Microbioogical Process Discussion

Calculation of Heat Sterilization Times for Fermentation MediaFRED H. DEINDOERFER1

Merck Sharp & Dohme Research Laboratories, Merck & Co., Inc., Rahway, New Jersey

Received for publication September 14, 1956

Sterilization of nutrient media is an operationessential to all industrial fermentation processes re-quiring pure culture maintenance. AMethods for steri-lizing nutrient media include 1) heating, 2) filtration,3) irradiations, 4) sonic vibration, and 5) exposure tochemical agents. Because heating the medium, usuallyby steam, is the most reliable and on a large scale theeasiest to control, it is the method of choice throughoutthe fermentation industry. The increasing availabilityof radioactive isotopes undoubtedly will stimulatefurther study of the use of gamma radiation for sterili-zation. However, a great deal of work is needed beforecommerically feasible gamma ray applications will beused in the fermentation industry. The other methods,although successful on a laboratory scale, present toomany operational drawbacks for large-scale use infermentation processes.How long should a nutrient medium be exposed at

high temperatures to achieve sterile conditions? Thisquestion arises often and usually is answered in bench-scale or pilot plant tests. Improper translation of thesetest results with large equipment may subject themedium to unnecessary overheating. A method bywhich minimum exposure time to achieve sterile condi-tions can be calculated from easily obtainable thermal-death relationships and the temperature conditions in aheat sterilization process is presented in this paper. Themethod can be used to correlate sterilization conditionsamong various sized fermentation vessels. It also per-mits evaluation of the temperature and retention timerelationship in continuous sterilizers. It need not berestricted to fermentation processes, but should beapplicable to any process involving a heat sterilizationor pasteurization operation.

Thermal Resistance of MicroorganismsThe ability of microorganisms to withstand heat is

much greater than that of other forms of life. Thermo-philes capable of even tolerating common heat sterili-zation conditions, such as exposure to steam at 250 Ffor 20 to 30 min, exist. Their occurrence in nutrientmedia, however, is rare. Relative resistances of severalmicroorganisms to moist heat are shown in table 1.

1 Present Address: E. R. Squibb & Sons, New Brunswick,New Jersey.

Bacterial spores are by far the most heat resistant formsof microorganisms.The thermal resistance of bacterial spores is in-

herently different among species and even among strainsof the same species. Besides thermal resistance inherentto spores of a particular species, variations can be causedby a number of environmental factors. These can begeneralized into two groups: 1) environmental factorsaffecting sporulation prior to sterilization, and 2)environmental factors affecting spores during thesterilizing heat exposure. The most important factorsin nutrient media sterilization fall in the latter categoryand include factors such as the pH during sterilizationand the osmotic nature of the media. The presence ofsuspended solids also affects sterilization by physicallyinsulating spores from heat exposure.

Thermal-Death Relationships of Bacterial Spores

Because bacterial spores are the most heat resistantforms of microorganisms, their germinated cells are themost frequent contaminants encountered in industrialfermentations due to improper sterilization. The ensuingdiscussion will deal, therefore, with the thermal-deathrelationships of these forms. Relationships between thenumber of viable spores and exposure time to heatdemonstrate a logarithmic rate of spore viability de-struction. Two typical survivor curves for spores ofBacillus stearothermophilus strain 1518, an organismoften used for sterilization studies in the food industry,are shown in figures 1A and lB. These curves wereobtained in buffer solutions and would differ from theirrespective curves in other media. Spore destruction atthe higher temperature is more than 400 times fasterthan at the lower temperature.

TABLE 1. Relative resistances of microorganisms to sterilizationby moist heat*

Organism ~~~~~~RelativeOrganism Resistance

Escherichia coli ............................. 1Bacterial spores ............................. 3,000,000Mold spores ................................. 2-10Viruses an(dbacteriophages ................. 1-5

* Rahn (1945).

