Allen Paul W. Industrial Fermentations

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Chapter 26.

Coffee and Cocoa.

Coffee.

Coffee is produced in Brazil, Central America, Hayti, West Indies,

Mexico, Jamaica, Arabia, Abyssinia, Liberia, and Dutch West Indies.

The quality of the product depends largely upon the planter's skill,

the soil, the olimate, and the preparation.

It is the opinion of many that some of the world's finest coffee

is produced in Brazil. The explanation given by E. L. Wright for the

superiority of Brazilian coffee is a combination of adaptable soil and

climate with a high order of intelligence on the part of the planters.

In the preparation of coffee the matured cherries are picked, placed

in bags and sent to the factory where preparation for market is com-

pleted. At the factory the fruit part, the outer parchment and the

silver lining are all removed either by the old dry method or the new

wet method. The wet method is used most. The first step is the

pulping process, after which the pulp and the greeds are separated by

flotation. Fermentation sets in amid the depulped seeds after several

days standing and separates the parchment and silver lining from the

seeds.

At the completion of fermentation the seeds are dried and

passed through a peeling machine which removes the parchments and

lining tissues.

The bacteria involved in the process of fermenting the parchment

coatings of ooffee seeds are similar to those of the fermentation which

goes on in flax retting.

Cocoa.

Cocoa beans are produced in Central America, South America,

Dutch East Indies, British West Indies', and West Africa. However,

Ecuador is the home of the cocoa tree which was discovered by the

Spaniards.

Cocoa beans have a bitter principle which is somewhat reduced

by a fermentation process. The quality of the cocoa made from the

beans depends to a considerable extent upon the success of this fer-

mentation. The cocoa beans grow in bunches of thirty to forty/ sur-

rounded by a soft husk or shell. The beans are collected and placed

in fermentation boxes with proper air vents. The sweet outer covering

of the beans begins to ferment immediately. This mucous lining of

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336 INDUSTRIAL FERMENTATIONS

the beans is liquefied and drips from the boxes. Heat of fermentation

develops in the boxes and the fermentation is controlled by observing

the temperature. When the fermentation has progressed to a proper

point the beans are removed, thoroughly scrubbed and dried.

Loew says, "the process of fermentation depends especially on yeast

cells which increase rapidly in the syrup of the cocoa bean, with the

formation of alcohol and CO

. Also bacteria take part in the fer-

m«itation, oxidizing the alcohol to AcOH. A rise in temperature

takes place and the slimy tissue surrounding the seeds comes off and

collects on the bottom of the vat. The removal of this tissue is the

chief end of the fermentation. It causes the seeds to be more easily

dried. An oxidation of the tannin takes place which causes the brown

color of the bean. The taste and the aroma depend on the fermentation

as well as on the roasting."

Chapter 27.

Drinking Water.

The Bacteriology of Drinking Water.

In supplying drinking water for use in cities, towns, railway

coaches, and steamboats, the object is that it shall be palatable and at

the same time safe, that is, free from disease germs. The task of sup-

plying potable water to cities grows harder as population increases, and

as previously safe sources of drinking water become polluted. The im-

portance of methods of water purification becomes more and more

emphasised. Also methods of determining the sanitary condition of

water becomes more vital to publio welfare.

Where a few years ago the water of our larger cities was grossly

polluted much of the time, now most cities have safe drinking water.

It was formerly considered that the drinking water of rural districts

was usually very good whereas now the amount of typhoid fever and

dysentery spread by drinking water in rural districts is one of our

great sanitary problems. City physicians have now come to expect

considerable typhoid fever in the fall when people return from their

vacations, "vacation typhoid."

There are more or less bacteria in all natural waters but the great

majority of these have no special health significance. For this reason

there is no attempt to obtain drinking water absolutely free from

bacteria. Water free from contamination by man or animals is con-

sidered safe as far as bacteria are concerned, but there is very little

such water.

Man is his own worst enemy as

far as bacterial contamination of

water is concerned and practically all bacteriologically unsafe water

is made so by contamination by human beings.

