GROWTH AND DIAGNOSTIC STUDIES

WITH NEISSERIA GONORRHOEAE

ROBERT LESLIE HOLLAND, B.S, IN E.E.

A THESIS

IN

MICROBIOLOGY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

// Accepted

August 1976

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ACKNOWLEDGEMENTS

I am greatly indebted to Professors Clsurence L.

Baugh, Jack Hayes and Jack Sevall for their tirae,

patience and helpful criticism in the direction and

preparation of this thesis.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

LIST OF TABLES • . . . . . • iv

LIST OF FIGURES V

CHAPTER

I . INTRODUCTION • • • • • • • 1

I I . MATERIALS AND METHODS • • • • • 18

l . RESULTS • • • • • • • • • 25

IV. DISCUSSION 51

V SUMMARY • . . . . . . . . 57

LITERATURE CITED 60

APPENDIX . . . . . . . . . . . . . . 70

iii

LIST OF TABLES

Table Page

1« Sites of infection and local

and general complications • • • • • . • • • • 5

2. Components of Neicarb medium . • • 19

J. Semi-solid medium for initial growth studies . • • • • . . • • • . . . . . 2 0

^. Liquid medium for growth curve

studies . • • • • . • . • • . . . . . • • • • 2 1

5. Carbon/Energy sources studied • • • • • • . . 2 2

6. Growth on semi-solid media . • • . • • . . . 2 6

7. Temperature effects on growth on semi-solid media . • • • . . • • . • • • • 2 7

8. Survival time with different carbon/energy sources . • • . . . . . • • • • 2 9

9. Papanicalaou and gram stain comparison • • • • • • . • • . . . . • • • . 5 0

iv

LIST OF FIGURES

Page

Figure 1. Growth of clinical isolate (2957) of Neisseria gonorrhoeae in a liquid growth medium with varying concentrat-ions of V-C-N inhibitor . • 30

Figure 2. Growth of clinical isolates (3092 and 3698) of Neisseria gonorrhoeae in a liquid growth medium with varying concentrations of glucose • • . . • • • • 3 ^

Fig\ire 3. Growth of clinical isolates (3092 and 3698) of Neisseria gonorrhoeae in liquid growth medium with glucose, sucrose and lactate as carbon/energy source . . . . . . . . . . . . . . . . . 3 ^

Figure k» Growth of clinical isolates (3092, 3225 and 3^13) of Neisseria ^onorrhoeae in liquid growth medium with glucose, fructose, propionate, raffinose, succinate or no addition as carbon/ energy source • • . • • . . . . • • • • • 3 8

Figure 5. Growth of clinical isolates (3092, 3225 and 3^13) of Neisseria gonorrhoeae in liquid growth mediura with glucose, rham-nose, xylose, aurabinose, malonate or inositol as carbon/energy source . • . • 40

Figure 6. Growth of clinical isolates (3092, 3225 and 3413) of Neisseria gonorrhoeae in liquid growth medium with combinations of glucose or sucrose with lactate as carbon/energy source • • • • • • • • • • 4 2

Figure 7» Growth of clinical isolates (3092, 3225 and 3413) of Neisseria ^onorrhoeae in liquid growth medium with combmations of glucose v/ith sucrose and with prop-ionate as carbon/energy source • • • • • 44

Figure 8^ Growth of clinical isolate (76I6) of Neisseria gonorrhoeae in a liquid growth medium with glucose or the combinations glucose plus xylose, glucose plus succinate or glucose plus lactate plus succinate • • • • 46

Figure 9. Growth of clinical isolate (7676) of Neisseria gonorrhoeae in liquid growth medium with glucose or glucose plus fructose as carbon/energy source • • • • • • • 48

vi

CHAPTER I

INTRODUCTION

Objectives. The purpose of this thesis was

essentially two fold. The first objective was to examine

the in vitro growth requirements and characteristics of

Neisseria gonorrhoeae, the etiologic agent of gonorrhea.

In particular, potential carbon and energy sources were

explored. A variety of different compounds were tried as

carbon/energy sources, first in semi-solid media, then

in broth cultures. In addition, combinations of sources

were studied. The effects of temperature on the bacteria's

growth with the different carbon/energy sources was also

studied.

The intent of this portion of the research was

both to more fully characterize the organism's growth

characteristics and to suggest a better clinical medium

for longer term survivability such as is necesseiry for

diagnosis following weekend incubation in small labor-

atories. Also, the organism's sensitivity to the inhib-

itor V-C-N, utilized in most clinical media, was studied.

The survivability of organisms in frozen storage was

also investigated.

The second objective was to evaluate the practic-

ality of using the Papanicalaou Smear as a screening tool

for gonorrhea. Cervical smears were compared by using

both the Papanicalaou and Gram stains.

The Disease. Gonorrhea is an acute infectious

disease involving primarily the mucous membranes of the

genitourinairy tract, rectum, cervix and occasionally the

eye with possible hematogenic spread to serous and

synovial membranes in other parts of the body (47)•

Gonorrhea is a disease of both sexes which is usually

transmitted by sexual intercourse (9).

It is an exceptionally prevalcnt disease throughout

the world, with the annual number of new cases exceeding

60-65 million (14, 38). In the United States nearly a

million cases are reported each year^ with possibly two

or three times as many unreported (13» 79)» Sexual

distribution for reported cases of gonorrhea is about

40^ female and 60^ male with the majority of these cases

occuring in the 15-24 year group (13)»

In the male, gonorrhea usually remains localized

in the urethra and connecting structures. Within 24

hours a purulent urethral discharge begins. If the

infection remains untreated, in 10 to 14 days the patient

may begin to experience dysuria and perhaps strangury.

In severe attacks there may be a few drops of blood at

the end of micturition (14, 47).

The most common site of infection in the feraale is

3

the cervix uteri (14, 47)• Ninety to ninety-five per-

cent of female patients with gonorrhea heæbor organisms

in the columnar epithelium of the cervix (11, 58). Ureth«

ral infection invariably spreads to the emall urethral

glands but rarely to the bladder. The vaginal membrane

is usually resistant to infection, Sometimes the mucous

membranes of the vagina are reddened and bathed in pus

from which the gonococci can be recovered.

The cervix shows endocervicitis which often results

in the formation of infected retention cysts, the

follicles of Naboth, which may be seen on the vaginal

portion of the cervix. The infection probably extends

over the surface of the endometrium to the Fallopian

tubes, although lymphatic spread is possible. In the

puerperium, the infecting organism may spread directly

to the uterine wall at the site of placental attachment,

causing parametratis (inflammation of the cellular tissue

around the ligaments near the uterus).

Once the infection reaches the Fallopian tubes,

the organism invades the lining epithelium and spreads

to the Bubmucosa. The tubes can then fill with pus,

restricting and distending them. The presence of the

infecting organism and the pus can lead to a variety of

other pelvic coraplications, including peritonitis and

pelvic abscess. In spite of treatment, often the

pelvic infections have permanent residual effects.

The incubation period may vary from 2-10 days,

though it is often less than five days. Occasionally,

incubation periods of a month or more have been described

but this could be because the symptoms did not attract

the patient's attention.

