes it is necessary to make a 
 test with a bottle of must in which a thermometer has been placed, in 
 order to determine how long it takes for the entire contents of the 
 bottle to reach the required temperature. It has been found at this 
 station, that although the must in the upper part of a quart cham- 
 pagne bottle reaches 75°C. (167°F. ) in eight minutes, when surrounded 
 
 DB, Double bottom. ST, Steam pipe. W, Water bath. T, Thermometer. 
 (Bottle, shows method of adjusting a cork holder of sheet metal.) 
 
 Fig. 4.— Cork Clamp and Pasteurizer for Must in Bottles. 
 
 by water at 85°C.(185°F.)it requires fifteen minutes before the must 
 at the bottom of the bottle acquires that temperature. The sterilization 
 in bottle should be conducted at a temperature at least 5°C. (9°F.) 
 lower than that reached in the continuous pasteurizer. Thus, if the 
 water in the first case was kept at 90°C.(194°F.) or 95°C.(203°F.) and 
 the must attained a temperature of 80°C, the water in the bottle 
 pasteurizer should be kept constantly at 85°C. and the time of 
 pasteurizing so chosen that the must in the bottles will attain a 
 maximum temperature of 75°C.(167°F.) If the final heating is 
 
11 
 
 higher than the first, it may cause a precipitation of solid matters which 
 will make the must cloudy in the bottles. 
 
 During this sterilization in bottle the corks are liable to be expelled 
 by the pressure developed. To prevent this they may be tied down 
 with strong twine; but it is a great saving of time and labor to use 
 some such contrivance as that illustrated in Fig. 4. 
 
 By this operation the must is thoroughly sterilized and will then 
 keep unchanged for years, or until the bottles are ODened. If, however, 
 the bottles are to be capsuled, or kept in a very damp place, there is 
 one other cause of spoiling that must be guarded against. However 
 carefully all the various operations are conducted, there are sure to be 
 mold spores on the upper surface of the cork. If this surface remains 
 dry these spores will not grow, and are harmless. But when the cork 
 is covered with a capsule the space between the capsule and the 
 surface of the cork finally becomes moist, and any spores there will 
 develop. Some molds have great penetrating power and may force 
 their way either through the cork, or between the cork and the neck 
 of the bottle (especially if the very best quality of corks has not been 
 used) and finally reach the must. The molds which enter in this way 
 do not, as a rule, grow into the liquid, on account of the small amount 
 of air present; but they make a moldy layer on top, which lessens the 
 selling value of the must, if it does not actually spoil it. This danger- 
 can be avoided by dipping the top of the neck of the bottle into a two 
 per cent, solution of bluestone and water, in such a way as to wet the 
 upper surface of the cork, before putting on the capsule. The same 
 object may be attained by dipping the neck, in the same way, into 
 very hot, melted paraffine. The bluestone acts by killing any spores 
 that maybe on the cork or which may find their way there later. The 
 heated paraffine kills the spores present, and prevents later infection 
 by completely covering the cork and keeping it dry. 
 
 The quality and character of the grape- juice prepared in this way 
 will vary greatly according to the variety of grape used ; and a pleasing 
 variety may be obtained by using, partially or wholly, grapes of 
 high aroma, such as Muscat, Isabella, etc. The color will, however, 
 always be white or yellowish except with a few grapes, such as the 
 Bouschets, which have pink or red juice. Red must, however, can 
 be obtained by a modification of the process described. If the must, 
 after it passes through the continuous pasteurizer, is allowed to come 
 out hot and flow into a vat containing the skins of red grapes, almost 
 any desired depth of color ma}^ be obtained, depending on the variety 
 of grape used and the time during which the hot must is left in 
 contact with the skins. Must prepared in this way, however, differs 
 in other respects than in color from the white must. Besides coloring 
 
. 12 
 
 matter various substances are extracted from the skins, the principal 
 being tannin. This makes the composition of the red must more like 
 that of red wine, though of course it still contains no alcohol. A 
 grape-juice of this character might appropriately be called "Unfer- 
 mented Wine," and would doubtless be useful in medicine, as it would 
 possess certain tonic properties not found in the white must. The 
 regular consumer, however, would in all probability generally prefer 
 the white must. 
 
 Grape-must, containing as it does generally from twenty to 
 twenty-four per cent, of sugar, is too sweet for many palates and 
 constitutions, but it may be diluted with water by the consumer to 
 any desired extent; and a mixture of equal parts of grape-must and 
 carbonated or mineral water makes a beverage much appreciated by 
 many people. In Europe a certain amount of sparkling grape- juice 
 is put up, i.e. grape- juice which has been carbonated, or charged 
 with carbonic acid gas. This, though an addition to the natural juice 
 of the grape, cannot be looked upon in any sense as a fraud or 
 adulteration, and makes the beverage more palatable to many; besides 
 if properly done it has no injurious effects on the health of the 
 consumer. 
 
 In conclusion the following brief summary of the main precautions 
 to be observed in the manufacture of unfermented grape-must may be 
 useful : 
 
 1. Only clean and perfectly sound grapes should be used. 
 
 2. The grapes should be picked and handled when cool. 
 
 3. The greatest cleanliness is necessary in every stage of the process. 
 
 4. All utensils and apparatus used should be cleaned and sterilized 
 immediately before using, and as short a time as possible after using. 
 
 5. The last sterilization should be at a temperature at least »5°C. 
 (9°F.) lower than the temperature used in the first sterilization. 
 
 6. Reliable thermometers should be used, and the temperature 
 watched very carefully.