Introduction

There are many bacteria which use the human body as a host, some with

negligible effects while others are more detrimental. Two of the bacteria which pose

severe threat to humans are Leptospira and Helicobacter. Much research has been done

and continues even today as it relates to the culturing of these bacteria. Most of these

works have been basically centred on culturing these bacteria in conventional nutrient-

rich media which have yielded success. Therefore, the thought of seeking alternative

media has never been seen as a priority.

While it is a fact that the culturing of these bacteria in nutrient rich media has

been relatively successful there are several factors which necessitate the procural of

alternative methods. These factors include: the high cost factor involved in culturing,

the time consuming element (4-6 months), inaccessibility of nutrient rich materials

(rabbit serum in case of Leptospira), and the need for a more expeditious approach in

combating the diseases caused by these bacteria (Wechter, 2007).

Today an ever increasing number of people suffer from Leptospirosis (caused by

Leptospira) and gastritis, stomach and peptic ulcers (caused by Helicobacter). As a

result of these phenomena medical science is seeking to understand more about these

bacteria in order that quicker diagnosis and treatment can be given to patients. It

therefore means that by developing alternative methods of culturing the probability

increases of controlling the diseases.

While some have cultured Leptospira and Helicobacter in a serum free medium

(yet nutrient rich), no seminal research has been done in the area of culturing them in a

1

nutrient limiting media (also known as oligophilic conditions). One might be tempted to

ask how is it possible that bacteria that normally cultured in nutrient rich media can be

grown in nutrient limiting media. The reality is both if these bacteria share similar

physiological niche in that the environment in which they are adapted to are generally

low in nutrients.

For instance, Leptospira lives in the proximal convoluted tubules of the kidney

where the available nutrients consist of water, sugar, salts, urea, soluble vitamins and

minerals. In the case of Helicobacter, it occupies the lining of the stomach walls where

it feeds off the nutrients provided by the dead white blood cells.

In sum, alternative methods are needed for the reliable cultivation, detection,

identification, and treatment of diseases caused by these bacteria. As stated before, the

currently used media are very cumbersome, time-consuming, and require a high level of

skill and experience to perform.

This research therefore aims to fulfil the following objectives:

To culture and successfully isolate Leptospira and Helicobacter under nutrient

limiting conditions, using Poor Ravan medium.

To use a serum-free culture media capable of growing Leptospira and

Helicobacter organisms.

To provide a cheap and easy method for detecting and characterizing

Leptospira and Helicobacter in a sample.

2

Review of Literature

Leptospira

Discovery of Micro-organism

The first description of Leptospira (although not called by that name then) was in

1812 by one of Napoleon’s troops while they were in war in Egypt. Later the illness

came to be known especially throughout Europe as‘bilious typhoid’ (Matthew et. al).

In 1886, Adolf Weil described Leptospirosis as a disease entity. As a tribute to

his work the disease was since called Weil’s disease.It was not until the second decade

of the 20th century that Leptospires were recognized by Inada and Ido in Japan and soon

after, independently, in Germany by Uhlenhuth and Fromme as the cause of the disease

that had been originally described by Weil (World Health Organization).

Taxonomy and Classification

Taxonomic Status:

Order: Spirochaetales

Family: Leptospiraceae

Genus: Leptospira

Species: L. interrogans

L. biflexa

3

Serological classification (Wolff and Broom):

Leptospira is divided into 2 species: L. interrogans and L.biflexa. L. interrogans

is pathogenic and causes diseases whereas L. biflexa is saprophytic which is found in

non-sterile envornoment and does not transmit diseases. The main difference between

these two is the former grows at 130C in the presence of 8-azaguanine and the latter fails

to form spherical cells in 1M NaCl. Both L. interrogans and L. biflexa are divided into

numerous serovars based on their antigenic composition.

