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Nutrient Media Bacterial Culture

Information on the Growth of Bacterial Cultures using Nutrient Media Broths and Culture Plates

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What is Nutrient Media- Definition?

"Chemically, like all other living cells, microorganisms consist of organic and inorganic nitrogen and mineral salts; it is therefore necessary in order to grow a microorganism, that these three classes of substances be made available, together with oxygen, which is an essential to the life of all living structures. Finally a certain amount of moisture is absolutely necessary." (Besson.)

A food prepared for the growth of microorganisms is given the general term nutrient medium. A large number of microorganisms will grow readily in or upon easily available nutrient media, as milk, bouillon, etc. Some microorgan isms have widely differing food requirements and need for growth nutrient media differing widely in their composition.

However, there are a few general rules that must be applied in the preparation of all nutrient media for the use of microorganisms. These are briefly, that:

Every culture medium must

1. Contain substances necessary for growth.
2. Be of suitable reaction.
3. Be contained in vessels which afford protection from contamination from outside sources.

Classification of Nutrient Media

Culture media may be classified as:

I. Natural Media as occurring in nature, e.g., milk, potato and other vegetables, meat and meat products, blood and blood serum, egg, soil, etc.

II. Prepared media, i.e., made in the laboratory. These are:

(a) Of unknown chemical composition; e.g., nutrient agar, gelatin, etc.

(6) Synthetic; i.e., chemical composition known, e.g., Giltay solution for denitrifying organisms.

Or as:

I. Liquid Media. These include:

A. Media made from animal tissue and fluids, e.g., nutrient broth, serum broth, carbohydrate broths, milk, blood, nitrate peptone solution, Dunham's solution.

B. Media made from vegetable tissue. Among these are: Malt extract (germinated barley), beer wort, yeast extract, hay infusion, natural fruit juices, wines (fermented fruit juices).

C. Synthetic media.

II. Solid Media. These mav be classified as:

A. Liquefiable, e.g., nutrient agar, nutrient gelatin.

B. Non-liquefiable, including: 1. Media liquid in a natural state but which, once solidified, cannot be liquefied by physical means, e.g., media prepared from albuminous fluids and tissues such as egg, blood serum, etc., or synthetic media solidified with sodium silicate.

2. Media which are solid in the natural state, e.g., vegetable media such as potato, carrot, banana, etc.

 

 

EXERCISE 3. TITRATION OF MEDIA

The titration of bacteriological media made from meat is an important step in their preparation, as microorganisms are sensitive to the reaction of the nutrient substrate.

Procedure. The following method is used for laboratory media, with the exception of milk, wort, cider, vinegar, fruit juices, etc. See p. 22.

1. Put 5 c.c. of the medium to be tested and 45 c.c. of distilled water in an evaporating dish.

2. Boil briskly one minute with constant stirring (to drive off all dissolved CO2 which registers as acidity).

$. Add 1 c.c. phenolphthalein solution for indicator.

4. Titrate while hot, preferably while boiling, with N/20 sodium hydroxide, or N/20 hydrochloric acid as the case de mands. ' A faint but distinct permanent rose color marks the end point. This color should remain permanent for five minutes.

5. Compute and record the reaction of the medium in degrees of Fuller's scale, which is the number of cubic centi meters of normal* acid or alkali present in 1000 cubic

* A solution is said to be normal when it contains 1 gram equiv alent of an acid or base in 1 liter.

A gram equivalent of an acid or a base is that quantity which is equivalent to or will neutralize 1 gram molecule of a mono-basic acid or of a mon-acid base.

The advantage of the system is that 1 c.c. of any normal solution will exactly neutralize or be exactly equivalent to 1 milligram equiva lent of any acid or base. (Noyes, Wm. A., Textbook of Chemistry, 1913, p. 184.)

TITRATION OF MEDIA 21

centimeters of the medium, using phenolphthalein as indi cator.

6. Alkaline media are denoted by placing a minus ( ) sign before the number of degrees of alkalinity; thus, 15 would indicate that the medium was 15 alkaline, or that 15 c.c. normal acid must be added per liter to neutral ize it.

Acid media are denoted by placing a plus. (+) sign before the number of degrees of acidity; thus, +15 would indicate that the medium was 15 acid or that 15 c.c. of normal alkali must be added per liter to neu tralize it.

Example.

Burette reading after titrating 5.4 c.c.

Burette reading before titrating 2.0 c.c.

Number of c.c. N/20 NaOH required

to neutralize the acid in 5 c.c. of

the medium .4 c.c.

If 5 c.c. of the medium (which is 1/20 of 100 c.c.) require

3. 4 c.c. of 1/20 normal NaOH to neutralize the acid pres ent, 100 c.c. of the medium would require 20X3.4 c.c. or 68 c.c. of 1/20 normal NaOH.

As a normal solution is 20 times the strength of a 1/20 normal solution, 100 c.c. of the medium would require 1/20 of 68 c.c. or 3.4 c.c. of normal NaOH for neutralization; and one liter or 1000 c.c. of medium would require 10X3.4

c. c. or 34 c.c. N/l NaOH for neutralization; i.e., the medium is 34 acid. Fuller's scale. This is the litre of the medium.

