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Three cases of Haemophilus parainfluenzae endocarditis in previously healthy patients were diagnosed in one six-month period. The presenting clinical picture was not typical of endocarditis. The initial physical examination and laboratory studies were normal except for hectic fever. Blood cultures were negative until subculture to chocolate agar and incubation in CO2 was done. Antibiotic susceptibility testing in vitro revealed that the penicillins, cephalothin, and gentamicin were active against the organisms isolated. It is recommended that blood cultures be subcultured to chocolate agar and incubated in CO2 whenever endocarditis is expected and the initial cultures are negative. Two of the patients were treated with ampicillin; the third patient received ampicillin, streptomycin and tetracycline. All patients were treated for six weeks and were cured.

Streptococcal and salmonella antisera inhibited carbohydrate metabolism for groups A, B, C, and D streptococci and group E salmonella, as measured by the formation of [ 14 C]dioxide from [ 14 C]glucose metabolism. For salmonella, the inhibition was type specific since group E salmonella were inhibited only by salmonella E antisera and not by anti-salmonella A or C 1 . For streptococci, quantitative differences were demonstrated, but major cross-reactivity was observed. At high concentrations, the antisera were bactericidal; at more dilute concentrations, for both salmonella and streptococci, carbohydrate metabolism was suppressed, but subculture on chocolate agar showed abundant growth. Cross-reacting antibodies could be absorbed by incubation with either antigen, e.g., streptococcal antisera versus heat-killed salmonella. The results suggest that the radiometric technique can be more sensitive than either capillary flocculation or visual detection of bacterial growth for detecting the inhibition of streptococci and salmonella by specific antibodies. The use of specific antisera may prove useful for bacterial species identification in an automated system for detection of bacterial growth.

— Occlusal fissures from unerupted third molars were implanted in occlusal fillings in molars of six dental students. Two experiments were performed consecutively in each tooth. After 7 d in the mouth the fissure was removed, opened and dropped into a prereduced suspending medium and the contents suspended by ultrasound. In phase contrast microscopy the microflora consisted of 81–93% cocci and 7–19% rods. No fusiforms, filaments, spirilla, spirochetes or yeasts were seen. In Gram-stained smears 81–96% of the bacteria were Gram-positive. The anaerobic total viable counts (AnVC) were 1–33×106 per fissure. Streptococcal counts on Mitis Salivarius Agar constituted 29–60% of AnVC. In differential counts, S. mutans constituted 0–40%, S. sanguis 0–78% and S. salivarius 0–63% of the colonies present. Lactobacilli grew on Rogosa SL Agar from five of the subjects. In four of these, lactobacilli constituted 0.002–4% of the total viable counts, whereas in one sample they made up 65%. Hemophili isolated on chocolate agar containing bacitracin constituted 0–15% (median 0.7%) of the AnVC. Although the large variation indicates each fissure to be a separate ecologic system, it may be concluded that the flora differs from that of 7-d dento-gingival plaque at least by the absence of fusiforms, filaments, spirilla and spirochetes. The proportional distributions of Streptococcal species also differ from those reported for approximal and buccal plaque as well as for saliva.

Krumwiede, Mishulow and Oldenbusch1 stated that the serum obtained from rabbits that had been injected with recently isolated pertussis strains grown on chocolate brown agar medium, agglutinated organisms grown on chocolate agar medium well and those grown on unadjusted blood agar but poorly. They found also that certain serums obtained from rabbits that had been injected with freshly isolated strains grown on unheated blood medium (Bordet-Gengou-Povitzky) agglutinated organisms grown on chocolate brown agar better than they did the homologous organisms grown on blood agar medium. They concluded that there were two types of H. pertussis?type A, where the strain serum agglutinated its homologous and heterologous organisms, and type B, where the strain serum agglutinated its homologous, but not its heterologous organisms. Their work was repeated elsewhere and confirmed when their cultures were used. In Paris,2 as well as in Copenhagen,3 no such comparable variation could be obtained from experiments with freshly isolated strains obtained from local sources, although it was admitted that the two American cultures acted like two separate types of organisms and in the manner described by Krumwiede et al. We grew freshly isolated organisms on 20*^ placental blood agar medium (Bordet-Povitzky modification)4 and the same organisms on the same stock medium, heating it to a chocolate brown color. The agglutinin titer of rabbit serum obtained after the injection of organisms continuously grown on 20(^ placental blood agar medium (Bordet) in the manner described previously5 was still high against the homologous organisms grown on blood agar (1:10240), but dropped markedly against 10 organisms grown on chocolate brown agar. In this paper, homologous organisms means the same organisms grown on the same medium; heterologous means the same organisms grown on different mediums. The results of our experiments are summarized in table 1.