221

F. H. DEINDOERFER

The curves in figures IA and lB represent first-orderreactions, where the rate of spore destruction is directlyproportional to the number of surviving spores. Thiscan be described mathematically as follows:

dNdO

spores of a particular species will be a function oftemperature only. It will behave similarly to other velo-city constants in its relation with temperature, quantita-tively expressed by the empirical Arrhenius equationas follows:

(1)k = Ae RT

where N is the number of surviving spores in the volumeunder consideration, 0 is the time of exposure, k is a

velocity constant, and dN is the rate of spore destruc-dO

tion.By integrating this equation, it can be modified to a

form that can be used to calculate the time for steriliza-tion at a particular temperature where the velocityconstant, k, is known. Then,

(3)

where A is a proportionality constant, R is the gasconstant, T is the absolute temperature, and ,u is anapparent activation energy for heat destruction of thespores. No theoretical significance need be attached tothis equation for this discussion. The conformity ofmany destruction rates to the Arrhenius equationprovides sufficient justification for its generalizationhere. In logarithmic form, equation 3 may be writtenas follows:

6=2.3l N1k N (2)

where N1 is the number of particular spores at the startof the heat exposure and N2 is the number of the samespores surviving at any time, 0, after exposure hasstarted. N2 in this relationship can never reach zero,but practikally this does not matter. For destruction ofspores wAith only one chance in one hundred of failing todestroy all spores, N2 should be set equal to 0.01.Higher degrees of confidence result from smaller valuesof N2.

In a given environment the velocity constant, k, for

05\

A) TEMPERATURE 220 F.REDRAWN FROM DATA

14 OF BALL (1943)

-J

\ k . . 00057 SEC.-'I102

z

* TIME MINUTES

logk= -- /+logA

For all practical purposes, log A may be treated as aconstant.2 Then equation 4 is of the form y = mx + b

2 The significance of the term, log A, is evident from exami-nation of the Eyring rate equation. See Johnson et al. (1954).

It includes an entropy term, which in cases of spore de-

struction is quite large. Occasionally, values of AS along withvalues of /Aare listed for thermal spore destruction. This com-pletely defines the velocity constant, k, as a function of tem-perature.

'OS

B) TEMPERATURE 268.7 F.REDRAWN FROM DATA

104 _ \OF STERN AND PROCTOR (1954)

-Iw

103

,o k- 0.25 SEC.-'0

z

(4)

-0- TIME MINUTES

FIG. 1. Survivor curves for spores of Bacillus stearothermophilus strain 1518 at two different temperatures

[VOL. 5222

STERILIZATION OF NUTRIENT MEDIA

and a plot of log k versus for spores of a particularT

species in a given medium should yield a straight line

having a slope equal to - 23RT* A typical plot is shown

in figure 2. Such plots are constructed by determiningthe velocity constant, k, at at least two temperatures.If there is doubt that the kinetics of the spore destruc-tion are such that the velocity constants are not relatedto temperature as in the Arrhenius equation, more kvalues should be determined. The subsequent treat-ment is not affected by another relationship, as long as

it is known. For spores of B. stearothermophilus strain1518, characterized in figure 2, the activation energy

for destruction is 67.7 Kcal/mole. Activation energiesand entropies for thermal destruction of other bacterialspores are listed in table 2.3

Batch Sterilization

The most common method of heat sterilizing nutrientmedia is the batch method. The medium ingredients are

charged directly into the fermentor. The fermentor andmedium are sterilized by heat transferred across thejacket and/or coil surfaces from condensing steam.Fermentors are usually of the geometric design shownin figure 3. The heat transfer surfaces are marked infigure 3 by heavy lines. The medium is agitated andoften steam is injected directly into the mediumthrough the air sparger to speed up the sterilization.

3Various other terms commonly are used to describe ther-mal behavior of bacterial spores during heat sterilization,especially in the food industry. The terms used in this articleare consistent with those used in chemical reaction kinetics.Some other terms and their relationship to the velocity con-

stant, k, are tabulated for reference purposes.

Term Relation Definition

kT The ratio of the velocity constant at aQio klTjo particular temperature to the veloc-

ity constant at a temperature tendegrees lower. This ratio is oftenfalsely assumed constant over theentire temperature range. It dimin-ishes as temperature is raised

D 2.3 Two synonymous terms representingk the time required for 90 per cent de-2.3 struction of a spore population at ak particular temperature, often called

the decimal reduction time

TDT 2 3 log N, Thermal death time, a term attachedk N2 to the time required for "complete"

destruction of spores in a particularenvironment. Algebraically, thisterm is meaningless unless N2 hassome finite value

F as above The thermal death time at 250 F

Injection of steam introduces some dilution to themedium, but this is taken into account during batchmake-up prior to sterilization.Batch sterilization conditions are often specified as a

holding period at a certain temperature. The heateffects involved during the time required to reach thedesired sterilizing temperature and the time required incooling down from this temperature are usually neg-

lected. In large-scale equipment, the rising and fallingtemperature portions of the heating cycle are muchlonger than the constant temperature portion.

