Allen Paul W. Industrial Fermentations

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DAIRY PRODUCTS

375

Pipettes of this type are now listed by all of the usual supply houses.

The pipettes shall be calibrated so as to deliver 1/100 cc, not to contain

1/100 cc. Because there are many inaccurately calibrated pipettes on

the market, the calibration of all pipettes shall be tested by weighing

the amount of milk discharged on cheinioal balances. The weight

for milk should be .0103 gram.

Brew discusses the advantages and disadvantages of the micro-

scopic and plate counts as follows:

DISADVANTAGES.

Direct

Microaoopio

Method.

Diffloult to measure

gmn.11

a quan-

tity of milk accurately.

The sample measured is too

Bin

all to

be representative.

Dead bacteria may be counted.

Error of count is great where bac-

teria are very few or many.

Cannot be used for quantitative work

when the bacteria are few in

number.

6. Many fields must be counted, because

of the uneven distribution, if an ac-

curate count is required.

Large, oompact clumps cannot be

counted.

8. Bacteria may be lost in process of

preparing slides.

Plate Method.

All bacteria do not grow on the

plates because of changes in food,

temperature relations, or other con-

ditions of environment.

The difficulty of breaking up the

clumps in the milk affects the accu-

racy of the count.

Requires from 2 to 6 days' incuba-

tion period.

Different species require different in-

cubation temperatures.

Gives no idea of the morphology of

the organisms present.

6. More apparatus required, therefore

more expensive. Technique com-

plicated and difficult for trained

bacteriologists to use in such a way

as to secure consistent results.

ADVANTAGES.

Direct

Microscopic

Method.

Less apparatus required, therefore

less expensive. Technique simple.

The results on a given sample can be

reported within a few minutes.

Shows the cell oontent, the presence

or absence of streptococci and other

important thingR necessary in esti-

mating the sanitary quality of milk.

Gives a better idea of the actual

number of germs present.

Plate Method.

Is necessary for isolation of pure

cultures.

Gelatin shows the liquefiers and, if

litmus is used, the acid-producing

bacteria.

Shows charaoter of growth.

Shows living organisms only.

Only a single pipette is needed in making a series of tests provided

this is kept clean while in use. In this kind of work cleanliness of

glassware is more important than sterilization. Clean towels may be

376 INDUSTRIAL FERMENTATIONS

used for wiping the exterior of the pipettes while making the micro-

scopio preparations, and their bores may be kept clean by rinsing them

in olean water between each sample. The small amount of water

left in the bore may be rinsed out in the milk sample under examina-

tion. This method of procedure, while adding a small number of

bacteria to each sample, introduces only a theoretical error, tests

showing that such bacteria cannot subsequently be deteoted, and

make no difference in the final result. After use, the pipettes should

be kept in a glass-cleaning solution, such as the commonly used

mixture of sulphuric aoid and potassium bichromate.

Routine laboratories will find it convenient to use larger micro-

aoopio slides than the ordinary 1 by 3 inch slide. The largest elides

that have been found to be conveniently examined with the use of a

mechanical stage are cut 2 by £¥% inches. Such slides may be stored

in ordinary card catalogue cases and may be very oheaply prepared

from thin window glass or old photographic negatives. A margin of

ground or etched glass on the longer edges of the slide about y± inch

in width allows lead pencil labeling. The margins may be ground

with an emery wheel, or they may be etched with hydrofluoric acid.

The cost of these home-made slides ought not to exceed 2 to 3 cents

each, whereas the similar slides listed by supply houses usually cost

much more than this'. A special guide plate (size 2 by 4^4 inches)

marked off with 16 square centimeter areas is also needed. This can

be obtained from regular supply nouses. Only one of these is needed

as it is used as a guide plate underneath the slides on which the milk

preparations are made.

Preparation o£ Films of Dried Milk.

