CUS AND GONOCOCCUS'
SADIE F. BAILEY
PathologicalLaboratories, University ofPittsburgh,Pittsburgh,Pennsylvania Receivedfor publication June 19, 1937
The meningococcus and gonococcus often give rise to lesions in the human being which are of the gravest import. It is, therefore, highly desirable that a differential diagnosis between thesetwo andother gram-negative cocci should be made as early as possible. Workers in hospital laboratories have encountered various difficulties in the practical differentiation of these or- ganisms. The difficulties lie mainly in the slowness of acid production on solid media in general use, and in the frequent failure of the organisms to multiply in fluid media. Numerous attempts have already been made to remedy these difficulties.
After aconsiderablestudyof thefactorsinfluencingthegrowth and growth products of Neisseria, a medium has been obtained which we believe offers a practical means by which an early differential diagnosis can be made.
The medium to be described consists of whole serum from which the fermentable substances and diastase have been re- moved and to which have been added a concentrated broth containing inorganic salts and a carbohydrate and indicator.
Differential identification can usually be established in 12 to 24hours.
REVIEW OF LITERATURE
Bumm (1885), who first successfully cultivated the gonococ- cus, grewit on solidified humanplacental blood. Later investi- gators added unheated proteins to agar for the cultivation of
1SubmittedtotheGraduate Schoolof theUniversityofPittsburgh inpartial fulfillmentoftherequirementsforthedegreeofMasterofScience.
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both the gonococcus and meningococcus. Dunn and Gordon (1905), Rundle, Mottram and Williams (1907), Symmers and Wilson (1907), Shennan and Ritchie (1908), Elser and Huntoon (1909), Swartz (1920), and Torrey and Buckell (1922) used hydrocele and ovarian fluids. Martin (1911), and Jenkins (1921) used human serum. Kinsella, Broun and Garcia (1923) used beef serum. Jenkins (1924), MacNaughton (1923), and Herrold (1928) utilized whole blood. North (1909) reported good results with gelatin-agar, and Vedder (1915) with corn- starch agar. Watabiki (1916) added whey to agar. Cole and Onslow (1916), on the other hand, reported best results with milk casein mixed with extract of pig pancreas.
In 1922 Erickson and Albertreportedthe use of beeftesticular blood-agar and in 1926 Pelouze and Viteri obtained excellent results with brain broth medium without addition of blood.
Cole and Lloyd (1916-17) used "tryptamine" blood extract in agar and in broth. They were particularly interested in the amino-acid content of themedium as werealso McLeod, Wheat- ley and Phelon (1927).
Although the type of protein utilized has been stressed by most investigators, Martin (1911), and Phelon, Duthie and McLeod (1927) indicated the importance of reaction of the medium. The latter authors called attention to the ill effects of unduly alkaline media on both the meningococcus and gonococcus. Wherry and Oliver (1916), and Chapin (1918) believed that an altered oxygen tension was also a major factor in thecultivation of gonococci.
Perhaps themedia most extensively used for differentiation of gonococcus and meningococcus are the following: the serum water medium ofHiss, thesemisolid agar of Torrey and Buckell (1922), the ascitic fluid of Elser and Huntoon (1909), Gradwohl (1926), and others, the sugar-free infusion-broth agar of Martin (1911), thehormone agar of Huntoon (1918), and the brain agar of Pelouze and Viteri (1926). These various media, with and without modifications, have been used in our laboratories for several years, but since growth requirements do not entirely parallel those of acid production they have all proved more or
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less unsatisfactory in our hands, chiefly because of irregular or tardyreactions. Likewise, the egg-white mediums of LeSoudier and Verge (1925) and of Roublot (1925) were tested as a basis for a differential medium but they could not be used because of interference with the action of the indicator.
It was evident, therefore, that none of the methods which we have tried were entirely satisfactory for diagnostic purposes, especiallyinthehands of the studentinbacteriology.
Some ofourpreliminaryobservations indicated that coagulated serum might possess the requisite qualities. These observations were extended in various ways and resulted in the development of the medium described in the following pages.
PREPARATION OF MEDIA
In these experiments, sera from beef, calf, lamb and hog were tested. Ingeneral, media preparedfromeach ofthe serayielded equally satisfactory results. Beef serum being more easily ob- tainable was, therefore, more extensively used. Blood for this purpose was collected in theusual manner and the serum sepa- rated from the clot.
