Lab. Virology MSc. Amaal M. K.

Complement Fixation Test(CFT)

Lab. Virology MSc. Amaal M. K.

Lab. Virology MSc. Amaal M. K.

FUNGI

• Uni or multicellular organisms

• Eukaryotes

• Defined nuclei

• Cell walls of carbohydrate and chitin

• Saprophytic or parasitic

• Vegetative and sexual reproduction

FUNGI IN MEDICINE

• Dermatophytes: grow on skin and hair

• Yeasts: grow on mucous surfaces and in the body

• Systemic infection: hyphae and yeasts

Fungul culture media

• Sabrouraud Dextrose Agar • Malt Agar (for mold and yeast) • Corn meal agar(for candida spp) • Potato Dextrose agar(PDA) to determine colonial

shape and colonial changes color • Brain Heart Infusion agar • Nutrient agar • Blood agar • Mycobiotic agar(contain anti fungul) • christensen´s Urea agar(for Cryptococcus neoformans) • Assimilation media

Important fungi stains

1-Lactophenol cotton blue

Phenol…..to killed bacteria

Lactic acid…. dissolved agar and seperate mycelia

Glycerin ….. Preservative

Cotton blue….. Blue dye

2-Giemsa stain

3-Indian ink

Important fungi samples

Skin, hair, nail scraping

Body fluid (CSF, urine, blood )

Smear (buccal cavity, bronchial cavity, sputum, vagina,…etc)

YEASTS

• Single cells

• Reproduce vegetative by budding

• Occasionally form pseudomycelium

• Sexual reproduction by forming ascospores within cell

• Candida, Malassezia, Cryptococcus, Histoplasma

CANDIDA

• 3-6 m, oval cells

• Gram positive

• Pseudohyphae

• Germ tubes

• Chlamydospores

• Grows at 37C on Sabouraud’s dextrose agar

• Creamy white 2mm colonies

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CANDIDA COLONIES

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PSEUDOHYPHAE, CANDIDA

DERMATOPHYTES

Septate branching hyphae

• Digest keratin

• Microconidia, arthrospores, macroconidia

• Grow on Sabouraud’s within 7-14 days at 28C

• by surface appearance and colour of underside

• Confirm by shape of macroconidia

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MACROCONIDIA

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MICROCONIDIA

ASPERGILLUS

• Septate branching hyphae

• Sporing heads or conidia in oxygen

• Conidiophore

• Aspergillus may have sexual stages

• Use colonial appearance, size and details of conidiophore to identify

ASPERGILLUS FUMIGATUS

• On food

• Spores infect young non-immune or immunosuppressed person

• Grows best on Sabouraud’s at 24-28C

• Star shaped colonies

• Green-blue with sporing heads

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ASPERGILLUS FUMIGATUS

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ASPERGILLUS CONIDIOPHORE

Colonial Morphology of Fungi

Cryptococcus neoformans

Candida albicans

Wangiella dermatitidis

Aspergillus fumigatus

T. menta-grophytes

Trichophyton tonsurans

Lab.1 Msc. Amaal M.K.

Safety Procedures and Precautions

1. Laboratory coats are worn.

2. Long hair is tied back away from the shoulders.

3. Working areas are kept clear of all unnecessary items.

4. Hand and bench tops are kept clean with disinfectant.

5. Nothing is placed in such as fingers, pencils or any subject. the mouth

6. Do not smoke, eat or drink in the laboratory.

7. Any student with afresh, unhealed cut, scratch, burn or other injury on

either hand should notify the instructor before beginning or continuing

with the laboratory work.

8. If you should spill or drop culture or if any types of accident occur, call

the instructor immediately.

9 .Unnecessary activities can cause accidents and promote contamination.

10. Before leaving the laboratory, carefully wash and disinfect your

hands.

Equipment needed for virology lab.

The virology laboratory should be provided with adequate equipment which

should take into account certain general principles, i.e. it should be:

Designed to prevent or limit contact between the operator and the

infectious material (e.g. biosafety cabinets, electronic pipetting aids, etc.)

