To cultivate and mass production of
Baker’s Yeast.
Introduction:
Bakers
yeast is a commercial preparation consisting of dried cells of one or more
strains of the fungus Saccharomyces cerevisiae, used as a leavening in
baking. Saccharomyces cerevisiae is known as top-fermenting yeast. It is
one of the major types of yeast used in the brewing of beer so called because
during the fermentation process it rises to top of the fermentation vessel.
Beers that use top-fermenting yeast are called ales, and for that reason these
yeasts are also sometimes called "ale yeasts". Top-fermenting yeasts
are unable to ferment some types of sugars, and the resulting beer is sweeter
and "fruitier".
Materials Required:
YPD (Yeast Extract-Peptone-Dextrose) Medium
/ Potato Dextrose Broth
Yeast Strain (Saccharomyces
cerevisiae)
Sugarcane Molasses
Procedure:
Strain Preparation/ Seed Culture
Preparation:
The seed culture was
developed by inoculating a single colony of S. cerevisiae into a growth medium. The culture was incubated at 37°C
for 24 h- 48 h at 150 rpm after adjusting the pH to 7.0.
Batch culture method
Shake-flask cultures Batch cultures in
250 mL Erlenmeyer flasks were applied with 100 mL malt or molasses-based
medium, inoculated with 2 mL pre-seed culture. The initial pH was adjusted to
4.5 and the flasks were incubated at 30 C with shaking at 150 rpm. Growth was
monitored by measuring the absorbance at 600 nm through using a
spectrophotometer by various time intervals.
Result:
The mass production of yeast in batch culture
method was observed and measures the cell mass by UV spectrophotometer.
Ref:
Ehab Ragheb El-Helow, Yasser Elbahloul,
Ebaa Ebrahim El-Sharouny, Sara Ramadan Ali & Atef Abdel-Mageed Ali (2015)
Economic production of baker's yeast using a new Saccharomycescerevisiae
isolate, Biotechnology & Biotechnological Equipment, 29:4, 705-713, DOI:
10.1080/13102818.2015.1038302
To study
the different routes of viral inoculation in the embryonated eggs.
Principle:
General Properties of Virus:
Viruses do not fall in the category of unicellular microorganism.
They lack cellular organizations and contain only one type nucleic acids,
either DNA or RNA.Viruses are obligate intracellular parasites and lack the
enzyme necessary for protein and nucleic acid synthesis. They depend on the
host machinery for their growth and survival. Unlike other microorganism,
complex processes are involved in their multiplication. Outside of the host
cells, viruses are inactive. However, inside living cells, viruses show some of
the characteristics of living things. Viruses are of medical importance because
they have the ability to cause a very large number of human diseases. Virus
diseases range from the minor common cold to sporadic and endemic diseases such
as mumps, hepatitis and so on.
Methods
for Cultivation of Virus:
Since the viruses are obligate intracellular parasites, they cannot be grown on
any inanimate culture medium. Viruses can be cultivated within suitable hosts,
such as a living cell. Generally three methods are employed for the virus
cultivation.
1. Inoculation of virus into animals.
2.
Inoculation of virus into embryonated eggs.
3.
Tissue culture.
noculation
of Virus into Embryonated eggs
Prior to the advent of cell culture, animal viruses could be propagated only on
whole animals or embryonated chicken eggs. Good pasture in 1931first used the
embryonated hen’s egg for the cultivation of virus. The process of cultivation
of viruses in embryonated eggs depends on the type of egg which is used. The
egg used for cultivation must be sterile and the shell should be intact and
healthy. A hole is drilled in the shell of the embryonated egg, and a viral
suspension or suspected virus- containing tissue is injected into the fluid of
the egg. Viral growth and multiplication in the egg embryo is indicated by the
death of the embryo, by embryo cell damage, or by the formation of typical
pocks or lesions on the egg membranes. An embryonated egg offers various sites
for the cultivation of viruses. The
different sites of viral inoculation in embryonated eggs are:
1. Chorioallantoic membrane(CAM)
2.
Amniotic Cavity
3.
Allantoic Cavity
4. Yolk
sac
Chorioallantoic Membrane(CAM) is
mainly employed in the growth of poxvirus. Virus growth and replication in the
CAM is indicated by visible lesions (pocks); grey white area in transparent
CAM. Herpes simplex virus is also grown. Each pock is derived from a single
virion. The morphology of the pocks may vary depending on the nature of the
virus. Under optimal conditions, each infectious virus particle can form one
pock. Hence this method is suitable for plaque studies. Herpes simplex virus
can also be inoculated via CAM.