Amixture of threeparts ofserumand one part ofsterile water (250 to 300 cc. ina 500-cc. Florence flask) was heatedin awater bath at 650C. for one hour, with frequent shakings, on two consecutive days. Enoughwater was kept in the bath to float the flask in order to prevent coagulation at the bottom. The mixture was cooled to 400C. or below and inoculated heavily with a 24-hour culture of Escherichia coli-communis. It was thenincubated at370C. for fourorfive days oruntil asecondary alkalinity of pH 7.7 to 7.9 had occurred. The mixture was again heated in the water bath at 65CC. forone hour to kill the organisms present. At this point the sterilized mixture could be stored in theicebox untilitwasconvenientto completeitsprepa- ration. The serum water mixture thus treated was found to be deficient in growth-promoting factors but was verysatisfactorily used as abasic mediumto which various substances were added inan attemptto secureamediumwhich wouldproduceluxuriant growth and a good differentiation, and would prolong the life
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of the organisms. Various materials were added to the basic medium, singly and in combinations, and the effects upon the cultures of meningococci and gonococci were studied. The chemicals studied most extensively were: potassium sulphate, magnesium sulphate, sodium sulphate, sodium sulphite, potas- sium chloride, calcium chloride, magnesium chloride, sodium nitrate, sodium bicarbonate, monobasic and dibasicpotassium phosphate and sodium phosphate.
Growth and acid production of both meningococci and gono- cocci were slightly accelerated by additions of sodium sulphate, potassium sulphate, potassium chloride, sodium nitrate and sodiumbicarbonate inamounts varying from0.1 to 0.3percent.
Of the combinations of salts studied, those which affected acid production most were mixtures of sodium or potassium sulphate with calcium chlorideand sodium bicarbonate orsodium nitrate.
In some instances we were able to detect acid production in media containing salts 4 to 12 hours earlier than in those con- tainingno salts. Further additions of meat extract, peptone or neopeptoneweretried. These latter substances so enhanced the growth of both organisms that a mixture of meat extract with one of the peptones has been employed, neopeptone being preferred.
Several indicators were tested, the most important being:
azolitmin, Andrade's, phenol-red, brom-thymol-blue, brom- cresol-purple, and cresol-red. Satisfactory results wereobtained with the last five but only when culture tubes were sealed with corks or rubber stoppers.
When theAndrade's indicator was employed, it was necessary to add 1 cc. to 100 cc. of medium. The phenol-red indicator required 5cc. of 0.04 per cent aqueous solution to each 100 cc. of medium. Cresol-red, brom-cresol-purple and brom-thymol-blue each gave optimum results by the addition of 2.5 cc. of 0.1 per cent aqueous solution to each 100 cc. of medium.
Brom-thymol-blue gave somewhat more satisfactory readings than the others and was, therefore, the indicator of choice.
In order to incorporate into the medium the most desirable ingredients studied, itwas found necessary to add to the serum-
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water mixture a special concentrated broth which was prepared as follows:
Water... 1000cc.
Neopeptone or peptone... 50 grams Meat extract... 10 grams Sodiumchloride... 10 grams Sodiumsulphate... 10 grams Sodiumnitrate... 5 grams
The mixture was heated until dissolved, reaction unadjusted, and then sterilized at 10 pounds pressure for 15 minutes.
The formula for the completed medium consisted of:
Serum-watermixture (as prepared above)... 800cc.
Concentrated broth (asprepared above)... 200 cc.
The reactionwas adjusted to pH 7.8.
Carbohydrate (glucose, maltose, etc.)... 10 grams Brom-thymol-blue0.1 per cent aqueoussolution... 25cc.
Sodiumhydroxide was used for adjusting the reactionwhen the mediumwas too acid. Additional peptonewas added when the medium was too alkaline. The medium was tubed in 5-cc.
amounts and coagulated in the inspissator. Inspissation of the mediumwasbestaccomplished by having theinitial temperature of the water in the inspissator jacketbetween 700 and 750C. and raisingthisto900C.in45minutesto one hour. This temperature was maintained until coagulation was completed. The time required to complete coagulation depended upon the number oftubes in the inspissator.
