Bull. Org. mond. Sante 1 1965, 32, 449-469 Bull. Wld Hlth Org.

Laboratory Diagnosis of Gonococcal Infections * ALICE REYN, M.D.'

CONTENTS

Page Pagp INTRODUCTION ...... 449 DRUG SENsiTIVITY DETERMINAnON ...... 462

COLLECTION AND HANDLING OF SPECIMENS MEDIA AND REAGENTS General remarks ...... 450 Preparation of broth ...... 462 Method of transportation ...... 451 Chocolate ascitic-fluid-agar ...... 463 HYL medium ...... 464 BACTERIOLOGICAL EXAMINATION Fermentation media ...... 464 . . . Microscopy and Gram-staining technique . . . 452 1. Danish fermentation medium .... 464 2. HAP medium ...... 465 Culture and isolation ...... 453 Stuart's medium with solid agar ...... 465 Control strains ...... 456 ...... 466 Fermentation tests ...... 457 Oxidase reagent ...... Diagnostic criteria ...... 458 Polymyxin B ...... 466 . Reporting of results ...... 458 Nystatin ...... 466 Reagents to be used in the Gram-staining technique 466 SEROLOGICAL EXAMINATION ANNEX. Reviewers ...... 467 General remarks ...... 459

Gonococcus complement-fixation reaction . . . 460 REFERENCES ...... 467

INTRODUCTION

The genus Neisseria comprises Gram-negative, organisms have been described more thoroughly than aerobic or facultatively anaerobic cocci, usually but the other members of the group, such as N. catar- not invariably arranged in pairs. The size is about rhalis, N. flavescens, N. sicca and N. flava (sub- 0.8,u by 0.6,u. They often grow poorly on ordinary species I-III). The classification of the Neisseria is media and they are frequently pathogenic. Few far from being complete and a taxonomic study of carbohydrates are fermented, indole is not produced this group is needed. and nitrates are not reduced. With a few exceptions, are N. gonorrhoeae (gonococcus) is the etiological catalase and oxidase abundantly produced; some agent of " gonorrhoea ", whereas N. meningitidis species are haemolytic. Neisseria are non-motile. (meningococcus) is the organism causing what is The most important members of the genus Neis- commonly called " epidemic cerebrospinal menin- seria are N. gonorrhoeae and N. meningitidis; these gitis ". The other less pathogenic or non-pathogenic members of the genus are most frequently found in * This is one of a series of studies on the laboratory diag- nosis of various diseases which, it is hoped, will eventually the respiratory tract and occasionally in the genital be revised and published in monograph form. An effort is regions. made to ensure that the diagnostic methods recommended in these studies are as internationally representative and In this paper, attention will be paid to the labora- acceptable as possible by securing the co-operation of a number of experts from different countries. A list of the tory identification of the gonococcus and reference reviewers of the study presented here is given in the Annex will be made to the other members of the genus only on page 467. To all these, and to the author herself, the World in connexion with differential diagnostic problems Health Organization is greatly indebted. - ED. 1 Director, Neisseria Department, Statens Seruminstitut, which may be of forensic significance, especially in Copenhagen, Denmark. the diagnosis of gonorrhoea.

1581 449 450 A. REYN

COLLECTION AND HANDLING OF SPECIMENS

GENERAL REMARKS available on the spot; in other cases plating is necessarily delayed for several hours or even several The material will most frequently be discharge days. If posting has to be delayed the specimens collected from the male or female urethra and the should be kept in the refrigerator or in some other cervix; specimens can also be taken from the rectal cool place until they are despatched. Transportation mucosa, prostate, vagina, conjunctiva, Bartholin's in temperate or hot climates for more than a few glands, Skene's ducts, joints, blood, spinal fluid and hours favours the growth of contaminants at the sediment from centrifuged urine. expense of the gonococci. Selective inhibition of the The material can be examined by direct microscopy contaminants during transportation by means of and by culture; for culture immediate plating is dyes (methylene blue, crystal violet, Nile blue A, preferable, but in most cases this is impossible. In etc.) or other chemical substances (thallium acetate) females, where the microscope findings are very or by antibiotics has been proved to be of little value unreliable, it is recommended that both culture and (Peizer & Steffen, 1947; Lagergren & Ouchterlony, microscopy be used. In male contacts (symptomless 1948; Bang, 1952). The lower limits of concentra- carriers) the culture is also of great importance. tions toxic to the contaminants are very close to those Culture is also much better as a test of cure than is the toxic to the gonococci. However, when sampling simple microscope examination of a slide. The conditions are very poor or when the patients are method of transport of the infected material plays a from a low social level the addition of inhibitors decisive role, especially when the transportation time to the primary isolation medium may be indicated. exceeds a few hours and the temperature is high. An exception to the general rule is polymyxin B When specimens are taken for both microscopy and which completely inhibits the growth of coliform culture the specimen taken 'first should be used for bacilli without harming the majority of gonococcal the culture because it is likely to contain more strains (Crookes & Stuart, 1959). However, at the material than the second specimen (Reyn et al., same time better conditions are provided for other 1960). contaminants, especially diphtheroids and Gram- positive rods.' In hot climates it is recommended Generally, wooden applicators covered with cot- that specimens be transported in vacuum bottles at ton wool are used to collect the material for micro- temperatures between 0°C and 4°C. scope and bacteriological examination. However, some kinds of wood are known to be toxic to the The sampling technique is of great importance but gonococcus and the use of aluminium wires has this is not the place for a detailed description of the therefore been recommended (Beakly, 1957). The techniques for taking samples from the various sites toxic effect of the swabs can be counteracted by of gonorrhoeal infection.2 Unnecessary contamina- boiling in buffer and impregnating with charcoal tion should be avoided and the sample should be as (Moffett et al., 1948); this procedure appears to act " rich " as possible in order to compensate for loss also upon the toxic fatty acids demonstrable in cer- during transportation. It is stressed that sterile tain batches of agar (Ley & Mueller, 1946). Many vaginal specula should be used for collecting speci- different types of collection outfit have been de- mens from the cervix; some lubricants are toxic and scribed. In this paper, the only method described in should be avoided. The cervical plug, if present, detail is the Stuart method (Stuart, 1946; Stuart et should be removed before the swab is taken. The al., 1954; Stuart, 1956) modified by Reyn et al. (1960) applicator should be inserted about 1 cm into the and by Ringertz (1960). The Stuart medium is a cervix. non-nutrient, reducing medium in which the oxygen tension can be checked by observing the 1 Recently, Thayer & Martin (1964) have proposed a change in the colour of the medium from colourless selective medium which contains, in addition to 25 IU of polymixin B, 10 ,Ag of ristocetin per ml. Ristocetin inhibits (reduced) to blue (oxidized). It should only be used many Gram-positive micro-organisms. In the author's in association with charcoal-impregnated swabs, laboratory, preliminary experiments with a selective medium which counteract toxic substances. Inmediate of similar composition have given very promising results. 2 For further details see US Public Health Service plating is possible only where laboratory facilities are (1962); Carpenter (1963). LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 451

FIG. 1 FIG. 2 STUART'S TUBE CONTAINING A CHARCOAL SWAB STUART'S TUBE WITH CARDBOARD CONTAINER

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'-:-. :.:..:., '...:.:..::"", ...... :: ...... -...... :. ,-...... ,'-... :'.' '::-... .: :::,.,:...... METHOD OF TRANSPORTATION' ...:... '.:.:.-'. .,.,...: .:. .: .:: The method of transportation described below was introduced by Stuart (Stuart, 1946; Stuart et al., 1954; Stuart,. 1956) and modified by Reyn et al. (1960). See also Ringertz (1960). The modified Stuart device consists of: (1) 15-mm x 150-mm tubes containing a 10-cm high column of solid Stuart's medium (for composi- tion see page 465). The labelled tubes are tightly closed with hydrophobic cotton-wool plugs and the date of preparation is stamped on the labels. (2) Cardboard tube containers. (3) Resistant, special coloured paper envelopes marked " Neisseria Department, Statens Serumin- stitut, K0benhavn ". (4) Charcoal-impregnated, sterile wooden appli- cators. (5) Forms on which to enter the patient's data. The Stuart's medium is dispensed while hot into the sterile cotton-wool-plugged tubes. After prepara- tion, the tubes contain a 10-cm column of medium, which should be colourless except for the uppermost half a centimetre, which will be blue owing to oxidation of the methylene blue. Oxidation will proceed during storage, and completely oxidized 1 As used at the Statens Seruminstitut, Copenhagen. 452 A. REYN the labelled tubes of media without breaking the posted, the specimens should be placed in cardboard sticks; the stoppers should be put in, leaving the containers (Fig. 2), which are then inserted in special upper ends of the sticks projecting about 1 cm above envelopes and they should be kept in a cool place if the edge of the tube as shown in Fig. 1. When posting is delayed.