Symptoms are absent or insufficient to attract

the patient's attention is up to 50-60^ of cases of

acute uncomplicated gonorrhea in the female (9, 14, 47,

56). Many of these women never seek treatment and are

often diagnosed only because their male contacts have

developed the disease. It is because of these cases

that a mass screening program or the incorporation of

gonorrhea testing into other routine work is essential.

The sites of infection and of local and general

complications are shown in Table 1.

Organism. Although first thought to be a symptom

of syphilis by Paracelsus (12), gonorrhea is caused by

Neisseria gonorrhoeae, first described by Albert Neisser

in 1879» He demonstrated the organism consistantly in ad-

ults and in smears from acute cases of vaginitis and

urethritis in adults and in smears from acute conjunctivit-

is of newboms (83). The organism was first cultivated by

Leistikow and Loeffler in 1882 (12) and three years later

by Bumm (14, 83). Bumm also successfully innoculated the

male urethra, producing characteristic symptoms and signs

of gonorrhea (14). The gonococcal complement-fixation test

TABLE 1

SITES OF INFECTION AND LOCAL AND GENERAL COMPLICATIONS^

Classification Sites of Infection

Local General Complications Complications

In Men

Anterior Urethra

Urethra

Posterior Urethra

Anorectum Conjunctivae Oropharynx

Tysonitis Paraurethr-al duct infection

Littritis Periurethr-al abscess Urethral stricture Cowperitis Prostatitis Prostatic abscess Vesiculitis Epididymitis Trigonitis

Arthritis Anterior uveitis Myocard-itis

Endocard-itis

Pericard-itis

In Women

In Children

Urethra

Cervix uteri

Anorectum Conjunctivae Oropharynx

Skenitis Bartholin-itis

Salpingo-oophoritis Parametritis Pelvic Abscess Pelvic peritonitis

Arthritis Anterior uveitis Myocard-itis

Endocard-itis

Mengitis

Vulva and Vagina Anorectum Conjunctivae Suppurative

panophthalraitis and ^ blindness * Data taken from King and Nicôl {i).

(GFT) was introduced by Muller and Oppenheim in I906.

Neisseria gonorrhoeae is a non-motile, non-spore-

forming, gram-negative coccus which grows in pairs or, on

occasion, in tetrads or clusters. The organisms are small

(ca. 0^8 by 0.6 micrometers) oval or spherical cocci with

flattened or slightly concave adjacent sides and resemble

a pair of kidney beans. They are parasites of the mucous

membranes of mammals (7). When observed in exudates or

cervical smeairs the organisms are often found inside poly-

morphonuclear leucocytes or associated with the surface

of polymorphs or other tissue, often in small matrix

arrangements (26, 75)«

The ultrastructure of Neisseria gonorrhoeae is

similar to other Neisseria species. The cell wall

contains a dense layer probably consisting of the peptid-

oglycan and an outer membrane. No capsule has been

convincingly demonstrated on gonococci. Fimbriae and

pili raay be present (71» 82). Pili appear:, to promote the

attachraent of virulent gonococci to huraan epithelial cells

and erythrocytes, enhancing the organisra's ability to \.

evade the host's defenses in tissue (6I).

Like most gram-negative bacteria, Neisseria

gonorrhoeae has an isoelectric point of about 5«0 (43).

An acidic change, greater than in most bacterial species,

occurs when iodine is added during Gram staining. This

change, attributed to the reaction of iodine with lipids

or reactive radicals such as the sulfhydryl groups of

proteins, probably accounts for some variability in the

decolorizing during Burke-Kopeloff-Beerman Gram staining

of laboratory cultures. The gonococcus can also be stain-

ed by polychrome stains such as Pappenheim-Saathoff methyl

green-pryonine (4). Eosin followed by methylene blue alone

yields good results. Intracellular granules can be seen in

stained preparations, but generally young cultures stain

evenly while older cultures (greater than 24 hours)

contain more involution forms which stain poorly.

On semi-solid media, colonies of gonococci are

small, round, convex, translucent, finely granular with

lobate margins and are greyish, with pearly opalescense

when viewed by transmitted light after 24-^8 hours incub-

ation.

After further incubation ajid serial passage, the

gonococci exhibit a number of colonial forms (32).

Kellog (35, 36) recognized four genetically determined

colonial types under standardized conditions. A trans-

parent medium with diffuse, angled light transmitted thr-

ough the medium from the bottora is necessary to observe

colonial characteristics. Type 1 colonies are small

(0.05 mm dia.), round, convex with entire edges, translu-

cent, dark colored, amorphous, slightly viscid and easily

emulsified. Type 2 colonies are similar to type 1, but

have sharper edges, are slightly crenated, have increased

8

intemal granularity, a friable consistancy, and are

better able to reflect light due to thicker reflective

surface film, Type 3 are larger (1 mm dia.), low convex,

flat edged, granular, viscid and brown colored. Type 4

colonies are similar but are amorphous and colorless.

Type 5 colonies have been described by Jephcott (32) to

be very shiny like type 2 but as big as a type 3. Easily

seen granules are present and they are brown in color.

Often a series of consecutive rings can be seen on the

surfaces. The edges aire clearly irregulaur and coarse.

Type 1 and Type 2 have been demonstrated to be virulent

orgsmisms, while the others appear avirulent (35» 36).

Five serological types have been demonstrated by

Hutchinson (30) in a manner similar to Lancefield's typ-

ing of streptococci but this method doesn't seem to have

much value beyond epidemiological tracings as subcultivat-

ion causes the loss of serological specificity.

Growth Characteristics. Neisseria gonorrhoeae is a

fastidious organism and is susceptible to raany toxic sub-

stances. Cultivation of the gonococcus on primary isolat-

ion is often difficult. The organism is fragile and does

not survive 24 hours in 12-79% of primary isolations (15,

28). Many types of transport and growth media have been

evaluated. Most of the existing knowledge of the organism's

metabolism has come from attempts to develop suitable

clinical media.

9

Neisseria gonorrhoeae can be grown on semi-solid

media with incubation temperatures between 30-38.5® C,

with optimura growth reported at 35-36® C (7)» The pres-

ence of moisture is important for good growth. Most

Neisseria gonorrhoeae strains require an atmosphere of

2-10% carbon dioxide or sraall quantities of suppleraental

bicarbonate for growth (24, 31, 72).

Early media were enriched by the addition of blood,

serum, ascitis and hydrocele fluid. Later modifications

included the use of peptone base, replacement of ascitis

fluid by yeast extract, the development of "chocolate

agar" to reraove some of the toxic blood products and the

use of purified agar (2, 8, 37). Other media were devel-

oped utilizing pea meal extract agar (21, 22), starch and

casamino acid (54), beef extract and heart infusion (51)»

and other growth enhancing factors (78).

Specific growth factors have been identified for

many strains. Glutamine and cocarboxylase (thiamine pyro-

phosphate) were found to be essential for growth in 10-15%

of the strains tested by Lankford and Snell (45) and

Lankford and Skaggs (44). Some strains were found to

require glutathione according to Gould (23). Griffin and

Racker (24) demonstrated a requirement for hypoxanthine

while showing that the use of yeast extract could be repl-

aced by a mixture of hypoxanthine, uracil and oxalacetate.

