razlog_radmila_1999_masters degree in technology homoeopathy in health and biotechnology

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Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujdigispace.uj.ac.za (Accessed: Date).

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A STUDY TO DETERMINE THE EFFECT OF HOMOEOPATHIC

BAPTISIA TINCTORIA (3CH, 15CH AND 30CH) AND

THUJA OCCIDENTALIS (3CH, 15CH AND 30CH) ON THE GROWTH

PRODUCTION OF STREPTOCOCCUS PYOGENES AND

CANDIDA ALBICANS RESPECTIVELY

By

Radmila Razlog

Student number: 9477637

A dissertation submitted in partial fulfilment for the Master's Degree in Technology,

Homoeopathy, to the Facultyof Health and Biotechnology at the Technikon

Witwatersrand, Johannesburg.

Submission approved for Examination:

Specialist Supervisor:

Mr. A van den Berg

Supervisor:

Dr. B.R van Olden

\ A

-,

Date ofsubmission:

DECLARATION

I, Radmila Razlog, declare that this work is original, that it has been conducted

without assistance and that it has not been submitted to any other institution for the

purpose ofobtaining a qualification.

···················o·········v···Radmila Razlog: Candidate Date

ii

Dedicatedto myparents, family and close friends for all their support and

encouragement

iii

ABSTRACT.

The aim of the study was to determine the effect of homoeopathic Baptisia tinctoria

(Jch, 15ch and 30ch) and Thuja occidenta/is (Sch, 15ch and 30ch) on the growth

production ofStreptococcus pyogenes and Candida albicans, It was hypothesised that

the homoeopathic treatment would decrease the growth production of these

organisms. Hence, it could possibly be used as complementary treatment in immuno

compromised, neonates, debilitated or chemotherapy patients for the successful

treatment of infections caused by the above organisms.

The experiments attempt to demonstrate the effects of homoeopathic remedies

Baptisia tinctoria and Thuja occidenta/is, in various potencies, on S. pyogenes and

C. a/bicans, being guided in the experiments by the similarity between the remedies

used and the respective microorganisms' disease pictures (54). The experiments were

conducted on twenty different strains of both S. pyogenes and C. a/bicans, which

were obtained from Lancet Pathology Laboratory. Pure cultures were then

subcultured aseptically. All experimentation was conducted in triplicate to order to

eliminate laboratory error. The two experiments conducted were the disc diffusion

and broth dilution methods. Discs impregnated with the homoeopathic remedies were

placed on agar, which had been inoculated with S. pyogenes and C. albicans

respectively. Zone diameters were then measured after 24 hours incubation. The broth

dilution method was used to determine which of the homoeopathic potencies were

fungistaticlbacteriostatic respectively and which were fungicidal/bacteriocidal

respectively, by measuring the optical density readings ofthe broths.

All the data obtained by the researcher during the experiment was then interpreted by

a two-way analysis ofvariance.

iv

, ACKNOWLEDGEMENTS

I express my sincere gratitude to the undermentioned for their assistance in the

execution ofthe study and the preparation ofthis dissertation.

Mr. Alan van den Berg '" .

Dr. Brian van Olden .

Mr. Alastair Campbell .

Mr. Neil de Villiers '" .

Mr. Greg Khoury .

Mrs. Michelle Knowles ,. '" '" .. , '" .

Mr. Miguel Ribeiro , .

Specialist Supervisor

Supervisor

Laboratory Technician

Statistics

Medical Technologist

Secretary ofBiotechnology

Proofreader

v

TABLE OF CONTENT

TITLE PAGE

DECLARATION

DEDICATION

ABSTRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENT

LIST OF FIGURES

LIST OF TABLES

n

111

IV

V

VI

Xl

xu

1.0

1.1

1.2

1.2.1

1.2.2

1.3

1.3.1

1.3.2

1.4

1.4.1

1.4.1.1

1.4.1.2

1.4.2

1.4.2.1

1.4.2.2

1.5

1.5.1

1.5.2

1.6

1.6.1

INTRODUCTION

Objective ..

Streptococcus pyogenes .

Morphology and Structure .

Pathogenesis and Pathology .

Candida albicans .

Morphology and Structure , .

Pathogenesis and Pathology .

Conventional Treatment .


Penicillin .

Toxicity/Side effects ofPenicillin .

Candida albicans .

Nystatin .

Toxicity/Side effects ofNystatin .

Drug Resistance .

Streptococcuspyogenes .

Candida albicans .

Bomoeopathy " .

SimiliaSimilibus Curentur .

vi

1

1

2

2

2

3

3

4

6

6

6

7

7

7

8

9

9

10

10

10

1.6.2

1.6.2.1

1.6.2.1.1

1.6.2.1.2

1.6.2.1.3

1.6.2.1.4

1.6.2.1.5

1.6.2.1.6

1.6.2.1.7

1.6.2.2

1.6.2.2.1

1.6.2.2.2

1.6.2.2.3

1.6.2.2.4

1.6.2.2.5

1.6.2.2.6

1.6.2.2.7

1.6.3

2.0

Homoeopathic Treatment , .

Baptisia tinctoria .

Source '" , .

Description and Habitat .

Parts Used ..

Prover

Appearance

Indications

Herbal Preparation

Thuja occidentalis

Source

Description

Parts Used

Prover .

Appearance , ,

Indications ,

Herbal Preparation .

Homoeopathic Philosophy .

MATERIALS AND METHODS

11

11

11

12

12

12

12

12

13

13

13

13

14

14

14

14

14

15

16

2.1

2.1.1

2.1.1.1

2.1.1.2

2.1.2

2.1.2.1

2.1.2.2

2.1.3

2.1.3.1

2.1.3.2

Materials , 16

Stock Cultures , 16

Streptococcus pyogenes , ... .. . ... ... ... ... ... .. . ... ... .. .. 16

Candida albicans . .. .. . ... . .. ... . .. .. . .. . . .. . .. . .. .. . . .. .. .. 16

Media 16

Streptococcuspyogel1es :....... 16

Candida albicans , . .. . .. .. . .. . ... . .. .. . .. . 16

Allopathic Medication .. . .. ... .. . . .. . .. . .. ... ... . .. . .. .. . . .. .. . . .. .. . .... 17

PenicillinG , , 17

Nystatin , 17

vii

.....................................

2.1.3.3

2.1.4

2.1.4.1

2.1.4.2

2.2

2.2.1

2.2.1.1

2.2.1.2

2.2.1.3

2.2.1.4

2.2.2

2.2.2.1

2.2.2.2

2.2.2.3

2.2.3

2.2.3.1

2.2.3.2

2.2.4

2.2.4.1

2.2.4.2

2.2.4.3

2.2.5

2.2.5.1

3.0

3.1

3.2

3.2.1

Bacitracin

Homoeopathic Treatment .

Baptisia tinctoria .

Thuja occidenta/is , .

Methods .

Preparation ofMedia .

Blood Agar for Streptococcus pyogenes ..

Sabouraud'sDextrose 4% Agar for Candida a/bicans ..

SerumBroth .

NutrientBroth .

Maintenance of Organisms .

Streptococclispyogenes , .

Candida a/bicans ..

Broth Subculturing .

Determination of Standard Curve .

SerialDilutions and Plating , .

Spectrophotometer Readings of Serial

Dilutions of Broths .

Disc Diffusion Method .


Candida a/bicans .

Zone measurement

Broth DilutionMethod .

Spectrophotometer Readings ..

RESULTS .

Standard Curve ..

Determination of Antimicrobial Activity .

Results of Streptococcus pyogenes and

Baptisia tinctoria treatmentgroups

viii

17

17

17

17

18

18

18

19

19

19

20

20

20

21

21

22

23

23

24

24

24

25

25

26

26

27

28

\

\

3.2.2

3.2.3

3.2.3.1

3.2.3.2

3.2.4.1

3.2.4.2

3.2.5

3.2.5.1

3.2.5.2

4.0

4.1

4.2

4.2.1.1

4.2.1.2

4.3.1

4.3.1.1

4.3.1.2

4.3

4.3.1

4.3.2

Results of Streptococcus pyogenes and Thuja

occidentalis treatment groups

Results ofDisc Diffusion Method

Streptococcus pyogenes disc diffusion method

treated with Penicillin G, Bacitracin and Baptisia

tinctoria

Streptococcus pyogenes disc diffusion method

treated with Penicillin G, Bacitracin and Thuja

occidentalis

Results of Candida albicans and Baptisia tinctoria

treatment groups

Results ofCandida albicans and Thuja occidentalis

treatment groups

Results ofDisc Diffusion Method

Candida albicans disc diffusion method treated

with Nystatin and Baptisia tinctoria

Candida albicans disc diffusion method treated

with Nystatin and Thuja occidentalis

DISCUSSION

Standard Curve

Broth Dilution Method

Baptisia tinctoria treatment groups .

Thuja occidentalis treatment groups .

Candida albicans .

Baptisia tinctoria treatment groups " .

Thuja occidentalis treatment groups .

Results ofDisc Diffusion Method .

Streptococcuspyogenes .

Candida albicans ..

ix

31

34

34

36

39

42

45

45

47

51

52

53

53

53

54

54

54

55

55

56

5.0

5.1

5.2

6.0

7.0

CONCLUSION

In Conclusion

Recommendation

APPENDICES

REFERENCES

x

57

57

57

59

85

LIST OF FIGURES

Fig 1.1

Fig 1.2

Fig 3.1

Fig 3.2

Fig 3.3

Fig 3.4

Fig 3.5

Fig 3.6

Fig 3.7

Fig 3.8

Fig 3.9

Fig 3.10

Basic structure ofPenicillin G .

Basic structure ofNystatin .

A graphic representation of the standard curve

correlating OD readings and cfu/ml ofS. pyogenes

A graphic representation of the standard curve

correlating OD readings and cfu/ml ofC. a/bicans

Bar graph showing the cfu/ml of S. pyogenes, in broth

dilution, when treated with Baptisia tinctoria and no

medication

Bar graph showing the cfu/ml of S. pyogenes, in broth

dilution, when treated with Thuja occidentalis and no

medication.

Bar graph of disc diffusion method displaying zone

diameters on S. pyogenes treated with Penicillin G,

Bacitracin and Baptisia tinctoria.


diameters on S. pyogenes treated with Penicillin G,

Bacitracin and Thuja occidenta/is.

Bar graph showing the cfu/ml of C. albicans, in broth

dilution, when treated with Baptisia tinctoria and no

medication.

Bar graph showing the cfu/ml of C. a/bicans, in broth

dilution, when treated with Thuja occidentalis and no

medication.


diameters on C. a/bicans treated with' Nystatin and

Baptisia tinctoria.


diameters on C. a/bicans treated with Nystatin and

Thuja occidenta/is.

xi

6

7

26

27

28

31

34

37

39

42

45

48

LIST OF TABLES

Table 3.1 ANDVA table of broth dilution displaying control and

Baptisia tinctoria treatment groups on the growth of

S. pyogenes reflecting mean values, standard deviations,

standard errors and confidence intervals. ......... 29

Table 3.2 The difference in median values of S. pyogenes treated

with Baptisia tinctoria. ......... 30

Table 3.3 Comparison of Baptisia tinctoria treatment groups,

difference ofranks and P-values ofS. pyogenes. ......... 30

Table 3.4 ANDVA table of broth dilution displaying control and

Thuja occidentalis treatment groups on the growth of

S. pyogenes reflecting mean values, standard deviations,

standard errors and confidence intervals. ......... 32


with Thuja occidenta/is. ......... 33

Table 3.6 Comparison of Thuja occidentalis treatment groups,

difference ofranks and P-values ofS. pyogenes. .......... 33

Table 3.7 ANDVA table of disc diffusion of S. pyogenes treated

with Penicillin G, Bacitracin and Baptisia tinctoria

displaying mean values, standard deviations, standard

errors and confidence intervals. ......... 35


with Penicillin G, Bacitracin and Baptisia tinctoria .......... 35

Table 3.9 Comparison of Baptisia tinctoria treatment groups and

Penicillin G displaying difference of ranks and P-values

ofS. pyogenes. ......... 39

Table 3.10 ANDVA table of disc diffusion of S. pyogenes treated

with Penicillin G, Bacitracin and Thuja occidentalis

displaying mean values, standard deviations, standard

errors and confidence intervals. ................ 38

xii

Table 3.11

Table 3.12

Table 3.13

The difference in median values ofS. pyogenes treated

with Penicillin G and Thuja oecidenta/is.

Comparison of Thuja occidenta/is treatment groups and

Penicillin G displaying difference of ranks and Psvalues

ofS.pyogenes.

ANOVA table of broth dilution displaying control and

Baptisia tinetoria treatment groups on the growth of

C. a/bieans reflecting mean values, standard deviations,

standard errors and confidence intervals.

Table 3.14 The difference in median values of C. albicans treated

with Baptisia tinetoria.

Table 3.15 Comparison of Baptisia tinetoria treatment groups,

difference of ranks and P-values ofC. albieans.

Table 3.16 ANOVA ofbroth dilution displaying control and Thuja

occidentalis treatment groups on the growth of

C. a/bieans reflecting mean values, standard deviations,

standard errors and confidence intervals.

Table 3.17 The difference in median values of C. albicans treated

with Thuja oecidenta/is.

Table 3.18 Comparison of Thuja oeeidenta/is treatment groups,

difference ofranks and Pvvalues ofC. albicans.

Table 3.19 ANOVA table of disc diffusion of C. a/bieans treated

with Nystatin and Baptisia tinetoria displaying mean

values, standard deviations, standard errors and

confidence intervals.

Table 3.20 The difference in median values ofC. a/bieans treated

with Nystatin and Baptisia tinetoria.

Table 3.21 Comparison of Baptisia tinetoria treatment groups and

Nystatin displaying difference of ranks and P-values of

C. a/bicans.

xiii

39

39

40

41

41

43

44

44

46

47

47

•

Table 3.22 ANOVA table of disc diffusion of e. albicans treated

with Nystatin and Thuja occidentalis displaying mean

values, standard deviations, standard errors and

confidence intervals.

Table 3.23 The difference in median values ofC, albicans treated

with Nystatin and Thuja occidentalis.

Table 3.24 Comparison of Thuja occidentalis treatment groups and

Nystatin displaying difference of ranks and P-values of

e. albicans.

xiv

49

49

50

'1.0 INTRODUCTION

Microorganisms are ubiquitous in and on the human body (1). From birth, people live in

a microbial biosphere composed of innumerable microorganisms representing types,

variants, strains and species (2). These temporary habitats of the dynamic microbial

environments include the larynx, trachea, bronchi, accessory nasal sinuses, oesophagus,

stomach, upper portion of the small intestines, upper urinary tract and the corresponding

distal areas of the male and female genital organs (2,5). The persistent finding of

numerous microorganisms in these inhabited areas of blood or in other sterile sites

provides a reliable marker for the imaginary line between health and disease (3). If an

individual becomes immuno-compromised, debilitated, or requires a drug regimen or

chemotherapeutic treatment, develops diabetes or acquires a prosthesis, the tendency to

develop infection caused by microorganisms is increased (2,4,7). These infections are

then treated with conventional drugs, but due to the use and abuse of antibiotics we find

ourselves having to contend with drug resistant organisms, allergic reactions and side

effects. The approach offered by homoeopathy is one of stimulation of the immune

system and provides a safe non-toxic alternative to the treatment of infectious diseases

(54).

1.1 Objective

The objective of this study is to determine the effect of homoeopathic treatments

Baptisia tinctoria (3ch, 15ch and 30ch) and Thuja occidentalis (3ch, 15ch and 30ch) on

the growth production of Streptococcus pyogenes and Candida albicans respectively.

The study will determine whether or not these homoeopathic substances display

antibacterial and/or antifungal properties.

1

1.2 Streptococcus pyogenes

1.2.1·Morphology and Structure

Streptococci are gram positive, chain-forming bacteria (6,8,9). They belong to the group

A f3-haemolytic streptococci, and are the main human pathogens associated with local

or systemic invasion and poststreptococcal immunological disorders (7). Phenotypic

criteria for classification of streptococci includes Lancefield serology and haemolytic

reactions (2). Strains of S. pyogenes typically produce clear zones of haemolysis, after

24h of incubation on blood agar, with large colony formation greater than O.5mm in

diameter (6,7). Colonies vary in colour from grey to whitish and usually glisten.

S. pyogenes is also PYR-positive (hydrolysis ofL-pyrrolidonyl-2-naphthylamide) and is

usually susceptible to Bacitracin (7,9).

1.2.2. Pathogenesis and Pathology

Streptococci colonise the skin and mucous membranes and can be isolated as part of the

normal flora of the alimentary, respiratory and genital tracts (2). A variety of distinct

disease processes are associated with S. pyogenes infection (7). The biologic properties

of the infecting organism, nature of the host's response and portal of entry all greatly

influence the pathological picture (7). The numerous virulence factors of S. pyogenes

allow it to produce a wide array of serious infections, including pharyngitis, upper

respiratory tract infections, wound and skin infections (impetigo, erysipelas), soft tissue

infections, necrotising fascitits, meningitis, puerperal sepsis, arthritis and Sydenham's

chorea (2,6,7,9). Infection with toxin-producing strains can result in Scarlet fever or more

serious toxic shock-like syndromes (6). Late complications include rheumatic fever,

endocarditis and acute glomerulonephritis (5,8). Septicaemia is a clinical .syndrome

whereby the microorganisms actively multiply in the bloodstream and may be as a

result ofS. pyogenes infection (6,9).

2

In most geographical regions S. pyogenes remains the major pathogenic bacterium'I

involved in throat disease, commonly referred to as strep sore throat (2,5,7). When it is

detected it is imperative to initiate therapy and prevent sequelae (2). S. pyogenes

pharyngitis is acquired by inhaling aerosols from an infected person, or less commonly,

by eating food contaminated with S. pyogenes (5). The mucous membrane of the throat

is protected by the washing action of saliva; hence bacteria which colonise the throat

must be able to adhere to the mucosal cells (5). Virulent S. pyogenes adhere to the

pharyngeal epithelium by means of lipoteichoic acid covering surface pili (7).

Streptococcus pharyngitis, in older children and adults, is characterised by pharyngeal

pain, inflammation and erythema with purulent exudate accompanied by fever and

anterior cervical adenopathy (4,7). With the most intense inflammation, tissues may

break down and form peri-tonsillar abscesses or Ludwig's angina, where massive

swelling of the floor of the mouth blocks air passages (7). In infants and small children,

the sore throat occurs as a sub-acute nasopharyngitis with a thin serous discharge and

little fever but with a tendency of the infection to extend to the middle ear, mastoids and

meninges (2,7). Suppurative sequelae of pharyngitis may result from the spread of

infection to contagious tissue or from bacteremic dissemination. Non-suppurative

sequelae include rheumatic fever and acute glomerulonephritis (2,4,5).

1.3 Candida albicans

1.3.1 Morphology and Structure

C. a/bicans is an oval, budding yeast, 2-3 x 4-6 nanometres, that produces pseudo

hyphae in culture, tissue and exudates (7). The heterogeneous genus Candida belongs to

the Cryptococcaceae family within the division Deutermycetes (Fungi Imperfecti).

