Laboratory Diagnosis of Gonococcal Infections

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

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

CONTENTS

Page

INTRODUCTION . ............... 449

COLLECTION AND HANDLING OF SPECIMENS

General remarks .... ........... 450Method of transportation . ........ . 451

BACTERIOLOGICAL EXAMINATION

Microscopy and Gram-staining technique . . . 452Culture and isolation . ....... . 453Control strains . ......... . 456Fermentation tests .... ......... . 457Diagnostic criteria .... ......... . 458Reporting of results ... ......... . 458

SEROLOGICAL EXAMINATION

General remarks .... ........... 459Gonococcus complement-fixation reaction . . . 460

PagpDRUG SENsiTIVITY DETERMINAnON .... . . . 462

MEDIA AND REAGENTS

Preparation of broth . . . . . . . . . . . . . 462Chocolate ascitic-fluid-agar . ....... . . 463HYL medium ..... ......... . . 464Fermentation media .... ...... . . . 464

1. Danish fermentation medium .... . . . 4642. HAP medium ...... . . . . . . . . 465

Stuart's medium with solid agar ..... . . 465Oxidase reagent .... ........ .. . 466Polymyxin B .... .......... . . 466Nystatin ..... ............ . 466Reagents to be used in the Gram-staining technique 466

ANNEX. Reviewers ...... . . . . . . . . 467

REFERENCES .................. 467

INTRODUCTION

The genus Neisseria comprises Gram-negative,aerobic or facultatively anaerobic cocci, usually butnot invariably arranged in pairs. The size is about0.8,u by 0.6,u. They often grow poorly on ordinarymedia and they are frequently pathogenic. Fewcarbohydrates are fermented, indole is not producedand nitrates are not reduced. With a few exceptions,catalase and oxidase are abundantly produced; somespecies are haemolytic. Neisseria are non-motile.The most important members of the genus Neis-

seria are N. gonorrhoeae and N. meningitidis; these

* This is one of a series of studies on the laboratory diag-nosis of various diseases which, it is hoped, will eventuallybe revised and published in monograph form. An effort ismade to ensure that the diagnostic methods recommendedin these studies are as internationally representative andacceptable as possible by securing the co-operation of anumber of experts from different countries. A list of thereviewers of the study presented here is given in the Annexon page 467. To all these, and to the author herself, the WorldHealth Organization is greatly indebted. - ED.

1 Director, Neisseria Department, Statens Seruminstitut,Copenhagen, Denmark.

organisms have been described more thoroughly thanthe other members of the group, such as N. catar-rhalis, N. flavescens, N. sicca and N. flava (sub-species I-III). The classification of the Neisseria isfar from being complete and a taxonomic study ofthis group is needed.

N. gonorrhoeae (gonococcus) is the etiologicalagent of " gonorrhoea ", whereas N. meningitidis(meningococcus) is the organism causing what iscommonly called " epidemic cerebrospinal menin-gitis ". The other less pathogenic or non-pathogenicmembers of the genus are most frequently found inthe respiratory tract and occasionally in the genitalregions.

In this paper, attention will be paid to the labora-tory identification of the gonococcus and referencewill be made to the other members of the genus onlyin connexion with differential diagnostic problemswhich may be of forensic significance, especially inthe diagnosis of gonorrhoea.

1581 449

450 A. REYN

COLLECTION AND HANDLING OF SPECIMENS

GENERAL REMARKS

The material will most frequently be dischargecollected from the male or female urethra and thecervix; specimens can also be taken from the rectalmucosa, prostate, vagina, conjunctiva, Bartholin'sglands, Skene's ducts, joints, blood, spinal fluid andsediment from centrifuged urine.

The material can be examined by direct microscopyand by culture; for culture immediate plating ispreferable, but in most cases this is impossible. Infemales, where the microscope findings are veryunreliable, it is recommended that both culture andmicroscopy be used. In male contacts (symptomlesscarriers) the culture is also of great importance.Culture is also much better as a test of cure than is thesimple microscope examination of a slide. Themethod of transport of the infected material plays adecisive role, especially when the transportation timeexceeds a few hours and the temperature is high.When specimens are taken for both microscopy andculture the specimen taken 'first should be used forthe culture because it is likely to contain morematerial than the second specimen (Reyn et al.,1960).

Generally, wooden applicators covered with cot-ton wool are used to collect the material for micro-scope and bacteriological examination. However,some kinds of wood are known to be toxic to thegonococcus and the use of aluminium wires hastherefore been recommended (Beakly, 1957). Thetoxic effect of the swabs can be counteracted byboiling in buffer and impregnating with charcoal(Moffett et al., 1948); this procedure appears to actalso upon the toxic fatty acids demonstrable in cer-tain batches of agar (Ley & Mueller, 1946). Manydifferent types of collection outfit have been de-scribed. In this paper, the only method described indetail is the Stuart method (Stuart, 1946; Stuart etal., 1954; Stuart, 1956) modified by Reyn et al. (1960)and by Ringertz (1960). The Stuart medium is anon-nutrient, reducing medium in which theoxygen tension can be checked by observing thechange in the colour of the medium from colourless(reduced) to blue (oxidized). It should only be usedin association with charcoal-impregnated swabs,which counteract toxic substances. Inmediateplating is possible only where laboratory facilities are

available on the spot; in other cases plating isnecessarily delayed for several hours or even severaldays. If posting has to be delayed the specimensshould be kept in the refrigerator or in some othercool place until they are despatched. Transportationin temperate or hot climates for more than a fewhours favours the growth of contaminants at theexpense of the gonococci. Selective inhibition of thecontaminants during transportation by means ofdyes (methylene blue, crystal violet, Nile blue A,etc.) or other chemical substances (thallium acetate)or by antibiotics has been proved to be of little value(Peizer & Steffen, 1947; Lagergren & Ouchterlony,1948; Bang, 1952). The lower limits of concentra-tions toxic to the contaminants are very close to thosetoxic to the gonococci. However, when samplingconditions are very poor or when the patients arefrom a low social level the addition of inhibitorsto the primary isolation medium may be indicated.An exception to the general rule is polymyxin Bwhich completely inhibits the growth of coliformbacilli without harming the majority of gonococcalstrains (Crookes & Stuart, 1959). However, at thesame time better conditions are provided for othercontaminants, especially diphtheroids and Gram-positive rods.' In hot climates it is recommendedthat specimens be transported in vacuum bottles attemperatures between 0°C and 4°C.

The sampling technique is of great importance butthis is not the place for a detailed description of thetechniques for taking samples from the various sitesof gonorrhoeal infection.2 Unnecessary contamina-tion should be avoided and the sample should be as" rich " as possible in order to compensate for lossduring transportation. It is stressed that sterilevaginal specula should be used for collecting speci-mens from the cervix; some lubricants are toxic andshould be avoided. The cervical plug, if present,should be removed before the swab is taken. Theapplicator should be inserted about 1 cm into thecervix.

1 Recently, Thayer & Martin (1964) have proposed aselective medium which contains, in addition to 25 IU ofpolymixin B, 10 ,Ag of ristocetin per ml. Ristocetin inhibitsmany Gram-positive micro-organisms. In the author'slaboratory, preliminary experiments with a selective mediumof similar composition have given very promising results.

2 For further details see US Public Health Service(1962); Carpenter (1963).

LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS 451

FIG. 1STUART'S TUBE CONTAINING A CHARCOAL SWAB

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0

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METHOD OF TRANSPORTATION'

The method of transportation described belowwas 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 tightlyclosed with hydrophobic cotton-wool plugs and thedate 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 uppermosthalf a centimetre, which will be blue owing tooxidation of the methylene blue. Oxidation willproceed during storage, and completely oxidized

1 As used at the Statens Seruminstitut, Copenhagen.

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A. REYN

the labelled tubes of media without breaking thesticks; the stoppers should be put in, leaving theupper ends of the sticks projecting about 1 cm abovethe edge of the tube as shown in Fig. 1. When

posted, the specimens should be placed in cardboardcontainers (Fig. 2), which are then inserted in specialenvelopes and they should be kept in a cool place ifposting is delayed.