4

Leptospiraceae

Turneria Leptospira

L.interrogans

serogroups

(>25)serova

rs (>250)

L. biflexa

serogroups (38)

serovars

(>60)

Leptonema

Genotypic Classification

Leptospiraceae

__________________________________

Leptospira Leptonema Turneria

L. borgpetersenii, L. interrogans, L. inadai, L. noguchi, L. Weillii, L. alexandri, L.

Wolbachii, L. meyeri, L. biflexa, L. santarosai, L. faini, L. parva, L. kirchneri

The above genotypic scheme distinguishes Leptospira based upon DNA

relatedness (Yasuda et. al, 1987).

Morphological characteristics

- Helical rods 6-12μm in length and 0.1μm in diameter.

- Flexible and corkscrew-shaped with each cell having 18 or more coils.

- One or both ends are characteristically hooked.

- Cell is encased in a 3-5 layer outer membrane or envelope. Beneath this outer membrane

are the helical peptidoglycan layer and the cytoplasmic membrane.

- Two flagella originating at each end of the cell lie between the outer membrane and the

peptidoglycan layer. The free ends extend toward the centre of the cell but do not

overlap.

- Basal bodies resemble that of Gram negative bacteria (Penn, 1990).

5

Movements of Leptopsira

According to Cox and Twigg, Leptospira undergoes at least four types of

motility:

1. Nontranslational: The extremities move in a cyclical motion while the other parts of the

body stay stagnant.

2. Translational: One end moves like a coil while the other end moves in an inconsistent

circular motion. Movement occurs towards the end showing helical motion.

3. Anchored: One end remains stationary while rest of body is in motion.

4. Shaking generally seen in semi-solid media (Cox and Twig 1974).

Epidemiology

Mode of transmissions can either be indirect through contact with some form of

contaminant in water, soil or urine of animals. (Turner et.al, 1967) Or it can be directly

through the bites of animals or passed on from mother to offspring (Shaked et. al, 1993).

Animals are often the primary host of Leptospira whereas human beings are the

accidental hosts.

The disease most affects people within the ages of 10-39 with higher prevalence

in men and persons engaged in farming, sewage disposal, laboratory and veterinary

work (Sanford, 1994).

Conventional nutritional requirements and environmental conditions

- Vitamin B1 and B12

- Long chain fatty acids bound to albumin

- Animal serum

6

- Nutrient rich supplements such as peptone, sodium pyruvate, glycerine, ammonium

salts, Sodium or Potassium, Calcium or Magnesium and Iron

- 5-fluoro-uracil for isolation from contaminated sources

- Temperature of 28-30oC

- Light protection

- pH 7.2-7.6

- Oxygen

- Amino acids such as L-asparagine (WHO)

Conventional forms of Media:

1. Liquid form

Liquid media are essential for the isolation of leptospires and for growing cultures.

Growth of leptospires in liquid media is indicated mainly by turbidity but sometimes by

a granular appearance on the bottom of the tubes in which they are growing, both of

which can be seen with the naked eye, but this should be confirmed by microscopic.

observation.

2. Semi-solid form

Semi-solid media contain 0.1–0.5 % agar (w/v). Such media are preferred for isolating

the various strains and for medium-term maintenance (up to several years). Growth is

readily initiated in these media and is usually easily visualized as one or more rings of

dense growth several millimetres below the surface of the medium (Coghlan, 1966).

7

3. Solid form

Solid media contain 0.8–1.3 % agar (w/v). The lower the concentration of agar, the

greater the tendency for leptospires to swarm across the plate and through the medium;

the higher the concentration, the smaller the colonies. (Johnson, 1964).

Types of conventional media containing sera:

1. Traditional media containing approximately 8–10% rabbit serum (Stuart, Korthof,

Fletcher, Vervoort, Schüffner. Rabbit serum contains the highest concentration of bound

vitamin B12, which is essential for the multiplication of leptospires.

2. The Tween 80/bovine serum albumin (BSA) medium of Ellinghausen & McCullough

and its modification by Johnson & Harris (EMJH). The BSA component of the medium

is the most expensive ingredient.