Constructed of materials that are impermeable to liquids, resistant to

corrosion.

Should be free of sharp edges, burrs and unguarded moving parts.

Designed, constructed and installed to facilitate simple operation and

provide for ease of maintenance, cleaning, decontamination and

certification testing. Glassware and other breakable materials should be

avoided wherever possible.

A list of essential and desirable equipment required for a virology laboratory

is listed below

Essential equipment

Biosafety cabinets – Three (one for handling cell cultures, one for

handling stock viruses and one for processing clinical specimens)

One incubator and two CO2 incubators (one for uninfected cell cultures

and the other for infected cell cultures)

-20 °C and –70 °C freezers

Inverted light microscope

Fluorescent microscope with photography attachments

Filtration apparatus for preparation of tissue/cell culture media

Refrigerate centrifuge

water bath

pH meter

Magnetic stirrer

Vortex mixer

Electronic balance for weighing chemicals

ElISA Reader and washer

Micropipttes ( 100µl, 200 µl, 20 µl)

Multi-channel pipettes – 8 and 12 channel pipettes (20-200 µl and 50-

300µl)

Autoclave – Two ( one for decontamination and one for sterilization)

Hot air oven for sterilizing glassware

One personal computer with printer, photocopier, fax machine and

telephone lines

PCR machine ( conventional and real-time)

Gel electrophoresis apparatus

UV trans illuminator

Ice-making machine

Liquid nitrogen containers

Water purification/distillation system, which provides high-grade water

suitable for tissue culture work

Glassware such as volumetric flasks, measuring cylinders, pipettes (1 ml,

2 ml, 5 ml and 10 ml), conical flasks, reagent storage bottles (50 ml,

100ml, 250 ml, 500 ml and 1000 ml)

Electric brushing machine and automatic pipette washer

Desirable equipments

Shaker water bath

Rocking platform

Ultracentrifuge

Lab.2 Virology MSc. Amaal M.K.

Collection and transport of viral specimens.

a) Specimen for detection of viruses should be collected as soon as

possible after the appearance of the symptoms that is when the

concentration of the virus is at its highest.

b) All specimens must be clearly labeled to identify their source (nose

swab, throat swab etc.). This is especially important when more than

one specimen is sent with one form.

c) All clinical specimens must be collected in clean sterile containers,

which must be properly sealed. The outer portion of the container

must not be contaminated.

d) Collect the specimen from the appropriate site within several days of

onset of clinical symptoms.

e) Collect an adequate amount of specimen. Inadequate amounts of

specimen may yield false-negative results.

General Criteria for Unacceptable Specimens

Specimens may be rejected for many reasons including the following:

1. Hemolysis.

2. Insufficient quantity.

3.Unlabeled specimen.

4. Prolonged transport.

Types of specimens that used for virus isolation

Blood: used to detect CMV, entero virus and adenoviruses. (5-10 ml) of

anticoagulated blood collected in a vacutaner tube is needed.

Urine: used to detect CMV, mumps, rubella, measles, polyoma viruses

and adenoviruses can be detected in urine. The best specimen is at least (10

ml) of a clean-voided first-morning urine.

Rectal swabs and stool specimens: Used to detect rotavirus, enteric

adenoviruses and entero viruses. Rectal swabs are collected by inserting a

swab (3-5) cm into the rectum to obtain feces.

Tissue: useful for detecting viruses that infect lung (CMV, influenza virus,

adenovirus), and gastrointestinal tract (CMV). Specimens are collected

during surgical procedures.

Nasopharyngeal swabs: are used for the detection of respiratory viruses

such as influenza virus A & B or parainfluenza virus. Throat swabs

Saliva

Cerebrospinal fluid

Specimen transport and storage:

1-All specimens collected for detection of viruses should be collected with

aseptic technique and transported immediately to the laboratory by using

transport media (in screw capped tube) and should be placed in ice,

specimens for viral isolation should not be kept at room or higher

temperature. Under unusual circumstances, specimens may need to be held

for days before processing. For storage up to 5 days, hold specimen at 4°C.

