Transcript Scientific Writing

MICR 306 Advanced Applications of
Viruses and Fungi:
PRACTICALS
Part A
Prof. J. Lin
University of KwaZulu-Natal
Westville campus
Microbiology Discipline
2014
School of Life Sciences
Practical
• Sit in the assigned location based on the your
student number.
• Carry your lab coat, permanent marker, match
and a lab book (A4 size)
• The students are requested to write down and be
familiar with the title, purpose of the practical and
the procedure before the practical.
• During the practical, write down each step clearly
in detail.
• Before leaving the lab, the assigned
demonstrator will check each lab notebook and
sign it to verify. (Consider as absent in the
practical if you do not have the signature)
Practical report mark
• The student write an comprehensive lab
report based on the departmental format.
The report should be attached with the lab
note after marking. (Microbiology
Practical Report from the Department)
 At the end of the semester, the notebook
will be evaluated as a part of the lab
report.
Report 1
Project I: Virus Culture in Embryonating
Chicken Egg (Practical 1 4)
Report 2
Project II: Enumeration of Somatic Phage of
Environmental Water samples
Report 3
Project III: Isolation, purification and
Identification of Fungal Cultures from seeds.
Project IV: Seedling Symptom Test
Virology Practical
Project I: Virus Culture in Embryonating Chicken Egg
i)
Candling and Inoculation of chicken egg.
1/08
ii)
Harvesting the Allantoic fluid
8/08
iii)
The Haemagglutination Test (HA)
15/08 iv)
The Haemagglutination Inhibition Test (HI)
22/08 Project II: Presence-Absence Spot Test for Coliphages
and Enumeration of Somatic Phage (Environmental
Water samples)
29/08
Theory Test 1 + record results
5/09
i)
Practical Test 1
ii)
Submitting the Assignment 1
iii)
Submitting the Project I report
25/07
Mycology Practical
12/9
19/9
26/9
3/10
Project III: Isolation, purification and Identification of
Fungal Cultures from seeds
Submitting the Project II report
Project III continued
Submitting the Assignment 2
Mid Term Break
Project III continued
Project IV: Seedling Symptom Test
10/10
Theory Test 2 (Main Hall)
17/10
Complete practical
Submitting Mycology Practical report
Practical Marks (20% CAM)
• Report 1 (12.5%) + Report 2 (12.5%)
• Report 3 (25%)
• Practical Tests (including all pre-tests) (50%)
Tutorial grouping
A. 205524290  210519852
(Monday 10:30 – 11:15, G4)
B. 210519862  211510458
(Monday 11:25 – 12:10, G4)
C. 211510550  211560578
(Wednesday 8:40 – 9:25, G4)
D. 212500850  212566989
(Thursday 1:15 – 2:00, G4)
Practical One
Virus Culture in Embryonating
Chicken Egg
Virus Culture in Embryonating
Chicken Egg
• Viruses can only replicate in living cells.
• Before cell culture was developed, fertile chicken
eggs were used to cultivate viruses in the
laboratory. The use of eggs for virus propagation
was first demonstrated by Woodruff,
Goodpasture and Burnet in 1930. Chicken
embryos continue to have certain uses in virology.
Host specific
• Under natural conditions, many viruses
are relatively host-specific. Moreover,
they may show a marked predilection for
certain tissues of the host such as nervous
tissue, epithelial tissue, etc. While a
number of viruses display host-specificity
and tissue affinity or "tropism“, the
majority can be adapted to foreign hosts
by passage.
• The cells and extra-embryonic membranes
of the chicken embryo provide varied
substrates that allow the growth of many
viruses. Because of the ability to alter
their tropism and to adapt to a new
host species, many viruses become
capable of growing in chicken embryo
tissues and may even attain a higher
concentration than in the tissues of the
natural host.
• Although many viruses are now cultivated in cell
culture, for some viruses no suitable cell culture
system exists and egg inoculation is the method
of choice.
• Influenza virus vaccines are still cultivated in
eggs, and hence people with egg allergies
cannot tolerate the influenza vaccines.
However, attempts to produce bird flu vaccines in
eggs have been unsuccessful, as these viruses
kill the embryo before sufficient virus can be
produced
Virus Culture in Embryonating
Chicken Egg
• For convenience, the mayxoma virus
grows well on the chorioallantoic
membrane, whereas the mumps virus
(like NDV) prefers the allantoic cavity.
The infection may produce a local tissue
lesion known as pock, whose appearance
often is characteristic of the virus.
Virus Culture in Embryonating
Chicken Egg (part 1)
• Candle the eggs, locate the air cell and mark the
base of the air cell.
• Disinfect an area about 3mm above the marked area.
• Use an 18 gauge needle to puncture the membrane
at the base of the air cell.