Figure 4 compares actual fermentor heating cycles forvarious sized vessels. Note that 110 F is the startingtemperature for the heating cycle. Batch make-up usingwarm water can save considerable time in the solution

-1f

I.-z

4n

z

0

3--

Fa

0-J

>

1i0

0.00135 000137 0.00139 0.00141 0.00143 0.00145 0.00147 0.00149

RECIPROCAL TEMPERATURE RT ~~~~~~~R

FIG. 2. Effect of temperature on the velocity constant fordestruction of spores of Bacillus stearothermophiluis strain1518.

TABLE 2. Activation energies and entropies for first orderdegradation of B-complex vitamins and death of bacterial

spores

Vitamin or Bacterial Spore Energy EntropyA AS

cal/mole cal/mole K

Folic acid ............................... 16,800* -14.1 td-Panthothenyl alcohol ....... ........... 21,000* 5. itCyanocobalamin ............. ............ 23,100* 2.2tThiamine hydrochloride .................. 26,000* 11.4tBacillus stearothermophilus strain 1518.... 67,700 105Putrefactive anaerobe NCA 3679 ..... .... 72,400t 1234Clostridium botulinum ......... ........... 82,l00t 160t

* Garrett (1956).t Calculated from data of Garrett (1956).t Levine (1956).

2231957]

F. H. DEINDOERFER

of batch ingredients and in heating time. If water at50 F were used in batch make-up it would have takenan additional 30 min to heat the medium in the 15,000-gal fermentor to 110 F. In order to avoid extending thegraphs unduly, 110 F was used as the finishing tempera-ture. Actually, temperatures below 200 F have littleeffect on the sterilization.

All the vessels characterized in figure 4 are reasonablygeometrically similar. Because of this congruency, theheat transfer area per unit volume decreases as vesselsize increases. Also, since the vessels operate at similarpower input per unit volume levels, the heat transfercoefficient also decreases as vessel size increases. This

STEAM INIAND

COOLINGOUTLE

RAW INGREDIENTSWATER

ACID OR BASE

LETS L {=

WATERETS

explains the different shapes of the heating cycles infigure 4. Coils are added to larger fermentors to provideadditional heat transfer surface. Very often, too, theamount of steam directly injected into the mediumper unit volume is increased for larger fermentors. De-spite this, however, the disadvantages of increased sizeare not fully compensated.

Obviously, the rising and falling portions of thesterilization cycle contribute significantly to fermentorand medium sterilization, especially in larger vessels. Amethod for determining the contribution of theseportions to the sterilization is suggested through theuse of the thermal-death relationships discussed earlier.

EXHAUST AIR OUTLET(STEAM VENT DURING STERILIZATION)

3 STEAM CONDENSATE OUTLETSAND

3 COOLING WATER INLETS

STERILE AIR INLET C

($TEAM DURNIN STERILIZATION)

FIG. 3. Geometric design of large-scale fermentors

o TimE MINUTES * TIME MINUTES

FIG. 4. Temperature rising and falling curves during sterilization of medium in various sized fermentors

224 [VOL. 5

STERILIZATION OF NUTRIENT MEDIA

The method involves a graphical integration of thevelocity constant over these portions of the cycle. k is afunction of T as shown in figure 2 and described inequation 3. T is a function of time, 0, as described by theheating cycle. Therefore, k is also a function of 0. Anaverage value of k for each portion of the cycle can beobtained by graphically integrating k over the timeperiod involved and dividing the integral by the timerepresented by the portion, as in equation 5 below.

2

lCv k dO (5)

kavg =1

02 - 01

It should be kept in mind that the heating cyclerising and falling curves for a vessel will vary with anumber of factors. These include the liquid physicalproperties of the nutrient medium such as density,viscosity, thermal conductivity, and specific heat;extent of fouling of the heat transfer surfaces; amountand enthalpy of steam sparged directly into the me-dium; medium charge volume; temperature andenthalpy of the heating steam and temperature ofthe cooling water in the vessel coils and jacket. For agiven process and given vessel these variables aremaintained reasonably constant, and good replicationin heating cycles occurs.The method is illustrated by way of the following

hypothetical example.Example 1. A 15,000-gal fermentor containing 12,000

gal of a penicillin production medium is to be sterilized.The medium contains 4 per cent corn steep liquor. Cornsteep provides an excellent source of contaminatingorganisms. Laboratory checks have shown bacterialcounts not exceeding 20 X 106 cells per ml in the

presterilized medium. For how long must the mediumbe sterilized at 250 F to be sure of sterilization in 999out of 1000 batches?

