3. Laboratory Safety

9. Monolayer culture
• Requires substrate or solid surface
for attachment and growth
: Anchorage dependent of growth
• Contact inhibition of movement Monolayer

10. Primary cell culture
+ enzymes
time

11. Subculture
enzymes
time

12. Transformation
loss of contact inhibition

13. Stationary cultivation

14. Culture flask
Culture Plate

15. Medium Constituents
Balance salt solution : Phosphate buffer, Mg , Ca
Inorganic ions and trace elements
: for membrane potential and osmotic pressure
: buffer
Energy source : glucose, glutamine
Amino acid : metabolism and biological synthesis

16. Role of Serum
• Buffer, Chelator, Carrier proteins
• Bind to toxin
• Protease inhibitor
• Promotes attachment of cell to substratum
• Source of Intermediate metabolites,
hormone and growth factor

17. Primary cell culture and Establishment cell line
• Preparation of cell suspension from intact tissue
. Single cell preparation
: use mechanical, Chemical, and/or enzymatic method
. Disaggregate or dissociate cell
: cutting, homogenizing, rotary shaker, vortex,
pipette

18. • Preparation of cell suspension from intact tissue (cont.)
Enzymes
• Trypsin (crude)
: from cattle and pig’s pancrease

19. • Preparation of cell suspension from intact tissue (cont.)
. Removal of tissue and place in Isotonic
(or growth medium with antibiotic)
. Trim off unwanted parts and cut into smaller pieces
. Dissociate cells using enzyme
. Remove large debris and wash cells

20. • Preparation of cell suspension from intact tissue (cont.)
. Suspend cells in growth medium accordingly
. Pipette into culture vessel and incubate

21. Common Cell line (cont.)
• HeLa
cancer tissue:cervical carcinoma
: harbors HPV type genome
• Vero
: from African green monkey kidney
: preparation of Poliovirus vaccine

22. Work in Safety cabinet Class-II
UV
ethanol for decontamination
Culture medium and Reagent
1.PBS
2. Growth medium
: %FCS-DMEM
3. Trypsin-Versene

23. Subpassage
Remove growth medium
Wash with PBS
Detach cell with Trypsin-Versene
Remove TV

24. Refresh with growth medium
Mix cell by pipette
Count and calculate number of cell
: Hemacytometer

25. Detection of virus in cell culture:
•Cytopathic effect -CPE:changed monolayer
architecture, the entire cell/ nucleus thickening and
shrinking (pycnosis), inclusion bodies formation, giant cell
(syncytia)formation, nucleolar displacement,rearangement
and margination of the nuclear
chromatin,vacuolization,the type of CPE depends on virus
specifity and the kind of cells, polio virus, adeno
virus, HSV, CMV;
•Viral hemagglutination–direct reaction with red
cells, influenza;
•Hemadsorption –adsorption of erythrocytes to infected
cells;parainfluenza, influenza
•Plaques effect –clear areas appear in culture (PFU)due to
cell lysis;

26. Hemagglutination test: method
1:8
1:2 1:2 1:2 1:2 1:2
virus
serial dilution
8 16 32 64 128 256
mix with red
blood cells
side view
top view
Titer = 32 HA units/ml

27. Hemagglutination assay: influenza virus
Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the
top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coat
wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last
dilution that shows complete hemagglutination activity. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters
Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)

28. Plaque
Assays

29. Quantitation of viruses
a. TCID50(50% Tissue culture infected dose
): Infections dose of 50% CPE of tissue cultures
caused by viral minimum infected dose is called
TCID50
b. PFU : under controlled condition, single
plague can arise from a single infection virus
particle, termed a plague-forming unit.

30. Plaque assay: method
1:100
1:10 1:10 1:10 1:10 1:10
virus
serial dilution
10-2 10-3 10-4 10-5 10-6 10-7
plate 1 ml
plaques
(100,000) (10,000) (1000) 100 10 1
Titer = 1 x 107 pfu/ml

31. Virus cultivation 2. Inoculation of embryonated chick eggs
•
USE OF FERTILE EGGS
Largely replaced by use of TC
• Still useful in cultivation and detection of some viruses, flu
• Three parts of the egg are of use
– A The amniotic cavity:
• Surrounds the embryo & lined by a single layer of epithelial cells.
• The amniotic fluid bathes the external surface of the embryo and comes into contact with
the respiratory and alimentary tracts.
– B The allantoic cavity:
• Comprises an outgrowth of the hind-gut of the embryo and is lined with endoderm.
• Both are useful for the cultivation of viruses, particularly
• orthomyxoviruses (e.g., influenza) and paramyxoviruses (e.g., mumps).
Membranes major source of cells in which virus growth occurs, but the embryo may also become
infected.
• The allantoic cavity is routinely used because it is technically much simpler to inoculate.
• However, some viruses (e.g., human influenza isolates) may need to be egg-adapted by growth in the
amniotic cavity before they will grow efficiently in the allantoic cavity; the reason for this is not
known.
C The chorio-allantoic membrane : The membrane consists of an outer layer of stratified epithelium
which constitutes the respiratory surface of the egg, and an inner layer of endoderm (the lining of
the allantoic cavity). The membrane may be used as a cell sheet provided it is first dropped away
from the shell membrane. Dermatropic viruses (poxviruses and some herpes viruses) will grow on
this membrane, and at low concentrations, will give discrete foci of infection which consist of
centres of cell proliferation and necrosis (pocks). The membrane may therefore be used to assay
these viruses. In addition, different viruses cause pocks of different colour and morphology, and
this is of diagnostic value for distinguishing between different poxviruses.

32. Growth of virus on embryonated eggs
Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1

33. •production of pocks or plaques on chorioallantoic
membrane(herpes, smallpox, vaccinia);
•development of hemagglutinins in embryonic
fluid(influenza);
•death of embryo -encephalitis viruses;
Convenient, inexpensive, the viral suspension
injected into the fluid of the egg, viral
growth is detected by death of the
embryo, or lesion on the membrane of
the egg.
It is the most common method for viral
growth and isolation , and used for
preparation of viral vaccine.