BACTERIOLOGICAL EXAMINATION

MICROSCOPY AND GRAM-STAINING TECHNIQUE easily decolorized and that short Gram-negative rods (E. coli, etc.) may also be mistaken for gono- The most primitive laboratory method is the cocci (Reyn, 1951). Badly performed Gram-staining microscope examination of a stained smear of is no better than staining with methylene blue. discharge. To a great extent the success of this At the Statens Seruminstitut in Copenhagen, the method depends upon the way in which the smear is fixed smears of discharge or of watery suspensions collected and prepared. Though apparently simple, of pure cultures are (1) stained with 0.2% aniline the method of preparation nevertheless deserves crystal violet for one minute, (2) treated with Lugol's close attention; it should be borne in mind that iodine solution for one minute, (3) decolorized with charcoal-impregnated swabs should be avoided in 96 % ethanol for half a minute, (4) rinsed in running the preparation of smears. Smears should be made tap water for about five seconds, (5) counterstained as thin and as even as possible. Before posting or with 0.1 % phenolized fuchsin for one minute, (6) staining, the smears should be exposed either to air- rinsed in running tap water for about five seconds drying and gentle flame-fixation or to air-drying plus and (7) dried with blotting paper. (For the composi- methanol fixation for not less than two minutes and tion of the reagents, see page 466.) preferably for 10 minutes. Fixation with methanol is Classically, in acute male gonorrhoea, large num- much better than flame-fixation in the case of bers of polynuclear leucocytes containing Gram- protein-rich smears from patients with a florid negative, coffee-bean-shaped diplococci are ob- gonorrhoea. The general rule must be that the served. In some cases, only a few of the leucocytes diagnosis of gonorrhoea by microscopy is made only show one or two typical intracellular pairs and in after careful Gram-staining; the latter may be other cases all the cocci are extracellular. In chronic performed after previous staining with 1 % methylene male and female gonorrhoea, it is often impossible blue, provided the immersion oil has been completely to find any typical gonococcus pairs, especially in removed by means of xylol. The Gram-staining the cervical secretion. technique may be any one of the usually recom- Stained films of pure cultures reveal the gonococci mended methods, but it is important to be familiar as roundish cocci, typically but not invariably with the method in question and to control arranged in pairs. After 18 to 24 hours' growth, the the various ingredients for precipitation and size is uniform and the stain is taken up quite infection. evenly. After 48 hours' growth, size and staining are Methylene blue without subsequent Gram-staining more variable and lysis is often observed. is still used even by specialists, who believe that by Cellular morphology is progressively altered by this means they are able to distinguish between contact with penicillin, both in vivo and in vitro. N. gonorrhoeae, staphylococci, Escherichia coli and Smears of exudate taken during the first four or five other Gram-negative, more or less well-defined, hours of penicillin therapy show gonococcal forms short, plump rods. This is impossible. Even after that progressively enlarge and decrease in stain- Gram-staining it may be impossible, although this ability until none can be detected. Similar mor- method is more reliable than simple methylene blue phologic changes are observed during chloromycetin staining. The reliability of the Gram procedure therapy but not during sulfonamide treatment. depends to a great extent on the staining technique; it In conclusion it is emphasized that it is impossible is very difficult to obtain the right degree of de- to diagnose gonorrhoea with certainty by microscopy colorization, especially when many slides of varying alone. Clinical evidence and history must also thickness are stained at the same time. It is empha- be taken into account, but even then there remains a sized that old staphylococcus cultures are very wide margin of error. LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 453

CULTURE AND ISOLATION media are sometimes more costly than fresh media, Most strains of gonococcus do not grow in veal but the cost should be considered in relation to or beef broth or on simple broth-agar plates. other relevant factors, such as number of technicians Sensitivity to inhibition, rather than complexity of and laboratory facilities. However, the dehydrated nutritional requirements may account for much of media are not always quite as good as the fresh the fastidiousness of the gonococcus. The beneficial media and further experiments are needed. The effect of the addition of charcoal, starch, albumin, addition of inhibitors (dyes or antibiotics) to the serum or ascitic fluid to the medium is generally culture media is not generally recommended (Gould, ascribed to a detoxifying action on inhibitors present 1944; Gould et al., 1944; Hill et al., 1948). A high in the agar or in the peptone, but fresh serum and yield of positive cultures may be obtained by using ascitic fluid also contain factors essential for the two or more plates, one (or more) containing a growth of certain strains. In semi-solid broth-agar, selective agent and one without it. most laboratory strains grow well in a layer just In the gonococcus laboratory of the Statens below the surface. The pH of the medium should be Seruminstitut a soft chocolate ascitic-fluid-agar adjusted to between 7.2 and 7.6. Synthetic media medium was formerly used for primary isolation. have been described (Welton & Scherp, 1944; Welton Recently, the ascitic fluid has been replaced by a et al., 1944; Gould et al., 1944; Hill, 1948; Hill et al., combination of yeast and liver autolysates and the 1948, 1949), but these are not suited for primary heated blood by 1 % haemoglobin (M0ller & Reyn, isolation of N. gonorrhoeae. 1965).2 This so-called HYL (Haemoglobin-Yeast- The CO2 tension is of decisive importance for Liver) medium is also a " chocolate " medium and successful primary isolation and a certain degree of for convenience both media are described in detail moisture is also favourable to growth (McLeod et al., in a later section (page 463). The turbid, brownish 1934; McLeod, 1947; Reymann, 1941, 1944; Mueller medium is dispensed into aluminium Petri dishes et al., 1942; Hill et al., 1948; Morton, 1945; Griffin 9 cm in diameter and about 1.5 cm deep, or into & Racker, 1956; Griffin & Rieder, 1957). It is similar dishes made of plastic and purchased in a impossible at the present time to prescribe the ideal sterile condition; the layer of medium should be not gonococcus medium, one of the difficulties appa- less than 5 mm. Polymyxin B sulfate is added and rently being that the growth requirements and under certain conditions nystatin is also added. resistance to toxic substances vary from one strain to another (Lankford & Snell, 1943; Lankford et al., Inoculation and incubation ofprimary cultures 1943; Gould, 1944; Lankford & Skaggs, 1946). Immediately upon arrival the swabs are streaked Numerous types of " enriched " media have been on one half of a plate, care being taken to transfer as described, most of which contain a broth-agar base much material as possible to the plate.3 The inocu- supplemented with heated blood (or haemoglobin), lum is spread as shown in Fig. 3 by means of two and/or yeast extract, serum, ascitic fluid and albumin. separate platinum wires (gauge 0.7 mm) about 8 cm A chemical supplement, containing glucose, glu- long with closed loops 2-3 mm in length. All plates tamine and cocarboxylase was described by Lank- should be examined for surface contamination ford (1950). before use. The result of the spreading is shown in The choice of medium depends upon the local Fig. 4. situation; in larger laboratories with easy access to The inoculated plates are incubated for about 24 fresh blood (horse, beef, rabbit or sheep) and ascitic hours at 35°-36°C in closed glass or plastic jars fluid and with centralized media production facilities containing a moist atmosphere with about 8% of it is natural and relatively cheap to use these products CO2 (see Fig. 5). as they are. In smaller laboratories, it is practical to The glass jars have a capacity of about 4.5 litres use the commercially available dehydrated complete and have cut collars which fit into metal lids; be- media or dehydrated media bases.' Dehydrated tween the collar and the lid a ring of rubber is inserted. The plates are placed on a perforated 1 Bacto-G C Medium Base enriched with haemoglobin and Bacto-Supplement B or C may well be used (see Difco Manual of Dehydrated Culture Media and Reagents for 'See the article on page 471 of this issue. Microbiological and Clinical Laboratory Procedures, 9th ed., Before inoculation urines, spinal and synovial fluids are Detroit, Mich., Difco Laboratories, 1953, p. 122). Bacto- centrifuged. Blood cultures are made by introducing 5 ml of Supplement C is the same as Bacto-Supplement B but blood into flasks containing ox-heart broth enriched with 1 % without the addition of glucose. of glucose and 25 % of ascitic fluid. 454 A. REYN

FIG. 3 DIAGRAM ILLUSTRATING METHOD OF INOCULATION

1: Streaking with swab. 2 & 3: Spreading with bent platinum needles.

