The set of reagents “Nutrient medium for the isolation of gonococci, dry” (GNK agar) is intended for microbiological purposes. It is used for isolating gonococcus when studying infected material, as well as as a medium for cultivating and storing gonococcus in research work.
The reagent kit is available as a set of two lyophilized components:

• basics of the nutrient medium - lyophilisate from a volume of 100 ml in a bottle - 1 piece;
• additive to the base of the nutrient medium - lyophilisate from a volume of 24 ml in a bottle - 1 pc.

Compound: basis of the nutrient medium - rabbit meat extract, dry enzymatic peptone for bacteriological purposes, sodium chloride, baker's yeast autolysate, orotic acid, microbiological agar. Additive to the base of the nutrient medium - normal liquid horse whey for bacteriological nutrient media, baker's yeast autolysate, aminopeptide for microbiological nutrient media.

Preparation: 100 ml of sterile purified water is added to the bottle with the base of the nutrient medium and kept for (30±5) minutes, then placed in a boiling water bath until the agar is completely melted, after melting the agar is kept in a water bath for no more than 5 minutes. Add 24 ml of sterile purified water to the bottle with the addition of nutrient medium and leave for (20±2) minutes until complete dissolution at a temperature of 20-25°C. The dissolved additive is transferred to a dissolved base cooled to a temperature of (50±2)°C. The medium is stirred, 3 ml are poured into sterile test tubes and mowed. 0.5 ml of a sterile solution of sodium chloride 0.9% is added to each test tube to moisten the medium. The medium can also be poured 10-15 ml into sterile Petri dishes. Test tubes or dishes with nutrient medium are placed for (24±1) hours in a thermostat at a temperature of (37±1)°C to control sterility. The prepared nutrient medium is stored at a temperature of (6±2)°C for up to 7 days.

Operating principle: the set of reagents should ensure the growth of each test strain of Neisseria gonorrhoeae "Loshakov" and Neisseria gonorrhoeae "Denisov" when inoculating 0.5 ml of microbial suspension from a dilution of 10"5 after 24 - 48 hours of incubation at a temperature of (37 ± 1) °C in the form transparent or translucent colorless colonies ranging in size from 0.5 to 2 mm with smooth edges.

Carrying out analysis: The results of inoculation are recorded after 24, 48 and 72 hours of incubation at a temperature of (37±1)°C. When inoculating the secretions of the genitourinary organs of patients with gonorrhea, the growth of gonococcus on the nutrient medium should be observed after 24-72 hours. If there is no growth of gonococcus, keep the crops in a thermostat for up to 7 days.

Packaging: 0.25 kg polyethylene jars for preparing 3.6 liters of medium.

Shelf life - 1 year.

The price is given including VAT per 1 kg.

Gonococci are bean-shaped, arranged in the form of diplococci, surrounded by a microcapsule, do not have flagella, and do not form spores, similar to meningocci. The cell wall has an outer membrane, the proteins of which are divided into three groups according to their functional significance. Gonococci are characterized by the presence of pili, which differ from each other in their antigenic properties (16 antigenic variants). Gonococci are cultivated on nutrient media containing native protein (blood serum, ascitic fluid). They grow better at 3-5% CO2. Transparent colonies with smooth edges form on ascitagarus. Of carbohydrates, only glucose is fermented, catalase and cytochrome oxidase are formed - enzymes typical of Neisseria.

Antigens

The antigenic structure of gonococci is variable. This is due to the presence of numerous antigenic variants of pili, which are formed during the development of infection.

Pathogenicity and pathogenesis

Gonococci attach to the cylindrical epithelium of the urethra, the vaginal part of the cervix, rectum, conjunctiva of the eye, as well as sperm and protozoa (Trichomonas, amoeba). Adhesion occurs due to pili and proteins of the outer membrane of the cell wall. A characteristic feature of gonococci is their ability to penetrate leukocytes and multiply in them. The lipooligosaccharide part of the cell wall has a toxic effect. Capsular polysaccharides inhibit phagocytosis. Connecting with the villi of the cylindrical epithelium of the urethral mucosa, and in women, the endocervical canal, gonococci penetrate into the cells with the participation of proteins of the outer membrane of the cell wall. This leads to the development of acute urethritis, cervicitis and damage to the cervix, appendages (tubes, ovaries) in women, seminal vesicles, and prostate gland in men. With extragenital localization, gonococci can damage the rectum and tonsils, and also cause blenorrhea (conjunctivitis) in newborns. Infection occurs during the passage of the birth canal of a mother with gonorrhea.

Immunity

With gonorrhea, a humoral immune response occurs. However, the resulting antibacterial antibodies do not have protective properties. During the course of the disease, IgA is formed, which suppresses the attachment of the pathogen's pili to the cells of the urethral mucosa. However, they are not able to protect the mucosa from subsequent infection by other generations of gonococci, which is associated with a change in their antigenic structure. This leads to reinfections and relapses, as well as the disease becoming chronic.

Gonococcal infections

The causative agent of gonorrhea and blenorrhea N.gonorrhoeae (preliminarily classified as gonococcus) belongs to the family Neisseriaceae, genus Neisseria. In smears from patient secretions, gonococci have the shape of coffee beans, are gram-negative, and are located in pairs both inside leukocytes (incomplete phagocytosis) and outside the cells. According to their morphological characteristics, they are very similar to meningococci. Gonococci are characterized by polymorphism - there are small and large cells, rarely rod-shaped. They are very picky about nutrient media. They grow better on media containing blood, serum, and ascitic fluid. Gonococci contain protein and polysaccharide antigens, according to which they are divided into 16 serovars, but they are not yet determined in routine bacteriological laboratories. For the microbiological diagnosis of gonococcal infections, bacterioscopic, bacteriological, serological and allergic methods are used.