The peroxide-forming bacteria have been shown by McLeod and Gordon' to be the pneumococci, streptococci, lactic acid bacilli, certain sarcinal forms and the anaerobes. These organisms also fail to produce catalase, thereby permitting an accumulation of peroxide to which their delicacy in subculture is attributable. Exclusion from air and light, and growth in mediums containing tissue fluids which supply catalase, or the addition of certain reducing substances such as cystein or sodium thioglycollate, are therefore necessary for the growth and preservation of these organisms. Demonstration of peroxide production by bacteria growing in liquid medium can be made by the peroxidase test.2,3 If only small quantities are formed, however, and at irregular intervals (as is the case with the streptococci) this test is unreliable, as shown by McLeod and Gordon4 who recommended chocolate agar on which the cumulative action of very small quantities of peroxide is manifested by a green discoloration. Penfold5 was the first to show that on benzidine blood agar peroxide-producing pneumococci and streptococci produced a black or brown pigment which he concluded was formed by the oxidation of the amino bodies of the benzidine by peroxide produced by the organisms. Peroxidase, contained in blood, was necessary for the test. Nonperoxide-producing organisms did not give the reaction. Although Penfold found that all strains of pneumococci and streptococci tested gave a positive reaction with benzidine, certain streptococcal strains gave variable results. This variability was shown by some strains giving all black colonies at one time and, at other times, a mixture of black and white colonies. Attempts to develop permanent black and white cultures of these strains were unsuccessful, and he concluded that the power to form peroxide is an erratic function of certain strains of hemolytic streptococci. Avery and Morgan,' using the peroxidase test on broth cultures, reported peroxide production by all pneumococcus cultures and by 15 of 23 strains of hemolytic streptococci. The observation of McLeod and Gordon7 that streptococci produced peroxide at a later period than pneumococci was confirmed by the former workers who showed that in

Several points of bacteriologic interest were brought out during the 1920 epidemic of influenza at the University of Wisconsin. During this epidemic, bacteriologic examination was made of the nasopharyngeal flora of 79 patients suffering from influenza, 15 patients in the hospital for other illnesses, a group of 26 medical students, a few of whom subsequently came down with influenza, and 9 patients with pulmonary tuberculosis. The medium used was for the most part heated blood agar (so-called "chocolate agar") made from a beef infusion basis, 5 Jo defibrinated sheep blood and Witte's peptone (purchased before the war) and adjusted to a Ph of 7.4-7.6. In some instances unheated blood agar and Avery's oleate medium of the same H-ion concentration were also used. In addition to the plates from the nasopharyngeal swabbings, blood cultures and sputum examinations were made from some of the patients. In all instances the plates were streaked at the bedside, and the sputum examinations and animal inoculations were made without any material loss of time. An ordinary bent wire applicator was employed to obtain the material from the nasopharynx, and with reasonable care no contaminations were obtained by striking the swab against the tongue or other parts of the mouth.

Summary(1) Typical polar granules appear as dense spherical masses, or possibly plates, in electron micrographs of unstained preparations of C. diphtheriae grown on blood infusion agar. (2) In addition to polar granules, needle-like crystals appear in electron micrographs of unstained preparations of C. diphtheriae cells grown on potassium tellurite chocolate agar. (3) The needle-like crystals, as well as the black color, of cell masses of C. diphtheriae grown on potassium tellurite chocolate agar disappear upon the addition of small amounts of bromine water. It is inferred, therefore, that the black color is due to the tellurium metal which occurs in the form of needles. (4) It is to be further inferred that the tellurite ion is able to diffuse through the cell wall and is reduced with the precipitation of tellurium metal within the cell boundaries. (5) With the aid of the electron microscope it is now possible to obtain pictorial records of the location of sites of certain chemical reactions incident to...

