Viral Growth Curve & Growth Cycle

Viral Growth Curve & Growth Cycle

Viral Growth Curve:

Graphical representation of replication of virus is called viral growth curve.

Phases of viral growth curve:

1. Striking : in this stage virus is reducing .the viruses are going to eclipse period .
2. Latent period: In this stage there is no evidence of virus. Because in this stage virus completely goes to eclipse period.
3. Appearance: Again the presence of virus demonstrated.
4. Rising period: Very rapidly number of viruses is increased. In this stage release of virus occurs.

Stages of the viral growth cycle:

Attachment and penetration by parental virion

Uncrating of the viral genome

Early viral m RNA synthesis

Early viral protein synthesis

Viral genome replication

Late viral m RNA synthesis

Progeny virion assembly

Virion release from cell.

Viruses

Viruses

Viruses are particles composed of an internal core containing either DNA or RNA (but not both) covered by a protective protein coat.  Viruses are small sized living organisms, they reproduce by replication and responsible for a wide range of infection.

Examples : Pox virus,

-          Hepatitis virus.

-          Polio virus.

-          Herpes virus.

General Characters:

-          Very small in size about 18 to 300 nanometer (mm)

-          Must grow inside the living host cell.

-          Contain either DNA or RNA.

-          Ribosome absent.

-          Energy yielding enzyme absent.

-          Inactivated by50 -60 °C for 30 minutes.

-          They can’t grow on inanimate culture media.

-          Optimum Ph  for viruses lies between 5 to 9

-          Multiply by replication.

-          Viruses are generally resistant to antibiotics but sensitive to interferon’s

Morphology :

1. Shape: May  be spherical, coiled, filamentous,
2. Size : From 18-300  nm.
3. Structure : Viruses consists of-

a.       Core- contain either DNA or RNA.

b.      Capsid –A  protein coat, capsid with capsomeres.

c.       Envelope-Outer part of capsid composed of lipoprotein , certain virus have envelope.

4. Optimum Ph : viruses lies between Ph-5 to 9.
5. Inactive : By heat at 50-60°C  for 30 minutes.
6. Multiply ; By replication.

Classification:

1. According to nucleic acid:

1. DNA Viruses

Examples:

-Pox virus.

- Parvo virus.

- Papova virus

- Herpes virus.

-Adeno virus etc.

2. RNA Viruses

Examples:

-          Rabies virus

-          Reo virus

-          Retro virus

-          Dengue virus.

-          Toga virus etc.

2. On the basis of strandedness:

1. Double stranded viruses

Examples:

-Pox virus

- Papova virus.

- Herpes virus.

- Adeno virus

- Rota virus.

- Small –pox virus etc.

2. Single  stranded viruses

Examples:

-Rabies virus

- Retro virus

- Dengue virus

- Toga virus etc.

C. On the basis of host range:

1. Animal viruses e.g. Rabies virus.

2. Plant viruses e.g. Tobacco Mosaic Virus (TMV)

3. Bacteria viruses(Bacteriophage)e,g T₂ Phage.

4. Human viruses e.g. polio virus.

Virology

Virology   

The    branch   of microbiology   that    deals with the study of viruses. It is the specialized subject which discuss about the cultivation, identification, destruction and exploration of viruses and related topics.

Branches of virology:

Based on application:

ð  Clinical virology – it deals with human pathogenic virus.

ð  Veterinary virology – it deals with animal pathogenic virus.

ð  Plant virology-      It deals with plant pathogenic virus.

Application of virology:

ð  In medical science.

ð  In veterinary science.

ð  In pharmaceutical science.

Steps in viral pathogenesis:

1. Attachment and entry within cells: Viruses usually replicate at the primary site of entry, eg. Respiratory tract, gastrointestinal tract, conjunctiva. Some viruses may be directly introduced in blood , e.g. hepatitis B virus, HIV.
2. Viral spread : After primary replication at the site of entry certain viruses spread and produce disease at a distant site, e.g. Polio virus , which enter through the gastrointestinal track but produces disease of  the central nervous system (CNS) Viraemia is the presence of virus in blood.
3. Cell tropism: Viruses exhibit organ and cell specificities due to presence of specific cell surface receptors, e.g. Enter viruses enter through the gastrointestinal tract but cause central nervous system disease.
4. Cell injury: The change depends on the virus and type of the cell.