When N/20 acid or alkali and a 5 c.c. portion of medium (in 45 c.c. of distilled water) are used, each 1/10 of 1 c.c. corresponds to 1 Fuller's scale.

Adjustment of Reaction. If it is desired to leave the medium with a, e.g., +15 reaction, we have:

22 GENERAL MICROBIOLOGY

Acidity of the medium

(+34) 3.4 c.c. per 100 c.c. of the medium

Desired acidity (+15) . . 1.5 c.c. per 100 c.c. of the medium Amount of normal alkali

to be added 1.9 c.c. per 100 c.c. of the medium

or 10X1.9 c.c. = 19 c.c. N/l NaOH per 1000 c.c. of medium

Since normal solutions are of equal strength by volume, that is, 1 c.c. of N/l acid will just neutralize 1 c.c. of N/l alkali, it will readily be seen that if 15 c.c. N/l NaOH are required to neutralize the acid present in 1 liter of medium, then there must be present in that liter exactly 15 c.c. of N/l acid, or we should say the reaction is ( + 15) fifteen degrees acid. For any other degree of acidity add enough normal alkali to reduce the acidity to the point desired.

The reaction of a medium changes somewhat after its neutralization, especially during sterilization, but also upon standing afterward at ordinary temperature. This change is toward an increased acidity, and is most marked in media rich in dextrose. Consequently it is necessary to determine the titre of a medium at the time it is used rather than to quote figures obtained before sterilization.

MILK, CIDER, VINEGAR, WORT, AND FRUIT JUICES

Procedure. 1. Into an evaporating dish measure 5 c.c. of the medium to be tested, by means of suitable pipette. Make up to 50 c.c. with distilled water.

Do not heat. The above media should not be heated before titration, as they contain volatile acids or other organic substances which may register as acid and which may be driven off by boiling,

2. Add 1 c.c. phenolphthalein solution.

3. Add, gradually, from an accurate burette, IN/20 NaOH until the first permanent pink appears.

4. Note the amount of NaOH required for the titration.

5. Always run duplicates.

MILK 23

6. Record as degrees of acidity the number of c.c. of N/l NaOH which would be required to neutralize one liter of medium.

MILK

Milk is valuable as a nutrient medium for microorganisms because: It is a natural nutriment and almost ideal for a large number of microorganisms. Its composition, averag ing 3.40% fat, 3.50% casein and albumen, 4.50% milk sugar, 0.75% ash, 87.75% water, is an evidence that it furnishes food in an excellent form for most microorganisms.

The biochemical activities of many bacteria reveal them selves definitely in the changes which milk, especially litmus milk, undergoes. Many of these changes are seen macro scopically. Some of these are:

(a) Acid Production. The lactose, C^IfeOn (milk sugar), is first inverted, forming two hexose molecules, 1 mol. Dextrose and 1 mol. Galactose.

And each molecule of hexose yields two molecules of lactic acid:

hexose = lactic acid.

C 6 H 12 6 = 2CH 3 CH(OH)COOH.

The blue litmus is turned red.

(b) AlKali Production. Litmus becomes darker blue. This change very often accompanies peptonization.

(c) Reduction (Decolorization of Litmus). This is due to the reduction of the coloring matter (litmus). Many microorganisms secrete enzymes which produce hydrogen. The hydrogen combines with the litmus, reducing it to its leuco-compound (colorless) . (Methylen blue becomes color less under like conditions.) That this is a reduction and not a destruction may be demonstrated by shaking the decolorized culture with a few cubic centimeters of hydro gen peroxid. The bacteria which decolorize the litmus also

24 GENERAL MICROBIOLOGY

reduce the hydrogen peroxid to E^O and nascent oxygen which reoxidizes the reduced litmus (showing by the reac tion of the milk the type of microorganisms present). Re oxidation takes place slowly under natural conditions. Reduction may take place when milk is acid, alkaline or neutral.

(d) Curdling through Acid Production. The casein, like most proteins, is amphoteric, i.e., it is capable of reacting both as a weak acid and a weak base. The otherwise insoluble casein is found to be in the milk in a partially dissolved state (colloidal), due to its combination with the calcium salts: the calcium that was formerly combined with the casein, through the production of acid by certain micro organisms, now combines with the lactic acid; as a result the casein precipitates, causing curdling (coagulation). Lit mus is turned decidedly red. Milk having an acid curd will titrate above +50.

(e) Rennet Curd. Coagulation may also take place when the medium is neutral or only slightly^ acid. This pro duction of curd is due to a rennet-like enzyme produced by microorganisms, and is similar to the action of the rennet used to curdle milk in cheese factories.

Many spore-forming species are found under the group of rennet-producing organisms. Rennet curd is usually followed by peptonization.

(/) Peptonization. The curd produced by acid or ren net-forming microorganisms may gradually disappear, leav ing only a whey-like liquid. This is caused by certain bacteria which produce proteolytic enzymes that digest the curd and render it soluble. This liquefaction of solid pro teins like gelatin, fibrin, boiled egg white, milk curd, etc., is due to two groups of enzymes, pepsin and trypsin.