Taking material for research

In order to carry out bacteriological and bacterioscopic diagnostics with dignity and good quality, it is important to correctly take clinical material. As a rule, it should be carried out by a doctor. In men, the secretions of the urethra, paraurethral ducts, rectum are examined, and, if indicated, material from the oropharynx, as well as the secretion of the prostate gland after its massage. You can also examine sediment and “threads” of urine, but gonococci are detected much less frequently in them. Before taking material from the urethra, the patient should not urinate for 4-5 hours, and not use antimicrobial drugs and disinfectant solutions. The external opening of the urethra is first wiped with a sterile cotton swab moistened with a 0.85% sodium chloride solution, then with a dry swab. Smears are made not from manure, which flows freely, but from material taken by scraping from the urethral mucosa with a bacteriological loop or a special Volkmann spoon. For minor discharge, it is necessary to perform a preliminary urethral massage. In women, material is taken from the urethra, paraurethral passages, cervix, rectum, and, if indicated, from the oropharynx. First, the vagina is cleaned of secretions, the urethra is massaged, and the material is removed by scraping with a bacteriological loop or a Volkmann spoon. The cervix is ​​first wiped with a sterile cotton swab to remove the mucus plug. Discharge from the cervical canal is taken with a bacteriological loop or tweezers. Material from the distal rectum is taken using a Volkmann spoon in a blind manner, i.e. without any preparation of the patient, or using a recoscope or rectal speculum. In this case, the material being studied is taken directly from the visible site of the lesion. With oropharyngeal gonorrhea, mucus They are taken from the oropharynx with sterile cotton swabs on special holders made of steel wire. To diagnose lenorrhea, the conjunctival secretion is removed with a bacteriological loop. Rarely, gonorrhea is complicated by gonosepsis, endocarditis, or arthritis. Then the material for juslidzhenya is blood or synovial fluid. Taking into account the high sensitivity of gonococci to temperature fluctuations, the materials under study are delivered to the laboratory in special thermoses or bags with a heating pad.

Bacterioscopic examination

Bacterioscopic examination is the most common, although less sensitive method of laboratory diagnosis of gonorrhea and blenorrhea compared to the isolation of actual cultures. This is especially true for the chronic course of the disease, when the test material contains a small amount of gonococci. However, with the correct collection of material, repeated examinations of patients, the use of provocation methods, and qualified assessment of smears, bacterioscopic examination quite often makes it possible to quickly and correctly diagnose the disease. Two thin, uniform smear preparations are made from the material being studied. One is stained with methylene blue, the second is stained using the Gram method. In the absence of methylene blue, one smear can be stained with a 1% aqueous solution of crystal violet or 0.5% solution of brilliant green for 1 minute. A conclusion about the presence of gonococci is made based on their properties: gram-negative color, diplococcal structure, shape of coffee beans, location inside leukocytes. Under the influence of antibiotics and other chemotherapy drugs, as well as in chronic gonorrhea, the morphology and color of gonococci can change. Individual cells acquire different shapes and sizes (the so-called Asch forms). In addition, the test material may contain gram-negative cocci similar to gonococci from the genus Veillonella. This to some extent limits the diagnostic value of the primary microscopy method. The best and most reliable results are obtained by the immunofluorescence method. Thin smears from the patient's secretions are fixed in the burner flame. Fluorescein isothiocyanate-labeled anti-gonococcal serum is applied to them for 1 hour at 35 ° C in a humid chamber. After this, the smears are washed twice with a buffer solution, buffered with glycerol are applied and covered with coverslips. When gonococci interact with labeled antibodies, a characteristic glow around the bacterial cells is visible under a fluorescent microscope.

Bacteriological research

Indications for isolating pure cultures of gonococci are repeated negative bacterioscopy results, the presence of microorganisms suspicious for gonococci, but not morphologically identified, as well as for reliably establishing the cure of the disease. It is very important to place the crops in the thermostat immediately. If it is impossible to carry out cultures at the site of collection of the material, you can hang a cotton swab into a test tube with Stewart's transport medium, which ensures the preservation of the viability of gonococci during delivery to the laboratory. Cultures are carried out according to the standard scheme in one of the special nutrient media in test tubes or Petri dishes (CDS, Bailey , blood or serum agar, dry nutrient medium of the Kharkov enterprise "Biolek" for the production of bacterial and medicinal preparations). For diagnostic cultivation of gonococci in many countries, “chocolate” agar is also used. The best media are those based on rabbit meat agar or fresh bovine hearts. Adding 20 units/ml of polymyxin and 2 μg/ml of lincomycin to them significantly increases the frequency of inoculation of gonococci, since these drugs inhibit the growth of other bacteria. Before sowing, all media are heated in a thermostat for 15-20 minutes. Dishes and test tubes with cultures are placed in desiccators, where an atmosphere of 20% CO2 is created. Colonies usually grow within 18-24 hours, but late growth is also possible. Then the crops are kept in a thermostat (in a desiccator!) for up to 8 days, checking the appearance of growth daily. The grown colonies of gonococci have a round, slightly convex shape, smooth edges, a shiny surface, and a mucous consistency. They are transparent, like drops of dew, almost colorless, although whitish variants can also occur. The resulting colonies are examined macroscopically and microscopically. In smears, gonococci are located in pairs, tetrads and clusters. Typical colonies are subcultured onto serum agar slants to isolate a pure culture. The final identification is carried out taking into account the morphological, cultural, enzymatic and antigenic properties. Biochemically, gonococci are little active. On whey media with 1.5% of various carbohydrates, they decompose only glucose, but not maltose and sucrose. The oxidase activity of isolated cultures is determined by applying a 1% solution of dimethylparaphenylenediamine to the colonies (after microscopy). Oxidase-positive colonies first turn red and later turn black. Differentiation of gonococci from other species of the genus Neisseria is of particular importance in the diagnosis of oropharyngeal gonorrhea. As is known, on the mucous membrane of the tonsils, mouth and nasopharynx there is always a large number of gram-negative Neisseria - representatives of the normal human microflora. Reliable methods for identifying gonococci are immunofluorescence, latex and coaglutination reactions, as well as determination of enzymatic properties. It is imperative to carry out a qualitative determination of the sensitivity or resistance of microorganisms to antibiotics using the agar diffusion method using disks. In order to increase the frequency of finding gonococci in smears during primary microscopy and more reliable isolation of pure cultures, especially in cases of sluggish, chronic course of the disease, methods of provoking gonorrhea are used , that is, an artificial exacerbation of the pathological process, as a result of which a larger number of gonococci appear in the secretions. The main of these methods are: a) chemical - instillation of a 0.5% solution of silver nitrate into the urethra in men, lubrication of the cervical canal with a 2-5% solution of silver nitrate; b) mechanical - insertion of a direct bougie into the urethra for 10 minutes, or anterior urethroscopy; c) biological - intramuscular injection of gonovaccine in an amount of 500 million microbial bodies or pyrogenal 200 MTD; d) nutritional - consumption of salty, spicy foods; e) thermal - warming the genitals with an inductothermic current; f) physiological - taking smears during menstruation. It is even better to combine several methods of provocation, for example, chemical, nutritional and biological. Recently, polymerase chain reaction has been used to more reliably identify the causative agent of gonorrhea. It allows you to identify the pathogen in cases of chronic gonorrhea, when bacterioscopic and bacteriological examination does not give positive results.

