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 .
A.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. B.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.