BACTERIOLOGICAL EXAMINATION

MICROSCOPY AND GRAM-STAINING TECHNIQUE

The most primitive laboratory method is themicroscope examination of a stained smear ofdischarge. To a great extent the success of thismethod depends upon the way in which the smear iscollected and prepared. Though apparently simple,the method of preparation nevertheless deservesclose attention; it should be borne in mind thatcharcoal-impregnated swabs should be avoided inthe preparation of smears. Smears should be madeas thin and as even as possible. Before posting or

staining, the smears should be exposed either to air-drying and gentle flame-fixation or to air-drying plusmethanol fixation for not less than two minutes andpreferably for 10 minutes. Fixation with methanol ismuch better than flame-fixation in the case ofprotein-rich smears from patients with a floridgonorrhoea. The general rule must be that thediagnosis of gonorrhoea by microscopy is made onlyafter careful Gram-staining; the latter may beperformed after previous staining with 1 % methyleneblue, provided the immersion oil has been completelyremoved by means of xylol. The Gram-stainingtechnique may be any one of the usually recom-

mended methods, but it is important to be familiarwith the method in question and to controlthe various ingredients for precipitation andinfection.

Methylene blue without subsequent Gram-stainingis still used even by specialists, who believe that bythis means they are able to distinguish betweenN. gonorrhoeae, staphylococci, Escherichia coli andother Gram-negative, more or less well-defined,short, plump rods. This is impossible. Even afterGram-staining it may be impossible, although thismethod is more reliable than simple methylene bluestaining. The reliability of the Gram proceduredepends to a great extent on the staining technique; itis very difficult to obtain the right degree of de-colorization, especially when many slides of varyingthickness are stained at the same time. It is empha-sized that old staphylococcus cultures are very

easily decolorized and that short Gram-negativerods (E. coli, etc.) may also be mistaken for gono-cocci (Reyn, 1951). Badly performed Gram-stainingis no better than staining with methylene blue.At the Statens Seruminstitut in Copenhagen, the

fixed smears of discharge or of watery suspensionsof pure cultures are (1) stained with 0.2% anilinecrystal violet for one minute, (2) treated with Lugol'siodine solution for one minute, (3) decolorized with96% ethanol for half a minute, (4) rinsed in runningtap water for about five seconds, (5) counterstainedwith 0.1 % phenolized fuchsin for one minute, (6)rinsed in running tap water for about five secondsand (7) dried with blotting paper. (For the composi-tion of the reagents, see page 466.)

Classically, in acute male gonorrhoea, large num-bers of polynuclear leucocytes containing Gram-negative, coffee-bean-shaped diplococci are ob-served. In some cases, only a few of the leucocytesshow one or two typical intracellular pairs and inother cases all the cocci are extracellular. In chronicmale and female gonorrhoea, it is often impossibleto find any typical gonococcus pairs, especially inthe cervical secretion.

Stained films of pure cultures reveal the gonococcias roundish cocci, typically but not invariablyarranged in pairs. After 18 to 24 hours' growth, thesize is uniform and the stain is taken up quiteevenly. After 48 hours' growth, size and staining aremore variable and lysis is often observed.

Cellular morphology is progressively altered bycontact with penicillin, both in vivo and in vitro.Smears of exudate taken during the first four or fivehours of penicillin therapy show gonococcal formsthat progressively enlarge and decrease in stain-ability until none can be detected. Similar mor-phologic changes are observed during chloromycetintherapy but not during sulfonamide treatment.

In conclusion it is emphasized that it is impossibleto diagnose gonorrhoea with certainty by microscopyalone. Clinical evidence and history must alsobe taken into account, but even then there remains awide margin of error.

452


CULTURE AND ISOLATION

Most strains of gonococcus do not grow in vealor beef broth or on simple broth-agar plates.Sensitivity to inhibition, rather than complexity ofnutritional requirements may account for much ofthe fastidiousness of the gonococcus. The beneficialeffect of the addition of charcoal, starch, albumin,serum or ascitic fluid to the medium is generallyascribed to a detoxifying action on inhibitors presentin the agar or in the peptone, but fresh serum andascitic fluid also contain factors essential for thegrowth of certain strains. In semi-solid broth-agar,most laboratory strains grow well in a layer justbelow the surface. The pH of the medium should beadjusted to between 7.2 and 7.6. Synthetic mediahave been described (Welton & Scherp, 1944; Weltonet al., 1944; Gould et al., 1944; Hill, 1948; Hill et al.,1948, 1949), but these are not suited for primaryisolation of N. gonorrhoeae.The CO2 tension is of decisive importance for

successful primary isolation and a certain degree ofmoisture is also favourable to growth (McLeod et al.,1934; McLeod, 1947; Reymann, 1941, 1944; Muelleret al., 1942; Hill et al., 1948; Morton, 1945; Griffin& Racker, 1956; Griffin & Rieder, 1957). It isimpossible at the present time to prescribe the idealgonococcus medium, one of the difficulties appa-rently being that the growth requirements andresistance to toxic substances vary from one strain toanother (Lankford & Snell, 1943; Lankford et al.,1943; Gould, 1944; Lankford & Skaggs, 1946).Numerous types of " enriched " media have beendescribed, most of which contain a broth-agar basesupplemented with heated blood (or haemoglobin),and/or yeast extract, serum, ascitic fluid and albumin.A chemical supplement, containing glucose, glu-tamine and cocarboxylase was described by Lank-ford (1950).The choice of medium depends upon the local

situation; in larger laboratories with easy access tofresh blood (horse, beef, rabbit or sheep) and asciticfluid and with centralized media production facilitiesit is natural and relatively cheap to use these productsas they are. In smaller laboratories, it is practical touse the commercially available dehydrated completemedia or dehydrated media bases.' Dehydrated

1 Bacto-G C Medium Base enriched with haemoglobinand Bacto-Supplement B or C may well be used (see DifcoManual of Dehydrated Culture Media and Reagents forMicrobiological and Clinical Laboratory Procedures, 9th ed.,Detroit, Mich., Difco Laboratories, 1953, p. 122). Bacto-Supplement C is the same as Bacto-Supplement B butwithout the addition of glucose.

media are sometimes more costly than fresh media,but the cost should be considered in relation toother relevant factors, such as number of techniciansand laboratory facilities. However, the dehydratedmedia are not always quite as good as the freshmedia and further experiments are needed. Theaddition of inhibitors (dyes or antibiotics) to theculture media is not generally recommended (Gould,1944; Gould et al., 1944; Hill et al., 1948). A highyield of positive cultures may be obtained by usingtwo or more plates, one (or more) containing aselective agent and one without it.

In the gonococcus laboratory of the StatensSeruminstitut a soft chocolate ascitic-fluid-agarmedium was formerly used for primary isolation.Recently, the ascitic fluid has been replaced by acombination of yeast and liver autolysates and theheated blood by 1% haemoglobin (M0ller & Reyn,1965).2 This so-called HYL (Haemoglobin-Yeast-Liver) medium is also a " chocolate " medium andfor convenience both media are described in detailin a later section (page 463). The turbid, brownishmedium is dispensed into aluminium Petri dishes9 cm in diameter and about 1.5 cm deep, or intosimilar dishes made of plastic and purchased in asterile condition; the layer of medium should be notless than 5 mm. Polymyxin B sulfate is added andunder certain conditions nystatin is also added.

Inoculation and incubation ofprimary culturesImmediately upon arrival the swabs are streaked

on one half of a plate, care being taken to transfer asmuch material as possible to the plate.3 The inocu-lum is spread as shown in Fig. 3 by means of twoseparate platinum wires (gauge 0.7 mm) about 8 cmlong with closed loops 2-3 mm in length. All platesshould be examined for surface contaminationbefore use. The result of the spreading is shown inFig. 4.The inoculated plates are incubated for about 24

hours at 35°-36°C in closed glass or plastic jarscontaining a moist atmosphere with about 8% ofCO2 (see Fig. 5).The glass jars have a capacity of about 4.5 litres

and have cut collars which fit into metal lids; be-tween the collar and the lid a ring of rubber isinserted. The plates are placed on a perforated

'See the article on page 471 of this issue.Before inoculation urines, spinal and synovial fluids are

centrifuged. Blood cultures are made by introducing 5 ml ofblood into flasks containing ox-heart broth enriched with 1%of glucose and 25 % of ascitic fluid.