FIG. 4 GROWTH OF NEISSERIA GONORRHOEAE AND STAPHYLOCOCCUS ALBUS AFTER 48 HOURS' INCUBATION

This photograph demonstrates the effect of the spreading method Illustrated in Fig. 3. The organisms were Inoculated on a " chocolate " ascitic medium in an aluminium Petri dish, shown natural size. LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 455

FIG. 5 JARS USED FOR INCUBATION OF INOCULATED PLATES

Left: Glass jar, metal lid, clamp and rubber ring. Inside the jar is a perforated metal support with a tube containing sulfuric acid; pellets of sodium bicarbonate can be seen on the bottom. Right: Plastic Jar with lid open. Beside It Is the plastic rest with tube containing sulfuric acid. support holding a tube containing 7 ml of molar A CO2 atmosphere can also be obtained by the H,SO4: a calibrated spoon containing about 1.5 g of " candlelight method " (Ferguson, 1945) or from an NaHCO3 is put in the bottom of each jar. The lid is iron tank containing a mixture of compressed air closed by means of a screw clamp; it is fitted with a and CO2. However, in the author's laboratory it was valve. After closing the lid with the valve open, the found, for some unknown reason, that the CO2 jar is tilted to allow the H2SO2 to combine with the supplied from iron tanks (especially when dispensed NaHCO3; when the CO2 evolved has pushed out into stainless steel containers instead of glass jars) the superfluous air, the valve is closed. was less favourable to the growth than the CO2 The chemical process that takes place in the jar supplied from H2SO4+NaHCO3. If the CO2 is is as follows: supplied in one of the ways mentioned above, a 2 NaHCO3+H2SO4=Na2SO4+2 H20+2 CO2. humified atmosphere should be obtained by means From this equation the amounts of H2SO4 and of moistened cotton-wool pads. NaHCO3 can be calculated for other volumes and Recently, acid-resistant polyvinyl chloride jars other concentrations (1 gram molecule=22.4 litres). have been used. They are much lighter than the It is most practical to use a constant amount of glass jars, they do not break, and it is not necessary H,SO4, which will determine the amount of CO2, to close their smoothly fitting lids by means of provided an excess of NaHCO5 is used. The amount screw clamps. The arrangement for filing with should be calculated making allowance for the CO2 is the same as that used in the glass jars. The fact that the jar will normally be full of plates. lids (without valves) are put on just after the CO2 has 456 A. REYN been produced by mixing the H2SO4 with the microscope with about 30-fold magnification. NaHCO3. Material from one or a few colonies is picked up by After 18-24 hours the plates are removed from the a thin, straight, pointed 0.6-mm platinum needle jars. Usually, the plates are inspected in artificial and spread on a " chocolate " agar plate. The use light with the naked eye. The typical round colonies of a microscope is very helpful and allows a rapid are 0.5 to 1 mm in diameter; they are convex, greyish isolation of pure cultures. One of the characteristics and glistening, with smooth edges; on further of 24 hours' gonococcus colonies is that they look incubation the colonies increase in size and develop much darker than those of the most common con- a roughened surface with crenated edges. It is taminants. If a stereomicroscope is not available, impossible to give an exact description of the colonies the use of a good loupe and a good light source is because their appearance varies with the medium, indispensable. After inspection, all plates (positive and particularly with the strain. The size and and negative) are returned to the incubator for appearance vary greatly with the age of the culture, another 24 hours' growth without CO2. the degree of crowding, and with the colony type. For ordinary subculturing a 0.6-mm-gauge plati- If the plate is crowded with large numbers of colonies num wire, 9-10 cm long, with closed loop is used or contaminants, the colonies will be small; if the and care is taken to avoid clonal selection by dipping colonies are well isolated they should be large. The the needle into several colonies before spreading on a size may vary from less than 1 mm to about 5 mm in fresh plate. If the 24-hour growth is rich, an attempt diameter.' The consistency of the growth is often is made to test for fermentation. viscid; this is most pronounced after 48 hours' Next day the " negative " plates are re-inspected; growth. In laboratories in which the identification suspicious-looking colonies are tested with oxidase of gonococci is a rare event it is recommended that reagent and, when positive, subcultured within 30 control strains should be kept for comparison. In minutes. Stained films (of the respective colonies) the author's laboratory, about 80% of the total are examined under the microscope. After 48 hours number of positive cultures are detected after 18-24 the gonococcus colonies appear less " typical " under hours. However, the longer the transportation time the low-power microscope; they appear lighter, the later will the colonies grow out, resulting in flatter and less circular, with a tendency to irregular delayed detection. edges. If the growth of gonococcus-like colonies is abundant, one drop of oxidase reagent (page 466) CONTROL STRAINS is added; if the colonies quickly turn deep purple Control (and other) strains can be kept as lyophi- and if the colour is maintained for more than half lized cultures, preferably in a refrigerator or in an a minute the reaction is considered positive and a incubator, as cultures in semi-solid nutrient agar Gram-stained smear is prepared. Colonies of other covered by mineral oil. Lyophilized cultures keep micro-organisms may also react with the oxidase for years, whereas subculturing is needed every reagent. Most of these are easily distinguished from third month for the cultures kept in nutrient agar gonococcal colonies, but notably species of Alkali- under mineral oil. Heavily infected charcoal swabs genes and Haemophilus and also of other Neisseria in Stuart tubes kept in a cool place preserve the may be impossible to distinguish on the basis of gonococci for about six weeks (Korner, 1963). colony form and oxidase reaction. If the growth is Stock cultures can also be prepared as follows. scarce, the oxidase reagent can be used by applica- The strains are grown on " chocolate " agar slopes tion of a few drops on a filter paper which is streaked for 24 hours in a CO2 jar. One ml of a 30 % solution with small amounts of growth. In this way several of inactivated horse, rabbit or sheep serum in cultures can be tested at a time on one piece of trypticase soy broth (BBL) is used to wash the paper. growth from the agar surface. The washings are Subcultures are made from all plates from which dispensed in portions of 0.2-0.4 ml into a large Gram-negative diplococci or cocci are found on number of prelabelled 13-mm x 100-mm cotton- microscopy. If there is heavy contamination typical stoppered sterile tubes (ink covered with self- colonies are picked out under a low-power stereo- adhesive plastic tape is suitable for labelling). The cotton stopper is cut off at the tube lip and a 1 Recently, Kellogg et al. (1963) observed four morpho- logically distinct clonal types in N. gonorrhoeae cultures. sterile rubber stopper inserted, pushing the cotton Virulence in man was found to be linked to clonal type 1. some way into the tube. The tubes are frozen in a LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 457 dry-ice/alcohol bath at - 70°C and stored in a dry- isolation. Most of these strains are easily distin- ice chest until needed. The frozen suspension is guished from true gonococci by their cultural and thawed in a water-bath at 37°C and promptly biological characteristics (see accompanying table). streaked on plates for recovery.' Formerly, 1-2 % of the "positive" gonococcus- like cultures examined in this laboratory showed an FERMENTATION TESTS absence of glucose fermentation in spite of satisfac- tory growth on the fermentation medium. This was In laboratories with some experience in the the situation until June 1957. At that time the num- identification of gonococci and meningococci it ber of non-fermenting, atypical strains showed a suffices to use fermentation plates of glucose and sudden increase. Not only was glucose fermentation maltose only, because these sugars will permit the absent or weak, but the growth was poor on the differentiation between N. gonorrhoeae, N. menin- routine medium used for primary isolation. It is gitidis and N. catarrhalis, provided the colonies are emphasized that freeze-dried strains isolated before typically non-chromogenic (Wilson & Miles, 1955; June 1957 grew well on the " old " media and showed Breed et al., 1957). It is preferable to use a solid typical fermentation. Serological tests indicated that medium on which contamination can be directly these atypical strains could be considered as true observed. In the Neisseria Department of the gonococci. However, as soon as the media were Statens Seruminstitut, translucent fermentation changed by using beef-heart broth instead of beef- plates are used; they are rather heavily inoculated, meat broth in the medium for primary isolation and and 6-8 strains are tested on one plate. The plates subculture, and placenta or beef-heart broth instead are incubated for about 24 hours in CO2 jars, then of beef-meat broth in the fermentation medium, removed and left on the table for about half an hour both growth and fermentation of the " atypical " with the lids open in order to allow the CO2 to strains became typical. This event is reported to escape from the medium. The fermentation medium demonstrate the great variability of the growth hitherto used by the author is described on page 464. requirements of gonococci (Reyn & Bentzon, 1961). Juhlin (1963) has recently introduced a medium The frequency of non-fermenting strains is now that allows growth from a very small inoculum, and below 0.05 0. very few strains fail to grow on it or to ferment Polymorphous Gram-negative rods, growing in glucose. gonococcus-like colonies and with a fermentation If there is typical acid production the inoculated pattern similar to that of gonococci may be a source areas of the glucose plates turn yellow; however, the of error. In most cases, howener, the oxidase- intensity of the acid production varies and strains reaction will be negative. Growth at 22°C on with weak or absent acid formation are occasionally unenriched medium will rule out N. gonorrhoeae. found. In typical cases, the maltose plates turn On careful microscope examination of successive purple on the growth areas. In order to detect conta- 16-18 hours' pure subcultures it will be found that mination, Gram-stained smears should be prepared either long rods will gradually predominate or the from the fermentation plates in all doubtful cases. single elements will appear oval and be orientated If the growth is weak or the acid formation is not in a pattern different from the typical coffee-bean very marked, it may help to incubate the glucose pairing. Large cocci or diplococci, which are evenly plate for another 24 hours. and deeply stained by the counter stain, require For the fermentation tests it is important that the special attention. When it is found that the isolated growth should be well supported; the use of ascitic micro-organism shows a high degree of penicillin fluid from patients treated with antibiotics should be resistance one should be even more suspicious. avoided. In the author's laboratory some batches of Further study of this group-or of these groups- phenol red have been found inhibitory. Another of micro-organisms is needed with a view to develop- source of error is the use of ascitic fluid from patients ing an improved classification. Several authors have with uncontrolled diabetes. called attention to these sources of error which play Neisseria other than gonococci are occasionally a comparatively greater role in smaller laboratories isolated from the genito-urinary tract; the incidence or in localities where these micro-organisms are depends on how much attention is paid to their relatively frequent (De Bord, 1939, 1943; Aiken et al., 1956; Cary et al., 1958; Gangarosa & Cary, 1 For further details see US Public Health Service (1962). 1960; Sampson et al., 1961; Svihus et al., 1961). 458 A. RELYN