3. Enriched media. To increase the growth of more fastidious leptospires such as serovar

hardjo, media can be enriched by adding serum (e.g. 1–4% fetal calf serum (FCS) and

rabbit serum) or other ingredients such as

lactalbumin hydrolysate, superoxide dismutase and pyruvate (Ellis, 1986). EMJH

medium is often enriched by adding 1% rabbit serum and 1% FCS.

4. Selective media with 5-fluorouracil (and/or other antimicrobials such as neomycin,

nalidixic acid, actidione, sulfadiazol, rifampicin, amphotericin B). These additives may

suppress the growth of contaminating bacteria in non-sterile clinical samples, while

leaving leptospires unaffected but they may also cause some reduction in the growth of

leptospires. This is particularly true of sulfadiazol.

8

Serum-free Media

1. Low-protein or protein-free media, often used for the preparation of vaccines

(Coghlan, 1966).

2. A serum-free media for culturing spirochetes developed and patented by Wechter

Stephen R.

Diagnostic methods

1. Direct microscopy: Microscopy is performed on urine, and blood specimen and even

bronchoalveolar lavage fluid. Since Leptospira cannot survive in acidic urine, the

sample must be neutralized before microscopy (Babudieri, 1961).

2. Serological Tests

The serological tests seek to detect antibodies and also serovars. Two of the more

common tests which are done are Enzyme Linked Immunosorbent Assay (ELISA) and

Microscopic Agglutination Test (MAT). ELISA involves the detection of antigen-

antibody system using enzyme linked antihuman antibody and a suitable substrate

(Terpstra, 1985). MAT is carried out by using live cultures of various serovars of L.

interrogans. Equal volume of antigen is added to serum dilutions and agglutination is

observed under darkfield microscope (Babudieri, 1961).

3. Molecular Methods

The two more common molecular methods for detecting leptopsires are

Polymerase Chain Reaction (PCR), and DNA-DNA hybridization. PCR method

involves in-vitro amplification of target DNA sequence brought about by thermostable

DNA polymerase. There are several limitations of PCR: technique is expensive and

complicated, contamination of test samples may lead to false results and also PCR is not

9

able to identify the infecting serovar. Hybridization occurs when nucleotide sequence in

a probe is used to detect a complementary sequence in a test sample (Terpstra, 1986 ).

Work done on growth of Leptospira in vitro

In 1967, Russell and Harris attempted to identify the differentiating

characteristics between pathogenic and saprophytic leptopsire by growing them at low

temperatures using nutrient rich medium (rabbit serum). They tested the response of 20

pathogens and 30 saprophytes at temperatures of 13oC-30oC. At 30oC all organisms

grew, however, only saprophytic grew at 13oC. They discovered that the pathogenic

leptospira grows best at higher temperature unlike the saprophytic (Russell and Harris,

1967). In as much as these researchers have discovered that pathogenic leptospira

grows better at higher temperatures than saprophytic, the media used for growth is still

nutrient rich.

Helicobacter

Discovery of organism

The presence of spiral-shaped micro-organisms in the stomach mucosa was

described almost 100 years ago. Their presence was not really taken seriously until the

late 1970’s when John Warren, a pathologist in Perth, Western Australia, noted the

appearance of spiral bacteria overlaying gastric mucosa and mostly over-inflamed tissue.

Warren and Barry Marshall cultured these organisms in 1982 from eleven patients with

gastritis. They were able to demonstrate a strong association between the presence of

Helicobacter pylori and the finding of inflammation on gastric biopsy (Marshall, 1989).

10

Originally called Campylobacter pyloridis, the name was changed to

Campylobacter pylori and then later to Helicobacter pylori as specific morphologic,

structural and genetic features indicated that it should be placed in a new genus(Marshall

and Warren, 1984).