1. Cell necrosis.
2. Proliferation of the cell followed by necrosis.
3. Proliferation of cell.
4. There is no change.

Virus shedding: The virus is shaded into the environment

Pseudomonas

Pseudomonas

Pseudomonas is a genus of gammaproteobacteria, belonging to the family pseudomonadaceae containing 191 validly species.

Characteristics of Pseudomonas:

ð  Gram –negative

ð  Rod shaped .

ð  One or more polar flagella, providing motility.

ð  Aerobic.

ð  Non –spore forming .

ð  Don not ferment glucose.

ð  Oxides positive.

ð  Catalase positive.

ð  Produce pigments (pyocyanin.pyvirdin)

ð  Non –invasive.

ð  Opportunistic pathogens.

ð  Fruity odor in culture.

Pathogen city of Pseudomonas:

Pseudomonas species cause infection in hospitalized patients such as-

1. Urinary tract infections, after catheterization.
2. Respiratory tract infection –pneumonia.
3. Wound infection (specially burns)
4. Sepsis.
5. Necrotic skin lesions.
6. Infection of ear –Otitis externa.
7. Corneal infection in contact lens users.

Steps:

1. Specimen collection :

-  Wound swab.

-  Urine

-  Pus.

2. Microscopic examination:

Gram staining: Gram –negative rod shaped bacilli.

3.Isolation & Culture:

- Inoculate the specimen on a plate of blood agar media or Mac Conkey’s agar media.

- Incubate at 37°C  for over night.

- Characteristics colonies are forming like.

=> Bluish colonies for pyoverdin pigment.

=> Greenish colonies for pyoverdin pigment.

=> Dark red colonies for pyorubin pigment.

=> Black colonies for pyomelanin pigment.

4. Biochemical tests:

=> Catalase—Positive

=> Oxidase- positive.

=> H₂S –Negative.

=> Gas-Negative.

Spirochetes & Treponema palladium

Spirochetes

A microscopic bacterial organism in the spirohaetaceae family spirochetes, have  a worm –like spiral-shaped form and wiggle vigorously when viewed under a microscope treponema palladium, the cause of syphilis ,is a particularly well known member of spirochete,

Classification of Spirochetes :

1. Pathogenic for humans : Three genera.

1. Treponema-causes syphilis, bejel , yaw and  painta,
2. Borrelia –causes relapsing fever and lyme disease.
3. Leptospira-causes leptospirosis (wails disease)

2. Communal and saprophytes: communal of the mouth and genital tract.

Treponema palladium:

Disease: syphilis-the great pox:

Morphology:

1. T. palladium is about 5-15 ×0.2 µm
2. It is a flexuous helix having evenly distributed 6-12 coils.
3. It is actively motile-rotating and slow bending motility often forming a complete circle.

Staining : staining by silver impregnation causes thickening of the organism and can be seen by dark field microscope. It can not be stained by gram method.

Treponema: three species are of medical importance-

1. Teponema palladium-causes syphilis and bejel.
2. Treponema preemie- causes yaws.
3. Treponema carteum –causes pinta.

Pathogen city: T.pallidum causes

1. Syphilis: Syphilis is a sexually transmitted disease(STD)

Syphilis are two types:

a.       Sexually acquired (venereal )syphilis.

b.      Congenital syphilis.

2. Bejel : bejel group of diseases, or endemic syphilis or non –venereal syphilis .

Transmission of syphilis :

1. Sexual contact with an infected person.
2. Transplacental transmission from infected mother to newborn.
3. From transfusion of infected blood
4. Intravenous drug abusers who share a common needle.
5. Accidental transmission in laboratory workers.

Stages of acquired syphilis:

1. Primary syphilis
2. Secondary syphilis.
3. Latent syphilis.
4. Tertiary syphilis.

Laboratory diagnosis of syphilis:

Principle: Laboratory diagnosis of syphilis consists of direct demonstration of treponemas by microscopic examination and serological test,

Steps:

-          From lesion fo primary syphilis(hared chancre) and secondary syphilis (condylomata lata).

-          Serum .

-          CSF.

Macroscopic  examination:

1.Dark ground illumination (DGI)

Findings: Cork screw movement of T.pallidum.