As it is impossible to ascertain the actual bacteriological condition

of large amounts of water it has become a custom to depend upon

indications and to condemn drinking water which according to bac-

teriological tests is

dangerous. The presence of B. coli of human origin

in water is quite generally aocepted as a serious indication that the

water is not safe to drink. The B. coli test was first advocated by

Theobald Smith, who considered that in so far as B. coli accompany

the feces of all warm blooded animals, water containing it in abundance

may logioally be considered to be contaminated with feces. Later it

was found that B. coli or coli-like baoteria exist even in uncontami-

nated fields of grain. This fact led to a very intensive study of this

337

338 INDUSTRIAL FERMENTATIONS

group of bacteria. Winslow described B. coli as aerobio non-spore-

fonning bacilli producing acid and gas in dextrose and lactose media.

While a great amount of work has been done concerning the colon

group of baoteria there is still much confusion as to the identification

of fecal and non-fecal strains of B. coli. Grossly polluted water is

easily identified by the presumptive test but waters on the margin

of allowed contamination give trouble.

Lactose bile broth thought to be favorable to B. coU but not to

other organisms was advocated as a test for B. coli but it was found

that the sodium taurocholate of bile inhibited nearly y% of the typical

coli organisms as well as miscellaneous water organisms and it ia

generally accepted that the use of lactose is more satisfactory in

making the presumptive test.

Work in the direction of identification of fecal and non-fecal B. coli

has been done by Rogers, Clark and Lubs. They found that the

carbon dioxide hydrogen ratio differs for fecal and non-fecal B. coli,

the non-fecal coli producing carbon dioxide and hydrogen in the ratio

of 2 to 1.

In using methyl red to distinguish fecal and non-fecal B. coli it

was found that the fecal organisms give an acid (red) color, while the

non-feoal B. coli give an alkaline (yellow).

According to Levine, the methyl red* test checks with the Voges-

Proskauer reaction. The test for B. coli of fecal origin is the develop-

ment of a red ooloration in the medium after twelve hours incubation.

Methyl red medium is made by adding methyl red to a phosphate

broth and Voges-Proskauer medium is made by adding 10% KOH

solution to the phosphate broth.

Many different devices and treatments for the purification of drink-

ing water have been tried out during the last thirty-five years. Some

of the more important processes used in America for reduction of

bacterial content are:

Soreening.

Sedimentation.

Filtration.

Coagulation.

Chlorination.

Use of ozone.

Use of violet ray.

Screening through fine wire cloth removes much objectionable nin-

terial which carries bacterial life or furnishes food for bacteria.

Sedimentation is the adoption of a natural method of water purifi-

cation. By reducing the rate of flow of water sediment is allowed to

settle out of water. With the removal of sediment large numbers of

bacteria are carried down.

The use of filtration of water in modern water plants is another

illustration of the adoption of one of nature's methods of purifying

water. When water passes through a considerable thickness of fine

DRINKING WATER 339

sand it becomes purified, because microorganisms adhere to the sand

partioles or are caught between them. In the operation of rapid

mechanical filters in water purification plants, "the filter beds are so

arranged that when they have become clogged with material removed

from the water they can be washed and sorubbed by rapid reverse

currents which polish the Band and gravel vigorously.

Slow sand filters are large beds of sand through which the water

passes slowly. These beds are cleaned occasionally by draining and

removing the upper surface of slime and dirt.

Coagulation as a method of bacterial water purification may be

very efficient. The material most generally used in producing the

"flock" is either iron sulphate or alum. Many different kinds of

mechanisms have been used for adding the chemical to the water in

definite amounts so that no excess of chemical will remain after the

flock has been formed and has precipitated.

The destruction of bacteria in water by the use of ohlorine has

become very important in recent years. Bacteria are very sensitive

to chlorine and are killed by very small amounts. The ohlorine is

added to the water in different ways as in the form of calcium hypo-

chlorite or as a liquid chlorine. By carefully controlling the amount

of chlorine added to water a large percentage of the bacteria are killed

and still very little or no free ohlorine remains in the water. This

result is due to the fact that chlorine unites with organic matter when-

ever it comes in contact with it.