The pepsin of the animal body acts only in an acid medium (present in the stomach).

The trypsin of the animal body acts only in alkaline medium (present in the intestine).

PREPARATION OF LITMUS MILK 25

The pepsinand trypsin-like enzymes produced by micro organisms cannot be thus separated by their activity in a medium of certain reaction; this varies with the species of microorganism and with environmental conditions. Pep tonization of milk usually takes place in a neutral, slightly alkaline, or more infrequently slightly acid reaction.

Some organisms peptonize milk without forming a rennet curd.

(g) Gas Production. This is characterized by the for mation of gas bubbles in the milk, and is generally accom panied by the formation of acid curd. Very commonly the curd shrinks, causing extrusion of whey.

EXERCISE 4. PREPARATION OF LITMUS MILK

Apparatus. Fresh separated or skimmed milk; titra tion apparatus; N/20 NaOH; phenolphthalein (indicator); 5 c.c. pipette; azolitmin, 2% solution; filling funnel; pinch cock; sterile test tubes; apparatus for steam sterili zation.

Method. 1. Fresh separated or skimmed milk should be used. Whole milk is undesirable on account of its fat content.

2. Titrate and record the reaction of the milk. If the milk titrates above 17 acid, the reaction must be adjusted to +15. Sour, curdled or uncurdled milk, after neutrali zation, does not make a desirable nutrient medium for microorganisms, therefore, milk whose titre is above 20-25 acid should be discarded.

Fresh milk varies in acidity from 12 to 18. Milk with an acidity above 18 to phenolphthalein will not give a satisfactory blue color with azolitmin, as at 18 it begins to show the acid coloration.

3. Add 2% of a standard solution of Kahlbaum's azo litmin. Litmus or azolitmin is added merely as an indi cator and should be of sufficient strength so as not to dilute the milk to any extent.

26 GENERAL MICROBIOLOGY

4. Mix the milk and the azolitmin thoroughly and tube, using approximately 8 c.c. of the litmus milk in each tube.

Note. Care should be taken to prevent the milk from coming in contact with the top of the tubes, as it will cause the cotton fibers to adhere to the tube. This may be avoided by the use of a " filling funnel."

5, Sterilize by heating in flowing steam for twenty minutes on four successive days. Milk is difficult to sterilize, owing to the resistant spores which are frequently present. If it is desired to sterilize a larger bulk than in tubes, the time of heating should be lengthened.

Caution: Overheating tends to change (caramelize) the milk sugar. The color of the azolitmin may also be destroyed. These changes are not desirable.

EXERCISE 5. PREPARATION OF GLYCERIN POTATO

A number of chromogenic and pathogenic organisms thrive especially well on media containing glycerin. The manner in which glycerin favors the growth of these organ isms is not known, but in some instances it seems to be directly utilized for the construction of fat (Bact. Tubercu losis) .

Apparatus. Large healthy potatoes; cylindrical potato knife, or cork borer; ordinary knife; tumbler; sodium car bonate, 1 : 1000 solution; glycerin, 5% solution; large sterile test tubes, or Roux potato tubes; absorbent cotton or short glass rod; 1 c.c. pipette; distilled water; apparatus for steam sterilization.

Method. 1. Carefully clean one or two large potatoes.

2. By means of a cylindrical potato knife or cork borer, cut cylinders of potato, 4 to 6 cm. Long and 1.5 to 1.8 cm. In diameter. With an ordinary knife, halve each cylinder by a diagonal cut so that each piece resembles in shape an agar slant. Remove any portions of the skin on these pieces.

3. Place in a tumbler and soak in a dilute (1 : 1000) solution of sodium carbonate* for twenty-four hours only.

* Sodium carbonate is used to neutralize the natural acids of the potato.

PREPARATION OF GLYCERIN POTATO

27

4. Transfer the pieces to a 5% solution of glycerin in water for a further twenty-four hours only.

5. Place in sterile tubes prepared as follows: Select extra large test tubes 1.5 to 2 cm. In diameter and clean and dry them. Place a small piece of absorbent cotton or glass rod 0.5 cm. X2.5 cm. In the bottom of each. Plug with cotton and sterilize in the usual way. (Roux tubes need only to be cleaned and sterilized.)

Just before introducing the pieces of potato, add about 1 c.c. of distilled water to each tube, using a pi pette. The potato should not touch the water.

6. Sterilize by heating at 100 G. on four successive

days for twenty minutes each day.

FIG. 8. Potato Tubes. (Orig. Northrup.)

Caution: The time stated in 3 and 4 must be strictly adhered to, else the potatoes will have to be discarded on account of contamina tion with resistant spore-forming organisms.

REFERENCE

SMIRNOW, M. R.: The value of glycerinated potato as a culture medium. Cent. F. Bakt., II Abt., Bd. 41, p. 303.

28 GENERAL MICROBIOLOGY

EXERCISE 6. PREPARATION OF MEAT INFUSION

Meat infusion is the foundation of the ordinary nutrient media, as broth, gelatin and agar, and also of a large number of special nutrient media, as sugar broths, etc.