Serological diagnosis

Serological diagnosis of gonorrhea is carried out relatively rarely, mainly in its chronic course, when bacterioscopic and bacteriological studies do not give positive results. In modern conditions, enzyme immunoassay, RNGA and Bordet-Gengou reactions (BRS) are carried out. The antigens for these reactions are: heat-killed polyvalent gonococcal vaccine, ultrasound-inactivated vaccine, protein and polysaccharide fractions of gonococci, as well as pyridine antigen. ELISA and RNGA are highly specific and reliable serological reactions. Compared to the past, RSK has somewhat lost its role. It has no practical value in the diagnosis of acute gonorrhea, since it is treated before the formation of a significant amount of antibodies. It is generally unsuitable for establishing the reliability of a cure. The Bordet-Gengou reaction is important in the serodiagnosis of chronic gonorrhea, especially in its complicated forms (gonosepsis, metritis, arthritis, prostatitis, etc.). The diagnostic value of allergy tests is somewhat devalued by the fact that they are positive for many years after gonorrhea. To set them up, 0.1 ml of fresh gonococcal vaccine (100 million microbial cells in 1 ml) is injected intradermally. After 24 hours, hyperemia is observed, sometimes with swelling in the center.

Treatment and prevention

For chemotherapy of gonorrhea, antibiotics are used: beta-lactams (penicillins, cephalosporins) and other antibiotics. Vaccinal prevention of gonorrhea is not carried out due to the lack of effective vaccines. To prevent blenorrhea, all newborns are given a solution of one of the listed antibiotics instilled onto the conjunctiva of the eye.

Neisseria gonorrhoeae belongs to the family Neisseriaceae, genus Neisseria. Gonococci were discovered by Neisser in 1879 and the entire family was named after him.

Morphology. Gonococci are diplococci consisting of two bean-shaped cocci lying concave to each other (reminiscent of coffee beans). The size of gonococci is 1.2-1.3 × 0.7-0.8 microns. They are polymorphic; along with large ones, there are very small, irregularly shaped L-form bacteria. Gonococci are immobile and do not spore. A capsule-shaped substance is found in the pathological material (pus). Gram negative. Under the influence of drugs and other substances, they quickly change: gram-positive forms appear. In pathological material they are located intracellularly (in a leukocyte), but can be outside the cell. They can be found in the form of individual cocci (see Fig. 4).

Cultivation. Gonococci are aerobes. Very demanding on nutrient media. They grow on media containing native protein (human) - blood, serum, at a temperature of 37 ° C and a pH of 7.2-7.4. The media must be freshly prepared and moist. Sowing should be done immediately after taking the material. On a serum medium, gonococci form small colonies 1-2 mm, transparent, shiny with smooth edges, resembling dew drops. There is no hemolysis in blood medium. In whey broth they give a slight turbidity and a film that settles to the bottom of the test tube. If growth is poor after 24 hours, the crops are left in the thermostat for the second day.

Enzymatic properties. Saccharolytic properties are weakly expressed. Gonococci break down only one sugar - glucose, producing acid. They do not have proteolytic properties.

Toxin formation. The cell wall of gonococci contains a toxic substance - lipopolysaccharide (little studied).

Antigenic structure. The antigenic structure is heterogeneous and easily changes under the influence of environmental factors. There is no generally accepted division of gonococci into serovars and serotypes.

Resistance to environmental factors. In the external environment, gonococci are not very stable. At a temperature of 56-60° C they die. At a temperature of 40° C, their viability decreases sharply. Low temperatures and drying quickly destroy them. But they remain in pus for up to 24 hours. Disinfectant solutions - 1% phenol solution, mercuric chloride 1:1000 kill gonococci within a few minutes. Gonococci are especially sensitive to silver salts - a 1% solution of silver nitrate kills them immediately. UV rays kill them within minutes.

Animal susceptibility. Animals are not sensitive to gonococcus. However, intraperitoneal injection of gonococcal toxin into white mice causes their death.

Sources of infection. A person with gonorrhea.

Transmission routes. Contact household (sexual), less often through contaminated objects (towel, sponges, etc.).

Diseases in humans. Gonorrhea and blenorrhea.

Pathogenesis. The natural host of gonococci is a sick person. Gonococci penetrate through the mucous membranes of the urethra (in women, the urethra and cervix). The pathogenicity factor of gonococci is the presence of pili, which, connecting with the microvilli of the cylindrical epithelium, facilitate the penetration of the gonococcus into the epithelial cell, causing an acute inflammatory process in the mucous membrane.

Clinically, gonorrhea is manifested by pain during urination, discharge of pus from the urethra and vagina. The disease is acute, but sometimes becomes chronic. Gonococci can cause gonorrheal conjunctivitis - blenorrhea (purulent inflammation of the mucous membrane of the eyes in newborns). Gonococci rarely penetrate from the urethra to other organs, but sometimes they can cause arthritis, endocarditis, etc.

Immunity. There is no natural resistance to gonococci. The transferred disease also does not create immunity. The observed phagocytosis is incomplete.