After a thorough shaking of the sample, 0.01 c.c. of milk or cream

shall be deposited under a clean glass slide by means of the pipette

above described. Spread the drop of milk uniformly over an area of

one square centimeter by means of a clean, stiff needle. This may be

most conveniently done by placing the slide upon the guide plate just

described, or upon any other form of guide plate of glass or paper

which is ruled in square centimeter areas. The marks showing

through the glass serve as guides. After spreading the preparation

shall be dried in a warm place upon a level surface protected from

dust. In order to prevent noticeable growth, this drying must be

accomplished within five to ten minutes; but excessive heat must be

avoided or the dry films may crack and peel from the slide in later

handling.

After drying, the Blides are to be dipped in xylol, or any other

suitable fat solvent, for a sufficient time to remove the fat (at least

one minute), then drained and again dried. After this, the slides

are to be immersed in 90 per cent grain or denatured alcohol for one

or more minutes, and then transferred to a solution of Loeffler's

methylene blue prepared as follows: Saturated alcoholic solution of

DAIRY PRODUCTS 377

methylene blue, 30 c.c; caustic potash in a 0.01 per cent solution

100 o.o.

Some of the methylene blue sold since the war has been found to

be unsatisfactory in that solutions dissolve the milk films or wash

them with an even blue color in which the bacteria fail to show

distinctly. In some cases these troubles can be corrected by adding

enough sodium carbonate to the liquid stain to make a .01 to .03 per

cent solution; but it is better to secure a fresh supply of methylene

blue from a satisfactory source. Old or unaltered stains are to be

aVoided as they may contain troublesome precipitates.

The slides are to be left in the stain until overstated. They are

then to be rinsed in water and decolorized in alcohol. The decoloriza-

tion takes from several seconds to a minute or more, during which

time the slide Bhould be under observation, in order that the de-

.colorization may not proceed too far. When properly decolorized the

background of the fil™ should show a faint blue tint. Poorly stained

slides may be decolorized and restained without apparent injury.

After drying, the slides may be examined at once, or they may be

preserved indefinitely.

Standardization of the Microscope.

The microscope used must be so adjusted that each field covers a

certain known fraction of the area of a square centimeter. This ad-

justment is simple if a micrometer slide, ruled in hundredths" of a

millimeter, is at hand (sometimes called a stage micrometer as it is

used under the objective on the stage of the microscope). The

microscope should have a 1.9 mm. (1/12 inch) oil immersion lens,

and an ocular giving approximately the field desired (for example a

6.4 x ocular). It should also be fitted with a mechanical stage. If

the large slides described above are used, this must be a special stage

allowing a larger area of the slide to be examined than can be

examined with the usual meohanical stage.

To standardize the microscope, place the stage micrometer on the

stage of the microscope, and by selection of oculars or by adjustment

of the draw tube, or both, bring the diameter of the whole microscopic

field to .205 mm. When so adjusted, each field of the microscope

covers an area of approximately

1/30000

cm. (actually

1/3028

cm.)

This means that the dried milk solids from

1/300,000

part of a c.c of

milk are visible in each field of the microscope. Therefore if the

bacteria in one field only are counted, the number found should be

multiplied by 300,000 to give the estimated number of bacteria per

cubic centimeter. In practice, however, more than a single field is

examined so that the number used for multiplication is smaller

than this.

As the microscopic examinations must be made with greater care

where the bacteria are relatively few in number, it is required that,

in grading low count milk, a special ocular micrometer with a circular

378 INDUSTRIAL FERMENTATIONS

ruling divided into quadrants shall be used. In using this miorometer,

the microscope shall be 00 adjusted that the diameter of the circle on

the eye piece micrometer shall be .146 mm. In ttjis case the amount

of dried Tniilr solids examined in each field of the microscope is

1/600,000 part of a o.c. of milir. The limitation of the examination of

the slide to the central portion of each field, avoids UBing the margins

of the field where definition is hazy, and lessens the danger of over-

looking bacteria. Likewise the magnification used is greater than

that used where the whole field is examined.

Counting and Grading Milk.