Taxonomic status and Classification

The genus Helicobacter presently comprises 18 validly named species and two

Candidatus species, a designation adopted by the International Committee on

Systematic Bacteriology to record the properties of putative procaryotic taxa that are

incompletely All Helicobacter species are characterized as fastidious, and most are

associated with gastric or extragastric diseases. (Solnick and Vandamme, 2001-tax

described of hel).

Morphological characteristics

- 0.2 to 1.2 μm in diameter and 1.5 to 10.0 μm long

- S-shaped bacterium with multiple, polar sheathed flagella(1-20).

- The cellular morphology may be curved, spiral, or fusiform.

- Periplasmic fibers or an electron-dense glycocalyx or capsule-like layer has been

observed on the cellular surface of several species

- The spiral wavelength may vary with the age, the growth conditions, and the species

identity of the cells. In old cultures or those exposed to air, cells may become coccoid

(Solnick and Vandamme, tax. of hel)

- H. pylori in vivo and under optimum in vitro conditions is an S-shaped bacterium with 1

to 3 turns, 0.5 ×5 μm in length, with a tuft of 5 to 7 polar sheathed flagella

11

Motility of Helicobacter

Helicobacter cells are motile, with a rapid cork-screw-like or slower wave-like motion

due to flagellar activity. They are often found within the lining of the stomach walls they

have become acclimatized to the stomach’s acidity (Goto, 1998).

Nutrient requirements and environmental conditions

- serum rich

- Temperature of 30-37oC

- Microaeropilic conditions (5-15% O2, 5-10% CO2, 85% N)

- Nutrient rich supplements: ferrous sulphate, mucine, sodium pyruvate, whole blood,

amino acids, sodium and potassium chloride, thiamine, hypoxanthine, zinc, magnesium,

isovitale X, hemin, cyclodextrin and cholesterol.

- pH 4.5-9

- Water activity (Aw)>0.98 (Battles, 1995).

Types of Medium containing serum

-Columbia blood agar plates

-moist Trypticase soy agar

blood agar plates

-brucella blood agar with TVP (trimethoprim,

vancomycin, polymyxin)(helicobacter 1)

Campylobacter agar (Becton Dickinson, Sparks, MD) containing 10% defibrinated

sheep blood (Quad Five, Ryegate, MT) (CBA)(nutritional req)

12

Serum-free medium

The growth of the gastric pathogen Helicobacter pylori in the absence of serum

remains challenging, and nutritional requirements have only partially been defined. H.

pylori grows in the chemically defined medium F-12, but not in other tissue culture

media examined. H. pylori has surprisingly few absolute requirements for growth: 9

amino acids, sodium and potassium chloride, thiamine, iron, zinc, magnesium,

hypoxanthine, and pyruvate. These data suggest that H. pylori and other Helicobacter

species are not as particular as previously thought.

The data also suggest that chemically defined media described herein could yield

the growth of a wide range of Helicobacter spp., allowing a more detailed

characterization of Helicobacter physiology and interactions with host cells. Nutritional

requirements and antibiotic resistance patterns of several other Helicobacter species

revealed that all except H. felis grew in serum-free, unsupplemented F-12.

Identification and Diagnostic Methods:

In taxonomic practice, the reference method for the delineation and identification

of bacterial species is determination of the level of DNA-DNA hybridization. Strains

are considered to belong to a single species if their whole genome DNA-DNA

hybridization level is about 70% or greater. The fastidious growth characteristics of

many Helicobacter species hamper the isolation of sufficient quantities of highly

purified high molecular weight DNA required for these hybridization experiments. Yet,

a number of DNA-DNA hybridization studies have been performed within and between

Helicobacter species (Wayne, 1987).

13

Protein Electrophoresis

It is not practical to implement DNA-DNA hybridizations in a routine laboratory

or to use it for routine identification in a reference laboratory. The comparison of whole-

cell protein patterns obtained by highly standardized sodium dodecyl sulfate-

polyacrylamide gel electrophoresis has proven to be extremely reliable to screen and

identify large numbers of strains. Numerous studies revealed a correlation between high

similarity in whole-cell protein content and level of DNA-DNA hybridization.