Allantoic Cavity is the most popular and
simple method for viral inoculation. Allantoic inoculation is employed for the
growth and replication of the influenza virus for vaccine production. This will
provide a rich yield of influenza and some paramyxoviruses. Other allantoic
vaccines include Yellow fever and rabies vaccines. Duck eggs provide a better
yield of rabies virus and were used for the preparation of the inactivated
non-neural rabies vaccines. But they need a longer incubation period than
embryonated hen’s egg. Most of avian viruses can be isolated using this method.
Amniotic Cavity: The amniotic sac is employed
inoculated for primary isolation of influenza a virus and the mumps virus.
Growth and replication of virus in egg embryo can be detected by haemagglutination
assay.
Yolk Sac: It is also a simplest method for growth
and multiplication of virus. Mostly mammalian viruses are isolated using this
method. Immune interference mechanism can be detected in most of avian viruses.
This method is also used for the cultivation of some bacteria like Chlamydiae
and Rickettsiae.
Materials Required:
1. Eggs: 9-day old or 10-day old embryonated
eggs.
2. Egg
shell punch/Carborundum disc
3.
Cotton
4.
Spirit
5. 1ml
disposable syringe
6.
Stationery tape (also called cello or sticky tape)
7.
Viral suspension in Beijenox bottle
8.
Biohazard
Procedure:
Swab the
end of the eggs to be inoculated with 70% ethanol. Allow the alcohol to
evaporate.
Place
used cotton wool in discard tray.
Candle
the egg with high intensity LED torch and mark “X” over the embryo’s eye.
Draw a
line on the shell marking the air space.
With a
sterile egg shell puncher or carborundum disc pierce a hole in the end of the
egg at the marked inoculation site.
Attach a
needle to 1 ml syringe.
Draw
0.1ml of inoculums (Viral suspension) into 1 ml syringe.
Place
the needle through the hole in the eggshell keep the needle and syringe
vertical. The needle will need to penetrate approximately 16 mm into the egg to
reach specific site of inoculation.
Inject
0.1 ml of inoculums into the egg.
Withdraw
the needle from the egg.
Seal the
hole in the shell with stationery tape or melted wax.
Discard
the used needles and syringes in a biohazard box.
Place
the inoculated eggs into a second incubator. Check the temperature and humidity
of incubator.
In the
case of amniotic inoculation, when the needle reaches the embryo a thrust will
strike the embryo and the embryo will move away from the needle. Then inject
the virus suspension.
Expected Results:
The virus suspension are succesfully inoculated into the eggs. The
growth of the virus can be detected by visible pocks in the egg.
To
demonstrate the ability of bacteriophage to replicate inside a susceptible host
cell.
To
determine the concentration of phage particles in a suspension.
Principle:
Bacteriophages
(phage) are obligate intracellular parasites. They multiply inside a bacterium
by making use of some or all of the host (ie., bacteria) biosynthetic
machinery (viruses that infect bacteria are known as bacteriophage). They enter
the bacterial cell by ‘landing’ on the cell wall and injecting their DNA into
the bacterial cytoplasm. After entry, the phage DNA acts as a template for
production of phage proteins. These proteins replicate the phage and subjugate
the cell, eventually causing lysis and death of the host cell. A
bacteriophage particle is even harder to see than a bacterium. Viruses are
beyond the limits of resolution of the light microscope and can be seen only
with electron microscopes. Fortunately, we can use a technique very similar to
the colony-counting technique used to measure the number of bacteria to count
phage particles, known as the plaque assay. Lytic phages are enumerated by this
method.
The plaque assay is
originally a virological assay employed to count and measure the infectivity
level of the bacteriophages. But later, it was applied to measure and count the
mammalian viruses as well. This assay is the most widely used technique for the
isolation of virus and its purification, and to optimize the viral titers. The
basis of plaque assay is to measure the ability of a single infectious virus to
form a “plaque” on a concurrent monolayer culture cells. A plaque is developed
as a part of infection of one cell by a single virus particle that is followed
by the replication of that virus, and finally, the death of the cell. The newly
replicated virus particles will later infect and then kill surrounding cells.
Materials Required:
Cultures:
24- hour nutrient broth cultures of Escheria coli B and T2 coli
phage.