Under these directions sufficient meat infusion is pre pared to make 1 liter each of nutrient broth, gelatin and agar.

Apparatus. 1.5 kilograms (3 Ibs.) Finely chopped fresh lean beef; 1500 c.c. tap water; 3.5 liter agateware pail; large funnel; ring stand; clean cloth; 1 liter measuring cup; three sterile 1 liter Erlenmeyer flasks; refrigerator; apparatus for steam sterilization (autoclav preferable).

Method. 1. To 1.5 kilograms of finely chopped, fresh lean beef in a 3.5 liter agateware pail, add 1500 c.c. of tap water,* mix thoroughly and allow to stand in a cool place (refrigerator preferred) for sixteen to twenty-four hours only.

2. Set up a large funnel in a ring stand and place a piece of clean cloth in the funnel. Place a measuring cup under the funnel.

3. Strain the meat infusion through clean cheesecloth, thoroughly pressing out all the juice. 1.5 liters should be recovered. If any loss occurs make up to 1500 c.c., using tap water.

This resulting sanguineous fluid contains the soluble albumins of the meat, the soluble salts, extractives and coloring matter, chiefly hemoglobin.

4. Place 500 c.c. of meat infusion in each of three sterile 1 liter Erlenmeyer flasks. Replace the plugs, and heat in the autoclav at 120 C. for thirty minutes. This is a safer procedure than heating for a longer time in flowing steam.

During this heating the albumins coagulable by heat are precipitated.

It has been found necessary and also more convenient to prepare and sterilize meat infusion before proceeding with the preparation of the different media in which it is used,

* Approximately 500 c.c. of water to each pound of meat.

PREPARATION OF NUTRIENT BROTH 29

on account of the resistant spore-forming organisms which are almost universally present in the chopped meat; economy of time also is a consideration. Unless sterilized immedi ately, meat infusion decomposes quickly owing to the abundance and diversity of the microflora acquired during the various processes of preparation for market.

Infusion made from freshly chopped lean beef will vary in acidity between +15 and +25 Fuller's scale. If the reaction is markedly lower or higher, microbial action is taking place, which is, or may be, injurious to the food value of the medium in which the meat infusion is used.

The infusion contains very little albuminous matter and consists chiefly of the soluble salts of the muscle, certain extractives, and altered coloring matters along with slight traces of protein not coagulated by heat.

EXERCISE 7. PREPARATION OF NUTRIENT BROTH

Nutrient broth is the standard liquid employed for cul tivating microorganisms. It is practically a beef tea con taining peptone. Peptone, a soluble protein not coagulable by heat, is added to replace the coagulated albuminous substances which precipitate when the meat infusion is sterilized. Salt is added to take the place of the phosphates and carbonates, some of which are precipitated on adjusting the acidity of the medium by sodium hydroxide.

The reaction of ordinary nutrient media is adjusted to about +15 with phenolphthalein as indicator, as it is found that most microorganisms grow best on a medium neutral or slightly alkaline to litmus.

When it is required that nutrient media be clear, egg albumen reduced to a smooth paste with water (or the well beaten white of an egg) is added. By coagulation, the egg albumen removes mechanically all small particles in suspen sion which otherwise would pass through the filter paper.

30 GENERAL MICROBIOLOGY

This process is most efficient when the egg albumen coagu lates slowly.

As egg albumen begins to coagulate at about 57 C. it is absolutely imperative for good results that the medium be cooled to 40-50 C. before the addition of egg albumen.

Although egg albumen contains small amounts of sol uble matter not coagulable by heat, as sugar, extractives and mineral matter, all of which will serve as microbial food, its purpose in nutrient media is primarily for its clari fying action.

Apparatus. 500 c.c. sterile meat infusion; 500 c.c. tap water; 10 gms. Peptone, Witte's; 5 gms. Salt; 10 gms. Egg albumen (or one egg); 3.5 liter agate-ware pail; titration apparatus; N/20 NaOH; N/l NaOH; phenolphthalein (indicator); distilled water; 5 c.c. pipette; large stirring rod; coarse balances; large gas burner; large funnel; plaited filter paper; filling funnel; sterile test tubes; sterile 1 liter flask; apparatus for steam sterilization-.

Method. 1. Put the contents of a flask of meat infusion (500 c.c.) in an agate pail and add 500 c.c. of tap water.

2. Add 1% of Witte's peptone and 0.5% of salt.

3. Add 10 gms. Of egg albumen which has been well mixed with 100 c.c. of tap water. (Put the egg albumen in a tumbler and add enough water to form a paste. Stir until smooth. Then add the remaining water. One egg* well beaten may be substituted.) Mix all thoroughly.

4. Heat in flowing steam for forty-five minutes or in the autoclav at 120 C. for thirty minutes.

5. Titrate with N/20 NaOH.

6. Adjust the reaction of the medium to +15 with normal NaOH or normal HC1. Retitrate and adjust again if necessary.

7. Counterpoise and note the weight.

8. Boil fifteen minutes over a free flame, stirring con stantly.

* It is not necessary to add water to the egg.