• Use 1ml tuberculin syringe with 25 gauge needle and
deposit 0.2 ml of live NDV….. (see practical notes)
• Seal the hole in the egg with melted paraffin wax.
• Incubate the eggs at 37 0C in an incubator containing
a beaker of water to maintain proper humidity.
Virus Culture in Embryonating
Chicken Egg (part 2)
• Candle the eggs (to eliminate
nonspecific deaths)
• Disinfect the entire area of the shell
covering the air well.
• Cut away the shell, covering the air cell,
with a pair of scissors to within 5-10
min of the base of the air cell.
Virus Culture in Embryonating
Chicken Egg (part 3)
• Carefully remove the membrane lining
the base of the air cell and using a
Pasteur pipette, aspirate the allantoic
fluid.
• Pool the allantoic fluid containing NDV
and centrifuge at 3000 rpm at 40C for 15
min to remove debris.
• Freeze 2 ml aliquots of allontoic fluid in
Bijou bottles. (for next week)
Hemagglutination Test
Purpose
1) To determine the titre of the virus in the
allantoic fluid from eggs inoculated with
NDV
2) To determine the titre of the antigen to
be used as standardized antigen in the
HI test.
 The Ag is diluted to provide 10 HA units
of activity per 40 µl of the Ag suspension
(standardized Ag).
Hemagglutination Test
Procedure
1) Dispense 40 ml of diluent into two sets of 11
wells of the micro-titre plate.
2) Dispense 40 ml of undiluted antigen into 1st
well, mix well and transfer 40 ml from well 1 to
well 2. Repeat  1:2 to 1: 2048
3) Dilute 5 fold of antigen using diluent. Repeat
step 2  1:10 to 1: 10240
4) To well 12, add 40 ml of diluent only.
5) Add 40 ml of 2% CRBC to all wells. Mix well
and leave for 60 min.
6) Record the end point as the high dilution of
antigen which provides complete agglutination
of CRBC.
Hemagglutination Assay
• The Hemagglutination Assay (HA) is a
quantification of viruses by hemagglutination.
• Some viral families have surface or envelope
proteins, that are able to agglutinate (stick to)
human or animal red blood cells (RBC) and
bind to its N-acetylneuraminic acid.
• In contrast to or LD50, HA does not give any
measure of viral infectivity, because no virus
replication is required in this assay.
• It is an easy, simple and rapid method and can
be applied to large amounts of samples.
Hemagglutination Assay
• All strains of Newcastle disease virus will
agglutinate chicken red blood cells.
This is the result of the haemagglutinin
part of the haemagglutinin/neuraminidase
viral protein binding to receptors on the
membrane of red blood cells. The linking
together of the red blood cells by the viral
particles results in clumping.
Hemagglutination
NDV
Hemagglutination
Hemagglutination Assay
No
Yes
Hemagglutination Assay
• The detailed conditions depend on the type of
virus. Some viruses bind RBCs only at certain
pH values, others at certain ionic strengths.
• A virus dilution (eg. 2-fold from 1:2 to 1:2048)
will be applied to an RBC dilution for approx. 3060 min, often at 40C, because else viruses with
neuraminidase activity will detach the virus from
the RBCs. Then the lattice forming parts will be
counted and the titre calculated.
• Virus concentration in virions per milliliter
 HA units
HA units
SERUM DILUTION
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
1:2048
LOG TITRE
1
2
3
4
5
6
7
8
9
10
11
512 HA units
Hemagglutination Inhibition Assay
• The hemagglutination assay may be
modified to include the addition of an
antiserum. By using a standard amount of
virus, a standard amount of blood cells,
and serially diluting the antiserum, one can
identify the minimum inhibitory
concentration of the antiserum (the
greatest dilution which inhibits
hemagglutination).
Standardised Antigens
SERUM DILUTION
1:2
1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
1:2048
LOG TITRE
1
2
3
4
5
6
7
8
9
10
11
10 HA units
?
512 HA units
Haemagglutination Inhibition (HI)
• Mechanism: The antigen in HI tests is simply a
solution of the antigenic particles (usually a
virus) which is capable of inducing the reaction
of haemagglutination when mixed with a
suspension of red blood cells. This agglutination
is not an antigen/antibody reaction but, rather
is the attachment of viral particles by their
receptor sites to more than 1 cell. As more
and more cells become attached in this manner
agglutination becomes visible. The presence
and concentration of antibody is measured
by its ability to inhibit the agglutination at
various dilutions.
Haemagglutination Inhibition (HI)
Most HI tests carried out in routine poultry serology
use chicken erythrocytes. It is important to carry
out at least three wash cycles to ensure that the
final CRBC suspension is free of antibody and
other serum proteins. A fourth cycle is advisable
if the HI titres in the source birds are 1:128 or
higher. After washing the red cells are resuspended in PBS at a standard concentration.