Microscopic observation ofC. albicans will reveal short hyphae that are not constricted

at the junction of the blastoconidium and the germ tube (2). On Sabouraud'sagar

incubated at room temperature soft, white-to-cream coloured, round colonies with

unbroken edges and a yeast odour develop (7). Several species of Candida, most

noticeably C. albicans, are diploid (16,17).

3

1.3.2 Pathogenesis and Pathology

C. albicans is the specie most commonly isolated from patients with nearly all forms of

candidiasis (2). C. albicans was once thought to be non-pathogenic, but has now been

considered as the most common cause of fungal infections (l0,11). Contributing to its

high association with disease is its high prevalence in the healthy population (2). It is a

member of the normal flora of the mucous membranes and often colonises the mouth,

respiratory, gastro-intestinal-and female genital tract of healthy individuals (4). In such

locations it may gain dominance, by overgrowth and/or invasion, and be associated

with pathogenic conditions (2). The higher incidence of Candida infections has been

attributed to the increased use of antibacterial agents, the HIVIAIDS pandemic, and the

rapidly expanding number of chemically induced immuno-suppressed and oncology

patients (20,21,22). As a result of improved management protocols, HWIAIDS, cancer,

and transplantation populations now survive longer and become highly susceptible to

life threatening fungal infections (23). Lesions in the throat caused by C. albicans

accompany a variety of drug regimens (overuse of broad-spectrum antibiotics) and may

also occur in patients with debilitating and neoplastic diseases, chemotherapy, immuno

suppression and neonates; and commonly designates as thrush. Thrush may involve the

oral mucosa (Candida stomatitis) or female genital tract (Candida vaginitis) and

presents with small white, irritating, itchy curd-like plaques (11). The ability of

C. albicans to form hyphae is believed to be important for colonisation and invasion of

the oral and vaginal mucosa (10). The oral mucosa and tongue plaques are surrounded

by a margin of erythema and may bleed (5,30). Chronic mucocutaneous candidiasis

(CMC) is characterised by chronic and recurrent infections of the skin, mucous

membranes and nails (18). Oral thrush is often the first manifestation of CMC. Many

afflicted patients have associated endocrinopathy or autoimmune phenomena. The

primary immune defect usually involves either the thymus-dependent lymphocytes or

the monocyte/macrophage system (18,19). Antifungal therapy is only palliative in CMC,

and because of the high incidence of morbidity and mortality from noncandidal

infections, it is clear that further research efforts should be continued toward the goal of

restoring immunocompetence in these patients (19). Mucocutaneous oral candidiasis has

4

· garnered much attention because an especially noxious form that affects the mouth,,

tongue and oesophagus is very common amongst patients infected with HIV. In general,

it appears that granulocytopaenia or defective phagocyte function promotes systemic

disease, whereas reduced T-cell/macrophage function promotes mucocutaneous disease

(19). Oral candidiasis is an opportunistic infection, considered as a defining illness for

HIVIAIDS (13,14), the oesophageal candidiasis manifesting clinically as pain or

difficulty on swallowing (4,8). It may present in the prodromal stages of HIVIAIDS or

may accompany later stages, infecting 80% of patients at some time during their illness

(8).

Yeast are emerging as important 'hospital acquired' pathogens (nosocomial), and add

significantly to the morbidity, mortality and economic burden caused by the underlying

disease alone (25,26,27). The attributable mortality associated with bloodstream

infections resulting in septicemia caused by Candida has increased significantly (15). In

survivors, candidemia also prolongs the length of hospital stay by 30 days above that

required treating the patients' underlying conditions (26,27). Other underlying conditions

that predispose C. albicans infection include burns, trauma, malnutrition, diabetes,

neutropeunia, pregnancy, renal failure, haemolytic malignancies high oestrogen levels

or organ transplantation (2,4). Apart from bloodstream invasion C. albicans may

produce thrombophlebitis, endocarditis, infection of the eyes (7,11), perineal contact

dermatitis in nappy-wearing infants (6) or virtually infect any tissue or organ when

introduced intravenously (intravenous catheter or intravenous drug abuse) (4,7).

C. albicans appears to possess a number of virulence attributes that may promote

successful parasitism causing a variety of superficial infections of moist epidermal

surfaces, most commonly the sub-mammary folds and the groin (2,7). C. albicans

infection may be a secondary invader of the lungs, kidneys and other organs where a

preexisting disease is present (11).

5

1.4 Conventional Treatment

1.4.1 Streptococcuspyogenes

1.4.1.1 Penicillin

The penicillins are derived from moulds ofthe genus Penicillium, and are obtained from

the extraction-of submerged cultures grown in special medium. All the penicillins have

the same basic structure (7). The basic nucleus ofpenicillin is 6-aminopenicillanic acid,

which consists of a thiazolidine ring linked to a B-Iactamgroup. This latter ring carries

a secondary amino group. The side-chain substituents at R1 determine the main

antibacterial and pharmacological characteristics of each particular penicillin (12).

o S1\ »<: /CH3

R-C-NH-CH-CH C

I I I"CH3

O=C-N-CH-COOH

Side chain (R)

</-CHf

Fig. 1.1 Basic structure ofPenicillin G .

Penicillin G has the highest activity against gram positive bacteria, and is the most

widely used natural penicillin (2,7). Depending on the concentration of the drug at the

site of infection and the susceptibility of the infectious organism, Penicillin G is either

bacteriostatic or bactericidal. Penicillin G has the ability to inhibit bacterial cell wall

synthesis through inhibition of a. membrane bound transpeptidase enzymes, thereby

preventing cross-linkage of peptidoglycan chains, which are necessary for bacterial cell

wall strength and rigidity (31,33). Weakening of the cell wall and consequent lysis

results in cell death (37,40).

6

Penicillin is distributed throughout most of the body and passes the placental barrier. It ..is effective against young rapidly dividing organisms and has little effect on mature

resting cells (31). Antimicrobial drugs have no effect on established glomerulonephritis

and rheumatic fever (1).

1.4.1.2Toxicity/Side effects of Penicillin.

Penicillin is a potent sensitizing agent (31). Common reactions to penicillin include

allergic skin rashes, exfoliative dermatitis, erythema multiforme (rarely Stevens

Johnson's Syndrome (9) ), hives, pruritis and wheezes (2,30,31). Diarrhoea, abdominal

cramps, nausea and vomiting, bloating, flatulence and fever are also possible side

effects (35). Renal and hepatic complications may also occur. Hypersensitivity reactions

are reported to be on the increase in paediatrics (31). Sensitivity reactions may be

immediate (within 20 minutes) or delayed (as long as several days or weeks after

initiation of therapy) (30,31). Severe anaphylactic reactions, which can be

fatal, may occur in previously sensitised patients re-challenged with penicillin.

Cases of laryngeal oedema, laryngospasm, prostration, bronchospasm, hypotension,

vascular collapse and death have also been reported (29,31). Penicillin G can cause

neurotoxicity including myoclonic twitching, neuromuscular irritability, seizures and

hallucinations (31,33). All the penicillins may cause interstitial nephritis on an allergic

basis. Neutropaenia, thrombocytopaenia, bone marrow depression, and anaemia are

other rare complications of penicillin treatment (2,31). Other adverse effects include

super-infection of the oropharynx with C. albicans, particularly in the debilitated,

immuno-compromised and elderly (31,34).

1.4.2 Candida albicans

1.4.2.1 Nystatin

Nystatin is a natural polyene, antifungal antibiotic derived from Streptomyces noursei

and is both fungistatic and fungicidal against all species of Candida (29,41). It is

indicated for Candida infections of the skin, gastrointestinal tract, vagina and mouth (6).

Structurally, it is a tetraene linked to the amino sugar mycosamine (12) (see fig. 1.2).

7

OH

oyOyCH3

HoyOHNH:

Fig. 1.2 Basic structure ofNystatin

Its structure and mode of action in similar to that of Amphotercin B. The polyene

antibiotics include about fifty antifungal treatments produced by Streptomyces species.

Nystatin and Amphotericin B are the only two used clinically (29). Nystatin acts by

binding sterols and disrupting membrane function (6). It binds to the fungal cell

membranes resulting in altered cellular permeability and leakage ofpotassium and other. .

essential intracellular components (31).

1.4.2.2 Toxicity/Side effects ofNystatin

Nystatin has few toxic symptoms, but side-effects include gastrointestinal symptoms

namely epigastric distress, nausea and vomiting, diarrhoea and other rare irritations (31).

A principaladverse effect is nephrotoxicity and hypokalaemia. Fever, chills and rigor

may follow administration of Nystatin (18). It is too toxic for systemic use but it is

useful against mucocutaneous candidiasis (29). Nystatin is not absorbed from the

gastro-intestinal tract, so oral use is useful for alimentary tract infection and local

application for oral thrush or vaginitis (29,30). Unabsorbed Nystatin is excreted in the

faeces (31).

8

1.5 Drug Resistance

Antibiotics have reduced the mortality of infectious diseases but not the prevalence of

certain infections, namely those caused by S. pyogenes and C. albicans (2,32).

Antimicrobial resistant organisms will becomemore prominent during the 1990's, but

the impact of these resistant organisms is difficult to qualify (38). Antibiotic resistance

severely limits the number of agents that physicians can use to treat infections (36,37).

To circumvent antibiotic resistance, clinicians may be forced to use antimicrobial agents

that are more expensive, more toxic, or less efficacious than standard drugs (32).

Furthermore, infections caused by resistant organisms may become difficult or

impossible to treat; hence, morbidity and mortality rates, length of hospital stay, and

cost may be increased (26,39). To meet the challenges and responsibilities of this unique

position effectively it is necessary to assess and if necessary, to redesign our strategy

(39). Thus, flexibility in the selection of treatment may be necessary (42).

1.5.1 Streptococcus pyogenes

Group A 6-haemolytic streptococci are usually sensitive to Penicillin G. However,

antimicrobial resistance is becoming widespread among a variety of clinically

significant bacterial species (32). Use and abuse of antimicrobial agents encourages the

evolution of bacteria towards resistance, often resulting in therapeutic failure (2, 36).

This evolution is due to the emergence of changing resistance mechanisms and to the

spread of well characterised mechanisms of resistance to the majority of bacterial

species (2,6,7). Bacterial resistance can be intrinsic or acquired. Intrinsic delineates the

spectrum of activity of the antibiotic (2). Treatment failure has been ascribed to

tolerance, a situation in which the bacterial growth is inhibited by penicillin, but the

drug's bactericidal activity is greatly reduced (7). Acquired resistance results from the

mutation in a gene located in the host chromosome or a plasmid or from acquisition of

new genetic material (2,4). Another possible explanation for treatment failure, especially

in pharingitis, is the production of an enzyme penicillinase (fJ-/actamase) producing

9

bacteria at the site of infection (2,7). It can hydrolyse the B-Iactam ring ofpenicillin to,form penicilloic acid, a substance that has no antibacterial activity (29).

In spite of reports of resistance in certain isolates, Penicillin G remains the drug of

choice for treatment of infection caused by S. pyogenes (29,31,35). Uncertainty about the

efficacy ofpenicillin alone for effective treatment of life threatening infections has led

to the use of synergistic combination of drugs, although effective, but with many side

effects (31). Strains displaying resistance to penicillin only, as well as to penicillin and

to other antibiotics has been encountered for S. pyogenes infections (2,31).


Systemic fungal infections continue to present major problems due to clinical

resistance, microbiological resistance, emergence of new pathogens and involvement of

more immunocompromised patients. Polyene-resistant Candida is due to decreased or

absent ergosterols in the fungal cell membranes. Resistant C. albicans organisms are

uncommon but have been described (18). Clinical resistance to Nystatin is defined as the

absence of clinical response to the treatment. Clinical resistance develops especially in

patients with profound immunosuppression or advanced fungal lesions (19).

1.6 Homoeopathy

1.6.1 Simi/ia Similibus Curentur

Homoeopathy is a therapeutic method that clinically applies the Law of Similars and

uses medicinal substances in weak or infinitesimal doses (43,45,53). The Law ofSimilars

was first recorded by Hippocrates and developed into a system of medicine by Dr

Samuel Hahnemann, which states as follows "Similia SimitibusCurentur" translated as

'let likes be cured by likes' (49,54). A pharmacological substance causes a set of

symptoms characteristic of this substance when the said substance is administered to a

healthy person; this experiment is referred to as a proving and the symptoms obtained

are referred to as pathogenetic symptoms (43). When the symptom picture produced by

10

the diseased patient is matched to the remedy that produces a similar symptom picture.in a healthy person, cure is initiated (52). Homoeopathy acts together with the body's

reactions and stimulates the defence mechanisms ofthe body so as to make the response

to the given stimulus more effective (46). Hence, the fundamental principle of the Law

ofSimilars states the parallel action between the toxicological action of a substance and

its therapeutic action to establish resonance between patient and remedy (43,45).

Hahnemann used minimal doses of medicine prepared according to his own method of

potentisation. He demonstrated that the more diluted and dynamised (by the process of

succussion) a remedy is, the more efficacious and penetrating in its action it will be and

that the quantity of drug required is inversely proportional to its similarity with the

symptoms ofthe patient (45).

Laboratory research is able to demonstrate the biological activity of homoeopathic

remedies. It is therefore possible to access changes on microorganisms that cannot be

explained as a placebo response. However, in order to restore the patient to health in the

most rapid and gentle manner, a remedy must be selected that concentrates on similar

symptoms.

1.6.2 Homoeopathic Treatment

1.6.2.1 Baptisia tinetoria

The native Americans, who also used it as a dye, first discovered Baptista tinctoria's

medicinal properties. Hence, the name Baptista, from the Greek word meaning "I dip or

immerse" (46). Before the discovery of antibiotics Baptisia was the remedy prescribed

for cases oftyphoid fever (46,48,55). Baptisia tinetoria in low potencies produces a form

of antibodies, agglutinins, to the typhoid bacillus. Thus it raises the natural bodily

resistance to the invasion of the bacillary intoxication, which produces the typhoid

syndrome (48) .

1.6.2.1.1 Source

Baptisia tinetoria is native to United States and Canada, and is a member of the

Leguminosae family. The plant is also commonly referred to as Wild Indigo (46,47).

11

1.6.2.1.2 Description and Habitat, .The plant is found growing in dry woods or fields, often in a hilly region. The plant

bears yellow flowers on a spike; the root is fleshy and brown to black in colour. The

whole plant emits an unpleasant odour if bruised (50).

1.6.2.1.3 Parts Used

The fresh root, including the bark of the plant, is used to prepare the mother tincture.

From the mother tincture further dilutions ofthe remedy are obtained (46,47).

1.6.2.1.4 Prover

Short provings of Baptisia tinctoria, made by seven persons, may be found in the fifth

and seventh volumes of the Northern American Journal of Homoeopathy (1857 and

1859) (50).

1.6.2.1.5 Appearance

The typical state of Baptisia tinctoria may be seen in serious infection. The face is

characterised by a drunken or besotted expression (44,48). Usually the remedy will

appear in septic conditions, but is also indicated in less severe infections especially

those accompanied with fever (51). Generally, there is a rapid onset of symptoms, with a

sensation ofbruised pains and the patient feels uncomfortable in any position (44,50).

1.6.2.1.6 Indications

Homoeopathic treatment Baptista tinctoria is indicated for dusky red inflammation or

ulcers in the throat (51). The mouth is ulcerated and in a foul state (50). The gums are

dark red or purple in colour and may ooze blood (48,55). The tongue may be coated

yellow, or brown down the centre with red glistening edges and is often studded with

aphthous ulcers. The tongue feels thick and burnt with slurred speech (48,51). There is

excessive secretion of thick ropy saliva. The patient cannot swallow liquids due to the

painful spasmodic phenomena in the throat and the oesophagus (50). Baptisia tinctoria is

also indicated for puerperal fever and a rapid onset of septic conditions (48,51)

12

1.6.2.1.7 Herbal Preparation; I

Wild Indigo is considered wherever there is a focused infection. It is especially useful in

the treatment of infection ofthe nose and sinuses. It is taken internally as a mouth wash

to heal mouth ulcers, gingivitis and help in the control of pyorrhea. Externally an

ointment will help infected ulcers. Its action is anti-microbial, anti-catarrhal, febrifuge,

hepatic and alterative. Systemically it may be helpful in the treatment of enlarged lymph

nodes and also reduces fever. (47).

1.6.2.2 Thuja occidentalis

.The generic name of this conifer tree, Thuja, is derived from the Greek word "thero"

meaning to fumigate or to sacrifice. Although Native Americans used the twigs and

leaves to treat malaria, coughs, gout and rheumatism, extracts of the tree have never

been used in orthodox medicine (46).

Most homoeopathic research conducted on Thuja occidentalis has been directed

towards the treatment of skin warts.

1.6.2.2.1 Source

Thuja occidentalis is a member of the Coniferae family. It is a smallish tree found

growing in cool, damp regions and long rocky banks of North America, England and

Italy (46,54).

1.6.2.2.2 Description

Thuja occidentalis, the so-called white cedar, or Arbor vitae Tree of Life, is an

evergreen conifer and not a true cedar (50). The wood of this tree is very resistant to

decay with a fragrant and light odour and hence the misnomer of it being a cedar. The

tree with its tapered and somewhat twisted trunk may reach the height of forty-five feet.

Along the stem are resinous callosities which look like oozing warts (54). The foliage is

very suggestive of the cypress with its green, adpressed, flattened and imbricated scales

(50).

13

L~.2.2.3 Parts Used

The mother tincture is prepared from the fresh green twigs of the tree which are

pounded into a pulp to make the homoeopathic remedy (46). Further dilutions are then

prepared in alcohol from the mother tincture (50).

1.6.2.2.4 Prover

Thuja occidentalis was proved by Hahnemann in 1819, and is said to have been one of

his most favourite remedies (46).

1.6.2.2.5 Appearance

In appearance the Thuja patient is fleshy and rounded with lax musculature and large

glands. Thin limbs, waxy greasy skin and irregular teeth are listed as characteristic in

the Thuja subject. The patient may generally look sickly (44,50).

1.6.2.2.6 Indications

Thuja occidentalis is indicated for a left-sided sore throat (50) and small pustules or

ulceration occurring on the lips and in the mouth (48). The mouth is very dry, feels as if

full of blisters or as if burnt (55). There is a raw feeling on the pharynx causing a

constant desire to swallow for relief. The tongue remains clean despite digestive upset,

but the tip ofthe tongue may be excessively sore with white blisters on the side close to

the root (44,48,50). Arbor Vitae has a specific antibacterial action and is indicated for

bleeding fungal growths (48,54).

1.6.2.2.7 Herbal Preparation

When used as a herbal extract Thuja has been found to cause severe side effects such as

uterine contractions leading to possible abortion in pregnant women. These side effects

however, are not observed when taken in homoeopathic preparation. Thuja's main

action is due to stimulating and alterative volatile oils. It has a role in the treatment of

uterine and skin complaints. It also acts as an expectorant and diuretic (47).

