Lesson N 3: LABORATORY DIAGNOSIS OF MENINGOCOCCAL AND GONOCOCCAL INFECTION1.Scientifically methodical ground of themeNeiss...
III S. pneumoniae, and a third fraction with which the specificity of meningococci is associated.According to the Internat...
Morphology. Gonococci are morphologically similar to meningococci. The organism is a paired,bean-shaped coccus, measuring ...
direct method the organisms under test are exposed to the action of fluorescent antibodies specific togonococci. In the in...
5. Students Practical activities:          1. To study morphology of meningococci and gonococci in museum smears (incomple...
Prior to the use of sulpha nil amide drugs and antibiotics, it is necessary to determine the serovar ofthe meningococcus r...
heated in an incubator. To facilitate better growth of the gonococci, the inoculated plates are placed intoan exsiccator w...
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Bohomolets Microbiology Lesson #3


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Bohomolets Microbiology Lesson #3

  1. 1. Lesson N 3: LABORATORY DIAGNOSIS OF MENINGOCOCCAL AND GONOCOCCAL INFECTION1.Scientifically methodical ground of themeNeisseriacae are Gram-negative diplococci. Although difficult to differentiate on morphological andcultural character, these two pathogens are associated with entirely different diseases. Neisseriameningitidis is the cause of a range of diseases, of which acute purulent meningitis (variously calledepidemic cerebrospinal meningitis, cerebrospinal fever or, because of a purpuric rush is sometimespresent, spotted fever) and an acute septicemic illness with a petechial rash but without meningitis arecommonest. About one-third of cases of meningococcal disease present as septicemia, most other beingof meningiti. The term meningococcal infection embraces these and other syndromes associated with theorganism. N. gonorrhoeae is the cause of sexually transmitted disease gonorrhea. In the newborn maygive rise to a purulent conjunctivitis, ophthalmia neonatorum and in young girls a vulvovaginitis.2.Educational purpose STUDENTS MUST KNOW: 1. Structure, cultivation and biological properties of meningococci. 2. Antigenic structure, serogroups and serovars of meningococci. 3. Epidemiology of diseases. The clinical forms of meningococcal infections, pathogenesis.Immunity. 4. Laboratory diagnosis of meningococcal infections. 5. Specific prophylaxis and therapy of meningococcal diseases. 6. Structure, cultivation and biological properties of gonococci. 7. Pathogenesis and diseases in man. Immunity. 8. Laboratory diagnosis of gonococcal infections. 9. Prophylaxis and treatments of gonorrhea. STUDENTS SHOULD BE ABLE TO: – read the result of complement fixation test for serological diagnosis of gonorrhea; – make smear and stain it by Gram’s and Neisser’s method; – value incomplete phagocytosis of gonococci and memingococci; – value of microbial growth on different media; – carry out determining of microbial sensitivity to antibiotics; – create the schemas of diagnosis of meningococcal and gonococcal infections.3.Chart of topic content. Meningococci. The meningococcus (Neisseria meningitidis) was isolated from the cerebrospinalfluid of patients with meningitis and studied in detail in 1887 by A. Weichselbaum. At present theorganism is classified in the genus Neisseria, family Neisseriaceae.Morphology. The meningococcus is a coccus 0.6-1 mcm in diameter, resembling a coffee bean, and isfound in pairs The organism is Gram-negative. As distinct from pneumococci, meningococci are joinedlongitudinally by their concave edges while their external sides are convex. Spores, capsules and flagella are not formed. In pure cultures meningococci occur as tetrads (infours) and in pus they are usually found within and less frequently outside the leukocytes In culturesmears, small or very large cocci are seen singly, in pairs, or in foursCultivation. The meningococcus is an aerobe or facultative anaerobe and does not grow on commonmedia. It grows readily at pH 7.2-7.4 on media to which serum or ascitic fluid has been added. Optimumtemperature for growth is 36-37 °C and there is no growth at 22° C. Growth is facilitated by 5-10 per centCO2 and high humidity. On solid media the organisms form fine transparent colonies measuring 2-3 mmin diameter. In serum broth they produce turbidity and a precipitate at the bottom of the test tube, andafter 3-4 days, a pellicle is formed on the surface of the medium. Meningococci can be adapted to simplemedia by repeated subculture on media with a gradual change from the optimum protein concentration tomedia containing a minimal concentration of proteins. Fermentative properties. Meningococci do not liquefy gelatin, cause no change in milk, andferment glucose and maltose, with acid formation. Virulenсe factors. Meningococci have 3 important virulence factors: polysaccharide capsulethat enables organism to resist phagocytosis, endotoxin, which causes fever, shock, and other, andimmunoglobulin A(Ig A) protease, which , by cleaving sescretory Ig A, helps the bacteria to attach to themembranes of respiratory tract. Antigenic structure and classification. Meningococci were found to contain three fractions:carbohydrate (C) which is common to all meningococci, protein (P) which is found in gonococci and type 1