GELATIN 31

9. Counterpoise and restore any loss by evaporation with distilled water.

10. Filter while boiling hot through plaited filter paper just previously washed with 1/2 liter of boiling water.

11. Pass the filtrate through the same paper till it is bright and clear.

12. Fill thirty sterile test tubes, using approximately 8 c.c. of this medium for each tube. Put the remaining broth in a large, sterile flask.

13. Heat the test tubes and contents in flowing steam twenty minutes on three successive days.

14. To sterilize a large flask of broth, heat for twenty minutes four days in succession.

GELATIN

Gelatin is one of the tools of the microbiologist. As such, it serves two purposes: as a solid culture medium, a technical device by which the isolation of a single species of microorganism is made possible, and, to those organisms which secrete proteolytic enzymes, it serves as a nitrogenous food material.

Gelatin bears the distinction of being the first substance used for a solid culture medium. This medium was origi nated in 1882 by Robert Koch and has since revolutionized the science of microbiology. Prior to the introduction of solid media, the isolation of a single species of microorganism involved much difficulty and almost always a certain measure of uncertainty. To quote from Jordan: " It cannot be a mere coincidence that the great discoveries in bacteriology followed fast on the heels of this important technical improvement, and it is perhaps not too much to claim that the rise of bacteriology from a congeries of incomplete although important observations into the position of a modern biologic science should be dated from about this period (1882)."

Koch's first plates were made by pouring the liquefied

32 GENERAL MICROBIOLOGY

nutrient gelatin upon sterile, flat pieces of glass. The student on becoming familiar with the difficulties of pre paring satisfactory plates with the use of the " Petri dish " will appreciate those met with in Koch's first gelatin plates.

Gelatin is a protein, i.e., a nitrogenous food material. It contains as its essential elements carbon, hydrogen, oxygen, and nitrogen (other elements, however, such as sulphur, phosphorus, etc., may be present). Its empirical formula according to Schiitzenberger and Bourgeois is C7eHi24N24O29, but such a formula only gives information of the chief constituents and allows one to form some idea of the huge size of the molecule; no idea of the structure of the molecule is given. However, by digesting with dilute sulphuric acid, gelatin breaks down in the same way as the proteins, yielding glycin, leucin and other fatty amino-acids.

Gelatin is an animal protein, but does hot occur as gelatin in the animal tissues. It exists there as the albu minoid collagen which is the principal solid constituent of fibrous connective tissue, being found also, but in smaller percentage, in cartilage, bone and ligament. Collagen from these various sources is not identical in composition and gelatin varies correspondingly, e.g., gelatin from cartilage differs from that of other sources in that it contains a lower percentage of nitrogen.

Gelatin, the body resulting from the hydrolysis of collagen, is also an albuminoid. (Hofmeister regards this hydrolysis as proceeding according to the equation :

collagen + water = gelatin

but in dealing with substances of such variable composition,

empirical formulae of this kind have no great significance).

Commercially, it is prepared from certain kinds of bones

and parts of skin. These are selected, washed and extracted

GELATIN 33

by water and with a dilute acid (hydrochloric), with rela tively little exposure to heat, so that as few as possible of the fluid disintegration products of the stock are formed and the jellying power of the resultant solution is not destroyed.

The term gelatin is derived from the Latin verb gelare, to congeal, and calls to mind the principal attribute of this substance, that of its stiffening or jellying property.

Gelatin belongs to that interesting class of substances called colloids. It is a typical example of the class, and exhibits the characteristic properties of the class. Colloids, in marked contrast to crystalloids, do not crystallize, do not readily diffuse and are impermeable to each other. The ultimate particles of colloids are much smaller than what we would ordinarily term a physical subdivision, but rather larger than chemical molecules; the diameter of the smallest particles in a colloidal solution, e.g., red colloidal gold, which have been counted by means of the ultra-micro scope, is 6 millimicrons or 6 thousandths of a micron. A micron is one thousandth of a millimeter. (Bacteria are much larger, the smallest visible by means of the ordinary microscope being from 0.3 to 1.0 micron in diameter.) Consequently their reactions stand midway between the physical and the chemical changes of matter, as may be seen by considering the properties of gelatin.

Gelatin will absorb a considerable quantity of warm water (it is almost insoluble in cold water) and swells up, yielding a jelly which, upon application of heat, melts to a viscous, sticky solution that gelatinizes again upon cooling. The name of hydrogel is applied to colloids showing this property. Ordinary gelatin media for microbiological work contain 12% to 15% gelatin. When dried at medium tem peratures, gelatin can again be redissolved and redried in definitely. From this property it is called a reversible colloid to distinguish it from other colloids which, when their physical state is once changed, are insoluble, e.g., casein and silicic acid.

34 GENERAL MICROBIOLOGY

If superdried at about 130 C., or superheated when in the gelatinous state either for a short time at a temperature above 100 C., or for a long time at 100 C., as in inter mittent sterilization, the gelatin is so modified that its redissolving or resolidifying power respectively is lost. In superdrying, the loss of the redissolving property is laid to the too close contact of the constituent particles, a change in the physical state; in the superheated gelatin, the loss of the resolidifying power is probably due to the disintegra tion of the gelatin molecule, a more purely chemical phenomenon. This loss of the gelatinizing property is also caused by the enzymic activities of many microorganisms and is also a disintegration process.