14

1.6.3 Homoeopathic Philosophy

The end result of treatment, according to the doctrine ofhomoeopathy, results in a strict

individualization of the treatment (54). It constitutes an individual terrain therapy and

the homoeopathic substance is a specific stimulus to the organism. Allopathy employs

the use of medicines that have a derivative action or action which is contradictory to the

patient's symptoms (43). According to the doctrine of homoeopathy, a disease is not a

living entity in the body nor a physical disturbance provoked by material causes. This

materialistic conception of disease inevitably leads to therapies which provoke the

disappearance of apparent causes, or of the products of the disease or of its ultimate

results, without correcting the individuals vital harmony (54). For homoeopathy, disease

is a vital reaction of a susceptible person when faced with a noxious or pathogenic

agent. The physical pathological disturbances are the last stages of a process which only

differs from the functional physiological disturbances by its intensity (45,52).

Homoeopathy does not choose to combat the causes of a disease, nor its ultimate results

or organic lesions, but tries to correct the disturbed vital energy (52,53).

Laboratory research will demonstrate the effects of homoeopathic remedies, in various

potencies, on different microorganisms, being guided in the experiments by the

symptom similarity between the remedies used and the disease picture. Therefore

research will determine whether homoeopathic substances have a dual role to play in

infection. Firstly, stimulating the ill person to combat the disease picture and to correct

vital harmony, and secondly, have a direct action upon the causative microorganism.

During this constructive relationship there should be no suppression, no introduction of

side-effects, no interference from masking medication and no failure to remove the

mechanical obstruction to cure (54). Homoeopathy teaches a philosophy of health,

disease and ofcure. According to that philosophy, human beings are not healthy if they

have only managed to eliminate the pathological symptoms but are still being disturbed

by strong emotional feelings. People are healthy only if they Jive in a harmonious state

ofmind and their organs are physiologically balanced (45).

15

2.0 MATERIALS AND METHODS• I

The following applied to all experiments conducted during this study:

[J Aseptic conditions/techniques were adhered to at all times

[J All experiments were conducted in vitro in triplicate

2.1 Materials

2.1.1 Stock Cultures

2.1.1.1 Streptococcus pyogenes

S. pyogenes was ordered from Lancet Pathology Laboratory. Twenty different isolates

were obtained and stored under refrigeration at 4-5°C.

2.1.1.2 Candida a/bicans

Twenty different isolates of C. albicans were ordered from Lancet Pathology

Laboratory and stored under refrigeration at 4-5°C.

2.1.2 Media


The choice of media for S. pyogenes was blood base agar, supplied by Biolab,

supplemented with horse blood, obtained from the South African Institute of Medical

Research. Serum broth was the liquid broth of choice for the optimal growth of

S. pyogenes in liquid medium. This broth was obtained from the South African Institute

ofMedical Research.

2.1.2.2 Candida albicans

The choice for storage and preparation of the twenty C. a/bicans isolates was solid

medium Sabouraud's dextrose agar 4%, supplied by Oxoid. Sabouraud's dextrose agar

is used in the isolation, identification and the cultivation of yeast and fungi.

16

Sabouraud's dextrose and Chloramphenycol agar plates, obtained from the South

'African Institute of Medical Research, were used to conduct the disc diffusion test on

C. a/bicans. The choice of liquid medium for the optimal growth of C. a/bicans was

nutrient broth, supplied by Biolab.

2.1.3 Allopathic Medication

2.1.3.1 Penicillin G

Conventional antibiotic medication Penicillin G has an antimicrobial action on

S. pyogenes. Penicillin G was obtained from Mast Diagnostics, impregnated onto 6mm

discs.

2.1.3.2 Nystatin

Conventional antibiotic medication, namely Nystatin 0.04 units, was obtained from

Mast Diagnostics. Nystatin discs had been purchased impregnated onto 6mm discs,

commercially prepared.

2.1.3.3 Bacitracin .

Bacitracin was used in the identification of S. pyogenes, because this organism is

sensitive to Bacitracin. Bacitracin was obtained from Mast Diagnostics, impregnated

onto 6mm discs.

All the discs were stored under refrigeration.

2.1.4 Homoeopathic Treatment

2.1.4.1 Baptisia tinctoria

For the purpose of this research, Baptisia tinctoria was obtained in three potencies: 3ch,

15ch and 30ch.

2.1.4.2 Thuja occidenta/is

Thuja occidenta/is was obtained in three potencies: 3ch, 15ch and 30ch.

17

Each potency, ofBaptisia ttnctoria and Thuja occidentalis respectively, was obtained in, .sOml amber bottles from Natura. Each potency was dispensed in 20% alcohol and

stored at room temperature away from direct light.

Blank discs, obtained from Mast Diagnostics, were purchased to impregnate the

homoeopathic substances.

2.2 Methods

2.2.1 Preparation ofMedia

2.2.1.1 Blood Agar for Streptococcus pyogenes

The blood that is incorporated into the blood base medium is an enrichment ingredient

for the cultivation of S. pyogenes. The blood also permits the demonstration of the

haemolytic properties of the organism. Growth and survival of an organism is greatly

influenced by the pH of the environment and this differs depending on the organisms

requirements. Despite this diversity, S. pyogenes can grow at the generality made at pH

7.3 after autoclaving.

• Blood agar base' powder was suspended into distilled water and boiled to dissolve

the powder.

• The solution was autoclaved at 121°C for IS minutes, and then allowed to cool to

50°C.

• Two sample plates were poured to ensure consistency of the blood base.

• To prepare the blood agar 2sml of sterile horse blood was added aseptically, in the

laminar flow unit, to sOOml blood agar base and gently rolled on a flat surface to

mix the blood base with the blood.

• Labeled petri dishes were then placed onto the laminar flow bench and

approximately 14ml of blood agar was poured intoeach petri dish. Air bubbles that

formed whilst pouring the agar were flamed from the surface of the blood agar with

a bunsen burner.

• The solidified plates were then sealed in plastic bags and stored at 4-SoC in the

refrigerator.

18

2.2.1.2 Sabouraud's Dextrose 4% Agar for Candida a/hieans'j

The preparation ofthis agar is necessary for the sub-cultivation ofC. albicans.

• Sabouraud's dextrose powder was suspended into distilled water.

• The solution was then poured into a sterile metal jug, placed onto a hot-plate to boil

and stirred frequently until completely dissolved.

• . The solution was autoclaved at 121°C for 15 minutes, and then allowed to cool to

5ere.• Two sample plates were poured.

• The labeled petri dishes were then placed on the disinfected surface of the laminar

flow bench. Approximately 14ml of Sabouraud's dextrose agar was poured into

each petri dish.

• The solidified plates were then sealed in plastic bags and stored at 4-5°C in the

refrigerator.

2.2.1.3 Serum Broth

Serum broth was prepared by adding 10% of sterile serum to sterile nutrient broth.

Serum broth tubes where stored at 4-5°C until required.

2.2.1.4 Nutrient Broth

Nutrient broth forms the basis of most media. The Nutrient broth that was used

contained an extract of meat (lg), sodium chloride (5g), yeast extract (2g) and added

peptones (5g) per litre preparation. Nutrient broth was used for the cultivation of

C. a/hicans.

To prepare the nutrient broth:

• The powdered broth was added to distilled water and mixed well.

• 4.500 of nutrient broth was then distributed into glass test tubes, supported in test

tube racks, with a mechanically aspirated pipette. .

• The filled test tubes were then autoclaved at 121°Cfor 15 minutes.

• The cooled test tubes were packed and sealed for storage until they were needed.

19

2.~.2 Maintenance ofOrganisms

Microorganisms are transferred from one medium to another by subculturing. This

technique is important in maintaining stock cultures as well as in microbial procedures

(10).


Stock cultures of twenty different isolates of S. pyogenes required subculturing onto

blood agar plates or broth tubes, every 5-7 days. Appropriately labeled blood agar plates

were placed onto a sterile work surface. With a sterile inoculating loop, a single discrete

colony ofS. pyogenes was removed from the stock culture. Each colony was presumed

to be a pure culture, consisting exclusively of the descendants of a single cell. A septic

transfer onto the blood agar medium was made. The four-way streak plate inoculation

method was used as a rapid qualitative isolation method. It is essentialJy a dilution

technique involving spreading the discrete colony over the surface of the agar plate.

Bacitracin sensitivity was tested to verify that the organism was indeed a group A

B-haemolytic streptococcus. The plates were incubated for 24h at 3'?C and thereafter

sealed and stored in arefrigerator at 4-5°C.

All twenty isolates were also frozen in serum broth and stored in the freezer at -SoC,

ensuring their further availability.

2.2.2.2 Candida a/bicans

Stock cultures of the twenty different isolates of C. a/bicans were subcultured every

5-7 days onto Sabouraud's dextrose 4% agar. Prior to inoculation the appropriately

labeled plates, that were stored under refrigeration, where allowed to equilibrate to

room temperature and the surface of the agar to dry. With a sterile inoculating loop, a

single discrete colony of C. a/bicans was removed from the stock culture. An aseptic

transfer for each isolate was made onto fresh Sabouraud's dextrose 4% agar medium.

The streak plate method wasalso used to spread the discrete colony of C. a/bicans over

the surface ofthe dextrose agar.

20

To ,ensure that no contamination had occurred and for verification of the organism, a

Nystatin disc was placed in the centre of the agar plate. The plates were then incubated

for 24h at 3'JOC, thereafter they were sealed and stored in a refrigerator at 4-5°C.

All twenty isolates were also frozen in nutrient broth and stored in the freezer at -SoC,

ensuring their further availability.

2.2.2.3 Broths Subculturing

It was necessary that the stock cultures of C. a/bieans and S.pyogenes were transferred

into nutrient broth and serum broth respectively.

• A single discrete colony of each isolate was selected from the fresh stock culture,

with a sterile inoculating loop, and transferred into 10ml ofbroth.

• Immediately prior to the wire loop being introduced into the broth, the mouth of the

test-tube was flamed to prevent contamination.

• Each test-tube was capped closed and placed in the incubator for 24h at 37°C.

• The broth subcultures were then removed from the incubator and placed into the

refrigerator until required.

2.2.3 Determination of Standard Curve

I

A standard curve of the optical density (OD) readings, set at 540 nanometers (nm), of

each respective organism against colony forming units per millilitre (cfulml) was

determined. This straight line graph was formulated using twenty different clinical

isolates of each organism. Serum broth and nutrient broth were used in the

determination ofthe standard curve for S. pyogenesand C. a/bieans respectively. Serial

dilutions ofeach organism were performed and the dilutions were plated out in order to

determine the cfulml. Similar procedures were employed to determine the standard

curve and conversion formulae for S. pyogenesand C. a/bieans respectively.

21

2.2.3.1 Serial Dilutions and Plating."

• Strain IA ofthe broth stock culture was prepared and constituted a pure culture.

• Six other broth tubes were then labeled from IA number 2 through to IA number 7

and placed in ascending numerical order in a test-tube rack.

• Strain IA of the organism was vortexed to ensure even distribution of the cells in

the culture broth.

• With a mechanical aspirated pipette, calibrated at Iml, an aseptic transfer of Irnl

was made from the culture broth (IA no. I) to broth IA no.2. The pipettes tip was

disregarded and replaced with a sterile tip. The culture of the organism had therefore

been diluted 10 times.

• Test-tube labeled IA no.2 was then vortexed and lrnl from Al no.2 transferred

aseptically with a pipette to test-tube Al no.3. The culture had therefore been

diluted 100 times.

• This procedure was repeated to obtain.a 10 000, 100 000 and I 000 000 dilution, in

triplicate, for each isolate.

• Agar plates IA no. I, IA no.2 through to IA nO.7 were appropriately labeled.

Sabouraud's dextrose agar was used to plate out C. a/bicans and blood agar for

S.pyogenesrespectively.

• Then returning to broth IA no.l, with a sterile pipette tip, O.lml of broth was

transferred to the agar plate labeled I A nO.I.

• For inoculation purposes a sterile L-shaped glass rod was used. By lightly touching

the sterile L-shaped rod to the surface of the agar medium and moving it back and

forth the culture was dispersed over the agar.

• Then 0.1 ml of broth IA no.2 was transferred with a sterile pipette tip to the agar

plate labeled IA no.2. and the culture was spread over the surface ofthe agar.

• After transferring the broths from labeled test-tubes IA nO.3 through to IA nO.7

onto the respective agar mediums, all the plates were then incubated in an inverted

position for 24h at 3T'C.

22

2.2.3.2 Spectrophotometer Readings of Serial Dilution ofBroths~ ,

Spectrophotometer readings were done to determine the turbidity in each broth dilution,

ofeach isolate, prepared in triplicate. The density of the cell suspension is expressed as

absorbance or optical density (OD).

• The spectrophotometer was turned on 10-15 minutes prior to use, set to absorbancy

readings at 540nm.

• 1m1 of sterile serum broth or nutrient broth was aspirated with a mechanical pipette

to calibrate readings .for S. pyogenes and C. albicans respectively.

• Broth 1A no.7 (the greatest dilution) of the organism was first vortexed and 1ml of•

the broth was transferred, with a mechanical aspirated pipette and sterile tip, to the

cuvette. The cuvette was placed into the tube holder, the cover closed and the

reading on the galvanometer recorded.

• The cuvette was removed from the holder and rinsed clean with distilled water.

• Broth1A no.6 of the organism was then vortexed and 1ml of broth was transferred

to the cuvette and the reading on the galvanometer recorded.

• This procedure was then repeated in descending numerical order, from 1A nO.5

through to IA no. I for S. pyogenes and C. albicans respectively..• All the readings were recorded for statistical purposes.

2.2.4 Disc Diffusion Method

A standardised filter-paper disc agar diffusion procedure known as the Kirby-Bauer

method was used to determine the drug susceptibility of S. pyogenes and C. albicans

respectively. This method allowed for the rapid determination of the efficacy of a drug

by measuring the diameter zone of inhibition that resulted from the diffusion of the

agent into the medium surrounding the disc (2,9).

23

2.2.4.1 Streptococcus pyogenes'.1

Prior to use, the blood agar plates were transferred to the incubator for 10 minutes at

37°C to dry the moisture that developed on the surface of the medium whilst in storage

in the refrigerator. The appropriately labeled agar plates were inoculated with the

twenty different isolates of S. pyogenes, in triplicate, using the L-shaped glass rod to

ensure confluent growth. Blank discs were placed into sterile containers, in a laminar

flow unit, and impregnated with Baptisia tinctoria in 3ch, 15ch and 30ch respectively

and Thuja occidentalis in 3ch, 15ch, 30ch respectively. The discs were then aseptically

applied to the surface of the agar plates at well-spaced intervals with a pair of sterile

forceps and gently pressed down onto the media to ensure that the discs adhered. All the

plates were then incubated at 3rC for 24h.

2.2.4.2 Candida albicans

The medium chosen to conduct the disc diffusion method on C. albicans was

Sabouraud's dextrose and Chloramphenycol agar. The appropriately labeled agar plates

were inoculated with the twenty different isolates of C. albicans, in triplicate, using the

L-shaped glass rod to ensure confluent growth. The blank discs were placed into sterile

containers and impregnated with Baptisia tinctoria in 3ch, 15ch and 30ch respectively

and Thuja occidentalis in 3ch, 15ch and 30ch respectively. The discs were aseptically

applied to the surface of the agar plates at well-spaced intervals. All the plates were then

incubated at 3?OC for 24h.

2.2.4.3 Zone Measurement

Following 24h incubation of the plates at 37°C, the size of the zones around the discs of

Baptisia tinctoria 3ch, 15ch and 30ch and Thuja occidentalis 3ch, 15ch and 30ch were

determined. The zones of inhibition that formed around the Nystatin, Penicillin G and

Bacitracin discs were also measured. The diameter of the zones were measured in

millimetres with a calibrator. The size of the zones were all recorded for statistical

analysis.

24

2.2.5 Broth Dilution Method

Culture broths of the twenty different isolates of S. pyogenes and C. albicans were

prepared Serum broth media was used for S. pyogenes and nutrient broth media for

C. albicans.

The procedure for both C. albicansand S.pyogenes was the same.

• O.sml of the culture broth was obtained from stain IA no.1 with a mechanical

aspirated pipette, and added to 4.sml aseptic broth, followed by the addition of

O.sml distilled water. This constituted the control broth for the organism and was

prepared for all twenty isolates ofeach organism, in triplicate.

• O.sml of the of the culture broth was obtained with a mechanical aspirated pipette,

and added to 4.sml aseptic broth, followed by the addition of O.sml Baptisia

tinctoria 3ch.

• The procedure was then repeated for Baptisia tinctoria l Sch and 30ch, and 11111ja

occidentalis3ch, l Sch and 30ch.

• All the broths were then incubated at 37°C for 24h.

2.2.5.1 Spectrophotometer Readings

Following 24h incubation of the broths the optical density (00) readings, at s40nm, of

S. pyogenes and C. albicans treated with Baptisia tinctoria 3ch, lsch and 30ch and

Thujaoccidentalis 3ch, Isch and 30ch, were determined.

• 1ml of the broth containing the organism and distilled water was aspirated with a

mechanical pipette and transferred to the cuvette, placed into the tube holder and the

reading determined.

• lml ofthe broth containing the organism and homoeopathic remedy in potency was

aspirated with a mechanical pipette and transferred to the cuvette, placed into the

tube holder and the reading determined.

• This was repeated until all the readings for the respective organisms had been

determined.

• All results were recorded for statistical analyses.

25

3.0 RESULTS

3.1 Standard Curve

The standard curves, illustrated in figures 3.1 and 3.2, correlate the optical density (00)

readings with colony forming units per millilitre (cfu/ml) of Streptococcus pyogenes

and Candida albicans respectively. The correlation ofthese two parameters allowed for

conversion of the 00 readings obtained from the broth dilution method to determine

cfulml. Results used for plotting the respective graphs are available in Appendix A and

Appendix B respectively.

1.5

1.2e+5 •1.Qe+5 •8.Qe+4

~ 6.Qe+4

,4.Qe+4

2.Qe+4

O.Qe+O

0.0 0.5 1.0

Q>tica1 density

• q,tical densityv Colooyf~ units--- Plot 1Lin::arregressioo- Plot 1ConfJderx:e irmval95%

I Figure 3.1 A graphic representation of the standard curve correlating 00 readings and

cfulml ofS. pyogenes.

The correlation coefficient for S. pyogene!' t'=O,87 and the formula to determine colony

forming units (cfu/ml) =4,87 x 1040 0 -1931,46.

26

2.5e+5

•2.0e+5

1.5e+5

1 1.0e+5<J

5.0e+4

O.Oe+O

-5.0e+4

0.0 0.2 0.4 0.6 0.8 1.0

Optical density

• Optical density v Colony fonningUDits--- Plot 1Linearregression- Plot 1Continence interval 95%

Figure 3.2 A graphic representation of the standard curve correlating OD readings and

cfu/ml ofC. a/bieans.