A. REYN

FIG. 3DIAGRAM ILLUSTRATING METHODOF INOCULATION

1: Streaking with swab.2 & 3: Spreading with bent platinumneedles.

FIG. 4GROWTH OF NEISSERIAGONORRHOEAE ANDSTAPHYLOCOCCUS ALBUS AFTER48 HOURS' INCUBATION

This photograph demonstrates theeffect of the spreading methodIllustrated in Fig. 3. The organismswere Inoculated on a " chocolate "ascitic medium in an aluminium Petridish, shown natural size.

454

LABORATORY DIAGNOSIS OF GONOCOCCAL INFECTIONS

FIG. 5JARS 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 molarH,SO4: a calibrated spoon containing about 1.5 g ofNaHCO3 is put in the bottom of each jar. The lid isclosed by means of a screw clamp; it is fitted with avalve. After closing the lid with the valve open, thejar is tilted to allow the H2SO2 to combine with theNaHCO3; when the CO2 evolved has pushed outthe superfluous air, the valve is closed.The chemical process that takes place in the jar

is as follows:2 NaHCO3+H2SO4=Na2SO4+2 H20+2 CO2.From this equation the amounts of H2SO4 and

NaHCO3 can be calculated for other volumes andother concentrations (1 gram molecule=22.4 litres).It is most practical to use a constant amount ofH,SO4, which will determine the amount of CO2,provided an excess of NaHCO5 is used. The amountshould be calculated making allowance for thefact that the jar will normally be full of plates.

A CO2 atmosphere can also be obtained by the" candlelight method " (Ferguson, 1945) or from aniron tank containing a mixture of compressed airand CO2. However, in the author's laboratory it wasfound, for some unknown reason, that the CO2supplied from iron tanks (especially when dispensedinto stainless steel containers instead of glass jars)was less favourable to the growth than the CO2supplied from H2SO4+NaHCO3. If the CO2 issupplied in one of the ways mentioned above, ahumified atmosphere should be obtained by meansof moistened cotton-wool pads.

Recently, acid-resistant polyvinyl chloride jarshave been used. They are much lighter than theglass jars, they do not break, and it is not necessaryto close their smoothly fitting lids by means ofscrew clamps. The arrangement for filing withCO2 is the same as that used in the glass jars. Thelids (without valves) are put on just after the CO2 has

455

456 A. REYN

been produced by mixing the H2SO4 with theNaHCO3.

After 18-24 hours the plates are removed from thejars. Usually, the plates are inspected in artificiallight with the naked eye. The typical round coloniesare 0.5 to 1 mm in diameter; they are convex, greyishand glistening, with smooth edges; on furtherincubation the colonies increase in size and developa roughened surface with crenated edges. It isimpossible to give an exact description of the coloniesbecause their appearance varies with the medium,and particularly with the strain. The size andappearance vary greatly with the age of the culture,the degree of crowding, and with the colony type.If the plate is crowded with large numbers of coloniesor contaminants, the colonies will be small; if thecolonies are well isolated they should be large. Thesize may vary from less than 1 mm to about 5 mm indiameter.' The consistency of the growth is oftenviscid; this is most pronounced after 48 hours'growth. In laboratories in which the identificationof gonococci is a rare event it is recommended thatcontrol strains should be kept for comparison. Inthe author's laboratory, about 80% of the totalnumber of positive cultures are detected after 18-24hours. However, the longer the transportation timethe later will the colonies grow out, resulting indelayed detection.

If the growth of gonococcus-like colonies isabundant, one drop of oxidase reagent (page 466)is added; if the colonies quickly turn deep purpleand if the colour is maintained for more than halfa minute the reaction is considered positive and aGram-stained smear is prepared. Colonies of othermicro-organisms may also react with the oxidasereagent. Most of these are easily distinguished fromgonococcal colonies, but notably species of Alkali-genes and Haemophilus and also of other Neisseriamay be impossible to distinguish on the basis ofcolony form and oxidase reaction. If the growth isscarce, the oxidase reagent can be used by applica-tion of a few drops on a filter paper which is streakedwith small amounts of growth. In this way severalcultures can be tested at a time on one piece ofpaper.

Subcultures are made from all plates from whichGram-negative diplococci or cocci are found onmicroscopy. If there is heavy contamination typicalcolonies are picked out under a low-power stereo-

1 Recently, Kellogg et al. (1963) observed four morpho-logically distinct clonal types in N. gonorrhoeae cultures.Virulence in man was found to be linked to clonal type 1.

microscope with about 30-fold magnification.Material from one or a few colonies is picked up bya thin, straight, pointed 0.6-mm platinum needleand spread on a " chocolate " agar plate. The useof a microscope is very helpful and allows a rapidisolation of pure cultures. One of the characteristicsof 24 hours' gonococcus colonies is that they lookmuch darker than those of the most common con-taminants. If a stereomicroscope is not available,the use of a good loupe and a good light source isindispensable. After inspection, all plates (positiveand negative) are returned to the incubator foranother 24 hours' growth without CO2.

For ordinary subculturing a 0.6-mm-gauge plati-num wire, 9-10 cm long, with closed loop is usedand care is taken to avoid clonal selection by dippingthe needle into several colonies before spreading on afresh plate. If the 24-hour growth is rich, an attemptis made to test for fermentation.Next day the " negative " plates are re-inspected;

suspicious-looking colonies are tested with oxidasereagent and, when positive, subcultured within 30minutes. Stained films (of the respective colonies)are examined under the microscope. After 48 hoursthe gonococcus colonies appear less " typical " underthe low-power microscope; they appear lighter,flatter and less circular, with a tendency to irregularedges.

CONTROL STRAINS

Control (and other) strains can be kept as lyophi-lized cultures, preferably in a refrigerator or in anincubator, as cultures in semi-solid nutrient agarcovered by mineral oil. Lyophilized cultures keepfor years, whereas subculturing is needed everythird month for the cultures kept in nutrient agarunder mineral oil. Heavily infected charcoal swabsin Stuart tubes kept in a cool place preserve thegonococci for about six weeks (Korner, 1963).

Stock cultures can also be prepared as follows.The strains are grown on " chocolate " agar slopesfor 24 hours in a CO2 jar. One ml of a 30% solutionof inactivated horse, rabbit or sheep serum intrypticase soy broth (BBL) is used to wash thegrowth from the agar surface. The washings aredispensed in portions of 0.2-0.4 ml into a largenumber of prelabelled 13-mm x 100-mm cotton-stoppered sterile tubes (ink covered with self-adhesive plastic tape is suitable for labelling). Thecotton stopper is cut off at the tube lip and asterile rubber stopper inserted, pushing the cottonsome way into the tube. The tubes are frozen in a


dry-ice/alcohol bath at - 70°C and stored in a dry-ice chest until needed. The frozen suspension isthawed in a water-bath at 37°C and promptlystreaked on plates for recovery.'

FERMENTATION TESTS

In laboratories with some experience in theidentification of gonococci and meningococci itsuffices to use fermentation plates of glucose andmaltose only, because these sugars will permit thedifferentiation between N. gonorrhoeae, N. menin-gitidis and N. catarrhalis, provided the colonies aretypically non-chromogenic (Wilson & Miles, 1955;Breed et al., 1957). It is preferable to use a solidmedium on which contamination can be directlyobserved. In the Neisseria Department of theStatens Seruminstitut, translucent fermentationplates are used; they are rather heavily inoculated,and 6-8 strains are tested on one plate. The platesare incubated for about 24 hours in CO2 jars, thenremoved and left on the table for about half an hourwith the lids open in order to allow the CO2 toescape from the medium. The fermentation mediumhitherto used by the author is described on page 464.Juhlin (1963) has recently introduced a mediumthat allows growth from a very small inoculum, andvery few strains fail to grow on it or to fermentglucose.