CULTURAL AND BIOLOGICAL CHARACTERISTICS OF DIFFERENT SPECIES OF NEISSERlA a

Fermentation Growthplain agaron plain Species Colony characteristics in brief agar l|dextrose |maltose |sucrose laevulose mannitol lactose 220C 370C

N. gonorrhoeae Both more or less greyish white, N. gonorrhoeae often more viscid + _ _ _ _ - - - than N. meningitidis which Is easily emulsified. N. meningitidis N. meningitidis colonies larger than N. gonorrhoeae + + _ _ _ _ - colonies, both variable in size.

N. catarrhalis Colonies variable, may be smooth and translucent or hard, opaque - - _ _ _ - + + and irregular.

N. flavescens Colonies golden yellow, glistening, - _ _ _ _ - + similar to N. meningitidis.

N. flava (sub- Colonies vary from greenish yel- + + F - :1: species 1, 11, 111) low to slightly yellow.

N. sicca Colonies usually hard, wrinkled, + + + + _ - + + opaque with greyish yellow pig- ment.

a Further information can be found in Wilson & Miles (1955), B (1957) and Cruickshank (1960). i= acid formation or growth varying, preponderantly positive. T = acid formation or growth varying, preponderantly negative

DIAGNOSTIC CRITERIA if swabs other than the charcoal-impregnated appli- cators have been used. In some cases, it may be As a general rule, a culture isolated from the convenient to report the transportation time. It is genito-urinary tract may be considered and reported recommended that copies be kept of all reports and as a gonococcus, provided the following criteria have that a system of checking be set up to ensure that all been met: extra remarks are given on both the reports and the (1) the colony morphology is typical; copies. (2) the colony is oxidase-positive; Cultures showing no growth of gonococci after 48 hours' incubation should be reported as " negative" (3) microscope examination of a pure culture or " no growth of gonococci ". shows typical Gram-negative diplococci; Cultures fulfilling the diagnostic criteria given (4) only glucose is fermented. above should be reported as " positive " or " growth Cultures that fulfil criteria 1 to 3 but do not fer- of gonococci ". Sometimes it is necessary to specify ment glucose or any of the sugars listed in the above that Gram-negative oxidase-positive diplococci were table may be identified as " gonococcus-like " if no found but that the strain was lost on subculture, or growth is obtained at 22°C. that growth of " gonococcus-like " micro-organisms It is stressed that gonorrhoea cannot be excluded with atypical fermentation was observed. In such unless repeated cultures show negative results. cases, the report should contain a suggestion that the test be repeated. If it takes more than five days to REPORTING OF RESULTS obtain a pure culture for the fermentation tests, a preliminary report should be made and care should Reports should contain all the identification marks be taken to ensure that the final report is also given. given on the forms accompanying the samples. In addition to " negative " and " positive " findings Missing information or errors should be noted. it is absolutely necessary to report " overgrown " or Furthermore, notice should be taken of whether the "unsatisfactory " cultures. A suggestion that the transportation tubes are beyond their expiry dates or test be repeated should be made as well. Cultures LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 459

should be designated as " overgrown " when it is gonococci, except for the addition to the plates of evident that any gonococci that might be present polymyxin B and perhaps also of ristocetin. Most of would have had no chance to develop in the presence the substances that have hitherto been shown to be of the overgrowing micro-organisms, which in turn inhibitory to the swarming of Proteus are too toxic may originate either from the patient or from the to N. gonorrhoeae and therefore useless. PNPG/1 medium. If overgrowing micro-organisms show forms the only exception (Jungfer, 1958). Two confluent growth in the fourth quadrant of the plate plates are used, one with the addition to the medium it is obvious that the plate is " overgrown ", but it is of60,ugof PNPG/1 per ml and one without PNPG/1. sometimes very difficult to distinguish between In most laboratories it will be impossible to deter- " overgrowth " and " no growth ". Each laboratory mine in detail the nature of the overgrowing orga- must develop its own style of reporting, balancing nisms. It is, however, recommended that it should be the risk of reporting too many " false negative " specified if Proteus is encountered. If not specified, results against that of reporting too many " over- the repeated reporting of " overgrown " cultures grown " cultures. In general, overgrowth by Proteus, may discredit the method. In certain periods, over- E. coli or yeast originates from the patient, and this is growth by soil bacteria or by moulds, both origina- also true of most of the cases in which B. subtilis is ting from contamination during the preparation of observed. Apart from spreading the swab material, the plates, may destroy the cultures. Such over- very little can be done to prevent overgrowth of growth may be effectively inhibited by the addition contaminating bacteria without damaging the of nystatin (for details, see page 466).

