Sanitary I Bacteriology...SANITARY BACTERIOLOGY In 1882, Koch devised a new technique for the separation ancl identification of bacteria, and this date marks the begin ning of bacteriology

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International Correspondence Schools Scranton, Pa.

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Sanitary Bacteriology . PREPARED ESPECULLY fOR BOYE STUDY

B,,

MAJOR l\1. J. BLEW S.,:>;ITARY EXGI XEER

EDITION 2

CopJrilbt. 1931, 1!131, by bTUIIATIOJrAL TunOOit COMPAIIY. Cop)'ript ia Great Britaia. All rights r~ned

31M8 Printed in U. S. A.

1943 Edition

SANITARY BACTERIOLOGY Serial 30-18 Edition 2

BACTERIA

DEVELOPMENT OF BACTERIOLOGY

INTRODUCTION

1. Definition.-Bacteriology is the science that deals with the smallest and simplest forms of living things, which are known as bacteria, every bacterium (singular of bacteria) consisting of a single cell. A great deal of discussion has taken place as to the position of bacteria in the scale of life. Bacteria possess properties of both the plant and animal kingdoms and therefore are considered as being between the two.

2. Origin of Bacteriology.-The belief that there existed invisible living organisms had been expressed many times before the invention of the microscope, wh:ch is an instrument used for viewing things too small to be seen by the human eye alone, but bacteriology had its beginning with the development of that instrument. Near the end of the seventeenth century Anton Van Leeuwenhoek, a skilled lens maker of Holland, gave the first definite evidence of the existence of minute living things when he examined, under a microscope, tartar scraped from his teeth and found tiny objects moving about in a lively manner. His discoveries passed almost unnoticed for nearly a century before others took up the work, which finally led to the brilliant researches of Pasteur and Koch. The work of Pasteur placed bacteriology among the natural sciences and gave it a great significance for all mankind.

SANITARY BACTERIOLOGY

In 1882, Koch devised a new technique for the separation ancl identification of bacteria, and this date marks the beginning of bacteriology as a true biological science. Since then, the science has developed rapidly in medicine and the arts, until today it plays one of the most important roles in our life.

USES OF BACTERIOLOGY

3. Medical Bacteriology.- In medical bacteriology, the main consideration is given to the effects produced by bacteria and their secretions upon the human body; to the methods by which bacteria enter the body; to the distribution of these disease-producing organisms in the body; and to the natural defensive agencies developed .by the body to resist them. Nearly all of the early work on bacteria concerned their relationship to disease, and this branch of the science will probably always be the most important one.

4. Sanitary Bacteriology.- There is no sharp distinction between medical bacteriology and sanitary bacteriology. This latter phase of the science deals with the avenues by which disease-producing bacteria leave the body of a sick person to enter the outer world; with their manner of living in air, water, food, and soil ; and with their means of entering the bodies of well persons. Sanitary bacteriology is also concerned with bacteria in their helpful relationship to putrefaction and decomposition. Bacteria disintegrate and destroy dead animal and vegetable matter, breaking down the complex organic molecules into simple substances containing nitrogen, sulphur, and carbon that are available for plant food. Other bacteria effect certain valuable changes in inorganic substances, such as nitrog~n and sulphur; t~us, some bacteria are capable of taking up mtrogen from the an and converting it into a form that is later utilized by the plant on which the bacteria grow.

5. Biological Significance.-The discoveries made in bacter_iology have revolutionized the idea of mankind concerning the umverse and man's relationship to it. Before the middle of the ninct~enth -;:entury, most of the common phenomena of everyday hfe, Sl'ch as decay and fermentation, were misunderstood.

SANITARY BACTERIOLOGY 3

The science of bacteriology has given the world a sane theory of disease and has placed the processes of decay and growth in their true relationship. Life on this planet would be impossible without the natural processes of certain forms of bacteria that are constantly decomposing dead organic matter, rendering it harmless, and producing materials that can be utilized by plant life.

6. Bacteriology in the Arts.-Such bacteria as are engaged in the production of available plant food are of great value to the farmer and fruit grower. Other bacteria give distinctive Aavors to cheese, butter, milk, and buttermilk. Bacteria play an important part in preparing flax, cu~ing tobacco, tanning hides, preserving food, and making vinegar, wine, and bread. The successful application of bacteriology to industry now occupies a prominent place in scientific research.

CHARACTERISTICS OF BACTERIA

MORPHOLOGY OF BACTERIA

7. Important Characteristics.- Before the applications of bacteriology can be understood, it is necessary to study t~e str~cture, shape, size, method of development, and physJOiogtcal requirements of the bacteria. The first three items, namely, structure, shape, and size, are commonly included in the single term 11wrplrology.

Bacteria differ in size and shape, in method of reproduction, and in other respects, and their characteristics can only be determined by the use of a high-power microscope. Bacteria may resemble each other greatly in structure and still be different in biochemical behaviour, that is, bring

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about different chemical reactions as a result of their life processes. Therefore, the biochemical and physiological behaviour ir used to identify and dassify them.

8. Cell Structure.- l\finute as bacteria are, some details of their structure can be determined by viewing them through a high-power microscope. As shown in Fig. 1, the cell of a

4 SANITARY BACTERIOLOGY

bacterium consists mainly of a semiliquid inner substance a, known as the cell substauce, or e11toplasm; the membraue, or ectoplasm, b; and the capsule, c.

A great deal of discussion has taken place among scientists as to the chemical nature of the cell substance. Ordinarily, the bacterial cells stain uniformly, which indicates that the cell substance is uniform in composition. On the other hand, some bacteria develop spots or granules when stained in a special manner. Some of these granules have been shown to be composed of fatty material and others of a form of starch called glycogen.

FIG. 2 (d.)

The cell membrane is the means of holding the semiliquid cell substance together as a unit. It is probably only a modified portion of the cell substance.

The capsule originates in the outer portion of the cell membrane. On the application of a special stain, most bacteria show the capsule as a sort of halo about the cell body. Certain bacteria develop capsules to a greater degree than others, and are referred to as being capsulated bacteria.

9. Composition of Bacteria.-Bacteria contain approximately 85 per cent water, and 15 per cent ash or mineral matter. ~he ash is largely composed of phosphoric acid, sulphur, potassmm, sodium, calcium, and chlorides. l\Iagnesium, iron,


and silicon are present in smaller quantities. Some higher forms of bacteria have considerable iron in them. Others, especially beggiatoa, which is found in water that is contaminated by sewage, contain much sulphur. Cellulose is absent, but a similar carbohydrate known as hemicellulose is often present. Starch and many protein-like substances have been observed.

10. Forms of Bacteria.-Bacteria are very simple in form. There are three main types: the coccus (plural cocci), shown in Fig. 2 (a), which is spherical; the bacillus (plural bacilli), shown in view (b), which is rod-shaped; and the spirillum (plural spirilla), shown in (c), which is spiral-shaped. There also exist somewhat higher forms of organisms called trichomycetes, shown in (d), which are larger and which develop filaments and branches. The forms of certain bacteria suggest that they are transitional between the other groups. Some bacteria are difficult to classify. A few common types of bacteria are as follows:

Coccus, or spherical form : Meningococcus (organism causing meningitis) Pneumococcus (organism causing pneumonia) Staphylococcus (cocci appearing in clusters) Streptococcus (cocci appearing in chains) Sarcina (cocci appearing in bundles)

Bacillus, or rod-shaped organism: Bacillus subtilis (hay bacillus) Bacillus anthracis (anthrax germ) Bacillus tuberculosis ( tubercule bacillus) Bacillus typhosus (typhoid-fever germ) Bacillus prodigiosus (organism that causes red spots on

bread and other carbohydrate foods) Spirillum, or spiral-shaped organism:

Spirillum cholerae (germ of Asiatic cholera)

The word bacillus is commonly abbreviated B. Thus, bacillus subtilis is written B. subtilis.

Under favorable conditions of life, each kind of bacteria regenerates and develops true to form. One scientist tabulated


833 bacilli, 96 spirilla, and 343 cocci; and another tabulated 1,325 bacteria altogether. \¥hen bacteria are grown under adverse conditions of life, their form sometimes changes so much that they are difficult to recognize. Some bacteria are more subject to the formation of degenerative, or involution, forms than others.

11. Size.-The unit for measuring bacteria is the micron, which is .001 millimeter, or about~ inch. A micron is generally represented by the Greek letter p. (pronounced either mew or moo). Bacteria vary greatly in size; some are so small that they resemble dots even under the highest magnification and some are even capable of passing unchanged through the compact pores of a porcelain filter. Dimensions of certain typical bacteria are listed in Table I.

TABLE I

DIMENSI ONS FOR T YP I CAL BACTERIA

Species

Meningococcus .... . .. . . . Staphylococcus ..... . .... . Streptococcus ........... . Typhoid bacillus ........ . Colon bacillus . ......... . Average bacillus .. .... .. . Influenza bacillus ....... . Anthrax bacillus ........ . Tetanus bacillus ........ . Tubercule bacillus ...... .

Length, in Microns

1 to3 2to4

2 .s

4.5 to 10 2to5 2to4

Diameter, in Microns

1 .7 to .9

1 .5 to .8 .4 to .7

.5 2

Ito 1.25 .3 to .5 .3 to .5

12. Motility.- The power of movement in bacteria depends on the flagella, which are long, fragile filaments originating in the cell membrane, as shown in Fig. 3. The number and location of flagella vary uniformly with the kind of bacteria under consideration. Some have a flagellum at one end as in view (a), and others have a flagellum at each end as in (b) ; some have a ~u ft of flagella from one end, as in (c), or from both ends, as m (d); and some, such as typhoid bacilli, have flagella extend-


ing from both the sides and the ends of the bacterial cell as shown in (e). Practically none of the cocci are motile, but most of the bacilli and spirilla exhibit motility.

REPRODUCTION OF BACTERIA

13. Cell Multiplication.-The multiplication of bacteria takes place by simple cell division, or fission, the original cell being divided into equal parts. Each of these newly formed -:ells develops to the maximum size, which is constant for its

(a) (b)

(c) (d)

FIG. 3

species, and in turn divides. This process is called vegetative reproduction. Bacilli and spirilla always divide at right angles to the long axis of the cell, as shown in Fig. 4 (a) and (b). Some bacilli cling together after cell division takes place and are known as strepto-bacilli. The cocci may divide to form Q.

line, a plane, or a solid. If cell division is in a line and the cell~ cling together so that a chain is formed, as shown in view (c), the coccus is caJied a streptococctts. \Vhen cell division occurs irregularly in all directions in a plane, flat sheets of bacteria are formed, somewhat resembling a cluster of grapes, as in view (d), and the coccus is called a slaphylococcus. \¥hen the cell divides in three directions at right angles, a cubical formation occurs resembling a bale or packet, as in view (e), and the type of coccus is called sarciua.