Solution. For sake of illustration, assume the curvein figure 2 characterizes the most heat resistant bacterialspores in the penicillin medium. Also, let the heatingcycle of the 15,000-gal fermentor shown in figure 4represent the heating cycle of the fermentor underconsideration. 220 F is chosen as the minimum lethaltemperature. This is an arbitrary choice, of course.Actually lower temperatures are lethal but theirrelative lethality is small and neglecting them does notintroduce any significant error.

Since the percentage of the laboratory bacterialcount that is contributed by the most heat resistantspores was not determined, assume that the entirecount was contributed by these spores. This is equiva-lent to adding a safety factor in the calculation.Then

N1 = (20 X 106 spores/ml)(3.78 X 103 ml/gal)(12 X 103 gal)

= 9.07 X 1014 spores

The chance for failure has been set at one in a thou-sand so that N2 = 0.001 spore.The velocity constants at the temperatures along the

rising portion of the sterilization cycle in figure 4 aredetermined from figure 2. These values are plottedversus the time corresponding to the temperaturesalong the cycle as in figure 5A. The procedure is re-peated for the falling portion of the cycle in figure 5B.Note that the time of each of these portions below 220 Fis not shown on the figures. Graphical integration of thearea under the curves in figures 5A and 5B, and solu-

A ) RISING PORTION OF CYCLE

f kd - o. 331

2kav90.335 0.0112 SEC.-l

~~~~I I I I0 5 10 15 20 25 31

8) FALLING PORTIONOF CYCLE

fkde 0.087kav 0.0870,0066 SEC. 1

______ I I.0 0 S 10

0 TIME MINUTES

FIG. 5. Velocity constant variation over rising and falling temperature portions of sterilization cycle

.024

-10I- .020z

z0

.016

0-Iw .012

.008o

.004

i-

225

F. H. DEINDOERFER

tion of equation 5, yields values of kavg for each of theseportions of the cycle. For 29.5 min of the rising portion,kavg is equal to 0.0112 sec-1 and for 13.2 min of thefalling portion, kavg is equal to 0.0066 sec-'.

Therefore, for portions of the heating cycle totalling42.7 min, a kavg of 0.0098 sec-' can be used to calculatethe contribution of these portions to the sterilization.Using equation 2, the population remaining if thesetwo periods followed each other can be calculated.

9.07 X 1014log- N2

(0-0098 sec') (60 sec/min) (42.7 min)2.3

N2 = 1.14 X 10' spores

The N2 just calculated becomes the new N1 to be usedin equation 2, this time to calculate the time thefermentor must be held at 250 F to reduce the popula-tion to the desired N2 of 0.001.

2.3 1.14 X 1030 = log0 (0.0265 sec-) (60 sec/min) 1 X 10-3

0 = 8.8 min

TABLE 3. Time at sterilization temperature to achieve the samedegree of sterilization in different sized fermentors

Fermentor Size Total Time of Heating Time at 250 FCycle Above 220 F

gal m# mn50 28.0 17.5150 33.7 12.6

1,500 41.3 11.315,000 51.5 8.8

RAW INGREDIENTSWATER

ACID OR BASE

MAKE -UPTANK

Thus, only 8.8 min at 250 F are required for thesterilization of the fermentor.For comparison, similar calculations have been

made for the other sterilization cycles depicted infigure 4. The results are listed in table 3. As vessel sizeincreases, the respective contributions of the rising andfalling portions of the cycle increase, and consequentlythe time required at so-called sterilization temperaturedecreases.

Heat Effects on Nutrients

The time required for batch sterilization is notusually the optimum time of heating when productyield is considered. The thermal effects on nutrientquality of the medium must also be taken into account.Sometimes, productivity is increased by prolongedheating of the medium. In most media, however, thedeleterious effects of extensive heating are more ap-parent than any beneficial effects, and overheating of themedium must be minimized.An easily destroyed nutrient quality might be any one

of the B-complex vitamins. Vitamin degradation isknown to occur rapidly at high temperatures. Al-though figures for vitamin destruction in fermentationmedia are not published, Garrett (1956) has studiedvitamin stability at elevated temperatures in liquidpreparations. He found activation energies of from16,800 to 26,000 calories/mole for destruction of severalB vitamins. The vitamins investigated by Garrett arelisted with their activation energies and entropies intable 2. The activation energies and entropies for thevitamins are much lower than for the three bacterialspores also listed.