SEROLOGICAL EXAMINATION

GENERAL REMARKS Grimberg, 1935; Dmitriw & Demidova, 1935; Casper, 1937; Tauber, 1959, 1960; Tauber & Russell, Serological examination can be used in two ways, 1960, 1961). A thermostable antigenic factor either to classify a culture or to test a patient's serum common to all gonococci has been identified by for antibodies. Neither of these procedures is much Reyn (1947, 1949a, c); several type-specific (or strain- used in the diagnosis of gonorrhoea. In the majority specific) antigenic substances (both thermostable and of cases the bacteriological examination suffices. thermolabile) were also demonstrated. Using agglu- Antibody is rarely present in acute cases. Sero- tination, Wilson (1954) demonstrated eight heat- logical testing has a certain diagnostic value in stable antigens, four of which behaved as group chronic infections (pelvic inflammations, prostatitis, antigens. In addition to the complex antigenic arthritis, etc.) and when bacteriological examination pattern, cross-reactions with other Neisseria are is inconvenient or impossible. However, serological often found; this applies mainly to N. meningitidis methods are not quite specific and antibodies tend and N. catarrhalis. In addition some strains of to persist for many years in spite of clinical cure. Pasteurella multocida show cross-reactions. The Despite much effort, serological investigation of cross-reactions depend on antigenic substances the gonococcus has not been very rewarding. On closely related to thermostable antigens (Reyn, 1947, culture, the gonococci undoubtedly change their 1949a; Wilson, 1956). serological characteristics quite readily and partly or Simple slide or tube agglutination tests are not very completely lose their surface antigens; this makes useful since many strains are auto-agglutinable when serological classification difficult (Uroma, 1943; suspended in saline. A number of precipitation and Reyn, 1947, 1949a, b, c; Chanarin, 1954; Wilson, flocculation methods have been described (Saint- 1954). Antigenic substances of different chemical Prix, 1950a, b); however, the only serological method specificity are responsible for the antibodies formed in common use is the complement-fixation reaction in gonorrhoeal infections. Antigens of poly- (Cohn, 1937; Le Minor, 1948; Price, 1949; Car- saccharidal, lipopolysaccharidal and proteinic nature penter, 1963). Further references to serological have been prepared, but further research is necessary methods will be found in the review by Scherp (Boor & Miller, 1934, 1939, 1944; Mutermilch & (1955). A fluorescent antibody technique (Deacon et 460 A. REYN al., 1959, 1960; Deacon, 1961; Harris et al., 1961) co-carboxylase. Eighteen-hour cultures are har- has been worked out for the identification of vested in physiological saline and suspended to a N. gonorrhoeae in smears. However, this technique density of about 2 x 1010 organisms per ml; from requires that the worker be familiar with the use of each of the suspensions aliquots are taken out and the ultraviolet light microscope and that he should submitted to microscopic and serological examina- have a basic understanding of the principles govern- tion before the various suspensions are mixed to ing fluorescent antibody techniques. Thus, this very form the polyvalent antigen. The antigen is dis- promising method cannot as yet be recommended pensed in l-ml or 2-ml amounts and heated to 56°C outside highly qualified laboratories, especially as for 20 minutes; it is now ready for use and can be certain specificity and sensitivity problems still stored at - 10°C for several years. The harvested, remain to be solved (Danielsson, 1963; Lind, 1963). unheated material is stored at about - 10°C until There have, however, been recent indications that the results of the microscopic and serological tests the specificity problems are being overcome.' are available. The following criteria should be The relative efficiencies of the culture method and fulfilled before the suspensions are mixed: (1) no the fluorescent gonococcal antibody technique have contamination, (2) no anti-complementary effect, been found to vary from one laboratory to another; (3) a " standard " degree of reaction with pools of for instance, one laboratory has claimed a superiority positive sera, (4) negative reaction with a pool of of 12% for the culture method while another has negative sera. claimed a superiority of more than 100% for the To obtain an estimate of the anti-complementary so-called " delayed fluorescent-antibody test " (Price, effect, the individual suspensions are tested in 1964). This variation is most probably due to lack complement-fixation experiments with a constant of uniformity in performing the conventional dose of antigen and two or three different comple- culture method. ment concentrations; suspensions only slightly anti- A rapid staining technique has been described by complementary are included in the antigen. A Kellogg & Deacon (1964). Very recently, Hess et al. suitable antigen concentration is usually obtained (1965) have described how the immunofluorescent by dilution of the stock antigen to give about technique can also be used indirectly as a means of 5 x 107 organisms per test-tube. The antigen is detecting antigonococcal antibody in human sera. diluted in S0rensen's phosphate buffer, pH 7.38. Finally, Reising & Kellogg (1964) have proposed The total volume is 0.3 ml and the quantity of serum a precipitin test for the detection of gonococcal in the first tube is 0.025 ml, in the second 0.025 ml antibodies. Preliminary findings with this test diluted 1 in 3, in the third 0.025 ml diluted 1 in 9 indicate that the virulent gonococcus contains a and so on. The 100% haemolytic unit of comple- protein antigen which is species specific. However, ment is determined in the presence of antigen and the test has not yet been evaluated in large-scale one unit is added per test-tube. experiments with respect to false positive and false Fixation is allowed to take place for three-quarters negative results. of an hour at room temperature and for three- quarters of an hour at 37°C in a water-bath; 0.2 ml GONOCOCCUS COMPLEMENT-FIXATION REACTION of a 2.5 % suspension of sensitized sheep-blood cells (three haemolytic doses) are then added to the tubes The following method was developed at the Statens which are re-incubated at 37°C for an additional Seruminstitut, Copenhagen (Kristensen, 1930; hour. The degrees of haemo ysis corresponding to Reyn, 1947, 1949a), the procedure being as follows: the different serum dilutions are estimated by The antigen is prepared from a mixture of about comparison with a scale of haemolysis prepared 30 fresh strains and seven serologically " known" from tubes with complete haemolysis. The results stock strains.2 The strains are grown on an ascitic are given as degrees of potency; these are calculated agar medium of the same composition as the fermen- after the method given by Kristensen (1930), the tation medium but without sugar, phenol red and degree of potency being defined as that value of n which whensubstitutedinthe formulaa= 0.025 x 3-n/3 1 US Public Health Service (1962); Brown et al. (1963); gives a value for a exactly equal to the volume of Moore et al. (1963); Shapiro & Lentz (1963); Lind (1964); serum containing enough antibody to give a mini- Price (1964). mum 60 haemolysis. Further details 2 Available in a freeze-dried state from the Neisseria reaction, i.e., % Department, Statens Seruminstitut, Copenhagen, Denmark. are given by Reyn (1949a). LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 461

The day-to-day variations in the test are counter- potency (0.111 log10 values) and it is proposed that acted by means of daily titration of six control sera the standard deviation of the difference between two for each of which an average " standard " value has different antigens should not exceed 1.4 degrees of been determined. The average amount by which potency (0.223 log10 values). This would mean that the titres of the control sera differ from their antigens giving differences greater than ± 2.8 degrees standard values is the correction to be applied to the of potency in more than 5 % of the sera would not be values for the other sera. accepted. Before a new antigen is accepted for use the The sensitivity of the complement-fixation reaction following comparisons with the old one are made: described is so adjusted that only about 30% of (1) The approximate titre of the new antigen is female cases with uncomplicated, acute gonorrhoea determined in preliminary experiments. As a rule, and about 90% of cases with complicated, chronic a fair degree of similarity between the results with gonorrhoea (male and female) show positive results. the new and the old antigen can be obtained after In spite of clinical cure the reaction may remain three to four days on which 10 positive sera of positive for several years and in the individual case varying potency are tested quantitatively against it is difficult to judge the specificity of a positive different dilutions (three per day) of the new antigen. reaction. The diagnostic value of a sero-reaction depends on its sensitivity and specificity under the (2) Having determined the approximate titre, the special local conditions under which it is intended to two antigens are used in parallel titration of about be used. If the incidence of previous and present 1000 sera picked out at random among the sera sent infection is high, many apparently non-specific in for gonococcus complement-fixation. This will reactions will be found and the diagnostic value in give a comparison of about 75-100 sera that react the individual case will be small. If, however, the positively with either or both of the antigens. incidence of gonorrhoea is low and confined to The new antigen is accepted for use if the average certain patient categories, the diagnostic value is serum titre difference between the two antigens does greater. A survey of positive reactions obtained in a not exceed ± 0.3 degrees of potency (±0.048 log10 medical ward in Copenhagen shows a certain trend values). If a difference in the same direction is regu- for the conditions in Denmark (Bang & Krag, 1942). larly observed for several days, another antigen It was found that 7.2 % of the males and 6.2% of the dilution is used and the experiments are started females were positive; by dividing the material into again. groups according to the history and the clinical signs In the Neisseria Department of the Statens of gonorrhoeal infection it was found that the Seruminstitut, a new antigen is made once a year. degree of specificity increased with increasing Care should be taken to avoid a gradual change in strength of the reaction. In 63.4% of the sera sensitivity as a result of repeatedly accepting antigens showing one degree of potency (serum concentration with a positive (or negative) difference. < 1:12) and in 84.7% of the sera showing three degrees of potency (serum concentration < 1: 36) (3) One thousand sera from blood donors (with- either the corresponding patients were known to be out clinical or serological evidence of syphilis) are suffering from gonorrhoea or their case histories tested with the " final " antigen titre. The number of made it highly probable that they had previously positive reactions should preferably not exceed five suffered from this disease. per thousand. Strong cross-reactions are usually found in patients The above procedure is related to the average with meningococcus infection. Positive reactions sensitivity or strength of the antigen; another ques- have frequently been observed in patients suffering tion is whether the standard deviation of the differ- from chronic bronchitis especially in those with ences should also be taken into consideration. The bronchiectasis, possibly due to infection with standard deviation of repeated titration of the same N. catarrhalis or other Neisseria species sharing antigen is estimated to be about 0.7 degrees of antigens with N. gonorrhoeae. 462 A. REYN