As soon as tubes were cooled enough to permit handling they were removed from the inspissator, placed in an autoclave in slanted position and heated for 30 minutes at two pounds pres- sure. They were then removed from the autoclave, incubated overnight at 370C. and re-heated for 30 minutes at two pounds pressure. This was sufficient to sterilize the medium. The tubes were then stoppered tightly with sterile corks or rubber stoppers and stored at roomtemperature in the dark, since light slowly decolorizesthe indicator.
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A mediumprepared andstored in this manner for longer than one year continues to give excellent results.
GENERAL RESULTS
The fermentation reactions of 56 strains of gonococci and 51 strains of meningococci have been repeatedly studied on this medium. Thirty-nine strains of the former and 41 strains of the latter were recently isolated from hospital cases. The re- maining strains were from stock cultures. The carbohydrates foundto be of mostvalue in thedifferentiation ofmeningococci and gonococci were those commonly used with other methods referred to, namely, glucose and maltose in 1 per cent concentra- tion. As the work progressed, other carbohydrates were tested but these added nothing of practical value to the information obtained by the use of the two mentioned. In cultures of meningococci which were tightly stoppered, fermentation oc- curredinglucose andmaltosein from4to 16hours withadefinite change in color of the indicator.
Sealedcultures ofgonococcifermented glucosein 8to24hours.
No further changeoccurred in culturesof either organism during the first four days, but on the fifth day both gonococci and meningococci produced acid in mannitol in all strains studied.
This reaction was probably due to impurities in the carbohy- drates. Nofermentationwasobtainedinsucrose, lactose, salicin, sorbitol, dulcitol, mannose, levulose and galactose by either organism. Prolonged incubation produced little or no color change inuninoculated controls and in those cultures with non- fermented sugars.
Other investigators have reported the increased amount of growthproduced by the gonococcus when tubes are sealed prior to incubation. We noted, however, that when shallow stabs were made into the medium rather than surface inoculation alone, acid appeared almost as early as when the tubes were sealed.
Sealing the tubes did not favorably influence the growth of meningococci, but when cottonplugs were employed all cultures of this organism produced a secondary alkalinity in fermented
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sugars within 48 hours. Three strains of gonococci which grew very luxuriantly produced a slight secondary alkalinity in glucose medium. With the remaining 53 strains studied, the acidity was maintained over a period of weeks. Two factors are concerned withproduction of these phenomena. These are oxygen tension andthe prevention of escape of any gas or volatile acid. Qualita- tive analysis, similar to that employed by Phelon, Duthie and McLeod (1927), except that barium hydroxide was substituted for soda lime in the last two tubes, gave a positive test for carbon dioxide. Negative results were obtained in the tests for volatile acids. Loss of the carbon dioxide probably explains the secondary alkaline reactions, althoughthepossibility of ammonia production was not ruled out.
Six strains of Micrococcus catarrhalis, three strains of Micro- coccuspharyngissiccusandeight strainsofchromogenicgram-neg- ative diplococciwere studied for comparisonwith the meningoc- occic and gonococcic strains. No carbohydrateswere fermented byMicrococcus catarrhalisintubes pluggedwith cotton, butwhen culture tubes were sealed, even those controls to which no car- bohydrate and no salts were added showed an acid reaction.
Thislatter phenomenon was present with all strains of Neisseria studied except meningococci and gonococci. Therefore, these mediums can be used to differentiate only the latter two or- ganismsfromeach otherand from others of this group.
DISCUSSION
For the differentiation of microorganisms, morphological, cul- tural, serological and fermentative methods are commonly employed. At present no one of these alone is sufficiently comprehensive. Fermentation, together with morphology, is probably most frequently used inroutine diagnosis. Fermenta- tion still presents many difficulties in the Neisseria. These bacteria have become so highly differentiated that the physical andchemicalpropertiesof the mediummustbecarefullyadjusted in order to permit optimal conditions for growth and ready utilization of carbohydrates for acid production.
The two organisms under study have such a high degree of
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specialization that they are capable of utilizing only certain proteins. In contradistinction to all other bacteria studied, including several members of the streptococcic, staphylococcic, diphtheria and colon-typhoid groups, the meningococci and gono- cocci, in sealed cultures, produced no change on the coagulated blood serumwhich had been depleted of fermentable substances andto which had been added anindicator but no carbohydrate.