The packed red cells should be re-suspended at
least at 0.75%, since lower erythrocyte
concentrations tend to be associated with more
variable HA values for the antigen and hence
affect HI results (McMullin, 1979)
Haemagglutination Inhibition (HI)
Methods:
The level of antibody to NDV is determine
by this test.
Haemagglutination Inhibition (HI)
Procedure
1) Dispense 40 ml of diluent into two sets of 12
wells of the micro-titre plate.
2) Dispense 40 ml of antiserum to NDV into 1st
well, mix well and transfer 40 ml from well 1 to
well 2. Repeat to well 11 1:2 to 1: 2048
3) Add 40 ml of standardized antigen to 1st – 11th
wells. Mix well and wait for 60 min.
4) Add 40 ml of 2% CRBC to all wells. Mix well and
leave for 60 min.
5) When the last (12th) well “buttoned out”. Record
the end point as the high dilution of serum which
produces complete inhibition of agglutination of
CRBC.
Haemagglutination Inhibition (HI)
Water Quality
To protect public health, many indicator
organisms have been proposed as all these
suggest the presence of human pathogens.
Diarrhoea  faecal contamination
MICR 307, membrane filtration technique
Water Quality Analysis
• Physico-Chemical Parameters
– T, pH, total C, N, P, BOD, COD
– Heavy Metals, toxic chemicals
• Microbiological Parameters
– Bacteriological (Membrane filtration technique)
• THC , TC, FC, E. coli, Salmonella pp., V. cholerae
 40% diarrhoeal cases due to unknown
causes in 2003  Enteric viruses
…….
Virological parameters
• Somatic coliphage, F-RNA coliphage …..
• Adenoviruses, Enterioviruses, rotavirus…….
• it is imperative to be able to detect the human
enteric viruses directly in water quality monitoring
 Technical problems  Virus ID
• The survival and incidence of bacterial viruses
(phages) in water environments resemble (ideally)
that of human viruses more closely than most other
bacterial indicators commonly used.
Virus indicators
• Somatic coliphage
• F+RNA coliphage
Somatic coliphages
• The term covers a wide range of phages.
They attach to the bacterial cell wall and,
in optimal conditions, may lyse the host
cell in 20–30 min.
• Myoviridae, Siphoviridae, Podoviridae &
Microviridae
E. coli WG5
E coli replication affected by a range of
factors
• The densities of both phages and their
host bacteria
• The physiological status of the host
• The presence of suspended particles and
other bacteria in water.
Presence-Absence Spot Test
• The procedure will involve a single layer of
agar prepared according to the below
procedure where the hosts are spread
onto the pre-prepared plates. Thereafter
10-50 μℓ of sample water (depending on
the water sources) will be spotted onto the
plates and incubated at 37°C for 24 hours.
Enumeration of somatic
coliphages
• Environmental water samples
 Filtered through 0.22 µm membrane
 Filtrate  concentrated
• Different media/Different hosts (2 plates)
– E. coli B4 (host T4 phage),
– E. coli WG5 (host somatic phage)
 specificity
Each plate  different bacteria sp.
(T4, WG5)
30 µl
30 µl
T4 phage
30 µl
Concentrated
Water
Somatic
phage
30 µl
Filtered
water
Enumeration of somatic
coliphages
• Double layers techniques  host bacteria
+ magnesium chloride + molten agar
medium to the water sample  pour the
total volume of the mixture into plates.
• Plaque formation (zones of bacterial host
lawn clearing)  expressed as plaque
forming units (PFU) / 100 mL.
Plaque Titration of Phage Lysate
• To quantitate the number of phage
particles in a given suspension, dilutions of
the suspension are overlayed on agar
surfaces in the presence of high
concentrations of the susceptible bacteria.
• After incubating for bacterial growth,
phage activity is evidenced by small clear
areas (plaques) in the otherwise smooth
film of bacterial growth.
Procedure
• Agitate the tube carefully by shaking to distribute
the indicator bacteria and phage uniformly
throughout the agar and quickly pour the mixture
over the surface of the bottom agar of the
nutrient agar plates provided.
• Rapidly spread the molten top agar over the
surface of the plate by tipping the plate slightly,
using a rotary motion.
• Pour a duplicate plate of dilution  10-3.
Procedure
• Repeat steps 2 and 3 with the 10-0  10-5
dilutions.
• After adding 0,2 ml bacteria, transfer the
contents of the remaining tube onto the last petri
dish. This petri dish will contain no phage
and will therefore serve as a control (one per
bench).
• After allowing the top agar to solidify, invert and
incubate the plates overnight at 37°C.
 Read your plates on Monday
Count the number of plaques
Titre (n) = y/vx
Where:
y = number of plaques,
v = volume plated,
x = dilution of sample
If the host is partially resistant to the phage then the plaque
may be uniformly turbid (for example, if 10% of the cells
survive phage infection).