Boor (5) reported than some strains of Neisseria pjonorrhoeae

10

require a cystine concentration of 0.025-0.075%.

Many of these facts were linked together in the

development of chemically defined media. Kenny et al. (37)

utilized a medium using Medium 199 without bicarbonate

(GIBCO), cocarboxylase, L-glutamine, glucose, and ferric

chloride, Hunter and McVeigh (29) have developed one using

inorganic salts, glucose, guanine, L-cystine, L-isoleucine,

L-leucine, L-proline, L-threonine, L-valine, cytosine,

B-vitamin supplement, L-arginine and L-aspartic acid. They

also showed conventional agar to be inhibitory to growth

but purified agar was suitable. Similar media have been

developed by other workers (16).

Catlin (8) has presented a nutritional profile for

Neisseria gonorrhoeae with the ultimate goal of auxotyping

different strains. The mediura was similar to that of

Hunter and McVeigh above. Catlin also showed that all

strains tested grew in the absence of hemin and histidine,

asparagine, tryptophan, tyrosine and phenylalanine, threo-

nine, glutamine and spermine. In sorae strains, one or

more of them stimulated growth.

Elmros (15) has shown that low pH (5»0-6,0), Cu'*"*',

Mn**"*", Mg"'"'*', Ca**"*", and sucrose (10%) increases survivability

by preventing autolysis, although these cannot be consid-

ered true essentials for growth.

The present most coramonly used laboratory mediura was

developed by Thayer and Martin (48, 50, 73. 74). Their

11

medium was a modification of Christensen and Schoenlein's

(10) "chocolate agar" to which they added the antibiotics

vancomycin, nystatin, and colistin. The addition of these

antibiotics was the main advantage to the Thayer-Martin

medium.

Vancomycin inhibits the incorporation of amino acids

into the peptidoglycan and causes accumulation of UDP-Mur-

NAc-peptides in Gram-positive organisms (20, 66)^ inter-

fering directly with the release of the phospholipid carrier

preventing transport of new material during cell-wall

synthesis, Nystatin alters the permeability of the membr-

ane inhibiting the uptake of glycine (18). Nystatin is

active against yeasts, many fungi and other eucaryotic

cells. Colistin (polymyxin E) affects the membrane function

through interactions with the lipopolysaccharide comp-

onents of Gram-negative cell walls (19). Colistin is eff-

ective against Pseudomonas, Escherichia, Enterobacter,

Salmonella, Klebsiella, Hemophilus and Shigella species.

A slight modification of Thayer-Martin media,

Transgrow, is used in most diagnostic facilities for the

transport of primary cultures. Transgrow incorporates a

carbon dioxide supplemented atmosphere within the culture

bottle (49). It also has aji increased agar concentration

(1% to 2%), the glucose concentration was increased from

0.1% to 0.25%, and triraethoprim lactate is added to reduce

the growth of Proteus species.

ÍP'

12

Talley and Baugh (72) have improved on the Thayer-

Martin and Transgrow media in the development of Neicarb

which eliminates the carbon dioxide atmosphere by the

incorporation of sodium bicarbonate.

Metabolism. In all of these studies mentioned thus

far, the primary energy source has been glucose. Glucose

is catabolized aerobically by the Enter-Doudoroff and

Pentose Phosphate pathways (84% and 16% respectively) in

this organisra (53)» The Embden-Meyerhof-Pamas pathway

appears not to function. The metabolism of glucose app-

arently occurs in two steps. During active growth, glucose

is broken down to acetate and carbon dioxide. Following

glucose depletion, acetate is oxidized to TCA intermed-

iates. During this period little or no growth occurs.

According to Morse (52), only glucose, pyruvate,

and lactate are utilized efficiently as a source of energy

^y Neisseria gonorrhoeae. His studies make no mention,

though, of the compounds which were tested and found unsuit-

able as energy sources.

Tonkazy and Pelczar (76) showed, in 1953» that

araong the TCA cycle corapounds only those from -keto-

glutaric acid through pyruvate were oxidized in whole cell

experiments, although fumerate sometiraes showed a high

rate. They also showed that of all known amino acids only

the two forms of glutamic acid were oxidized rapidly.

Hebler and Morse (25) have since taken Neisseria gonorr-

^

13

hoeae grown in a glucose-lactate medium and, before

assaying for enzymes, allowed the cells to metabolize

acetate for a short time. In these cells, all of the

normal TCA enzymes were found. He also demonstrated that

glutamine entered the cycle at -ketoglutaric acid but

that this was not reversible.

Winter and Morse (81) have demonstrated several

non-heme iron centers, cytochrome c, two b cytochromes,

and cytochrome o as the terminal oxidase in the respiratory

chain of Neisseria gonorrhoeae.

Bergey's Manual (7) describes Neisseria gonorrhoeae

as able to grow sinaerobically although Williams and Wende

(80) and James-Holmquest et al. (31) were unable to

obtain growth under anaerobic conditions. In these studies,

an atmosphere high in hydrogen and carbon dioxide could

account for the lack of growth.

Diagnosis. Diagnosis of gonorrhea in the male is

based on history, clinical signs and the demonstration of

gram-negative diplococci, intracellular in white blood

cells in exudates from obvious lesions. In the female,

culture is normally necessary to elucidate the presence of

Neisseria Ronorrhoeae in addition to history and signs.

Cultures and smears are first examined microscopic-

ally for the presence of grara-negative, "coffee-bean"

shaped diplococci. There is some debate as to the efficacy

of using Gram's staining as a diagnostic test in addition

14

to clinical history and evidence in female gonorrhea.

There are many who will base a positive or negative diagn-

osis of gonorrhea on symptoras and direct microscopy only

(42, 69). On subsequent controls of therapeutic results,

the absence of symptoras in combination with a negative

direct smear has usually been considered sufficient to

declare the patient non-infectious (39, 42). Many consider

that although this procedure may be partly justified in

male patients, it has not been satisfactory in females (55,

59» 63). In fact, it has been highly unproductive (34, 64).

According to Juhlin (33)» the discrepancy between these

viewpoints is the reason why, lately, culture has been

little used for the confirmation of successful treatment

of gonorrhea.

Lodin ( 6) presented data in 1964 on 107 females

where direct microscopy was positive and culture negative

in 24.3%, Prasier (59) in 1968 conducted a study in which

he showed only 5% of the 211 diagnosed positive female

gonorrhea cases had positive gram-stained smears with

negative culture growth where 31% bad culture growth with

negative smears. In 1972, Phillips (60) studied 2557 cases

of female gonorrhea in all common sites and found only

8% where culture was negative and gram-staining indicated

positive. With 2 ^ of the cases, the organism could not

be identified microscopically with the gram stain but were

culture positive.

15

The difficulty with microscopic examinations is that

the gonococcus is either not present in smear preparations

or because of the variety of bacteria including Morexella

urethralis, Neisseria sicca and other gram-negative diplo-

cocci may be present in the female cervix creating a

microscopic picture resembling gonorrhea (57).

The use of fluorescent antibody techniques (FAT)

have proved valuable in diagnosis, especially in women,

though less sensitive than culture (65). Although rauch

has appeared in the literature in recent years, the use of

FAT has not increased significantly in clinical laboratories.