Gelatin possesses a liquefaction point which, however, varies considerably under different conditions. Ordinarily, media containing 12% to 15% gelatin will liquefy or melt at a temperature in the vicinity of 24 to 26 C., solidify ing again at 8 to 10 C. to a clear, transparent jelly. As a consequence, gelatin media may be employed only for organisms which do not require a higher temperature than 22 to 24 C. for development. Overheating in the process of preparation or sterilization will cause a considerable lowering of the liquefaction point, perhaps ultimately so low that the medium will be liquid at room temperature (20 to 21 C.) It will readily be seen how the latter gelatin medium could not handily be used for the isola tion of organisms. A few data will assist in fixing this in mind.

The solidifying property of gelatin varies in inverse proportion with the time of heating during the process of sterilization; its liquefying point is lowered on an average of 2 C. for each hour of heating at 100 C. This makes clear why such care must be taken in the preparation of a gelatin medium, in the fractional sterilization of this medium in streaming steam, and why immediate cooling is necessary aftei; each fractionation in the process of its preparation.

GELATIN 35

Although temperatures above 100 C. are much more destructive to the solidifying property than that of 100 C., it is possible to sterilize a medium containing 12% to 15% of gelatin in the autoclav (7 to 8 Ibs. Pressure) at 112 to 113 C. for twenty minutes or at 15 Ibs. Pressure (120 C. for five minutes) without impairing its usefulness as a solid culture medium.

This use of steam under pressure (dry steam) is almost necessary in the case of a gelatin medium to effect sterili zation, since gelatin, from its source, method of preparation, and later liabilities to contamination, is almost certain to contain or bear upon its surface a large number of very resistant spores. Heating at 100 C. for thirty minutes on three or even four or five consecutive days is not always efficient, as these spores do not always germinate within twenty-four hours after heating and, referring to the data above, it is readily seen that the lowering of the lique faction point is not to be considered as negligible in the process of intermittent sterilization.

Gelatin possesses another property which renders it valuable for bacteriological work: i.e., in gelatin plate cultures no water of condensation ordinarily collects on the cover of the Petri dish (as with agar) later to drop on the surface of the gelatin and thus obliterate forms of colonies and cause isolated colonies to become contaminated with neighboring ones. The storing of this medium either in test tubes or in plates, sterile or inoculated, is thus rendered much more simple than with agar.

REFERENCE

VAN DERHEIDE, C.C.: Gelatinose Losungen und Verflussigungspunkt der Nahrgelatine, Arch. F. Hyg., Bd. 30, 1897, pp. 82-115.

36 GENERAL MICROBIOLOGY

EXERCISE 8. PREPARATION OF NUTRIENT GELATIN

Apparatus. 500 c.c. sterile meat infusion; 500 c.c. tap water; 150 gms. Gelatin; 10 gms. Peptone, Witte's; 5 gms. Salt; 10 gms. Egg albumen (or one egg); water bath; thermometer; 3.5 liter agate ware pail; long heavy stirring rod; titration apparatus; N/20 NaOH; N/l NaOH; phenolphthalein (indicator); distilled water; coarse bal ances; large gas burner; large funnel; plaited filter paper; filling funnel; sterile test tubes; sterile 500 c.c. Erlenmeyer flasks; apparatus for steam sterilization; running-water bath or refrigerator.

Method. 1. Put the contents of a flask of meat infusion (500 c.c.) in an agate pail and add 500 c.c. of tap water.

2. Add 15% gelatin, 1% Witte's peptone, and 0.5% salt to the mixture.

3. Heat this mixture in a water bath to dissolve the gelatin, peptone and salt, stirring occasionally.

4. Cool to 40-50 C. This is imperative.

5. Then add 10 gms. Of egg albumen which has been well mixed with 100 c.c. of tap water. (Put the egg albumen in a tumbler, add enough water to form a paste and stir until smooth; then add the remaining water. One egg well beaten may be substituted.) Mix all thoroughly.

6. Heat in flowing steam for forty-five minutes or in the autoclav at 105 C. for thirty minutes.

7. Titrate with N/20 NaOH.

8. Adjust the reaction of the medium to +15 with normal NaOH or normal HC1. Re titrate and adjust again if necessary.

9. Counterpoise and note the weight.

10. Boil fifteen minutes over the free flame, stirring con stantly.

11. Counterpoise and restore any loss by evaporation with distilled water.

12. Filter while boiling hot through plaited filter paper

AGAR 37

just previously washed with 1/2 liter boiling water. Pass the filtrate through the same paper until it is bright and clear.

13. Fill thirty sterile test tubes, using approximately 8 c.c. of medium for each tube. Divide the remainder into two equal portions and place in sterile 1/2 liter Erlenmeyer flasks.

14. Heat in flowing steam twenty minutes on three successive days.

15. Cool the gelatin in a running-water bath, immediately after each heating. Care must be taken to heat the gelatin as little as possible, since part of the solidifying power of gelatin is lost with each application of heat.