2.Direct florescent antibody (DFA)test.

Findings : Typical fluorescent stained spirochetes.

Serological tests:

1. Non –treponemal regain tests.

-          VDRL.

2. Treponemal detection

-          TPHA

-          FTA-ABS

-          ELISA.

Clostridium

Clostridium

Spore forming bacteria:

The spore –bearing organisms are gram positive, non-acid fast, may be motile , aerobic or anaerobic and may be capsulated or non –capsulated.

Classification of spore – forming bacteria:

Spore forming bacteria three types:

Spore forming bacteria

1.  Gram –positive bacilli.         2. Gram –positive cocci              3. Gram-negative coccobacilli.

-          Bacillus anthraces.               –Sporosarcina spp.                           –Coxiella burnetti.

-          Bacillus subtilis

-          Clostridium tetani.

Clostridium tetani:

Habitat: Soil is the usual habitat. In man, they found in intestine.

Morpholoty:

Shape                   : Rod shaped with rounded end.

Size                      : 2-5 µm

Flagella                : pertrichous flagella present.

Spore                   : Present.

Capsule                : Absent.

Motility               : Motile.

Staining               : They are gram –positive bacilli & non-acid fast.

Cultural character:

1. Anaerobic
2. Optimum temperature: 37°C
3. Growth on:

1. Agar streak : colonies are thin transparent film.
2. Anaerobic agar plate culture: light ,flocky & cloudy colonies,
3. Agar stab : surface growth negative , growth occur along the line of inoculating wire.
4. Blood agar: Haemolysis occur.
5. Brain meat medium : Digestion of the medium slowly & blackening.
6. Cooked meat medium  slight turbid growth with gas formation.
7. Coagulated agar medium :liquefaction of medium.
8. Gelatin stab : liquefaction & blackened.
9. Litmus silk: precipitation of casein.
10. Serum agar: Small & transparent colonies.
11. On blood agar: Haemolysis colonies.
12. On cooked meat medium .

1. Turbidity of the medium.
2. Blackening of meat.

O. On Loffler’s serum slope: softened.

Biochemical character:

1. Sugars are not fermented.
2. Milk is not coagulated .
3. In dole is produced.
4. Gelatin is liquefied.
5. Coagulated serum is softened.
6. Iron- gelatin is blackened.

Clinical features: 

After incubation period: 6-10 days-

1. Lock jaw (trismus )due to masseter muscle spasm.
2. Rises sardonic us due to spasm of facial muscle.
3. Opistohotonus due to marked trunk muscle spasm.

Laboratory diagnosis of tetanus:

Principle: Finding out of the causative organism confirms the diagnosis of tetanus.

Steps:

1. Specimen collection : Wound swab .
2. Microscopic examination : On Gram staining .

Finding: Gram –positive bacilli (tennis racket appearance)

3. Isolation &identification from culture:

1. On Robertson’s cooked meat media.

Finding: turbidity of medium& blackening of meat.

2. On blood agar: Haemolysis colonies.
3. On Loffler’s serum slope: softened.

4. Other tests:

-          Agglutination test.

-          Toxigenicity test.

Animal inoculation test.

Vireo

Vireo

Vibrionaceae: Vireo is a genus of gram –negative bacteria possessing a curved rod shape, several species of which can cause food borne infection, usually associated with eating undercooked seafood, typically found in salt in salt water . the name vibreo derives from fillips pacing who isolated microorganisms , he called ‘’Vibrions’s from cholera patients in 1854.

Classification of vireos:

1. Vireo cholera.
2. Other vireos e.g. V. parahaemolyticus . V . fluvialis.

Vireo cholera:

Habitat         : Intestine of man.

Morphology :

Shape           : Curved comma.

Size              : 15 to 30 µm

Flagella        : Present, flagellated.

Spore           :  Absent, non-sporing.

Capsule        : Absent, non-capsulated.

Motility       : Motile,

Staining       :

They are gram negative and non-acid fast.

Cultural characters of Vireo cholera:

1. Aerobic.
2. Optimum temperature: 37°C 
3. Optimum Ph : 8-8.4
4. Growth :

ð  On Mac Conkey’s agar and DCA: Colonics are pale.

ð  On Monusr’mdeia: Colonies are dark with chacteristic hollow around them.