STERILE

STEAM MEDIUM

UNSTERILE I

MEDIUM WATER

FIG. 6. Steam-injection type of continuous sterilizer

226 [VOL. 5

STERILIZATION OF NUTRIENT MEDIA

The conditions in a batch sterilization carried out at250 F cannot always achieve sterilization withoutimpairing nutrient quality of the medium. Higher tem-perature-shorter time batch sterilizations can be carriedout, but fermentation vessels are usually limited tooperation at not more than 30 psig (274 F) by designrestrictions.

Continuous Sterilization

A sterilization method becoming increasingly popularand having several advantages over the conventionalbatch sterilization is continuous sterilization. Contin-uous sterilization of media for riboflavin fermentationshas been reported by Pfeifer et al. (1950) and forpenicillin fermentations by Whitmarsh (1954).

In one type of continuous sterilization, preheatedunsterile medium passes through an injection heater inwhich steam is introduced. The vigorous and almostinstantaneous mixing obtained raises the medium tosterilization temperature immediately. This tempera-ture is maintained for the required amount of time in aninsulated retention tube through which the hot mediumflows. A diagram of a typical continuous sterilizer isshown in figure 6. The hot medium passes through aheat exchanger where it is cooled to below its flash pointas it preheats unsterile medium. Final cooling to processtemperature is accomplished in the fermentor.The activation energy of bacterial spore destruction

is much higher than the activation energy of simplerchemical reactions. It is, therefore, an advantage to usea high temperature-short exposure time sterilizationoperation whenever nutrient degradation occurs to theextent that it lowers the process yield. Continuoussterilization not only overcomes unfavorable nutrientdestruction, but has a number of operational ad-vantages. Pfeifer and Vojnovich (1952) point out theseadvantages in an excellent paper on continuous sterili-zation. Operating conditions employed by these authorsfor continuous sterilization of several types of mediaare shown in table 4. In a batch sterilization, probably

TABLE 4. Operating conditions employed forcontinuous sterilization of mnedia*

Media Used Suspended Solids pH Temra- Time

%t70 F min

Riboflavin........... 1.8 corn steep liquor 4.5 275 4Cyanocobalamin ..... 4.0 soybean meal 4.5 325 13

and distillers'solubles

Acetone, butanol ...1 1.8 ground corn 6.5 275 3Sodium gluconate 0.4 corn steep liquor 4.5 275 5Itaconic acid ........0 .2 corn steep liquor 6.1 300 5Fungal amylase...... 4.0 distillers' sol- 5.0 325 13

ubles and3.0 ground corn

* Pfeifer and Vojnovich (1952).

only those sterilizations at 275 F could be approached,if at all, in conventional fermentors.The calculation of sterilization conditions for con-

tinuous operation is simplified by almost instantaneousrising time to sterilization temperature and a rapidcooling period thereafter. For this type of sterilization,only the velocity constants of the most resistant sporespresent in the medium and the total initial bacterialconcentration need be known to perform the calcula-tion. Usually, in production operations the sterilizer isof a fixed length. Different retention times are achievedby controlled pumping. If temperature conditions arespecified, the retention time can be calculated easilyfrom equation 2. If process conditions dictate a certainflow rate, as may be the case in continuous fermenta-tions, any modification in sterilizer operation will haveto be made temperature-wise. Consider the followingexample.Example 2. Process conditions are such that a reten-

tion time of 5 min is required in the sterilization of asodium gluconate production medium containing 0.4per cent corn steep liquor. Plate counts of 3 X 106bacteria per ml in the presterilized medium have beendetermined in the laboratory. At what temperatureshould the sterilizer be operated to achieve a steriliza-tion having 99 per cent certainty of being successful?One hundred thousand gallons of medium are to besterilized in this manner.

Solution. For sake of illustration, the curve in figure2 again will be assumed as characterizing the most heatresistant spores in the medium. The initial populationagain will be assumed as entirely consisting of thisspecies.Then

N1 = (3 X 106 spores/ml)(3.78 X 103 ml/gal)(1 X 101 gal)

= 1.13 X 1015 spores