SUMMARY: The morphological and cultural characteristics of seven recently isolated strains of Bacterium melaninogenicum closely resembled those given by previous authors. Fermentation reactions were unsatisfactory, owing to the production of acid in basal media without added carbohydrate. The growth of two strains studied in detail was greatly facilitated by X factor and to a much smaller extent by V factor; it was best on chocolate agar. The strains were non-pathogenic in mice. Serologically, they behaved alike in precipitin and complement-fixation tests. There was insufficient information to classify them into subtypes. The black pigment, extracted by a new technique which avoids the use at any stage of alkalis, proved to be haematin (not melanin), united in the cells with a bacterial protein to form a parahaematin. Crystalline pyridine haemochromogen and haemin have been prepared from the isolated pigment. The haemin when coupled with renatured ox globin gave reconstituted methaemoglobin, oxy- and carboxy-haemoglobins. Ultra-violet and visible spectral absorption data support the identification of the pigments. In particular, the possible reduction of the vinyl groups of the porphyrin nucleus by the organism, leading to meso- or deuterohaematin, has been excluded. It is suggested that the organism be assigned to the genus Rusiformis, and the specific name be altered from melaninogenicus to nigrescens, making Fusifomis nigrescens

Summary 1. The susceptibility of infants to infection is apparent from the age distribution of the various etiologic types of meningitis. The majority of cases of each type occur during the first two years of life. 2. The frequency of the occurrence of influenzal meningitis is pointed out. 3. The characteristics of the spinal fluid are discussed for the various types of meningitis from the point of view of practical etiologic diagnosis. 4. The superiority of chocolate agar as a routine spinal fluid culture medium is emphasized. 5. Summaries of nine cases of meningitis due to various members of the colon group of bacilli are presented. In addition one case due to the Friedlander bacillus is presented.

Background : Cholelithiasis is one of the most common digestive surgical disorders. The natural history of gallstone development is unknown. Bacteria are found in high concentration in bile and stone. It is difficult to ascertain whether bacterial infection of the bile arose before stone formation or vice versa. Materials & methods : Prospective study was carried out in 30 cholecystectomy specimens of patients with cholelithiasis collected from 15th January 2005 to 24th July 2005 in Department of Pathology, Kathmandu Medical College Teaching Hospital. The samples were collected in a sterile vial containing 0.5% saline. A small piece of gallbladder was taken in Blood agar and Chocolate agar media for micro anaerobic culture at 370C. The growth of the organism was observed after 48 hours of incubation. Gram stain and various biochemical tests were performed for the confirmation of Helicobacter hepaticus and its sensitivity pattern was studied. Histologically Helicobacter hepaticus was demonstrated in formalin fixed tissue sections using Warthin Starry Silver stain. Results : A total of 76.66% cases showed growth positivity. Nitrofurantoin was found to be the most sensitive drug (95.7%) for Helicobacter hepaticus followed by Ciprofloxacin (91.3%), Cephalaxin (91.3%), Certriaxone (91.3%), Ofloxacin (82.6%), Amikacin (65.2%) and Norfloxacin (60.9%). Helicobacter hepaticus was least sensitive to Amoxycillin (57%). Conclusion: From this study, it is quite apparent that Helicobacter hepaticus infection is commonly associated (76.66%) with cholelithiasis. If the patients are treated with the sensitive antibiotics regularly and the infection, that is the number one cause for the precipitation of bile leading to the formation of gallstones, can be controlled, the incidence of cholelithiasis can be reduced so that the health care expenditure related to gallbladder disease can be reduced markedly. Key words: Cholelithiasis Helicobacter hepaticus; WSS stain DOI: 10.3126/kumj.v7i2.2704 Kathmandu University Medical Journal (2009) Vol.7, No.2 Issue 26, 125-128

It is desired to report the development of a transparent, reproducible medium, containing no heat-labile material such as serum, which is sufficiently stable to withstand autoclave sterilization and storage, and which will support satisfactory primary growth of either meningococci or gonococci. The latter were used as the test organisms during the experimental work, since a constant source of primary cultures of the meningococcus is ordinarily not available. The similarity between the two organisms, and the reputedly greater difficulty expected in the cultivation of the gonococcus, indicated that a medium suitable for this organism should prove equally satisfactory for the meningococcus.As a basis for comparison, the Difco “Proteose No. 3 Chocolate Agar” has been used. This medium has been adopted as a standard in certain laboratories for the diagnosis of gonorrhoea1 and one of us (J.H.) was thoroughly familiar with its use.The plan as formulated in beginning the experiments was essentially that followed ...