  2. 2. III S. pneumoniae, and a third fraction with which the specificity of meningococci is associated.According to the International Classification, four groups of meningococci are distinguished, groups A,B, C, and D. Recently the number of types has increased to seven, but only the first two are dominant. The organisms are characterized by intraspecies variability. A change of types takes place atcertain times. Resistance. The meningococcus is a microbe of low stability, and is destroyed by drying in a fewhours. By heating to a temperature of 60° C it is killed in 10 minutes, and to 80 °C, in 2 minutes. Whentreated with 1 per cent phenol, the culture dies in 1 minute. The organism is very sensitive to lowtemperatures. Bearing this in mind, test material should be transported under conditions which protect themeningococcus against cooling. Pathogenesis and diseases in man. People suffering from meningococcal infection and carriersare sources of diseases. The infection is transmitted by the air-droplet route. The causative agent islocalized primarily in the nasopharynx. From here it invades the lymph vessels and blood and causes thedevelopment of bacteriemia. Then as a result of metastasis the meningococci pass into the meninges andproduce acute pyogenic inflammation in the membranes of the brain and spinal cord (nasopharyngitis,meningococcaemia, meningitis). The disease usually arises suddenly with high temperature, vomiting, rigidity of the occipitalmuscles, severe headache, and increased skin sensitivity. Later paresis of the cranial nerves develops dueto an increase in the intracranial pressure. Dilatation of the pupils, disturbances of accommodation, aswell as other symptoms appear. A large number of leukocytes are present in the cerebrospinal fluid, andthe latter after puncture escapes with a spurt because of the high pressure. In some cases meningococcal sepsis develops. In such conditions the organisms are found in theblood, joints, and lungs. The disease mainly attacks children from 1 to 5 years of age. Before the use ofantibiotics and sulphonamides the death rate was very high (30-60 per cent). The population density plays an important part in the spread of meningitis. During epidemicoutbreaks there is a large number of carriers for every individual affected by the disease. In non-epidemicperiods the carrier rate increases in the spring and autumn. Body resistance and the amount and virulenceof the causative agent are significant. Depending on these factors, the spread of infection is eithersporadic or epidemic. Meningitis can also be caused by other pathogenic microbes (streptococci, E. coli, staphylococci,bacteria of influenza, mycobacteria of tuberculosis, and certain viruses). These organisms, however,cause sporadic outbreaks of the disease, while meningococci may cause epidemic meningitis. Immunity. There is a well-developed natural immunity in humans. Acquired immunity isobtained not only as a result of the disease but also as the result of natural immunity developed during themeningococcal carrier state. In the course of the disease agglutinins, precipitins, opsonins, andcomplement-fixing antibodies are produced. Recurring infections are rare. Laboratory diagnosis. Specimens of cerebrospinal fluid, nasopharyngeal discharge, blood, andorgans obtained at autopsy are used for examination. The following methods of investigation are employed: (1) microscopic examination ofcerebrospinal fluid precipitate; (2) inoculation of this precipitate, blood or nasopharyngeal discharge intoascitic broth, blood agar, or ascitic agar; identification of the isolated cultures by their fermentative andserologic properties; differentiation of meningococci from the catarrhal micrococcus (Branhamellacatarrhalis) and saprophytes normally present in the throat. The meningococcus ferments glucose andmaltose, whereas Branhamella catarrhalis does not ferment carbohydrates, and Neisseria sicca fermentsglucose, levulose, and maltose; (3) performance of the precipitin reaction with the cerebrospinal fluid. Treatment. Antibiotics (penicillin, oxytetracycline, etc.) and sulphonamides (streptocid,methylsulphazine) are prescribed. Prophylaxis is ensured by general sanitary procedures and epidemic control measures (earlydiagnosis, transference