Download - Lab dig virus
![Page 1: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/1.jpg)
Lab Diagnosis Of Viral Diseases
Prabin ShahBScMLT, MSc(Biochemistry)
![Page 2: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/2.jpg)
17/04/2023 Dept of Oral Pathology, TNGDC, Chennai
Introduction
Types of Viral Lab Diagnosis
CONTENTS
![Page 3: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/3.jpg)
What is a virus?
It is a segment of either RNA or DNA protected by a protein coat and in some families of viruses a host derived envelope with attached viral proteins
It lacks: - Protein synthesizing machinery- Energy producing system- No mitochondria- No stores of amino acids, nucleotides
energy rich molecules It is a compulsory intra cellular parasite
![Page 4: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/4.jpg)
Diagnostic Methods in Virology
1. Direct Examination
2. Indirect Examination (Virus Isolation)
3. Serology
![Page 5: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/5.jpg)
Direct Examination
1. Antigen Detection immunofluorescence, ELISA etc.
2. Electron Microscopy morphology of virus particles
immune electron microscopy
3. Light Microscopy histological appearance
inclusion bodies
4. Viral Genome Detection hybridization with specific nucleic acid probes
polymerase chain reaction (PCR)
![Page 6: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/6.jpg)
Indirect Examination
1. Cell Culture cytopathic effect (CPE) haemadsorption
immunofluorescence
2. Eggs pocks on CAM
haemagglutination
inclusion bodies
3. Animals disease or death
![Page 7: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/7.jpg)
Serology
Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.
Classical Techniques Newer Techniques
1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay
![Page 8: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/8.jpg)
Specimens for Routine Tests
Clinical Category Blood Throatswab
Faeces CSF Other
1. Meningitis + + + +2. Encephalitis + + + + Brain biopsy3. Paralytic disease + + + +4. Respiratory illness + + Nasopharyngeal aspirate5. Hepatitis +6. Gastroenteritis +7. Congenital diseases + Urine, saliva8. Skin lesions + + Lesion sample e.g. vesicle
fluid, skin scrapping9. Eye lesions Eye swab10. Myocarditis + Pericardial fluid11. Myositis + +12. Glandular fever +13. Post Mortem + Autopsy
After use, swabs should be broken into a small bottle containing 2 ml of virus transport medium.Swabs should be sent to the laboratory as soon as possible without freezing. Faeces, CSF, biopsy orautopsy specimens should be put into a dry sterile container.
![Page 9: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/9.jpg)
Electron Microscopy
106 virus particles per ml required for visualization, 50,000 - 60,000 magnification normally used. Viruses may be detected in the following specimens.
Faeces Rotavirus, Adenovirus
Norwalk like viruses
Astrovirus, Calicivirus
Vesicle Fluid HSV
VZV
Skin scrapings papillomavirus
![Page 10: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/10.jpg)
Electronmicrographs
Adenovirus Rotavirus
(courtesy of Linda Stannard, University of Cape Town, S.A.)
![Page 11: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/11.jpg)
Inclusion bodies
Negribodies
Negribodies
neuron
Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 39.9)
![Page 12: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/12.jpg)
Immune Electron Microscopy
The sensitivity and specificity of EM may be enhanced by immune electron microscopy. There are two variants:-
Classical Immune electron microscopy (IEM) - the sample is treated with specific anti-sera before being put up for EM. Viral particles present will be agglutinated and thus congregate together by the antibody.
Solid phase immune electron microscopy (SPIEM) - the grid is coated with specific anti-sera. Virus particles present in the sample will be absorbed onto the grid by the antibody.
![Page 13: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/13.jpg)
Immunofluorescence
• Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes.
• Immunofluorescent labeled tissue sections are studied using a
fluorescence microscope.
• Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
![Page 14: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/14.jpg)
Direct IF Indirect IF
![Page 15: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/15.jpg)
![Page 16: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/16.jpg)
ELISA
![Page 17: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/17.jpg)
Embryonated Egg
Cell Lines/ Tissue cultures
Animal inoculation
Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Primary
Diploid/ Secondary
Continuous
Suckling mice
Virus Culture
![Page 18: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/18.jpg)
Embryonated Hen’s Egg
• Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
• Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
• Amniotic cavity – primary isolation of Influenza virus
• Yolk sac – Chlamyadia, Rickettsiae & some viruses
![Page 19: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/19.jpg)
Growth of virus on embryonated eggs
Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1
![Page 20: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/20.jpg)
Embryonated Hen’s Egg
![Page 21: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/21.jpg)
Types of Tissue Culture
Organ culture
Explant culture
Cell culture
![Page 22: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/22.jpg)
Cell Culture
• Tissues Individual cells trypsin & mechanical shaking
• Cells are washed, counted & suspended in a growth medium.