Further identification of Neisseria gonorrhoeae is

achieved through characteristics of the cultured organism,

presence of certain enzymes, sugar fermentation reactions

or fluorescent antibody staining. Neisseria gonorrhoeae

produce acid from glucose but not from maltose, fructose,

sucrose, or starch as opposed to other Neisseria which

present different fermentation pattems.

Neisseria gonorrhoeae and other Neisseria species

produce tho enzyme oxidase or indophenol oxidase which in

the presence of air acts on certain aromatic amines to

produce color changes. The color change, from pink to raag-

enta to purple or black, produced by an indicator such as

N, N, N', N'-tetramethyl-£-phenylenediamine dihydrochloride

(1%) is readily observed and used as a confirmatory test.

16

With federal funding for public health prograras

declining (1), support for routine culture of women in

public clinics is diminishing. For this reason, a simpler

and less expensive method of screening for gonorrhea is

necessary (3)» The center of screening programs will

probably be the asymptomatic female carrier, although there

are some asymptomatic males. At present, no true screening

test exists^

In developing better diagnostic tools, Lagerholm

and Lodin (41) analized the cytology of scrapings from

urethral smears in male gonorrhea patients. Ward sind

Watt (77) also examined the relationship between infecting

bacteria and mucosal cells. These studies and those by

Heller (25) showed that the organism is often found in

close association with the mucosal cells of the urethra

and vaginal duct. Ward and Watt (77) consider the adher-

ance to these cells to be directly related to virulence.

Since there is a close relationship between the

organism and mucosal tissue, iraproved diagnostic samples

are obtained by scraping the surface with an endometrical

currette. Shapiro (68) demonstrated this by inserting a

Novak endometrical currett into the cervical os to the

endocervix in non-pregnant women. Both by scraping and

deeper sampling he was able to increase his diagnostic

sensitivity by 13-20%.

17

Some VD clinics, in Great Britain, include screening

for cervical carcinoma as a routine in addition to gonorr-

hea testing (17). This screening is accomplished by taking

cervical scrapings with wooden spatulas, staining them by

the method of Papanicolaou, and examining them with a

standard light raicroscope (27). Heller (25) took advantage

of the simultaneous testing and the fact that metaplastic

and parabasal cells have been described in association

with gonorrhea infections ( O) to look for gonococci in

Papanicolaou smears. In this prelirainary study, about 70%

of the slides examined had raetaplastic cells present with

gonococci in a mosaic arrangement situated on a different

plane of focus from the nucleus of the cells. This would

suggest that the Papsinicolaou sraear could be used for

screening woraen for gonorrhea or the presence of the carrier

state during routine pelvic examinations. Since Heller's

work appears to be a promising screening tool, this study

further investigated its potential by comparing Gram and

Papanicolaou staining for the presence of gram-negative

intracellular diplococci in white blood cells.

CHAPTER II

MATERIALS AND METHODS

Cultures. Primary cultures of Neisseria gonorrhoeae

were obtained from clinical specimens from the Lubbock

(Texas) City-County Health Department. All cultures were

confirmed as Neisseria gonorrhoeae by Gram stain, positive

oxidase reaction and fermentation of glucose. Primary

cultures were transferred to either Neicarb (below) or

liquid culture medium and incubated at 35^ C for 24-it'tt hours.

Cultures for growth studies were taken from these first-

pass subcultivations.

Samples for the diagnostic studies were taken by

staff members at the Venereal Disease Clinic of the Lubbock

(Texas) City-County Health Department in accordance with a

mutually agreeable protocol (see Appendix). Tissue scrap-

ings were taken from female patients using established

procedures for cervical sampling for Papanicolaou smears.

Tissue from the cervix uteri was obtained with a wooden

"mutton-bone" spatula. Samples were then smeared on two

prelabelled glass slides. Two samples were taken and

altemately placed on slides for gram staining and for

Papanicolaou staining. Those to be stained by the method

of Papanicolaou were immediately sprayed with a cytology

fixative. Both were then refrigerated until staining.

18

19

Women selected for this sample were mostly positive

gonorrhea patients, confirmed by culture and clinical

observation.

Cultures for frozen storage studies were prepaured

by first transferring a suitable colony to 1 ml of liquid

growth medium and then treating it on a Vortex-Geni

(Model K-550-6) to disperse clumps. One-half milliliter of

this suspension was then added to 4 ml of Skim Milk Medium

(DIFCO) in one dram screw-cap vials and frozen at -90® C.

Growth Media. Cells were grown on a variety of media.

Neicarb was prepared in 1 or 2-ounce tightly capped pre-

scription bottles (Table 2).

TABLE 2

COMPONENTS OF NEICARB MEDIUM *

Component Amount/liter

GC Agar Base (DIFCO) 72.0 gm Hemoglobin 20.0 gm IsoVitaleX 10.0 ml V-C-N 10.0 ml NaHCO-j (7.5%) 30.0 ml

^lsoViialeX (BBL), V-C-N (BBL) and NaHCO^ were added aseptically after basic medium was auto-claved.

Initial growth studies were accomplished on hemoglobin

and glucose-free Neicarb suppleraented with the carbon/energy

source being studied. Glucose-free medium was achieved by

20

using sterile distilled water in place of the diluent

fluid for reconstituting the IsoVitaleX. The diluent

fluid is a 10% glucose in water solution (Table 3)« Growth

was studied in 1 or 2-ounce prescription bottles.

TABLE 3

SEMI-SOLID MEDIUM FOR INITIAL GROWTH STUDIES *

Components Amount/liter

GC Agar Base (DIFCO) 72.0 gm IsoVitaleX (Glucose-free) 10.0 ml V-C-N 10.0 ml NaHCO^ (7.5%) 30.0 ml

*lsoVitaleX (Glucose-free)(BBL), V-C-N (BBL), NaHCOo and the sterile carbon/energy source were added aseptically after basic medium was auto-claved. Final pH was adjusted to 7»0 with 2N NaOH.

Further growth studies were accomplished in a liquid

mediura described in Table 4. One hundred milliliters of

medium were used in 500 ml growth flasks for growth studies.

The different carbon/energy sources studied in both

semi-solid and liquid cultures are listed in Table 5.

Combinations of these sources were also studied.

Growth Studies. For initial studies, cell suspen-

sions of Neisseria gonorrhoeae were prepared in liquid

growth medium and treated on a Vortex-Geni (Model K-550-G)

to disperse any cell clumps.

21

TABLE ^

LIQUID MEDIUM FOR GROWTH CURVE STUDIES *

Components

Polypeptone (BBL) Corn Starch K HPOzi. KH2POZ1 NaCl IsoVitaleX (Glucose-V-C-N NaHCO^ (7*5%)

-free)

Amount/liter

15.0 gm 1.0 gm ^.0 gm 1.0 gm 5tO gm 10.0 ml 5.0 ml 10.0 ml

NaJÍCO and sterile carbon/energy source were added aseptically after basic mediura was auto-claved. Final pH was adjusted to 7.0 with 2N NaOH.