DRUG-SENSITIVITY DETERMINATION

Until recently, determination of the sensitivity to Ringertz, 1961; Gjessing & 0degaard, 1962; Ranta- antibiotics was not very much used in connexion salo et al., 1962). Completely resistant strains have with routine culture of gonococci. However, the not been isolated from patients. However, about number of strains of gonococcus showing decreased 25 % of strains isolated in Denmark since 1957 need sensitivity to antibiotics makes the development of a more than 0.225,ug of penicillin per ml for complete recommended reference method desirable. The use in vitro inhibition and this is only two to three times of reference strains would be a further aid in making the average serum concentration obtained after the comparison of results obtained in different 300 000 units of aqueous procaine penicillin G. laboratories more meaningful. The WHO Expert Geographical differences may, of course, be en- Committee on Antibiotics in its second report (1961) countered (Reyn, 1962, 1963). Streptomycin- stated that " a standard method of test is required" resistant strains have been isolated and an increased and that " stocks of lyophilized cultures should be failure rate of streptomycin treatment has been re- kept, in order that future direct comparisons may be ported by Alergant (1958), Reyn (1961), Durel et al. possible between strains of gonococcus isolated at (1961) and Roiron et al. (1961). different times ". Under the auspices of WHO a A tablet method and a plate dilution method for group of laboratory experts is now doing the pre- sensitivity determination of penicillin, streptomycin paratory work for the establishment of such a and tetracycline were described by Reyn et al. in reference method. The preliminary results of this 1958, and a plate dilution method was described in study are referred to in the first report of the WHO more detail by Reyn et al. in 1963. These methods Expert Committee on Gonococcal Infections (1963). can easily be applied to the determination of sensi- The aim of this work is to agree on a procedure in tivity to other antibiotics. The tablets should be which the inhibiting concentration of antibiotic is chosen so as to give zones of a suitable size (Lund measured by a plate dilution method. et al., 1951; Lund, 1953). The concentrations in the Carefully controlled diffusion tests might be solid medium used in the dilution method should preferred for local use. This conception is supported be so adjusted that the change from full to no by the results obtained in a recent collaborative growth for the majority of the strains is likely to study of different methods by Denmark, Finland, occur in the middle of the series of concentrations Norway and Sweden (Reyn et al., 1963). The employed. If, for some reason, the agar cup tech- results obtained in this study indicate the possi- nique, the disc method, the gradient plate technique, bility of converting a diffusion result read in or tube dilution methods with fluid or semi-fluid millimetres to a concentration obtained by a dilution medium are found more convenient, they may well method. be used. However, regardless of the method it is The great majority of cases are cured by penicillin, important to know something about its variation streptomycin or tetracycline; however, it has been under the local conditions. demonstrated both in vitro and in vivo that the Direct sensitivity determination from swabs is penicillin sensitivity is decreasing (Jensen, personal possible only in a few cases and it is preferable to use communication, 1957; Cradock-Watson et al., 1958; pure cultures from which the size of the inoculum Curtis & Wilkinson, 1958; King, 1958; Scamberg et can be controlled. In order to avoid selection, not al., 1958; Reyn et al., 1958; Hirsch & Finland, 1960; less than 10 colonies (if possible) should be used for Cole et al., 1961; Reyn, 1961; Thayer et al., 1961; the preparation of the pure cultures.

MEDIA AND REAGENTS

PREPARATION OF BROTH broth-agar base for later use. Suitable amounts may several at low Containers used in the preparation of broth, etc. be kept for months temperatures. should be of stainless steel, glass or some other inert Erlenmeyer flasks are suitable for this purpose. material. It is practical to prepare stock batches of Broth formula: chopped meat, 500 g; tap water, LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 463

1000 ml; peptone, 10-15 g; 1 Na2HPO4.12 H20, 2 g; rated from the broth and the peptone and salts are and NaCI, 3 g. added with subsequent boiling for 15 minutes; the The " meat " can be veal or beef-heart or human pH is adjusted to about 8.1. After autoclaving the placenta tissue (see below), which should be freed pH should be about 7.2. from fat and tendinous tissue. After cutting and chopping, the meat is mixed with half the water and CHOCOLATE ASCITIC-FLUID-AGAR placed overnight in the cold. Next morning it is boiled for 15 minutes and the soup is collected, using This is prepared by the addition of horse blood a sieve. The meat is boiled again for 10 minutes and ascitic fluid to a broth-agar base consisting of together with the other half of the water and the 2.4% of Danish AKI agar in beef-heart broth with two soups are then combined. After addition of pep- 1 % of peptone (" Orthana " special), 0.3 % of NaCI tone and salts the broth is heated to boiling, during and 0.2% of Na2HPO4.12H20. which the pH is adjusted to about 8.1 by means of The base is dispensed in 540-ml amounts in 5N NaOH. The broth should boil for not less than Erlenmeyer flasks and kept cool; before further 10 minutes and for at least 5 minutes after the last preparation it is melted and kept at 80°C. 60 ml of addition of sodium hydroxide. By means of distilled defibrinated horse blood are added. The mixture is water the original volume (plus an extra 5% to kept at 80°C for 15 minutes and shaken frequently; counteract the autoclaving effect) is restored. The it is cooled to 560C and mixed with 300 ml of sterile broth is cleared by paper filtration, the agar2 melted ascitic fluid 3 (30 %), which is brought to 56°C before in the hot broth and boiled for 20-25 minutes. The mixing. The ascitic fluid causes a rise in pH which hot broth-agar is then filtered by suction and dis- should be compensated for by adding 0.5N HCI pensed in Erlenmeyer flasks. It is sterilized by auto- until the pH is about 7.1-7.2. It is false economy to claving at 120°C for 20 minutes or by heating for pour plates too thinly. Immediately afterwards, 20 minutes on a boiling water-bath (" steaming ") plates 4-5 mm thick are poured into 9-cm wide on three days in succession. After sterilization the aluminium or plastic Petri dishes. If ascitic fluid is pH should be about 7.2. difficult to obtain the amount can be reduced to If the broth is prepared for later use it should be 150 ml (15 %) without appreciable loss in the quality sterilized by Seitz filtration and kept in the cold in of the medium. It is also possible to replace the Erlenmeyer flasks until the agar is added, followed ascitic fluid entirely by 100 ml (10%) of sterile horse by autoclaving as described above. serum. Plates should be stored in the refrigerator Autoclaving is more deleterious than " steaming" and should be used within 10 days to 2 weeks. and it also has a more acidifying effect than has The finished medium is a soft agar and should be " steaming ". handled and inoculated with care. Placenta broth is prepared from fresh human The surface moisture may be removed without placentas (no antiseptics) which are freed of the undue danger of contamination by inverting the umbilical cords and the coverings. They are cut into plate in the incubator, lifting the bottom out of the pieces and rinsed in cold tap water. top, and resting it on the edge of the top (agar After coarse chopping in a machine, two litres of surface always downwards) until all droplets of tap water are added per kg, the crude broth being moisture on the agar surface have disappeared. The kept at about +4°C until the following day, when excessive moisture produced by condensation and by it is boiled for 15 minutes. The placenta tissue is sepa- hysteresis of the agar is neither necessary nor desir- able for growth of the gonococcus, provided the