In previous papers I 1 have shown that the hemolytic streptococci from early typical cases of scarlet fever produced no color on sheep blood agar heated to 90 C. (chocolate agar) or rarely a slight greening after several day's growth, in contrast with the vivid green developed by "normal" strains of S. erysipelatis and S. epidemicus. On this medium "normal" colonies of S. scarlatinae, examined with a culture microscope, appeared slightly granular and conical. Although cultures of scarlatinal streptococci isolated as long as eight years remained stable, others changed and formed colonies that differed from the "normal" type in shape and often in production of color on chocolate agar. The cocci from such atypical colonies were also changed immunologically (opsonic test) and no longer acted like the original scarlatinal group. The atypical colonies observed in old cultures of scarlatinal streptococci were granular convex, and large, moist, smooth convex, both turning chocolate agar green, and small, smooth convex and flat, rough colonies with irregular edges, neither producing green. By transplanting in dextrose broth or by aging, the cocci from these atypical colonies were reverted to the nongreening slightly granular conical colonies typical of S. scarlatinae. The reverted cocci acted immunologically like specific scarlatinal streptococci. Morphologically, on solid mediums, the cocci from dissociated and "normal" strains appeared as small, round cocci in short chains and looked alike, except those from rough colonies, which were larger and showed giant coccoid and pyriform bodies and bacillary-and filamentous-forms and grew in long, twisted chains. The cocci from all the strains grew diffusely in dextrose broth, except those from rough colonies, which produced a granular growth. The cocci from rough colonies were avirulent, while those from other colonies generally killed mice in twenty-four hours.

Vaginal and cervical specimens taken for the detection of gonorrhea frequently contain Candida albicans . Thayer-Martin medium containing nystatin (VCTN) is unsatisfactory because nystatin fails to selectively inhibit C. albicans . Thayer-Martin medium containing no nystatin was prepared with varying concentrations of amphotericin (VCTA) added, and this latter medium was compared with VCTN for its ability to selectively inhibit varying numbers of C. albicans . The effect of amphotericin on the recovery of varying numbers of Neisseria gonorrhoeae was also studied. VCTN failed to inhibit 10 1 to 10 6 C. albicans . VCTA suppressed growth of up to 10 6 C. albicans . The optimal concentration of amphotericin B appeared to be 100 μg/ml. VCTA containing 100 μg of amphotericin B per ml (VCTA 100) was not inhibitory for N. gonorrhoeae when a dense population was applied with a replicator. However, it was slightly inhibitory when 10 1 to 10 2 N. gonorrhoeae were applied over the entire surface of an agar plate, although the colony count on VCTA 100 was more than 50% of the counts on chocolate agar and 75% of the counts on VCTN. These properties of VCTA 100, when stored at 4 C, were stable for up to 2 weeks.

Susceptibility of 64 clinical isolates of Neisseria gonorrhoeae to metronidazole was determined by the agar dilution technique using Mueller-Hinton and chocolate agar media. All isolates were resistant to metronidazole at 12.5 μg/ml in either medium. This lack of activity of metronidazole against N. gonorrhoeae would suggest that the ability to recover N. gonorrhoeae in patients being treated with metronidazole for concurrent Trichomonas vaginalis infections should not be hampered.

The incorporation of trimethoprim into existing selective media using either saponin-lysed blood agar or chocolate agar as the base is suggested for the routine isolation of the gonococcus. The principal advantage over other selective media is the inhibition of Proteus mirabilis and other commensal organisms as a consequence of which multiple swabs can be plated out onto a single selective plate irrespective of source, while the recovery rate of the gonococcus from these specimens is increased. The relative merits of both basic media are briefly discussed.

