A patient was suffering from hepatorenal syndrome. A urine sample was obtained from the patient and was examined under the microscope. Which of the following technique is being used to make the organism visible?

(a) Dark field microscopyLEPTOSPIRALeptospira, comprises of two species:* L. interrogans (pathogenic for humans): Causes leptospirosis or Weil's disease involving liver and kidney.* L.biflexa (saprophyte)* Mode of transmission:It is zoonotic. Direct human-to-human transmission does not occur. It is transmitted by:0 Indirect contact with water, moist soil and wet surfaces contaminated with animal urine or0 Direct contact with urine and products of parturition, placenta of infected animals* Source: Although >100 animals can be infected; but important sources of infection are rats, dogs, cattle, and pigs.* 3R's: Important epidemiological determinants for leptospirosis include exposure to rodents, rain f all and rice field.Clinical ManifestationsThe incubation period is around 5-14 days. In general, the manifestations can be divided into two distinct clinical syndromes.* Mild anicteric febrile illness: Occurs in 90% of patients. It is biphasic; septicemic phase followed by immune phase* Weil's disease (Hepato-renal-hemorrhagic syndrome): It is a severe form of icteric illness, occurs in 10% patients. Typical biphasic course may not be present. It is more severe and fulminant Mild anicteric febrile illnessWeil's diseaseFirst stage 3-10 days (septicemic)Second stage 10-30 days (immune)First stage 3-10 days (septicemic)Second stage 10-30 days (immune)Clinical FindingsFeverMyalgiaHeadacheConjunctivalsuffusionAbdominal painPharyngeal erythema without exudatesVomitingMeningitisUveitis, optic neutiris, chorioretinitisRashFeverPeripheralneuropathyHigh grade feverJaundice and raised liver enzymesHemorrhages:* Pulmonary hemorrhage* Petechiae and purpura* Conjunctival hemorrhage* GI hemorrhageKidney: Raised serum urea and creatinine and Renal failureIsolationFrom blood and CSFFrom UrineBlood and CSFUrineSerum lgMAbsentPresentAbsentPresentAntibioticsSusceptible to antibioticsRefractory to treatmentSusceptible to antibiotcsRefractory to treatmentLaboratory Diagnosis* Specimens: CSF and blood (in first 10 days of infection) and urine (between 10-30 days of infection)Microscopy: Leptospira are extremely thin, hence seen under dark ground microscope.* They are tightly and regularly coiled, with characteristic hooked ends like umbrella handle.* They are highly motile; exhibit spinning and translational movements.* Isolation:0 Culture condition: Leptospira is obligate aerobe and slow growing. Incubated at 30degC for 4-6 weeks.0 Culture media: As Leptospira is highly fastidious, requires enriched media such as: EMJH medium- MC media Korthof's media* Serology for antibody detection: Antibody detection tests can be broadly classified into:0 Genus specific tests uses broadly reactive genus specific antigen prepared from non-pathogenic L.biflexa Patoc 1 strain. They cannot detect the infecting serovar.Various tests available are: Macroscopic slide agglutination test Microcapsule agglutination test (MCAT) Latex agglutination test, ELISA and ICT Serovar specific test: Microscopic agglutination test (MAT) detects antibodies against specific serovars of L. interroagans. It is the gold standard method and reference test for the diagnosis of leptospirosis.* Molecular methods:0 Targeted genes are 16S or 23SrRNA or IS 1533 insertion sequence.0 PCR detects early before seroconversion occurs. However, PCR is not serovar specific.0 PCR-RFLP or PFGE are the methods to determine the genomospecies of Leptospira.* Fame's criteria is WHO approved guideline used for the diagnosis of leptospirosis. It is based on clinical, epidemiological and laboratory findings.Treatment* Mild leptospirosis: Oral doxycycline (Alternatively Amoxicillin)* Severe leptospirosis: Penicillin is the DOC (alternatively ceftriaxone/cefotaxime)