of patients to hospital), appropriate sanitary measures in relation to carriers,quarantine in childrens institutions. Observance of hygiene in factories, institutions public premises, andlodgings, and prevention of crowded condition are also obligatory. An antimeningococcal vaccine derivedfrom the C/B serogroup is now under test. It contains specific polysaccharides. The incidence of meningitis has grown recently. The disease follows a severe course andsometimes terminates in death. Gonococci. The causative agent of gonorrhoea and blennorrhoea (Neisseria gonorrhoeae) wasdiscovered in 1879 by A. Neisser in suppurative discharges. In 1885 E. Bumm isolated a pure culture ofthe organism and studied it in detail. Gonococci belong to the genus Neisseria, familyNeisseriaceae. 2
  3. 3. Morphology. Gonococci are morphologically similar to meningococci. The organism is a paired,bean-shaped coccus, measuring 0.6-1 mcm in diameter. It is Gram-negative and occurs inside and outsideof the cells. Neither spores nor flagella are formed. Under the electron microscope a cell wall, 0.3-0.4mcm in thickness, surrounding the gonococci is visible. Pleomorphism of the gonococci is a characteristicproperty. They readily change their form under the effect of medicines, losing their typical shape, andgrowing larger, sometimes turning Gram-positive, and are found outside the cells. In chronic forms of the disease autolysis of the gonococci takes place with formation of varianttypes (Asch types). Usually gonococcal cells varying in size and shape are formed. The tendency towardmorphological variability among the gonococci should be taken into account in laboratory diagnosis. L-forms occur under the effect of penicillin. Cultivation. The gonococcus is an aerobe or facultative anaerobe which does not grow onordinary media, but can be cultivated readily on media containing human proteins (blood, serum, asciticfluid) when the pH of the media is in the range of 7.2-7.6. The optimum temperature for growth is 37° C,and the organism does not grow at 25 and 42° C. It is essential to provide 5-10 per cent CO2.It alsorequires an adequate degree of humidity. Ascitic agar, ascitic broth, and egg-yolk medium are the mostsuitable media. On solid media gonococci produce transparent, circular colonies, 1-3 mm in diameter.Cultures of gonococci form a pellicle in ascitic broth, which in a few days settles at the bottom of the testtube. Fermentative properties. The gonococcus possesses low biochemical activity and no proteolyticactivity. It ferments only glucose, with acid formation. Virulenсe factors The gonococci do not produce soluble toxin (exotoxin). An endotoxin isreleased as a result of disintegration of the bacterial cells. This endotoxin is also toxic for experimentalanimals. Pili are most of the important virulence factors, because they mediate attachment to mucosal celsurface and are antiphagocytic. Piliated gonococci are usually virulent, whereas nonpiliated strains areavirulent. Ig A protease can hydrolyze secretory Ig A, which could otherwise block attachment to themucosa. Gonococci have no capsules. Antigenic structure and classification. The antigenic structure of gonococci is associated withthe protein (O-antigen) and polysaccharide (K-antigen) fractions. No group specific or international typesof gonococci have been revealed. Gonococci and meningococci share some antigens in common. Resistance. Gonococci are very sensitive to cooling. They do not survive drying, although theymay live as long as 24 hours in a thick layer of pus or on moist objects. They are killed in 5 minutes at atemperature of 56 °C, and in several minutes after treatment with a 1 : 1000 silver nitrate solution or 1 percent phenol. Pathogenesis and diseases in man. Patients with gonorrhoea are sources of the infection. Thedisease is transmitted via the genital organs and by articles of domestic use (diapers, sponges, towels,etc). The causative agent enters the body via the urethral mucous membranes and, in women, via theurethra and cervix uteri. Gonorrhoea is accompanied by acute pyogenic inflammation of the urethra,cervix uteri, and glands in the lower genital tract. Often, however, the upper genito-urinary organs arealso involved. Inflammations of the uterus, uterine tubes, and ovaries occur in women, vulvovaginitisoccurs in girls, and inflammation of the seminal vesicles and prostata in men. The disease may assume achronic course. From the cervix uteri the gonococci can penetrate into the rectum. Inefficient treatmentleads to affections of the joints and endocardium, and to septicaemia. Gonococci and Trichomonasvaginalis are often found at the same time in sick females. The trichomonads contain (in the phagosomes)gonococci protected by membranes against the effect of therapeutic agents. Gonococcus