Storage for 6 or more days should be at – 20°C or preferably at – 70°C.

2-Some antibiotics (e.g penicillin, streptomycin, nystatin) should be added to

viral transport media (VTM), especially when contamination with microbial

flora is expected. Examples of successful transport media include Stuart’s

medium, Amie’s medium.

3-Centrifuge under refrigeration, the supernatant will contain the viruses.

4-Filter the supernatant to give crude liquid of viruses.

5-Detection of viruses in this crude liquid of viruses .

Viral transport media (VTM)is used to:

1. preserve viral infectivity within the specimen.

2. prevent specimen from drying.

3. stop the growth of bacteria and fungi.

VTM contains:

1. Isotonic solution containing protective protein such as bovine

serum albumin to promote viral stability and maintain viral

infectivity.

2. Antibiotics to control microbial contamination

3. Buffers to control the pH

Isolation and identification of virus

Techniques for diagnosing viral infection can be grouped into three

categories:

1. Direct examination of specimen

o Clinical specimen- examined directly for the presence of

Virus antigen

Virus particles

Viral nucleic acid

o Electron microscopy

o Light microscopy: histological appearance.eg. inclusion bodies.

o Antigen detection by immunofluorescence, ELISA ..

o Molecular techniques for direct detection of viral genomes.

2. Indirect detection (virus isolation).

Viruses require living metabolizing cell for grow, so they can be isolated in:

Cell culture.

Fertile hen's embryos.

Experimental animal.

3. Serology

Complement fixation test (CF).

Hemagglutination inhibition test (HI).

Neutralization test.

Agar gel immundefusion test (AGI).

Cultivation of Viruses • Virus cultivation is also referred to as

propagation or growth.

• Viruses are obligate intracellular parasites that require living cells in order to replicate.

• Cultured cells, eggs and laboratory animals may be used for virus isolation.

• Although embryonated eggs and laboratory animals are very useful for the isolation of certain viruses, cell cultures are the sole system for virus isolation in most laboratories.

1-Cultivation of Viruses in laboratory animals

• To find out the course of the disease, the

clinical symptoms, pathogenicity and

post-mortem lesions, the viruses have to

be inoculated in the laboratory animals.

• When viruses are grown in animals, the

animals should be healthy, disease free.

Inoculation routes of lab animals

• Intra Muscular(I.M)

• Sub Cutaneous(S.C)

• Intra Venous(I.V)

• Intra Peritoneal(I.P)

• Intra Nasal(I.N)

• Intra Cerebral(I.C)

Laboratory animals for

virological studies

• Mice

• Guinea pig

• Rabbits

• Monkey

• Hamsters

• Chickens

2-Cultivation of viruses in the fertile

egg

Chicken embryos are used almost exclusively because of their

1) Availability

2) Economy

3) Convenient size

4) Relative freedom from latent infection and extraneous

contamination. Eggs only from healthy, disease-free flocks should

be used.

5) Lack of production of antibodies against the viral inoculum.

Factors which affect the multiplication

of viruses in chicken eggs

1.Age of embryo

2.Route of inoculation

3.Concentration and volume of inoculum

4.Temperature and time of incubation after

inoculation.

Procedure of egg inoculation

1) Candle the egg and select an area of inoculation. In this

area make a pencil mark at the point of inoculation.

2) Drill a small hole through the shell at mark but do not

pierce the shell membrane.

3) Apply suitable disinfectant to the holes and allow

drying.

4) Inoculate the virus

5) Use Elmer's glue or wax to close hole.

Inoculation routes of embryonated eggs

A-The amniotic cavity

B-The allantoic cavity

C-The chorio-allantoic membrane

D- Yolk sac

The pathological changes on

embryonated eggs

1.Death of embryos.

2.Curling and dwarfing of embryos.

3.Hemorrhages of subcutaneous tissues.

4.Pock lesions on Chorioallantoic membrane

5.Development of inclusion bodies in the

cytoplasm or nucleus of infected cells.