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14. R ate of D evelopment.-Cell division takes place yery rapidly and the offspring soon reach matu_rity. The hay bacillus may reach maturity and divide in 30 mmutes and the cholera spirillum in 20 minutes. If bacterial development were to proceed unchecked, the offspring from a single cell woultl weigh over 7,400 tons at the end of 3 days. Fortunately, bacteria are prevented from developing in strict geometric progression by many outside influences. One of the most powerful of these

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(a)

(c) (d) (e)

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influences is the fact that during the life of a bacterium it produces secretions and other injurious products which, when concentrated, react upon it as a poison. The lack of food and water, improper temperature, and the ravages of other bacteria and the higher forms of life also keep tremendous numbers of bacteria from forming.

15. Spore F ormation.-Some bacteria, when placed under conditions adverse to them, develop within the cell a round or oval spore, a structure somewhat resembling the seed of a plant. In Fig. 5 are shown spore formations in some bacilli, namely, B, subtilisin (a), B . tetani in (b), and B. anthracis in (c) . In each case, a is the spore and b the bacterial cell. In all e.xcept a few species of bacteria, when the spore matures, the cell wall surrounding it disintegrates and the spore is liberated. These spores are a resting stage produced from the cell substance for the purpose of carrying the bacteria through an unfavorable


situation. They are able to resist heat, drying, starvation, and chemical action. The spore may lie dormant and waiting for many .years, but when conditions are again suitable for bacterial growth, the spore develops into the true vegetative form for its species, and cell division begins by simple fission. It is a fortunate thing for the human race that practically none of the disease-producing bacteria form spores. Many of the bacteria found in sewage and soil are spore formers.

(§)~ ~~ b a

%~ .~00G !!@:::::::, % %a ~~~~ (~ (a} (c)

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REQUIREMENTS FOR L IFE

16. Food S upply.- Bacteria are able to utilize as food most organic matter, especially complex nitrogenous or carbonaceous substances, such as protein, albumen, peptone, starch, sugar, and dextrin ; and also some mineral salts such as nitrates, nitrites, sulphates, phosphates, carbonates, and ammonium compounds.

Bacteria that live and acquire their food supply within the body of a living animal of a higher type are called parasitic bacteria, or parasites. If they cause sickness and death to the animal, they are said to be pathogenic. If they do not cause sickness, they are 11011-patlwgenic. Bacteria that live on dead o rganic matter are said to be saproph)•fes.

The distinction between parasites and saprophytes is not definite, as many bacteria are able to exist on either Jiving or dead organic matter. \Yater and soil bacteria are usually distinctly saprophytic. l\Iost pathogenic bacteria are distinctly parasitic.

17. Necessity for Moisture.-Practically all bacteria are destroyed rapidly by drying or desiccation, although their spores may surviVe. Pathogenic bacteria are especially susceptible to


drying, although a few, such as bacillus tuberculosis, possess a gelatinous capsule which enables them to withstand desiccation for long periods of time. The spores of the anthrax bacillus will develop into vegetative forms after being dried for 10 years, but the vegetative forms are rapidly killed by drying.

18. Necessity for Oxygen.-1\Iost bacteria are capable of growing under ordinary atmospheric conditions, but some few do best in an environment devoid or nearly devoid of oxygen.

Those bacteria requiring free or atmospheric oxygen are called aerobic bacteria or aerobes. ).lost bacteria occurring naturally in soil, water, and air, and most disease-producing organisms are aerobic. Many bacteria found in sprinkling filters, contact beds, and activated sludge are of this type.

Bacteria growing only in an atmosphere practically devoid of free O:l\.--ygen are called anaerobic bacteria or anaerobes. They are able to obtain such oxygen as they require from their food supply. Many of the bacteria involved in the digestion of sewage solids are anaerobic. The important anaerobic pathogens are B. tetani, causing lockjaw, and B. botulinus, which causes meat poisoning.

Behveen the aerobes and anaerobes are organisms, called facultative a11aerobcs, that are usually aerobic but can exist and multiply in an atmosphere devoid of free oxygen. Many of the bacteria found in uncontaminated soil and water are of this type.

EFFECTS OF ENVIRONMENT ON DEVELOPMENT

19. Temperature. - The three important temperatures, which may be recorded for each species of bacteria, are the minimum, the maximum, and the optimum. The minimum temperature is the lowest at which development can take place; the maximum temperature is the highest at which growth can occur; and the optimum temperature is the most advantageous for the profuse and rapid development of the vegetative form of the bacteria. The range of temperature for bacteria living normally in an animal body is much narrower than for those living naturally in soil and water. In the laboratory, soil and water bacteria are cultured, that is, allowed to reproduce in a suitable

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substance called a culture medium, at either 20° or 37° C. Those that live in an animal body are normally cultured at or near the normal temperature of the body, which is 37° C. The approximate minimum, optimum, and maximum temperatures for various bacteria are given in Table II, together with their usual habitats, or places of abode.

20. H eating.-The vegetative form of bacteria is usually destroyed by heating in the presence of moisture for 10 minutes at 60° C. The temperature must be raised to 170° C. and that

TABLE II

CRIT ICAL TEMPERATURES AND HABITATS FOR B ACTERIA

Species of Bacteria

B. phosphorescens ..... B. subtilis ········ ······

B. anthracis · ··········

B. tuberculosis ········ B. thermophilus ·······

Temperatures Dtgrees Centigrade

}[inimum Optimum \Iaximum

0 20 37 6 30 50

1-1 37 45

29 38 42 42 68 72

Habitat

Decaying wood Hay, dust, univer-

sally present Animal body, r ichly

fertilized soil Animal body Manure piles, etc.

temperature held for an hour to insure destruction of bacteria when the atmosphere is dry. Steam under pressure must be used to kill spores. A pressure of 15 pounds per square inch, which is the pressure of steam at a temperature of 122° C., is usually maintained for from 10 to 20 minutes. The killing temperatures, or thermal death points, for some bacteria are given in Table III.

21. F reezing.- Freezing is much less destructive to bacterial life than heat. It is true that most pathogenic bacteria die off rapidly when exposed to freezing, but some of them last for many months. The typhoid bacillus has been identified in ice from the St. Lawrence River 7 months 'l.fter being cut, following the outbreak of an epidemic of typhoid fever. Ice made


from contaminated water is therefore potentially dangerous, even though there have been relatively few cases of infection actually traced to it.

22. Light.-Sunlight is one of the most powerful germicidal, or germ-killing, agents known, and is nature's own method of destroying disease organisms. Ultra-violet rays have been used in water purification. The rays from an electric lamp act in the same manner as S!lnlight, although not so intensely.

TABLE III

THERMAL DEATH POINT FOR BACTERIA AFTER 10 MINUTES• CONTACT

Speeics

Spirillum cholerae ............ ..... . ............ . Anthrax bacillus .................. ." ............. . Anthrax spores ....... ....... ... ... ... ........ . . . Typhoid bacillus ...... .................. . ....... . Tuberculosis bacillus .. . ......................... . Common sewage organisms ..... . ............... .

Temperature Degrees Centigrade

58 60

100 60 60 58

23. Reaction of Medium.-The reaction, or the degree of alkalinity or acidity, of a medium is of great importance in the growth of bacteria. i\Iost organisms grow best on a medium that is neutral or faintly acid, but some bacteria require a medium that is distinctly acid, and others require one that is distinctly alkaline. The acidity or alkalinity may not be sufficiently strong to sterilize the medium entirely and prevent growth of hacteria, and yet may be strong enough to inhibit their development and cause a false impression of their biological activity.

The reaction of a medium is determined by adding to it in definite proportions a prepared indicator solution, and comparing the color of the mi>..1:ure with a series of standard color solutions. By the use of a properly graded series of color solutions, the reaction of any medium can be readily ascertained. To represent relative reactions, the standard color solutions are numbered from 1 at the acid end to 10 at the alkaline end, the neutral point being about 6.8.


An indicator solution is prepared by dissolving in water eith~r a small amount of a weak organic acid that changes color ~~~

the presence of a stronger alkali, or a small amount of an alkali that changes color in the presence of a stronger acid. The materials commonly used in these solutions are brom-thymol blue, phenol red, and cresol red.

24. Concentration of Food Supply.-Bacterial growth is most rapid in dilute solutions of the food supply for the reason that bacteria require soluble food. Insoluble substances are first rendered soluble or partly so by secretions, called ~nzymes, pr~duced by the bacteria for that purpose. Bactenal growth IS

retarded when the food supply is too concentrated, even though the food itself is of suitable nature for bacteria. For this reason desiccation is an excellent method of preserving foodstuffs. '

BACTERIAL METABOLISM

PHYSICAL EFFECTS OF BACTERIAL ACTION

25. General Remarks.-Bacteria, like the higher forms of life assimilate food material so that it is incorporated in the cell substance. On the other hand, they use up or tear down some of that living substance in giving off energy in the form of heat, light, and in some cases motion. Part of the food acted on by bacteria is transformed into chemi:al substances that differ widely from the original food matenal. The processes of building up and tearing down cell substances that co~stitute the general life activities of a cell are l<nown as metabolism.

26. Production of Heat.-Some bacteria generate heat during the pursuit of their vital processes. T~1e .spontane~us heating of damp hay, dead grass, or a manure pile IS a reaction of this nature. Bacteria that generate heat are called thermogenic bacteria.

27. Production of Light.-Certain types of bacteria produce light in the form of phosphorescence. Sea water often displays phosphorescence from bacterial origin. Phosphorescence also occurs on the bodies of dead fish and animals. These bacteria are said to be photogenic bacteria.


BIOCHEMICAL EFFECTS OF BACTERIAL ACTION

28. Pigmentation.-~Iany bacteria when grown in masses produce pigment. Nearly all the colors of the rainbow are developed, namely, violet, blue, green, orange, red, and yellow. Some of the pigments are soluble and some are insoluble. In some cases a type of bacterium produces two pigments, one of which is soluble and one insoluble. Bacteria producing color are said to be chromogeuic.

29. Enzymes.-Bacteria secrete many substances, called enzymes, or ferments, which are responsible for most of the results attributed to bacterial action. Among these enzymes are the substances that sour milk, decompose foods, digest sewage solids, and assist in the decomposition of organic matter. Reactions, such as putrefaction, digestion, and fermentation are {'aused by enzyme action. Enzymes possess powerful activity along certain lines which are important in the life processes of the bacteria themselves. UsualJy, enzymes are poisonous to the bacteria that produce them and also to other bacteria. Sometimes, enzymes or the results of their action are beneficial to other bacteria.

30. Gas Formation.-Certain bacteria develop gas as a by-product of their metabolism. Such bacteria are called aeroge11s. If they are aerobic bacteria, the gas leaves the surface of the medium unnoticed. If the bacteria are anaerobic and develop colonies in the deeper parts of the medium, the gas bubbles can be seen caught in the medium about the colony. The formation of gas is often used to distinguish one organism from another.