A series of strains of hemolytic streptococci and mucoid encapsulated streptococci was obtained from the throats of scarlet fever patients at Cook County Contagious Hospital.Laboratory studies were made on a group of these strains to determine. (1) the results of their growth on chocolate agar, (2) their effect on the sugars sorbitol and trehalose and (3) the influence of mouse passage on capsule formation and colony characteristics.Using chocolate agar Tunnicliff1 found that hemolytic streptococci of scarlet fever produced no color change in this medium except occasionally a slight greening after several days'growth. The colonies almost always were conical in shape and slightly granular. Occasionally, however, a strain dissociated and produced atypical colonies that were large, moist, smooth and convex and turned chocolate agar green. These atypical colonies were not scarlatinal by the opsonic, agglutination or toxin neutralization tests.2,3

Objective: To study the prevalence of streptococcal pharyngitis in pediatric patients presenting with sore throat. Methods: A total of 100 patients aged 5.15 years were enrolled in the study. Verbal consent was obtained to collect specimens. The specimens were collected with the help of sterile cotton tipped swab, cultured in blood agar and chocolate agar as per the guideline. Streptococcus pyogenes was identified on the basis of Gram.s stain and its sensitivity towards 0.04 units of bacitracin disc. Antimicrobial susceptibility testing of the isolates was performed using Kirby Bauer disc diffusion method. Finding: Out of 100 patients which were included, 54 were male and 46 were female. GAS was isolated from 5 patients, of which 3 were male and 2 were female. Of the 5, S. pyogenes strains isolated, 2(66.6%) were found to be resistant to cotrimoxazole. Ciprofloxacin resistance was seen in one isolate. No resistance was detected in gentamycin, cefalexin, penicillin, and erythromycin. Conclusion: The results show that the prevalence of streptococcal pharyngitis is 5% among OPD cases, further study should be conducted in the community to know the actual prevalence of streptococcal pharyngitis. Keywords: Pharyngitis; Prevalence; Streptococcus pyogenes DOI: 10.3126/hren.v8i2.4418 Health Renaissance , May-Aug 2010; Vol 8 (No.2):90-92

Blood was cultured in two vaccum bottles containing Columbia broth with sodium polyanethol sulfonate and CO 2 . Filtered air was admitted to one bottle, and the bottles were incubated at 35 C until growth was detected or for a maximum of 7 days. Bottles were examined daily for macroscopic growth. Gram stains were made routinely on the 1st, 4th, and 7th days, and samples were routinely subcultured to sheep blood agar (incubated in GasPak jar) and chocolate agar (incubated in CO 2 ) on the 1st and 4th days of incubation. Of 1,127 positive blood cultures, 65% were first detected by macroscopic examination, 23% were first detected by Gram stain, and 12% were first detected only by subculture.

Media for isolation of Haemophilus sp. from the conjuctiva were compared in an oasis in southern Tunisia where severe trachoma and seasonal epidemic purulent conjunctivitis are common. Of 89 children tested, IsoVitaleX-supplemented chocolate agar yielded Haemophilus in 87%, plain chocolate agar in 75%, sheep blood agar with a stab of Staphylococcus epidermidis in 74%, and Fildes medium in 58%. Since other microbial pathogens are easily identified in the modified blood agar, it was the most useful single medium.

CEPHALORIDINE, a semisynthetic derivative of cephalosporin, has been demonstrated to have in vitro activity against Hemophilus influenzae . 1,2 Nonmeningeal infections with H influenzae have been successfully treated. 3 Few reports of its clinical use in meningitis are available, but significant spinal fluid penetration has been demonstrated in the presence and absence of meningeal inflammation. 4,5 This report describes the use of cephaloridine in the treatment of three infants with H influenzae meningitis and provides data on the spinal fluid penetration, effectiveness, and toxicity of this antibiotic at high dosage levels in meningeal disease. Materials and Methods Three infants with H influenzae meningitis were treated with cephaloridine on the Pediatric Service, Mercy Hospital, Baltimore. Hemophilus influenzae was initially isolated on chocolate agar from the cerebrospinal fluid (CSF) or blood or both of each patient. The minimum inhibitory concentration (MIC) of cephaloridine for each organism was determined by the tube dilution

Walsh, Martin J. (Communicable Disease Center, Atlanta, Ga.), Bobby C. Brown, Leonard Brown, and Carl I. Pirkle . Use of the chick embryo in maintaining and restoring virulence of Neisseria gonorrhoeae . J. Bacteriol. 86: 478–481. 1963.—Data based on its capabilities of producing acute urethritis in human male volunteers showed that Neisseria gonorrhoeae rapidly decreased in virulence after repeated subculture on chocolate agar medium. Cultures of a particular strain (GCM13) in the allantoic cavity of a developing chick embryo maintained virulence after 82 successive transfers. Another strain (GCF62), after loss of virulence through repeated subculture on chocolate agar, was again able to produce infection after 15 consecutive transfers in chick embryo. Thus, chick embryo apparently had the capability not only to maintain the virulence factor but also to reconstitute virulence. It is postulated that chick embryo serves as a selective medium for the growth of virulent gonococci, permitting rapid multiplication of these organisms and suppressing growth of the nonvirulent organisms.