However, this method is not appropriate for routine identification studies because it is

laborious, time-consuming, and technically demanding to run the patterns in a

sufficiently standardized way (Vandamme, 1996)

Cellular Fatty Acid Analysis

The total cellular fatty acid methyl ester composition is a stable parameter

provided that highly standardized culture conditions are used. Comparison of fatty acid

profiles is of little value if different culture conditions or extraction procedures are used.

However, it is a simple, inexpensive, and rapid method that is highly automated

(Godwin, 1989).

DNA Probes and PCR Assays

Specific oligonucleotide probes or PCR assays have been described for H. pylori

and several other Helicobacter species. It should be stressed that, because of the

constant developments in the taxonomy of Helicobacter species, none of these probes or

PCR assays have been fully evaluated against all species presently described (Page,

1996).

14

Invasive techniques

Histology

Histological examination of biopsy samples taken during endoscopy is usually

considered 'the gold standard'for the diagnosis of H.pylori. But owing to the patchy

distribution of H.pylori in gastric mucosa, the

biopsy-based tests may suffer from sampling error (Anderson, 1998). Furthermore,

histological examination is highly dependent on the experience of the pathologist, and

high inter-observer variation has been reported (Morris, 1989).

Rapid urease test (CLO test)

Biopsies of gastric mucosa are placed in a gel containing urea, and the

subsequent ammonia production causes a pH change, which is observed as a color

change. Besides suffering from biopsy sampling error, the CLO test depends greatly on

the pH of the media and the amount of the urea in the medium. These factors may vary

in different products and thereby influence the results obtained with other tests (Thijs,

1996).

Culture

Culture is the most specific diagnostic method for H.pylori infection but its

sensitivity is low. The role of culture for primary diagnosis is limited but it is an

important method as isolates for the traditional susceptibility testing are obtained.

Although routine susceptibility testing for H.pylori is not recommended, increasing

resistance rates to metronidazole and clarithromycin might make routine susceptibility

more popular (7).

15

Non-invasive techniques

Serological tests

Serological tests are based on the detection of specific anti-H.pylori IgG

antibodies in a patient's serum. While serological tests are simple and easy to perform,

they are not reliable tests for the diagnosis of H.pylori infection in elderly people

because of poor antibodyproduction, or for determination of eradication ofH.pylori,

since it remains positive for a long period despite adequate treatment (Newell, 1991).

Serological tests are not able to distinguish between active infection and a previous

exposure to H.pylori. Different commercial kits also have different levels of diagnostic

accuracy (range 68-82%) (Feldman, 1995).

Stool antigen test

An enzyme immunoassay, which detects the presence

of antigen in stool specimen, has recently become available. This assay has undergone

extensive testing for the initial diagnosis of the H.pylori infection and in the

confirmation of eradication after treatment. Several studies have suggested that

polyclonal antibody test is comparable to the ure breath test in the initial diagnosis of

H.pylori infection (sensitivity 93.2% and specificity (93.2%). It has been reported that

stool antigen test isles accurate than UBT in the post-treatment setting. Recently it was

been reported that monoclonal technique has higher sensitivity than the polyclonal one

especially in the post-treatment setting (Bilardi, 2002).

Urea breath tests

H.pylori produces urease, an enzyme that splits urea into ammonia and carbon

dioxide. The production of high amounts of urease by H.pylori has been used in the

16

development of urea breath tests . Patients ingest urea labeled 13C or 14C. Hydrolysis

of urea occurs within the mucous layer and results in the production of ammonia and

labelled CO2. Labelled CO2 diffuses into the blood vessel and can be detected in the

breath as a marker of infection (Christensen, 1992).

Materials and Methods

A total of fifteen reference serovars of leptospires will be used during this

research which will be obtained from the Leptospira Department of B.J.Medical

College. Out of these, eight serovars have been procured. The facilties at Abasaheb

Garware college will be used. The methodology follows a particular sequence: culturing

of Leptospira on nutrient limiting media, microscopy investigation, subculturing onto

nutrient limiting solid media, subculturing into conventional media, DNA isolation, PCR

assays, sequencing and data analysis.