The correlation coefficient for C. a/bicans "=0,78 and the formula to determine colony

forming units (cfu/ml) =1,79 x 1050 D -3847,73.

3.2 Determination ofAntimicrobial Activity

The efficacy of the medication was determined by the degree to which there was a

significant' difference between the growth of S. pyogenes and C. a/bicans respectively

when treated with homoeopathic Baptisia tinctoria 3ch, 15ch and 30ch and Thuja

occidenta/is 3ch, 15ch and 30ch. The homeopathic potencies were prepared in 20%

alcohol. In previous studies, it was determined that alcohol at 20% had no direct effect

on the readings obtained (personal commurdcation with supervisor).

27

Statistical tests were executed on ANOVA. Three statistical procedures were

conducted:

o One Way Analysis of Variance

o Kruskal-Wallis One Way Analysis of Variance on Ranks

o All Pairwise Multiple Compari son Procedures (Dunnett's Method)

3.2.1 Results ofStreptococcus pyogenes and Baptisia tinctoria treatment groups

Figure 3.3 illustrates the effect of homoeopathic Baptisia tinctoria 3ch, 15ch and 30ch

and no medication (control) on the growth production of S. pyogenes in brot h dilution.

Resu lts of all twenty strains of S. pyogenes 00 broth dilution readings are availabl e in

Appendi x C.

6OOX)

5CXXX)

4OOX)

"'" 3CXXX)....-s<:::o

2OOX)

100l)

0

TreatnUlt CIWpS

_ nulicatioo-control

_ Bqxisiatinaona3d1

Bqxisia tinaaia 1.'X:l1_ Bopasia tinaoia 3U:I1

Figure 3.3 Bar graph showing the cfu/ml of S. pyogenes, in broth dilution , when treated

with Baptisia tinctoria and no medication.

28

Upon further analysis of the statistical values and Figure 3.3, it was evident that there

was a statistical difference between the treatment groups on the twenty different isolates

of S. pyogenes and the isolates that contained no medication. The most significant

difference noted in Figure 3.3 was that of Baptisia tinctoria 3ch, when compared to

S. pyogenes control groups containing no medication. In this treatment group, Baptisia

tinctoria 3ch, the mean cfu/ml ofS.pyogenes was measured at 45900. This illustrated a

decrease when compared to the control group mean cfu/ml measuring 57100. Baptisia

tinctoria 15ch and 30ch treatment groups mean cfu/ml were determined to be 49100

and 52100 respectively.

Mean values for the treatment groups and control groups of S. pyogenes were used for

comparing the results. These results are indicated in table 3.1.

Table 3.1 ANOVA table of broth dilution displaying control and Baptisia tinctoria

treatment groups on the growth of S. pyogenes reflecting mean values,

standard deviations, standard errors and confidence intervals.

Key: Control= S pyogenescontaining no medication

3ch=Baptistatinctoria 3ch

15ch=Baptisiatinctoria 15ch

30ch=Baptistatinctoria 30ch

Control 3ch 15ch 30ch

Mean 5.71e+4 4.59e+4 4.91e+4 5.21e+4

StandardDeviation 4565.30 5136.66 5499.07 4782.73

StandardError 589.38 663.14 709.93 617.45

95% Confidence interval 1179.36 1326.96 1420.59 1235.53


When comparing the mean values noted in table 3.1 of S. pyogenes treated with

Baptisia tinctoria, it can be seen that Baptisia tinctoria 3ch, 15ch and 30ch displayed an

effect, ofvarying degrees, on the.growth production ofS. pyogenes in broth dilution.

29

One Way Analysis ofVariance Normality test failed (P=<0.0001).

According to the Kruskal-Wallis One Way Analysis of Variance on Ranks, the

difference in the median values among the treatment groups are greater than what

would be expected by chance; there was a statistically significant difference

(P=3.64E-036).

H= 179.9 with 6 degrees of freedom (P=<0.0001).

Table 3.2 The difference m median values of S. pyogenes treated with Baptisia

tinetoria.

Group Median 25% 75%

Control 56504.0 55184.0 57632.0

Baptistatinctoria 3ch 46969.0 42152.0 49585.0

Baptisiatinctorta 15ch 47946.5 44377.0 53423.0


The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a

comparison between the homoeopathic treatment groups vs S. pyogenes control,

determining the difference of ranks and P-values. Statistically significant readings

should be P<0.05.

Table 3.3 Comparison of Baptisia tinctoria treatment groups, difference of ranks and

P-values ofS.pyogenes.

Comparison Difference ofRanks P-value P< 0.05

Baptista tinctorta 3chvs control 13915.5 10.46 Yes

Baptistatinctorta 15ch vs control 10557.0 9.26 Yes

Baptisia tinetoria 30chvs control 7213.5 9.48 Yes

30

3.2.2 Results ofStreptococcus pyogenes and Thuja occidentalis treatment groups

Figure 3.4 illustrates the effect of homoeopathic Thuja occidentalis 3ch, 15ch and 30ch

and no medication (control) on the growth production of S. pyogenes in broth dilution.

Results of all twenty isolates of S. pyogenes OD readings of the broth dilution and

treatment groups are available in Appendix D.

axm ,------- - - - - ---,

icro

O--+--

• m:rlicaim-catJd• 7hgtuxxidndis 3ch

1hgtJ(xxidndis 15dl

• 1hgtuxxidndis 3Qh

Figure 3.4 Bar graph showing the cfulm l of S. pyogene , in broth diiution, when treated

with Thuja occidentali and no medication.

Upon further analysis of the statistical value and Figure 3.4. it wa evident that there

was only a minimal difference between the treatment group on the twenty different

isolates of . pyogenes and the i olates that contained no medication . The mo t

significant difference noted in Figure 3.4 was that of Thuja occidentalis 3ch when

compared to S. pyogene control group containing no medication. In thi treatment

group Thuja occidentalis 3ch cfulml of S. pyogene wa mea ured at 51600. This

illustrated a slight decrea e when compared to the control group mean cfulml 57100.

31

Thuja occidenta/is 15ch measured 55200 mean cfulml and Thuja occidenta/is 30ch.showed a very minimal decrease in cfulml measuring 57000 mean value. Hence, Thuja

occidenta/is 15ch and 30ch showed no antimicrobial activity.

When comparing the mean values for all the treatment groups and the control values of

the S. pyogenes isolates, it was noted that only Thuja occidenta/is 3ch had a slight

antimicrobial effect displayed in the cfulml. These results are indicated in table 3.4.

Table 3.4 ANOVA table of broth dilution displaying control and Thuja occidentalis

treatment groups on the growth of S. pyogenes reflecting mean values,


Key: Control= S. pyogenes containing no medication

3ch= Thuja occidentalis 3ch



, Control 3ch 15ch 30ch

Mean 5.71e+4 5. 16e+4 5.52e+4 5.70e+4

Standard Deviation 4565.30 4282.18 8514.17 5651.64

Standard Error 589.38 552.83 1099.17 729.62



One Way Analysis ofVariance Normality test failed (P=<O.OOOI).

The difference in the median values, according to ANOVA statistics, among the

, treatment groups are greater than what would be expected by chance; there was a

statistically significant difference (p=3.64E-036).

H= 179.9 with 6 degrees offreedom (p=<O.OOOI).

32

Table 3.5 The difference in median values of S. pyogenes treated with Thuja

oecidenta/is.


Control 56504.0 55184.0 57632.0

Thuja occidentalis 3ch 52445.5 48044.5 54572.5




comparison between the homoeopathic treatment groups vs S. pyogenes control,

determining the difference of ranks and P-values.

Table 3.6 Comparison of Thuja occidentalis treatment groups, difference of ranks and

P-values ofS. pyogenes.

Comparison Difference ofRanks P-value P<0.05

Thuja occidenta/is 3ch vs control 7940.0 8.36 Yes



According to the comparison P<O.05, values were significant for all treatment groups.

When considering the pattern of antimicrobial cell death of S. pyogenes, it is evident

that Thuja oeeidenta/is 3ch was most effective. Decreased growth production varied

with each potency. A lower degree of antimicrobial efficacy was noted for Baptisia

tinetoria 15ch and 30ch and Thuja oeeidenta/is 15ch and 30ch. No substantial results

were depicted to suggest antimicrobial activity was prominent with these potencies.

Baptisia tinetoria treatment groups showed a greater effectiveness on decreasing the

growth production, i.e. lower cfulml ofS. pyogenes, than Thuja oeeidenta/is treatment

groups. However, both treatment groups showed a statistically significant difference

greater than would be expected by chance.33

3.2 .3 Results of Disc Diffusion Method

3.2 .3.1 Streptococcus pyogenes disc diffusion method treat ed with Penicillin G,

Bacitracin and Baptisia tinctoria

Baptisia tinctoria 3ch and 15ch showed minimal areas of inhibition on S. pyogenes in

the disc diffusion method. Only a few random areas of inhibition were evident

around the discs impregnated with the se treatment groups . Baptisia tinctoria 30ch

demonstrated no zones of inhibition on S. pyogenes. Baptisia tinctoria 3ch displayed a

mean area of inhibition measuring OAOmm zone diameter. Baptisia tinctoria 15ch and

Baptisia tinctoria 30ch produ ced mean zones of inhibi tion measuring O.25mm and

O.OOmm respectively. Figure 3.6 illustrates the disc diffusion results. Penicillin G

showed the largest zone of inhibition measur ing 36 .32mm mea n value, followed by

Bacitracin with a zone of inhibition measuring 14.eSmm mean value.

40 -.--- - - - - - - - - - - ---,

35

30

b 25

lI 20~ '-' 15

10

5

oTrcatrrcnt Group;

_ PemcillinG_ Baitrocin

Bopasiatinaaia 3ch_ ~isia tinaaia 15ch

_ ~isia ti,uGia J(k;h I

Figure 3.5 Bar graph of disc diffusion method displa ying zone diameters on

S. pyogenes treated with Penicillin G, Bacitracin and Baptisia tinctoria .

3-l

The mean values were used for comparative purposes and can be seen in table 3.7.•

Results ofall the values are available in Appendix E.

Table 3.7 ANOVA table of disc diffusion of S. pyogenes treated with Penicillin G,

Bacitracin and Baptisia tinctoria displaying mean values, standard

deviations, standard errors and confidence intervals.

Penicillin G Bacitracin 3ch 15ch 30ch

Mean 36.32 14.88 0.40 0.25 0.00

Standard Deviation 3.80 1.62 1.77 1.36 0.00

Standard Error 0.49 0.21 0.23 0.18 0.00

95% Confidence interval 0.98 0.42 0.46 0.35 0.00

99% Confidenceinterval 1.31 0.56 0.61 0.47 0.00

When comparing the mean values noted in table 3.7 of S. pyogenes treated with

Baptisia tinctoria, it can be seen that Baptisia tinctoria 3ch and 15ch displayed an

effect, of varying degree, with the formation of zones inhibition on S. pyogenes.

Baptisia tinctoria 30ch displayed no zones of inhibition on S. pyogenes.


treatment groups are greater than what would be expected by chance; there was a

statistically significant difference (p=1.27E-094).

H= 457.2 with 7 degrees of freedom (P=<O.OOOI).

35

Table 3.8 The difference in median values of S. pyogenes treated with Penicillin G,,Bacitracin and Baptisia tinctoria.


PenicillinG 36.00 33.00 40.00

Bacitracin 15.00 14.00 16.00

Baptisia tinctoria 3ch 0.00 0.00 0.00




comparison between the homoeopathic treatment groups vs Penicillin G, determining

the difference ofranks and P-values. Statistically significant readings should be P<O.05.

Table 3.9 Comparison of Baptisia tinctoria treatment groups and Penicillin G,

displaying difference of ranks and P-values ofS.pyogenes.

Comparison J Difference ofRanks P-value P<0.05

Baptisia tinctoria 3ch vs Penicillin G 15868.00 27.80 Yes

Baptista tinctoria 15ch vs Penicillin G 16051.00 16.89 Yes

Baptista tinctoria 30ch, vs Penicillin G 16410.00 10.80 Yes

According to the comparison P<O.05, the comparison values were significant for the

homoeopathic treatment groups.

_ 3.2.3.2 Streptococcus pyogenes disc diffusion method treated with Penicillin G,

Bacitracin and Thuja occidentalis

Thuja occidenta/is produced minimal areas of inhibition on S. pyogenes in the disc

diffusion method. Only a few random areas of inhibition were evident around the discs

impregnated with Thuja occidenta/is 3ch. Within this treatment group a mean area of

inhibition measuring O.15mm zone diameter.

36

Penicillin G reflected the largest zone of inhibition with mean zone diameter of

15.94mm and Bacitracin a mean zone diameter of 4.14mm. Thuj a occidentalis 15ch

displayed a mean zon e diameter of O.OOmm and Thuja occidentalis 30ch O.OOmm.

Figure 3.9 illustrates the disc diffusion resu lts. The difference in the values among the

treatment groups were all greater than what would be expected by chance. Results of all

the values are available in Appendix F .

40 --r--- - - - - - - - - - - ---,

35

30

IJ 25

l1 20~ "-' 15

10

5

oTreatmnt groups

_ Penicillin G_ Baitraiin

~ 7111.ifo ocodentdis 3ch_ Thuja occideudis 15ch_ 7111.ljooccidaudis 30ch

Figu re 3.6 Ba r graph of disc diffu sion method displaying zone diameters on S. pyogenes

treated with Penicillin G, Bacitracin and Thuja occidentalis.

The mean values obtained were u ed for comparative purpo e . Analy i of the mean

values dep icted that Thuja occidentali 3ch, 15ch and 30ch displayed no antimi crobial

activity on S. pyogenes in the disc diffusion method . ean value obtained from the

disc impregnated with Penicillin G, Bacitracin and Thuja occidentalis treatm ent groups

can be seen in table 3.10.

37

Table 3.10 ANOVA table of disc diffusion of S. pyogenes treated with Penicillin G.

Bacitracin and Thuja occidentalis displaying mean values, standard

deviations, standard errors and confidence intervals.

PenicillinG Bacitracin 3ch 15ch 30ch

Mean 15.94 4.14 0.15 0.00 0.00

Standard Deviation 1.12 0.71 0.31 0.00 0.00

Standard Error 0.14 0.09 0.04 0.00 0.00





statistically significant difference (P=5.49E-056).

H= 272.8 with 6 degrees of freedom. (P=<O.OOOI)

Table 3.11 The difference in median values of S. pyogenes treated with Penicillin G,

Bacitracin and Thuja occidentalis.


Penicillin G 36.00 33.00 40.00

Bacitracin 15.00 14.00 16.00

Thuja occtdentalis 3ch 0.00 0.00 0.00

Thuja occidentalis15ch 0.00 0.00 0.00

Thuja occidentalis30ch 0.00 0.00 0.00


comparison between the homoeopathic treatment groups vs Penicillin G, determining

the difference ofranks and P-values. Statistically significant readings should be P<O.OS.

38

Table 3.12 Comparison of Thuja occidentalis treatment group and Penicillin G,

displaying difference of ranks and P-value of S. pyogenes.

Comparison Difference of Ranks P-value P<0.05

Thllja occidentalis 3ch vs Penicillin G 11739.0 20.6 Yes

Thuja occidentalis 15ch vs Penicillin G 14100 .0 10.6 Yes

Thuja occidentalis 30ch vs Penicillin G 14100.0 10.6 Yes

3.2.4 .1 Results of Candida albicans and Baptisia tinctoria treatment group

Figure 3.7 illustrates the effect of homoeopathic Baptisia tinctoria 3ch, 15ch and 30ch

and no medication on the growth production of C. albicans in bro th dilution. Re ult of

all twenty i olates of C. albicans 00 readings in broth dilut ion are avai lable In

ppendix G.

7aXX) ...,.---- - - - - - - - - - ---,

EIDJ)

EJ:rIJJ

4aXX)~'i5 DlX)

zrmioxo

O-+--

Figure 3.7 Bar graph hewing the cfu/ml of C albtcans. in broth dilut ion . \ hen treated

with Bapti ia tinctoria and no medicati on.

Upon further analysis of the statistical values and Figure 3.7, it was evident that there

was a difference in values between the treatment groups on the twenty different isolates

of C. albicans and the isolates that contained no medication. The most significant

difference noted in Figure 3.7 was that of Baptisia tinctoria 3ch when compared to

C. albicans control groups containing no medication. In this treatment group, Baptisia

tinctoria 3ch, cfulml of C. albicans was measured 11500 mean. This illustrated a

substantial decrease compared to the mean control group cfulml 58100. Baptisia

tinctoria 15ch cfulml mean value was determined to be 15900 and Baptista tinctoria

30ch cfulml 28100. Mean values for all the treatment groups and control values of

e. albicans were used for comparing the results. These results are indicated in

table 3.13.

Table 3.13 ANOVA table of broth dilution displaying control and Baptisia tinctoria

treatment groups on the growth of C. albicans reflecting mean values,


Control 3ch 15th 30th

Mean 5.81e+4 1.15e+4 1.59e+4 2.81e+4


Standard. Error 1209.39 543.61 711.03 1031.38



When comparing the mean values noted in table 3.13 of e. albicans treated with

Baptisia tinctoria, it can be seen that Baptista tinctoria 3ch, 15ch and 30ch displayed an

effect, ofvarying degrees, on the growth production oiC. albicans in broth dilution.

,

One Way Analysis ofVariance Normality test failed (P=<O.OOOI).

40

According to the Kruskal-Wallis One Way Analysis of Variance on Ranks, the

difference in the median values among the treatment groups are greater than what

would be expected by chance; there was a statistically significant difference

(P=5.44E-077).

H= 375.0 with 7 degrees of freedom (P=<0.0001).

Table 3.14 The difference in median values of C. albicans treated with Baptisia

tinctoria.


Control 58175.5 51821.0 64172.0


Baptisia tinetoria 15ch 14589.0 11635.5 18885.0



comparison between the homoeopathic treatment groups vs C. albicans control,


should be P<0.05.

Table 3.15 Comparison of Baptista tinctoria treatment groups, difference of ranks and

P-values ofC. albicans.

Comparison Difference of Ranks P P< 0.05

Baptisia tinetorla 3ch vs control 19795.0 13.03 Yes

Baptista tinctaria 15ch vs control 17419.5 13.10 Yes

Baptista tinctorta30ch vs control 12417.0 10.89 Yes

41

3.2.4.2 Re sults oiCandida albicans and Thuja occidentalis treatment group

Figure 3.8 illustrates the effect of homoeopathic Thuja occidentalis 3ch, 15ch and 30ch

and no medication on the growth production of C. albicans in broth dilut ion. Re ult of

all twenty isolates of C. albicans OD broth dilution read ing and treatment group are

available in Appendix H.