31. Acid and Alkali Formation.-Acid is produced by some bacteria and alkali by others during their growth. The presence of either acid or alkali can be determined by adding a sterile indicator solution .to the medium before inoculating it with the bacterial culture. Indicators such as litmus or bromthymol blue which are not poisonous to bacterial life may be used. When litmus is used, acid production by the bacteria


turns the medium red. The presence of alkali-forming bacteria is indicated by the medium turning blue. Litmus solution is often added to milk for the detection of acid formations.

32. Liquefaction.-Some bacteria possess the power to liquefy gelatin or coagulated proteins; they are called proteolytic bacteria. The proteolytic, or protein-digesting, power of bacteria is commonly tested in water analysis.

33. Peptoni'l:ation.-Bacteria in some cases possess the power to peptonize or digest milk proteins. The process is different for different species; some digest the casein with no apparent change in the milk; some gelatinize the milk; some coagulate it ; and some transform the milk into a transparent watery fluid. This action on milk is used extensively in differentiating bacteria; for instance, B. coli coagulates milk, while B. typhosus does not.

34. Putrefaction.-Putrefaction is largely a process of anaerobic decomposition. It is one of the most easily recognized reactions caused by bacteria, as it is usually accompanied by foul odors which betray the presence of decaying organic matter. The bacteria that cause putrefaction are known as saproge11ic bacteria. Among the gases produced by putrefaction are ammonia, hydrogen, carbon dioxide, carbon monoxide, methane, nitrogen, and hydrogen sulphide. Peptides, peptones, amides, and aromatic-odor-producing bodies, such as indo! and skatol, are also formed in putrefaction.

35. Production of Ptomaines.- The action of bacteria on complex nitrogenous food materials like meat, often produces in the process of decomposition intermediate products, called ptomaines, which are very poisonous to human beings. These products contain carbon, hydrogen, and nitrogen, and are known as diami11es. The first ptomaines isolated were cadaverine and putrescine, which were so called from the fact that they were isolated, respectively, from a decomposing human cadaver, or corpse, and a decomposing animal.

36. Product ion of Toxins.- Poisonous substances produced by localized bacterial action and having a specific reac-


tion on the entire animal body are called toxins. They are soluble and in many respects resemble the enzymes. Examples of toxins are the substances produced by the organisms causing tetanus, or lockjaw, and diphtheria. In neither case are the organisms founcf away from the site of the original infection, which is the throat in the case of diphtheria and an open wound in the case of tetanus. However, the toxins are carried away from the site of infection by the blood and attack the tissues of the body, causing sickness and often death.

Oeao' 0r¥'onk NollerB

37. Nitrogen Cycle.- Bacteria play an important part m the circulation of nitrogen in nature. Life on this planet would be impossible without the saprogenic bacteria, which putrefy the dead animal and vegetable matter and the excreta of living beings, breaking them up into· simple substances that are later made available for plant food, or are passed off into the air.

T he processes involved in the circulation of nitrogen constitute the so-called nitrogen cycle, which is represented diagrammatically in Fig. 6. Those processes that are dependent on bacterial action are indicated by means of heavy Jines, and those not directly dependent on such action are represented by light lines. \\'hen death occurs in either plant or animal life, as


indicated by AB in the diagram, the saprogenic bacteria act on the dead animal or yegetable matter and by means of their enzymes split up the animal or plant proteins into ammonia and other simple substances; this proce.ss, which is known as putrefacti()n, is represented in the diagram by line BC. Furthermore, the nitrogenous waste materials or urea that" result from the process of digestion in livrng animals are reduced by the saprogenic bacteria into ammonia and other simple substances, the process being known as ammonification. In the diagram, the process of digestion is represented by line AD, and the process of ammonification 'by line DC. The ammonia from the two processes may either unite with soil acids or pass off into the air. The ammonia that unites with soil acids is transformed by nitrifying bacteria into nitrites and eventuaLly into nitrates, which are available for plant food; this process, which is called nitrification, is indicated by line CE. hlost of these nitrates are speedily consumed by growing plants and are transformed into vegetable proteins. \\' hen an animal consumes plants, the vegetable matter is digested and the plant proteins are rebuilt into animal proteins, the cycle being thus completed.

There are important deviations from this cycle. In the absence of oxygen, or under anaerobic conditions, the nitrates and other nitrogenous compounds in the soil may be acted on by denitrifying bacteria and reduced by their enzymes to ammonia and free nitrogen, the process being known as denitrifiration and indicated by line EF in the diagram. On the other hand, there are other forms, known as nitrogen-fixing 'bacteria, that take the free ammonia and nitrogen from the air and change them into nitrates which are available for plant food; this process, called 11itrogen fixation, is represented in the diagram by line FG. The nitrogen-fixing bacteria are present in the nodules or roots of clover, peas, alfalfa, and beans, which are therefore excellent crops for reclaiming impoverished soil.

38. Sulphur Cycle.-Sulphur undergoes a cycle in nature similar to nitrogen in which bacteria play an important part. Sulphur is found in the tissues, fluids, and discharges from the animal and vegetable worlds; it is also found in soil and water


in both organic and inorganic forms. It is released from organic material by the process of putrefaction, usually with the production of foul odors. These odors are due to the presence of hydrogen sulphide and other volatile sulphur compounds. The hydrogen sulphide is transformed by aerobic sulphur bacteria into suiphuric acid and free sulphur. The sulphuric acid combines with alkaline earths to form sulphates; also, under the action of sulphur bacteria, the free sulphur is converted into sulphates. The sulphates in the soil are absorbed by plants and converted into plant tissue, and some of these plants in turn are eaten and assimilated by animals.

In the absence of oxygen, a reduction process may take place and the sulphates may be reduced by anaerobic bacteria to hydrogen sulphide.

39. ~a~bon Cycle.-Carbon passes through a cycle m nature stm1lar to that undergone by nitrogen, but bacteria do not pla.y s~ch a~ _important part in the cycle. In the process of resp1ratwn, hvmg animals inhale oxygen and exhale carbon dioxide. On the other hand, green plants absorb carbon dioxide from the air, transform the carbon into plant material and give off oxygen ; this process, which is carried out with th~ aid of sunlight, is known as photosynthesis. As the processes of respiration and photosynthesis are of opposite character there is a continuous transfer of carbon between plants and a1~imals w~ch goes on without the aid of bacteria. However, by th~ actJon of bacteria, the carbon in dead animal and vegetable matter and in the urea excreted by living animals is converted into carbon dioxide. Also, carbon dioxide is often the product of bacterial metabolism.

DISTRIBUTION OF BACTERIA

BACTERIA IN AIR

40. Number of Bacteria in Air.-Air is not a natural habitat. for bacteria, as it does not contain the necessary food and mOisture to make a suitable place for bacteria to thrive. Bact~ria that are found in air are always associated with dust particles or droplets of water.


The number of bacteria may or may not be proportional to the number of dust particles in the air. :More bacteria will be found in air over a fertile agricultural soil than in air over a clay or sandy soil. Air from city streets, schoolrooms, factories, and other places where people pass and congregate contains more bacteria than air over lakes, oceans, mountains, and deserts. Bacteria are not present in any appreciable quantity over wet surfaces because they are prevented by the water from

TABLE IV

NUMBER OF BACTERIA IN AIR

Locality

Outdoor air, Boston ............. .

Open air ••.• . ...•................ Open field .•. . .................... Sea coast . . . . ................... . Mountain altitude, 200 meters ... . hiont Blanc ..................... . Spitzbergen (Arctic region) ...... . Middle of Paris ..... . ........... . Paris street . .................... . Tailor's r oom in \Vhitechapel . .. . Boot workshop .................. .

Number of Bacteria per Cubic Meter

1~150

100-150 250 100

0 4-Jl

0 4,000 3,500

17,000 25,000

Observer

Sedgwick and Tucker

Fischer Uffelman Uffelman Pasteur Ellis Levin Ellis Fischer Ellis Ellis

nsmg. However, bacteria may be present over bodies of contarni_nated water, such as sewage, being carried with the spray commg from the surface when the wind velocity is high. The species of bacteria will always vary with the nature of the surroundings.

The spray sent into the air from the throat and mouth in talking and sneezing always contains bacteria, many of which are disease-producing. T his is one of the most fruitful methods of transmitting diseases, such as measles, mumps, scarlet fever, tu.be~culosis, and meningitis. Actual. contact with the freshly ehmmated spray must be had, however, to produce disease, because these bacteria do not travel far, and die rapidly in the


open air. Sewer gas is practically free from bacteria, although sewage itself is teeming \\ ith them. Bacteria may be sent into the a ir by the breaking of gas bubbles, ripples, etc., but they do not persist for long periods of time. Air is washed practically free from bacteria by rain or snow storms.

Bacteria in air suffer greatly by drying and the action of sunlight. In Table IV, which is taken from Microbiology by ~Iarshall, is shown the number of bacteria in a cubic meter of air in various localities.

41. Kinds of Bacteria in Air.-Bacteria m atr ca•1 be divided into the following groups :

1. Pathogenic bacteria. 2. Sarcinae. 3. Spore-forming bacilli.

Pathogenic bacteria get into the air from persons who are suffering from disease. In time of epidemics, conditions may become serious, but the number of pathogens ordinarily present in the air is small.

:Many chromogenic cocci are found in the air. Sarcinae also are found.

Bacillus subtilis, or the hay bacillus, is the chief spore-forming bacillus in the air. This organism and its spores cause much trouble in the bacteriological laboratory. Contaminations on plates are frequently found to be bacillus subtilis.

Other microscopic organisms that are found in air are yeasts and molds. They are larger than bacteria and higher up the scale of living things. Yeasts are not found in any appreciable amount, but molds are present in large quantities.

:BACTERIA I N SOI L

42. Non-Pathogenic Bacteria in Soil.- Bacteria are normally found in the soil. The number depends on the reaction of the soil. its temperature. its moisture content, and the nature ancl amount of food present. An aYerage total ranges from 3,000.000 to 25,000,000 .per gram of dry soil. Other microorganisms found in the soil are protozoa, !JL" minute animals, algae, molds, and other fungi.


The following types of non-pathogenic bacteria are normally found in soil :

(a) A group of very short bacilli most of which are not motile, and which either do not liquefy gelatin or do so slowly.

(b) A few short bacilli with flagella at each end, which liquefy gelatin rapidly, such as pseudomonas fluorescens.

(c) Actinomycetes, which are mostly pigment formers whose functions are little understood.