Prevention. Health education. Increasing the cultural and hygienic level. There is no specific prevention. To prevent blenorrhea, children must be injected into the conjunctival sac immediately after birth with 1-2 drops of a 30% albucid solution.

Treatment. Antibiotics (penicillin, bicillin, streptomycin, etc.). Sulfonamide drugs are also used. For the chronic form, gonococcal vaccine is used.

Control questions

1. Describe the morphological properties of gonococci.

2. What are the enzymatic activity and toxin production of gonococci?

3. What is the resistance of gonococci. To which drug are gonococci especially sensitive?

4. What diseases are caused by gonococci and their pathogenesis.

Microbiological examination

Purpose of the study: identification of gonococci and antigonococcal antibodies.

Material for research

1. Discharge from the urethral mucosa in men.

2. Discharge from the mucous membrane of the urethra and cervix in women.

3. Purulent discharge from the eyes.

4. Blood for serum.

Note. For bacterioscopic and bacteriological examination, material is taken: 1) before the start of antibiotic treatment: 2) no earlier than 10 days after the end of antibiotic treatment; 3) no earlier than 2 hours after the last urination; 4) no earlier than 2 hours after douching.

Basic research methods

1. Microscopic (mainly used for acute forms).

2. Microbiological.

3. Serological.

Progress of the study

Second day of the study

Take the crops out of the thermostat and inspect them. Studying colonies. They make smears. If there are suspicious gram-negative diplococci, colonies are subcultured onto slanted medium in test tubes (the medium must be freshly prepared and contain a sufficient amount of condensate) and tested for oxidase. To do this, a drop of 1% dimethylparaphenylenediamine solution is applied to the colony with a pipette; the colonies change color from dark brown to black.

Third day of the study

The cultures are taken out of the thermostat, smears are made from the slanted agar, stained with Gram and microscopically examined. Inoculate on Hiss media (lactose, glucose, mannitol and maltose). These carbohydrates should contain 30% of blood serum. The inoculated test tubes are placed in a thermostat.

Fourth day of research

Remove the tubes from the thermostat; if there is no growth, leave them in the thermostat for another 1-2 days. If there is growth, the results are taken into account (Table 28).

Serological diagnosis

Third week of illness. In the chronic course of the disease and in doubtful cases, RSC is performed using the patient’s serum (see Chapter 12). A killed culture of gonococci, which is prepared under industrial conditions, is used as an antigen. You can use the indirect hemagglutination reaction (see Chapter 12).

Control questions

1. What material is used to identify gonococci and how is it obtained?

2. How long after urination (or douching in women) can material be taken for research?

3. Which research method is the main one for the acute form and which one for the chronic form of gonorrhea?

4. When and what serological test is performed if gonorrhea is suspected?

5. What microorganisms need to differentiate gonococci from?

Get the medicine from your teacher. Study it and sketch the gonococci located inside and outside the leukocyte using Gram stain.

Culture media

Yolk medium. To 100 ml of MPA from rabbit meat add 15 ml of yolk (fresh chicken egg), 6 ml of phenol red indicator, 1.5 ml of sugar dissolved in 1 ml of sterile distilled water.

Nutrient medium ascites-agar. To the filtrate of the broth prepared from rabbit meat, add 2% agar, 1% peptone and 0.5% sodium chloride. Heat until the agar dissolves, set the pH to 7.4-7.5, and alkalize with 20% sodium hydroxide. The medium is brought to a boil, filtered, poured into sterile vials and sterilized in an autoclave for 15 minutes at 115°C.

Recipes for ascites-free culture media (MPA pH 7.4-7.5).

1) meat water from rabbit meat or ox hearts - 100 ml

casein hydrolyzate - 2 ml

yeast autolysate - 2 ml

cattle blood serum - 20 ml

2) meat water from rabbit meat or ox hearts - 100 ml

5% solution of hemohydrolysate - 2 ml

yeast autolysate - 2 ml

cattle serum - 20 ml

3) meat water from rabbit meat or ox hearts - 100 ml

chicken egg yolk - 10 ml

cattle blood serum - 20 ml

The growth of gonococci on these media is abundant. Gonococcus colonies can be detected using an oxidase test, in which they turn red, turning black.

The causative agent of gonorrhea is Neisseria gonorrhoeae , causing acute and chronic inflammatory lesions of the genitourinary organs, mucous membrane of the oropharynx and conjunctiva of the eye.

The material for research is purulent discharge of the genitourinary organs, rectum or conjunctiva of the eyes (with blenorrhea), articular and peritoneal exudate. Strictly regulated methods for laboratory diagnosis of gonorrhea are microscopic and bacteriological (cultural) examination. Methods for microbiological diagnosis of gonorrhea are reflected in scheme 18.

Scheme 18. Methods for microbiological diagnosis of gonorrhea

Microscopic method consists of examining smears from the test material, stained with Gram and methylene blue. When staining preparations for gonorrhea, a modification of the Gram method is used (gentian violet dye is replaced with crystal violet, and neutral red is used instead of fuchsin; the preparation is washed after each stage of staining). In smears of acute gonorrhea, a large number of polymorphonuclear leukocytes are found, inside and outside of which bean-shaped gram-negative diplococci are located; the accompanying microflora is either minimal or absent altogether (Fig. 25). In chronic gonorrhea, in some cases, a typical gonococcus is not detected in smears, or looks atypical in the form of small dust-like or large spherical formations; often 2 gonococcus cells are located at an angle to each other, and abundant accompanying microflora, leukocytes, epithelial and other cells are found. IFM can be used as an express diagnostic method.

Rice. 25. The causative agent of gonorrhea (Neisseria gonorrhoeae) in smears from the urethra of a patient with acute gonorrhea. Internally and extracellularly located grammatical diplococci, an abundance of leukocytes.