7rJ:J:1J -r----- - - - - - - - - -----,

ecocos:x:xx>

• rrcdictioo- co• 1hujo oajeJ:ntnhs:kh

7hujoeJaieJ:nrclhs 15ch7hujo ocadsuaas

Figure 3.8 Bar graph howing the cfu/ml of '. albicans, in broth dilution , when treated

with Thuja occidentalis no med ication .

pon further anal i of the tati tical alue and 'igure 3. , it i evident that there wa

a difference in value between the treatment group on the twent different i olate of

. albicans and the isolate that contained no medicat ion . The mo t ignificant

difference noted in Figure 3. wa that of Thuja occidentali 3ch, when compared to

C. albican control group containing no medicat ion. In thi treatment group Thuja

occidentali 3ch mean cfu/ml of C. albicans v.a mea ured at 2300. Thi illustrated a

con iderable decrease compared to the control grou p mean cfu/ml 58 100.

Thuja occidentalis 15ch demonstrated a decrease in mean cfu/ml to 55200 and 30ch

showeddecrease in cfulml measuring 54000.

When comparing the mean values for all the treatment groups, as well as for the control

of C. albicans, it was noted that Thuja occidentalis 3ch showed a slight antimicrobial

effect displayed in the cfu/ml. These results are indicated in table 3.16.

Table 3.16 ANOVA table of broth dilution displaying control and Thuja occidentalis

treatment groups on the growth of C. albicans reflecting mean values,

standard deviations, standard error values and confidence intervals.

Control 3ch 15ch 30ch

Mean 5.81e+4 323e+4 5.52e+4 5.40e+4

Standard Deviation 9367.93 1.02e+4 1.12e+4 1.23e+4

Standard Error 1209.39 1315.11 1450.]8 J584.47



One Way Analysis ofVariance Normality test failed (P=<O.OOOl).




H= 375.0 with 7 degrees of freedom (P=<O.OOOI).

43

Table 3.17 The difference In median values of C. a/hieans treated with Thuja

occidenta/is.


Control 58175.5 51821.0 64172.0

Thuja occtdentalis 3ch 31862.5 23986.0 41170.5




comparison between the homoeopathic treatment groups vs C. a/hieans control,


should be P<O.05.

Table 3.18 Comparison of Thuja occidentalis treatment groups, difference of ranks and

P-vaIues ofC. albicans.


Thuja occidentalis 3ch vs control 10928.0 11.50 Yes

Thuja occidentalis 15ch vs control 1352.5 3.55 Yes

Thujaoccidentalis 30ch vs control 2112.0 3.70 Yes

According to the comparison P<O.05 values were significant for all treatment groups.

When considering the pattern ofantimicrobial cell death ofC. albicans, it is evident that

Baptisia tinctoria 3ch and Thuja occidentalis 3ch were most effective. Decreased

growth production varied with each potency. The homoeopathic remedy, Baptisia

tinctoria treatment groups showed a greater effectiveness on decreasing the growth

production of C. a/bieans than Thuja oceidenta/is treatment groups. However, both

treatment groups showed a statistically significant difference greater than would be

expected by chance.

44

3.2.5 Results of Disc Diffusion ethod

3.2.5.1 Candida albicans disc diffusion method treated with 'ystatin and Bapti iia

tinctoria.

Baplisia tinctoria 3ch, 15ch and 30ch howed a minimal area of inhibition on

. albican in the di c diffu sion method . On ly a few random area of inhibition \ ere

evident around the discs impregnated with Bapti iia tinctoria treatment group . Baptista

tinctoria 3ch was the most effective and di played a mean area of inhibition mea uring

1.02mm zone diameter. Figure 3.9 illustrat es the di c diffu ion re ult . The difference in

value among the treatment group were all greater than what would be e pected by

chance. Nystatin produced the large t mean zone of inhibition mea urin g 23.4 3mm.

30

25

20~

I I 15 -

~10

5

0

ill

Baptuwlinc10na 3chBaptgo atJC10na IBaptisUl tinc10na JCkh

Figure 3.9 Bar graph of di c diffu ion method di pia ing zen diameter on C. albicans

treated with J } ' latin and Bapti ia lin toria.

The mean values were used for comparative purposes. The mean values and standard

error of results obtained from the disc impregnated with Nystatin and Baptisia tinctoria

treatment groups can be seen in table 3.14. Results of all the values are available in

Appendix I.

Table 3.19 ANDVA table of disc diffusion of C. albicans treated with Nystatin and

Baptisia tinctoria displaying mean values, standard deviations, standard

errors and confidence intervals.

Nystatin 3ch 15ch 30ch

Mean 23.436 1.02 0.38 0.26


Standard Error 0.39 0.34 0.22 0.18


99% Confidence interval 1.03 0.90 058 0.48

When comparing the mean values noted in table 3.14 of C. albicans treated with

Baptisia tinctoria, it can be seen that Baptisia tinctoria 3ch, 15ch and 30ch produced

small zones of inhibition that are not indicative of susceptibility of the organism to the

treatment. Baptisia tinctoria 15ch and 30ch displayed mean zones on inhibition

measuring 0.38mm and 0.26mm respectively. These results do not reflect an antifungal

action.

The difference in the median values, according to ANDVA statistics, among the



H= 349.0 with 6 degrees offreedom. (p=<O.OOOI)

46

Table 3:20 The difference in median values of C. albicans treated with Nystatin and

Baptisia tinctoria.


Nystatin 25.00 21.00 25.00





comparison between the homoeopathic treatment groups vs Nystatin determining the

difference of ranks and P-values.

Table 3.21 Comparison of Baptisia tinctoria treatment groups and Nystatin displaying

difference of ranks and P values of C. albicans.


Baptistatinctoria 3chvs Nystatin 11613.0 30.48 Yes

Baptistatinctorta 15ch vs Nystatin 12510.5 21.92 Yes

Baptistatinctoria 30chvsNystatin 12688.0 16.68 Yes

3.2.5.2 Candida albicans disc diffusion method treated with Nystatin and Thuja

occidentalis

Thuja occidentalis 3ch, 15ch and 30ch showed minimal areas of inhibition on

C. albicans in the disc diffusion method. Only a few random areas of inhibition were

evident around the discs impregnated with Thuja occidentalis treatment groups. Thuja

occidentalis 3ch was the most effective and displayed a mean area of inhibition

47

Thuja occidentalis 15ch displayed a mean zone diameter mea uring O.OOmm and 77l11ja

occidentalis 30ch O.OOmm. Nystatin reflected the largest zone of inhibition with mean

zone diameter of 23.43mm. The difference in the value among the treatment group

were all greater than what would be expected by chance. Re ult of all the value are

available in Appendix J.

30

25

20a~ 'a" 15a ..:.a sj,l

10

5

0

Tn.:: 1('

Figure 3.10 Bar graph of di diffu ion method di pia ing zone diameter on

. albicans treated with 'ystatin and 771Uja occidentalis.

The mean value obtained from the di c diffu ion m thod ere u ed for comparative

purpo e and the e value available in table 3.17. nal i of the re ult reflected that

Thuja occidentalis 3ch, 15ch and 30ch di pia ed no ignificant antimicrobial activit

on the growth production of C. albicans.

48

Table 3.22 ANDVA table of disc diffusion of C. albicans treated with Nystatin and

Thuja occidentalis displaying mean values, standard deviations, standard

errors and confidence intervals.

Nystatin 3ch 15ch 30ch

Mean 23.43 0.55 0.00 0.00


Standard Error 0.39 0.27 0.00 0.00



The difference in the median values, according to ANDVA statistics, among the



H= 349.0 with 6 degrees of freedom. (P=<O.OOOI)

Table 3.23 The difference in median values of C. albicans treated with Nystatin and

Thuja occidentalis.


Nystatin 25.00 21.00 25.00

Thujaoccidentalts 3ch 0.00 0.00 0.00


Thujaocctdentalis 30ch 0.00 0.00 0.00


.comparison between the homoeopathic treatment groups vs Nystatin, determining the

difference of ranks and P-values.

49

Table 3.24 Comparison of Thuja occidentalis treatment groups and Nystatin displaying

difference ofranks and P-values ofC. albicans.


Thuja occidentalis 3ch vs Nystatin 12688.0 16.68 Yes



According to the comparisonP<O.05 values were significant for all treatment groups.

so

4.0 DISCUSSION

Our modem day microbial biosphere is composed of innumerable infectious

microorganisms (2). Even with progress in the discovery of new and improved drugs.

the potentially dangerous situation of drug resistant microorganisms still exists. The

current antibiotic resistant crisis differs from those of the past because several

organisms are involved and there are no immediate solutions apparent in the short term

(42). The consequences of resistance include higher mortality and greater morbidity.

Infections with organisms resistant to microbial agents leads to longer hospitalisation

and greater expenses (2,26).

Efforts to reduce antibiotic resistance must address both the heavy use and ease of

spread. Although this measure has been advocated for years. most medical practitioners

still do not appear to understand either the nature of the crisis or how transmission and

drug use are contributing to resistance. Hand in hand with meticulous infection control

must go reductions in the appropriate use of antibiotics (42). Prolonged repetitive

courses of antibiotics. for example streptococcus sore throat or mild oral thrush. should

therefore be discouraged.

In most instances. the human body has within itself the ability to combat infectious

agents without the intervention of antibiotics (53). This is definitely not to say that

streptococcal endocarditis or septicaemia do not require antibiotic therapy. but merely

that pharyngitis or stomatitis do not. It must be borne in mind that drug resistance is due

to use and abuse ofantibiotics.

The body has the capability to respond to invasion by an infectious organism (43). The

defence mechanism will attempt to make the best possible response in order to restore

balance; hence providing the opportunity ofbringing the disturbed immune system back

to equilibrium (52). This response however may not be immediate and certain systems in

the body require stimulation in order to respond. Often, before the appropriate response

is made, the body is bombarded with a course ofantibiotics.

51

Orthodox medicine with its diverse chemico-synthetic medicines causes an alarming

phenomena contrary to the most elementary biological laws. Instead of raising the

individual's defences and improving the individuals homeostasis when the body is

faced with the aggravation factors, on the contrary, it provokes an immuno-suppression

(45). Furthermore with repetitive dosing and inappropriate use, resistance tends to

develop (2). For this reason the medical profession needs to consider and allow for

alte~ative approaches to combating diseases (42). One such alternative form of

treatment is homoeopathy. Homoeopathy is a therapeutic method, which clinically

applies the Law ofSimilars, to stimulate the immune system in an attempt to cure the

body from the morbific! infectious agent.

Numerous studies in the field of homoeopathy have demonstrated that homoeopathic

substances have a stimulatory effect. However, the purpose of this study is to determine

the direct effect of homoeopathic Baptisia tinctoria and Thuja occidentalts, in various

potencies, on the growth production of S. pyogenes and C. albicans respectively. The

study has determined whether Baptisia tinctorta (3ch, 15ch and 30ch) and Thuja

occidentalis (3ch,15ch and 30ch) demonstrate fungistatic! fungicidal or bacteriostatic!

bacteriocidal qualities.

4.1 Standard Curve

The standard curves for S. pyogenes and C. albicans were determined according to the

correlation of dilutions for each organism to determine the OD and colony formation

units (cfulml). Conversion formulae were obtained from the standard curve for each

organism. These formulae were employed to calculate colony forming units (cfulml) by

substitution of the 00 readings into the respective formulae, for each organism, when

treated with the Baptisia tinctoria (3ch, 15ch and 3Och) and Thuja occidentalis (3ch,

15ch and 30ch).

52

4.2 Broth Dilution Method


4.2.1.1 Baptisiatinctoria treatment groups

Baptisia tinctoria 3ch reflected an antimicrobial action upon the growth production of

S. pyogenes in the broth dilution method. Although these readings may not be

significant in orthodox medicine, it has indicated that the homoeopathic treatment has

had a definite action on the organism. Analysis of the results reflected that Baptisia

tinctoria 3ch displayed a slight bacteriostatic action. The mechanism of action of

orthodox medicine is different to that of homoeopathic medicine. Penicillin G has the

ability to inhibit bacterial cell wall synthesis through the inhibition of the membrane

bound enzymes which are necessary for bacterial cell wall strength and rigidity (31,33).

whereas homoeopathic remedies stimulate the immune system to overthrow the disease

condition. Analysis of the results ofBaptista tinctoria 15ch and 30ch illustrated that the

remedy, in these potencies, did not produce any bacteriostatic nor bacteriocidal activity

on S. pyogenes.

4.2.1.2 11III)a occidentalis treatment groups

11III)a occtdentalis 3ch reflected a slight inhibitory action on the growth production of

S. pyogenes. However. this action was so minimal that the results are not substantial to

conclude that the remedy has any bacteriostatic properties. 11III)a occidentalis 15ch and

30ch displayed no antimicrobial activity. There was no significant difference between

the control group and that of 11III)a occidentalis 15ch and 30ch. Hence. 171I1)a

occidentalis should not be prescribed as an antimicrobial treatment. The results however

do not rule out 111I1)a occidentalis in treatment of S. pyogenes because the

homoeopathic prescription is based on the totality ofpr~ntingsymptoms and may still

result in cure through the stimulation of the immune system (45).

S3


4.3.1.1 Baptisiatinctoria treatment groups

Baptisia tinetoria 3ch, 15ch and 30ch reflected an inhibitory action upon the growth

production of C. a/bieans, in the broth dilution method. There was a significant

difference between the treatment groups and the control group. The results imply that

Baptisia tinetoria, particularly in the 3ch potency, exerted a fungistatic action upon

C. albicans. Ifthese results are proved to be conclusive, homoeopathic intervention may

be possible as a first line treatment in C. a/bicans infections. It can be speculated that

the decrease in growth production of C. albicans is lowered sufficiently with Baptista

tinetoria 3ch, that the body's immune response may be strong enough to restore the

body to health. Homoeopathic medication is also compatible with allopathic

medication, and the solution may lie in using homoeopathic and allopathic treatments in

conjunction. Patients most commonly predisposed to C. albicans infections are those

who are imrnuno-compromised or debilitated (2). Homoeopathy is beneficial in these

cases because it stimulates the immune system, thus strengthening the overall health

status of the patient. This would be very beneficial to CMC patients who suffer from

recurrent candidiasis and require frequent courses of antibiotics. Immuno-compromised

or debilitated patients are also far too weak to still have to endure the side-effects as a

result of conventional treatment. Homoeopathy is well tolerated, suitable for all patients

and can be used in long-term therapy. It provides a safe, non-toxic alternative to the

treatment of infectious diseases (43,52,54). Homeopathy can also be used as

preventative treatment against hospital acquired pathogens.

4.3.1.2 Thuja occidentalis treatment groups

Interpretation of the results of Thuja oceidenta/is on the growth production of

C. albicans demonstrated that Thuja occtdentalis 3ch displayed an inhibitory effect.

There was a significant difference between Thuja occidentalis 3ch and the control

group. The medication has the ability to decrease the growth production of C. albicans

to almost half the cfulml when compared to that of the control. The decrease in cfulml

may be significantly lowered so as to allow the body's own immune system to

overcome the infection and restore the body to health. Thuja occidentalis 15ch and

30ch displayed no fungistatic nor fungicidal activity. There was no significant

difference between these treatment groups and the control group.

Discrepancies existed between the efficacy of the potencies of Thuja occidentalis and

Baptisia tinctoria respectively. This may be due to the fact that the 3ch potencies have

not surpassed Avogadro's number; which represents the number of molecules contained

in a molecule gram of substance (45). It is equal to 6.23xlO-2J (42). The molecule is

reputed to be non-divisible by the physical means used in the course of the dilution

process, therefore from the llch potency on there remains no more active substance in

the homeopathic dilutions (45). Hence, 15ch and 30ch potencies have surpassed

Avogadro's number and are said to contain no original substance.

4.3 Results of Disc Diffusion Method


The results of Baptisia tinctoria and Thuja occidentalis treatment groups were not

significant in the disc diffusion method. The discs that were impregnated with Thuja

occidentalis (3ch, 15ch and 30ch) and Baptista tinctoria (3ch, 15ch and 30ch) reflected

no clear zones of inhibition on S. pyogenes. The organism was not susceptible to the

homoeopathic treatments. It can therefore be speculated that the homoeopathic remedies

employed in the disc diffusion method displayed no bacteriostatic nor bacteriocidal

activity on S. pyogenes. Penicillin G proved to be effective as a bacteriostatic treatment

as the measured zones of inhibition were within the guidelines for susceptibility (2).

55

4.3.2 Candida a/bieans

Baptisia tinetoria (3ch, 15ch and 30ch) and Thuja oeeidenta/is (3ch, 15ch and 30ch)

treatment results, obtained from the disc diffusion, were not clinically significant. The

discs that were impregnated with homoeopathic substances reflected no clear zones of

inhibition on C. albieans. Hence, the homoeopathic substances employed in the disc

diffusion method displayed no fungistatic nor fungicidal action on C. albicans. Nystatin

proved to be effective as a fungistatic treatment as the measured zones of inhibition

were within the guidelines for susceptibility (2).

S6

5.0 CONCLUSION

5.1 In Conclusion

In conclusion, this research has served as an indication of the ability of Thuja------oeeidentalis-andBaptisia tinctoria to exert a direct antibacterial and antifungal effect on

;

the growth production ofS. pyogenes and C. a/hieans respectively. Insufficient in vitro

evidence was obtained to establish the mechanism of action of these remedies and

hence, it is advisable that further in vivo and in vitro studies be conducted before a

conclusion can be drawn about the exact mechanism of action of these homeopathic

treatments. More sensitive research techniques will therefore need to be employed in

order to verify these phenomena.

In addition, this research has added to the developing volume of evidence which

indicates that the efficacy of homoeopathy is not due to the placebo effect, and has

ultimately added to the data base of information available to explain the therapeutic

efficacy ofhomoeopathic remedies.

5.2 Recommendation

Further research needs to be conducted in order to determine whether the obtained

results with Baptista tinctorta treatment groups (particularly 3ch) on the growth

production of C. albicans are conclusive. In vivo studies may determine whether

intervention with these homoeopathic treatments may be possible as a first line

treatment in C. albicans infections in immuno-compromised, CMC or debilitated

patients that are allergic, non-responsive or display side-effects to conventional

treatment. To verify these results, more sensitive studies on C. albicans will be

required. Patient grouping (allopathic control and. allopathic and homoeopathic

treatment groups), can be conducted to determine whether intervention with

homoeopathic treatment is more effective in facilitating cure than with administration of

allopathic treatment alone.

57

Further studies would In addition reveal whether Thuja occidentalis and Baptisia

tinctoria could be used as an adjunctive therapy with antibiotics and for the removing of

symptoms that continue to linger post-infection.

Research should also be conducted on a larger pool of organism isolates employing a

wider range of homoeopathic remedies. This will reflect a more accurate statistical

picture of the antimicrobial qualities of homoeopathic substances. According to the

results obtained from this research, it is not advisable to conduct further

experimentation on higher potencies in order to determine whether these potencies

demonstrate a direct antimicrobial activity. However, further in vitro research can be

conducted on low potencies as it appears, as reflected in this research, that the lower

potencies are more effective in demonstrating antimicrobial activity.

58

APPENDIX A:

OD readings and cfulml used to plot standard curve ofS.pyogenes isolates according toa dilution scale.