Culturing Leptospira using Poor Ravan plates

The following serovars were acquired from the Leptospira Department of

B.J.Medical College: L. australis, L. autumnalis, L. icterohaemorrhagiae, L. Tarasorri

and L. Bataviae. These organisms were streaked from conventional medium (EMJH see

Appendix) directly onto Ravan medium agar plates in a 1:100 diluted or poor form

(glucose 5g, peptone 5g, yeast extract 5g, sodium acetate 5g, sodium citrate 5g, pyruvic

acid 2g, distilled water 11, pH 7-7.2, agarose 10g). These plates were all incubated at

37oC and growth was checked for each week using bright field microscopy.

17

Culturing Leptospira using a cyclic method

Six serovars of Leptospira were used thus far from B.J. Medical college for

using this technique. They are: L. patoc 1, L. patoc 2, L. autumnalis, L. Bataviae, L.

icterohaemorrhagiae and L. pyrogenes. One loopful of each was placed into 3ml 1:100

diluted Ravan broth medium and incubated at room temperature. Wet mounts were

prepared weekly for observing growth under dark field microscope. Morphology and

motility were carefully documented and cell count was taken. When >15 organisms can

be seen per field one loopful was subcultured onto diluted Ravan agar medium.

These plates were incubated at 28oC.When colonies were visible with the naked

eye, the colony morphologies were observed under bright field microscope. Wet mounts

were also prepared and the morphology and motility of the organisms recorded using

dark field microscope. When >15 organisms can be seen per field one single colony was

picked up and placed back into EMJH medium and incubated at room temperature.

Morphology and motility was carefully documented.

EMJH brothPR brothPR agarEMJH broth

DNA isolation

A loopful of cells were harvested and placed into PCR tube. 20ul of single

colony lyses solution (SCL) was added to the tube and kept at 55oC for 1 hour. Enzyme

activity was then inhibited at 85oC for 20 minutes. Contents were vortexed and

centrifuged at 5000rpm for 1 minute.

18

Amplification of 16S rRNA gene

Polymerase Chain Reaction amplification was conducted using the following

combinations of primers: FOD2 and RPP2, 16R1525 and 16F27, and 16S-For-primers

and 16S-Rev-. The following conditions were used: denaturation at 94oC for 3 mins,

94oC for 1 min, annealing at 60oC for 1 min and elongation at 72oc for 1 min. A total of

35 cycles will be performed followed by a further elongstion step at 72oC for 10

minutes. The purity of the amplified product was determined by electrophoresis in a 1%

agarose gel. DNA was stained with ethidium bromide and viewed under short-

wavelength UV light.

Sequencing methods

This method is yet to be conducted by the researcher. The purified DNA product

will be sequenced using sequencer to confirm whether the isolate see is the desired

strain.

Data analysis

Growth curve will be plotted using cell count. C

Results

The organisms cultured on PR agar medium only were seen as tightly coiled

appearing bumpy on the surface. Some had a motion which involved folding of

elongated organism into a ball like structure and rotating in a haphazard motion. Colony

morphology was also observed but only one type of each was seen. Contamination was

the main problem initially on PR plates.

19

The cyclic method was performed at the Leptospira Department in B.J. Medical

college. The results are recorded in Figure 2. A sketch graph of L. patoc 1, L. patoc 2, L.

autumnalis and L. Bataviae were plotted as shown in Figure 2.

L. patoc 1, L. patoc 2 , L. autumnalis and L. Bataviae were subcultured onto PR

agar plates and three distinct morphologies were recorded.

Figure 1: Growth and Morphology of Leptospira serovars in PR broth medium.