The number of fields of the microscope to be examined varies

with the character of the milk, and with the character of the data

desired. Experience has shown that where the purpose is primarily,

to detect and eliminate the worst milk from ordinary market milk

supplies, it is entirely permissible to use the entire field of the

microscope for examination. At leaBt thirty representative fields of

the microscope should be examined for each sample of milk. .Where

the average number of individual bacteria (not groups of bacteria) is

less than 24 bacteria per 30 fields {% of a bacterium per field), it may

be assumed that the milk will ordinarily give an official plate count

of less than 60,000 per c.c. Where the number is less than 80 in 30

fields (average of less than 2% per field) it may be assumed that

the official plate count will be less than 200,000 per c.c. Where less

than 800 per 30 fields (average of less than 26% per field) it may be

assumed that the official plate count will not exceed one to two

million per c.c.

Where counts are made in or^er to enforce more stringent stand-

ards,

as at Grade A plants or as a basis for premiums on milk giving

an official plate count of less than 10,000 per c.c. the special eyepiece

micrometer described above shall be used and the microscope so

adjusted that only the central portion of each field is examined for

counting. Where less than 4 bacteria are found in 60 fields (average

of less than %

of a bacterium per field) it may be assumed that the

milk would ordinarily give an official plate count of less than 10,000

per c.c. The grading of milk of this type must be done with especial

oare as persons inexperienced with microscopic work have been found

readily to confuse extraneous objects with bacteria, in milk containing

very few organisms. Where the number is less than 24 per 60

fields (average of less than % a bacterium per field), it may be

assumed that the official plate count will be less than 60,000 per c.c.

Where the number is less than 80 per 60 field (average of less than

VJ&

bacteria per field), it may be assumed that the official plate count

will be less than 200,000 per c.c. Where a number is less than 800

per 60 fields (average of less than 13^ bacteria per field), it may be

assumed that the official plate count will be less than one or two

million.

DAIRY PRODUCTS 379

The standards given are computed (with the exception of the

poorest grades') on the assumption that the official plate count will

normally average V± of the total number of individual bacteria present.

As many cases will be found which diverge markedly from the

average, it is self-evident that this average represents only an ap-

proximation to the real conditions in any specific case, so that in some

cases the microscopio grading will be more severe than that based

on the plate counts, and vice versa. There is still a lack of sufficient

data from which to judge fairly which system of grading is the more

accurate. The indications are, however, that where the work is done

with equal

RTHI]

and care, and the allowances indicated are made, a

reasonably close agreement in grade will be secured. This

fact is

highly reassuring as to the general accuracy of both systems of

grading.

In the routine grading of milk by the microscopic method it is not

expected that exact counts will be made. A high grade milk will show

field after field on the microscope in which no bacteria are seen, while

a poor grade of milk will show numerous bacteria in every field

examined. It is only where the number of bacteria present is close

to the border line between grades that counts need to be made. The

examination, however, must be sufficiently thorough to make sure

of the grade as speoified above.

In order to insure careful work in grading, it is required that

laboratories conforming to standard procedure shall preserve micro-

scopic preparations until a reasonable period has elapsed after the

reports are rendered to the person or persons whose milk has been

examined. It is an excellent custom occasionally to have the grading

done by one analyst repeated by a second analyst, particularly in

those cases where punitive actions are to be based on the reports

made.

Common Sources of Error in Counts.

Routine microscopic counts, like all bacterial counts, are to be

regarded as estimates of numbers only. They oannot be made with

absolute accuracy even with the most careful technique. Errors will

arise from inaccuracies in measurement of the minute quantities of

milk examined at any one time, from faulty staining or preparation

of slides, from mistakes in observation and the like. These limita-

tions, while important, are not difficult to overcome in sufficient

measure to make microscopic grading a satisfactory method of con-

trolling the quality of unpasteurized milk. As it is only in this way

that counts of the bacteria themselves can be made, it must be

recognized that accurately carried out microscopic counts of individual

bacteria give the truest picture of the actual conditions of raw milk

that can be obtained with any technique.

Where there is reason to expect the presence of large numbers of

dead organisms, as for example in pasteurized milk, it is improper to

place reliance upon microscopic counts. Valuable information may,

380 INDUSTRIAL FERMENTATIONS

however, sometimes be obtained by making both plate and microscopic

counts from samples of pasteurized milk.