Isolate·:: :1!\1i!]':9 D ~;1:crulml

la,,'·'Pure 1,172 38500

1st j 0,206 3850

2nd 0,055 220

3rd 0,033 55

4th 0,016 27

5th 0,011 8

6th 0,009 °IbPure 1,168 44800

1st 0,25 4480

2nd 0,06 236

3rd 0,038 66

4th 0,022 30

5th 0,02 25

6th 0,007 2

IePure 1,159 49800

1st 0,236 4980

2nd 0,052 216

3rd 0,036 78

4th 0,021 30

5th 0,016 J3

6th 0,005 °%aPure 1,225 50900

1st 0,246 5090

2nd 0,074 298

3rd 0,037 72

4th 0,03 48

5th 0,027 40

6th 0,019 30

2b· »,

Pure 1,191 29600

1st 0,21 2960

2nd 0,035 72

3rd 0,028 52

4th 0,026 33

5th 0,025 12

6th 0,01 6

IsOlate:; :· .. ·OD :dulml

zePure 1,258 530001st 0,242 5300

2nd 0,039 140

3rd 0,034 92

4th 0,019 36

5th 0,022 30

6th 0,011 5

3aPure 1,19 51500

1st 0,224 5150

2nd 0,078 270

3rd 0,038 76

4th 0,015 20

5th 0,01 126th 0,006 73bPure 1,166 77400

1st 0,228 77402nd 0,043 208

3rd 0,039 134

4th 0,022 42

5th 0,016 10

6th 0,014 6

3cPun:: 1,206 608001st 0,246 60802nd 0,048 1563rd 0,033 1064th 0,022 50

5th 0,011 86th 0,009 °b.Pure 1,018 848001st 0,212 84802nd 0,068 1463rd 0,042 1554th 0,028 385th 0,024 °6th 0,013 °

Isolate 'cOD :·dulml4bPure 1,214 985001st 0,25 98502nd 0,073 2903rd 0,044 1684th 0,031 525th 0,026 146th 0,012 °4ePure 1,198 96500

1st 0,278 96502nd 0,06 2103rd 0,048 1724th 0,036 48

5th 0,025 146th O,OIl 10

SaPure 1,089 700001st 0,213 70002nd 0,045 320

3rd 0,039 1084th 0,03 40

5th 0,024 30

6th 0,01 °5bPure 1,042 47500

1st 0,23 4750

2nd 0,081 3103rd 0,036 644th 0,029 315th 0,017 196th 0,013 7sePure 1,123 391001st 0,241 39102nd 0,047 2023rd 0,03 584th 0,022 305th 0,015 146th 0,011 °

59

Sa! ';H,:OD :·cfulmlPure 1,201 469001st 0,21 46902nd 0,042 2583rd 0,031 684th 0,014 185th 0,009 06th 0,009 06b';::",,......._....

Pure 1,128 338001st 0,263 33802nd 0,048 2163rd 0,029 46

4th 0,026 325th 0,013 12

6th 0,008 2

6cPure 1,2 50400

1st 0,203 50402nd 0,048 208

3rd 0,032 804th 0,024 255th 0,015 156th 0,011 4

7aPure 1,168 444001st 0,236 4440

2nd 0,051 168

3rd 0,035 724th 0,027 36

5th 0,024 30

6th 0,015 4

7bPure 1,176 72400

1st 0,227 7240

2nd 0,049 208

3rd 0,04 124

4th 0,031 46

5th 0,018 16

6th 0,02 30

7e:':::':,<:Pure i.u 35200

1st 0,222 3520

2nd 0,04 144

3rd 0,024 56

4th 0,022 30

5th 0,014 10

6th 0,008 0

Sa OD crulmlPure 1,201 469001st 0,21 46902nd 0,042 2583rd 0,031 684th 0,014 185th 0,009 06th 0,009 08bPure 1,209 531001st 0,226 53102nd 0,055 2423rd 0,039 82

4th 0,023 325th 0,017 306th 0,012 0

BePure 1,144 524001st 0,258 52402nd 0,042 1683rd 0,036 884th 0,025 345th 0,02 306th 0,01 09.Pure 1,189 114100Ist 0,272 114102nd 0,073 2623rd 0,043 2024th 0,026 385th 0,019 306th 0,013 09bPure 1,228 611001st 0,227 61102nd 0,058 2023rd 0,039 1024th 0,026 305th 0,019 166th 0,013 09c: ,', "Pure 1,3 442501st 0,198 44252nd 0,05 2053rd 0,032 684th 0,019 145th 0,02 186th 0,009 0

lOa OD dulmlPure 1,063 31200

1st 0,198 31202nd 0,041 1243rd 0,026 504th 0,023 345th 0,02 176th 0,016 4lObPure 1,177 444001st 0,209 44402nd 0,061 1983rd 0,038 694th 0,034 555th 0,022 306th 0,012 0

lOe:Pure 1,15 651001st 0,204 65102nd 0,06 2223rd 0,04 1084th 0,036 685th 0,018 126th 0,02 18llaPure 1,154 461001st 0,213 46102nd 0,042 1623rd 0,037 764th 0,029 535th 0,014 156th 0,01 4llbPure 1,208 542001st 0,216 54202nd 0,04 1843rd 0,039 904th 0,024 395th 0,018 96th 0,012 6HePure 1,11 352001st 0,222 35202nd 0,04 1443rd 0,024 564th 0,022 305th 0,014 106th 0,008 0

60

12a, );jECii1:0D d'ulmlPure 0,998 470001st 0,226 47002nd 0,041 1903rd 0,035 754th 0,026 485th 0,017 126th O,OIl 3Ubi·

Pure 1,176 504001st 0,206 50402nd 0,048 1883rd 0,026 824th 0,025 405th 0,02 326th 0,007 0

12cPure 1,11 611001st 0,265 61102nd 0,045 2023rd 0,037 1024th 0,022 305th 0,01 10

6th 0,015 14

61

APPENDIXB:

OD readings and cfulml used to plot standard curveof C. a/bieans isolates according toa dilution scale.

Isolate·;' mmm,QD m~¢fUIml

1a:::···

Pure 0,482 890501st 0,098 89052nd 0,035 3613ed 0,028 1424th 0,025 805th 0,013 56th 0,011 31bPure 0,52 906501st 0,9 90652nd 0,032 3733ed 0,026 1444th 0,023 55

5th 0,018 146th 0,016 4

1ePure 0,485 961001st 0,092 9610

2nd 0,036 3023rd 0,028 162

4th 0,022 42

5th 0,02 38

6th 0,018 6

ZaPure 0,489 115000

1st 0,059 11500

2nd 0,033 300

3ed 0,029 200

4th 0,021 38

5th 0,013 2

6th 0,009 0

2bPure 0,548 ooסס10

1st 0,081 toooo2nd 0,032 290

3ed 0,028 1714th 0,03 166

5th 0,015 20

6th 0,002 0

Isolate;. ',··::::·'OD /dulml2e •••.. ,.:

Pure 0,52 81000Ist 0,079 81002nd 0,038 4003rd 0,028 1224th 0,015 285th 0,014 66th 0,021 323aPure 0,514 1041001st 0,084 104102nd 0,031 3323ed 0,029 1754th 0,019 365th 0,013 66th 0,014 8JbPure 0,389 739001st 0,057 73902nd 0,034 3083ed 0,022 1174th 0,011 115th 0,015 136th 0,005 03cPure 0,456 734001st 0,062 73402nd 0,035 3683ed 0,028 1104th 0,015 125th 0,018 306th 0,009 04aPure 0,434 1201001st 0,082 120102nd 0,048 3823ed 0,03 2024th 0,019 425th 0,008 76th 0,008 4

Isolate .·'.'OD "dulml4bPure 0,444 893001st 0,074 89302nd 0,047 4963rd 0,027 1294th 0,017 315th 0,008 86th 0,005 04ePure 0,415 891001st 0,089 89102nd 0,032 4023rd 0,03 1384th 0,024 425th 0,01 66th 0,009 2SaPure 0,539 1010001st 0,106 101002nd 0,052 4803rd 0,026 1544th 0,018 525th 0,009 IS6th 0,011 6SbPure 0,509 960001st 0,094 96002nd 0,031 4203ed 0,026 1504th 0,022 325th 0,014 06th 0,011 0sePure 0,51 925001st 0,089 92502nd 0,035 2703ed 0027 1584th 0,023 805th 0,014 166th 0,008 5

62

6a.:, ;,mrOD Ud'ulmlPure .0,395 895001st 0,09 89502nd 0,028 2903rd 0,023 150

4th 0,018 355th 0,008 26th 0,009 96b'[;·:.Pure 0,415 810001st 0,089 81002nd 0,031 2203rd 0,028 1404th 0,026 1105th 0,018 306th 0,004 0

6cPure 0,412 47800

1st 0,085 47802nd 0,046 356

3rd 0,026 60

4th 0,011 2

5th 0,013 4

6th 0,009 0

7aPure 0,586 161000

1st 0,134 16100

2nd 0,088 960

3rd 0,034 226

4th 0,033 186

5th 0,031 136

6th 0,021 32

7bPure 0,57 216000

1st 0,133 21600

2nd 0,08 1320

3rd 0,049 300

4th 0,029 150

5th 0,039 225

6th 0,014 14

7c .' OD .:dulmJPure 0,523 1495001st 0,126 149502nd 0,042 3503rd 0,04 2644th 0,02 605th 0,022 656th 0,018 208a ...

Pure 0,514 1038001st 0,11 103802nd 0,059 4963rd 0,038 1584th 0,011 285th 0,004 06th 0,008 68bPure 0,534 1225001st 0,111 122502nd 0,051 5503rd 0,029 1904th 0,02 385th 0,014 166th 0,008 6BePure 0,521 1420001st 0,132 142002nd 0,05 4803rd 0,038 2364th 0,015 155th 0,026 506th 0,013 69aPure 0,512 1063001st 0,088 106302nd 0,052 4663rd 0,035 1664th 0,02 425th 0,011 146th 0,01 10

9b OD dulmJPIue 0,502 91000

1st 0,085 91002nd 0,063 6203J"d 0,034 120

4th 0,026 825th 0,009 126th 0,007 49c:Pure 0,546 825001st 0,09 82502nd 0,046 4503rd 0,036 1204th 0,028 765th 0,016 306th 0,008 4lOaPure 0,47 273001st 0,088 27302nd 0,027 1463rd 0,016 404th 0,023 46Sth 0,008 126th 0,002 0JObPure 0,415 980001st 0,089 98002nd 0,035 3603rd 0,028 1604th 0,016 30Sth 0,008 66th 0,01 10JOePure 0,489 800001st 0,102 80002nd 0,04 3803rd 0,028 1224th 0,024 485th 0,016 336th 0,015 18

63

11a·;.;.· •••. •· tm1iss!tOD ""':du/mlPure 0,634 1290001st 0,136 129002nd 0,042 4803ed 0,03 2104th 0,016 375th 0,013 126th 0,012 10llb';

Pure 0,628 360001st 0,13 36002nd 0,04 3203rd 0,018 404th 0,016 305th 0,011 226th 0,01 6

HePure 0,529 875001st 0,125 87502nd 0,06 4903rd 0,028 126

4th 0,016 36

Sth 0,013 20

6th 0,01 1012aPure 0,527 530001st 0,122 5300

2nd 0,069 S603rd 0,017 50

4th 0,015 32

5th 0,014 226th 0,005 0

12b ".Pure 0,61 39800

1st 0,09 3980

2nd 0,039 296

3ed 0,02 SO4th 0,017 40Sth 0,015 30

6th 0,005 °

12c: OD cfulmlPure 0,562 730001st 0,115 73002nd 0,036 2603ed 0,026 1204th 0,02 485th 0,013 146th 0,011 12

64

APPENDIXC:

on readings and cfulml of S. pyogenes control isolates and isolates treated withBaptisiatinctoria 3ch, 15ch and 30ch in broth dilution

, Isolate,; ;;,)ijijEPD );::trwmlla">',::",

Control 1,119 527883ch 0,827 3850915ch 0,935 4379030ch 0,912 42665Ib ,'" 'Control 1,126 531303ch 0,827 3850915ch 1,117 5269030ch 1,119 52788IeControl 1,202 568463ch 0,789 3665115ch 0,982 4608830ch 1,168 551842aControl 1,219 576783ch 0,912 4266515ch 1,142 5391230Ch 1,081 509292bControl 1,197 566023ch 0,811 3772615ch 0,978 4589330Ch 1,058 498052<:Control 1,168 551843ch 0,78 3621115th 1,087 5122330Ch 1,116 526413aControl 1,2 567493ch 0,825 3841115th 0,929 4349730Ch 1,137 53668JlrEi'i;Xi",Control 1,209 571893ch 0,917 42910ISch 1,219 5767830ch 1,005 47213

bOJate,t; " -'on "dulml3<:Control 1,219 582453ch 1,105 5210315ch 1,002 4706630ch 1,306 619324aControl 1,168 551843ch 1,002 4706615ch 1,202 5684630ch 1,106 521524bControl 1,221 577753ch 0,853 3978015ch 0,925 4330130ch 1,094 51565ok

Control 1,25 591943ch 0,956 4481715ch 1,002 4706630ch 1,205 56993SaControl 1,118 527393ch 1,006 4726215th 1,131 5337430ch 1,135 53570SbControl 1,821 871153ch I,ll 5234815th 1,112 5244530ch 1,117 52690seControl 1,203 568953ch 1,058 49805ISch 1,282 6075830ch 1,305 618836._+~ __:Control 1,146 541083ch 1,011 47506ISch 1,082 5097830ch 1,117 52690

Isolate :: ,:, OD dulml6bControl 1,179 557223ch 1,084 5107615ch 1,185 5601530ch 1,119 527886cControl 1,168 551843ch 1,018 4784915ch 1,181 5581930ch 1,111 523967aControl 1,179 557223ch 1,084 5107615ch 1,085 5112530ch 1,186 560647bControl 1,168 551843ch 1,018 4784915ch 1,181 5581930ch 1,111 523967cControl 1,204 569.J43ch 1,005 4721315ch 1,102 5195630ch 1,199 56700SaControl 1,177 556243ch 1,082 5097815th 1,133 5347230ch 1,192 563578bControl 1,183 559173ch 0,971 45550ISch 1,194 5645530ch 1,196 56553Se'"Control 1,208 571403ch 0,905 42323IScb 1,008 4736030cb 1,158 S4695

65

9a:··· ;)1;]if'OD :Ic:fuImlControl 1,192 563573ch 1,037 4877815ch 1,033 4858230ch 1,015 477029b; ...

Control 1,195 565043ch 0,935 4379015ch 0,988 4638230ch 1,257 595369c:Control 1,192 563573ch 0,856 3992715ch 0,963 4515930ch 1,202 56846lOaControl 1,216 575313ch 0,914 4276315th 1,079 5083230ch 1,069 50343lObControl 1,22 577273ch 1,098 5176115ch 1,182 5586830ch 1,071 50440lOeControl 1,1OS 521033ch 0,893 4173615th 1,001 4701730ch 1,085 S1125

11.Control 1,258 595853ch 1,139 5376615ch 1,011 4750630ch 1,09 51370neControl 1,143 539613ch 0,898 41981IScll 0,911 4261630ch 1,002 47066

11ei--"";Control 1,258 59585

3ch 0,898 41981ISch 0,879 4105230ch 1,253 59340

12a .\OD dulmlControl 1,159 547443ch 1,134 5352115ch 1,185 5601530ch 1,188 5616212bControl 1,138 537173ch 0,809 3762915ch 0,806 3748230ch 0,963 4515912cControl 1,36 645733ch 0,89 4159015ch 0,9 4207930ch 1,008 4736013aControl 1,204 569443ch 1,007 4731115ch 1,018 4784930ch 1,133 5347213bControl 1,1901 564553ch 1 4696915ch 1,002 4706630ch 1,216 5753113cControl 1,24 587053ch 1,036 4872915ch 1,1 5185930ch I 4696914.Control 1,206 570423ch I 46969ISch 1,002 4706630ch 1,12 52837J4bControl 1,209 571893ch 1,076 S0685ISch 1,201 5679730ch 1,2 5674914C;~:

Control 1,195 56S043ch 1,194 S64SSISch 1,271 6022030ch 1,198 S6651

ISa OD dulmlControl 1,211 572863ch 1,203 5689515ch 1,222 5782430ch 1,2 56749ISbControl 1,193 564063ch 0,908 4247015ch 1,022 4804430ch 1,197 566021StControl 1,287 610033ch 0,928 4344815ch 0,978 4589330ch 1,025 4819116aControl 1,192 563573ch 0,945 4427915ch 0,915 4281230ch 1,052 4951116bControl 1,203 568953ch 1,143 5396115ch 1,168 5518430ch 1,151 5435216cControl 1.258 595853ch 0.865 4036715ch 0,936 4383930ch 1,058 4980517.Control 1,159 547443ch 0,926 4335015ch 0,958 4491530ch 1,008 4736017bControl 1,125 530813ch 1 46969ISch 0,924 4325230ch 1,012 4755517c . i

Control 1,159 547443ch 0,954 4471915ch 1,069 5034330ch 1,023 48093

66

18a' ';:&,0D:;dulmlControl 1,191 563083ch 1,005 4721315ch 0,905 4232330ch 1,066 5019618b:"Control 1,19 562603ch 1,042 4902215ch 1,072 5048930ch 1,148 5420618c:,'L'Control 1,136 536193ch 0,934 4374115ch 0,91 4256830ch 1,001 4701719aControl 1,187 561133ch 1,005 4721315ch 1,049 4936530ch 1,056 4970719bControl 1,254 593893ch 1,147 5415715ch 1,025 4819130ch 1,102 5195619c:Control 1,296 614433ch 1,058 4980515ch 0,879 4105230ch 1,068 5029420aControl 1,201 567973ch 1,049 4936515ch 0,998 4687130ch 1,12 52837lObControl 1,25 591943ch 0,906 4237215cl1 0,923 4320330ch 0,879 410522OC,:l;:,Control 1,236 58509

3ch 0,89 41590lSch 0,924 4325230ch 1,047 49267

67

APPENDIXD:

OD readings and cfulml ofS. pyogenes control isolates and isolates treated with Thujaoccidentalis 3cll, 15ch and 30ch in broth dilution

ISOliteL sL[;:iOD .:::-db/mlla::>Control 1,119 527883ch 0,975 4574615ch 1,111 5239630ch 1,126 53130lb· ..•. '.Control 1,126 531303ch 1,147 5415715ch 1,135 5357030ch 1,189 56211IeControl 1,202 568463ch 0,928 4344815ch 1,369 6501330ch 1,247 590472aControl 1,219 576783ch 0,995 4672415ch 1,123 5298330ch 1,112 524452bControl 1,197 566023ch 0,97 4550215th 1,128 5322830ch 1,197 566022cControl 1,168 551843ch 0,903 4222515th 1,117 5269030ch 1,205 569933aControl 1,2 567493ch 0,997 46822ISch 1,244 5890030ch 1,204 569443bControl 1,209 571893ch 1,116 52641lSch 1,256 5948730ch 1,245 58949

IsOlate:·· ........ 'OD dulml3cControl 1,219 582453ch 1,203 5689515ch 1,258 5958530ch 1,245 589494aControl 1,168 551843ch 0,908 4247015ch 1,173 5542830ch 1,208 571404bControl 1,221 5m53ch 1,053 4956015ch 1,203 5689530ch 1,179 557224cConlrOl 1,25 591943ch 1,087 5122315th 1,369 6501330ch 1,456 69267SaControl 1,118 527393ch 1,058 4980515th 1,15 5430430ch 1,32 626175bControl 1,821 871153ch 1,041 4897315th 1,089 5132130ch 1,107 52201seControl 1,203 568953ch 1,007 47311ISch 1,216 5753130ch 1,208 571406aControl 1,146 541083ch 1,058 4980515th 1,15 5430430ch 1,11 52348

Isolate ,·.OD dulml6bControl 1,179 557223ch 1,015 4770215ch 1,133 5347230ch 1,313 622746cControl 1,168 551843ch 1,025 4819115ch 1,148 5420630ch 1,134 535217aControl 1,179 557223ch 1,019 4789815ch 1,15 5430430ch 1,133 534727bControl 1,168 551843ch 1,125 5308115th 1,148 5420630ch 1,134 535217cControl 1,204 569443ch 1,108 5225015ch 1,114 5254330ch 1,211 572868a

Control 1,177 556243ch 1,19 5626015th 1,195 5650430ch 1,175 555268bControl 1,183 559173ch 0,987 4633315th 1,102 5195630ch 1,144 54010BeControl 1,208 571403ch 1,078 5078315th 1,147 5415730ch 1,469 69903

68

9a·::' .: ,!fi"OD 0id'ulmiControl 1,192 563573ch 1,206 5704215ch 1,205 5699330Ch 1,202 568469b:Control 1,195 565043ch 1,058 4980515ch 1,208 5714030Ch 1,204 569449cControl 1,192 563573ch 1,001 4701715ch 1,234 5841130Ch 1,21 57238lOaControl 1,216 575313ch 1,006 4726215ch 1,154 5449930Ch 1,145 54059lObControl 1,22 577273ch 1,15 5430415ch 1,15 5430430Ch 1,185 56015lOeControl 1,105 521033ch 1,099 51810ISch 1,147 5415730Ch 1,369 6501311.Control 1,258 595853ch 1,129 532n

ISch 1,016 4n51

30Ch 1,163 54939llbControl 1,143 539613ch 1,094 5156515th 1,163 5493930ch 1,15 54304He:.

