collection and preservation of virus.pptx

collection and preservation of virus

1. Virology 3th class lab 2 Clinical Samples Collection & Preservation By : Assist. lecturer . Mawahb Hatem Mones

2. virus pathogenic sample. There are two types of virus samples:- 1-Fulid sample (Blood, s.f ,serum , c.s.f , urine ). 2- Solid sample (Lung, heart ,Liver , Kidney ,spleen , stool, )

3. Specimens sending to the viruses Laboratory. 1- Anticoagulated blood. 2- Urine Specimens 3-Skin scraping :in case infections caused by poxvirus and herpes virus. 4-Faeces : In case enterovirus infection caused by Rota virus. 5- Swabs: Collected a specimen from (nose, eye, trachea, vaginal, Rectal). 6- Organs (heart, spleen, liver, kidney, lung. In case of died animals it’s called (Autopsy). 7- Biopsy Specimen sending or part from infected organ to human or animal, when are living .

4. Transporting of specimens to the viruses Laboratory. Must be the following information when transport the pathogenic specimens to the Labor. 1- Specimens transport into strong a sterile screw cap tube or bottles which contains viral transport medium like 199 media add to mixture antibiotics to preserve the Specimens. 2- They should be transported insulated box that contains ice packs or ice cubes in plastic bags. 3- Using some time preservated substance like glycerin 50% or phosphate Buffer saline

5. Clinical Samples Collection 1 - Blood Specimens :Blood Include Serum or plasma sample could be obtained from venous blood, which can be performed by the laboratory personnel. For serum or plasma sample, first 2-3 ml of venous blood is collected using sterile syringe and needle from a patient putting into a clean, dry, sterile tube. Care must be taken to avoid hemolysis, since this may produce a false-positive result . Serum : is required, allow the whole blood to clot at room temperature for at least one hour and centrifuge the clotted blood for 10 minutes at 2000 rpm. Then transfer the serum to a labeled tube .

6. Plasma : sample is obtained by treating fresh blood with an anticoagulant, centrifuge and separate the supernatant. The specimen should be free from hemolyzed blood. Finally, the tube should be labeled with full patient's identification (Age, Sex, code no, etc.). The test should be performed within hours after sample collection, if testing cannot be performed immediately, serum may be stored between 2°C and 8°C for up to 72 hours. If this could not be done preserve it at -200°c

7. Blood separation Plasma is that part of the blood, which contains blood clotting agent called as fibrinogen. serum is the fluid part of the blood and does not contain clotting agent.

8. 2- Urine Specimens : The specimen should be collected at the onset of illness if congenital disease is suspected. - Examining 2 - 3 consecutive urine specimens. - (10 - 20 ml of urine) are collected in sterile containers and transported to the laboratory on wet ice or cold pack. , examples; polyoma virus 3-Stool Specimens: Stool specimens for viral isolation attempts should be collected as soon as possible, and usually no later than 7-10 days after onset of illness. Enteroviruses may be excreted for weeks so if infection with these viruses is suspected, stool specimens collected later than 10 days post onset may be collected .example ; Rota virus . 4- Saliva : collected by aspiration or expectoration into a sterile container also may be used for virus isolation . Ex ; corona virus 5-Semen Specimens : Semen specimens collected into sterile screw-cap jars should be sent to the laboratory on wet ice or cold pack

9. 6-Eye Specimens: A swab moistened in sterile saline is used to collect secretions from the conjunctiva. 7- Cerebrospinal Fluids (CSF): For virus isolation 3-4 ml of CSF should be collected no later than 7-10 days after onset of illness

10. 8-Cervical Specimens : The specimen is transported to the laboratory on wet ice or a cold pack. If it cannot be tested within 48 hours it should be frozen below - 70 C, Its can make it as a paraffin -wax block or pap smear for cytology and virus detection. 9-Vesicular Lesion Specimens: Vesicular fluids and cellular material from the base of lesions should be collected for virus isolation during the first 3 days of the eruption. Vesicles are washed gently with sterile saline and the vesicular fluids are aspirated with a 26-gauge or 27-gauge needle attached to a tuberculin syringe, or with a capillary pipette.

11. 12- Throat and Nasopharyngeal Specimens: Virus isolation is most successful if respiratory specimens are taken within the first 3 days of illness, and they should be collected no later than 5 days after onset. For virus isolation, swabs from both the throat and nasal passage should be collected. Note, respiratory specimens should not be frozen at -20 C temperatures as this will markedly reduce chances of isolating respiratory viruses. In genera freezing should be avoided if possible Example : Influenza viruses, Adeno virus, Corona virus and others.

12. Viruses preservation In general, DNA viruses are more stable than RNA viruses but both types are extremely stable and can be preserved relatively easily. Many viruses can be kept for months at refrigerator temperatures and stored for years at very low temperatures without the need for special preservatives or carefully regulated slow freezing techniques. Their simple structure, small size and the absence of free water are largely responsible for this stability. Viruses with lipid envelopes are often less stable than non-enveloped viruses at ambient temperatures but survive well at ultra-low temperatures or in the freeze-dried state

13. Methods for long-term virus preservation 1. Freeze dried preparations of virus can be maintained at 4°C in the dark and lower temperatures increase the storage time . 2. Virus is retained for very long periods in liquid nitrogen. However , most viruses will survive almost in liquid nitrogen . 3. Proteins are effective protectants for virus cryopreservation . The suspending medium of choice for many viruses is tissue culture medium containing added serum or other proteins, at concentrations up to or greater than 10 %

14. the proteins possible provide buffering capacity against pH changes, assist in colloidal dispersion of the virus particles and reduce or inhibit other processes that damage nucleic acids. Viruses contained in serum or tissues from human or animal specimens can be stored at ultra low temperatures without further treatment . 4 . High titer virus preparations are retain for long-term storage longer than a low titer preparation . 5 . Freeze drying ( lyphoilization ) viruses for long term preservation .This is probably the most satisfactory method of preserving viruses for very long periods

15. 6- It is good method to preserve small volumes of virus suspension. In general, virus infectivity is maintained more effectively when samples are preserved in small volumes because rapid freezing and thawing of a virus preparation is less harmful to the virus than slow freezing and thawing. 7- Viruses can be preserved for long periods as nucleic acid. The purified nucleic acid viral RNA and many DNA viruses This principle can be utilized to preserve these viruses for very long periods of time. The ethanol precipitated RNA and DNA can be stored almost indefinitely at 4°C (or lower temperatures) under ethanol. The ethanol is important for long term storage of RNA, to inhibit enzymes that breakdown RNA .