is responsible forgonorrhoeal conjunctivitis and blennorrhea in adults and newborn infants. Immunity. The disease does not produce insusceptibility and there is no congenital immunity.Antibodies (agglutinins, precipitins, opsonins, and complement-fixing bodies) are present in patientssera, but they do not protect the body from reinfection and recurrence of symptoms. Phagocytosis ingonorrhoea is incomplete. The phagocytic and humoral immunity produced in gonorrhoea is incapable ofproviding complete protection, so, in view of this fact, treatment includes measures which increase bodyreactivity. This is achieved by raising the patients temperature artificially. Laboratory diagnosis. Specimens for microscopic examination are obtained from the dischargeof the urethra, vagina, vulva, cervix uteri, prostate, rectal mucous membrane, and conjunctiva. The spermand urine precipitates and filaments are also studied microscopically, Smears are stained by Gramsmethod and with methylene blue by Loefflers method). Microscopy is quite frequently an unreliablediagnostic method since other Gram-negative bacteria, identical to the gonococci, may be present in thematerial under test. Most specific are the immuno-fluorescence methods (both direct and indirect). In the 3
  4. 4. direct method the organisms under test are exposed to the action of fluorescent antibodies specific togonococci. In the indirect method, the known organisms (gonococci) are treated with patients serum. Thecombination of the antibody with the antigen becomes visible when fluorescent antiserum is added. If diagnosis cannot be made by microscopic examination, isolation of the culture is carried out.For this purpose the test material (pus, conjunctival discharge, urine precipitate, etc.) is inoculated ontomedia. The Bordeux-Gengou complement-fixation reaction and the allergic test are employed in chronicand complicated cases of gonorrhea. Treatment. Patients with gonorrhoea are prescribed antibiotics (bicillin-6, ampicillin,monomycin, kanamycin) and sulphonamides of a prolonged action. Injections of polyvalent vaccine andautovaccine as well as pyrotherapy (introduction of heterologous proteins) are applied in complicatedcases. Improper treatment renders the gonococci drug-resistant, and this may lead to the development ofcomplications and to a chronic course of the disease. Prophylaxis includes systematic precautions for establishing normal conditions of everyday andfamily life, health education and improvement of the general cultural and hygienic standards of thepopulation. In the control of gonorrhoea great importance is assigned to early exposure of sources ofinfection and contacts and to successful treatment of patients. The prevention of blennorrhea is effected by introducing one or two drops of a 2 per cent silvernitrate solution into the conjunctival sac of all newborn infants. In certain cases (in prematurely borninfants) silver nitrate gives no positive result. Good results are obtained by introducing two drops of a 3per cent penicillin solution in oil into the conjunctival sac. The gonococci are killed in 15-30 minutes. In spite of the use of effective antibiotics the incidence of gonorrhoea tends to be on the increasein all countries (Africa, America, South-Eastern Asia, Europe, etc.). The number of complications hasalso increased: gonococcal ophthalmia of newborn infants (blennorrhea), vulvovaginitis in children, andinflammation of the pelvic organs (salpingitis) and sterility in women. The rise in the incidence ofgonorrhoea is caused by social habits (prostitution, homosexualism, etc.), inefficient registration ofindividuals harbouring the disease, deficient treatment, and the appearance of gonococci resistant to thedrugs used. The WHO expert committee has recommended listing the gonococcal infection among infectiousdiseases with compulsory registration and making a profound study of the cause of the epidemic characterof gonococcal diseases in certain African countries. Stricter blennorrhea control measures, andelaboration of uniform criteria of clinical and laboratory diagnosis, and treatment of gonococcal infectionand more efficient methods for determining the sensitivity of circulating gonococci to various drugs arealso recommended by the committee.4. Student’s independent study program 1. Structure and biochemical properties of meningococci. Cultivation. Virulenсe factors andpathogenicity of causative agents. Resistance in an external environment. 2. Characterize antigenic structure, serogroups and serovars of meningococci. 3. Epidemiology of diseases. The clinical forms of meningococcal infections, pathogenesis.Immunity. 