Media:
Tryptone agar plates, Tryptone soft agar tubes (2ml/tube) and
tryptone broth tubes (9ml/tube)
Equipments:
·Bunsen burner
·Water bath
·Thermometer
·1-ml sterile pipettes
·Sterile Pasteur pipettes
·Mechanical pipetting devices
·Test tube rack
·Glassware marking pencil
Media Preparation:
a) Tryptone agar:- Add 10g
Tryptone, 0.01-0.03M Calcium chloride (reagent), 5g Sodium chloride and 11g
agar in 1L of water. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. Autoclave at 121°C for 15 minutes.
b)
Tryptone Broth:-Prepared as above without the addition of agar in the medium. c)
Tryptone soft agar:-Add 10g Tryptone, 5ml Potassium chloride
and 9g Agar in 1L of water. Heat with frequent agitation and boil for 1 minute
to completely dissolve the powder. Autoclave at 121°C for 15 minutes.
Procedure:
Since viruses can grow to incredibly high concentrations, we need
to dilute them in order to count them effectively. Perform dilution of the
bacteriophage culture .
Label all dilution tubes and media as follows. Each tube represents a ten-fold
dilution of the virus
a) Five tryptone soft agar tubes : 10-5,
10-6, 10-7, 10-8, 10-9
b) Five tryptone hard agar plates : 10-5, 10-6,
10-7, 10-8, 10-9
c) Ten tryptone
broth tubes : 10-1 through
10-10
Serial Dilution
·Put on gloves, fill 9 ml of tryptone broth to ten culture tubes
labeled as 10-1 through 10-10. These tubes will be
used for viral serial dilutions.
·Take 1 ml of the phage culture stock that you want to titer and
transfer it to the tube titled 10-1 with a pipette. Mix the
tube well. This is your first ten-fold dilution.(ie; a 1 in 10 dilution)
·Take 1 ml of the mixed culture from your tube labeled 10-1 and
transfer it with a new pipette to the next tube, labeled 10-2 .
Mix this tube as well.
·Continue this pattern to create a serial dilution series. You will
end up with 9 tubes of 9 ml and 1 tube of 10 ml. The viral loads in your tubes
will be diluted anywhere from 10 times (your first tube) or 100 times (your
second tube) to ten billion times (your final tube).
Preparing Plates
·Take five tubes of tryptone soft agar and five Petri plates
labeled as 10-5 through 10-9
·Place the five labeled soft tryptone agar tubes into a water bath.
Water should be of a depth just slightly above that of the agar in the tubes.
Bring the water bath to 100˚C to melt the agar. Cool and maintain the melted
agar at 45˚C. This will ensure that your agar does not solidify in the tubes
before you have a chance to pour it into the petri dishes.
·Aseptically transfer two drops of Escheria coli B culture with a
Pasteur pipette to the agar and mix it gently. These are the bacteria that will
be killed, allowing you to count the number of virus particles in a particular
solution.
·Add 0.1 ml of each serial dilution to its corresponding soft agar
tube while the tubes are still in the hot water bath. For example, 0.1 ml of
your 10-5 serial dilution should go into the soft agar tube
labeled 10-5.
·Using separate Pasteur pipettes and sterile pipette tips, repeat
the previous step for the tryptone broth phage dilution tubes labeled 10-6through
10-9.
·Mix the tubes well and then pour each tube into the Petri plate
with the corresponding label. This will create a thin layer of agar that has
been inoculated with bacteria and viruses in each plate. Incubate all plate
cultures in an inverted position for 24 hours at 37 ˚C
Counting
and Calculating Titers
The viral titer is a quantitative measurement of the biological
activity of your virus and is expressed as plaque forming units (pfu) per
ml.
To calculate the viral titer,
·Take your plates out of the incubator and examine them. You should
see cloudy areas throughout the plate where bacteria have grown, except for
small clear spots called plaques. These plaques are patches of dead bacteria,
and each plaque represents one virus.
·Find a plate that has between 30 and 300 plaques and count the
exact number of plaques on that plate.
·Then use the following formula to determine the titer (pfu/ml) of
your viral stock.
Where, d = dilution
v = volume of diluted virus added to the plate
Sample
calculation:
• An average of 50 plaques formed in the 1:10,000 dilution
wells
• Volume of diluted virus added: 0.2 ml
Result:
The titer value of Phage in the given sample is
___________pfu/ml