• Growth medium – Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator
![Page 23: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/23.jpg)
Cell Culture• Routinely used for growing viruses• Classified into 3 types:
– Primary cell culture – normal cells freshly taken from body & cultured, limited growth1. Rhesus monkey kidney2. Chick embryo fibroblast3. Human amnion cell culture
– Diploid cell strains – cells of single type (fibroblast cells) that can be subcultivated for limited number of times, mostly 501. WI-38: human embryonic lung cell2. HL-8: Rhesus embryo cell
![Page 24: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/24.jpg)
– Continuous cell lines – malignant cells, indefinite subcultivation1. HeLa: Human Ca of cervix cell line2. HEP-2: Human epithelioma of larynx3. Vero: Vervet monkey kidney4. McCoy, Detroit-6, BHK-21, KB
![Page 25: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/25.jpg)
Cell Culture Bottles / Tubes
![Page 26: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/26.jpg)
Detection of virus growth in cell cultures
1. Cytopathic effects (CPE) – morphological changes in cultured cells, seen under microscope, characteristic CPE for different groups of viruses
2. Metabolic Inhibition – no acid production in presence of virus
3. Hemadsorption – influenza & parainfluenza viruses, by adding guinea pig erythrocytes to the culture, erythocytes are absorbed on surface of cells
![Page 27: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/27.jpg)
Cytopathic Effect
Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells . (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)
![Page 28: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/28.jpg)
Hemadsorbtion
add red blood cells
![Page 29: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/29.jpg)
Haemadsorption
Syncytial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet. (courtesy of Linda Stannard, University of Cape Town, S.A.)
![Page 30: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/30.jpg)
4. Interference – growth of a non cytopathogenic virus can be tested by inoculating a known cytopathogenic virus: growth of first virus will inhibit the infection by second
5. Transformation – oncogenic viruses induce transformation & loss of contact inhibition – growth appear in a pile-up fashion producing microtumors
6. Immunofluorescence – test for viral Ag in cells from viral infected cultures.
![Page 31: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/31.jpg)
Transformationloss of contact inhibition
![Page 32: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/32.jpg)
Problems with cell culture
• Long period (up to 4 weeks) required for result.• Often very poor sensitivity, sensitivity depends on a
large extent on the condition of the specimen.• Susceptible to bacterial contamination.• Susceptible to toxic substances which may be present in
the specimen.• Many viruses will not grow in cell culture e.g. Hepatitis
B, Diarrhoeal viruses, parvovirus, papillomavirus.
![Page 33: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/33.jpg)
Rapid Culture Techniques
Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. The CMV DEAFF test is the best example, whereby
• The cell sheet is grown on individual cover slips in a plastic bottle.
• Following inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days.
• The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorescence.
![Page 34: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/34.jpg)
DEAFF test for CMV
(Virology Laboratory, Yale-New Haven Hospital)
![Page 35: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/35.jpg)
Animal inoculation
• Useful for study of pathogenesis, immune response, epidemilogy and oncogenesis
• Earlier human volunteers and monkey were used• Suckling mice are used (arbo, coxsackie)• Mice is inoculated (intracerebral, subcutaneous,
intraperitoneal, intranasal)• Growth of virus is indicated by death, disease or
viable lesion
![Page 36: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/36.jpg)
SEROLOGY
![Page 37: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/37.jpg)
Serology
Criteria for diagnosing Primary Infection
• 4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera
• Presence of IgM• Seroconversion• A single high titre of IgG (or total antibody) - very
unreliable
Criteria for diagnosing Reinfection
• fold or more increase in titre of IgG or total antibody between acute and convalescent sera
• Absence or slight increase in IgM
![Page 38: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/38.jpg)
– Primary and secondary responses to viral infections• IgM (1st exposure)• IgG (2nd exposure)
Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.
![Page 39: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/39.jpg)
Serological Test
Solid-Phase ImmunoassaysEnzyme immunoassay Passive latex agglutinationImmunofluorescence immunoassay Passive hemagglutinationChemiluminescence immunoassay
ImmunoflourescenceIndirect immunofluorescence NeutralizationAnticomplement immunofluorescence
![Page 40: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/40.jpg)
Agglutination AssaysPassive latex agglutinationPassive hemagglutinationHemagglutination inhibition
Complement fixation
Neutralization
Immunoblotting
![Page 41: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/41.jpg)
Viral Hemagglutination• Hemagglutination
– Originally seen with the Influenza virus by Hirst in 1941.– A convenient method of detection & assay of Influenza
virus. – Due to the presence of Hemagglutinin spikes on the
surface.
• Reversal of hemagglutination – Elution– Due to the presence of Neuraminidase enzyme, Receptor
Destroying Enzyme (RDE)– Destruction of receptor – reversal of hemagglutination –
release of virus from the red cell surface– Found only in Myxoviuses.