The use of ozone, violet rays, etc., has not become extensively used

because of the expense of these methods. However, these methods

have been used on board vessels with considerable success.

Methods * of the American Public Health Associations for testing

for the presence of members of the B. coli group in water are as

follows:

It is recommended that the B. coli group be considered as including

all non-spore-forming bacilli which ferment lactose with gas formation

and p*ow aerobically on standard solid media.

The formation of 10 per cent or more of gas in a standard lactose

broth fermentation tube within 24 hours at 37 degrees C. is presump-

tive evidence of the presence of members of the B. coli group, since

the majority of the bacteria which give such a reaction belong to this

group.

The appearance of aerobic lactose-splitting colonies on Endo or

eosin methylene blue plates made from a lactose broth fermentation

tube in which gas has formed, confirms to a considerable extent the

presumption that gas-formation in the fermentation tube was due to

the presence of members of the B. coli group.

To complete the demonstration of the presence of B. ooli as above

•The methods for water analysis given here are taken from the Standard

methods of American Publio Health Association. Fifth Edition, 1923, and I wish

to give acknowledgement here of their splendid work which means so much to

the publio health.

340 INDUSTRIAL FERMENTATIONS

defined, it is necessary to show that one or more of these aerobic plate

colonies consists of non-spore-forming bacilli which, when inoculated

into a lactose-broth fermentation tube, form gas

. It is recommended

that the standard tests for the B. coli group be either (a) the Pre-

Bumptdve, (b) the Partially Confirmed, or (c) the Completed test as

hereafter defined, each test being applicable under the circumstances

specified.

A. Presumptive Test.

Inoculate a series of fermentation tubes with appropriate

graduated quantities of the water to be tested. In every fermentation

tube there must always be at least three times' as much medium as

the amount of water to be tested. When necessary to examine larger

amounts than 10 cc. as many tubes as necessary shall be inoculated

with 10 cc. each.

Incubate these tubes at 37° C. for 48 hrs. Examine each tube

at 24 and 48 hours and record gas-formation. The records should be

such as to distinguish between:

(a) Absence of gas-formation.

(b) Formation of gas occupying less than ten per cent (10%) of

the olosed arm.

(o) Formation of gas occupying more than ten. per cent (10%) of

the closed arm.

More detailed records of the amount of gas formed, though de-

sirable for purposes

of study, are not necessary for carrying out the

standard tests prescribed.

The formation within 24 hours of gas occupying more than 10

per cent (10%) of the closed arm of fermentatipn tube constitutes a

positive presumptive test.

If no gas is formed in 24 hours, or if the gas formed is leers than

ten per cent (10%), the incubation shall be continued to 48 hours.

The presence of gas in any amount in such a tube at 48 hours con-

stitutes a doubtful test, which in all cases requires confirmation.

The absence of gas formation after 48 hours incubation con-

stitutes a negative test. (An arbitrary limit of 48 hours' observation

doubtless excludes from consideration occasional members of the B.

coli group which form gas very slowly, but for the purposes of a

standard test the exclusion of these occasional slow gas forming

organisms is considered immaterial.)

Partially Confirmed Test.

Makeone or more Endo or eosin methylene blue plates from

the tube which, after 48 hours' incubation, shows gas formation from

the smallest amount of water tested. (For example, if the water has

been tested in amounts to 10 cc, 1 cc, and 0.1 cc

, and gas is formed

in 10 cc and 1 cc not in 0.1 cc, the test need be confirmed only in the

1 oc amount.)

Incubate the plates at 37 degrees C, 18 to 24 hrs.

DRINKING WATER 341

If typical colon-like colonies have developed upon the plate

within this period, the confirmed test may be considered positive.