Bacteriological method. Gonococcus is highly sensitive to various unfavorable factors, so cultural testing must be carried out immediately after collecting the material. Sowing is carried out on special nutrient media. The basis of these media is MPA from rabbit meat with the addition of yeast autolysate, casein hydrolysate or hemohydrolysate and bovine whey. Ascitic fluid and egg yolk are not currently used as additives to the culture medium for gonococcus. The second type of culture media is selective and includes, in addition to the above components, antibiotics (polymyxin, lincomycin, and for the diagnosis of pharyngeal gonorrhea - orotic acid). Crops are grown at 37 0 C for 24-72 hours in an atmosphere with a high CO 2 content. Colonies of gonococci are round, transparent, in the form of dew drops. Colonies typical for gonococci are subcultured into test tubes with a gonococcus culture medium to obtain pure cultures, which are identified by morphological and saccharolytic properties on “variegated” media (semi-liquid agar with whey and carbohydrate) or using microtest systems. In smears from pure cultures, gonococci are not located in pairs, but separately. Biochemically, gonococci are inactive and ferment only glucose with the formation of acid and produce oxidase. To detect oxidase, a test with a 1% aqueous solution of dimethylparaphenylenediamine or diethylparaphenylenediamine is used, which colors gonococcal colonies first pink, then red, and after a few minutes - black. The sensitivity of gonococci to antibiotics and its beta-lactamase activity are also studied.

Serological method(determination of antibodies to gonococcus in the blood of patients using RSK and ELISA) is not strictly regulated, does not have diagnostic value, is not a method for monitoring the effectiveness of treatment, and is not currently used in real practice.

Gene diagnostics. It is aimed at detecting specific fragments of gonococcal DNA using PCR and can be used as an additional highly informative method for diagnosing gonorrhea.

Independent work of students

1. Microscopic method- examination of purulent discharge from the urethra of a patient with suspected gonorrhea (demonstration microslide, Gram stain).

2. Bacteriological method. Study of cultural . the causative agent of gonorrhea on a nutrient medium for isolating gonococci, on which gonococci form round, transparent colonies in the form of dew drops. Enzymatically, gonococcus is inactive and only ferments glucose to form acid. Take into account the results of sowing gonococcus on the “variegated” row (demonstration).

Diagnosis of gonorrhea

Without testing for gonorrhea, no modern doctor will make a diagnosis. It doesn’t matter what symptoms the patient has - gonorrhea, like many other infections, is determined primarily by laboratory methods. And timely testing, together with a competent choice of the method that is best suited in each specific case, play a decisive role in preventing serious complications.

Several methods are used for laboratory diagnosis of gonorrhea:

• smear microscopy for Neisser gonococcus (bacterioscopic method);
• bacterial culture for gonorrhea (gonococci);
• serological tests (serodiagnosis);
• enzyme immunoassay (ELISA);
• PCR (polymerase chain reaction);
• rapid tests for gonorrhea.

The most accurate and common of them are tank culture and PCR. Let's look at each in detail to understand why.

How is a microscopic test for gonorrhea done?

Microscopy of a smear from the urethra or vagina (cervix) is the simplest method of preliminary diagnosis of gonorrhea. The principle of the study is simple. The smear is stained and examined under a microscope. At the same time, attention is paid to the presence of double cocci with a coffee bean-shaped shape characteristic of gonococci.

With any inflammation, the smear contains a large number of leukocytes - blood cells responsible for protecting the body. With gonorrhea, their number is also increased, but this fact cannot confirm the diagnosis. The presence of white blood cells only indicates inflammation, but does not indicate what infection caused it.

Leukocytes and gonococci in gonorrhea can be determined in a general urine test (found in the sediment).

The advantage of the method is its simplicity and speed. And the main problem is low accuracy. You can complete the analysis in 10-15 minutes, but the probability that the result is correct is 50%. A similar appearance is characteristic of almost all Neisseria, including non-pathogenic ones. Therefore, microscopy is used only for screening, to understand whether there is anything foreign in the smear. If bacteria similar to gonococci are detected, other, more accurate tests are performed: culture, PCR or blood test using ELISA.

How is bacterial culture for gonococci performed?

Unlike microscopic examination of a smear, tank culture is an informative method. Gonococci grow only on special media; their colonies have a characteristic appearance and color. In addition, different types of bacteria have different abilities to process certain substances (in particular, sugars). Taken together, these characteristics help identify both the species and subspecies of the microbe. The accuracy of the method is 90%.

If gonococci are found, then in the laboratory the sensitivity to antibiotics will be determined on the same colonies. Then the doctor will know exactly which medicine is stronger in each specific case. Therefore, it makes sense to carry out the analysis, even if another method has clearly determined the presence of infection.

The disadvantage of the method is its duration. The time it takes to prepare such an analysis for gonorrhea can range from several days to several weeks.

In what cases is serodiagnosis of gonorrhea carried out?

To fight the disease, the immune system produces antibodies - special protective proteins. They are specific to each infection they must fight. Doctors went from the opposite - if there are antibodies (effect), then there must be bacteria (cause). The principle of the serological method is to observe the reaction of the patient’s blood, containing antibodies to gonococci, with antigens to these antibodies contained in the diagnostic kit. Antibodies and antigens are attracted to each other like the poles of a magnet and stick together. Because of this, a precipitate forms in the solution.

Serological methods are rarely used to diagnose gonorrhea. Mainly of interest to scientists. Previously, they were widely used to detect gonorrhea, but they were abandoned due to low accuracy and the dependence of the result on subjective factors.

The waiting period for test results for gonorrhea using serological methods is several hours. Information content – ​​from 60 to 75%, depending on the diagnostic set of antigens. Serological tests do not detect the disease in the first weeks after infection - the body needs time to accumulate antibodies in the blood.

How to detect gonorrhea using enzyme immunoassay

Enzyme-linked immunosorbent assay or ELISA is a method that has replaced serological diagnostic methods. In the same way, it allows you to determine in the blood certain classes of antibodies specific for gonorrhea, but more precisely. Able to diagnose both acute (several days after infection) and chronic gonorrhea. Widely used for diagnosis and screening.