If there is typical acid production the inoculatedareas of the glucose plates turn yellow; however, theintensity of the acid production varies and strainswith weak or absent acid formation are occasionallyfound. In typical cases, the maltose plates turnpurple on the growth areas. In order to detect conta-mination, Gram-stained smears should be preparedfrom the fermentation plates in all doubtful cases.If the growth is weak or the acid formation is notvery marked, it may help to incubate the glucoseplate for another 24 hours.For the fermentation tests it is important that the

growth should be well supported; the use of asciticfluid from patients treated with antibiotics should beavoided. In the author's laboratory some batches ofphenol red have been found inhibitory. Anothersource of error is the use of ascitic fluid from patientswith uncontrolled diabetes.

Neisseria other than gonococci are occasionallyisolated from the genito-urinary tract; the incidencedepends on how much attention is paid to their

1 For further details see US Public Health Service (1962).

isolation. Most of these strains are easily distin-guished from true gonococci by their cultural andbiological characteristics (see accompanying table).

Formerly, 1-2% of the "positive" gonococcus-like cultures examined in this laboratory showed anabsence of glucose fermentation in spite of satisfac-tory growth on the fermentation medium. This wasthe situation until June 1957. At that time the num-ber of non-fermenting, atypical strains showed asudden increase. Not only was glucose fermentationabsent or weak, but the growth was poor on theroutine medium used for primary isolation. It isemphasized that freeze-dried strains isolated beforeJune 1957 grew well on the " old " media and showedtypical fermentation. Serological tests indicated thatthese atypical strains could be considered as truegonococci. However, as soon as the media werechanged by using beef-heart broth instead of beef-meat broth in the medium for primary isolation andsubculture, and placenta or beef-heart broth insteadof beef-meat broth in the fermentation medium,both growth and fermentation of the " atypical "strains became typical. This event is reported todemonstrate the great variability of the growthrequirements of gonococci (Reyn & Bentzon, 1961).The frequency of non-fermenting strains is nowbelow 0.05 0.Polymorphous Gram-negative rods, growing in

gonococcus-like colonies and with a fermentationpattern similar to that of gonococci may be a sourceof error. In most cases, howener, the oxidase-reaction will be negative. Growth at 22°C onunenriched medium will rule out N. gonorrhoeae.On careful microscope examination of successive16-18 hours' pure subcultures it will be found thateither long rods will gradually predominate or thesingle elements will appear oval and be orientatedin a pattern different from the typical coffee-beanpairing. Large cocci or diplococci, which are evenlyand deeply stained by the counter stain, requirespecial attention. When it is found that the isolatedmicro-organism shows a high degree of penicillinresistance one should be even more suspicious.Further study of this group-or of these groups-of micro-organisms is needed with a view to develop-ing an improved classification. Several authors havecalled attention to these sources of error which playa comparatively greater role in smaller laboratoriesor in localities where these micro-organisms arerelatively frequent (De Bord, 1939, 1943; Aikenet al., 1956; Cary et al., 1958; Gangarosa & Cary,1960; Sampson et al., 1961; Svihus et al., 1961).

CULTURAL AND BIOLOGICAL CHARACTERISTICS OF DIFFERENT SPECIES OF NEISSERlA a

Growth onFermentation plain agarSpecies Colony characteristics in brief

plain 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 Iseasily emulsified. N. meningitidis

N. meningitidis colonies larger than N. gonorrhoeae + + _ _ _ _ -colonies, both variable in size.

N. catarrhalis Colonies variable, may be smoothand 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), Bi= acid formation or growth varying, preponderantly positive.T = acid formation or growth varying, preponderantly negative

DIAGNOSTIC CRITERIA

As a general rule, a culture isolated from thegenito-urinary tract may be considered and reportedas a gonococcus, provided the following criteria havebeen met:

(1) the colony morphology is typical;(2) the colony is oxidase-positive;(3) microscope examination of a pure culture

shows typical Gram-negative diplococci;(4) only glucose is fermented.Cultures that fulfil criteria 1 to 3 but do not fer-

ment glucose or any of the sugars listed in the abovetable may be identified as " gonococcus-like " if nogrowth is obtained at 22°C.

It is stressed that gonorrhoea cannot be excludedunless repeated cultures show negative results.

REPORTING OF RESULTS

Reports should contain all the identification marksgiven on the forms accompanying the samples.Missing information or errors should be noted.Furthermore, notice should be taken of whether thetransportation tubes are beyond their expiry dates or

(1957) and Cruickshank (1960).

if swabs other than the charcoal-impregnated appli-cators have been used. In some cases, it may beconvenient to report the transportation time. It isrecommended that copies be kept of all reports andthat a system of checking be set up to ensure that allextra remarks are given on both the reports and thecopies.

Cultures showing no growth of gonococci after 48hours' incubation should be reported as " negative"or " no growth of gonococci ".

Cultures fulfilling the diagnostic criteria givenabove should be reported as " positive " or " growthof gonococci ". Sometimes it is necessary to specifythat Gram-negative oxidase-positive diplococci werefound but that the strain was lost on subculture, orthat growth of " gonococcus-like " micro-organismswith atypical fermentation was observed. In suchcases, the report should contain a suggestion that thetest be repeated. If it takes more than five days toobtain a pure culture for the fermentation tests, apreliminary report should be made and care shouldbe taken to ensure that the final report is also given.

In addition to " negative " and " positive " findingsit is absolutely necessary to report " overgrown " or"unsatisfactory " cultures. A suggestion that thetest be repeated should be made as well. Cultures

458 A. RELYN


should be designated as " overgrown " when it isevident that any gonococci that might be presentwould have had no chance to develop in the presenceof the overgrowing micro-organisms, which in turnmay originate either from the patient or from themedium. If overgrowing micro-organisms showconfluent growth in the fourth quadrant of the plateit is obvious that the plate is " overgrown ", but it issometimes very difficult to distinguish between" overgrowth " and " no growth ". Each laboratorymust develop its own style of reporting, balancingthe risk of reporting too many " false negative "results against that of reporting too many " over-grown " cultures. In general, overgrowth by Proteus,E. coli or yeast originates from the patient, and this isalso true of most of the cases in which B. subtilis isobserved. Apart from spreading the swab material,very little can be done to prevent overgrowth ofcontaminating bacteria without damaging the

gonococci, except for the addition to the plates ofpolymyxin B and perhaps also of ristocetin. Most ofthe substances that have hitherto been shown to beinhibitory to the swarming of Proteus are too toxicto N. gonorrhoeae and therefore useless. PNPG/1forms the only exception (Jungfer, 1958). Twoplates are used, one with the addition to the mediumof60,ugof PNPG/1 per ml and one without PNPG/1.

In most laboratories it will be impossible to deter-mine in detail the nature of the overgrowing orga-nisms. It is, however, recommended that it should bespecified if Proteus is encountered. If not specified,the repeated reporting of " overgrown " culturesmay discredit the method. In certain periods, over-growth by soil bacteria or by moulds, both origina-ting from contamination during the preparation ofthe plates, may destroy the cultures. Such over-growth may be effectively inhibited by the additionof nystatin (for details, see page 466).

SEROLOGICAL EXAMINATION

GENERAL REMARKS

Serological examination can be used in two ways,either to classify a culture or to test a patient's serumfor antibodies. Neither of these procedures is muchused in the diagnosis of gonorrhoea. In the majorityof cases the bacteriological examination suffices.Antibody is rarely present in acute cases. Sero-logical testing has a certain diagnostic value inchronic infections (pelvic inflammations, prostatitis,arthritis, etc.) and when bacteriological examinationis inconvenient or impossible. However, serologicalmethods are not quite specific and antibodies tendto persist for many years in spite of clinical cure.

