Agar Plates Bacterial Culture

Information on the Growth of Bacterial Cultures using Agar Culture Plates

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What is Agar - Definition?

Agar or agar-agar (from a Malay word meaning " vegetable"), 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.

Background and History on Microbiology using Agar Nutrient Broth and Plates

Payen, a French chemist -(about 1859), obtained the agar jelly from the seaweed, Gelidium corneum, in the following 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, 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 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.

Advantages and Disadvantages as Agar as a Nutrient Media

Difficulties are encountered in the preparation of a solid culture medium from agar, due to its slow solubility, viscosity 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 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, during 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. 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.

 

 

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

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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.

 

 

 

References for Agar Bacterial Culture

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.