The advantages of the method are sufficient accuracy (75-85%), reasonable cost. Disadvantage: results may be distorted for subjective (spoiled reagents, human factor) and objective (presence of immunodeficiency, immune diseases) reasons. How long it takes to do a blood test for gonorrhea using ELISA depends on each specific laboratory and reagents (from 1 to 5-6 days).

Test for gonorrhea using PCR method

The polymerase chain reaction method is the crowning achievement in the field of disease diagnosis. He is very accurate and specific. The creator of the method, Kary Mullis, received the Nobel Prize in Medicine in 1993. The principle of the method is based on the detection of the genetic material of the pathogen (DNA of neisseria gonorrhoeae) in the studied samples of biological material. To conduct the study, you can use any biological material - blood, serum, secretions from the genital tract, urethra, smears and scrapings. The material may be contaminated with pus, exudate, urine and secretions, but this does not affect the effectiveness of the analysis.

The material is searched for the sequence of genes characteristic of the pathogen. If found, then this is a clear sign of infection. The PCR test for gonorrhea has the highest information content and specificity. Its accuracy is about 95%. The duration of the analysis is from one to several days, depending on the laboratory and reagents.

Express tests for gonorrhea at home

The most popular manufacturer of express tests is STD Test Express

Express tests for gonorrhea have been developed especially for people who are sexually active. It is based on a principle similar to ELISA (the reaction of gonococcal antigen with antibodies from material taken from a person with suspected disease). Samples of blood, secretions from the genital tract, urethra, and rectum are suitable for testing.

The main advantage of express tests is speed. The procedure takes 10-20 minutes. The disadvantage of the method is frequent false-positive and false-negative results, dependence on the correct testing, distortion of results due to violation of storage rules. Therefore, it is better to look into the nearest laboratory.

Let's sum it up

In most cases, gonococci are transmitted along with other sexually transmitted infections. If you suspect a disease, you need to be tested for all possible diseases. Therefore, it is optimal to start with a PCR analysis - it is prepared quickly, and one smear can be simultaneously examined for different bacteria and viruses.

It makes sense to do a tank culture if gonorrhea is already known to confirm the diagnosis and determine sensitivity to antibiotics.

Can gonorrhea not show up in tests? False negative results occur in the most accurate methods. Antibiotics and other medications, characteristics of the patient’s body, chronic infection and other factors can change the appearance and shape of gonococci, turning them into the so-called L-forms. In difficult cases, the doctor prescribes a gonorrhea provocation - a deliberate weakening of the immune system in order to stimulate the infection to manifest itself. The home method of provocation is simple and pleasant - drink beer and take a hot bath, and is recommended for everyone who is about to undergo a smear test for culture.

Culture for gonorrhea

Gonorrhea is a sexually transmitted disease that is one of the most common among the population.

The disease is characterized by inflammation of the urinary system. A common cause of infection is promiscuity without the use of protective equipment.

Both men and women can become infected.

Important! Gonorrhea, without timely treatment, contributes to the development of infertility.

Today we will tell you when and how to take cultures for gonococcal infection, and will help you understand the test results.

Culture for gonorrhea: indications

From the moment of infection to the first visible signs, it usually takes from 3 to 15 days. May be asymptomatic.

The following symptomatic signs are characteristic:

• Purulent discharge;
• Redness and swelling of the urethra;
• Burning and itching in the genital area;
• Pain in the lower abdomen;
• Possible increase in body temperature;
• Painful urination.

If you notice these symptoms, you should seek help from a doctor as soon as possible. The indication for examination is gonococcal infection in a sexual partner.

Attention! The transition of the disease to chronic can lead to a number of complications and infertility.

Preparation: culture for gonorrhea

Any biological material can be used for bacteriological research.

Before the study, a fence is taken:

• Skin;
• Sperm;
• Oral mucosa, gastrointestinal tract;
• Scraping of the genitourinary tract.

Bacteriological culture is prescribed by a urologist or gynecologist.

To obtain a reliable result, you should know the rules for preparing for bacteriological inoculation.

If your doctor has prescribed a smear test, you must:

• Do not have sexual intercourse two days before the examination;
• Do not douche;
• Carry out genital hygiene without intimate hygiene products;
• 2-3 hours before taking the material, refrain from going to the toilet;
• Stop taking medications one week before the test.

In women, a smear is taken in the first days after the end of menstruation.

The crops are placed in a thermostat at optimal humidity and temperature conditions. For gonococcus, it is necessary to maintain a temperature within 37 degrees. Afterwards, questionable colonies are studied.

Species features of colony growth on agar:

• Colorless or with a yellow tint;
• Not big;
• The surface is smooth and shiny;
• Slightly convex.

This method of research is quite informative. The downside is that it takes a lot of time to do research. Using bacteriological testing, the sensitivity of a microorganism to antibacterial drugs can be determined. This is necessary for the effectiveness of further treatment.

Throat culture for gonorrhea

If you suspect a gonorrheal process in the throat, you should contact an ENT specialist or venereologist. You should definitely take a swab from the throat, tonsils or pharynx.

At the same time, a test is taken from the genitourinary organs. A test for gonococcal infection should be taken before starting treatment. After treatment you should undergo a follow-up examination.

Culture for gonorrhea from the eye

The affected eyelid swells and becomes bluish-red. In the process of large discharge of pus, the eyelid may stick together.

Soreness, itching and burning appear. The discharge from the eye is taken for analysis. A PCR study and bacteriological culture are carried out with further testing for sensitivity to antibiotics.

Culture of prostate secretion for gonorrhea

Using prostate massage, prostate secretions are collected.

This is a clear liquid that is inoculated on special nutrient media. After one or two days, colonies of microorganisms can be identified. An antibiogram is performed to determine an effective drug for further treatment.

Culture for gonorrhea from the anus

Several methods are used to collect material. In the first case, a cotton swab is passed along the perianal folds. Afterwards they are placed in a sterile container. The second method is to take a scraping.

Time frame for preparing cultures for gonorrhea

Laboratory tests can take from five to nine days.

This depends on the type and methodology of the analysis performed. The result of a microscopic analysis can be obtained in about half an hour.