" ."'

Control 1,258 595853ch 1,245 5894915ch 1,202 5684630ch 1,222 57824

12a OD au/mlControl 1,159 547443ch 1,128 5322815ch 1,131 5337430ch 1,147 5415712bControl 1,138 537173ch 1,002 4706615ch 1,1 5185930ch 1,106 5215212cControl 1,36 645733ch 1,123 5298315ch 1,205 5699330ch 1,207 57091IJaControl 1,204 569443ch 1,153 5445015ch 1,166 5508630ch 1,214 57433IJbControl 1,194 564553ch 1,206 57042ISch 1,153 5445030ch 1,166 55086IJcControl 1,24 587053ch 1,158 54695ISch 1,129 532n30ch 1,145 5405914.Control 1,206 570423ch 1,211 57286ISch 1,198 5665130ch 1,879 8995214bControl 1,209 571893ch 1,16 54793ISch 1,248 5909630ch 1,245 5894914cControl 1,195 565043ch 1,168 5518415ch 1,785 8535530ch 1,185 56015

ISa OD dulmlControl 1,211 572863ch 1,209 5718915ch 1,202 5684630Ch 1,168 55184ISbControl 1,193 564063ch 1,195 5650415ch 1,151 5435230Ch 1,135 53570lScControl 1,287 610033ch 1,128 5322815ch 1,213 5738430ch 1,203 5689516aControl 1,192 563573ch 1,089 5132115ch 1,188 5616230Ch 1,196 5655316bControl 1,203 568953ch 1,172 55379ISch 1,148 5420630Ch 1,124 5303216cControl 1,258 595853ch 1,147 5415715ch 1,587 7567330Ch 1,164 5498817.Control 1,159 547443ch 1,125 53081ISch 1,149 5425530ch 1,13 5332617bControl 1,125 530813ch 1,125 5308115ch 1,111 5239630Ch 1,136 5361917cControl 1,159 547443ch 1,278 6056315ch 1,144 5401030ch 1,174 5S4n

69

18a .•.. ::("OD .·dulmlControl 1,191 563083ch 1,108 5225015ch 1,184 5596630ch 1,15 5430418bControl 1,19 562603ch ,1,176 5557515ch 1,161 5484130ch 1,12 5283718cControl 1,136 536193ch 1,14 5381515ch 1,147 5415730ch 1,137 5366819aControl 1,187 561133ch 0,923 4320315ch 1,178 5567330ch 1,184 5596619bControl 1,254 593893ch 1,025 4819115ch 1,087 5122330ch 1,125 5308119cControl 1,296 614433ch 1,106 5215215ch 1,187 5611330ch 1,298 6154120aControl 1.201 567973ch 1,147 5415715ch 1,099 5181030ch 1,21 5723820bControl 1,25 591943ch 1,124 53032ISch 1,157 5464630ch 1,269 60123

20c OD dulmlControl 1,236 585093ch 1,188 5616215ch 1,168 5518430ch 1,209 57189

70

APPENDIXE:

Disc diffusion method displaying zone diameters, measured in millimetres (rnm), on

S. pyogeneswhen treated with Penicillin G, Bacitracin and Baptista tinctoria 3ch, 15ch

and 30ch.

Isolate PeniciJDn G Bacitracin Baptisia Baptisla BaptisiaZOne diameter ZOne diameter tinaoria 3ch tinaori« 15ch tindOM 30ch

la 33mm lSmm 9mm 0 01b 37mm I6mm 0 0 0Ie 37mm 17mm 0 0 02a 43mm 16mm 0 0 02b 43mm lSmm 0 0 7.Smm2e 4lmm ISmm 0 0 03a 40mm 14mm 0 0 03b 40mm 17mm 0 0 03e 39mm 17mm 0 0 04a 40mm 18mm 0 0 04b 39mm 14mm 0 0 04c 37mm 16mm 7.Smm 0 0Sa 39mm 16mm 0 0 0Sb 41mm 17mm 0 7.Smm 0Se 40nun lSnun 0 0 06a 41mm lSmm 0 0 06b 4lmm 14mm 0 0 06c 40mm ISmm 0 0 07a 36nun 14mm 0 0 07b 3Snun 17mm 7.Smrn 0 07e 37mm ISmm 0 0 08a 3Snun 17mm 0 0 08b 36mm I5nun 0 0 08c 37mm ISmm 0 0 09a 37mm 14mm 0 0 09b 39nun 13mm 0 0 0ge 39mm ISmm 0 0 0lOa 43mm ISmm 0 0 0lOb 42mm lSmm 0 0 0lOe 4lmm 16mm 0 0 011a 37mm 14mm 0 0 011b 38mm 12mm 0 0 0He 39mm lSmm 0 0 0118 43mm 14mm 0 0 012b 41mm 13mm I) 0 012c 38mm 12mm 0 0 0

71

13a 36mm 13mm 0 0 013b 39mm 15inrn 0 0 013c 39mm ISmm 0 0 014a 37mm 16mm 0 0 014b 41mm 16mm 0 7.Smrn 014c 41mm 15mm 0 0 015a 40mm 17mm 0 0 015b 41mrn 17mrn 0 0 015c 37mm 17mm 0 0 016a 36mrn 13mm 0 0 016b 39mrn 13mm 0 0 016c 37mm 14mm 0 0 017a 41mm 15mrn 0 0 017b 38mm ISmm 0 0 017c 36mm lSmm 0 0 0183 37mm 16mm 0 0 018b 39mm 15mm 0 0 018c 39mm 15mm 0 0 019a 38mm 17mm 0 0 019b 42mm 17mm 0 0 019c 39mm 16mm 0 0 020a 41mm 17mm 0 0 020b 39mm 16mm 0 0 020C 41mm 17mm 0 0 0

ri

APPENDIXF:

Disc diffusion method displaying zone diameters, measured in millimetres (mm), on

S. pyogenes treated with Penicillin G. Bacitracin and Thuja occidentalis 3ch, 15ch and

30ch.

Isolate Penicillin G Bacitracin Thuja Thuja ThujaZonediameter Zonediameter occidentalis . ocddentalis occidentalis

3th 15th 30thla 33mm 15mm 0 0 0Ib 37mm 16mm 0 0 0Ie 37mm 17mm 0 0 02a 43mm 16mm 0 0 02b 43mm ISmm 0 0 02c 41mm 15mm 0 0 03a 40mm 14mm 0 0 03b 40mm 17mm 0 0 03c 39mm 17mm 0 0 04a 40mm J8mm 0 0 04b 39mm 14mm 0 0 04c 37mm 16mm 0 0 0Sa 39mm 16mm 0 0 0Sb 4lmm 17mm 0 0 0Se 40mm J5mm 9mm 0 06a 4lmm 15mm 0 0 06b 4lmm 14mm 0 0 06c 40mm 15mm 0 0 07a 36mm 14mm 0 0 07b 35mm 17mm 0 0 07e 37mm 15mm 0 0 0Sa 35mm 17mm 0 0 08b 36mm 15mm 0 0 08c 37mm 15mm 0 0 09a 37mm 14mm 0 0 09b 39mm 13mm 0 0 09c 39nun 15mm 0 0 0lOa 43mm 15mm 0 0 0lOb 42nun ISmm 0 0 0lOe 41mm 16mm 0 0 0lla 37mm 14mm 0 0 0lIb 38mm 12mm 0 0 0He 39mm 15mm 0 0 0118 43mm 14mm r) 0 012b 41mm 13mm 0 0 012c 38mm 12mm 0 0 013a 36mm 13mm 0 0 0

73

--------_ .......

13b 39mm ISmm 0 0 013c 39mm ISmrn 0 0 014a 37mm 16mrn 0 0 014b 41mm 16mm 0 0 014c 41mm ISmrn 0 0 0ISa 40mm 17mm 0 0 0ISb 41mm 17mm 0 0 0ISc 37mm 17mm 0 0 016a 36mm 13mrn 0 0 016b 39mm 13mm 0 0 016c 37mm 14mm 0 0 017a 41mm ISmm 0 0 017b 38mm ISmrn 0 0 017c 36mm ISmrn 0 0 0lSa 37mm 16mm 0 0 018b 39mm ISmm 0 0 018c 39mm ISmm 0 0 019a 38mm 17mm 0 0 019b 42mm 17mm 0 0 019c 39mm 16mm 0 0 020a 41mm 17mm 0 0 020b 39mm 16mm 0 0 020c 41mrn 17mm 0 0 0

74

APPENDIXG:

OD readingsand cfulmlofC. albicanscontrol isolatesand those treated withBaptisiatinctoria 3ch, 15chand 30ch in broth dilution

Isolate,- ':::"OD ')·d'ulmJ1aControl 0,335 561173eh 0,057 6355

15eh 0,122 1799030ch 0,156 24076Ib .Control 0,351 589813eh 0,056 6176

15eh 0,128 19064

30ch 0,167 26045

IeControl 0,342 57370

3eh 0,068 8324

15ch 0,086 11546

30ch 0,245 40007

2aControl 0,341 57191

3eh 0,086 11546

15eh 0,107 15305

30ch 0,183 28909

2bControl 0,398 67394

3eh 0,097 13515

15eh 0,163 25329

30ch 0,155 23897

2cControl 0,38 64172

3ch 0,101 14231

15th 0,084 11188

30ch 0,148 22644

3.Control 0,288 47704

3eh . 0,058 6534

15ch 0,068 8324

30ch 0,26 42692

3bControl 0,398 67394

3ch 0,086 11546

15th 0,111 16021

30cb 0,246 40186

Isolate '- ·OD du/ml3eControl 0,326 54506

3eh 0,07 8682

15eh 0,082 1083030ch 0,253 41439

4aControl 0,447 761653eh 0,1 1405215ch 0,18 28372

30ch 0,156 24076

4bControl 0,436 74196

3eh 0,064 7608

15eh 0,067 8145

30ch 0,182 28730

4c

Control 0,456 77776

3ch 0,087 1172515eh 0,104 14768

30ch 0,169 26403

SaControl 0,358 602343eh 0,152 23360

15th 0,147 2246530ch 0,199 31773

5bControl 0,368 62024

3eh 0,131 1960115th 0,151 2318130ch 0,187 29625

5cControl 0,458 781343eh 0,087 1172515th 0,079 1029330ch 0,105 149476.Control 0,36 605923ch 0,063 742915th 0,093 J279930ch 0,215 34637

Isolate OD dulml6bControl 0,244 398283ch 0,123 1816915ch 0,146 2228630ch 0,228 36964

6cControl 0,34 570123eh 0,07 868215th 0,185 2926730Ch 0,205 32847

7.Control 0,44 749123ch 0,061 707115eh 0,133 1995930Ch 0,144 219287b

Control 0,317 528953eh 0,092 1262015ch 0,152 2336030Ch 0,148 22644

7cControl 0,38 641723eh 0,065 7787

15th 0,143 2174930Ch 0,2 319528aControl 0,322 537903ch 0,093 1279915th 0,084 1118830ch 0,272 448408bControl 0,319 532533eh 0,094 1297815th 0,167 2604530ch 0,206 33026kControl 0,387 654253eh 0,078 1011415th 0,1 1405230ch 0,19 30162

75

9a ';;'_':OD ,cfulmlControl 0,351 589813ch 0,154 2371815ch 0,167 2604530ch 0,187 2%259b:Control 0,377 636353ch 0,108 1548415ch 0,188 2980430ch 0,197 314159cControl 0,366 616663ch 0,109 1566315ch 0,117 1709530ch 0,187 29625lOaControl U,31 516423ch 0,081 1065115ch 0,089 1208330ch 0,092 12620lObControl 0,385 650673ch 0,053 563915ch 0,094 1297830ch 0,146 2228610eControl 0,344 5n283ch 0,122 1799015ch 0,101 1423130ch 0,21 33742ltaControl 0,277 457353ch 0,068 832415ch 0,109 1566330ch 0,171 26761libControl 0,401 679313ch 0,128 1906415ch 0,106 1512630ch 0,149 22823ueControl 0,265 435873ch 0,075 957715ch 0,078 1011430ch 0.2 31952

12a OD cfulmlControl 0,331 554013ch 0,084 1118815ch 0,113 1637930ch 0,18 2837212bControl 0,314 523583ch 0,12 1763215ch 0,126 1870630ch 0,165 2568712eControl 0,365 614873ch 0,07 868215ch 0,151 2318130ch 0,102 1441013a

Control 0,335 561173ch 0,069 850315ch 0,084 1118830ch 0,164 2550813bControl 0,274 451983ch 0,081 1065115ch 0,087 1172530ch 0,121 17811IJcControl 0,258 423343ch 0,062 725015ch 0,088 1190430ch 0,197 3141514.Control 0,285 471673ch 0,087 1172515ch 0,108 1548430ch 0,136 2049614bControl 0,246 401863ch 0,084 1118815ch 0,114 1655!J30ch 0,143 2174910k ,-

Control 0,346 580863ch 0,08 1047215ch 0,087 1172530ch 0,122 17990

ISa OD dulmlControl 0,34 570123ch 0,13 1942215ch 0,122 1799030ch 0,256 41976ISbControl 0,367 618453ch 0,089 1208315ch 0,055 599730ch 0,175 274771StControl 0,369 622033ch 0,07 868215ch 0,081 1065130ch 0,159 2461316aControl 0,347 582653ch 0,058 653415ch 0,09 1226230Ch 0,106 1512616b

\·'::ontrol 0,309 514633ch 0,063 742915ch 0,089 1208330ch 0,147 2246516cControl 0,312 520003ch 0,102 1441015ch 0,135 2031730ch 0,147 2246517.Control 0,257 421553ch 0,068 832415ch 0,088 11904JOch 0,249 4072317bControl 0,302 502103ch 0,074 939815ch 0,11 1584230ch 0,14 2121217cControl 0,258 423343ch 0,085 1136715ch 0,089 1208330ch 0,102 14410

76

18a· ,':OD .: cfulml

Control 0,348 58444

3ch 0,079 10293

15ch 0,11 15842

30ch 0,207 33205

18b:.·Control 0,381 64351

3ch 0,075 9577

15ch 0,082 10830

30ch 0,272 44840

18c .....Control 0,302 50210

3ch 0,076 9756

15ch 0,098 13694

30ch 0,187 29625

19aControl 0,38 64172

3ch 0,067 8145

15ch 0,08 10472

30ch 0,139 21033

19bControl 0,402 68110

3ch 0,088 11904

15ch 0,108 15484

30ch 0,156 24076

19cControl 0,3 49852

3ch 0,083 11009

15ch 0,153 23539

30ch 0,14 21212

20.Control 0,293 48599

3ch 0,112 16200

15ch 0,079 10293

30ch 0,128 19064

10bControl 0,367 61845

3ch 0,078 10114

15th 0,084 11188

30ch 0.209 335632Oc:'.•

Control 0,352 59160

3th 0,079 10293

15ch 0,087 11725

30ch 0,148 22644

77

APPENDIXH:

OD readings and cfulml of C. a/bicans control isolates and isolates treated with Thujaoccidentalis 3ch, 15ch and 30ch in broth dilution

Isolate·.··. i.E':OD jdulml1a ••.••

Control 0,335 561173ch , 0,185 2926715ch 0,314 5235830ch 0,323 53969lb·:Control 0,351 589813ch 0,24 3911215ch 0,282 4663030ch 0,307 51105IeControl 0,342 573703ch 0,156 2407615ch 0,276 4555630ch 0,369 622032aControl 0,341 571913ch 0,109 1566315ch 0,334 5593830ch 0,293 485992bControl 0,398 673943ch 0,168 26224ISch 0,291 4824130ch 0,286 473462eControl 0,38 641723eh 0,274 4S19815ch 0,332 SS5SO30ch 0,365 614873aControl 0,288 477043ch 0,148 2264415ch 0,311 5182130ch 0,321 536113bControl 0,398 673943ch 0,26 4269215ch 0,357 6005S30ch 0,426 72406

Isolate OD du/ml3c: .Control 0,326 545063ch 0,154 2371815ch 0,378 6381430ch 0,319 532534aControl 0,447 761653ch 0,148 2264415ch 0,453 7723930ch 0,46 784924bControl 0,436 741963ch 0,178 2801415ch 0,455 7759730ch 0,468 799244eControl 0,456 777763ch 0,286 4734615ch 0,4 6775230ch 0,398 67394SaControl 0,358 602343ch 0,164 25508ISch 0,324 5414830ch 0,439 74733SbControl 0,368 620243ch 0,211 33921l5ch 0,246 4018630ch 0,287 47525seControl 0,458 781343ch 0,158 24434l5ch 0,369 6220330Ch 0,408 691846.Control 0,36 605923th 0,277 4573515ch 0,306 50926.lOch 0,27 44482

Isolate OD dulml6bControl 0,244 398283ch 0,222 3589015ch 0,249 4072330ch 0,159 246136c:Control 0,34 570123ch 0,237 3857515ch 0,354 5951830ch 0,3 498527.Control 0,44 749123ch 0,198 3159415ch 0,464 7920830ch 0,222 358907bControl 0,317 528953ch 0,259 4251315ch 0,46 7849230ch 0,41 695427c:Control 0,38 641723ch 0,17 2658215ch 0,338 5665430ch 0,286 473468aControl 0,322 537903ch 0,263 4322915ch 0,268 44124.lOch 0,271 446618bControl 0,319 532533ch 0,187 29625l5ch 0,439 7473330ch 0,323 53969BeControl 0,387 654253ch 0,3 49852l5ch 0,338 S665430ch 0,288 47704

78

9a.···· .~<:;OD ..• culml

Control 0,351 58981. 3ch 0,248 40544

15ch 0,439 7473330ch 0,456 777769b·

Control 0,377 63635

3ch 0,33 5522215ch 0,321 53611

30ch 0,358 60234

9cControl 0,366 61666

3ch 0,255 41797

15ch 0,306 50926

30ch 0,323 53969

lOa

Control 0,31 51642

3ch 0,26 42692

15ch 0,336 56296

30ch 0,347 58265

lObControl 0,385 65067

3ch 0,145 22107

15ch 0,402 68110

30ch 0,357 60055

lOeControl 0,344 57728

3ch 0,247 40365

l5ch 0,357 60055

30ch 0,315 52537

11.Control 0,277 45735

3ch 0,264 43408

15ch 0,378 63814

30ch 0,392 66320

Ub

Control 0,401 67931

3ch 0,111 16021

ISch 0,364 61308

30ch 0,437 7437511«: .