16. To sterilize a large flask of nutrient gelatin, heat for twenty minutes on four days in succession.

AGAR

Agar or agar-agar (from a Malay word meaning " vege table "), the substance which is used in preparing one kind of solid culture medium for bacteriological work, is a pro duct prepared from various seaweeds found near the Indian Ocean and in Chinese and Japanese waters. This type of seaweed has several common names, as Ceylon or .Jaffna moss, Bengal isinglass, etc. Various species are used for food and the trade is considerable.

Payen, a French chemist -(about 1859), obtained the agar jelly from the seaweed, Gelidium corneum, in the fol lowing manner : The seaweed was allowed to stand for some time in a cold dilute solution of hydrochloric acid; the acid was removed by rinsing several times with water, then the seaweed was placed in a cold dilute solution of ammonia; next the ammonia was removed by repeated rinsing with cold water. During this process, the seaweed lost 53% of its weight in mineral salts, coloring matter, and organic constituents. The remaining portion was boiled in water,

38 GENERAL MICROBIOLOGY

during which process the vegetable jelly was extracted. The solution so obtained was poured off, leaving the useless sediment behind. This jelly is the same in composition as that existing in the vegetable tissues; it has not been changed chemically, as is collagen in the preparation of gelatin. The commercial agar is most probably prepared by evaporating this solution to dryness by different means.

Agar usually comes into the hands of the bacteriologist as long, slender, grayish-white strips, or as blocks, or more especially in recent years, in the form of a gray-white pow der of European manufacture.

Agar, in contrast with gelatin, is a carbohydrate, i.e., it consists of a combination of carbon, hydrogen and oxygen only. Traces of nitrogen are present as impurities. The above qualitative determinations of its elementary constit uents were made by Payen, by Parumbaru and by Hueppe, who made their determinations on agar from different sources. As far as can be ascertained, its empirical formula has not yet been investigated to any extent.

Like gelatin, however, agar is a reversible colloid. It soaks up in cold water, dissolves in hot water after a long boiling to a tasteless and odorless clear solution, and solid ifies upon cooling to a more or less opaque jelly. Its watery solution is neutral or nearly neutral to phenol phthalein; still, a drop or two of twentieth normal sodium hydrate is sufficient to make the pink color perceptible.

The colloidal properties of agar are not destroyed by a long-continued heating at a high temperature, nor by the action of ordinary microorganisms as are those of gelatin. The above properties, however, are influenced and may be wholly impaired by the reaction of the liquid in which the agar is dissolved.

The reaction of the liquid, i.e., whether it is acid or alkaline, influences the agar as to its solubility, solidity, color, transparency, filterability and amount of condensa tion water. If agar is dissolved in a liquid of an acidity

AGAR 39

equivalent to 0.1% HC1, the agar dissolves very readily, filters quickly, the resultant filtrate being a light yellow, transparent, slippery, watery solution which does not solidify upon cooling. If a smaller percentage of hydro chloric acid is used, solidification occurs (below 40 C.) but the jelly will not " stand up " and is therefore useless for agar slant or plate cultures. A large amount of con densation water is present also.

If agar is dissolved in a weak alkaline or neutral broth, a thick, reddish-brown, viscous liquid is obtained which filters slowly and solidifies quickly at 40 C., to a very solid, opaque, dry jelly, having but little condensation water; it retains its shape well in slants and in plates. Thus the value of the agar as a solid culture medium is raised or lowered according to the cjegree of alkalinity or acidity.

It must be noted in addition, however, that when once the solidifying property of agar is destroyed by the presence of an excess of acid in its solution, this property can never be regained by neutralization with alkali; the acid per manently destroys the reversibility of the colloid.

The melting-point of agar (of 1.5% in neutral solution) is 97 C. and although its solidifying point is at 40 C., when once it has solidified it will stand up in the thermostat at a temperature of 50 C. For bacteriological purposes, only that form of agar can be used which remains fluid at from 38 to 40 C. Agar which remains fluid only at a temperature above this point would be too hot when in a fluid state for use; the vitality of organisms introduced would be impaired or destroyed by the high temperature.

Difficulties are encountered in the preparation of a solid culture medium from agar, due to its slow solubility, vis cosity and consequent slow filterability. Its solution (digestion) is effected, as mentioned above, by a long heating in a water-bath, steam sterilizer, autoclav, or over a free flame. The length of time required for complete digestion depends upon three things: The reaction of the

40 GENERAL MICROBIOLOGY

liquid in which the agar is dissolved, the per cent content of agar, and the method of dissolving. The influence of the reaction of agar solutions has been treated above. For general culture use, however, ordinary agar is made +15 Fuller's scale (agar solidifies with difficulty above +30 Fuller's scale).

One per cent agar is much more easily soluble under equal conditions than a higher per cent. One and one half per cent is the amount used in ordinary agar media, giving a somewhat stiffer and thus more desirable jelly.