22

TABLE 5

CARBON/ENERGY SOURCES STUDIED

Components

Glucose (IX) Glucose (0.1 X) Glucose (0.01 X) Glucose (0.001 X) Sucrose Fructose Lactose Mannitol Sorbitol Inositol Arabinose Xylose Pyruvate (Sodium Salt) Galactose Lactate (Sodium Salt)(l X) Lactate (Sodium Salt)(0.1 X) Maltose Tartrate (Potasium sodium salt) Malonic Acid Propionate (Sodium Salt) Succinate (Sodium Salt)

Amount/liter

5.0 0.5 0.05 0.005 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.5 5.0 5.0 5.0 5.0 5.0

gm gm gm gm gm gm gm gm gm gm gm gm gra gm gm gm gra gm gm gm gm

23

Two loopfuls were inoculated on previously prepared

aemi-solid media which had been preheated to 35® C. The

organisms were spread over the surface of the media with

the inoculating loop. The cultures were then incubated

at 35® C (or as specified). Growth was based qualitatively

on surface growth.

For growth maintenance studies, one loopful of the

cell suspension was placed on the lower surface of pre-

pared preheated (35® C) medium in 2-ounce prescription

bottles and incubated at 35® C at the vertical position.

Subsequent streaks from the initial growth were made at

Z^ hour intervals for five days.

For later studies, cell suspensions were prepared

by transferring primary cells to liquid growth medium with

glucose (0.5%) and growing for 24 hours. One milliliter

of this suspension was inoculated into 500 ml screw-top

nephlo-culture flasks containing 100 ml of preheated (35® C)

liquid media. The cultures were incubated at 35° C on a

controlled environmental incubator-shaker at 200 RPM

(New Brunswiclc Scientific Model G25). Growth was measured

turbidimetrically with a Klett-Summerson Colorimeter

(Model 900-3) with a number 42 filter.

Papanicolaou Stainin^« Slides for staining using

Papanicolaou's method were sprayed with a cytology fix-

ative at the time the samplc was taken. Subsequent stain-

ing was a four minute hematoxylin regressive Papanicolaou

24

stain (62). After staining, slides were cover-slipped,

preserved in Histoclad (Clay Adams) and screened with a

standard light microscope.

Gram Staining. Slides prepared for gram staining were

refrigerated and stained within 48 hours. staining was

accomplished using the Burke-Kopeloff-Beerman modification

of the gram stain (34). After staining, slides were

cover-slipped, preserved in Histoclad (Clay Adams) and

screened with a standard light raicroscope.

CHAPTER III

RESULTS

Growth Studies on Semi-Solid Media. Studies with H C W M M U a

the various carbon/energy soxirces were first performed on

semi-solid media. Clinical isolates 1735» 1773 and 76I6

were used for preliminary studies followed by isolates

2326, 3001 and 7676. Growth obtained with these organisms

is summarized in Table 6. Especially good growth was

obtained at 24 hours on media using glucose, sucrose,

xylose and pyruvate alone and the combinations of pyruvate

plus sucrose plus 0.05% glucose, pyruvate plus 0.5%

glucose and pyruvate plus succinate. Poor growth was noted

with no additional carbon/energy source, mannitol and

tartrate. In all cases, no visable growth was noted at

24 or 48 hours on media with fructose as the priraary carbon/

energy source.

Further studies with semi-solid media investigated

the ability of the organism to grow at various temperatures.

A summary of these studies is found in Table 7« In all

cases, the best growth was obtained at 35® C. At 30® C

media with sorbitol, inositol, succinate and tartrate

supported growth the same as at 35® C. Media with the rest

of the carbon/energy sources showed less growth at 30^ C

than at 35° C, with no growth observed for xylose and

25

TABLE 6 26

GROWTH ON SEMI-SOLID MEDIA •

Carbon/Energy Sources

Growth on Isolate 1735 1773 7676 2326 3001 7616

V.

1. None 2. Glucose 3. Glucose (0.05%) 4. Sucrose 5. Lactate 6. Maltose 7. Maltose +

Glucose (0.05%) 8. Pyruvate 9. Rhamnose 10. Tartrate 11. Malonaté 12. Arabinose 13. Inositol 14. Fructose 15. Propionate 16. Propionate (0.25%) 17. Propionate (0.75%) 18. Propionate (1.0%) 19. Propionate (0.5%) + Glucose (0 20. Propionate (0.25%) + Glucose ( 21. Propionate (0.75%) + Glucose ( 22. Propionate (1.0%) + Glucose (0 23. Raffinose 24. Succinate 25. Mannitol 26. Xylose 27. Sorbitol 28. Pyruvate + Sucrose + Glucose ( 29. Pyruvate + Glucose (0.05%) ++ 30. Pyruvate + Lactate + Succinate 31. Pyruvate + Succinate 32. Succinate + Lactate 33« Pyruvate + Lactate

+ +++ +++ ++ ++ ++

+++ ++

«

+ +++

++ ++ ++

+++ ++

-

+ +++

+++

++ +

++ +

++

++

+ +++

+++ ++ ++

+++ ++ ++ ++ ++ ++

-

+

+ +++

+++ ++ ++

+++ ++ +

++ ++ ++ -

++

+++

++ + + +

0.5%) 0.5%) .5%)

0.05%) ++

++ +

++ ++

++ +++ ++

+++

++ + +

+++ +

+++ +++ +++ ++ ++ ++

+

++ -

+ +++ ++

+++ +++

• Incubated at 35° C. + Poor Growth, ++ Moderate Growth, +++ Excellent Growth.

27

TABLE 7

TEMPERATURE EFFECTS ON GROWTH ON SEMI-SOLID MEDIA

Carbon/Energy Source

Temperature 22® 250 300 35®

1. Glucose

2. Raffinose

3» Tartrate

4. Sorbitol

5. Xylose

6. Succinate

7. Malonate

8. Arabinose

9. Rhamnose

10. Inositol

11. Propionate

12. Sucrose

++

+

+

++

-

+

++

-

+

++

+

++

+++

++

+

++

++

+

++

+

++

++

++

+++

28

arabinose at 30® C.

Survival Time with Different Carbon/Energy Sources.

Clinical isolates 7618 and 7619 were studies on semi-solid

raedia with the different carbon/energy sources for maximum

survival time at 35° C. The results aire summarized in

Table 8. Isolates grown on media containing lactate,

pyruvate, sucrose, inositol, sorbitol and tartrate remained

viable longer than media with glucose, with the best

results obtained from lactate and sucrose. Isolates grown

on media containing malonate as carbon/energy source

remained viable for a shorter time than those grown on

glucose. The remainder of the carbon/energy soxirces

demonstrated survivability comparable to glucose.

V-C-N Concentration Studies. The effects of various

concentrations of V-C-N inhibitor is summarized in Figure 1*

Concentrations greater than 5 ml/liter have significant

effect of the growth of Neisseria gonorrhoeae. At

10 ml/liter, maximum growth was rauch less and was achieved

a few hours earlier. With 20 ml/liter, maximum growth was

very much lower and death phase began almost immediately

after log growth.

Growth StudJes in Liquid Media. Those carbon/energy

sources which supported good growth on semi-solid media

were studied in broth cultures with clinical isolates 3092,

3225, 3413. 3698 and 7616.