PCR is performed in no more than one day, and express analysis can also be performed. The PCR palette allows parallel testing for the presence of concomitant infections.

Culture for gonorrhea to determine the effectiveness of treatment

The absence of symptomatic signs of gonorrhea and negative laboratory tests is a natural result of treatment. Control is carried out on the 10th day after discontinuation of antibacterial drugs.

The study includes examining the smear three times under a microscope and inoculating the material once on a nutrient medium.

Men are required to take the test once, while women take it three times. After two weeks of discontinuation of treatment, during and after menstruation.

If there are no symptoms and negative tests are obtained, the person is healthy. If the results are positive, but no symptoms are observed, this indicates a hidden process.

The course of treatment should be repeated. Negative results and the presence of signs of inflammation indicate the presence of another type of pathogen in the body.

Culture for gonorrhea: which doctor should I contact?

Cultures for gonorrhea can be prescribed by appropriate specialists - a gynecologist, urologist, venereologist.

The examination can be done at any public or private clinic. If you need urgent test results, we recommend contacting a private laboratory.

Remember! If the first symptoms appear, immediately consult a specialist doctor.

Culture for gonorrhea: price

The cost of the analyzes depends on the method of studying the material.

Prices will vary between 150-2000 rubles. Bacteriological sowing in the pricing policy will be approximately 800-1300 rubles.

If you need to perform a culture test for gonorrhea in Moscow, please contact our clinic. Our own laboratory allows us to conduct research quickly, efficiently and inexpensively. When you receive the results, you can consult a venereologist.

For preventive purposes, barrier contraception should be used. It is better to prevent the development of the disease than to treat it later.

If you suspect gonorrhea, contact a competent venereologist.

Use: microbiology, diagnosis of gonorrhea, nutrient medium for the isolation and cultivation of gonococcus. The essence of the invention: the nutrient medium contains as a nutrient base pancreatic hydrolyzate of fish meal and hydrochloric acid hydrolyzate of casein, as a growth stimulant and source of vitamins - autolysate of baker's or brewer's yeast, a growth stimulator of hemophilic microorganisms. The medium additionally contains glucose, microbiological agar, polymyxin-B sulfate, lincomycin hydrochloride and distilled water. The medium ensures the growth of gonococcus of museum strains and from material from patients with gonorrhea, preserving the typical morphology of the microorganism, which contributes to its widespread use in practical healthcare in the diagnosis and treatment of sexually transmitted diseases. 3 tables