(d) Large spore-forming bacilli like B. subtilis, B. mycoides,

and B. cereus. (e) Cocci of many kinds. (f) Spirilla. (g) Organisms of fecal origin like B. coli. (II) Nitrogen-fixing forms, such as azotobacter and rhizo-

bium. (i) (j)

Sulphur bacteria, as thiobacteriales: Nitrifying bacteria.

( k) Denitri fying bacteria. These bacteria are responsible for a number of reactions that

are being carried on in the soil, such as nitrification, ammonification, denitrification, and nitrogen fixation. Most of these forms have but little bearing on sanitary bacteriology.

43. Pathogenic Bacteria in Soii.-Pathogenic bacteria a re not found in the soil nonnally, but tetanus, anthrax, and gas gangrene may all be contracted from highly fertilized soils. Parasitic diseases such as hookworm and tapeworm are more apt to be spread through infected soil than diseases from pathogenic bacteria, especially in warm climates. In modern warfare, with its high explosives and methods of conduct, tetanus and gas gangrene are very dangerous.

:BACTERI A I N W ATER

44. Classification of Bacteria in Water.-Bacteria occurring in water may be classified as follows :

(a) Bacteria produci11g fluorescence. (b) Bacteria producing colored colonies (violet, red, and

yellow) .

22

(c) (d)

SANITARY BACTERIOLOGY

Bacteria resembling B. coli (intestinal types). Bacteria of the proteus group (hay bacillus) .

(e) Non-gas-forming, non-chromogenic, non-spore-forming rods which may or may not produce acid in milk and liquefy gelatin, and whose colonies do not resemble B. proteus. This type is prominent in natural water.

(f) Spore-forming bacteda of the subtilis type. (g) White, yellow, and pink cocci. (h) Aliens or temporary dwdlers. From the point of view of the sanitary engineer, a more con

venient classification is as follows: (a) Jatural water bacteria, as pseudomonas fluorescens. (b) Unobjectionable aliens, mainly from the soil, such as

B. mycoides. (c) Objectionable aliens, such as the organisms of the coli

aeroge,es group. This group includes B. coli, which are generally excreted with bowel discharges, and Aerobacter aerogenes (A. aerogenes), which normally live in soil and on vegetables.

45. Rain Water.-Theoretically, rain water is the purest of waters. The only bacteria it ordinarily carries are few in number and mostly those attached to the dust particles collected from the air. Rain water is usually stored in cisterns and, although it may .be pure at its source, it is easily contaminated by surface washings, leakage, and drainage.

46. S~ace Water.-Ponds, lakes, and reservoirs are the most satisfactory water supplies. It is advisable that such waters be kept pure by strict control of the watershed, rather than by resort to purification later. Large lakes and rivers are commonly used as the dumping grounds for raw sewage, and water from them must be treated before it is safe for drinking.

47. Self-Purification of Surface Water.-It was erroneously believed in the past that surface water in a flowing stream purified itself in flowing the distance of 7 miles, but this is not the case. Self-purification depends on the following factors : Sedimentation, sunlight, temperature, diminution of food supply, changed food supply, the presence of other organisms

SAKITARY BACTERIOLOGY 23

unfavorable to bacteria, and possibly the action of oxygen of the air. Another factor is the bacteriophage, or bacteria-toxic substance, which occurs naturally in water and rapidly causes the destruction of bacteria. Plain sedimentation removes great numbers of bacteria, because most of them are attached to partides of solid matter suspended in flowing water, which subside, or settle, when the velocity of flow is sufficiently reduced. \Vater in a subsidence basin will be much more liable to purify itself than water in a rapidly flowing stream. The sdf-purification of water is determined not so much by the distance of flow as by the velocity of flow and the concentration of contamination.

48. Melting snow or heavy rain always causes a marked increase in the number of bacteria in surface water, because more surface drainage is received from the ground. On the other hand, surface water that is normally high in bacteria from sewage may contain fewer bacteria at the time of hard rains, because of simple dilution.

49. Storage of Water.-\Vhen surface water is stored, all the agencies acting on it are intensified. A greater percentage of reduction in bacteria follows the storage of a highly polluted river water than the storage of a relatively unpolluted water.

Bacteria are removed from drinking water supplies by sedimentation; coagulation with alum, which enmeshes the bacteria and causes them to subside; filtration; and chlorination of the final filter effluent to destroy aJI intestinal organisms that may have passed through the previous processes.

50. Ground Water.-Water collected in shallow wells is usually contaminated with surface drainage, and is only good for use when the wells are properly placed and properly inspected. Artesian wells, deep wells, and springs may supply good water free from injurious bacteria when the water filters through sandstone rock. On the other hand, it is often dangerously contaminated when it flows through limestone. The action of water on limestone over long periods of time has in many instances worn channels through which water flows unimpeded. This water carries many surface bacteria, and may include those

282C-6

24 SA~ITARY BACTERIOLGY

of intestinal ongm. \Vater filtered through earth is relatively free from bacteria and the number of bacteria in soil decreases rapidly with increasing depth. However, a deep well may contain the same bacteria as a surface stream.

BACTERIA Hf SEW AGE

51. Important Sewage Bacteria.-Sewage is the waterborne filth from communities. It contains all the soluble and suspended waste from city life, such as dish water, laundry water, excreta, decomposing vegetables, trade waste from various types of industry, and floor and street washings. Bacteria of all kinds may be present, but pathogens other than intestinal forms are not common, because they die rapidly outside the human body. Enormous quantities of bacteria varying from 322 billion to 25,652 billion per person are eliminated daily and are carried off in sewage. \Vhen a person has an intestinal disease, such as typhoid, dysentery, or cholera, the fecal discharges become a prolific source of danger to the community. Most of the bacteria eliminated daily by each individual die rapidly on leaving the body, but sewage always contains from a few thousand to many millions of bacteria per milliliter or cubic centimeter. One of the predominating bacteria is B. coli, and the presence of this organism in water is taken as an indication of pollution by sewage. It is estimated that from 42 billion to 583 billion B. coli are eliminated daily by each person. The presence of streptococci in water has been considered an indication of recent pollution by sewage. Many cases of skin infection and sore eyes are traceable to bathing in water contaminated by sewage.

Contaminated water contains all the common soil, water, air, and intestinal organisms at times. The members of the proteus group, B. mesentericus, B. coli, A. aerogenes. B. subtilis, B. typhi, streptococcus pyogenes, spirillum cholera, and the anerobes B. welchii and B. tetani are the most common forms. B. fluorescens liquefacines has been suggested as being an indicator of unpolluted water. The soil and water bacteria found in sewage are largely responsible for the purification that takes place in sewage treatment plants.


52. Aerobic Bacteria in Sewage.-Oxidation in sprinkling· filters, contact beds, and activated sludge plants is primarily due to the action of aerobic bacteria. They oxidize aminonia to nitrites, nitrites to nitrates, sulphur to sulphates, and marsh gas to <:arbon dioxide.

53. Anaerobic Bacteria in Sewage.-The anaerobes in sewage are largely saprogenic and accomplish the putrefaction of protein matter, producing simple water-soluble molecules. Nitrogen, ammonia, peptone, and amino acids are thus formed. Other anaerobes decompose grease and fat, forming alcohol and acids.

54. Bacteria in Sludge.-Siudge is the solid matter allowed to settle from industrial waste water or sewage. It contains most of the bacteria originally present in sewage. The predominating type of bacteria in freshly settled sludge is of the B. coli, or acid-forming, type. As the solid matter slowly decomposes, the B. coli disappear in great numbers and there appear alkali-forming anaerobes, which are active agents in sludge-digestion processes. Most pathogens, including the intestinal ones, are absent in digested sludge, but the eggs of animal parasites and the spores of pathogenic anaerobes still exist.

LABORATORY PRACTICE

P RINCIPAL FEATU RES

55. T ests for Bacteria.-\Vater and sewage are examined to determine the number and general nature of the bacteria they contain. !\o attempt is made to isolate and identify all the types of bacteria present, as this would be a long and timeconsuming operation. The presence of members of the coli-aerogenes group is taken as the indication of contamination of a water supply. In water or sewage treatment, the presence or absence of members of the coli-aerogenes group is taken as the indication of the degree of purification effected at different stages of thr treabnent process.

56. Sterilizati<m and Disinfection.-Before attempting to 5tudy bacteria, a knpwledge of the principles underlying steril-


"ization and disinfection is necessary. By disinfectivn is meant the destruction of pathogenic bacteria only, but the sterilization of an object kills all living things in it. Germicides kill bacteria, whereas antiseptics only hinder their development. Deodorants may or may not destroy bacteria. A chemical may act as antiseptic in one concentration, or solution of one strength, and as a sterilizing agen. in another.

57. I mportance of Sterilization.-Success in bacteriological work depends on maintaining an exact technique which is more or less peculiar to this science. In order to obtain correct results, it is important to prevent contamination by outside organisms. Hence, the media and the glassware and other apparatus required for the work should be sterile and they should be

F1c. 7

very carefully manipulated so as to maintain sterility. However, sterility is difficult to produce and still more difficult to maintain because of the presence of bacteria everywhere about us, as on our hands, on table tops, and in dust

58. Inhibition of Growth.-Some chemicals such as chloroform, toluene, xylene, and thymol act as preservatives in inhibiting or preventing the growth and reproduction of bacteria. Such chemi~ls, which are known as inhibiting agents, are used for preservmg samples containing organic matter such as sewage, sludge, food, and animal tissue.

APPARATUS

DESCRIPTION OF APPARATUS

. 59. Petri Dishes.-In Fig. 7 is shown a petri dish, which •s much used in bacteriological laboratories; it is a round dish a ?£ g!ass or unglazed porcelain, with a flat bottom about 4 inches m. d1ameter and straight sides about i inch high. It is provided ~\·at~ a cover b ~h~ped like the dish and large enough to fit over ~ts sades. _Petn d1shes are used in water examinations for countmg bactena and for the isolation of specific bacterial colonies.

I


60. Test Tubes.-Ordinary glass tubes, such as the one shown in Fig. 8 (a), which are open at the top and provided with rounded bottoms, are known as test tubes. \Vhen thoroughly cleaned and sterilized, test tubes are used for holding liquid and solid bacterial media, as a in view (b). The mouths of tubes are plugged with non-absorbent cotton b to prevent the contamination of their contents with living organisms from the outside. The cotton is exposed to the Aarne of a Bunsen burner before being used, to kill any bacteria contained in it

(a} (b) (b) F1c. 8 Ftc. 9

61. Fermentation T ubes.-Fermentation tubes are used to observe gas formation by bacteria. Some fermentation tubes are made of an open bulb and a closed vertical arm, as in Fig. 9 (a). Such tubes are expensive, hard to clean, and easily broken. Hence, for practical work, a straight tube called the Dunham tube, which is shown in view (b), is recommended as being more satisfactory. The Dunham tube is made of two test tubes, a large one a and a much smaller one b, which is inverted inside the large one.