29

TABLE 8

SURVIVAL TIME WITH DIFFERENT CARBON/ENERGY SOURCES

Carbon/Energy Source

1. Glucose

2. Lactate

3. Pyruvate

4. Sucrose

5. Succinate

6. Arabinose

7. Sorbitol

8. Inositol

9. Tartrate

10. Rhamnose

11. Propionate

12. Raffinose

13. Xylose

14. Malonate

Days 1

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Since 2

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

2+

Initial 3

2+

4+

4+

4+

2+

2+

4+

4+

4+

2+

2+

2+

2+

2+

.•L.H- - ± J

Inoculation 4 5

m>

4+

2+

4+

-

-

2+

2+

2+

-

•»

-

-

TJ'i" L - . ..

-

2+

2+

2+

-

-

-

•w

-

«»

-

-

-

30

Figure 1. Growth of clinical isolate (2957) of Neisseria p;onorrhoeae in a liquid growth medium with varymg concentrations of V-C-N inhibitor.

Cultures were incubated at 35® C in screw-cap nephlo-culture flasks in a controlled environmental shaker-incubator at 200 RPM.

r

Symbolsi • 0 ml/liter

^ 2 ml/liter

I © 5 ml/liter

Q 10 ml/liter

A 20 ml/liter

31

400

300

200

100

'E

••-•

O cc CJ

10

0 8 16 24 32

TIME (Hours)

32

Growth with various concentrations of glucose was

explored with isolate 3698. The best growth was obtained

with a final glucose concentration of 0.5%. Successively

lower maximal growth was obtained down to 0.0005% glucose,

as shown in Figure 2.

With the other isolates the growth with various

other carbon/energy sources was studied. The best growth

was achieved using glucose, sucrose or lactate. These

results are found in Figure 3. The other energy sources

all had similar growth patterns as shown in Figures 4 and 5.

The maximum growth obtained was much less than with

glucose, sucrose or lactate. Maximum growth from the

greatest to the least with the various carbon/energy

sources wasi raffinose, rhamnose, malonate, arabinose,

xylose, inositol, propionate, no additional source,

fructose and succinate. Xylose and propionate both showed

log growth similar to the others, but the stationary

phase was much shorter and the declining death phase began

sooner. The rate of growth during log phase was similar

with all the carbon/energy sources. The lag phase was

similar for all, except with arabinose aná with no addit-

ional carbon/energy source which exhibited lag phases

about 2 hours longer than the others.

Figures 6, 7, 8 and 9 show the growth obtained with

combinations of carbon/energy sources, All the combinations

used grew about the same with the exception of propionate

33

plus 0.05% glucose and fructose plus 0.5% or 0.05%

glucose. The growth obtained in all combinations

paralleled growth with 0.05% glucose alone, except when

used with propionate which had growth less than with

' either alone and with fructose which showed a slight

reduction of growth with the combination.

Papanicolaou and Gram Staining Comparisons. Cervical

smears from twenty-six positive female gonorrhea patients

were stained by both Gram's stain and the method of

Papanicolaou. Of those gram stained, seventeen (65.4%)

smears had an overwhelming number of small, gram-negative

diplococci, both intracellular and extracellular. In

eight cases (30.8%) there was a wide variety of micro-

flora with some gram-negative diplococci, some intra-

cellular. In one case (3.8%) no gram-negative diplococci

were convincingly demonstrated.

Smears stained by Papanicolaou's method were of

poorer resolution which made identification of any micro-

flora difficult. Bacteria either stain poorly or not at

all. By adjusting the plane of focus away from the nucleus

of the tissue cells present in the smear, small areas of

what appeared to be groupings of diplococci could be seen

in eight (30.8%) smears. These areas usually appeared

to be associated with the tissue or polymorphonuclear

leucocytes. In the remainder of the slides, evidence of

Neisseria ^onorrhoeae was not present. These results

34

•'J:

Figure 2. Growth of clinical isolates (3092 and 3698) of Neisseria gonorrhoeae in a liquid growth medium v/ith varymg concentrations of glucose.

Cultures were incubated at 35® C in screw-cap nephlo-culture flasks with the addition of varying concentrations of glucose in a controlled environmental shaJker-incubator at 200 RPM.

r >

• Symbolsi • 0.5% glucose m

r

s ® 0.05% glucose r

1 A 0.005% glucose a

@ 0.0005% glucose

^ 0.0% glucose

35

C

h-

o cc

TIME (Hours)

36

Figure 3. Growth of clinical isolates (3092 and 3698) of Neisseria gonorrhoeae in liquid growth medium with glucose, sucrose or lactate as carbon/energy source.

Cultures were incubated at 35° C in screw-cap nephlo-culture flasks with the addition of different carbon/energy sources in a

5 controlled environmental shaker at 200 RPM.

- Symbolsi • Glucose (0.5%)

l 0 Sucrose (0.5%)

» Q Lactate (0.5%)

37

400

300

200

100

c

O QC O

10 .

0

I

TIME (Hours)

38

t

Figure 4. Growth of clinical isolates (3092, 3225 and 3413) of Neisseria gonorrhoeae in liquid growth medium with glucose, fructose, propionate, raffinose, succinate or no addition as carbon/energy source.

Cultures were incubated at 35® C in screw-cap nephlo-culture flasks with the addition

r of different carbon/energy sources in a H controlled environraental shaker at * 200 RPM. n c Symbolsi ^ Glucose (0.5%)

a Fructose (0.5%)

A Propionate (0.5%)

^ Raffinose (0.5%)

K Succinate (0.5%)

• No Addition

39

to +j 'E D 4-»

O tr

0 10 15

TIME (Hours)

20 25

• ; ' . ; •.

40

> 1 .• s * » ^. Í ( . 1 . . ' 1

• • ' ! .• • i

V {

I

r > >

n C 3

Figure 5. Growth of clinical isolates (3092, 3225 and 3413) of Neisseria gonorrhoeae in liquid growth medium with glucose, rhamnose, xylose, arabinose, malonate or inositol as carbon/energy source.

Cultures were incubated at 35® C in screw-cap nephlo-culture flasks with the addition of different carbon/energy sources in a controlled environmental shaker-incubator at 200 RPM.

Symbolsi x Glucose (0.5%)

• Rhamnose (0.5%)

1 ® Xylose (0.5%)

Arabinose (0.5%)

Malonate (0.5%)

Inositol (0.5%)

41

400

300

200

100

tn

"E D 00

X

O ÛC (D 10

10 15

TIME (Hours)

42

Figure 6. Growth of clinical isolates (3092, 3225 and 3413) of Neisseria gonorrhoeae in liquid growth medium with combinations of glucose or sucrose with lactate as carbon/energy source.

Cultures were incubated at 35® C in screw-cap nephlo-culture flasks with the addition of combinations of different carbon/energy sources in a controlled environmental

Í incubator-shaker at 200 RPM. >

5 Symbolsi • Lactate (0.5%) + Glucose (0.05%)

3.

Lactate (0.5%) + Glucose (0.005%)

Lactate (0.5%) + Sucrose (0.05%)

^ 3

C

+•>

.2

X h-

o cc o

15

T I M E (Hours)

r ..

' . , . ' • • •

• I ,

k V i. • J í.