ð  On alkaline peptone water: vireo grows in 6 hours forming a pellicle on the surface.

ð  On gelatin stab: liquefaction occurs at the top and spreads downwards in a funnel shaped form.

Classification of Vireo cholera:

V. cholera is classified into  V, cholera ol and V, cholera non-01 serogroups or serotypes on O antigens (lipopolysaccharide).

1. V. cholera o1 are further classified into serotypes and biotypes:

a. serotypes : indaba, ogawa and Hikojima.

b. Biotypes: Classical biotype and EI Tor biotype.

2. Vibrio cholera non o1 : serogroup or serotypes 02 to 0139 have been isolated.

Biochemical behavior of Vireo cholera:

1. They ferments various sugars with acid formation but no gas.
2. It is non-lactose fermented & non-urea splitter.
3. In dole is produced and nitrate is reduced .Cholera –red reaction is positive.
4. VP reaction is negative with V. cholera (classical ) but is positive with EI Tor (biotype)

Steps;

1. Specimen collection:

-          Stool

-          Vomit.

-          Rectal swab.

2. Microscopic examination:

1. Gram staining:

Gram –negative comma shaped bacilli.

2. Hanging drop preparation: Typical motility (a fish in stream appearance).
3. Dark ground illumination (DGI) shooting star in dark sky appearance.

3. Isolation and culture:

-          Specimens inoculate in alkaline peptone water as selective and enrichment media.

-          Incubate at 37°C for overnight.

-          Streak on Mansur’s media and citrate-Bilesuctose agar (TCBS agar)

-          Characteristic colony will be appeared,

Findings:

On Mansur’s media:

ð  Small black colonies surrounded by characteristic hollows.

On TCBS agar ; Large yellow colored colony.

4. Biochemical test: Oxides-positive.
5. Serological test:

-          Slide agglutination test (mostly used)

-          Indirect haemagglutination test

-          ELISA.

Klebsiella

Klebsiella

The genus klebsiella belongs to the tribe Klebsiellae, a  member of the family Enterobacteriaceae.

The organisms are named after Edwin Klebs, a 19^th century German Microbiologist. Klebsiellae are non-motile, rod –shaped, gram –negative bacteria with a prominent polysaccharide capsule.

Classification of Klebsiella:

Synonym	Pathogen city
K. Pneumonia.	UTI Bacteremia. Hemorrhagic necrotizing consolidation of lung.
K. ozaenea	Ozena in nasal mucosa.
K. rhinoscleromatis.	Rhinoscleroma-destructive granuloma of nose & pharynx.
K. oxytoca	UTI.

Cultural characters of Klebsiella :

1. Aerobic.
2. Optimum temperature: 37°C.
3. Optimum Ph: 7.4.
4. Culture:

ð  On blood agar: Colonies are large& mucoid.

ð  On Mac Conkey’s agar : Colonies are large, mucoid  due to capsular substance.

Laboratory diagnosis of Klebsiella:

Steps:

1. Specimen collection:

ð  Throat swab.

ð  Sputum.

ð  Urine.

2. Microscopic examination:

Finding : Gram negative capsulated rod shape bacilli.

3. Isolation and identification from culture:

1. The materials are streaked on blood agar and Mac-Conkey’ s agar media
2. Incubated at 37°C for over night .
3. Large& mucoid colonies are picked up.
4. Biochemical behavior of Klebsiella is noted.

4. Perform  antibiogram  of the sensitivity tests.

Proteus

Proteus

Diseases: UTI, Wound infections.

Proteus species are part of the Enterobacteriaceae family of gram negative bacilli

Proteus organisms are implicated as serious causes of infections in humans .they are widely distributed in nature and are associated with variety of infections in man , like wound infections and urinary tract infections.

Characteristics of Proteus:

1. Gram-negative rods.
2. Facultative anaerobic.
3. Lactose non-fermenter,oxidase-negative.
4. Produce the enzyme , phenylalanine dreaminess.
5. Very motile & produce swarming growth on blood agar
6. Produce the enzyme , urease’

The following are the members of the genus : proteus.

1. Proteus vulgar is
2. Proteus mirabilis.
3. Proteus moraine.
4. Proteus Ruggeri

Diseases caused by Proteus.

ð  Urinary tract infections (UTI)

ð  Bacteremia.