![Page 42: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/42.jpg)
Hemagglutination (HA) Test
+
RBC Suspension HA Virus Settling Pattern
![Page 43: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/43.jpg)
![Page 44: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/44.jpg)
Hemagglutination-Inhibition (HI) Test
+
RBC Suspension HA Virus Settling Pattern
+
Antibody
![Page 45: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/45.jpg)
ELISA (ELISA) can detect unknown Ag or Ab by
direct or indirect means. A positive result is visualized when a
colored product is released by an enzyme-substrate reaction.
• Uses an enzyme as the label
– Reaction of the enzyme with its substrate produces a colored product indicative of a positive test
• Most common form of ELISA is used to detect the presence of antibodies in serum
![Page 46: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/46.jpg)
ELISA Procedures
Figure 5-19a
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition . ASM Press, 2000.
Figure 5-19b
![Page 47: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/47.jpg)
© Hank Morgan/Science Photo Library/Photo Researchers, Inc.
HIV ELISA test.
![Page 48: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/48.jpg)
Western Blot
HIV-1 Western Blot• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive
Dr.T.V.Rao MD 48
![Page 49: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/49.jpg)
Figure 5.21b: The structure of HIV-1.
Image courtesy of Bio-Rad Laboratories
Figure 5.21c: The typical results of a Western blot testing patient serum for HIV-1 antibodies.
(c)(b)
![Page 50: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/50.jpg)
Figure 5.21a: The basic principles behind the Western blotting procedure. Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition. ASM Press, 2000.
![Page 51: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/51.jpg)
Neutralization Test
1 2 3
B
Following virus isolation:
1.Divide culture yield into small volume in a set of test tubes
2. Prepare the panel of antisera against which the virus isolate is to be challenged
3. To each test tube add one antisera and leave one as a virus control and one as serum control
![Page 52: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/52.jpg)
• Incubate for one hour then inoculate each into cell culture tubes, incubate and observe daily.
![Page 53: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/53.jpg)
2 31
Principle of Neutralization test
![Page 54: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/54.jpg)
54
Complement fixation: Principle
Complement fixation tests detect lysins- Ab that fix complement and can lyse target cells. Involves mixing test Ag and Ab with complement and then with sensitized sheep RBCs. If complement is fixed by the Ag-Ab, the RBCs remain intact and the test is positive. If RBCs are hemolyzed, specific Ab are lacking and the test is negative.
![Page 55: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/55.jpg)
Results and Interpretations:• No haemolysis is considered as a positive test. • haemolysis of erythrocytes indicative of a negative test.
1 2 3 4
A
B
• Microtiter plate showing Haemolysis (Well A2, A3, A4 and B4) and No Haemolysis (Well A1, B1, B2 and B3)
![Page 56: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/56.jpg)
MOLECULAR TECHNICS
![Page 57: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/57.jpg)
Polymerase Chain Reaction • PCR allows the in vitro amplification of specific target
DNA sequences by a factor of 106 and is thus an extremely sensitive technique.
• It is based on an enzymatic reaction involving the use of
synthetic oligonucleotides flanking the target nucleic sequence of interest.
• These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC), annealing of primers to their complementary sequences (50oC), and primer extension (72oC) result in the exponential production of the specific target fragment.
![Page 58: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/58.jpg)
• Further sensitivity and specificity may be obtained by the nested PCR
• Detection and identification of the PCR product is usually carried out by
agarose gel electrophoresis,
hybridization with a specific oligonucleotide probe,
restriction enzyme analysis, or DNA sequencing.
![Page 59: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/59.jpg)
• Advantages of PCR:
– Extremely high sensitivity, may detect down to one viral genome per sample volume
– Easy to set up
– Fast turnaround time
• Disadvantages of PCR
– Extremely liable to contamination
– High degree of operator skill required
– Not easy to set up a quantitative assay.
![Page 60: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/60.jpg)
Schematic of PCR
Each cycle doubles the copy number of the target
![Page 61: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/61.jpg)
Other Newer Molecular Techniques
• Commercial proprietary techniques such as LCR, NASBA, TMA depend on exponential amplification of the signal or the target.
• PCR and related techniques are bound to play an increasingly important role in the diagnosis of viral infections.
• DNA chip is another promising technology where it would be possible to detect a large number of viruses, their pathogenic potential, and their drug sensitivity at the same time.
![Page 62: Lab dig virus](https://reader035.vdocuments.site/reader035/viewer/2022062420/55d02a31bb61eb9b608b4845/html5/thumbnails/62.jpg)
References
• BAILEY & SCOTT`S DIAGNOSTIC MICROBILOGY-12th Edition
• TOPLEY & WILSION`S Textbook of Microbiology
• Ananthanarayan and Paniker`s Textbook of Microbiology- 8th Edition
• Koneman`s Color Atlas and Textbook of Diagnostic Microbiology- 6th Edition