Agar is digested most rapidly over a free flame. If not heated sufficiently, after the filtration and sterilization of the agar by the intermittent method, a flocculent precip itate frequently appears in the previously clear medium. This can be made to disappear in most cases by subjecting to the temperature of the autoclav (120 C. 15 Ibs.).

Agar for culture media should be entirely clear when liquid, and homogeneously opaque-translucent when solid; it should have a translucence sufficient to allow deep colonies on plates or stab cultures to be observed readily; it should not contain flocculent material, sediment, or pieces of cotton or filter paper, as these hinder typical colony development of microorganisms and, to the inexperienced, may some times be mistaken for colonies.

In the first methods ever used for making agar culture media, instead of filtering the hot agar through filter paper, absorbent cotton, or asbestos, it was allowed to cool, dur ing which process the sediment settled to the bottom; when solid the sediment was cut off. This method was not desirable, as the clearness of the resultant agar would depend upon the rate of cooling; the slower the cooling, the more completely would sedimentation take place.

Agar is not a food for microorganisms in general, i.e., it is not affected by the digestive enzymes of most bacteria, as is gelatin. However, a few bacteria are known which have the power of liquefying agar, among which are B.

PREPARATION OF NUTRIENT AGAR 41

gelaticus n. sp. (gran) and Bad. Nenckii, both of which are found, as would be expected, in sea water. This compara tive inertness of agar renders it valuable for the preparation, of solid synthetic media, the value of which may be en hanced by subjecting the commercial agar to natural fermentation during which process any traces of avail able food substances are used up by the microorganisms present. (Beijerinck.)

Agar is of special use in bacteriological work in which the cultivation of microorganisms must be conducted at a temperature above the melting-point of gelatin. This feature has made possible the great strides that have been taken in medical bacteriology, as many pathogenic bacteria can be isolated and grown only with difficulty at tempera tures below that of the body.

REFERENCES

SMITH, ERWIN F.: Bacteria in Relation to Plant Diseases. Vol. I,

pp. 31-36. Several illustrations. SCHULTZ, N. K.: Zur Frage von der Bereitung einiger Nahrsubstrate.

Cent. F. Bakt. I. Orig., Bd. 10, 1891, p. 57.

EXERCISE 9. PREPARATION OF NUTRIENT AGAR

Apparatus. 3.5 liter agate-ware pail; 15 gms. Agar; 10 gms. Peptone; 5 gms. Salt; 10 gms. Egg albumen (or one egg); 500 c.c. sterile meat infusion; 500 c.c. tap water; titration apparatus; N/20 NaOH; N/l NaOH; phenol phthalein (indicator); distilled water; large funnel; plaited filter paper; filling funnel; sterile test tubes; sterile liter flask; coarse balances; large gas burner; 1 liter measuring cup; apparatus for steam sterilization.

Method. 1. In a 3 liter agate ware pail place 15 gms. Of agar in 500 c.c. of tap water.

2. Wash the agar well, separating the shreds and squeez ing it through the hands.

3. Decant the dirty water, measuring the amount poured

42

GENERAL MICROBIOLOGY

off; replace with the same amount of clean tap water.

Repeat.

4. Dissolve over a free flame and boil for five minutes, stirring constantly. The solu tion must be entirely free from lumps of agar.

5. Add 1% Witte's peptone and 0.5% salt to the boiling agar.

6. To 500 c.c. of meat in fusion add 10 gms. Of egg al bumen which has been well mixed with 100 c.c. of tap water. (Put the egg albumen in a tumbler and add enough water to form a paste. Stir until smooth and then add the remain ing water. One egg; well beaten,

FIG. 9. Hot Water Funnel for may be substituted.) Mix all Filtering Agar or Gelatin. Thoroughly

7. Pour the melted agar mixture slowly into the meat infusion, stirring constantly. Heat in the autoclav at 120 C. for forty-five minutes or for an hour in flowing steam.

Note. The time for this heating may be lengthened to advantage, but never shortened. If agar has not been heated sufficiently before filtration, a flocculent precipitate will form in the tubes upon heating in flowing steam. In most cases this may be caused to disappear by heating for a short time in the autoclav at 15 Ibs.

8. Titrate with N/20 NaOH.

9. Adjust the reaction of the medium to +15 with normal NaOH or normal HC1. Retitrate and readjust the reaction if necessary.

10. Counterpoise and note the weight.

11. Boil fifteen minutes over a free flame, stirring con stantly,

DUNHAM'S PEPTONE SOLUTION 43

12. Counterpoise and make up any loss in weight with boiling distilled water.

13. Filter boiling hot through plaited filter paper just previously washed with boiling water. Pass the filtrate through the same paper until clear.

14. Fill 60 to 70 sterile test tubes, using approximately 8 c.c. of the medium for each tube.

15. Heat in flowing steam twenty minutes on three suc cessive days.

16. At the end of the final heating, place the tubes of agar in an inclined position to solidify (do not allow the medium to touch the plug) so that a large surface is pre sented for the cultivation of microorganisms. These are called agar slants.

Note. If agar tubes are to be used only for agar slants, less of the medium is needed in the tube than when they are to be used for plating.

17. To sterilize a large flask of agar, heat for thirty minutes on four successive days.