Post on 11-Apr-2015
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
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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.
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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
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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.
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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).
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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
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- 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).
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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.
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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
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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).
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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
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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)
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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).
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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).
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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).
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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
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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.
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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.
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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.
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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
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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
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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
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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
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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.
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