Seventeen recent isolates from a variety of human clinical sources of organisms designated as HB-1 according to the subgrouping of King and Tatum11,12 were studied with respect to their morphologic, cultural, and biochemical characteristics, as well as their experimental pathogenicity in mice. These nonfermentative, Gram-negative rods produced striking, dry, flat, radially-spreading colonies consisting of a clear, moist center circled by a highly refractile, speckled, pearl-like zone of growth resembling mercury droplets, surrounded in turn by an outer nonrefractile perimeter of spreading growth. Growth of all HB-1 strains was enhanced by the concomitant presence of X factor (hemin) and Co2, but growth was not influenced by V factor (NAD). However, on brain–heart infusion agar alone, in the absence of hemin, growth was obtained under anaerobiosis which was comparable to that observed on blood and chocolate agar. Biochemically, all strains were nonfermentative, and only oxidase and nitrate were positive. All of the isolates were susceptible to most of the commonly employed antibiotics with the exception of lincomycin and oxacillin. Experimental mouse pathogenicity could not be demonstrated, but microorganisms of the HB-1 group may conceivably possess pathogenic potential, as evidenced by their derivation from human pathologic sources.

Attempts to reproduce the Shwartzman phenomenon with B. pertussis have been reported by several authors. (Gross,1 Shwartzman,2 Mishulow, Mowry and Scott.3) However, the potency of the nitrates seemed to have varied for unknown reasons and always remained low as compared to similar preparations from B. typhosus,4 meningococcus5 and other organisms.6During some work with serum neutralizations of B. pertussis toxic substances it occurred to the author of this paper to employ Toomey and McClelland's brain veal infusion medium7 for the preparation of the toxic factors necessary for the production of the Shwartzman phenomenon.In these experiments the brain medium was prepared essentially according to the method of Toomey and McClelland with various peptones (neopeptone, proteose-peptone (Difco), Witte's peptone). The H-ion concentration was adjusted either to pH 7.3 or 7.8. The media were seeded with 24-hour-old cultures on “chocolate agar” slants. The strain used was subcultured for at least 3 successive days ...

In previous papers1 I show that hemolyzing streptococci from scarlet fever, which form a specific immunologic group, produce a slightly granular conical colony2 on heated blood agar (chocolate agar), which is not changed in color, in contrast to those peculiar to erysipelas and septic sore throat, which turn this medium green. According to McLeod and Gordon3 the greening of chocolate agar is probably due to the production of hydrogen peroxide. Chocolate agar is prepared by adding 8 cc. of defibrinated sheep blood to 150 cc. of melted and slightly cooled Liebig' s beef extract plain agar, pn 7.0, heated to 90 C, and cooled to 50 C. and pouring the mixture into petri dishes. Freshly poured plates are streaked and either incubated at 37 C. for twenty-four hours and then left at room temperature four or five days, or incubated at 34 C, since the green color is produced slowly by hemolyzing streptococci and as a rule not above 34 C. Dextrose and lack of oxygen inhibit greening. Saelhof4 found that differences in color can be produced by using pure hemoglobin and that a more marked greening of heated hemoglobin agar by hemolyzing streptococci occurred with sheep and rabbit hemoglobin than with horse and human. Some of the colonies of dissociating scarlatinal streptococci, including mucoid, granular convex and conical, turn chocolate agar green but lose this power when reverted to cocci forming conical colonies, immunologically specific for scarlet fever. Stabilized rough colonies do not color chocolate agar. During the course of scarlet fever, not only conical colonies not changing chocolate agar, but non-specific granular, smooth convex and conical colonies are observed which turn chocolate agar green.6 The cocci from these colonies when reverted to S. scarlatinae, colonially and immunologically, lose their ability to change the color of chocolate agar.