Despite much effort, serological investigation ofthe gonococcus has not been very rewarding. Onculture, the gonococci undoubtedly change theirserological characteristics quite readily and partly or

completely lose their surface antigens; this makesserological classification difficult (Uroma, 1943;Reyn, 1947, 1949a, b, c; Chanarin, 1954; Wilson,1954). Antigenic substances of different chemicalspecificity are responsible for the antibodies formedin gonorrhoeal infections. Antigens of poly-saccharidal, lipopolysaccharidal and proteinic naturehave been prepared, but further research is necessary(Boor & Miller, 1934, 1939, 1944; Mutermilch &

Grimberg, 1935; Dmitriw & Demidova, 1935;Casper, 1937; Tauber, 1959, 1960; Tauber & Russell,1960, 1961). A thermostable antigenic factorcommon to all gonococci has been identified byReyn (1947, 1949a, c); several type-specific (or strain-specific) antigenic substances (both thermostable andthermolabile) were also demonstrated. Using agglu-tination, Wilson (1954) demonstrated eight heat-stable antigens, four of which behaved as groupantigens. In addition to the complex antigenicpattern, cross-reactions with other Neisseria areoften found; this applies mainly to N. meningitidisand N. catarrhalis. In addition some strains ofPasteurella multocida show cross-reactions. Thecross-reactions depend on antigenic substancesclosely related to thermostable antigens (Reyn, 1947,1949a; Wilson, 1956).Simple slide or tube agglutination tests are not very

useful since many strains are auto-agglutinable whensuspended in saline. A number of precipitation andflocculation methods have been described (Saint-Prix, 1950a, b); however, the only serological methodin common use is the complement-fixation reaction(Cohn, 1937; Le Minor, 1948; Price, 1949; Car-penter, 1963). Further references to serologicalmethods will be found in the review by Scherp(1955). A fluorescent antibody technique (Deacon et

459

460 A. REYN

al., 1959, 1960; Deacon, 1961; Harris et al., 1961)has been worked out for the identification ofN. gonorrhoeae in smears. However, this techniquerequires that the worker be familiar with the use ofthe ultraviolet light microscope and that he shouldhave a basic understanding of the principles govern-ing fluorescent antibody techniques. Thus, this verypromising method cannot as yet be recommendedoutside highly qualified laboratories, especially ascertain specificity and sensitivity problems stillremain to be solved (Danielsson, 1963; Lind, 1963).There have, however, been recent indications thatthe specificity problems are being overcome.'The relative efficiencies of the culture method and

the fluorescent gonococcal antibody technique havebeen found to vary from one laboratory to another;for instance, one laboratory has claimed a superiorityof 12% for the culture method while another hasclaimed a superiority of more than 100% for theso-called " delayed fluorescent-antibody test " (Price,1964). This variation is most probably due to lackof uniformity in performing the conventionalculture method.A rapid staining technique has been described by

Kellogg & Deacon (1964). Very recently, Hess et al.(1965) have described how the immunofluorescenttechnique can also be used indirectly as a means ofdetecting antigonococcal antibody in human sera.Finally, Reising & Kellogg (1964) have proposeda precipitin test for the detection of gonococcalantibodies. Preliminary findings with this testindicate that the virulent gonococcus contains aprotein antigen which is species specific. However,the test has not yet been evaluated in large-scaleexperiments with respect to false positive and falsenegative results.

GONOCOCCUS COMPLEMENT-FIXATION REACTION

The following method was developed at the StatensSeruminstitut, Copenhagen (Kristensen, 1930;Reyn, 1947, 1949a), the procedure being as follows:The antigen is prepared from a mixture of about

30 fresh strains and seven serologically " known"stock strains.2 The strains are grown on an asciticagar medium of the same composition as the fermen-tation medium but without sugar, phenol red and

1 US Public Health Service (1962); Brown et al. (1963);Moore et al. (1963); Shapiro & Lentz (1963); Lind (1964);Price (1964).

2 Available in a freeze-dried state from the NeisseriaDepartment, Statens Seruminstitut, Copenhagen, Denmark.

co-carboxylase. Eighteen-hour cultures are har-vested in physiological saline and suspended to adensity of about 2 x 1010 organisms per ml; fromeach of the suspensions aliquots are taken out andsubmitted to microscopic and serological examina-tion before the various suspensions are mixed toform the polyvalent antigen. The antigen is dis-pensed in l-ml or 2-ml amounts and heated to 56°Cfor 20 minutes; it is now ready for use and can bestored at - 10°C for several years. The harvested,unheated material is stored at about - 10°C untilthe results of the microscopic and serological testsare available. The following criteria should befulfilled before the suspensions are mixed: (1) nocontamination, (2) no anti-complementary effect,(3) a " standard " degree of reaction with pools ofpositive sera, (4) negative reaction with a pool ofnegative sera.To obtain an estimate of the anti-complementary

effect, the individual suspensions are tested incomplement-fixation experiments with a constantdose of antigen and two or three different comple-ment concentrations; suspensions only slightly anti-complementary are included in the antigen. Asuitable antigen concentration is usually obtainedby dilution of the stock antigen to give about5 x 107 organisms per test-tube. The antigen isdiluted in S0rensen's phosphate buffer, pH 7.38.The total volume is 0.3 ml and the quantity of serumin the first tube is 0.025 ml, in the second 0.025 mldiluted 1 in 3, in the third 0.025 ml diluted 1 in 9and so on. The 100% haemolytic unit of comple-ment is determined in the presence of antigen andone unit is added per test-tube.

Fixation is allowed to take place for three-quartersof an hour at room temperature and for three-quarters of an hour at 37°C in a water-bath; 0.2 mlof a 2.5% suspension of sensitized sheep-blood cells(three haemolytic doses) are then added to the tubeswhich are re-incubated at 37°C for an additionalhour. The degrees of haemo ysis corresponding tothe different serum dilutions are estimated bycomparison with a scale of haemolysis preparedfrom tubes with complete haemolysis. The resultsare given as degrees of potency; these are calculatedafter the method given by Kristensen (1930), thedegree of potency being defined as that value of nwhich when substituted in the formula a= 0.025 x 3-n/3gives a value for a exactly equal to the volume ofserum containing enough antibody to give a mini-mum reaction, i.e., 60% haemolysis. Further detailsare given by Reyn (1949a).


The day-to-day variations in the test are counter-acted by means of daily titration of six control serafor each of which an average " standard " value hasbeen determined. The average amount by whichthe titres of the control sera differ from theirstandard values is the correction to be applied to thevalues for the other sera.

Before a new antigen is accepted for use thefollowing comparisons with the old one are made:

(1) The approximate titre of the new antigen isdetermined in preliminary experiments. As a rule,a fair degree of similarity between the results withthe new and the old antigen can be obtained afterthree to four days on which 10 positive sera ofvarying potency are tested quantitatively againstdifferent dilutions (three per day) of the new antigen.

(2) Having determined the approximate titre, thetwo antigens are used in parallel titration of about1000 sera picked out at random among the sera sentin for gonococcus complement-fixation. This willgive a comparison of about 75-100 sera that reactpositively with either or both of the antigens.The new antigen is accepted for use if the average

serum titre difference between the two antigens doesnot exceed ± 0.3 degrees of potency (±0.048 log10values). If a difference in the same direction is regu-larly observed for several days, another antigendilution is used and the experiments are startedagain.

In the Neisseria Department of the StatensSeruminstitut, a new antigen is made once a year.Care should be taken to avoid a gradual change insensitivity as a result of repeatedly accepting antigenswith a positive (or negative) difference.

(3) One thousand sera from blood donors (with-out clinical or serological evidence of syphilis) aretested with the " final " antigen titre. The number ofpositive reactions should preferably not exceed fiveper thousand.The above procedure is related to the average

sensitivity or strength of the antigen; another ques-tion is whether the standard deviation of the differ-ences should also be taken into consideration. Thestandard deviation of repeated titration of the sameantigen is estimated to be about 0.7 degrees of

potency (0.111 log10 values) and it is proposed thatthe standard deviation of the difference between twodifferent antigens should not exceed 1.4 degrees ofpotency (0.223 log10 values). This would mean thatantigens giving differences greater than ± 2.8 degreesof potency in more than 5 % of the sera would not beaccepted.The sensitivity of the complement-fixation reaction

described is so adjusted that only about 30% offemale cases with uncomplicated, acute gonorrhoeaand about 90% of cases with complicated, chronicgonorrhoea (male and female) show positive results.