Since one chance in one hundred was chosen as themargin of failure, N2 is equal to 0.01. Knowing the re-quired retention time, equation 2 can be used to solvefor the necessary velocity constant.

2.3 1.13 X 1015300 1 X 10-2

= 0.131 sec-1

The calculated value of k is used to read the requiredsterilization temperature from figure 2. For k = 0.131

1sec- T = 0.1382. Thus, T = 724 R and a sterilization

temperature of 264 F is required.To achieve the same sterilization at 250 F, a retention

time of 24.8 minutes would be required. Figure 7illustrates the relationship between time and tempera-

1957] 227

F. H. DEINDOERFER

ture to achieve the above sterilization. Also listed intable 5 are the relative effects of an adverse chemicalreaction destroying a hypothetical vitamin during thesterilization, the degradation of which is characterizedby an activation energy of 22.6 Kcal/mole, one-thirdthat of the activation energy for bacterial spore destruc-tion. Even for the retention time and temperature usedin example 2, the vitamin was almost completelydestroyed. Much of the vitamin quality can be retained,however, by higher temperature-shorter retention timesterilizations.

go-2 Il

10.2

40

2

w

o2~

0.00129 0.00131 0.00133 0.00135 0.0057 0.00139 000141 0.00143

RECIPROCAL TEMPERATUREI

0~~~~~~~~~~~~~

FIG. 7. Time-temperature relationship to achieve the samedegree of sterilization in a continuous sterilizer.

TABLE, 5. Time-temperature relationship and its effect on vitamincontent in a continuous steri,lization

Relative Retention ofTemperature Time Original Vitamin

Content*

F min %

250 24.8 0.0265 4.1 0.0280 0.72 2.3295 0.14 28310 0.029 64325 0.0061 89

* k for vitamin destruction assumed equal to k for sporedestruction at 250 F.

Design MethodThe use of well-known thermal behavior characteris-

tics of bacterial spores and the temperature character-istics of the sterilization cycle in calculating the timefor sterilization of fermentation medium has beenillustrated. This method offers an approach to thecorrelation of sterilization conditions among varioussized fermentation vessels and between temperatureand retention time in continuous sterilizers. The advan-tages of continuous sterilization have been pointed outin a sterilization where nutrient damage occurs.The steps involved in calculating process conditions

for sterilization operations can be summarized asfollows:

(1) Periodically determine the thermal-death relation-ship of the most heat resistant bacterial spore in themedium to be sterilized.

(2) Determine the initial population concentration ofthe medium and choose a confidence level for thesterilization. An added safety factor is introduced byassuming that the total population consists entirelyof the most heat resistant spores.

(3) Calculate the contribution to the sterilization ofthe rising and falling portions of the sterilization cycle;this step is unnecessary for steam injection type con-tinuous sterilizations.

(4) Calculate the time required to hold the mediumisothermally at the highest temperature chosen for thesterilization. For continuous sterilizations, the time ofexposure is often chosen, and calculations are carriedout to find the required temperature.

REFERENCESBALL, C. 0. 1943 Short-time pasteurization of milk. Ind.

Eng. Chem., 35, 71-84.GARRETT, E. R. 1956 Prediction of stability in pharmaceu-

tical preparations. II. Vitamin stability in liquid multi-vitamin preparations. J. Am. Pharm. Assn., 45, 171-178.

JOHNSON, F. H., EYRING, H., AND PILISSAR, M. J. 1954 TheKinetic Basis of Molecular Biology, p. 220. John Wiley& Sons, New York, N. Y.

LEVINE, S. 1956 Determination of the thermal death rateof bacteria. Food Research, 21, 295-301.

PFEIFER, V. F., TANNER, F. W., VOJNOVICH, C., AND TRAUF-LER, D. H. 1950 Riboflavin production by fermentationwith Ashbya gossypii. Ind. Eng. Chem., 42, 1776-1781.

PFEIFER, V. F. AND VOJNOVICH, C. 1952 Continuous sterili-zation of media in biochemical processes. Ind. Eng.Chem., 44, 1940-1946.

RAHN, 0. 1945 Physical methods of sterilization of micro-organisms. Bacteriol. Reviews, 9, 1-47.

STERN, J. A. AND PROCTOR, B. E. 1954 A micro-method andapparatus for the multiple determination of rates of de-struction of bacteria and bacterial spores subjected toheat. Food Technol., 8, 139-143.

WHITMARSH, J. M. 1954 Continuous sterilization of fermen-tation media. J. Appl. Bacteriol., 17, 27.

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