The invention relates to the field of medical microbiology and can be used in the diagnosis of gonorrhea. Leading foreign companies ("Oxoid", "Gibco", "BRL", etc.) produce nutrient media for the cultivation of gonococcus based on meat digests and fermentolysates with the addition of sterile native blood. Among domestic preparations, the following are known: nutrient medium "Arginine-agar", diagnostic , dry, produced by NPO Nutrient Media, Makhachkala, which consists of the following components, g: peptone 8.0-12.0; arginine 0.07-0.3; cystine 0.007-0.03; glutamic acid 1.0-1.8; EKD 4.0-7.0; thiamine bromide 0.002-0.007; iron nitrate 0.002-0.01; potassium chloride 0.07-0.2; sodium chloride 5.0-8.0; ammonium chloride 0.9-2.0; magnesium sulfate 0.4-0.9; glucose 4.0-7.0; starch 0.5-2.0; Tris buffer 1.0-2.5; agar 13-17.0; bovine serum 153.0-255.0; polymyxin-M sulfate 15000-25000 units; lincomycin hydrochloride 0.001-0.003; distilled water up to 1 liter. The disadvantage of this medium is: low sensitivity, instability of results on the intensity of gonococcus growth. Lyophile-dried medium for the isolation of gonococcus (produced by the Scientific Research Institute of VSA, St. Petersburg) consisting of a medium base and an additive in the following ratio of components, g; is shown in Table 1. The disadvantage of this medium is: the instability of the results on the intensity of gonococcus growth and sensitivity. In practice, laboratory-prepared ascites-free media are mainly used from extracts of fresh rabbit meat and fresh bovine hearts with the addition of peptone and 20% cattle blood serum or sterile native blood. The lack of the required quantity of domestic nutrient media for the diagnosis of gonorrhea, as well as their quality, causes criticism from the practical health service. The objective of the invention is to create a dry nutrient medium that ensures stable results in terms of growth intensity and increased sensitivity, not inferior to commercial media for the diagnosis of gonococcus, and to use non-food products for its production. The problem is solved by balancing the component composition of the medium containing a nutritious protein base, autolysate of baker's or brewer's yeast, agar, sodium chloride, inhibitors of foreign microflora lincomycin hydrochloride and polymyxin-B sulfate; as a nutritional base it contains pancreatic hydrolyzate of fish feed meal and hydrochloric acid hydrolyzate of casein , in addition, it additionally contains a growth stimulator of hemophilic microorganisms (enzymatic hydrolyzate of black albumin), glucose, to increase the stability of results when examining patients with chronic gonorrhea, it is possible to add cattle blood serum to the medium (5-10)% with the following content of components, g/ l: pancreatic hydrolyzate of fish meal 22.0-28.0 acid hydrolyzate of casein 2.8-3.2 autolysate of baker's or brewer's yeast 0.8-1.2 growth stimulator of hemophilic microorganisms 2.0-10.0 glucose 9.0 -11.0 sodium chloride 2.9-3.1 microbiological agar 12.0-15.0 polymyxin-B sulfate 15000-25000 Units
lincomycin hydrochloride 0.001-0.003
distilled water up to 1 l
pH 7.3+0.2
The quantitative ratio of the components of the nutrient medium and its pH provide an increased level of intensity of gonococcus growth and sensitivity of the medium. The nutrient medium is prepared as follows: weighed portions of the main ingredients are added to a flask with distilled water. The medium is thoroughly mixed and, closing the flask with a cotton-gauze stopper, sterilizes at (115-119) o C for 30 minutes. then cooled to a temperature of (45-50) o C and sterile, having previously dissolved in sterile saline. solution, add weighed amounts of polymyxin-B sulfate and lincomycin hydrochloride. The medium is mixed and poured into sterile Petri dishes. For biological control of culture media, a culture of Neisseria gonorrhoeae museum strains and material from gonorrhea patients were used. All crops were cultivated at a temperature of (37+0.5) o C in an atmosphere of 10% CO 2 and high humidity for 24-48 hours: data in table 1. Example 1. The proposed medium was tested by cultivating Neisseria gonorrhoeae strains and on clinical material on a nutrient medium of the following composition, g:
pancreatic hydrolyzate of fish meal 22.0
baker's yeast autolysate 0.8
hydrochloric acid hydrolyzate of casein 2.8
growth stimulator of hemophilic microorganisms 2.0
glucose 9.0
sodium chloride 2.9
microbiological agar 12.0
polymyxin-B sulfate 15000 units
lincomycin hydrochloride 0.001
distilled water up to 1 l
pH 7.3+0.2
The results were recorded after 24-48 hours of cultivation. Example 2. The proposed medium was tested by inoculating Neisseria gonorrhoeae strains and using clinical material on a nutrient medium with the optimal content of components of the following composition, g:
pancreatic hydrolyzate of fish meal 25.0
baker's yeast autolysate 1.0
hydrochloric acid hydrolyzate of casein 3.0
growth stimulator of hemophilic microorganisms 5.0
glucose 10.0
sodium chloride 3.0
microbiological agar 13.0
polymyxin-B sulfate 20000 units
lincomycin hydrochloride 0.002
distilled water up to 1 l
pH 7.3+0.2
The results were recorded after 24-48 hours of cultivation. Example 3. The proposed medium was tested by inoculating Neisseria gonorrhoeae strains and using clinical material on a nutrient medium with the maximum content of components of the following composition, g:
pancreatic hydrolyzate of fish meal 28.0
baker's yeast autolysate 1.2
hydrochloric acid hydrolyzate of casein 3.2
growth stimulator of hemophilic microorganisms 10.0
glucose 11.0
sodium chloride 3.1
microbiological agar 15.0
polymyxin-B sulfate 25000 units
lincomycin hydrochloride 0.003
distilled water up to 1 l
pH 7.3+0.2
The results were recorded after 24-48 hours of cultivation. Example 4. The proposed medium was tested by inoculating Neisseria gonorrhoeae strains and using clinical material on a nutrient medium with a violation of the quantitative ratio of the components of the following composition, g:
pancreatic hydrolyzate of fish meal 31.0
baker's yeast autolysate 1.4
hydrochloric acid hydrolyzate of casein 3.4
growth stimulator of hemophilic microorganisms 15.0
glucose 13.0
sodium chloride 3.5
microbiological agar 20.0
polymyxin-B sulfate 30000 units
distilled water up to 1 l
pH 7.3+0.2
The results were recorded after 24-48 hours of cultivation. Example 5. The proposed medium was tested by inoculating Neisseria gonorrhoeae strains and using clinical material on a nutrient medium with a violation of the quantitative ratio of the components of the following composition, g:
pancreatic hydrolyzate of fishmeal 18.0
baker's yeast autolysate 0.5
hydrochloric acid hydrolyzate of casein 2.5
growth stimulator of hemophilic microorganisms 0.5
glucose 5.0
sodium chloride 2.5
microbiological agar 10.0
polymyxin-B sulfate 10000 units
lincomycin hydrochloride 0.005
distilled water up to 1 l
pH 7.3+0.2
The results were recorded after 24-48 hours of cultivation. Table 2 provides a comparative description of the growth of gonococci from clinical material and museum strains on the proposed and known media after 24-48 hours of growth. The morphology of colonies and stained smears on all tested media is identical to that typical for gonococcus. Table 2 shows that the proposed medium is superior to commercial media in terms of gonococcus growth intensity and sensitivity. Violation of the quantitative ratio of the components of the environment leads to a sharp deterioration in the microbiological indicators of the quality of the proposed environment. The proposed medium makes it possible to maintain the viability of Nisseria gonorrhoeae strains (both freshly isolated from patients and museum specimens) during passaging for 9 months. Table 3 presents data on the viability of the Neisseria gonorrhoeae culture when cultivated and stored on solid media,
From Table 3 it can be seen that the proposed nutrient medium is suitable for diagnostic purposes in identifying patients with acute and chronic gonorrhea, as well as for obtaining Neisseria gonorrhoeae biomass for creating gonovaccines. Information sources
1. Handbook Culture Media (1982)
2. The manual of microbiological culture media (Gibco BRL 1988)
3. The Oxoid manual of cultural media, ingredients and other laboratory services. (Oxoid Limited 1979)
4. Manual of BBL products and laborator procedures. 1991. 5. Author. Certificate No. 1451167, born. Gadzhieva et al. “Nutrient medium for the isolation of gonococci.” 6. FS 42-146 VS-88 Nutrient medium for the isolation of gonococcus, dry.

Claim

1 Nutrient medium for the isolation and cultivation of gonococcus, containing a nutrient base, baker's or brewer's yeast autolysate, microbiological agar, sodium chloride, an inhibitor of foreign microflora and distilled water, characterized in that as a nutrient base it contains pancreatic hydrolyzate of fish meal and hydrochloric acid hydrolyzate of casein , additionally contains a growth stimulator of hemophilic microorganisms, glucose, and as an inhibitor of foreign microflora polymyxin-B sulfate and lincomycin hydrochloride in the following ratio of components, g/l: 3 Pancreatic hydrolyzate of fish meal7 22 283 Hydrochloric acid hydrolyzate of casein7 2.8 3.23 Bakery autolysate or brewer's yeast7 0.8 1.23 Growth stimulator of hemophilic microorganisms7 2 103 Glucose7 9 113 Sodium chloride7 2.9 3.13 Microbiological agar7 12 - 153 Polymyxin-B sulfate, Unit7 15000 250003 Lincomycin hydrochloride7 0.001 0.0033 B Distilled water7 Up to 1 l3 pH7 7.3 + 0.2

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