44

m

Figure 7. Growth of clinical isolates (3092, 3225 and 3413) of Neisseria gonorrhoeae in liquid growth medium with combinations of glucose with sucrose and with propionate as carbon/energy source.

Cultures were incubated at 35® C in screw-cap nephlo-culture flasks with the addition of combinations of different carbon/energy sources in a controlled environmental

^ incubator-shaker at 200 RPM.

1'

Symbolsi # Sucrose (0.5%) + Glucose (0.05%)

^ Propionate (0.5%) + Glucose (0.05%)

45

0 10 15 20

TIME(Hours)

30

46

. 4

r. ' i

'* r< 't

l. i:i \ "

^

. - . f

T.í •• .'.n

•"*

f . , • > < ? . y \

=f". •• s -

Figure 8. Growth of clinical isolate (76I6) of Neisseria gonorrhoeae in liquid growth medium with glucose or the combinations glucose plus xylose, glucose plus Buccinate or glucose plus lactate plus succinate.

Cultures were incubated at 35^ C in screw-cap nephlo-culture flasks with the addition

;* of combinations of different carbon/energy > sources in a controlled environmental •* incubator-shaker at 200 RPM. m S Symbolsi • Xylose (0.5%) + Z Glucose (0.05%)

< 5 @ Succinate (0.5%) +

Glucose (0.05%)

B Glucose (0.05%)

• Lactate (0.5%) + Succinate (0.5%) + Glucose (0.05%)

c

O cc o 10

47

100

JQ)

10 15 20 25 30

TIME (Hours)

48

0) asoq.onaj Q

(%^*0) asoq.onjtá + (5^^0*0) osoonxo ^

(%í*0) 9soq.onjy[ + (2éí*0) asoonxo x

($éíO*0) asoonxo ©

(%í'0) asoonxo • isioqni/Cs

•MH 002 íí- ae3{Bt{s-aoíi.BqnouT XT3q.U9uiuoaT«Aue paxxoaq.uoo B UT saoanos

iC9j8ua/uoqaBO q.uaaej:jTp jo suoTí).BÛTqraoo jo uoxq.TppB auq. iiq.TM SJISBXJ aanq.x^ô-0IM<Í9U dBO

-Maâos UT 0 Q C Q- peq.BqnouT eaaM saanq.xno

•aoanos iCSaaua/uoqaBo SB asoq.onaj snxd osoonx^ ao asoonxS q-îM mnTpam

i(q.Moa3 p-cnbTX u" aBaoqaaouoa BTaassTaM j o (9<i9^) *aq.Bxos X^^TUTXO JO Mq.Moao »6 aan^xá

49

c

I H

O cc o

0 10 15

TIME (Hours)

20 25 30

TEXAS TECU LIBRARY

50

are summarized in Table 9.

TABLE 9

PAPANICOLAOU AND GRAM STAIN COMPARISON

Presence of Gonorrhea-like Stain Organisms

Gram Stain 17 (65.4%) 8 (30.8%) 1 (3.8%) Papanicolaou Stain 0 8 (30.8%) 18 (69.2%)

Oxidase Testing. Attempts to incorporate a test

for oxidase into the grara staining procedure using

N, N, N*I N'-tetramethyl-£-phenylenediamine dihydrochloride

were unsuccessful. The test reagent was tried in all

steps of the Burke-Kopeloff-Beerman and Hoeker modificat-

ion of the gram stain but was either washed off or

crystalized, destroying a useful stain. At no time was a

color change indicating the presence of oxidase observed.

Frozen-Storage Studies. Two clinical isolates,

2959 and 3001, were frozen and recovered successfully at

intervals spanning over two months. Colonies recovered

appeared to be of a different morphology than those taken

for freezing. Colonies selected from Neicarb v/ere small,

convex and easily emulsified. Frozen cultures regrovm on

Ncicarb had colonies which v/ere much larger, convex and

mucoid.

CHAPTER IV

DISCUSSION

Initial growth studies on semi-solid media demon-

strated that Neisseria gonorrhoeae can grow with a variety

of compounds as the primary carbon/energy source.

Excellent growth occured with glucose, sucrose, pyruvate

and xylose on semi-solid media. Good growth occured with

a number of other carbon/energy sources including lactate,

rhamnose and raffinose. The growth curves established

from growth in liquid media quantified the growth from

these carbon/energy sources. As previously reported by

Morse and Bertenstein (52), excellent growth occured using

glucose, lactate and pyruvate. Surprisingly good growth

also occured using sucrose. Sucrose (0.5%) supported

growth comparable to 0.05% glucose. Glucose comtaraination

could not account for this level of growth, as the

maximum glucose present, according to the manufacture

(Matheson, Coleman and Bell), in the sucrose used would

result in a final glucose concentration of only 0.00025%.

Growth on peptone without any additional carbon/

energy source was also demonstrated. Although growth

was poor using peptone alone, it was better than with

fructose or succinate.

51

52

In media with fructose plus 0.5% and 0.05% glucose,

growth of Neisseria gonorrhoeae is reduced due to an

inhibitory effect of the fructose (see Figiire 9). The

inhibition is not as profound as found in liquid media with

propionate plus 0.05% glucose (see Figure 7). In this case

the propionate had the effect of reducing growth to levels

lower than when grown with either alone. In fact growth

with propionate plus 0.05% glucose showed that the propion-

ate inhibited growth to a level lower than growth on pept-

one alone. When grown on semi-solid media with varying

concentrations of propionate (see Table 6), Neisseria

^onorrhoeae was inhibited by concentrations greater than

0.75% when used along with 0.5% glucose.

The dependance on temperature for in vitro growth

oí Neisseria ^onorrhoeae was also studied. On semi-solid

media using glucose, raffinose, tartrate, malonate or

inositol as carbon/energy source, growth was the same at

both 30° C and 35° C. For all the other carbon/energy

sources growth was best at 35^ C. Isolates using xylose

or arabinose failed to grow at 30° C. No growth occured

using any carbon/energy source at 25° C or 23° C.

Bergey's Manual (7) records the optimum growth temperature

for Neisseria ^onorrhoeae as 35-36 C, but this optimum

refers to growth using only glucose as the primary

carbon/energy source. Results from these studies suggest

53

that for carbon/energy sources such as tartrate, malonate

or inositol, the optimum could be slightly lower or have

a slightly wider range.

When cell suspensions of Neisseria gonorrhoeae,

inoculated onto semi-solid media containing the various

carbon/energy sources, are restreaked from the initial

growth at one day intervals the length of time the

organisms remained viable was examined. On semi-solid

media using glucose as the primary carbon/energy source,

as found in most clinical media, the organisms remain

viable for only 2-3 days. This time prevents accurate

diagnosis in small laboratories for cultures taken on

Friday when the laboratory is to be closed over the week-

end. On media with lactate, pyruvate or sucrose as the

carbon/energy source, survivability time increases to

4-5 days. It appears that media for primary isolation of

Neisseria gonorrhoeae could be more useful, especially for

small laboratories or clinics, if they utilized lactate,

pyruvate or sucrose in place of glucose. Growth in liquid

growth media for these same carbon/energy sources

indicate that the lag phase and rate of growth during log

phase is comparable to a medium using glucose as carbon/

energy source. Thus a semi-solid media with lactate,

pyruvate or sucrose replacing glucose would also be suitable

for larger laboratories or small laboratories which read

cultures at 24-48 hours.

