MANUFACTURE NATURAL MEDIA PLASMA AND SERUM

By:
Name : Dion Satrio Pambudi
Student ID : B1B015018
Group :I
Subgroup :1

PRACTICAL REPORT OF ANIMAL TISSUE CULTURE

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO

2017
 I. INTRODUCTION

A. Background

Cell culture is one of major techniques in the life sciences, is the general term
used for the removal of cells, tissues or organs from an animal or plant and their
subsequent placement into an artificial environment conducive to their survival and/or
proliferation. Basic environmental requirements for cells to grow optimally are:
controlled temperature, substrate for cell attachment, and appropriate growth medium
and incubator that maintains the correct pH and osmolality. The most important and
crucial step in cell culture is selecting appropriate growth medium for the in vitro
cultivation. A growth medium or culture medium is a liquid or gel designed to support
the growth of microorganisms, cells, or small plants. Cell culture media generally
comprise an appropriate source of energy and compounds which regulate the cell
cycle. A typical culture medium is composed of a complement of amino acids,
vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones,
and attachment factors. In addition to nutrients, the medium also helps maintain pH
and osmolality (Meenakshi, 2013).
Serum serves as a source of amino acids, proteins, vitamins, carbohydrates,
lipids, hormones, growth factors, inorganic salts, trace elements, and other compounds.
It also improves the pH-buffering capacity of the medium and helps to reduce shear
stress (ie, physical damage that is caused by pipette manipulation and stirring).
Furthermore, serum alters the conditions at a culture substratum, allowing the adherent
cells to readily proliferate there. Fetal bovine serum (FBS) is the most popular and
widely applicable serum today. Other kinds of serum are used in certain situations,
including calf serum (CS) and horse serum. Researchers can select an appropriate
serum type on the basis of its characteristics. Fetal bovine serum generally is rich in
growth factors and contains low levels of -globulins (which have a cell growth-
inhibitory activity) (Meenakshi, 2013).
Foreign substances from unidentified sources can contaminate a culture medium,
thus possibly affecting the empirical results. Typically, those contaminants include
viruses, bacteria, mycoplasma, and endotoxins. There are, however, other types of
contaminants, like plasticizers that might be eluted from plastic instruments or trace
elements, even in water (Takatori et al., 2012). These substances also can affect the
cells in culture (Hamilton & Ham, 1977). It also was reported that some toxic
substances are eluted from the microfilters that are used for sterilization (Harrison et
al., 1990). Some of this contamination seems to be even inevitable, but care must be
taken to minimize it in order to make culture experiments reliable and highly
reproducible. Thus, researchers may consider practicing the sterile technique strictly
and selecting culture instruments carefully (Meenakshi, 2013).
B. Purpose

The aims of this practical class are students know how to make natural media in
the form of plasma and blood serum from chicken and fish.
C. Benefits

The benefit of this practical class are:

1. Students know how to make natural media plasma and serum from chicken and
fish blood.
2. Students know the method how to take plasma and serum from chicken and
fish blood.
3. Students know the aceptic technique to make plasma and serum from chicken
and fish blood.
 II. MATERIALS AND METHODS

A. Materials

The materials used in this practice are alcohol 70%, Heparin/EDTA, tissue, male
chick, nilem fish.
The tools used in this lab activity are table lab, centrifuge, micro centrifuge tube
1,5 ml, syringe, surgery tools, erlenmeyer, Bunsen burner, refrigerator, pipette transfer,
marker, label.
B. Methods

The method used in this practical class are:

1. Injection, syringe, and plastic tube needles are given heparin (EDTA) before
blood is taken to obtain plasma. The heparin (EDTA) level in the tube is taken
into account so that the levels in the blood taken are no more than 0.2 mg.
Taking blood to get serum does not use anticoagulant.
2. Chickens tied on the operating table, wings stretched. The feathers in the elbow
are plucked until a large vein is visible.
3. The skin is rubbed with 70% alcohol, injected needle syringe into the vein.
4. The obtained blood is transferred into a centrifuge tube. The volume of blood
taken recorded.
5. The tube is inserted into the centrifuge and rotated at a speed of 2000 - 2500
rpm for 5 minutes.
6. The clearest layer is transferred to the plastic tube with aseptic technique near
the Bunsen burner. Plasma is stored at a temperature of 4o C.
7. Steps 2-4 were repeated with an injection syringe without heparin or EDTA for
serum preparation.
8. Let the blood shrink about 30 minutes.
9. Centrifuge at a speed of 2000 rpm for 5 minutes, transferred the clearest layer
into new tube and storage it in refrigerator with 4oC or -20oC.
10. Volume serum and plasma noted, and the color which is obtained are observed.
 III. RESULT AND DISCUSSION

A. Result

Table 1. Entourage Data of Plasma and Serum

Media Purpose of Initial Initial

Group Final Colour Final Volume
Source Making Colour Volume

Fish Serum Dark red 0.43 ml Dark Orange 0.1 ml

Fish Serum Dark red 0.45 ml - -

1 Fish Plasma Dark red 1 ml Red 0.2 ml

Chicken Serum Red 0.05 Dark Yellow 0.02 ml

Chicken Plasma Dark red 0.35 Orange 0.05 ml

Fish Serum Dark red 0.4 ml Red 0.12 ml

Fish Serum Dark red 0.3 ml Red 0.05 ml

2 Fish Plasma Dark red 0.3 ml Dark red 0.04 ml

Chicken Serum Dark red 0.4 ml Light yellow 0.06 ml

Chicken Plasma Dark red 0.94 ml Orange 0.2 ml

Fish Serum Dark red 0.5 ml Dark red 0.02 ml

Fish Plasma Dark red 0.3 ml Light red 0.03 ml

3
Chicken Serum Dark red 0.2 ml Orange 0.04 ml

Chicken Plasma Dark red 0.94 ml Light yellow. 0.1 ml

 2

Figure 1. Chicken Blood After Centrifugation

Figure Details:
1. Plasma (0.35 ml)
2. Serum (0.05 ml)

Figure 2. Fish Blood After Centrifugation 1

Figure Details:
1. Serum (0.43 ml)
2. Serum (0.45 ml)
3. Plasma (1 ml)
 4

Figure 2. Natants

Figure Details:
1. Chicken Serum (0.02 ml)
2. Chicken Plasma (0.05 ml)
3. Fish Serum (0.1 ml)
4. Fish Plasma (0.2 ml)
 B. Discussion