4. Laboratory diagnosis of epidemic cerebrospinal meningitis: a – describe the features of receipt of material for examination; b – microscopical method of diagnosis; c – characteristic of main stages of bacteriological method of diagnosis; d – role of serological tests (precipitation test, counter immunoelectrophoresis, radioimmunemethods) as express methods of diagnosis of epidemic cerebrospinal meningitis. 6. Specific prophylaxis and therapy of meningococcal diseases. 7. Structure, staining, biochemical properties of gonococci. Cultivation. 8. Virulence factors of gonococci and mechanism of their penetration into organism. 9. Resistance of gonococci. 10. Pathogenesis and diseases in man. Immunity. 11. Bacterioscopic method of diagnosis of gonorrhea. The method of receipt of tested material. 12. Value of bacteriological method of diagnosis of gonorrhea. 13. Diagnostic value of a Bordeux–Gengou test. 14. Main methods of provocations in diagnosis and treatment of gonorrhea. 15. Prophylaxis and treatments of gonorrhea 4
  5. 5. 5. Students Practical activities: 1. To study morphology of meningococci and gonococci in museum smears (incompletephagocytosis). 2. To study peculiarities of gonococci and meningococci growth on nutrient media. 3. To familiarize with biological preparations, which are used for diagnosis, specific prophylaxisand treatment of gonococcal and meningococcal diseases. 4. To describe the features of growth of bacteria on blood agar, make smear, stain it by Gram’stechnique, create scheme of identification of isolated streptococci culture. 5. To continue diagnostic of staphilococcal infection. Examine bacterial growth in MPA.Subcultivate culture in Hiss’s media, blood agar and plasma. Start determining of isolated culturesensitivity to antibiotics and phagotype. MENINGOCOCCAL INFECTION. Meningococcal infection is caused by meningococci(Neisseria meningitidis). The material to be tested is secretions from the nasal portion of the throat,cerebrospinal fluid, blood, and scrapings from elements of the haemorrhagic rash on the skin. Cerebrospinal fluid is collected into a sterile tube to be inoculated onto nutrient media or to bepromptly sent (without allowing it to cool down) to the laboratory. This requirement is necessitated bythe fact that meningococci are very sensitive to temperature fluctuations. Mucosal secretions in the nasal portion of the throat are collected with a special swab bent at adefinite angle. The best results are obtained when the nasopharyngeal mucus is immediately streaked ontosolid nutrient media. To achieve the maximal separation of bacterial cells, 2-3 plates with medium areutilized. If the material is to be studied 3-5 hrs after the collection, it is inoculated onto a liquid nutrientmedium (casein hydrolysate of fermentative splitting, which contains 1.5 g/1 of amine nitrogen and 250U/ml of ristomycin) and then placed in a 37 °C water bath. Thereafter, it is streaked onto serum agar andplaced into an incubator. Bacterioscopic examination of cerebrospinal fluid and blood permits detection of the causativeagent. If the cerebrospinal fluid looks like pus, smears are prepared without its preliminary treatmentwhereas in the presence of only mild turbidity the cerebrospinal fluid is centrifuged and the deposit isused to make smears. The latter are stained with aniline dyes (aqueous solution of basic fuchsine,methylene blue) since the Gram staining method is associated with alteration in the formed elements ofthe cerebrospinal fluid and a large number of artefacts. Meningococci appear as bean-shaped diplococcisituated within the leukocyte cytoplasm and touching each other with concave edges. A tender capsule isquite a frequent finding. In meningococcaemia meningococci may be demonstrated in blood smears. Athick-drop (film) preparation is made, stained for 2-3 min with aqueous solution of methylene bluewithout fixation, washed in tap water, and dried in the air. On a light blue background of the preparationone can see dark blue leukocytes with numerous small dark-blue cocci arranged in clusters, pairs, andsingly in and around leukocytes. Rapid diagnosis is performed by means of gel precipitation, counter-immunoelectrophoresis withgroup precipitating antisera or radioimmunoassay and based on the detection in the patients cerebro-spinal fluid or blood of the specific meningococcal antigen. Bacteriological examination. The cerebrospinal fluid or its sediment is cultured simultaneouslywith conducting bacterioscopic study. The meningococcus grows on special nutrient media containingnative protein (serum broth and agar). One can also use Hottingers agar containing 0.15 per cent ofinsoluble starch, which does not change the cultural, fermentative, and agglutinating properties of thecausative agent. It is preferable that the cerebrospinal fluid be cultured after centrifugation at 3500 X g forfive minutes. Some 0.3-0.5 ml of the material is taken from the bottom and 2-3 