54

In another part of this study, the effects of

different concentrations of the commonly used inhibitor

V-C-N was studied. Concentrations of 0-5.0 ml/liter of

V-C-N have little effect on the growth of Neisseria

gonorrhoeae. At 10 ml/liter of V-C-N inhibitor growth

is significantly reduced. At 20 ml/liter, growth is poor

with the death phase beginning almost immediately

following log growth. It appears that the concentration

of 5.0 ml/liter of liquid growth media, as used in all

broth cultures in these studies, is the maximum amount

of V-C-N that ghould be used for good recovery of

Neisseria gonorrhoeae.

During frozen-storage studies, the use of Skim

Milk medixjm without the addition of glycerine appeared

to be satisfactory for storing cultures of Neisseria

gonorrhoeae over extended periods. Although colonial

morphology changed from small, convex and easily

emulsified colonies to ones which were large, convex and

mucoid when frozen cell suspensions were regrown, no

attempt was made to properly type either colony.

Results of these studies also indicate that it is

not feasable to use the Papanicolaou smear as a screening

tool for gonorrhea. The Papanicolaou staining procedure

does not adequately stain bacteria and results in a slide

of much lower resolution. The simplier gram stain is

far more valuable and much more clearly shows the micro-

55

flora on the cervix. Twenty-five cases (96.2%) showed

gram-negative diplococci using gram staining while only

eight (30.8%) Papanicolaou slides had any diplococci

present. Diplococci seen in the Papanicolaou smears

were difficult to observe and identify.

It would be more valuable for clinical screening to

make two slides at the time the Papanicolaou smear is made,

just as in this study, and stain one by the Burke-

Kopeloff-Beerman modification of the gram stain than it

would be to use the Papanicolaou stain to identify diplo-

cocci. Although this does not directly incorporate gon-

orrhea screening into an already routine procedure, it

would provide a quick technique to be performed during

the same sampling procedure. Praiser (59) and Phillips

(60) showed, in clinical diagnosis, between 24 and 31%

of the positive feraale gonorrhea cases tested v/ere

culture positive but were negative using gram staining

of cervical smears. Thus it is indicated that a raore

sensitive screening test is necessary. Data from these

investigations indicate that screening Papanicolaou

smears is not more sensitive technique, as suggested by

Heller (25). Although gram staining of cervical smears

appears more useful than the Papanicolaou staining

procedures, a more valuable microscopic tool should

involve specific fluorescent antibody procedures (65).

Also from the studies presented here, no practical

56

method was found for incorporating into the gram stain

a test for the enzyme oxidase. No location in, or

modification of, the procedure was successful in

detecting oxidase. Since the enzyme is normally detected

on the cell surface or extracellularly, the washing

procedure of the gram staining procediire tends to remove

most of the enzyme. If the enzyme was present on the

cell surface and reacted with the indicator, the cell

would most likely have been darkened, thus masking the

proper gram reaction. Microscopic techniques using

oxidase for confirmation of the presence of Neisseria

gonorrhoeae does not appear practical.

CHAPTER V

SUMMARY

This study demonstrated that in vitro Neisseria

gonorrhoeae can utilize a wide variety of compounds as

its primary carbon/energy source. The organism used

glucose, lactate and pyruvate with excellent growth.

Good growth also was obtained with sucrose as the carbon/

energy source. Other compounds found usable as carbon/

energy sources in order of maximum growth werei raffinose,

malonate, arabinose, xylose, inositol, propionate,

peptone alone, fructose and succinate.

Fructose, when used in conj\Jinction with glucose as

the carbon/energy source, was found to have a mild

inhibitory effect on the growth of Neisseria gonorrhoeae.

Propionate was found to be more inhibitory when used in

conjunction with glucose for the carbon/energy source.

A profound inhibition of the growth of Neisseria

gonorrhoeae results when propionate is added.

Preliminary studies on the dependance on temperature

for in vitro growth of Neisseria f!:onorrhoeae indicated

that the optimum temperature for growth on semi-solid

media utilizing most carbon/energy sources is near 35° C.

Growth with sorbitol, malonate, succinate, tartrate and

57

5Q

inositol was about the same at 30^ C and 350 C at 24 hours.

This implied that for these carbon/energy sources the

optimum may be slightly lower or have a broader range

than is reported for media with glucose.

With a medium utilizing glucose as the primary

carbon/energy source, Neisseria gonorrhoeae underwent

autolysis within 2-3 days. In these studies, it was

demonstrated that the use of sucrose, pyruvate or

lactate as the primary carbon/energy source in semi-solid

media prevented autolysis for about 5 days. This pro-

longed viability would provide small clinics and

laboratories the ability to collect samples on Friday

and read them on Monday without reducing diagnostic

accuracy. The lag and log growth phases are nearly

identical for in vitro growth with glucose, sucrose,

pyruvate and lactate, so there would be no loss in

diagnostic accuracy if the culture were read at shorter

intervals.

This study also demonstrated the effects of the

inhibitor V-C-N on in vitro growth of Neisseria

gonorrhoeae indicating that concentrations greater than

5 ml/liter in liquid growth media cause significant

reduction in maximum growth.

During frozen storage studies, it was deterrained

that Neisseria ^onorrhoeae can be stored at -90° C in

Skim Milk raedium without the addition of glycerine or

59

any other additives for prolonged periods with good

survivability.

The Papanicolaou smear was shown not to be a

valuable tool for the detection of gonorrhea in women. *

Bacteria in Papanicolaou smears are poorly stained and

difficult to identify. Two slides made from cervical

scrapings, one stained by Papanicolaou*s method for

cervical carcinoma and one stained by the Burke-Kopeloff-

Beerman modification of Gram's stain would provide a

more useful technique for carcinoma and gonorrhea

screening. The gram stain indicates the presence of

diplococci intracellular in white blood cells much more

clearly, and is a simple, quick procedvire.

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••mmmv^

APPENDIX

70

71

APPENDIX

PROTOCOL FOR PREPARATION OF PAPANICOLAOU SMEARS

Introduction. This study is designed to evaluate

the efficacy of combining the routine Papanicolaou smear

with a microscopic examination for Neisseria gonorrhoeae.

It is felt that the routine nature of the Papanicolaou

Test makes it an ideal time for simultaneous screening

for gonorrhea. These trials will serve as data for the

development of rapid, easily performed screening test in

asymptomatic OB patients.

Suggested Procedure. You will be given packets

of two slides, one labelled PAP and one G. For female

patients presenting themselves for gonorrhea testing,

an additional two specimens will be taken. Using stand-

ard procedures for taking cervical smears with a wooden

"mutton-bone" spatula, collect two samples and alternat-

ely place them on the two glass slides. Before the

sample on the slide labelled PAP can dry, spray with a

cytology fixative. Allow the G slide to air dry. Place

both slides back into the cardboard packets and place in

the refrigerator. Slide packets should be labelled v ith

an identification number corresponding to the patient so

culture results can be compared with the microccopic

findings.

/