If, however, no typical colonies have developed within 24 hours,

the test cannot yet be considered definitely negative, since it not in-

frequently happens that members of the B. coli group fail to form

typical colonies on Endo or eosin methylene blue plates, or that the

colonies develop slowly. In such case, it is always necessary to com-

plete the test as directed,under "C" 2 and 3.

C. Completed Test.

From the Endo or eosin methylene plates made as prescribed

under "B," fish at least two typioal colonies, transferring each to an

agar slant and a lactose broth fermentation tube.

If no typical colonies appear upon the plate within 24 hours, the

plate should be reincubated another 24 hours, after whioh at least two

of the colonies considered to be most likely B. coli, whether typical or

not, shall be transferred to agar slants and lactose broth fermentation

tubes.

The lactose broth fermentation tubes thus inoculated shall be

incubated until gas

formation is noted; the incubation not to exceed

48 hours. The agar slants shall be incubated at 37° C. for 48 hours,

when a microecopio examination shall be made of at least one culture,

selecting one which corresponds to one of the lactose broth fermentation

tubes which has shown gas-formation.

Hie formation of gas in lactose broth and the demonstration of

non-spore-forming bacilli in the agar culture shall be considered a

satisfactory completed test, demonstrating the presence of a member

of the B. coli group.

The absence of gas-formation in lactose broth or failure to demon-

strate non-spore-fonning bacilli in a gas-forming culture constitutes a

negative test.

In order that tests for B. coli may have quantitative significance,

the following general principles and ruleB should be observed:

Ordinarily not less than three portions of eaoh sample should be

tested, the portions being even decimal multiples or fractions of a

oubic centimeter; for example, 10 cc, 1 cc, 0.1 cc, .01 cc, etc. It is

essential that the dilutions should be such that the largest amount

gives a positive test (unless the water is such as to give negative tests

in 10 cc.) and the smallest dilution, a negative result. To insure this

result, it is often necessary to plant four or five dilutions, especially

in the examination of a sample of entirely unknown quality. The

quantitative value of a series of tests is lost, unless all or at least

a large proportion of the smallest dilutions tested have given negative

results.

In reporting a single test, it is" preferable merely to record results

as observed, indicating the amounts tested and the result in each,

rather than to attempt expression of the result in numbers of B. coli

342 INDUSTRIAL FERMENTATIONS

per cc. In summarizing the results of a series of tests, however, it is

desirable, for the sake of simplicity, to express the results in terms of

the numbers of B. coli per cc. or per 100 cc. To convert results of

fermentation tests to this form, the result of each test is recorded as

indicating a number of B. coli per cc. equal to the reciprocal of the

smallest decimal or multiple fraction of a cubic centimeter giving a

positive result. For example, the result: 10 cc. plus: 1 cc. plus; 0.1 cc.

minus; would be recorded as indicating one B. coli per cc. An ex-

ception should be made in the case where a negative result is obtained

in an amount larger than the smallest portion giving a positive result.

For example, in a result such as: 10 cc. plus; 1 cc. minus; 0.1 oo. plus.

In such case, the result should be recorded as indicating a number of

coli per cc. equal to the reciprocal of the dilution next larger than

the smallest one giving a positive test, this being a more probable

result.

Where tests are made in amounts larger than 1 cc. giving average

results less than one B. coli per cc. it is more convenient to express

results in terms of the numbers of B. coli per 100 cc.

The following table illustrates the method of recording and averag-

ing results of B. coli tests:

, Remit of

Tests

Amounts Designated—^ Indicated

Number of B. coli

10 cc. 1 cc. 01 cc. XII cc. per oc per 100 cc.

negative 01 10.

negative 1.0 100.

negative 10.0

1,000.

pluB 100.0 10,000.

plus 10.0

1,000.

Total (for estimating averages) 121.1 12,110.

Average of 5 tests 24.0 2,422.