drops are placed on thesurface of heated nutrient medium. The inoculated culture is incubated at 37 °C and in conditions ofelevated CO2 contents. To do it, place onto the lid of a sterile Petri dish a sheet of filter paper soaked with1.5-2.0 ml of 10 per cent pyrogallic acid and then cover it with a second sheet moistened with 1.5-2.0 mlof 20 per cent solution of sodium hydrocarbonate. The inoculated dish is covered with the lid containingthe paper sheets and inverted (lid downward). The remainder of the cerebrospinal fluid is utilized forcounter-immunoelectrophoresis. On the second day of incubation at 37 °C, the growth is studied for its cultural properties.Meningococci form small, round, convex, and transparent colonies. Smears made of these coloniesdisplay polymorphic diplococci and tetracocci. The microscopic picture is so diverse that it creates theimpression of unpure culture. The colonies are subcultured onto a serum agar slant. On the third day of investigation, the isolated culture is agglutinated with meningococcal sera. 5
  6. 6. Prior to the use of sulpha nil amide drugs and antibiotics, it is necessary to determine the serovar ofthe meningococcus responsible for the disease since treatment is based on specific meningococcal sera.The agglutination test in Nobles modification is currently employed for determining the meningococcalserovar with an epidemiological purpose. Three-drop portions of thick suspension of microorganisms arepoured into three test tubes, then three-drop aliquots of undiluted or diluted 1:10 meningococcal serum ofA, B, and C serovars are added to them. The mixture is shaken for 2-4 min, then 10-20 drops of isotonicsodium chloride solution are added to each test tube, and the results are read. To assay the fermentative activity of pure culture, it is transferred to media with lactose, glucose,maltose, sucrose, and fructose. Meningococci ferment glucose and maltose with the production of acid.The culture is also streaked onto a 5 per cent yolk agar and serum agar containing 5 per cent sugar. Aftera 48-hour incubation, 1 drop of Lugols solution is put on the surface of the grown colonies. Theappearance of brownish staining indicates polysaccharide splitting. Neisseria are identified by the oxidasetest which consists in the following. On the colony formed on the serum agar place a drop of the freshly-prepared 1 per cent solution of hydrochloric paradiethylphenylendiamine. As a result, colonies possessingoxidase activity turn pink and then black. Such colonies are transferred to a serum agar for furtherinvestigation. To differentiate between the meningococcus and non-pathogenic Neisseria (Neisseria catarrhalis),the ability of the latter to grow on simple nutrient media and to form colonies at room temperature (22°C) is utilized. To demonstrate the meningococcus in the blood, introduce 5-10 ml of blood obtained from a veinunder sterile conditions into vials with 50 ml of broth containing 0.1 per cent of agar-agar. Subcultureonto a serum agar 24 hours later. The procedures of isolation and identification of the cultures are thesame as in the examination of cerebrospinal fluid. Indirect haemagglutination with erythrocytes sensitized with group-specific polysaccharides isemployed for serological diagnosis. GONOCOCCAL INFECTION. The causative agent of gonorrhoea is the gonococcus (Neisseriagonorrhoeae), which is morphologically similar to the meningococcus. Bacterioscopic, bacteriological,and serological techniques are employed for the diagnosis of this disease. Bacterioscopic examination is the main method for diagnosing acute gonorrhoea and blennorrhea.The material for examination is taken from the urethra in the following manner: wipe the urethralopening with cotton wool moistened with sterile physiological salt solution, press with your finger ontothe posterior wall of the urethra in the outward direction (in females the forefinger is inserted into thevagina for this purpose), and express a drop of pus. The secretion from the prostate is obtained byprostatic massage. The secretion of the cervical mucosa is collected with a swab, following intravaginalintroduction of Cuscos speculum. In patients with blennorrhea conjunctival secretion is removed with aloop and spread over a glass slide. The preparation is stained with alkaline solution of methylene blue andwith the Gram stain (two smears). Upon microscopic examination gonococci appear as bean-shapedGram-negative diplococci positioned outside or inside the cells (neutrophilic granulocytes) similar tomeningococci. Grams staining allows differentiation of the gonococci from other bacteria. To ensure a moredistinct outline of the gonococci, smears should be fixed by dimethylsulphoxide (dimexide). Pourdimexide on the smear until it is completely dry and then stain it. Since