Control 0,265 43587

3th 0,22 35532

ISch 0,338 5665430ch 0,178 28014

12a on dulmlControl 0,331 55401

3ch 0,106 1512615ch 0,3 4985230ch 0,28 46272

12bControl 0,314 523583ch 0,158 2443415ch 0;£:)8 49494

30ch 0,318 53074

12cControl 0,365 61487

3ch 0,197 3141515ch 0,347 58265

30ch 0,258 42334

13aControl 0,335 56117

3ch 0,259 42513

15ch 0,298 49494

30ch 0,222 35890

13bControl 0,274 45198

3ch 0,153 23539

15ch 0,295 48957

30ch 0,3 49852

IJcControl 0,258 42334

3ch 0,108 15484

l5ch 0,308 51284

30ch 0,227 36785

14.Control 0,285 47167

3ch 0,123 18169

15ch 0,336 5629630ch 0,339 56833

14b

Control 0,246 401863ch 0,155 2389715th 0,304 S056~

30ch 0,258 42334

10kControl 0,346 580863ch 0,208 3338415th 0,334 5593830ch 0,25 40902

ISa on dulmlControl 0,34 57012

3ch 0,127 1888515ch 0,36 60592

30ch 0,3 49852

ISbControl 0,367 618453ch 0,103 1458915ch 0,315 52537

30Ch 0,268 44124IxControl 0,369 622033ch 0,11 1584215ch 0,302 5021030Ch 0,27 4448216a

Control 0,347 582653ch 0,194 3087815ch 0,327 5468530Ch 0,274 4519816bControl 0,309 514633ch 0,282 4663015ch 0,331 5540130Ch 0,305 50747J6cControl 0,312 520003ch 0,192 3052015ch 0,333 5575930ch 0,345 57907

17.Control 0,257 421553ch 0,219 3535315ch 0,227 3678530ch 0,255 4179717b

Control 0,302 S02103ch 0,178 2801415ch 0,389 6578330cb 0,369 6220317cControl 0,258 423343ch 0,262 4305015th 0,299 4967330ch 0,325 54327

79

18a"

,/:;on· dulmlControl 0,348 584443ch 0,283 4680915ch 0,344 5772830ch 0,421 7151118b:Control 0,381 643513ch 0,26 4269215ch 0,311 5182130ch 0,292 4842018e"·" ,.Control 0,302 502103ch 0,201 3213115ch 0,309 5146330ch 0,288 4770419aControl 0,38 641723ch 0,147 2246515ch 0,222 3589030ch 0,302 5021019bControl 0,402 681103ch 0,247 4036515ch 0,452 7706030ch 0,391 661411geControl 0,3 498523ch 0,227 3678515th 0,288 4770430ch 0,307 51105ZOaControl 0,293 485993ch 0,188 2980415th 0,251 4108130ch 0,297 4931520bControl 0,367 618453ch 0,222 3589015th 0,35 5880230ch 0,42 71332zOeControl 0,352 591603ch 0,218 3517415th 0,343 5754930ch 0,351 58981

80

APPENDIX I:

Disc diffusion method displaying zone diameters on C. albicans, measures in

miJlimetres (mm), when treated with Nystatin and Baptisia tinetoria 3ch, 15ch and

30ch.

Isolate Nystatin Baptisia Baptisia BaptisiaZone diameter tinaoria 3ch tinaoria 15ch tinaoria 30ch

Ia I4mm 0 0 0Ib 25mm 0 0 0Ie 25mm 0 0 02a I9mm 0 0 02b 19mm 0 8mm 02e 2lmm 0 0 03a 19mm 0 0 03b 2lmm 0 0 03e 19mm 0 0 04a 2lmm 8mm 0 04b 19mm 8mm 0 04c 22mm 0 0 0Sa 19nun 0 0 0nSb 23mm 0 0 0Sc 22mm 0 0 06a 22mm 0 0 06b 19mm 0 0 06c 23mm 0 0 07a 22mm 0 0 07b 2lmm 0 0 07c 20mm 0 0 0Sa 18mm 0 0 08b 19mm 7.Smm 0 08c 19mm 0 0 09a 21mm 0 0 09b 23nun 0 0 09c 22mm 0 0 0lOa 22mm 0 0 0lOb 21nun 0 0 0lOe - 19mm 0 0 0us : 20mm 0 7.5mm 0lIb 19nun 0 0 0lIe 20mm 0 0 0113 23nun 7mm 0 012b 23mm 0 0 012e 19nun 0 0 0

81

13a 20mm 0 0 013b 20mm 0 0 0l3e 18mm 0 0 014a 19mm 0 0 7.Smm14b 19mm 8mm 0 014e 20mm 0 0 0ISa 22mm 8mm 0 0ISb 21mm 0 0 0ISe 22mm 0 0 016a 21mm 0 0 016b 19mm 0 0 016c 20mm 0 0 017a 20mm 0 0 017b 19mm 0 0 017e 21mm 7mm 0 018a 20mm 0 0 0ISb 21mm 0 7rnrn 018c 19mm 0 0 019a 22mm 0 0 019b 20mm 0 0 8mrn19c 17mm 0 0 020a 17mm 0 0 020b 18mm 8mm 0 020c 19mrn 0 0 0

82

APPENDIXJ:

Disc diffusion method on C. albicans displaying zones of inhibition, measures in

millimetres (mm), when treated with Nystatin and Thuja occidentalis 3ch, 15ch and

30ch.

Isolate Nystatin Thuja Thuja ThujaZOne diameter occidentalis 3ch occidentalislSch occidentalis 30th

la 14mm 0 0 0Ib 25mm 0 0 0Ie 25mm 0 0 02a 19mm 0 0 02b 19mm 0 0 02e 21mm 0 0 03a 19mm 0 0 03b 21mm 0 0 03e 19mm 0 0 04a 21mm 0 0 04b 19mm 0 0 04c 22mm 0 0 0Sa 19mm 7.Smm 0 0Sb 23mm 0 0 0Se 22mm 0 0 06a 22mm 0 0 06b 19mm 0 0 06c 23mm 0 0 07a 22mm 0 8mm 07b 21mm 0 0 07e 20mm 0 0 0Sa 18mm 0 0 08b 19mm 0 0 08c 19mm 0 0 09a 21mm 0 0 09b 23mm 0 0 09c 22mm 0 0 0lOa 22mm 0 0 0lOb 2lmm 0 0 0lOe - 19mm 0 0 0lIa 20mm 0 -0 0lIb 19mm 7.5mm 0 0lIe 20tmn 0 0 0128 23mm 0 0 012b 23mm 0 0 012e 19mm 0 0 0

83

13a 20mm 0 0 013b 20mm 8mm 0 0Be 18mm 0 0 014a 19mm 0 0 014b 19mm 0 0 014e 20mm 0 0 0ISa 22mm 0 0 0ISb 21mm 0 0 0ISe 22mm 0 0 016a ,

21mm 0 0 016b 19mm 0 0 016c 20mm 0 0 017a 20mm 0 0 017b 19mm 0 0 017e 21mm 0 0 018a 20mm 0 0 018b 21mm 0 0 018c 19mm 0 0 019a 22mm 10mm 0 019b 20mm 0 0 01ge 17mm 0 0 020a 17mm 0 0 020b 18mm 0 7.Smm 020c 19mm 0 0 0

7.0 REFERENCES

1. Cappuccino J, Sherman N, 1996. Microbiology: A Laboratory Manual, 4th Edition.

The Benjamin! Cummings Publishing Company, Inc. California, New York,

Amsterdam, Bonn, Paris, Madrid, Sydney, Singapore, Tokyo. ppl,20S,211,381

382.

2. Murray P, Baron E,Pfaller M, Tenover F, Yolken R, 1995. Manual of Clinical

Microbiology, Sixth edition. ASM Press, Washington, D.C. pp5-8,17,169

176,299-302,723,731-732,1102.

3. Rosebury, T, 1981. Microorganisms Indigenous to Man. McGraw-Hili Book Co.,

Balimore. pp2-3.

4. Andreoli T, Carpenter C, Plum F, Smith L, 1986. Cecil Essential of Medicine,

low cost student edition. W.B Saunders Company, Philadelphia, London,

Toronto, Mexico City, Rio de Janeiro, Sydney, Tokyo, Hong Kong. pp60

61,233,526-527,559-560.

5. Salyers A, Whitt D, 1994. Bacterial Pathogenesis: A molecular approach. ASM

Press, Washington D.C. ppI2,36-37,110,332-334.

6. Inglis T, 1996. Microbiology and Infection. Churchill Livingstone, Edinburgh,

New York, London, Madrid, Melbourne, San Francisco, Tokyo. pp90-91,J5J,

191,230.

7. Jawets E, Melnick J, Adelberg E, 1995. Medical Microbiology, 20th edition.

Lange Medical Books, Norwalk, Connecticut. ppI49,153,196-199,545-547

8. Hope R, Longmore J, Hodgens T, Ramrakha P, 1995. Oxford Handbook of

Clinical Medicine. Oxford University Press, Oxford, New York, Tokyo.

pp214,222,304,532.

9. Collee J, Duguid J, Fraser A, Marmion B, 1989. Practical Medical Microbiology,

thirteenth edition. Churchill Livingstone, Edinburgh, New York, London. pp317

321,680-684.

85

10. Horan T, White J, Jarvis W, Emori T, Culver D, Munn V, Thornsberry C, Olson

D, Hughes J, 1986. Nosocomial infections surveillance 1984. Centers for Disease

Control Nosocomial Infection Surveillance, Morbid. Mortal. Weekly Rep.

35: 17S8- 29S8.

11. Odds F, 1988. Candida and Candidosis, 2nd edition. Bailliere Tindall, London.

pp37-42.

12. Hazen K, Brawner D, Riesselman M, Cutler J, Jutita M, 1991. Differential

adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse

tissue. Infect. Immun. 59: 907-912.

13. Phelan J, Saltzman B, Friedland G, Klein R, 1987. Oral findings in patients with

acquired immunodeficiency syndrome. Oral surg. 64:50-56.

14. Tavitian A, Raufman J, Rosenthal L, 1986. Oral candidiasis as a marker for

oesophageal candidiasis in the acquired immuno-deficiency syndrome. Ann.

Intern. Med. 104:54-55.

15. Edwards 1, Filler S, 1992. Current strategies for treating invasive candidiasis:

emphasis on infections in nonneutropenic patients. Clin. Infect. Dis.14 (Suppl. I)

:SI06-S113.

16. Rine 1, Hansen W, Hardeman E, Davis R, 1983. Targeted selection of

recombinant clones through gene dosage effect. Proc. Natl. Acad. Science. USA

80:6750-6754.

17. Whelan W, Kwon-Chung K, 1988. Auxotrophic heterozygosities and the ploidy

ofCandida. J. Med. Vet. Mycol. 26:163-171.

18. Domer J, Kehrer R, 1993. Introduction to Candida: systematic candidiasis, p. 49

116. In J.W. Murphy, H. Friedman, and M. Bendinelli (ed.), Fungal Infections

and Immune Responses. Plenum Press, New York.

19. Filler S, Edwards 1,1993. Chronic Mucocutaneous candidiasis, p. 117-133. In

. 1.W. Murphy, H. Friedman, and M. Bendinelli (ed.), Fungal Infections and

Immune Responses. Plenum Press, New York.

20. Sarasi G, Johnson P, 1992. Disseminated histoplasmosis in patients infected with

human immunodeficiency virus. Clin. Infect. Dis.14 (SuppI. 1):S60-S67.

86

21. Walsh T, Pizzo A, 1988.Treatment of systemic fungal infections: recent progress

and current problems. Eur. J. Clin, Microbiol. Infect. Dis. 7:460-475.

22. Anaissie E, Bodey G, Rinaldi M, 1989. Emergingfungal pathogens. Eur. 1. Clin.

Microbiol. Infect. Dis. 8:323-330.

23. Bullock W, Kozel T, Scherer S, Dixon D, 1993.Medical mycology in the 1990's:

involvementofNllI and the wider community. ASMNews 59:182-185.,

24. Poederly W, Kobayashi G, Herzig G, Medoff G, 1988. Amphotericin B- resistant

yeast infectionsin immunocompromised patients. Am. J. Clin. Microbiol. 24:600

606.

25. Townsend T, Wenzel R, 1981. Nosocomial bloodstream infections in a newborn

intensive care unit: a cross-matched control studyofmorbidity, mortality and risk.

Am. 1. Epidemiol. 114:73-80.

26. Wey S, Mori M, Pflaller M, Woolson R, Wenzel R, 1988. Hospital-acquired

candidemia; attributable mortality and excess length of stay. Arch. Intern. Med.

148:2642-2647.

27. Wenzel R, 1989. The mortality of hospital- acquired bloodstream infections: need

for a new vital statistic?Int. J. Epidemiol. 17:225-227.

28. Wey S, Mori M, Pflaller M, Woolson R, Wenzel R, 1989. Risk factors for

hospital- acquired candidemia: a matched case-control study. Arch. Intern. Med.

149:2349-2353.

29. Foster R, 1994. Basic Pharmacology, 4lh Edition. Butterworth Henemann,

Manchester,U.K. pp301,308-309

30. Dekker A, Dreyer A, Smit R, 1993. Pharmacist Initiated Therapy: Recognition

and treatment ofminorailments. pp92-93,238-241.

31. Spratto G, Woods A, 1997. Demar's A- Z NOR- 97. National Students Nurses

Association, USA ppl90-193,978-979,1024-1025.

32. Neu H, 1992.The crisis in antibioticresistance. Science257:1064-1073.

33. Waxman D, Strominger, 1983. Penicillin binding proteins and the mechanism of

action ofbeta-Iaetam antibiotics. Rev. Infect. Dis.5 (Suppl. 3) S407-S411.

34. Fass R, CopelanE, BrandtI, Moeschberger M, Ashton1, 1987.Platelet- mediated

bleeding causedby broad spectrumpenicillins. 1. Infect. Dis. 155:1242-1248.

87

35. Barlett J, 1982. Antibiotic- associated diarrhea. Clin. Infect. Dis. 15:573-579.

36. Cohen S, Hachler H, Levy S, 1993.Generic and Functional analysis of the

multiple antibiotic resistance. Bacteriol. 175:1484-1492.

37. Leonard S, 1992. Pharmacology, second edition. Harwal Publishing Company,

Media, Pennsylvania, New York, Toronto, Singapore. pp208-212,226.

38. Cohen M, 1992. Epidemiology of drug resistance: implication for a post

antimicrobial era. Science 257:1050-1055.

39. Gibbons A, 1992. Exploring new strategies to fight drug resistant microbes.

Science 257:1036-1038.

40. Theoharides T, 1992. Pharmacology. Library of Congress-in-Publication Data.

ppl0l-l03202-203.

41. Jones R, Sader H, 1993. The clinical impact of enterococcal resistance.

Challenges Infect. Dis. 1:1-6.

42. Murray B, 1994. Can antibiotic resistance be controlled? N. Engl. 1. Med.

330: 1229-1230.

43. Jouanny J, 1994. The Essentials of Homoeopathic Therapeutics. Edition Boiron,

France. ppll-12,14,20.

44. Jouanny J, 1994. The Essentials of Homoeopathic Materia Medica. Edition

Boiron, France. pp62,416-423.

45. Eizayaga F, 1991. Treatise on Homoeopathic Medicine. Ediciones Marecel,

Buenos Aires. ppI2,13,35-38,512-517.

46. Lockie A, Geddes N, 1995. The Complete Guide to Homoeopathy: The Principles

and Practice of Treatment. Dorling Kindersley Limited, London. ppI4,18

19,110,118-119.

47. Hoffmann D, 1996. The Complete Illustrated Holistic Herbal. Elements Books

Limited, Great Britain. pp67, I52, I84-I85.

48. Boericke W, 1995. Pocket Manual of Homoeopathic Materia Medica and

Repertory. B. Jain Publishers, New Delhi India. ppI03-105,643-649.

49. Hahnemann S. 1995. Organon ofMedicine. Jain Publishers, India.

50. Gibson D, 1996. Studies ofHomoeopathic Remedies. British Library Cataloguing

in Publishing Data, Great Britain. pp74-76,512-517.

88

51. Morrison R, 1993. Desktop Guide to Keynotes and Confirmatory Symptoms.

Hahnemann Clinic Publishing, California. pp54,387-389.

52. Vithoulkas, G, 1980. The Science of Homoeopathy. Grove Press, New York.

pp98,104.

53. Roy M, 1994. The Principles Of Homoeopathic Philosophy. Churchill

Livingstone, Edinburgh, London, Madrid, Melbourne, New York, Tokyo. ppl

4,10-11,17.

54. Hubbard E, 1990. Homoeopathyas Art and Science. Beaconfield Publishers LTD.

Beaconfield, Bucks, England. ppl,3-4,26,271.

55. Tyler M, 1996. Homoeopathic Drug Pictures. Indian Books and Periodical

Syndicate, New Dehli, India. pp111-115,819-829.

89

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