In spite of clinical cure the reaction may remainpositive for several years and in the individual caseit is difficult to judge the specificity of a positivereaction. The diagnostic value of a sero-reactiondepends on its sensitivity and specificity under thespecial local conditions under which it is intended tobe used. If the incidence of previous and presentinfection is high, many apparently non-specificreactions will be found and the diagnostic value inthe individual case will be small. If, however, theincidence of gonorrhoea is low and confined tocertain patient categories, the diagnostic value isgreater. A survey of positive reactions obtained in amedical ward in Copenhagen shows a certain trendfor the conditions in Denmark (Bang & Krag, 1942).It was found that 7.2% of the males and 6.2% of thefemales were positive; by dividing the material intogroups according to the history and the clinical signsof gonorrhoeal infection it was found that thedegree of specificity increased with increasingstrength of the reaction. In 63.4% of the serashowing one degree of potency (serum concentration< 1:12) and in 84.7% of the sera showing threedegrees of potency (serum concentration < 1: 36)either the corresponding patients were known to besuffering from gonorrhoea or their case historiesmade it highly probable that they had previouslysuffered from this disease.

Strong cross-reactions are usually found in patientswith meningococcus infection. Positive reactionshave frequently been observed in patients sufferingfrom chronic bronchitis especially in those withbronchiectasis, possibly due to infection withN. catarrhalis or other Neisseria species sharingantigens with N. gonorrhoeae.

461

A. REYN

DRUG-SENSITIVITY DETERMINATION

Until recently, determination of the sensitivity toantibiotics was not very much used in connexionwith routine culture of gonococci. However, thenumber of strains of gonococcus showing decreasedsensitivity to antibiotics makes the development of arecommended reference method desirable. The useof reference strains would be a further aid in makingthe comparison of results obtained in differentlaboratories more meaningful. The WHO ExpertCommittee on Antibiotics in its second report (1961)stated that " a standard method of test is required"and that " stocks of lyophilized cultures should bekept, in order that future direct comparisons may bepossible between strains of gonococcus isolated atdifferent times ". Under the auspices of WHO agroup of laboratory experts is now doing the pre-paratory work for the establishment of such areference method. The preliminary results of thisstudy are referred to in the first report of the WHOExpert Committee on Gonococcal Infections (1963).The aim of this work is to agree on a procedure inwhich the inhibiting concentration of antibiotic ismeasured by a plate dilution method.

Carefully controlled diffusion tests might bepreferred for local use. This conception is supportedby the results obtained in a recent collaborativestudy of different methods by Denmark, Finland,Norway and Sweden (Reyn et al., 1963). Theresults obtained in this study indicate the possi-bility of converting a diffusion result read inmillimetres to a concentration obtained by a dilutionmethod.The great majority of cases are cured by penicillin,

streptomycin or tetracycline; however, it has beendemonstrated both in vitro and in vivo that thepenicillin sensitivity is decreasing (Jensen, personalcommunication, 1957; Cradock-Watson et al., 1958;Curtis & Wilkinson, 1958; King, 1958; Scamberg etal., 1958; Reyn et al., 1958; Hirsch & Finland, 1960;Cole et al., 1961; Reyn, 1961; Thayer et al., 1961;

Ringertz, 1961; Gjessing & 0degaard, 1962; Ranta-salo et al., 1962). Completely resistant strains havenot been isolated from patients. However, about25% of strains isolated in Denmark since 1957 needmore than 0.225,ug of penicillin per ml for completein vitro inhibition and this is only two to three timesthe average serum concentration obtained after300 000 units of aqueous procaine penicillin G.Geographical differences may, of course, be en-countered (Reyn, 1962, 1963). Streptomycin-resistant strains have been isolated and an increasedfailure rate of streptomycin treatment has been re-ported by Alergant (1958), Reyn (1961), Durel et al.(1961) and Roiron et al. (1961).A tablet method and a plate dilution method for

sensitivity determination of penicillin, streptomycinand tetracycline were described by Reyn et al. in1958, and a plate dilution method was described inmore detail by Reyn et al. in 1963. These methodscan easily be applied to the determination of sensi-tivity to other antibiotics. The tablets should bechosen so as to give zones of a suitable size (Lundet al., 1951; Lund, 1953). The concentrations in thesolid medium used in the dilution method shouldbe so adjusted that the change from full to nogrowth for the majority of the strains is likely tooccur in the middle of the series of concentrationsemployed. If, for some reason, the agar cup tech-nique, the disc method, the gradient plate technique,or tube dilution methods with fluid or semi-fluidmedium are found more convenient, they may wellbe used. However, regardless of the method it isimportant to know something about its variationunder the local conditions.

Direct sensitivity determination from swabs ispossible only in a few cases and it is preferable to usepure cultures from which the size of the inoculumcan be controlled. In order to avoid selection, notless than 10 colonies (if possible) should be used forthe preparation of the pure cultures.

MEDIA AND REAGENTS

PREPARATION OF BROTH

Containers used in the preparation of broth, etc.

should be of stainless steel, glass or some other inertmaterial. It is practical to prepare stock batches of

broth-agar base for later use. Suitable amounts maybe kept for several months at low temperatures.Erlenmeyer flasks are suitable for this purpose.

Broth formula: chopped meat, 500 g; tap water,

462


1000 ml; peptone, 10-15 g; 1 Na2HPO4.12 H20, 2 g;and NaCI, 3 g.The " meat " can be veal or beef-heart or human

placenta tissue (see below), which should be freedfrom fat and tendinous tissue. After cutting andchopping, the meat is mixed with half the water andplaced overnight in the cold. Next morning it isboiled for 15 minutes and the soup is collected, usinga sieve. The meat is boiled again for 10 minutestogether with the other half of the water and thetwo soups are then combined. After addition of pep-tone and salts the broth is heated to boiling, duringwhich the pH is adjusted to about 8.1 by means of5N NaOH. The broth should boil for not less than10 minutes and for at least 5 minutes after the lastaddition of sodium hydroxide. By means of distilledwater the original volume (plus an extra 5% tocounteract the autoclaving effect) is restored. Thebroth is cleared by paper filtration, the agar2 meltedin the hot broth and boiled for 20-25 minutes. Thehot broth-agar is then filtered by suction and dis-pensed in Erlenmeyer flasks. It is sterilized by auto-claving at 120°C for 20 minutes or by heating for20 minutes on a boiling water-bath (" steaming ")on three days in succession. After sterilization thepH should be about 7.2.

If the broth is prepared for later use it should besterilized by Seitz filtration and kept in the cold inErlenmeyer flasks until the agar is added, followedby autoclaving as described above.

Autoclaving is more deleterious than " steaming"and it also has a more acidifying effect than has" steaming ".Placenta broth is prepared from fresh human

placentas (no antiseptics) which are freed of theumbilical cords and the coverings. They are cut intopieces and rinsed in cold tap water.

After coarse chopping in a machine, two litres oftap water are added per kg, the crude broth beingkept at about +4°C until the following day, whenit is boiled for 15 minutes. The placenta tissue is sepa-

l The precise quantity will depend on the brand andquality.

a The exact percentage depends on the batch and brandof agar; the agars should be soft. At the Statens Serum-institut, Danish AKI agar is used in the chocolate ascitic-fluidmedium. Selected batches of Japanese agar Kobe 1 are em-ployed in the fermentation medium. Preparations with AKIagar do not need to be filtered before autoclaving. The AKIagar is not a true agar; it resembles carrageen which is rich inmethyl groups. It solidifies at about 45°C (in the concen-tration used) whereas Japanese agar, for example, solidifiesat about 30°C. The pH also varies when different brands ofagar are used.

rated from the broth and the peptone and salts areadded with subsequent boiling for 15 minutes; thepH is adjusted to about 8.1. After autoclaving thepH should be about 7.2.