l The precise quantity will depend on the brand and s It is important to exclude the presence of antibiotics in quality. the ascitic fluid. When derived from diabetic patients it a The exact percentage depends on the batch and brand should be examined for glucose, especially when used in the of agar; the agars should be soft. At the Statens Serum- fermentation medium. Seitz filtration is necessary unless the institut, Danish AKI agar is used in the chocolate ascitic-fluid technique of collection is absolutely safe. In fermentation medium. Selected batches of Japanese agar Kobe 1 are em- tests and in experiments to determine the sensitivity to ployed in the fermentation medium. Preparations with AKI antibiotics, it is advisable to test the ascitic fluid before agar do not need to be filtered before autoclaving. The AKI use; if possible, fluid from the same patient should be used agar is not a true agar; it resembles carrageen which is rich in for long periods. Ascitic fluid and other biological materials methyl groups. It solidifies at about 45°C (in the concen- are expensive and difficult to procure. Recent experiments tration used) whereas Japanese agar, for example, solidifies indicate that simple autolysates prepared from fresh brewer's at about 30°C. The pH also varies when different brands of yeast and pig liver may replace the ascitic fluid, see " HYL agar are used. medium ", page 464. 464 A. REYN plates are incubated in a moist atmosphere. How- and then through a membrane filter 1120. It is ever, drying should not be continued until the surface dispensed in sterile bottles and stored at 4°C. becomes wrinkled by lines of stress; this indicates Liver autolysate ' that the agar is losing water, and should always be avoided. 1 kg of pork liver is chopped three times and While mixing and pouring the medium, care 700 ml of distilled water are added. The mixture is should be taken to avoid the promotion of excessive stirred and placed in a 50°C water-bath with subse- amounts of foam and bubbles. If bubbles do find quent slow heating to 75°C; this temperature is their way into the plates, they may be removed by maintained for one hour during which the prepara- flashing a Bunsen flame over the agar surface before tion is constantly stirred. After centrifugation, it is it has solidified. sterilized by filtration, using the same filters specified above. The pH is adjusted to 7.4-7.6. HYL 1 MEDIUM Formula for preparation of 100 ml ofmedium Haemoglobin 66 ml of broth-agar base prepared as described Powdered ERG.B6 (Riedel-De Haen AG, Seelze- under Chocolate ascitic-fluid-agar, 15 ml of sterile 6% Hannover) is mixed with a small amount of de- haemoglobin, 3 ml of a mixture of one part of yeast mineralized water to make a paste after which autolysate and two parts of liver autolysate, distilled sufficient demineralized water is added while stirring water to 100 ml. The brand of agar used should to give a 6% solution. (This will be clear and the pH fulfil certain requirements; in particular, it should be will be 7.9.) The solution is poured into screw-cap non-toxic when used in an amount resulting in a bottles and the screw-caps tightly closed. It is then suitable gel strength. A method of checking the gel heated in a steam-bath for 10 minutes and autoclaved strength has been published by Fulthorpe (1951); for 15 minutes at a pressure of 0.6 kg/cm'. When recently, the method was simplified by M0ller. heated in tightly closed bottles and stored at 4°C the To measure the toxicity, comparative experiments haemoglobin remains as an opalescent solution for should be done, using gonococcal strains with several months. differing growth requirements. Both countings of the viable units and measurements of the colony Yeast autolysate 2 sizes should be performed. The AKI agar, which Fresh brewer's yeast (extremely flocculent bottom in our hands has shown a low toxicity to N. gonor- yeast) 3 is obtained from a brewery. Water is added rhoeae, has a higher melting-point than have in excess and after some time the supernatant is Japanese and Spanish agars. Thus, an AKI agar discarded. After two more washings with water, base must be kept at about 70°C when mixed with the suspensions being neutralized each time by the heat-labile enriching substances. means of a bicarbonate solution to remove the resin A Spanish agar (brand unknown as yet) has also substances contained in the hops, the suspended been shown to be non-toxic and to give a suitable yeast is centrifuged and subsequently mixed with gel strength when used in a final concentration of an equal amount of water. For every 2.5 litres of about 0.65 %. suspension 80 ml of ethyl acetate are added and the The equipment for the preparation of the medium mixture covered by a tight lid and heated for one is simple and the ingredients needed for its prepara- hour in a 40°C water-bath. For practical reasons, tion are easily obtainable. The constituents of the the mixture is then moved into an incubator at 370C medium have not yet been analysed and defined until the next morning, when the pH is adjusted to chemically. It is possible that further analysis will about 7.4 using about 17-20 ml of SN NaOH per reveal a close similarity to the media previously litre. The suspension is centrifuged and the superna- described by Lankford and co-workers. tant yeast autolysate sterilized by filtration, first through a membrane filter S. S. G6ttingen 1118 FERMENTATION MEDIA 1. Danish fermentation medium " Haemoglobin, Yeast, Liver. Broth-agar base: 2.2 % of agar in human placenta Society for General Microbiology (1958). broth prepared with 1% of peptone and 0.5% of 'If it is impossible to obtain an extremely flocculent bottom yeast, lager beer type, it will be necessary to use 'Mo11er, Ake, personal communication, 1961. centrifugation at this point. 'As developed at the Statens Seruminstitut, Copenhagen. LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 465