62. Platinum Needle and Loop.-Platinum needles, like those shown in Fig. 10, are used to transfer bacterial colonies from one plate to another and from a plate to a culture tube.


A platinum needle is made of a thin platinum wire a sealed into a glass rod b. As shown at c, the end of the wire is often bent into the form of a circular loop about i inch in diameter which can be standardized and used for delivering definite quantities of liquid. In the illustration, the needles at·e shown inserted in holes of a wooden stand d, which is conveniently employed for keeping platinum needles.

63. Pipettes.-A pipette is a graduated glass measuring tube open on both ends that is used for delivering accurate portions of a liquid. Pipettes may be of any

FIG. 10

convenient size or shape, as long as they deliver the required amount of liquid. A common bacteriological form is shown in Fig. 11, which is graduated for practically its entire length.

64. Dilution Bottles.-Dilutions of water and sewage samples are made in tall bottles or in Erlenmeyer flasks, which are shown in Fig. 12 (a) and (b) , respectively. These bottles or flasks should be of sufficient capacity to hold twice the volume of water actually used. A ground glass stopper a may be provided, but preferably the bottle or flask should be plugged with cotton. In either case, care must be exercised to prevent contamination and to maintain a constant volume of diluting liquid. Fie. 11

65. Incubators.-An incubator is essentially an insulated . cabinet with means of applying heat and carefully regulating the temperature of its interior. It is used to store cultures under conditions ideal for promoting growth. Two types of incubaton: arc in common use, one maintaining a constant temperature of 37° C. and the other a temperature of 20° C. Heat may be

I


applied by gas, petroleum lamp, or electt·icity. \Vhen the temperature is to be maintained at 20° C., an alternate heating and cooling device is required.

In Fig. 13 is shown an electrically operated incubator. The outer shell consists of two layers of sheeting a sepat·ated by an insulating material b. The trays c hold the petri dishes d.

(b)

FlG. 12 FIG. 13

A thermometer e is placed in the top of the cabinet and is directly connected with an electric circuit f by means of which the temperature is automatically kept constant.

STERILIZATION OF APPARATUS

66. Methods of Sterilizatioo.- Sterilization may be accomplished by the use of chemical agents, by heat, or by filtration. Heat or chemical agents actually kill the bacteria, but filtration merely serves to remove the bacteria mechanically.

30 SANITARY BACTERI OLOGY

Ordinary fi lters of crushed stone, sand, gravel, cinders, slag, clinker, or glass beads do not remove bacteria to a point of sterility. The passing of a bacterial culture through an unglazed porcelain plate, however, will render it sterile.

67. S imple Methods of Ster ilization by H eat.--Stcrilization by heat may be accomplished in three ways, namely, by dry heat, by moist heat, and by flame. All are in common use in the laboratory, each being employed in the work for which it is best suited.

Fu;. 14

Glassware, pipettes, petri dishes, and such pieces of apparatus are best sterilized by dry heat. A hot-air oven or hot-air sterilizer, such as that shown in Fig. 14, which is similar to the oven of a cooking range, is used for this purpose. Material to be steri lized is placed on shelves a in the oven, and heated to between 150° and 180° C. for an hour. This method kills all bacteria and spores, but the heat is too great for material of either vegetable or animal origin.

Such articles as scissors, knives, and glassware can be sterilized by boiling in water for 30 minutes or more. This treatment does not always kill spores but it does kill all vegetative bacteria. It is a safe precaution against practically all pathogens except the tetanus bacillus.

Platinum needles and loops are sterilized by holding them in the flame of a Bunsen burner or an alcohol lamp. The necks


of test tubes and flasks are sterilized in the same manner. The cotton plugs that are inserted in the mouths of the tubes or flasks are momentarily ignited in the flame to destroy any bacteria that may have lodged in the cotton.

68. Arnold S terilizer.-In an Arnold sterilizer shown in ~ig. 15, sterilization is carried out by means of s~eaming, or hve, steam which is allowed to come in contact with the material that is to be sterilized. The pan a, which is partially fi lled with water, has a double bottom at b, the upper one being per forated

FIG. IS

so that the water can enter the lower portion between the bottoms. 'When heat is applied at c, the water in the lower portion of the pan evaporates and the steam escapes upwards through the central flue d. 'Vater then flows into the lower portion from the upper part and replaces that lost by evaporation. The steam passes through the holes e into the chamber f and finally escapes into the hollow casing by wey of the holes at g. The cabinet casing is double and the condensed steam returns by way of the hollow wall /1- to the pan a.

SAi>liTARY BACTERIOLOGY

" ' hen an Arnold sterilizer is used, the sterilization process is intermittent. If the material to be sterilized is heated in steam for 30 minutes, all the vegetable forms of bacteria are killed, but the more resistant spores are not harmed. If these spores are allowed to incubate, they will develop into vegetative forms. A second heating a day later will kill the newly developed vegetative forms. A second incubation and a third heating on the

Frc. 16

third day will insure complete sterilization. This method is used to sterilize materials that would be damaged by heating under pressure.

69. Autoclave.-Thc most satisfactory method for sterilizing materials that can stand moisture and also pressure is by means of an apparatus known as an autoclave. l\Iost culture media are sterilized in an autoclave. As shown in Fig. 16, it consists essentially of a large pressure compartment, or chamber, a, which can be closed and sealed so as to be air-tight. The material to be sterilized is placed on a tray b. The top door or

SAi\I"IT ARY BACTERIOLOGY 33

liJ c is opened by means of the handles d, the tt·ay is inserted in the chamber a, water is admitted to the chamber, and the lid is clamped down. Heat is then applied at e until steam forms and drives out the air through the stopcock f, which is left open until the chamber is filled with stt!am. All ports are finally closed and pressure is allowed to develop. The steam is kept at a pressure of 15 pounds per square inch for 10 to 20 minutes. The pressure of the steam is read directly from the gage g. The chamber a is provided with a safety valve II, which may be adjusted to the desired pressure.

70. Sterilization by Chemicals.-i\Iany chemical agents are used for sterilizing, among which are the following: The metallic compounds, such as mercuric chloride, copper sulphate, ferrous sulphate, and zinc chloride; the coal-tar products, such as carbolic acid, cresol, creolin, lysol, and chinosol; and the oxidizing agents, such as potassium permanganate, chlorine, sodium hypochlorite, calcium hypochlorite, iodine, hydrogen peroxide, oxygen, and ozone.

71. Disinfection.-The following considerations must be kept in mind in using a disinfectant :

l. The strength of solution required to offset the effect of dilution. If a disinfectant of known strength is put into an equal volume of material to be sterilized, its disinfecting power is cut in hal f.

2. The temperature. Disinfectants act more thoroughly and more readily in a hot solution than in a cold one.

3. The nature of the material being disinfected. A large percentage of organic matter in the material to be disinfected reduces the efficiency of the disinfectant.

4. The period of contact. At least an bout· should be allowed to complete the chemical reaction that takes place.

72. Cleaning New Glasswa.re.-New glassware should be cleaned by immersing it in the following cleaning solution:

Sodium bichromate . . . . . . . . . . . . . . . . . 52 parts Tap water . . . . . . . . . . . . . . . . . . . . . . . . 300 parts Commercial sulphuric acid .. .. .. .. . .. 460 parts

S.\~IT.\RY B.\CTERIOLOGY

Tl I • 1 t · di.ssolved in the water and the acid care-le >JC uoma e IS

f II . dd d t ·t After tubes and flasks have been washed and u ) a c o 1 . · 1· · 1 tl I I to 1· nsure the removal of excess ac1d or alka 1, nnsec toroug 1 y I I gcd \\•ith cotton. The plug should be compact t te\· are p ug

I. · h h to allow the tube to be suspended by the anc ug t enoug stopper.

CULTURE MEDIA

PURP OSE AND FORYS OF MEDIA

73. Use of Culture Media.-Before bacteria can be identified, they must be grown on a suitable_ materi~l that is specially prepared for the cultivation of bactena and IS called a culture

FIG. 17

medium (plural media), or simply a medium. Over 6,000 different media haYe been prepared for the isolation and study of special forms of bacteria, but comparatively few of them are in general use for water and sewage organisms. In Fig. 17, which is similar to Fig. 1-l: in Priuciplcs of Bacteriology by A. C. Abbott, is shown the appearance of colonies of bacteria grown on a medium in a petri dish; every independent spot or shape represents a separate colony, which has grown from a single bacterium.


74. Requirements of Media.-Any culture medium must fulfill the following requirements:

1. Moisture must be present. 2. Suitable food must be present for the growth of the bac

teria in question. 3. The reaction of the medium must be neutral or slightly

acid. This is a very important factor and causes considerable difficulty in the preparation of the medium; but, when some prepared medium is used, the reaction ",.;II almost always be found to be correct and no adjustment \\·ill be necessary.

4. The medium must be free from all forms of bacteria; hence, the medium and everything that comes in contact with it must be thoroughly sterilized. A medium may usually be sterilized by heating it in the autoclave at a pressure of 15 pounds for 15 minutes.

5. Oxygen must be available in the proper concentration. 6. The medium must be adapted to any peculiarities of the

bacteria to be studied; thus, sugar, blood, or body fluids must be present as required.

7. Natural conditions under which the bacteria develop best must be reproduced.

75. F orms of Media.-Culture media may be liquid, semisolid, or solid in form. Solid media furnish a means of separating bacteria. The most used media such as agar and gelatin are liquefied at will by gently heating in hot water, and are again solidified on cooling. Dyes are sometimes added to media to indicate the growth of particular strains of bacteria or to serve as inhibiting agents to all other strains than those whose development is desired.

76. Prepared Media.-The use of prepared culture media, which is authorized by the leading associations, lessens considerably the labor in preparing media. They are purchased in the solid state and are weighed out and dissolved in distilled water as required. In almost all cases the reaction of a prepared medium is correct without adjustment after sterilizing in the autoclave.


DESCRIPTIONS OF MEDIA

77. Nutrient Broth or Plain Bouillon.-Filtered beef broth containing a little peptone is known as nutrient broth or plain bouillon. It is the basis of practicaJly all standard media and is prepared by dissolving the following substances in 1 liter of distilled water:

Beef extract . . . . . . . . . . . . . . . . . . . . . . . . 3 grams Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams

The broth is placed in tubes or flasks as desired, and sterilized.

78. Nutrient Gelatin.-Gelatin is one of the commonest of solid media used for separating bacteria. It is prepared by dis

FIG. 18

solving the following ingredients in a liter of distilled water, with the use of as little heat as possible :

Beef extract . . . . . . . . . . 3 grams Peptone . . . . . . . . . . . . . . 5 grams Gelatin ............ .. 120 grams

After the material is in solution, it is filtered into tubes or flasks and sterilized. The gelatin is solidified by immersing the tubes or flasks in cold water for a few minutes, and is then stored in a refrigerator.