As has 'already been noted, other members of the Neisseria produced acid reaction in this medium. Based onthese data, it would appear that most other forms of bacteria are able to hydrolyze complex proteins into simpler compounds such as proteose, polypeptide, peptide, etc., and from amongthe simpler organic compounds certain fractions are utilized and others are eliminated as dissimilation products comparable to the findings ofKluyver (1931). Thepresence of thesedissimilation products may explain acid production in control tubes. On the other hand, meningococci and gonococci do not have the property of hydrolyzing complex proteins in order to secure the selective substances for growth. These two organisms can utilize only such simple protein substances as are directly available for assimilation.
The question of amino-acids has received consideration by various investigators in connection with this group of bacteria.
The value of Cole and Onslow's medium (1916) for the cultiva- tionof the gonococcus was largely attributed by Cole and Lloyd (1916-17) to its abundance of amino-acids. The latter investi- gators stated, "Thevalue of theadditionofpeptoneto anutrient medium is almost entirely due to the fact that all commercial samples contain small amounts of amino-acids in the free state which are directly available for assimilation." The fact that dialyzed trypsin digest failed to support growth of the gonococ- cus was attributed by Miller, Hastings and Castles (1932) to the removal of amino-acids.
McLeod, Wheatley and Phelon concluded that among the elements essential to the growth of the gonococcus were, "Some sources of amino nitrogen and salts such as are present in meat extract." They further stated, however, that large amounts of amino-acid inhibit growth.
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Whatever these growth-promoting substances may be, they are presentinpeptone, neopeptone and meat extract.
It is debatable whether such substances are amino-acids or nitrogen compounds of simpler constitution. That such simple compounds, as nitrates, may be directly usable for growth is indicated by the fact that among the various inorganic salts tested the greatest acceleration of growth was obtained by addition of sodium nitrate to the culture medium. These find- ings agree in part with those reported by Miller, Hastings and Castles (1932). They found bicarbonate to be essential to the growth of gonococci. No detailed study was made to ascertain whether this was due to the kation or anion or what part was played by valency. At present it is not clear how this result is brought about. Obviously osmotic pressure is increased and this may act in a greater utilization of available food material.
A more plausible explanationis thespecific effect of certainions on enzymes associated with growth and acid production. Nu- merous instances of the enhancing effect of ions in biochemical activitymight be cited. Waldschmidt-Leitz (1929) and Haldane in their studies on enzymic actionin theanimaland in theplant organism illustrated the effect of various inorganic salts on specific enzyme production. Glick and King (1932) showed the inhibiting effect of certain organic compounds on liver esterase.
Karstrbm (Stephenson, 1932) has established the fact that bac- terialspecies are not constantinregardto theirenzymic content, but vary sharply according to their nutritional environment, and that certain of the sugar-splitting enzymes are present in demonstrable amounts only if the organism has been grown in the presence of its specific substrate.
Oxygen tension
Thetwomain factorsinvolvedintheincreased acidproduction in sealed cultures are lowered oxygen tension and loss of any volatile substances. Experiments indicated that retention of carbon dioxide is probably the essential differential factor con- cerned in the meningococcus cultures. Ther6le played by each of these two factors ingonococcic cultures is not so easily deter- mined. Wherry and Oliver (1916) and Chapin (1918) claimed
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reduced oxygen tension to be an important growth promoting factor in thegonococcus. On theotherhand, TorreyandBuckell (1922), and Erickson and Albert (1922) believed that a reduced oxygen tension was not necessary. They attributed the in- creased growths obtained in partially anaerobic cultures to the changed physical conditions brought about by these methods, the retention of moisture being the condition most stressed.
The observations made in this study support the contention that an altered oxygen tension, such as can be produced by methods described, does enhance growth as well as prevent escapeofmoisture and volatile substances. Inunsealedcultures, growth along the edge ofthe water ofcondensationwas no more luxuriant than at any otherplace on the slant. As has already been noted, shallow stabs made at any place on the surface resulted in an early production of acid, indicating the presence ofgrowth.
SUMMARY
The preparation of a differential medium for the diagnosis of meningococcus and gonococcus has been described. Essential features of this medium are a combination of serum from which fermentable substances and diastases have been removed and to which have been added a concentrated broth containing certain inorganic salts. Having the culture tubes sealed during growth was found to be necessary in order to insure the continued presence of acidin the medium.
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