Mishulow, Mowry and Scott1 demonstrated that exotoxic filtrates could be obtained from whooping cough cultures. They planted their organisms on chocolate agar over which they had poured a small amount of horse serum-beef heart broth. The small amount of diluent made the harvest of toxin necessarily small. On the other hand, Craster and Smith2 mentioned that there was a signal absence of toxin formation in planted cultures, and Riesenfeld3 stated that there was no proof that this bacillus produced a toxin in ascitic broth. Besredka4 isolated toxic substances termed endotoxins from the dried and powdered organisms.In 1928, we found that pertussis bacillus produced a substance in culture growth, the exact nature of which was problematic. With the report of Mishulow, Mowry and Scott, we felt that further work should be done along this line. Since large amounts of toxin could not be practically produced on solid preparations, various liquid media were tried. We found that Pelouze's and Viteri's gonococcous med...

Our material comprises a study of 256 patients over a period of nine years in private and clinic practice. An additional group of 261 cases is reported from a national questionnaire relative to the use of sulfanilamide. DIAGNOSTIC METHODS Smears by Gram stain have been studied in all cases. Occasional disagreements with regard to diagnosis have been finally settled on the basis of cultures. We have learned to rely very strongly on the Douglas chocolate agar method and are at present using it as a routine. Criteria for cure have remained as formerly outlined 1 —exacting in the extreme. Such a program has, we believe, been instrumental in our satisfactory final figure for cure and our low figure for late recurrence. We prefer to regard recurrence and even possible reinfection as relapse, which of course increases our figure for average time before cure. METHODS OF TREATMENT Distention with Silver Nitrate

IN THE immunofluoreseent diagnosis of gonorrhea previously described (1-3), a direct and a delayed procedure for identifying gonococci in exudates have been used. When sufficient number of gonococci are present in the exudate, direct fluorescent antibody (FA) staining of smears permits detection and identification of the gonococci within less than an hour even when many contaminating bacteria are present. When the direct FA method is used, the presence of only a few gonococci in an exudate results in a 50 percent loss of sensitivity compared with results by the delayed FA method (1). By the delayed FA method, exudate collected with a swab is incubated 16 to 20 hours on chocolate agar slants, and gonococci too few to detect by the direct FA technique groiw to easily observable numbers. Rapid diagnosis of gonorrhea is often necessary, and the longer time required with the delayed FA method is undesirable. When the simpler, faster, direct FA procedure is used, differentiation of specific Neisseria gonorrhoeae fluorescence is difficult even when the organisms are present in sufficient numbers to permit identification. Various counterstains have been reported to increase the effectiveness of the FA method (4-6). These counterstains allow faster and more specific detection of microorganisms in smears by increasing the background contrast. Careful attention to the collection of specimens for direct smears is necessary for the successful detection of gonococci. It has been suggested (7) that the use of a bacteriological loop is preferable to a swab for the collection of specimens from female gonorrheal patients, particularly when a small amount of exudate is present. In this study the results of examination of smears by the direct FA (DFA) method and by the direct FA method using Flazo orange counterstain (FFA) were compared with the cultural results when specimens were obtained with a bacteriological loop.

In studying colonies of hemolytic streptococci on chocolate agar, I find that typical cultures from erysipelas produce a vivid green after from twenty-four to forty-eight hours' growth, while those from scarlet fever produce no color change except occasionally a slightly greenish tinge after several days' incubation. Twenty-five cultures from scarlet fever, thirteen from erysipelas and forty from other sources have been so examined. Most of these cultures have been examined immunologically as well, generally by the opsonic method but many also by agglutination and toxin neutralization tests to determine their specificity. The forty strains from sources other than scarlet fever and erysipelas did not belong immunologically to the scarlet fever group; some turned chocolate agar green, others did not. In examining cultures of scarlet fever and erysipelas streptococci isolated a long time, it is necessary to grow them on a blood agar plate to make sure that the original colonies

IN THE PAST, Neis8eria gonorrhoeae has been isolated on chocolate agar, and it has been taken for granted that the inclusion of hemoglobin in the medium was necessary. The purpose of this study was to determine whether the omission of hemoglobin from the medium would affect the growth of N. gonorrhoeae in primary isolates from discharges of patients. Growth of N. gonorrhoeae on the hemoglobin-free medium was compared with growth on the standard Thayer-Martin (T-M) medium containing hemoglobin.