CANDLING

BOX

Egg Holders

HARVEST OF EMBRYO

INFECTIOUS BRONCHITIS

IBV EMBRYOS

NDV – OCCIPITAL

HAEMORRHAGE

ND VIRULENT VIRUS

DUCK PLAGUE – VIRULENT

VIRUS

BLUE TONGUE VIRUS – CHERRY

RED EMBRYO

BLUE TONGUE VIRUS –

CHERRY RED EMBRYO

‘POCK’ LESIONS ON CAM

.الضاري مقارنة بأجنة مجموعة السيطرة تبين الآفات الموجودة عمى الأجنة الهالكة بفايروس نيوكاسل( 1)صورة رقم A- الحقنساعة من عممية ( 48)جنين بيضة هالك ومحتقن في أقل من. B - جنين بيضة السيطرة غير الهالك المحقون بدارئ الفوسفات.

3-Cultivation of Viruses in Cell culture

Cell culture preparation

• Most commonly used tissues for cell culture

preparations are kidneys and tests from young

animals like lambs, kids, calves and piglets.

• Chicken embryo kidneys and fibroblast cell

cultures are also commonly used.

• Tissue fragments are first dissociated, usually with the aid of trypsin or collagenase.

• The cell suspension is then placed in a flat-bottomed glass or plastic container (petri dish, a flask, a bottle, test tube) together with a suitable liquid medium and an animal serum.

• The cells will attach and spread on the bottom of the container and then start dividing, giving rise to a primary culture.

• Primary cultures are maintained by changing the fluid 2 or 3 times a week.

• When the cultures become too crowded, the cells are detached from the vessel wall by either trypsin or EDTA, and portions are used to initiate secondary cultures.

• Continuous cell lines consist of cells that have been immortalized,

either in the laboratory or in the body; they can be subcultured

indefinitely.

• Cells are cultured in media [such as Eagle's medium, Minimum

Essential Medium (MEM) and Hanks Medium (HM)] that provide

nutrients.

• Most media are supplemented with animal serum, which contains

substances that promote the growth of many cell lines.

• Other important roles for the medium are the maintenance of

optimum osmotic pressure and pH for the cells.

• Viruses can be cultivated in cells growing on the surface of a

variety of plastic vessels with the cells bathed in the growth

medium.

• Cells grow on a plastic or glass surface as a single layer of cells,

known as a monolayer.

• Contamination with bacteria and fungi can cause major problems

in cell culture work; in order to minimize these problems work is

normally done in a sterile cabinet and add antibiotics with

antifungal.

• Many cell types require a relatively high concentration of carbon

dioxide, which can be supplied in a special incubator.

Cell culture flasks, dishes and plates

Cell culture flasks, dishes and plates

Cell

culture

work

Types of cell cultures

1. Primary cells – These are normal cells obtained from

freshly killed adult animals. These cells can be

subcultured only once or twice e.g. Monkey Kidney.

2. Semi-continuous diploid cells – These are cells taken

from embryonic tissue, and may be passaged up to 50

times. e.g. fetal bovine kidney

3. Continuous cells – Make from cancer tissues of human or

animal tissue such as HeLa, Vero, and Hep2. These cells

are immortalized cells and may be passaged indefinitely.

Virus Neutralization Test (VNT)

Serological method that detects the presence of viral

neutralizing antibodies.

The antibodies bind to the viral particals, block viral

infectivity- no cell infection.

VNT are conducted by:

Mixing dilutions of antibodies (serum patients) with

standardized amount of virus, incubating them and cultured in

to cells, eggs or animals to have clear cytopathic effect

observation.

cytopathic effect (CPE) refers to structural changes in host

Procedure of Virus Neutralization Test (VNT)

First step:

Serial dilutions of serum (virus-neutralizing Ab)+ known virus.

Incubated for 1-2hr at 37 °C.

Second step:

Cell culture inoculated with mixture.

Seal the plate and incubated for 37°C.

Direct microscope.

No Ab (in serum) +virus no neutralization

CPE

Ab( in serum) +virus neutralization no CPE

Cell remain intact