CHOCOLATE ASCITIC-FLUID-AGAR

This is prepared by the addition of horse bloodand ascitic fluid to a broth-agar base consisting of2.4% of Danish AKI agar in beef-heart broth with1 % of peptone (" Orthana " special), 0.3% of NaCIand 0.2% of Na2HPO4.12H20.The base is dispensed in 540-ml amounts in

Erlenmeyer flasks and kept cool; before furtherpreparation it is melted and kept at 80°C. 60 ml ofdefibrinated horse blood are added. The mixture iskept at 80°C for 15 minutes and shaken frequently;it is cooled to 560C and mixed with 300 ml of sterileascitic fluid 3 (30 %), which is brought to 56°C beforemixing. The ascitic fluid causes a rise in pH whichshould be compensated for by adding 0.5N HCIuntil the pH is about 7.1-7.2. It is false economy topour plates too thinly. Immediately afterwards,plates 4-5 mm thick are poured into 9-cm widealuminium or plastic Petri dishes. If ascitic fluid isdifficult to obtain the amount can be reduced to150 ml (15 %) without appreciable loss in the qualityof the medium. It is also possible to replace theascitic fluid entirely by 100 ml (10%) of sterile horseserum. Plates should be stored in the refrigeratorand should be used within 10 days to 2 weeks.The finished medium is a soft agar and should be

handled and inoculated with care.The surface moisture may be removed without

undue danger of contamination by inverting theplate in the incubator, lifting the bottom out of thetop, and resting it on the edge of the top (agarsurface always downwards) until all droplets ofmoisture on the agar surface have disappeared. Theexcessive moisture produced by condensation and byhysteresis of the agar is neither necessary nor desir-able for growth of the gonococcus, provided the

s It is important to exclude the presence of antibiotics inthe ascitic fluid. When derived from diabetic patients itshould be examined for glucose, especially when used in thefermentation medium. Seitz filtration is necessary unless thetechnique of collection is absolutely safe. In fermentationtests and in experiments to determine the sensitivity toantibiotics, it is advisable to test the ascitic fluid beforeuse; if possible, fluid from the same patient should be usedfor long periods. Ascitic fluid and other biological materialsare expensive and difficult to procure. Recent experimentsindicate that simple autolysates prepared from fresh brewer'syeast and pig liver may replace the ascitic fluid, see " HYLmedium ", page 464.

464 A. REYN

plates are incubated in a moist atmosphere. How-ever, drying should not be continued until the surfacebecomes wrinkled by lines of stress; this indicatesthat the agar is losing water, and should always beavoided.While mixing and pouring the medium, care

should be taken to avoid the promotion of excessiveamounts of foam and bubbles. If bubbles do findtheir way into the plates, they may be removed byflashing a Bunsen flame over the agar surface beforeit has solidified.

HYL 1 MEDIUM

HaemoglobinPowdered ERG.B6 (Riedel-De Haen AG, Seelze-

Hannover) is mixed with a small amount of de-mineralized water to make a paste after whichsufficient demineralized water is added while stirringto give a 6% solution. (This will be clear and the pHwill be 7.9.) The solution is poured into screw-capbottles and the screw-caps tightly closed. It is thenheated in a steam-bath for 10 minutes and autoclavedfor 15 minutes at a pressure of 0.6 kg/cm'. Whenheated in tightly closed bottles and stored at 4°C thehaemoglobin remains as an opalescent solution forseveral months.

Yeast autolysate 2Fresh brewer's yeast (extremely flocculent bottom

yeast) 3 is obtained from a brewery. Water is addedin excess and after some time the supernatant isdiscarded. After two more washings with water,the suspensions being neutralized each time bymeans of a bicarbonate solution to remove the resinsubstances contained in the hops, the suspendedyeast is centrifuged and subsequently mixed withan equal amount of water. For every 2.5 litres ofsuspension 80 ml of ethyl acetate are added and themixture covered by a tight lid and heated for onehour in a 40°C water-bath. For practical reasons,the mixture is then moved into an incubator at 370Cuntil the next morning, when the pH is adjusted toabout 7.4 using about 17-20 ml of SN NaOH perlitre. The suspension is centrifuged and the superna-tant yeast autolysate sterilized by filtration, firstthrough a membrane filter S. S. G6ttingen 1118

Haemoglobin, Yeast, Liver.Society for General Microbiology (1958).

'If it is impossible to obtain an extremely flocculentbottom yeast, lager beer type, it will be necessary to usecentrifugation at this point.

and then through a membrane filter 1120. It isdispensed in sterile bottles and stored at 4°C.

Liver autolysate '1 kg of pork liver is chopped three times and

700 ml of distilled water are added. The mixture isstirred and placed in a 50°C water-bath with subse-quent slow heating to 75°C; this temperature ismaintained for one hour during which the prepara-tion is constantly stirred. After centrifugation, it issterilized by filtration, using the same filters specifiedabove. The pH is adjusted to 7.4-7.6.

Formula for preparation of 100 ml ofmedium66 ml of broth-agar base prepared as described

under Chocolate ascitic-fluid-agar, 15 ml of sterile 6%haemoglobin, 3 ml of a mixture of one part of yeastautolysate and two parts of liver autolysate, distilledwater to 100 ml. The brand of agar used shouldfulfil certain requirements; in particular, it should benon-toxic when used in an amount resulting in asuitable gel strength. A method of checking the gelstrength has been published by Fulthorpe (1951);recently, the method was simplified by M0ller.To measure the toxicity, comparative experimentsshould be done, using gonococcal strains withdiffering growth requirements. Both countings ofthe viable units and measurements of the colonysizes should be performed. The AKI agar, whichin our hands has shown a low toxicity to N. gonor-rhoeae, has a higher melting-point than haveJapanese and Spanish agars. Thus, an AKI agarbase must be kept at about 70°C when mixed withthe heat-labile enriching substances.A Spanish agar (brand unknown as yet) has also

been shown to be non-toxic and to give a suitablegel strength when used in a final concentration ofabout 0.65 %.The equipment for the preparation of the medium

is simple and the ingredients needed for its prepara-tion are easily obtainable. The constituents of themedium have not yet been analysed and definedchemically. It is possible that further analysis willreveal a close similarity to the media previouslydescribed by Lankford and co-workers.

FERMENTATION MEDIA

1. Danish fermentation medium "

Broth-agar base: 2.2% of agar in human placentabroth prepared with 1% of peptone and 0.5% of

'Mo11er, Ake, personal communication, 1961.'As developed at the Statens Seruminstitut, Copenhagen.


NaCI. No phosphate buffer is added (the pH isadjusted to 7.1-7.2). The quality of the peptone ishighly important and should be especially tested.The base is dispensed in 600-ml amounts and

mixed with 300 ml of ascitic fluid, 36 ml of phenolred 1: 500, 9 g of sugar (glucose or maltose) and9 ml of 0.1 % co-carboxylase.1The sugar is dissolved in a minimum of water

and either heated to 100°C for 10-15 minutes orsterilized by filtration. The co-carboxylase (Hoff-mann-La Roche) is dissolved in sterile water and,surprisingly, it can be added without sterilizing.4 g of phenol red (Merck) are dissolved in 160 ml of0.1N NaOH at 37°C with shaking. The volume isbrought up to 2000 ml with distilled water and thesolution is sterilized by autoclaving at 120°C for20 minutes. The constituents are mixed at about50°C, the pH is adjusted to about 7.4 with 5N HCI;by the following day it will have risen to about 7.8,which is suitable for the incubation in a CO2-atmosphere. The medium is poured into glass orplastic Petri dishes in a 4-5-mm layer.

2. HAP mediumThis is a new solid fermentation medium enriched

with haemin, recently introduced by Juhlin (1963).It is somewhat more complex than the Danishmedium, but has given very good results and is nowused in the Neisseria Department of the StatensSeruminstitut. The method of preparation is asfollows:

Placenta infusion: Fresh human placentas areground after removal of membranes and umbilicalcords. The amount thus obtained is weighed andmixed with twice as much distilled water and kept at4°C during the night. The next day the mixture isboiled for 20 minutes and paper-filtered, yielding aclear fluid which is dispensed into Erlenmeyerflasks and autoclaved at 120°C for 20 minutes.

Broth-agar base: Placenta infusion, 200 ml;sodium chloride (3% solution in placenta infusion),60 ml; disodium hydrogen phosphate dihydrate(2% solution in placenta infusion), 60 ml; ProteosePeptone No. 3 (Difco)2 (10% solution in placenta

I This comparatively high amount of co-carboxylase wasfound to be needed by Bang (personal communication, 1952),who at that time did not know the results obtained by Lank-ford & Skaggs (1946).

'Difco Manual ofDehydrated Culture Media and Reagentsfor Microbiological and Clinical Laboratory Procedures, 9thed., Detroit, Mich., Difco Laboratories, 1953, pp. 259 and290.

infusion), 60 ml; Sodium Alginate, BPC,3 0.6 g;Bacto Agar (Difco)2 6.0 g. These ingredients aremixed in each of four flasks and are then autoclavedat 120°C for 20 minutes. The broth-agar base iscooled to 55°C.