79. Nutrient Agar.-Nutrient agar is prepared by dissolving in 1 liter of distilled water the following ingred:ents:

Beef extract ....... . . Peptone ............ . Agar . . ............ .

3 grams 5 grams

15 grams

The material is dissolved, filtered, and tubed, similarly to gelatin, and is then sterilized. After sterilization, some of the tubes are allowed to cool in a slanting position because the slanting surface gives a larger area for bacterial growth. Nutrient agar slants are used e>..'tensively for carrying cultures from place to place, and for perpetuating them in the bacteriological laboratory. The growth of bacteria on an agar slant is illustrated in


Fig. 18, where a is a test tube, b the cotton plug, c the agar in the tube, and d the grm\"th of bacteria on the slanting surface of the agar.

80. Sugar Media. - It is common practice to add sugars of different kinds to media to study the fermentative reaction of bacteria. The two sugars in most common use arc lactose, or milk sugar, and dextrose. The sugar is generally added to a broth medium to study fermentation, but is sometimes added to a solid medium to study acid formation in the presence of an indicator.

Lactose broth is extensively used to determine the presence of members of the coli-aerogenes group in water. It is prepared by dissolving in a liter of water the following substances :

Beef e>..1:ract . . . . . . . . . . . . . . . . . . . . . . . . 3 gram~ Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams

The broth is distributed into fermentation tubes, and sterilized. Dextrose broth is prepared similarly to lactose broth, with the

substitution of 5 grams of dextrose for the lactose, the ingredients being as follows:

Beef extract . . . . . . . . . . . . . . . . . . . . . . . . 3 grams Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams De>.."trose . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams

Other sugar broths are prepared in a similar manner.

81. Eosin Methylene Blue Agar.-The medium known as eosin methylene blue, or E M B, agar is extensively used in testing for the presence of bacteria of the coli-aerogenes group and in distinguishing fecal from non-fecal bacteria of that group. The ingredients of this medium are: -·

Peptone . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Dipotassium phosphate . . . . . . . . . . . . 2 graq1s Agar . . . . . . . . . . . . . . . . . . . . . . . . . . I 5 grams Eosin ..... : . . . . . . . . . . . . . . . . . . . . . . .4 gram l\Iethylene blue ..................... 01 gram

3& SANITARY BACTERIOLOGY

T hese substances a re dissolved in a liter of distilled water and ::.tared in tubes or flasks. The tubes and flasks of E l\f B agar should be sterilized. The medium is fairly stable and can be safely stored on ice for a week or more.

82. Endo's Agar.-The following ingredients are used in preparing Endo's agar:

Peptone ......... ... .. . ......... . 10 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 grams Dipotassium phosphate . . . . . . . . . . . . 3.5 grams Sodium sulphite . . . . . . . . . . . . . . . . . . 2.5 grams Basic fuchsin . . . . . . . . . . . . . . . . . . . . .5 gram

T hese substances are dissolved in 1 liter of distilled water, placed in t ubes or flasks, and sterilized. T he sterilized medium is poured into petr i dishes and allowed to solidify. This medium is used extensively in testing for the presence of bacteria of the coli-aerogenes group and in differentiating between B. coli and B. typhosus.

83. Brilliant Green Lactose Bile.-The ingredients of bril-liant green lactose bile are:

Peptone . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . 20 grams Brilliant g reen . . . . . . . . . . . . . . . . . . .0133 gram

They are dissolved in 1 liter of distilled water, placed in fermentation tubes, and sterilized. This medium is used in testing for the coli-aerogenes group. The bile exerts an inhibitory effect upon bacteria that do not normally inhabit the human intestines.

84. Crystal Violet Lactose Broth.-The ingredients that a re dissolved in a liter of distilled water to form crystal violet lactose broth are :

Peptone . . . . . . . . . . . . . . . . . . . . . . . 5 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . 5 grams Dipotassium phosphate . . . . . . . . . . 5 grams Potassium dihydrogen phosphate . . gram Crystal violet ........ . ... ... . . .. 00143 gram


The medium is placed in fermentation tubes and sterilized. I t is often used instead of brilliant green lactose bile in testing for members of the coli-aerogenes group.

85. Fuchsin Lactose Broth.-To prepare fuchsin lactose broth, the following ingredients are dissolved in a liter of dist illed water :

Beef extract . . . . . . . . . . . . . . . . . . . . . . 3 grams Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams Basic fuchsin . . . . . . . . . . . . . . . . . . . . .015 gram

This mixture is poured into fermentation tubes and sterilized. I t is another substitute for brilliant green lactose bile in the test for members of the coli-aerogenes group.

86. Formate Ricinoleate Broth.-Another medium that ~ay be used in testing for members of the coli-aerogenes group ~s formate ricinoleate broth. This medium contains the following mgredients in a liter of distilled water :

Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams 'Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams Sodium formate . . . . . . . . . . . . . . . . . . . . 5 grams Sodium ricinoleate . . . . . . . . . . . . . . . . . . I gram

The mixture is distributed into fermentation tubes and is sterilized by heating it in the autoclave at a pressure of 11 to 13 pounds for 15 minutes. ·

87. Koser Citrate Medium.-In the preparation of the Koser citrate medium the following ingredients are dissolved in one liter of distilled water :

Sodium ammonium phosphate ... . . . Potassium dihydrogen phosphate .. . . l\1a~esium sulphate ... . ......... . Sodium citrate ............... . .. .

1.5 grams 1.0 gram .2 gram

3.0 grams

The mixture is poured into tubes and sterilized. This medium is used to distinguish between fecal and non-fecal bacteria of the colon group. Carbon compounds must be kept out of the medium, as they destroy its value.

282C-7


88. Lactose L itmus Agar.-Lactose litmus agar is prepared by dissolving the following ingredients in l liter uf distilled water and heating gently for a few minutes:

Beef extract . . . . . . . . . . . . . . . . . . . . . . 3 grams Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . 5 grams Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 grams Azolitmin . . . . . . . . . . . . . . . . . . . . . . . . 1 gram

The agar is placed in flasks or tubes and sterilized. All bacteria that ferment lactose are readily distinguished on this medium. The fermentation of lactose is accompanied by the production of acid which colors the litmus in the medium red.

OBSE RV ATION OF BACTE RIA

THE M ICROSCOP E

89. Description of Micr oscope.- T he microscope ts an instrument containing a series of fineJy-ground lenses which reproduce and magnify exact pictures of objects that are too small to be seen by the unaided eye.

The microscope, shown in Fig. 19, is composed of the two adjustable tubes a and the barrel b, within which are installed an eyepiece or ocular consisting of an upper, or eye, lens at c and a lower, or field, lens at d, and an object glass, or object ive, e, made up of three lenses placed very close together. The objective produces an enlarged image of the object under observation and the ocular remagnifies the image to the size observed by the eye.

T he microscope is supported by a U-shaped base f and a fixed upright cylinder g, which is hinged at h so that the microscope may be t ipped at an angle for purposes of observation. Above this hinge is another cylinder i arranged to hold the stage, or platform, j upon which rests the object k to be examined. In bacteriological work, the specimen of culture medium to be observed is smeared on a glass plate or slide. There is a central opening in the stage so that light may enter below the specimen. Under the opening are diaphragms I for controlling the


amount of light admitted to the specimen. In this particular instrument the diaphragm opening is adjusted by means of a small Je,·er 111.

The reflector 11 is an adjustable mirror under the stage for iJiuminating the specimen. It catches the light from a lamp or window and reflects it through the specimen and the. objective e. Connected with the diaphragms is a substage condensing apparatus o consisting of a system of lenses beneath the central opening of the stage. This condensing apparatus serves to condense the light from the reflector and focus it on the specimen.

90. About midway on the barrel of the microscope are two disks p, Fig. 19, which operate a rack-andpinion control for the coarse adjustment of the microscope. By turn- ~~::!§~ ing these disks the objective is brought to its· approximate position above the specimen. The cylinder ; contains a geared control for the fine adjustment of the microscope, this adjustment being made by means of the cone-shaped wheel q. It is very fir;;,"'=~~e."'<act and especially necessary for ..,_"'-""-'-...L..- .._:!l

high-power magnification. FIG. 19

Th~ microscope also has an arrangement r for changing the magnifying power of the objective. This arrangement, which is called the nose piece, is provided so that several objectives of different magnifying powers can be attached to the microscope and brought into use one at a time by rotating the collar on which they are placed.

In the examination of bacteria it is necessary to obtain a clearly defined image at high-power magnification. and the socalled oil-immersion objcclivc is used for this purpose. I t is


constructed so that it can be used only with a transparent medium through which tight passes at the same angle as through glass and which can therefore act as a homogeneous material with the glass of the slide bearing the object to be examined. Over the object is placed a drop of special oil, which has the same angle of refraction as glass, and the objective is lowered until it is immersed in the oil. The rays of light pass through the glass and oil in a straight line to the objective; hence, there is no loss of light by deflection, such as takes place when light passes from glass to air. Thus, more light passes through the

(a) b

(b)

FIG.20

objective and a clearer image of the object is produced at the eyepiece.

91. Han g i n g Dr op. The simplest method of observing bacteria is by the hanging drop. A microscope slide, Fig. 20 (a), with a concave depression a is used for this purpose. As shown in view (b), a drop o£ liquid b,known as a hang

ing drop and containing the bacteria to be examined, is placed on a cover slip c, which is a small, thin piece of clear glass about l to i inch square. Vaseline is then placed around the depression a and the cover slip c is placed on the slide with the droplet b suspended downward in the depression, as shown in crosssection in view (c). The bacteria can then be intensively studied under a microscope while they are alive. This method is used in studying motility, capsule and spore formation, form, and cell arrangement.

As the fluid containing the bacterial culture is transparent, the cover slip can be easily broken by the objective of the microscope before the observer is aware that the objective is touching it. Damage to the preparation and the lens may be prevented by gently moving the slide back and forth while the microscope is being adjusted.


92. Examination of Stained Specimen.-To examine a stained specimen under a microscope, the following procedure should be carried out:

1. The stained specimen is prepared on a slide and covered with a cover slip.

2. A smaU drop of oil is placed on the cover slip. 3. The slide is placed on the center of the microscope stage. 4. The oil-immersion objective is lowered with the coarse

adjustment until the objective barely touches the drop of oil.

5. The diaphragm opening and the inclination of the reflecting mirror are adjusted so that the light passes through the objective and the slide.

6. The eye is placed over the ocular, and the fine adjustment is moved gently to the right with the thumb and forefinger of the right hand, until the field appears somewhat colored.

7. With the thumb and forefinger of the right hand, the fine adjustment is carefully manipulated while the slide is constantly moved gently with the thumb and forefinger of the left hand. The fine adjustment may be moved slowly backward and forward while the slide is examined, until the proper section of the slide is found. The slide should then be fastened with clips provided on the stage for that purpose and left stationary during the study of the specimen.