Reports.

As only a few ordinances have yet been adopted in which both

official and microscopic count standards have been given, the form of

report used will need to be adapted to the circumstances under which

each laboratory is working. Specifio counts should not be given under

normal circumstances, and care should be take^i to avoid making finer

distinctions in grade than are justified by the acouracy of the grading,

A series of samples should be examined in all cases before rendering

judgment as to the quality of any milk supply.

Microscopic Colony Count (Frost Method) Provisional Method.

Although this technique is not recommended at tfrjg time as a

standard of official technique, it is described in this report because of

the need for more extensive comparative investigations upon which to

base judgment as to its real merits. The technique in question has

been described by its author as follows".

"An area of four square centimeters is marked off on an ordinary

microscopic slide with a wax pencil. The slide is sterilized in a flame,

and then 0.05 (%

)

-°.- °* &

mfik to be examined is placed on it

with an accurately calibrated pipette. An equal amount of sterile

nutrient liquefied agar, at 42-46 degrees C. is added and the twa drops

thoroughly mixed with a sterile loop and carefully spread over the

area marked off. The mixture is allowed to harden and then a 'little

plate' culture is formed.

"The bacteria in the milk are allowed to grow into colonies by

keeping the preparation in a moist sterile chamber for a few hours.

The period of incubation should be long enough to allow the bacteria

to grow into distinct colonies although they may not be visible to the

naked eye. In practice it seems best to allow eight hours at 37.5

degrees C. although good sized colonies are frequently formed in four

hours.

On the other hand, if more convenient, they may be allowed

to grow 16 or more hours before they are counted. In order to count

the colonies most readily the plates' are thoroughly dried at a little

less than 100 degrees C, treated with a 10 per cent solution of glacial

acetic acid in 95 per cent alcohol, and stained with a methylene blue

or carbolthionine solution about *4 its usual strength. In this way the

colonies are deeply stained -while the background is colorless.

"The colonies are counted under the microscope. Usually this can

be done with the 16 mm. {% inch) objective, although where the

colonies are small or very numerous the higher powers may be used,

e.g. the 4 mm. (% inch) or the 1.9 mm. (%

inch) oil immersion lens!

The factor needed to convert colonies per microscopic field into the

number of colonies that would develop per c.o. of milk must be

DAIRY PRODUCTS 381

determined for each microscope and each combination of lenses of the

same; but roughly, when a 10 x eyepiece is used, one colony under a

16 mm. lens

means 4,000 colonies per c.c. of milk, and the colonies in

an average field of a 4 mm. lens should be multiplied by 100,000, and

under a 1.9 mm. or oil immersion lens by 400,000. At least five

representative fields should be counted and averaged.

"Dilutions may be avoided with milks expected to contain several

million bacteria per o.o. by using 0.01 c.c. A drop of sterile milk

should be added before mixing with the agar in order that the oom-

position and consistency of the medium may not vary greatly from

that used with the ordinary dilution. Some special apparatus has been

found to be desirable."

This is essentially an agar plate technique in which the count of

colonies can be secured within a shorter period than that used in the

standard plating technique. This possibility makes it highly desirable

that further studies be made with it. Some comparative counts have

already been made by the author, and others who have used the

method report to the Committee that they find counts similar to those

obtained by the author. The fact that a microscope is used in making

the counts has caused some to oonfuse this technique with that just

described as the Breed method. The two are essentially different in

that in the one case, the actual bacteria are counted as they exist in

the milk, while in the other case the count is a count of colonies of

bacteria which have grown either from isolated bacteria or from

clumps of bacteria. The counts obtained by the Frost teohnique, like

those from the standard technique ought not to be described as •show-

ing the number of individual bacteria originally present in the milk.

Because the counts obtained with the Frost technique do not

agree exactly with standard counts', this technique cannot be recom-

mended as a standard technique at present. This fact ought not,

however, to be interpreted as expressing a view on the part of the

Committee that the counts obtained by experienced workers are any

less accurate than those obtained by the standard technique. Very

little data have thus been gathered even in comparative studies, and

none is of sufficient extent or accuracy to warrant making a mathe-

matical analysis of it in order to establish the true accuracy of the

counts.