the examined material may also contain other Gram-negative bacteria resembling thegonococci, both direct and indirect immunofluorescence methods are employed. In the directimmunofluorescence test the smears are treated with fluorescent antibodies against gonococci, in theindirect one, gonococci and the patients serum are used. Conjugation between the antibody and theantigen becomes evident when a fluorescent serum against human globulins is added. Bacteriological examination is carried out when the study of smears reveals either no gonococcior only their atypical, altered forms. In view of extreme sensitivity-of the gonococcus to temperature thematerial tested should not be transported. Moreover, the gonococcus is very sensitive to disinfectants, soit is advisable that 1 to 2 days before culturing the patients should temporarily discontinue the use ofdisinfectants and antibacterial drugs. The material is inoculated immediately after its collection onto plates with a protein-containingmeat-peptone agar. Ascitic-free media with casein digest, yeast autolysate, and native cattle serum arewidely utilized for this purpose. Inclusion into the nutrient medium of ristomycin and poIymixin M (10U/ml) significantly enhances gonococcal growth. Prior to inoculation, the nutrient medium should be 6
  7. 7. heated in an incubator. To facilitate better growth of the gonococci, the inoculated plates are placed intoan exsiccator with a CO2 concentration amounting to 10 per cent. A 24-hour incubation at 37 °C brings about the formation of transparent, with smooth edges,convex, mucoid colonies of the gonococcus, which resemble drops of dew. Pure culture is isolated andidentified. Biochemically, the gonococcus shows weak activity and breaks down only glucose with theformation of acid. To determine oxidase activity, the culture is introduced into yolk medium (to 100 ml ofprotein-containing meat-peptone agar add 1.5 g of glucose, 6 ml of phenol red solution, and 15 ml of eggyolk). Agglutination with specific serum does not always yield positive results because the gonococcushas many serovars and the serum may contain only low titres of the appropriate agglutinins. Serological diagnosis is resorted to in chronic gonorrhoea when the patient has no discharge, andbacterioscopic and bacteriological examinations are impossible. In such cases the complement-fixationtest with the patients blood serum or indirect immunofluorescence is used. A gonococcal vaccine or aspecial antigen prepared of killed (by variable methods, with antiformin being the most common one)gonococci is employed as the antigen.6.Control questions and test: Choose the correct answers: 1. Meningococci have such properties: a – gram-negative; b – lancet-shaped form; c – tetracocci; d– diplococci; e – stain with methylene blue. 2. Cultivation of meningococci: a – grow on МPA and in МPB; b – require the presence ofcarbohydrates; c – grow on media with serum; d – give good growth on Endo’s and Levin’s media; e – onagar with serum form fine transparent colonies. 3. For differentiation between meningococci and gonococci such tests are used: a – fermentation oflactose b – fermentation of glucose and maltose; c – morphological properties; d – motility; e – hemolysistest. 4. Antigenic structure of meningococci and their classification: a – they are classified intoserogroups A, B, C, D, Х; b – determination of serovars is necessary for treatment of the patients; c –most strains from outbreaks are A, B, C serovars; d – determination of serovars is necessary forepidemiological analysis; e – serovars are determined by surface antigens. 5. Choose diseases, which are caused by meningococci: a – epidemic cerebrospinal meningitis; b–nasopharyngitis; c – furunculosis; d – meningococcaemia, e-pneumonia. 6. Methods of laboratory diagnosis of meningococcal infections: a – bacteriological; b –serological; c – allergic; d – hemolysis test; e – inoculation of yolk salt agar. 7. In bacteriological diagnosis of an epidemic cerebrospinal meningitis such material is used: a –lavage waters of the stomach; b – liquor; c – samples of washings from skin surface; d – vomitivemasses; e – urine. 8. Main methods of diagnosis of epidemic cerebrospinal meningitis: a – biological; b – allergic; c– bacteriological; d – precipitation test; д – Coombs’ test. 9. Gonococci have such properties: a – paired bean-shaped cocci; b – lancet-shaped cocci; c –gram-positive; d – gram-negative; e – have fimbriae. 10. Cultivation and biochemical properties of gonococci: а – require special media; b – arecultivated on serumal and ascitic media; c – are cultivated on sugar MPA; d – fermentate glucose; e –fermentate glucose and maltose.7. List of literature: 1. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, part 2, Lugansk,2004,Chapter16, p.20-28. 7