NaCI. No phosphate buffer is added (the pH is infusion), 60 ml; Sodium Alginate, BPC,3 0.6 g; adjusted to 7.1-7.2). The quality of the peptone is Bacto Agar (Difco)2 6.0 g. These ingredients are highly important and should be especially tested. mixed in each of four flasks and are then autoclaved The base is dispensed in 600-ml amounts and at 120°C for 20 minutes. The broth-agar base is mixed with 300 ml of ascitic fluid, 36 ml of phenol cooled to 55°C. red 1: 500, 9 g of sugar (glucose or maltose) and Ascitic fluid: Each fresh batch is sterilized by 9 ml of 0.1 % co-carboxylase.1 filtration (Seitz filter EKS2) and compared with The sugar is dissolved in a minimum of water earlier batches as regards growth-stimulating effect and either heated to 100°C for 10-15 minutes or on strains of N. gonorrhoeae, Streptococcus pyogenes, sterilized by filtration. The co-carboxylase (Hoff- Haemophilus influenzae, and Diplococcuspneumoniae. mann-La Roche) is dissolved in sterile water and, Phenol-red (Merck): A 0.4% solution in distilled surprisingly, it can be added without sterilizing. water is autoclaved at 120° for 20 minutes. 4 g of phenol red (Merck) are dissolved in 160 ml of 0.1N NaOH at 37°C with shaking. The volume is Haemin, bovine (twice recrystallized) (Sigma): brought up to 2000 ml with distilled water and the A 0.2% solution is prepared by dispersing 0.15 g of solution is sterilized by autoclaving at 120°C for haemin crystals in 75 ml of distilled water and 20 minutes. The constituents are mixed at about adding 1.5 ml of 3N sodium hydroxide. The 50°C, the pH is adjusted to about 7.4 with 5N HCI; suspension is shaken well until the crystals have by the following day it will have risen to about 7.8, dissolved completely and a perfectly clear, dark- which is suitable for the incubation in a CO2- green solution is obtained. This solution is then atmosphere. The medium is poured into glass or sterilized by filtration (Seitz filter EKS2). plastic Petri dishes in a 4-5-mm layer. Sugar: A 20 % solution of dextrose, maltose, sucrose, or laevulose in placenta infusion. The 2. HAP medium solution is sterilized by filtration (Seitz filter EKS2). This is a new solid fermentation medium enriched HAP medium: Broth-agar base, 380 ml; ascitic with haemin, recently introduced by Juhlin (1963). fluid (preheated to 52°C), 180 ml; phenol-red (Merck) It is somewhat more complex than the Danish (0.4% water solution), 6 ml; haemin (Sigma) (0.2% medium, but has given very good results and is now water solution), 10 ml; sugar (20% solution in used in the Neisseria Department of the Statens placenta infusion), 30 ml. The sterile ingredients Seruminstitut. The method of preparation is as are mixed at 52°-55°C and the pH is adjusted to 7.7 follows: with 5N sodium hydroxide. The medium is poured Placenta infusion: Fresh human placentas are into plastic Petri dishes, filling each one to a depth ground after removal of membranes and umbilical of 4 mm. cords. The amount thus obtained is weighed and Co-carboxylase (B&rolase, Hoffmann-La Roche) mixed with twice as much distilled water and kept at is dissolved in a buffer consisting of 1.0 g of sodium 4°C during the night. The next day the mixture is acetate, 0.2 g of sodium hydroxide and 100 ml of boiled for 20 minutes and paper-filtered, yielding a sterile water to make a 0.I% solution. Of this clear fluid which is dispensed into Erlenmeyer solution, 6 ml are added to the HAP medium at the flasks and autoclaved at 120°C for 20 minutes. time when the broth-agar base, the ascitic fluid, and Broth-agar base: Placenta infusion, 200 ml; the other ingredients are mixed, giving a final sodium chloride (3 % solution in placenta infusion), concentration of about 0.001 % of co-carboxylase. 60 ml; disodium hydrogen phosphate dihydrate (2% solution in placenta infusion), 60 ml; Proteose STUART'S MEDIUM WITH SOLID AGAR Peptone No. 3 (Difco)2 (10% solution in placenta Formula 20% by I This comparatively high amount of co-carboxylase was Agar, 16 g;4 sodium glycerophosphate, found to be needed by Bang (personal communication, 1952), volume, 50 ml; thioglycolic acid, 80%, 0.95 ml; who at that time did not know the results obtained by Lank- ford & Skaggs (1946). 'Difco Manual ofDehydrated Culture Media and Reagents 'British Pharmaceutical Codex, London, The Pharma- for Microbiological and Clinical Laboratory Procedures, 9th ceutical Press, 1959, p. 675. ed., Detroit, Mich., Difco Laboratories, 1953, pp. 259 and ' The precise quantity of agar will depend on the brand 290. used and on the batch. 466 A. REYN

CaCl2, 10%, 1 ml; methylene blue, 0.1 %, 2 ml; Every morning, a fresh reagent is prepared by and distilled water to 1000 ml. It is recommended mixing one volume of the stock suspension with that all distilled water be passed through an ion- two volumes of distilled water. When fresh, it has exchange-resin column to remove free chlorine a very light purple colour (almost colourless); it is (Crookes & Stuart, 1959). A 1.5% "water base" slowly oxidized in air, turning a deeper and deeper is stored in 950-ml amounts. The base is melted and bluish purple until after several hours it is com- the ingredients are added with subsequent adjustment pletely oxidized, and is then useless. The gonococci to pH 7.4 by the addition of SN NaOH. When hot, produce an indophenol oxidase which enhances the the medium is dispensed in sterile hydrophobic oxidation of the reagent, so that on the culture cotton-wool-plugged 15-mm x 150-mm tubes form- plates the reaction takes place in about a minute ing a column 10 cm high. The tubes are steamed at instead of over the course of several hours. 100°C for one hour, and then cooled in cold water for 15 minutes (see also page 451).1 The tubes POLYMYXIN B should be stored in a cool dark place until required. Ordinarily, they will keep (i.e., will still contain a This reagent is used for the inhibition of coliform colourless layer more than 3 cm high) for at least bacilli. two months, provided that the thioglycolic acid A 0.1% stock solution of polymyxin B sulfate fulfils the criteria laid down in the United States (Pfizer) is prepared with sterile distilled water. The Pharmacopoea, vol. 15, p. 1070. unit/weight relation differs from one batch to the other. The stock solutions keep for at least two Preparation ofswabs weeks at about +4'C. The final concentration used For sampling, good quality absorbent cotton-wool in the medium for primary isolation is 25 IU per ml. and wooden swab sticks (see page 451) are used. Note: Gonococcus strains are very rarely suscep- The swabs should be carefully prepared, being tible to this drug (Crookes & Stuart, 1959). neither too small nor too thick, and the cotton-wool should fit smoothly to the end of the applicators. NYSTATIN Prepared swabs are boiled in S0rensen's phosphate buffer solution at pH 7.4 and immediately afterwards Nystatin 2 is used to inhibit the growth of moulds. dipped into a 1 % suspension of powdered charcoal Stock solution: 200 ml of 70 % ethanol plus 4 in water. After impregnation, the applicators are drops of 1 % sodium citrate are used to dissolve the dried, packed in two layers of paper, and sterilized content of a vial containing 500 000 units of nystatin. by autoclaving at 125°C for 20 minutes. The mixture is shaken for 30-60 minutes with sterile glass beads, resulting in a turbid suspension which is dispensed in suitable amounts for later use. This OXIDASE REAGENT suspension keeps for about two months at - 20°C. A stock suspension is prepared by the addition 1 ml is added per 100 ml of medium giving a final of 100 ml of concentrated ethanol to 1 g of dry, nystatin concentration of 25 IU per ml and an powdered tetramethylparaphenylenediamine hydro- ethanol concentration of 0.7%. This is used in all chloride. The substance is hydrophilic and plates in periods when moulds are disturbing. If a should be kept in sealed bottles or in a desiccator concentration of 25 IU per ml is insufficient, 40-50 over P205. The reagent kills the gonococci in about IU per ml may be added without any damaging 30 minutes. The dimethyl compound (p-amino- effect. dimethylaniline monohydrochloride) is cheaper but far more toxic; in addition it is more difficult to REAGENTS TO BE USED IN THE GRAM-STAINING observe the colour change to red instead of purple, TECHNIQUE'3 especially when " chocolate " plates are used. 0.2 % aniline crystal violet 1 In some cases, it may be found convenient after cooling Solution A: 0.2 g of crystal violet is dissolved in to remove the cotton-wool plugs one at a time, soak them 10 ml of 96% ethanol. in a hot mixture of 9 parts of vaseline and 1 part of solid paraffin (melting point 500-60°C) and replace them in the tubes. In this way, evaporation will be minimized-for I Mycostatin (Squibb) has been used in the Neisseria example, when the tubes are heated in order to reduce the Department of the Statens Seruminstitut, Copenhagen. medium (see also page 450). I As developed at the Stateas Seruminstitut, Copenhagen. LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 467

Solution B: 3.2 g of aniline are dissolved in 90 ml 0.1 % phenolized fuchsin. 1 g of basic fuchsin of distilled water, vigorously shaken for 15 minutes and 10 ml of 96% ethanol are mixed with 5 ml of and filtered through wet filter paper. liquid phenol (Danish Pharmacopoeia, 1948) and Solutions A and B are then mixed. The final pH is added to 90 ml of distilled water. Before use, this about 7.5. Thereagentkeeps for 3-4 weeks at 1 8°-25°C. stock solution is diluted 1: 9 with distilled water to Lugol's iodine solution. 1 g of iodine and 2 g of give a pH of about 4.8. potassium iodate are dissolved in 300 ml of distilled water; the pH will be about 4.8. 96 % ethanol.

Annex

REVIEWERS

Dr Sara E. Branham, 5307 Glenwood Road, Bethesda, Md., USA Professor C. E. Lankford, Department of Microbiology, University of Texas, Austin, Texas, USA Dr H. W. Scherp, Chief, Laboratory of Microbiology, National Institute of Dental Research, Department of Health, Education, and Welfare, Bethesda, Md., USA Dr J. D. Thayer, Assistant Chief, Microbiology Research, Venereal Disease Research Laboratory, Department of Health, Education, and Welfare, Communicable Disease Center, Atlanta, Ga., USA Dr A. E. Wilkinson, Director, Venereal Diseases Reference Laboratory, The London Hospital Research Laboratories, London, England

REFERENCES

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