Microscopic Count of Bacteria- (Allen.)

The Hydroxide Method of Counting Bacteria in Milk. A brass

centrifuge tube of 23 mm. diameter with a flat removable screw

bottom is used. Place 19 cc. of milk and 5 cc. of a water suspension

of aluminum hydroxide in the tube and shake thoroughly. Then drop

in a thick cover-glass of 22 mm. diameter and see that it settles to a

position flat on the bottom of the tube. Centrifuge sufficiently to

throw down the flock in a firm layer on the cover-glass. Immerse the

hydroxide film in xylol to dissolve out the fat globules. Next im-

merse the film in alcohol to remove the xylol. Then dry the film

382 INDUSTRIAL FERMENTATIONS

thoroughly to the cover-glass and stain with aqueous methylene blue

or other suitable stain. An average of one organism in the usual

1.9 oil immersion field gives a count of 600 bacteria per cubio centi-

meter of Tnilir The centrifuging can be done in the Babcock tester

along with butter fat tests.

The cover-glasB preparations not only show bacteria but also are

ideal for grading the milk as to insoluble dirt content.

Note: The aluminum hydroxide suspension should be of such a

dilution that a film of ideal thickness for observation of bacteria in

the microscope field is obtained. The flock should be made by bring-

ing together very weak solutions of aluminum sulphate and sodium

hydroxide both of which must be pure. The flock should be thoroughly

washed by a number of decantations after dilutions with distilled

water.

Butter.

Danish butter makers asked Dr. Storch in 1885 to aid them in

certain flavor troubles which were current in butter making. He

began a series of studies on the germ Ufe in cream for butter making

and found that the flavor of butter varies with the kind of germ life in

the sour cream. Storch then experimented with a small amount of

sour cream as a "starter" in making butter and tried to isolate the

ideal cream ripening organism from the finest samples of butter. In

this

he failed, but later isolated the organism from the best ripened

cream of the most successful creameries. The results of his in-

vestigations were published in 1890. Soon after this investigation the

pure culture manufacture of butter in creameries in the United States

began.

The next year Lunde showed that pasteurization at 185° F. in a

flash pasteurizer was

desirable in that it killed all tuberculosis or-

ganisms and at the same time reduced the bacterial life in the cream

to a point at which the "butter starter" when added would dominate

the fermentation.

H. W. Conn and H. Weigmann during 1890 to 1897 carried on much

work which showed to the creamery men that the use of "butter-

starters" for ripening cream reduced butter making to a scientific

basis.

It was this introduction of pasteurization and "butter-starters"

combined with the Babcock test, which made it possible for creameries

to be successfully established throughout dairy regions and to develop

new dairy regions.

Conn held that the aroma of butter is due to the splitting up by

bacteria of the nitrogenous portion of the butter, while Weigmann

held that the aroma of butter is due to the esters formed during

ripening of the cream.

Off flavors in butter have been studied extensively but this subject

is found to be a very hard one to reduce to demonstrable facts. The

chemist is inclined to .hold that rancidity in butter is due to light and

oxidation while the baoteriologist is inclined to ascribe ranoidity to

DAIRY PRODUCTS 383

bacterial action in butter. C. A. Brqwn defines rancidity as any

chemical or physical change from the normal in the character of the

butter fat.

The temperature of pasteurization of cream in Europe is 180° F.

while lower temperatures are extensively used in this country. Rogers,

Berg, and Davis say that in the continuous pasteurization of sweet

cream for butter making, a temperature not lower than 166° F. nor

higher than 175° F. should be used. Examination of the butter after

storage indicates that pasteurization at 150° F. or lower leaves in the

cream some factor causing a deterioration of the butter. The flavors

of butter made from cream pasteurized at 180° F. is somewhat affected

by heat.

E. S. Guthrie (1917) says as a summary to hie experiments with

butter that chemical changes in the butter were very slight when

biological agents were held in check, that enzymic development pro-

duced no rancidity and that exposure of butter fat to high tempera-

tures,

light and air, did not cause rancidity. In this work rancidity in

butter was considered as a butyric acid flavor.