8. After the examination is complete, the objective should be raised, cleaned thoroughly with a fine silk cloth or lens paper, and put away in a box to preserYe it from injury.

STAINING J.r.ETHODS

93. · Common Stains.-In order to bring out details of bacterial structure and to render the study of bacteria more accurate, stains are used. Although many stains haYe been developed, a few standard ones will answer for all ordinary pur~oses. Three of the most useful stains are dilute carbol-fuchsm, or Ziehl-Neelson, stain; alkaline methylene blue, or Loeffler's, stain ; and the Gram stain.

Carbol-fuchsin, or Ziehl-Neelson, stain consists of the fol· lowing substances :


Saturated alcoholic solution of basic fuchsin . . 10 milliliters 5-per cent water solution of carbolic acid .... 100 Imlliliters

Alkaline methylene blue, or LoefHer's, stain is composed of the following substances:

Saturated alcoholic solution of methylene blue 30 milliliters Potassium hydroxide, 1 to 10,000 solution .... 100 milliliters

A 1 to 10,000 solution of potassium hydroxide is made by adding 2 drops of a 10-per cent potassium hydroxide solution to 100 milliliters of distilled water.

Gram's stain consists of two solutions. Solution 1, or aniline gentian violet solution, is made up of the following:

Aniline oil water . . . . . . . . . . . . . . . . . . . . . . . . 75 milliliters Saturated alcoholic solution of gentian violet 25 milliliters

The aniline oil water is prepared by dissolving 2 milliliters of aniline oil in 100 milliliters of distilled water, agitating the solution, and then filtering it.

Solution 2, or Gram's iodine solution, is composed of the following ingredients:

Potassium iodide . . . . . . . . . . . . . . . 2 grams Iodine . . . . . . . . . . . . . . . . . . . . . . . . 1 gram Distilled water . . . . . . . . . . . . . . . . 300 milliliters

94. Staining With Carbol Fuchsin or Alkaline Methylene Blue.-In order to obtain satisfactory results with carbolfuchsin or alkaline-methylene-blue stains, the following instructions should be carefully carried out:

A small amount of the bacterial culture is transferred to a cover slip and then dried in the air or heated gently in the warm air above a Bunsen flame or an alcohol burner. After the moisture has evaporated, the slip is slowly passed through the flame of a Bunsen burner or an alcohol lamp, thus causing the material to adhere firmly to the glass or become fixed. Next, a few drops of the stain are applied and the slip is allowed to remain for a length of time varying with the stain used and the culture of bacteria. The e.xcess stain is then washed from the cover slip with distilled water, and the cover slip is mounted in place on a microscope slide, Canada balsam being used to fasten it.


The sample is ~xamined first with the low power of tht microscope, then with the high power, and finally with the oil immersion.

95. Use of Gram's Stain.-The use of Gram's stain is one of the most important staining methods and considerable practice is required before it can be done satisfactorily. The procedure is as follows: A small amount of bacterial culture is fixed on a cover slip as in the preceding article and stained with the aniline gentian violet solution for It minutes. The stained specimen is washed in distilled water and the iodine solution is applied until the bacterial material turns purplish black, which requires about lf minutes. Some bacteria are colored by the stain while others are not. To distinguish the stained bacteria, 95 per cent alcohol is added and a·llowed to stand for 2 minutes. This decolorizes the medium but does not remove the color from stained bacteria. The cover slip is washed with distilled water, dried, and mounted on the microscope slide for examination. A sharp contrast can be made by counter-staining the medium with a second stain, such as Bismarck brown.

96. Differentiation by Gram's Stain.-Bacteria which are stained a deep violet by the Gram stain are said to be Gram positive. Those which do not take the stain are called Gram 11egaiivc. Some of the common Gram-positive bacteria are B. anthracis, B. tuberculosis, B. subtilis, B. tetanus, B. aerogenes capsulatus, streptococcus pyogenes, staphylococcus pyogenes, pneumococcus, micrococcus tetragenous. Some of the Gram-negative organisms are: B. typhosus, B. coli, B. dysenteriae, B. proteus, spirillum cholerae, meningococcus, gonococcus.

EXAM IN AT ION OF WATER FOR BACTERIA

PRELIMINARY EXPLANATIONS

97. Procedure in Examination.-The e.'<amination of water for bacteria consists o£ two main parts: ( l) the determination of the total number of bacteria per milliliter of the water; and (2) the determination of the presence or absence in the water of bacteria of the coli-aerogenes group. The organisms that


are now designated as the coli-aerogenes group were formerly referred to as the B. coli group, because of the predominance of B. coli in this group. However, the term B. coli applies strictly to the bacteria known as Escherichia coli, which are normally found in the feces of human beings and other war~blooded animals, and it does not include A. aerogenes, whtch are normally found in surface soil and on vegetables. The members of the coli-aerogenes group are not themselves harmful, but they indicate the possible presence of dangerous types of intestinal bacteria.

The procedure in a bacteriological ex~minatiot! of ~vater <:<>:1-sists of the following operations: samplmg, platmg, mcubatmg, counting, and reporting the results. The determinatio~ of the number of bacteria is only of relative value, for there ts no method of obtaining the absolute number. However, t~e relative number of bacteria is all that is necessary from a santtary standpoint.

98. Collecting and Storing Sam.ples.- A water sample must be collected in a clean, sterile, glass-stoppered bottle, usually of 2-ounce or 8-ounce capacity. Care must be exercised to obtain a sample that is representative and to protect it from contamination after collection. Samples should not be taken from a faucet until all the water in the service pipe has been wasted, nor from a pump or hydrant until the connections have been emptied. If a lake or stream is being sampled, the bottle should be plunged beneath the surface and some precaution taken to prevent foreign material such as leaves and surface dust from getting into the bottle.

Immediately after the bottle is filled, the stopper should be replaced, covered with a doth, paper hood, or tin-foil, and tied in place. Samples should be e--xamined as soon as possible after collection, for bacteria increase rapidly in samples, especially if they are allowed to become warm. \i\fhen it is impossible to examine a sample at once, it should be stored at a temperature between 6° and 10° C. The period allowed between the time of filling a sample bottle with water and the time of making the bacteriological examination should preferably be not more


than 6 hours for impure waters nor more than 12 hours for relatively pure waters.

99. Making Dilutions.-For bacteriological e-xamination, the sample is usually diluted by mixing 1 milliliter with either 9 or 99 milliliters of sterilized tap water. The tap water contained in a dilution bottle is sterilized in tl1e autoclave at a pressure of 15 pounds for 15 minutes. Then the sample of water to be examined is shaken thoroughly, and 1 milliliter is taken out with a sterile pipette and placed in the dilution bottle containing 9 or 99 milliliters of sterilized water, as required. The dilution water and the original sample are well mixed by shaking. For highly polluted water and sewage, further dilutions may be made in the same way. Dilutions should be such as will produce a count of from 30 to 300 colonies of bacteria per milliliter, and the determination of the proper dilution necessary is based on experience.

100. P lating.-\Vhen the culture medium is liquefied nutrient agar or gelatin, 1 milliliter of the diluted sample is withdrawn with a sterile pipette and placed in a sterile petri dish. Then 10 milliliters of the medium is added to the sample in the petri dish and the contents are mixed thoroughly by tilting the dish from side to side. After mixing, the dish is cooled, the agar is allowed to solidify, and the dish is placed in the incubator. The petri dish is often called a plate and this process is known as plating. Plates of Endo's and E M B agar are first prepared by pouring the liquid medium into the sterile petri dish and allowing it to solidify, and then inoculating it by streaking the surface with some of the sample by means of a platinum loop.

Plates should be prepared in duplicate. The medium is diluted too much for good results if more than one milliliter of diluted sample is used, and the resulting mixture will not solidify on cooling.

101. Incubating.-Agar plates are usually incubated for 24 hours at 37° C. The atmosphere of the incubator must be dark, moist, and well-ventilated, and the ten1perature must be


constant. If the temperature is zoo C., incubation should proceed for 48 hours.

All gelatin plates, after inoculation, must be incubated at 20° C. This low temperature makes a longer incubation period necessary, and gelatin plates are usually incubated for 48 hours. Test tubes and plates are held for 5 days to determine the liquefying action of the bacteria.

BACTERIAL COUNT

102. Counting Bacteria.-After the bacteria have been inoculated into a solid medium and allowed to incubate, they multiply very rapidly, so that, after incubation for 24 hours, the offspring from each separate bacterium originally present in the sample will form a colony of many thousands of bacteria which can be readily seen by the naked eye. It is these colonies that are counted and not the individual bacteria originally present in the sample.

For convenience in counting the bacteria, the plate is usually divided into sections, such as quarters or eighths of the whole area. The colonies appearing in a few sections are counted, an average obtained, and the result multiplied by the number of such sections in the plate. The bacterial count, or the number

TABL E V

NUMBERS USED I N REPORTI NG B ACTERIAL COUNTS

(This tabulation r~fcrs to colonies either on agar at 37" C. or on gelati" at zo• C.}

Number of Colonies Counted per Milliliter

1 to SO 51 to 100

101 to 250 251 to 500 SOl to 1,000

1,001 to 10,000 10,001 to 50,000 50,001 to 100,000

100,001 to 500,000 500,001 to 1,000,000

1,000,001 to 10,000,000

Xumber of Colonies to be Reported

As found Nearest 5 Nearest 10 Nearest 25 Nearest 50 Nearest 100 Nearest 500 Nearest 1,000 Nearest 10,000 Nearest 50,000 Nearest 100,000


of bacteria per milliliter of the original sample, is found by multiplying the number of colonies counted for the whole plate l>y 10 or 100, depending on whether 9 or 99 milliliters of tap water was used in making the original dilution.

103. Reporting Results.-Errors in sampling, plating, and counting make any attempt at close tabulation of bacterial counts false and misleading. Hence, to avoid false accuracy, results are reported as indicated in Table V.

E XAMI NATI ON FOR MEMBERS OF COLI· AEROGENBS GROUP

104. Tests f or Presence of Coli-Aerogenes Group.-All bacilli that are Gram-negative, cause fermentation of lactose with the formation of gas, grow on ordinary media in the presence of free oxygen, and do not form spores are classed as members of the coli-aerogenes group. There are means of distinguishing between the fecal B. coli and the non-fecal aerogenes, but tlus is not usually done in ordinary water examination, because the presence of either type in water is sufficient caus6 for looking upon the supply with suspicion. The entire test is composed of three parts: (a) the presumptive test, (b) the confirmed test, and (c) the completed test.