The above method of expressing results is not mathematically

altogether correct. The average number of B. coli per cc. as thus

estimated is not precisely the most probable number calculated by

application of the theory of probability. To apply this theory to a

correct mathematical solution of any considerable series' of results

involves, however, mathematical calculations so complex as to be

impracticable of application in general practice. The simpler method

given is therefore considered preferable, since it is easily applied, and

the results so expressed are readily comprehensible.

In order that results as reported may be checked and carefully

valuated, it is necessary that the report should show not only the

average number of B. coli per cc. but also the number of samples

examined; and, for each dilution, the total number of tests made, and

the number (or per cent) positive.

Methods of the American Publio Health Association for differentiat-

ing fecal from non-fecal members of the B. coli group.

(1) At least 10 cultures should be used. If possible these should

be subcultures from plates made direct from the water since all of

plus.

plus

negative

plus

negative

plus

negative

DRINKING WATER 343

the cultures obtained by plating from fermentation tubes may be

descendants of a single cell in the water. If oultures from water plates

are not available those obtained from plates made as prescribed under

above may be used.

(2) Inoculate each culture into dextrose potassium phosphate broth,

adonite broth, and gelatin. For additional confirmatory evidence

inoculation may be made into tryptophane broth, and saccharose

broth.. The dextrose broth must be incubated at 30 degrees. Other

sugar broths may be incubated at 30 degrees or 37 degrees as con-

venient. Gelatin should be incubated at 20 degrees".

(3) After 48 hours record gas formation in adonite and saccharose

broths. Determine indol formation in tryptophane broth by adding

drop by drop, to avoid mixing with the medium, about 1 oc. of a two

per oent alcoholic solution of p-dimethyl amido-benzaldehyd, then a

few drops of concentrated hydrochlorio acid. The presence of indol

is indicated by a red color which is soluble in chloroform. There

may be some unconverted tryptophane still present whioh will give

a distinctly blue color-which is insoluble in chloroform. A mixture

of the two will be either blue or violet. If from such a mixture

of colors' the red of indol be extracted with chloroform, proof of the

presence of indol will be complete.

(4) After 5 days apply methyl red test and Voges-Proskauer test

to dextrose broth.

Methyl Red Test*

Indicator solution—Dissolve 0.1 gram methyl red in 300 cc. alcohol

and dilute to 600 co. with distilled water.

Procedure in

test—1.

To 5 cc. of each culture add 5 drops of methyl

red solution.

Record distinct red color as methyl red -|-, distinct yellow color

as methyl red —, and intermediate colors as ?.

Voges-Proskauer Test.

To the remaining 5 cc. of medium add 5 cc. of a ten per cent solu-

tion of potassium hydroxide. Allow to stand over night. A positive

test is indicated by an eoein pink color.

(5) Gelatin tubes should not be pronounced negative until they

have been incubated at least 15 days.

The following group reactions indicate the source of the culture

with a high degree of probability:

Methyl red +

Voges-Proskauer — ..

Gelatin —

Adonite —

-B.

coli of fecal origin.

Indol, usually +

flaocharose, usually — __

*NOTB:

See A. P. H. A. Standard Methods of Water Analysis (1923), p. 107,

for preparation of media.

344

INDUSTRIAL FERMENTATIONS

Methyl red — ....

Voges-Proskauer +

Gelatin —

Adonite +

Indol, usually — .

Saccharose +

Methyl red —

Voges-Proakauer +

Gelatin—

Adonite —

Indol, usually — .

Saccharose +

Methyl red — —

Voges-Proskauer +

Gelatin +

Adonite +

Indol, usually — .

Saccharose -f

•B.

aerogenes of fecal origin.

-B.

aerogenes, probably not of feoal origin,.

•B.

cloaosB, may or may not be of feoal origin.

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Breed, R. 8., and Dotterrer, W. D. The number of colonies allowable on

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Browne, W. W. A comparative study of the Smith fermentation tube and

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Science n.s., 42, 1915, 71.

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Sciences, 6, 1916, 483.

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Cobb,

Williams and Letton. Drinking water on interstate oarriers. Reprint No.

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Public Health Repts., 31, 1916, 2845-67.

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