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 [Unpublished]: University of Johannesburg.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).
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 .
Streptococcus pyogenes .
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 .
Streptococcus pyogenes .
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.
Bar graph of disc diffusion method displaying zone
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.
Bar graph of disc diffusion method displaying zone
diameters on C. a/bicans treated with' Nystatin and
Baptisia tinctoria.
Bar graph of disc diffusion method displaying zone
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
Table 3.5 The difference in median values of S. pyogenes treated
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
Table 3.8 The difference in median values of S. pyogenes treated
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).
1.5.2 Candida albicans
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
2.1.2.1 Streptococcus pyogenes
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).
2.2.2.1 Streptococcus pyogenes
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
99% Confidence interval 1568.89 1765.24 1889.79 1643.61
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
Baptistatinctoria 30ch 52518.5 49609.0 56113.0
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,
standard deviations, standard errors and confidence intervals.
Key: Control= S. pyogenes containing no medication
3ch= Thuja occidentalis 3ch
15ch= Thuja occidentalis 15ch
30ch= Thuja occidentalis 30ch
, 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
95% Confidence interval 1179.36 1106.22 2199.48 1460.00
99% Confidence interval 1568.89 1471.59 2925.94 1942.22
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.
Group Median 25% 75%
Control 56504.0 55184.0 57632.0
Thuja occidentalis 3ch 52445.5 48044.5 54572.5
Thuja occidentalis 15ch 54401.0 53423.0 56870.5
Thuja occidentalis 30ch 56015.0 53839.0 57262.0
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.
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
Thuja occidenta/is 15ch vs control 2936.5 5.14 Yes
Thuja occidenta/is 30ch vs control 970.5 2.55 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.
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=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.
Group Median 25% 75%
PenicillinG 36.00 33.00 40.00
Bacitracin 15.00 14.00 16.00
Baptisia tinctoria 3ch 0.00 0.00 0.00
Baptisia tinctoria 15ch 0.00 0.00 0.00
Baptisia tinctoria 30ch 0.00 0.00 0.00
The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a
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
95% Confidence interval 0.29 0.18 0.08 0.00 0.00
99% Confidence interval 0.38 0.25 0.11 0.00 0.00
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=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.
Group Median 25% 75%
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
The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a
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,
standard deviations, standard errors and confidence intervals.
Control 3ch 15th 30th
Mean 5.81e+4 1.15e+4 1.59e+4 2.81e+4
Standard Deviation 9367.93 4210.78 5507.58 7989.00
Standard. Error 1209.39 543.61 711.03 1031.38
95% Confidence interval 2420.04 1087.78 1422.79 2063.82
99% Confidence interval 3219.34 1447.06 1892.71 2745.47
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.
Group Median 25% 75%
Control 58175.5 51821.0 64172.0
Baptisia tinctoria 3ch 10561.5 8324.0 13246.5
Baptisia tinetoria 15ch 14589.0 11635.5 18885.0
Baptisia tinctoria 30ch 27119.0 22465.0 32936.5
The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a
comparison between the homoeopathic treatment groups vs C. albicans control,
determining the difference of ranks and P-values. Statistically significant readings
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
95% Confidence interval 2420.04 2631.57 2901.86 3170.57
99% Confidence interval 3219.34 3500.74 3860.31 4217.76
One Way Analysis ofVariance Normality test failed (P=<O.OOOl).
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=5.44E-077).
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.
Group Median 25% 75%
Control 58175.5 51821.0 64172.0
Thuja occtdentalis 3ch 31862.5 23986.0 41170.5
Thuja occidentalis 15ch 55490.5 50031.0 59786.5
Thuja occidentalis 30ch 51821.0 46809.0 61845.0
The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a
comparison between the homoeopathic treatment groups vs C. a/hieans control,
determining the difference of ranks and P-values. Statistically significant readings
should be P<O.05.
Table 3.18 Comparison of Thuja occidentalis treatment groups, difference of ranks and
P-vaIues ofC. albicans.
Comparison Difference ofRanks P-value P<0.05
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 Deviation 3.00 2.62 1.69 1.40
Standard Error 0.39 0.34 0.22 0.18
95% Confidence interval 0.78 0.68 0.44 0.36
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
treatment groups are greater than what would be expected by chance; there was a
statistically significant difference (P=2.53E-072).
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.
Group Median 25% 75%
Nystatin 25.00 21.00 25.00
Baptistatinctoria 3ch 0.00 0.00 1.00
Baptistatinctoria 15ch 0.00 0.00 0.00
Baptistatinctoria 30ch 0.00 0.00 0.00
The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a
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.
Comparison Difference ofRanks P-value P<0.05
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 Deviation 3.00 2.09 0.00 0.00
Standard Error 0.39 0.27 0.00 0.00
95% Confidence interval 0.78 0.54 0.00 0.00
99% Confidence interval 1.03 0.72 0.00 0.00
The difference in the median values, according to ANDVA statistics, among the
treatment groups are greater than what would be expected by chance; there was a
statistically significant difference (P=2.53E-072).
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.
Group Median 25% 75%
Nystatin 25.00 21.00 25.00
Thujaoccidentalts 3ch 0.00 0.00 0.00
Thuja occidentalis 15ch 0.00 0.00 0.00
Thujaocctdentalis 30ch 0.00 0.00 0.00
The All Pairwise Multiple Comparison Procedures (Dunnett's Method) depicted a
.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.
Comparison Difference ofRanks P-value P<0.05
Thuja occidentalis 3ch vs Nystatin 12688.0 16.68 Yes
Thuja occidentalis 15ch vs Nystatin 13050.0 9.81 Yes
Thuja occidentalis 30ch vs Nystatin 13050.0 11.45 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 Streptococcus pyogenes
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 Candida albicans
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
4.3.1 Streptococcus pyogenes
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
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