Serovar Day Organism count

Morphology and motility

L. patoc 1 6 1 Sluggishly motile48 (subcultured) 4 Motile59 3 Sluggishly motile73 7 Non- motile

L. patoc 1 (1st sub) 11 12 Motile; small and short25 50 Motile

L. patoc 2 6 1 Sluggishly motile47 6 Motile63 5 Motile

L. autumnalis 6 15 Non-motile47 11 Motile73 3 Motile

L. Bataviae 30 15 Motile43(subcultured) Autoclumping57 10 Motile25 15 Short and motile

L. Bataviae (1st sub) 11 15 Short and motile25 15

L. icterhaemorrhigaie

11 2 Motile

25 2 MotileL. pyrogenes 11 1 Motile

25 0

20

21

Time (Days)

Number of orgs./field

Main type of morphology of colonies:

Colony type 1:

- Translucent

- no filaments inside

- smooth egdes

- brownish in colour

- circular form which tend to spread along streak lines

- most common in 1st and 2nd streak

Colony type 2:

- black colour

- Rarely seen inside colony type 1

- hairy like projections

- rough egdes

- circular form

- occurred only in 3rd and 4th streak mostly

- very few in number compared to other two types

22

Colony type 2:

- translucent with filaments inside, usually along streak line and spreading

- smooth edges

- brownish in colour

- occurs in all streak lines

PCR amplification from the initial batch grown on PR plates have only been

successful with 16R1525 and 16F27. Good primers still need to be identified for

improving PCR methods.

Discussion

In the first culturing technique employing only PR agar medium, colonies did not

seem to strive to well due to contamination problems. As compared to growth on agar

plates in the cyclic method, small colonies are very much distinct and with very little

contamination. This could have been due to simple difference in environmental

conditions or maybe.

Under bright field microscope, seem to be spreading more from observation but

distinct colony types were not identified. This again could have been due to

contamination since in both cases, morphology was observed using bright field

microscopy.

The morphology of the colony seem to change with time. Colony type 1 seem to

precede before colony type 3. However , colony type 2 occurs most times isolated and

23

its difficult to draw conclusions at this time as to whether it’s a contaminant or another

morphological form of colony for Leptospira.

Rate of growth varies. Some strains grow well and multiply rapidly from the

start; some appear to multiply fast for a few days and then become static and inert; in

others small numbers of lively leptospires appear but seem to multiply very slowly. L.

patoc 1 and L. patoc 2 seem to grow very slowly whereas L. autumnalis and L. Bataviae

seem to grow faster as shown from graph.

In broth, Leptospira serovars maintained there morphology and there

motility clearly. Variations like short size and autoclumping was also seen when

organisms was in adapting phase or probably in death phase. On plates, thus far all

organisms seen on wete mount ate short and most are motile. The Soilid medium seem

to inhibit there growth and also cause changes in morphology was seen under brightfield

microscope from those grown on PR agar only.

The reason to use the cyclic method is first to help the organism to adapt to

nutrient limiting conditions while still maintain a fluid environment using PR broth. This

is then followed by subculturing onto PR agar medium so the organisms will not be

subjected to the shock of change in medium and form of medium but only form of

media. Colonies are then subcultured from here back into conventional medium for just

confirmation that it is indeed leptospira that is growing and not some contaminant.

Other confirmation methods are also employed such as sequencing so that

further confirmation can be made.

Theoretically, if an organism grows in nature, it can be cultured if its

physiological niche is perceived and duplicated under in vitro conditions. It is

24

established that the nutrient concentrations in commonly used laboratory media are

several-fold higher than those present in the natural environment, specially the aquatic

habitat2. It is further revealed that a predominant group, i.e. oligophiles in the natural

bacterial population from both aquatic and terrestrial habitats does not grow on

conventional media but forms distinct colonies on 100-fold diluted versions of such

media3–8.

Oligophilic ‘k-selected’ bacteria are adapted to grow in nutrient poor environments.

These are characterized by slow growth and form small microscopic colonies. In their

natural environments nutrients are limited, meaning that these bacteria cannot reproduce

indefinitely.

25