105. Presumptive Test.-In the presumptive test, a number of fermentation tubes containing lactose broth are sterilized and then inoculated with a definite quantity of the water being examined. The tubes are incubated for 24 hours at a temperature of 37° C. and are then inspected for the formation of gas. Usually one to five tubes are used, a (]ifferent quantity of water being introduced in each ; common amounts are 10 milliliters, 1 milliliter, and .1 milliliter. The volume of lactose broth should be at least twice that of the water sample introduced into the tube.

In Fig. 21 the fermentation tube a is partly filled with liquid lactose broth b, and plugged with cotton c. The apparatus is then sterilized in the autoclave. During sterilization, the medium is forced up into the closed or inverted arm of the tube and that arm is completely filled by it. \\' hen the tube is


"inoculated, the bacteria from the sample mix with the medium enter the closed arm, and multiply. '

If gas is formed by the bacteria, it rises, pushing down the medium, and is trapped in the closed arm at d. This gas is ~eadily distinguished and is measured by Frost's scale e, which JS placed behind the tube as shown in Fig. 21. Here the volume of gas is about 18 per cent of the volume of the closed arm.

FIG. 21

If the gas formed after incubating for 24 hours occupies 10 per cent or more of the closed arm of the fermentation tube tl t • "d I 1e est IS ~onst ered a positive presumptive test for the presence of the coh-aerogenes group. HO\vever, absolute proof of the presence of organisms of this group depends on the results of the completed test. If the amount of aas formed is less than 10 pe t "f b r cen • or 1 no gas is formed, the results of the test are doubtful, and the tubes should be placed in the incubator for


an additional 2~ hours. In case there is some gas, but less than 10 per cent, at the end of the additional incubation, the test is reported as a doubtful presumptive test and the confirmed test must be made. The absence of gas at the end of 48 hours' incubation constitutes a 11egative presumptive test, which is always considered sufficient to indicate the absence of the coliaerogenes group.

The presumptive test is sufficient only when it is definitely negative. When the test is positive or doubtful, the presence of the coli-aerogenes group must be further confirmed. However, in field work, or in cases of emergency or disaster, it is unnecessary to complete the test for the presence of the coliaerogenes group, and the water may be reported unsafe upon the formation of any gas within 24 hours in lactose-broth fermentation tubes.

106. Confirmed Test.-The confirmed test for members of the coli-aerogenes group may consist of either of two procedures. In one method, a few drops, or more if desired, of the liquid from a fermentation tube that has shown gas in the presumptive test is transferred to a second fermentation tube containing any one of the following liquid confirmatory media : brilli·ant green lactose bile, crystal violet lactose broth, fuchsin lactose broth, or formate ricinoleate broth. For the purpose of this test, all four media are equivalent. For a particular water, it is desirable to choose the medium that seems to give the best correlation between the results of the confirmed test and the .results of a series of completed tests. The contents of this second tube are incubated for 48 hours at 37° C. If no gas is formed, the confirmed test is negative, and it may be concluded that no members of the coli-aerogenes group are present and the water is safe. However, the formation of gas in this tube at any time withjn the 48 hours indicates the possible presence of the coli-aerogenes group. For absolute proof of their presence, it is necessary to continue with the completed test.

In the other method for making the confirmed test, some of the liquid from a fermentation tube that has shown gas in

52 SANITARY BACfERIOLOGY

the presumptive test is removed by means of a platinum wire and is streaked on one or more plates containing E i\I B agar or Endo's agar. These plates are allowed to incubate for 18 to 24 hours at 37° C. If the plates show typical coli-aerogenes colonies, which are generally dark red or purple at the center and have a pink halo, the confirmed test is considered positive. Such a result indicates the probable presence of members of the coliaerogenes group, and the completed test should be made. If no typical colonies are formed on the plate, the test cannot be considered negative until the completed test is made. Thus, where agar plates are used for the confirmed test, it is always desirable to make the completed test.

107. Completed Test.-The completed test is used to show whether or not the gas-forming organisms are Gram-negative non-spore-forming bacilli. This is done by streaking one or more plates of Endo's orE M B agar with liquid from the tubes that contained gas in either the presumptive or the confirmed test. The plates are incubated for from 18 to 24 hours at 37° C. This results in a condition similar to that obtained in the confirmed test when the solid medium is used. Colonies of bacteria that are typical of the coli-aerogenes group are removed ; or, if there are no such typical colonies, those considered most likely to be coli-aerogenes are removed. In either case, both lactose-broth fermentation tubes and tubes containing agar are inoculated with bacteria from suitable colonies. The lactose broth is incubated at 37° C. until the formation of gas is noted or until 48 hours elapse. The agar is incubated for 24 hours at 37° C. If no gas is formed in the fermentation tubes, the test is negative. In case gas is formed, it is necessary to examine the agar cultures for spores and to apply the Gram stain to at least one such culture. If all the bacilli are Gram positive or if some are Gram negative but are spore formers, the test is negative even though gas was formed.

108. Number of Coli-Aerogenes Group.-The relative number of members of the coli-aerogenes group present in water is the reciprocal of the volume in milliliters of the smallest portion of water tested showing positive results. If a sample of

SANITARY BACfERIOLOGY 53

water was planted in lactose broth with the result that the coliaerogenes group was present in 10-, 1,- and .!-milliliter portions but absent in the portions of .01 and .001 milliliters, the number of members of the coli-aerogenes group in the sample would be reciprocal of .1, or 10, per milliliter of sample. This number is sometimes called the B . coli index.

RECOMMENDED STANDARDS

109. The American Public Health Association furnishes a complete scheme for the e.."\.cunination of water in its standard methods of water analysis.

The United States Treasury Department standard, as recommended for water used in interstate traffic, is as follows:

(a) Total bacterial count shall not exceed 100 bacteria per milliliter of water. Plates shall be prepared in duplicate, and incubated for 24 hours at 37° C.

(b) Not more than one out of five 10-milliliter portions shall show the presence of the coli-aerogenes group as demonstrated by standard methods for water analysis.

(c) It is recommended that one 1-milliliter and one .1-milliliter portion be inoculated into lactose broth for the purpose of indicating the extent of pollution.

The Bureau of Chemistry condemns oysters that show the presence of B. coli in three out of five .!-milliliter portions.

FURTHER EXAMINATI ONS

110. Isolating Bacteria.-To isolate bacteria, a tube of nutrient agar is melted by heating in boiling water until the contents are thoroughly liquefied. A loopful of the material to be examined is placed in the agar, care being taken that the contents of the tube do not become contaminated in the process and that the agar is cooled sufficiently to prevent killing the bacteria in the culture. The sample and the agar are mixed thoroughly and one loopful of the mixture is transferred to a second tube of liquid agar; after the contents of the tube are mi..,.ed. a loopful is transferred to a third tube of agar. Then the contents of the second and third tubes are poured into sterile petri dishes. The diluting and mixing separates the bacteria in the


culture so that individual bacteria will be separated when poured into a petri dish. \~'hen the bacteria are allowed to incubate, each bact!'!rium will form an isolated colony, which can be picked off with a platinum loop and examined.

111. Distinguishing Fecal from Non-Fecal Bacteria of the Coli-Aerogenes Group.-The members of the coli-aerogenes group are not differentiated in sanitary e-xaminations of water. Use must be made of their biochemical characteristics to identify them, which requires special media and considerable e-xperience. On E M B agar, B. coli show a dark center and have a metallic luster. Typhoid or para-typhoid bacilli produce transparent colorless colonies. Non-fecal bacteria of the coli-aerogenes group are much larger than B. coli and have a tendency to run together ; their centers are usually purple and not so dark as those of B. coli, and the metallic luster is only occasionally noticed.

Colonies resembling B. coli on Endo's agar or on E l.I B agar are carefully picked off with a sterile platinum loop. inoculated into sterile tubes of Koser citrate medium, and incubated for 24 to 48 hours at 37° C. The tubes should be kept for a few days. Fecal types of the coli-aerogenes group fail to develop in this medium, while the aerogenes develop readily. Turbidity in the tube indicates bacterial growth.

112. Distinguishing Between B. Coli and B. Typhosus. To distinguish between B. coli and B. typhosus, the material to be examined is streaked across the surface of solid Endo's agar in a petri d ish. Colonies of B. typhosus appear as clear, colorless, glistening droplets against the faintly pink medium. On the other hand, B. coli form colonies that are intensely red and color the medium red in the vicinity of each colony.

SANITARY BACTERIOLOGY Serial 3048 Edition 2

E XAMINATION QUESTIONS

Notice to Students.-Study tlze InstruarotJ Paper tllorottgllly before yo1e atlempt to a11-S111er tlu;se questw11;s. Read each question carefully and be sure you understand tt; t/um wnte the best a11-S111er yo11 can. Wilen yo11r a11swers are completed, exami1l~ t11~11J closely, correa all the errors you caiJ find, atzd see that every questton 1s answered; llretJ mail your work to tiS.

(1) (a) What are the three main types of bacteria and what is the general shape of each type? (b) What is a bacteriai spore and under what conditions is it formed?

(2) What are (a) enzymes, (b) toxins, and (c) ptomaines?

(3) (a) Do bacteria generally thrive in air? Give reasons. (b) How do pathogenic bacteria generally get into the air?

(4) (a) \Vhat factors are conducive to self-purification in bodies of surface water? (b) \i\ihy is an artesian well Rowing through limestone liable to prove a dangerous source of water supply?

.(5) State brieRy how bacteria play an important part in the nitrogen cycle.

(6) (a) \i\fhat is the difference between sterilization and disinfection? (b) Why is sterilization very important in bacteriological laboratory work?

. (7) _(a) 0 f what use are culture media? (b) What essenbat reqmrements must be fulfilled by a culture medium ?

(8) (a) "What is the main advantage of an oil-immersion objective? (b) How is such an objective used in the examination of bacteria ?


(9) (a) State briefly how the presumptive test is carried out in examining water for the presence of members of the coliaerogenes group. (b) Under what special conditions may doubtful results of that test be considered sufficient?

( 10) (a) If a sample of water is introduced in lactose broth and the presence of members of the coli-aerogenes group is established in the 10-, 1-, .1-, and .01-milliliter portions, but not in the .001-milliliter portion, what is the B. coli index of the sample? (b) Describe the method employed for determining whether B. typhosus is present in feeal bacteria.

( 11) Explain the bacterial difference between freshly settled sludge and digested sludge.

( 12) (a) Mention the operations that are necessary in bacteriological examination of water to determine the total number of bacteria. (b) What procedure is usually followed in counting bacteria? (c) If the average bacterial count per milliliter is found to be 75,400, what number should be reported?

Mail your work on this lesson as soon as you have finished it and looked it over carefully. DO NOT HOLD IT until another Jesson is ready.

Documents

Sanitary I Bacteriology...SANITARY BACTERIOLOGY In 1882, Koch devised a new technique for the separation ancl identification of bacteria, and this date marks the begin ning of bacteriology