ð  Abscess

ð  Pyemic abscess

ð  Wound infections.

ð  Pneumonia.

ð  Pyemia.

ð  Septicemia.

Enter toxin mediated

Pathogenesis of diarrhea by E. coli (Enter toxin mediated)

Diagnose urinary tract infection (UTI) caused by E. coli.

Steps:

1. Specimen:

ð  Clean catch mid-stream urine.

ð  Suprapubic puncture in infants.

2. Microscopic examination:

Wet preparation of centrifuged deposit , it helps in provisional diagnosis.

Finding : Pus cell > 5 per high power field (HPF) is significant to diagnose of UTI.

3. Isolation and identification from culture:

Process:

1. Mix the urine by rotating the container .
2. Using a sterile calibrated wire loops, inoculate a loopful of urine in media like

ð  Blood agar.

ð  Mac-Conkey’s agar media.

3. Incubate the plates aerobically at 37°C for over night.

Colony coutn:

• 10⁵/ ml of Gram –negative bacilli is  significant
• In case of supra-pubic puncture, only one colony of Gram-negative bacilli is significant.

4. Biochemical tests: Identification is also done by standard bio-chemical tests like-

i.KIA medium

Slant           =y²

Butt            =y

Gas             = ±

H₂S            =Negative(-)

ii.MIU medium

Motility      =   +¹

Indole         =    +³

Urease        =(-)  

Escherichia coli

Escherichia coli

E.coli is the most common cause of urinary tract infection and gram negative rod sepsis. It is one of the two important causes of neonatal meningitis and the agent most frequently associated with ‘’traveler’s diarrhoea’’a watery diarrhea .some strains of E. coli are entero hemorrhagic and cause bloody diarrhea.

Morphology of Escherichia coli:

1. Gram negative rod.
2. Capsulated by slime layer.
3. Motile with peritrichous flagella
4. Facultative anaerobe and aerobe.
5. Lactose fomenters i.e. form pink colonies in Mac-Coney’s agar media.

Cultural Characters of Escherichia coli:

1. On blood agar: Large, grayish-white, opaque, moist ,circular colonies , some stains produce hemolytic colonies.
2. On Mac-Conkey agar: Colonies are rose pink which indicates that the organism is lactose fermenter.
3. DCA,CL D agar & SS  Agar: No or poor growth:
4. Agar streak: white, circular, smooth & white to yellowish white in colour.
5. Eosine-methylene blue agar: Circular moist colony.
6. Gelatin stab: Good growth along the line of inoculation % no liquefaction.
7. Litmus mile: Acid & gas produce by media coagulates.
8. Endomedium: Circular, smooth , red colonies with metallic sheen.
9. Broth medium: Rapid growth with the formation of pellicle %   a    slight slimy sediment.
10. Potato medium: Grayish to yellowish brown growth.
11. Wilson & Blair medium :No growth,

Biochemical reactions: 

E.coli is lactose fermented and produce acid & gas.

• Diseases caused by E.coli:

E.coli causes diseases both within and outside the enteric tract.

1. Urinary tract infections (UTI): 

Cystitis, pyelonephritis(by certain O –serotype).

2. Systemic infections :

=> Wound infection.

=> Pneumonia, septicemia, abscess,

=> Neonatal meningitis.

=> Disseminated intravascular coagulation(DIC)

=> Biliary tract infections

3. Intestinal tract infections : Diarrhoea,dysentery.

Classification of Escherichia coli:

Classification of E coli on the basis of diarrhea production:

1. Enteropathogenic E. coli (EPEC) : Dysentery characterized by bloody diarrhea.
2. Enterotoxigenic E . coli (ETEC) : Traveler’s diarrhea and infantile enteritis by LT& ST.
3. Vero toxin producing E.coli (VTEC) :

ð  Bloody diarrhea.

ð  Hemorrhagic colitis.

ð  Hemolytic uremic syndrome.

4. Enter invasive E.coli (EIEC) Infantile diarrhea by invasion.
5. Enteroaggregasive E.coli (EAggE): Persistent diarrhea.

Mechanism of diarrhea by E.coli:

Some species of E.  coli (ETEC) cause watery , non –bloody diarrhea and some species (EPEC,EHEC) cause bloody diarrhea.