One of the commonest of undesirable flavors is the fishy flavor.

This

trouble is thought by some to be due to high acidity and the

presence of oxygen. Fishy flavor was attributed by O'Callaghan to

Odium Lactis. Weigmann thought fishy flavor was due to over-

working and from use of salt high in magnesium.

The production of butyric acid by fermentation was first observed

by Marcharid in 1840. Some twenty years later Pasteur explained the

production of butyric acid from lactose sugar by the following

reaction:

0u + H

O = 4C

Lactic acid

= 2H

+ 2CO

+ C^O,,

Butyric acid

LaFar says' B. Clostridium butyrioum consists of a number of closely

allied but nevertheless distinct species which produce butyric acid.

He says that no sharply drawn line can be made between this group

and the potato bacillus.

According to LaFar, Granulobacter, often called the true butyric

acid bacillus or B. butylicus, possesses the faculty of storing up within

its'

cells interior granulose. From glucose this organism produces

butyl alcohol, carbon dioxide, hydrogen, and butyric acid.

Perdrin says that B. butyricus anaerobically causes with glucose

the following reaction:

«6C

+ 42H

O = 156H

+ 114CO

+ 39C

+ 36C

followed by 46C

+ 18H

O = 112H

+ 94CO

+ 15C

38C;H

and then simplification of the reaction as follows:

lfl

= 2H

+ 2CO

384 INDUSTRIAL FERMENTATIONS

Ice Cream and Ices.

Back in the middle of the eighteenth century in Italy .the art of

making "sorbetto" was carried to a high point of perfection. The art

loving Italians of that period were famed for their tastes for these

delicacies. They were made in colors to imitate flowers, fruits, build-

ings",

and statues. Wiley says these dishes were called "sorbetto" in

Italy, "glace

" in France and "ices" in England. In this country most

frozen dishes are called ice cream.

It is said that Dolly Madison invented American ice cream.

Thyra Winslow writing in the Illustrated World says that while this

is not true, it is no doubt true that the first ice cream served as part

of a dinner was at the White House during the administration of

President Madison. It is said' that the guests were so delighted with

the new dish that they all wished to imitate it in their own homes.

Since this introduction of ice cream, it has increased in popularity

and has grown in use steadily.

Thyra Winslow says

that the first ice cream of the Philadelphia

type was made in London in about 1800 by a confectioner named

Gunton. She says that Gunton's methods of freezing were very orude.

Some years later the method of making ice cream was greatly facili-

tated by the invention of the ice cream freezer by Nancy Johnson,

the wife of an American Naval Officer.

Frandsen and Markham in their book on ice cream and ices men-

tion the fact that a New York newspaper, The Post Boy, in 1786,

contained an advertisement for ice cream. This no doubt was an

advertisement for the Neapolitan type of custard cream which was

made in the home for years before the introduction of the Philadelphia

type.

For many years the Philadelphia type was popular as a delicacy

only in the home and only on special occasions. It was perhaps little

thought of as having food value. A little later caterers began to use

it considerably and by 1850 the wholesale manufacture of ice cream

was undertaken in Baltimore. Jacob Fussell, a milk dealer of Balti-

more, began to practice using up his

extra sweet cream by manufactur-

ing it into ice cream. He succeeded to the extent that he later went

out of the milk business entirely and became exclusively an ice cream

manufacturer, establishing plants also in Washington, Boston, and

New York City.

Brazelton, an employee of Fussell's, a little later, established ice

cream plants in St. Louis, Cincinnati, and Chicago. It is interesting

to note that J. M. Horton, who died in 1914, bought Fussell's New

York ice cream plant. Horton was then a young man of 22. This

same ice cream plant was estimated in 1910 at $600,000. Horton left

an estate of $4,000,000. These facte give a clear idea of what the ice

cream factory of to-day must be to compete in the large city trade.

This enormous ice cream business has been pretty largely developed

by use of one type, the Philadelphia ice cream, although the increase