Ascitic fluid: Each fresh batch is sterilized byfiltration (Seitz filter EKS2) and compared withearlier batches as regards growth-stimulating effecton strains of N. gonorrhoeae, Streptococcus pyogenes,Haemophilus influenzae, and Diplococcuspneumoniae.

Phenol-red (Merck): A 0.4% solution in distilledwater is autoclaved at 120° for 20 minutes.Haemin, bovine (twice recrystallized) (Sigma):

A 0.2% solution is prepared by dispersing 0.15 g ofhaemin crystals in 75 ml of distilled water andadding 1.5 ml of 3N sodium hydroxide. Thesuspension is shaken well until the crystals havedissolved completely and a perfectly clear, dark-green solution is obtained. This solution is thensterilized by filtration (Seitz filter EKS2).

Sugar: A 20% solution of dextrose, maltose,sucrose, or laevulose in placenta infusion. Thesolution is sterilized by filtration (Seitz filter EKS2).HAP medium: Broth-agar base, 380 ml; ascitic

fluid (preheated to 52°C), 180 ml; phenol-red (Merck)(0.4% water solution), 6 ml; haemin (Sigma) (0.2%water solution), 10 ml; sugar (20% solution inplacenta infusion), 30 ml. The sterile ingredientsare mixed at 52°-55°C and the pH is adjusted to 7.7with 5N sodium hydroxide. The medium is pouredinto plastic Petri dishes, filling each one to a depthof 4 mm.

Co-carboxylase (B&rolase, Hoffmann-La Roche)is dissolved in a buffer consisting of 1.0 g of sodiumacetate, 0.2 g of sodium hydroxide and 100 ml ofsterile water to make a 0.I% solution. Of thissolution, 6 ml are added to the HAP medium at thetime when the broth-agar base, the ascitic fluid, andthe other ingredients are mixed, giving a finalconcentration of about 0.001 % of co-carboxylase.

STUART'S MEDIUM WITH SOLID AGAR

Formula

Agar, 16 g;4 sodium glycerophosphate, 20% byvolume, 50 ml; thioglycolic acid, 80%, 0.95 ml;

'British Pharmaceutical Codex, London, The Pharma-ceutical Press, 1959, p. 675.

' The precise quantity of agar will depend on the brandused and on the batch.

466 A. REYN

CaCl2, 10%, 1 ml; methylene blue, 0.1 %, 2 ml;and distilled water to 1000 ml. It is recommendedthat all distilled water be passed through an ion-exchange-resin column to remove free chlorine(Crookes & Stuart, 1959). A 1.5% "water base"is stored in 950-ml amounts. The base is melted andthe ingredients are added with subsequent adjustmentto pH 7.4 by the addition of SN NaOH. When hot,the medium is dispensed in sterile hydrophobiccotton-wool-plugged 15-mm x 150-mm tubes form-ing a column 10 cm high. The tubes are steamed at100°C for one hour, and then cooled in cold waterfor 15 minutes (see also page 451).1 The tubesshould be stored in a cool dark place until required.Ordinarily, they will keep (i.e., will still contain acolourless layer more than 3 cm high) for at leasttwo months, provided that the thioglycolic acidfulfils the criteria laid down in the United StatesPharmacopoea, vol. 15, p. 1070.

Preparation ofswabsFor sampling, good quality absorbent cotton-wool

and wooden swab sticks (see page 451) are used.The swabs should be carefully prepared, beingneither too small nor too thick, and the cotton-woolshould fit smoothly to the end of the applicators.Prepared swabs are boiled in S0rensen's phosphatebuffer solution at pH 7.4 and immediately afterwardsdipped into a 1% suspension of powdered charcoalin water. After impregnation, the applicators aredried, packed in two layers of paper, and sterilizedby autoclaving at 125°C for 20 minutes.

OXIDASE REAGENT

A stock suspension is prepared by the additionof 100 ml of concentrated ethanol to 1 g of dry,powdered tetramethylparaphenylenediamine hydro-chloride. The substance is hydrophilic andshould be kept in sealed bottles or in a desiccatorover P205. The reagent kills the gonococci in about30 minutes. The dimethyl compound (p-amino-dimethylaniline monohydrochloride) is cheaper butfar more toxic; in addition it is more difficult toobserve the colour change to red instead of purple,especially when " chocolate " plates are used.

1 In some cases, it may be found convenient after coolingto remove the cotton-wool plugs one at a time, soak themin a hot mixture of 9 parts of vaseline and 1 part of solidparaffin (melting point 500-60°C) and replace them in thetubes. In this way, evaporation will be minimized-forexample, when the tubes are heated in order to reduce themedium (see also page 450).

Every morning, a fresh reagent is prepared bymixing one volume of the stock suspension withtwo volumes of distilled water. When fresh, it hasa very light purple colour (almost colourless); it isslowly oxidized in air, turning a deeper and deeperbluish purple until after several hours it is com-pletely oxidized, and is then useless. The gonococciproduce an indophenol oxidase which enhances theoxidation of the reagent, so that on the cultureplates the reaction takes place in about a minuteinstead of over the course of several hours.

POLYMYXIN B

This reagent is used for the inhibition of coliformbacilli.A 0.1% stock solution of polymyxin B sulfate

(Pfizer) is prepared with sterile distilled water. Theunit/weight relation differs from one batch to theother. The stock solutions keep for at least twoweeks at about +4'C. The final concentration usedin the medium for primary isolation is 25 IU per ml.

Note: Gonococcus strains are very rarely suscep-tible to this drug (Crookes & Stuart, 1959).

NYSTATIN

Nystatin 2 is used to inhibit the growth of moulds.Stock solution: 200 ml of 70% ethanol plus 4

drops of 1% sodium citrate are used to dissolve thecontent of a vial containing 500 000 units of nystatin.The mixture is shaken for 30-60 minutes with sterileglass beads, resulting in a turbid suspension whichis dispensed in suitable amounts for later use. Thissuspension keeps for about two months at - 20°C.1 ml is added per 100 ml of medium giving a finalnystatin concentration of 25 IU per ml and anethanol concentration of 0.7%. This is used in allplates in periods when moulds are disturbing. If aconcentration of 25 IU per ml is insufficient, 40-50IU per ml may be added without any damagingeffect.

REAGENTS TO BE USED IN THE GRAM-STAINING

TECHNIQUE'3

0.2 % aniline crystal violetSolution A: 0.2 g of crystal violet is dissolved in

10 ml of 96% ethanol.

I Mycostatin (Squibb) has been used in the NeisseriaDepartment of the Statens Seruminstitut, Copenhagen.

I As developed at the Stateas Seruminstitut, Copenhagen.


Solution B: 3.2 g of aniline are dissolved in 90 mlof distilled water, vigorously shaken for 15 minutesand filtered through wet filter paper.

Solutions A and B are then mixed. The final pH isabout 7.5. Thereagent keeps for 3-4 weeks at 1 8°-25°C.

Lugol's iodine solution. 1 g of iodine and 2 g ofpotassium iodate are dissolved in 300 ml of distilledwater; the pH will be about 4.8.

0.1 % phenolized fuchsin. 1 g of basic fuchsinand 10 ml of 96% ethanol are mixed with 5 ml ofliquid phenol (Danish Pharmacopoeia, 1948) andadded to 90 ml of distilled water. Before use, thisstock solution is diluted 1: 9 with distilled water togive a pH of about 4.8.

96 % ethanol.

Annex

REVIEWERS

Dr Sara E. Branham, 5307 Glenwood Road, Bethesda, Md., USAProfessor C. E. Lankford, Department of Microbiology, University of Texas, Austin, Texas, USADr H. W. Scherp, Chief, Laboratory of Microbiology, National Institute of Dental Research, Department of Health,

Education, and Welfare, Bethesda, Md., USADr J. D. Thayer, Assistant Chief, Microbiology Research, Venereal Disease Research Laboratory, Department of

Health, Education, and Welfare, Communicable Disease Center, Atlanta, Ga., USADr A. E. Wilkinson, Director, Venereal Diseases Reference Laboratory, The London Hospital Research Laboratories,

London, England

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Laboratory Diagnosis of Gonococcal Infections