Natural media are the natural sources of nutrient sufficient for growth and
proliferation of animal cells and tissue. The natural media, which promote growth in
animal cell, are of three types. They are: Coagula - plasma clots, Biological fluids -
Serum, Tissue extract - embryo extract. Plasma provides a complete nutrient in which
cells could service and multiply in a similar way in which they multiply in body. It
also provides a matrix for new cells during the repair of injury in the body. It provides
a mean of conditioning to the surface of glass for better attachment of cells. It protects
cells and tissues from excessive traumatic damage during subculture. It provides
protection to certain extent to the sudden changes in the environment at times of fluid
change. Plasma is usually taken from a male fowl and heparin is added to prevent the
coagualation of blood. Chicken plasma is preferred to mammalian plasma as it is clear,
solid coagulum even when diluted several time (Manjula, 2007).
Serum, a mixture of hundreds of proteins, contains various factors needed for
proliferation of cells in culture. For example, it contains insulin, a hormone required
for growth of many cultured vertebrate cells, and transferrin, an iron - transporting
protein essential for incorporation of iron by cells in culture. Serum buffers the cell
culture system against a variety of perturbations and toxin effect such as pH change,
presence of heavy metals, proteolytic activity and endotoxins. Besides serum, other
biological fluids used as natural media are; amniotic fluid, pleural fluid, aqueous
humour (from eye), serum ultrafilterate, insect haemolymph, coconut milk, etc
(Manjula, 2007).
Synthetic media are prepared artificially by adding several nutrient (organic and
inorganic), vitamins, salt, O2 and CO2 gas phases, serum proteins, carbohydrates,
cofactors, etc. Different artificial media have been formulated in order to provide a
balanced salt solution for immediate survival with specific pH and osmotic pressure.
For prolonged survival of cultured cells by providing suitable organic compounds and
salt solution supplemented with serum, to make the cultured cells to perform
specialized functions and also for getting continuous cells (Manjula, 2007).
The advantage of serum in media, serum is an effective growth promoting
supplement for all types of cells because of its complexity and multiplicity of growth
promoting, cell protection, and nutritional factors that it contains, natural clot serum
stimulate cell proliferation, and protease inhibitors. Disadvantages of Serum in media
are, serum is a major route for contaminating agents, its difficult due to batch to batch
variation in serum, changing serum batch requires fresh testing. Serum provides
carriers or chelators for labile or water-insoluble nutrients, hormones and growth
factors, protease inhibitors, and binds and neutralizes toxic moieties (Manjula, 2007).
Plasma blood taken as much as 1 ml and serum 0.43 ml and 0.45 ml before
centrifugation. Plasma blood taken as much as 0.35 ml and serum in get as much as
0.05 ml before in centrifugation. After the blood in centrifugation was obtained serum
chicken 0.02 ml and chicken plasma 0.05 ml. Centrifuged fish blood serum 0,1 ml and
plasma 0,2 ml. Most of the plasma and serum obtained from chicken blood and fish
are slightly red, only serum that obtained from chick with orange-color. The parameter
for plasma or serum is it good or not are ; clear, yellow, orange, light red, dark red.
According to Marvin 2007, the color of plasma or serum may turn black because of
haemolysis, i.e rupture of red blood cells, either due to improper handling of blood
samples, pathogenic infection, or disease in donor individuals, from blood samples
taken. If serum or plasma is cloudy, it could be due to lipid in blood or bacterial
contamination. In our opinion, cell lysis is most likely due to improper handling, given
that we had to repeatedly take blood from donor animals and it took a long time to
isolate the blood.

(Tatsuma & Yuta, 2017).

 Collection of blood from chicken, Venipuncture of the cutaneous ulnar or
brachial veins (wing veins) is superficial and easily visualized. Therefore, bleeding
from these veins is usually the simplest and best method for obtaining blood from
turkeys, chickens, and most fowl under field conditions. This is especially when the
bird is to be returned to the flock. Expose the vein to view by plucking a few feathers
from the ventral surface of the humeral region of the wing. The right jugular vein is
usually larger. Place the needle at a slight angle, bevel up, against the vein. Puncture
the vein and slowly withdraw blood. Remove the needle and apply pressure to the vein
for a few seconds (Muhammad et al., 2013).
Blood is a commonly used biofluid for biomarker discovery because it is a data-
rich source containing several thousands of hydrophilic and hydrophobic metabolites
that likely reflect many complex biological process in the body (Wishart et al., 2013).
Age-associated differences in lipid metabolite levels were more prevalent in females
than in males. Many TGs, in particular, showed age-associated differences in females
but not in males. This observation suggests that the decrease in estrogen secretion
following menopause affects the metabolism of several lipoproteins, resulting in an
increase in the levels of TGs in the elderly. Estrogen may be involved in regulation of
the metabolism of lipoproteins such as low-density lipoprotein (Masaki et al., 2014).
 III. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result can be concluded that

1. Natural media are the natural sources of nutrient sufficient for growth and
proliferation of animal cells and tissue.
2. Collection of blood from chicken, Venipuncture of the cutaneous ulnar or
brachial veins (wing veins), and collection of blood from fish can be done from
caudal fin vein.
B. Suggestion

There is no suggestion, but for myself its hard for me to find the vein blood
from caudal fin in fish.
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