Introduction

solution to&balanced
(BSS) simple salt
growth
media
By- Ankush, Janvi, & Muskan

Balanced Salt Solution (BSS)

1. What is balanced salt solution (BSS)
2. What are the functions of BSS?
3. Composition of BSS?
4. How does BSS work?
5. Applications of BSS.
6. During tissue disaggregation or dispersal, what
kind of BSS is prefered? /what modifications are
preferred in this case?
7.General protocol for BSS preparation.

• What is balanced salt solution (BSS)

An isotonic solution of inorganic salts present in

approximately the correct physiological
concentrations.
BSS are primarily composed of inorganic salts. It
is a solution made to a physiological pH and
isotonic salt conc.
 Sometimes, sodium bicarbonate, glucose and
HEPESbuffer may also be added to BSS.

• What are the functions of BSS?

The important functions of balanced salt solutions
are listed hereunder:
• Supply essential inorganic ions. Provide the
requisite pH.
• Maintain the desired osmolality.
• Supply energy from glucose.

• Composition of BSS?

• How does BSS work?

• Na & K regulate tonicity and permeability.

• Ca & Mg maintain the integrity of cell membrane
and internal structure.
• Phosphate (HPO4 – H3PO4) and/or bicarbonate
(HCO3-) control the hydrogen ion concentration
through their buffering effect.
• BSS may also include glucose which provides
readily available energy source for cells.
• Applications of BSS.
i. BSS is used as diluent for concentrates of
amino acids ans vitamins to make complete
media. (Basically BSS forms the basis of
many complete media)
ii.As an isotonic wash or dissection medium
iii. For short incubation up to about 4 hrs
(usually with glucose present)

• During tissue diaggregation or dispersal, what

kind of BSS is preferred?/What modifications
are preferred in this case?
PBS without Ca2+ and Mg2+ i.e. PBS Solution A or
D-PBSA is used for this purpose. As Calcium and
Magnesium determines cell-cell interaction and
cell-matrix interaction. Basically Ca & Mg
promote cell adhesion.

• General protocol for BSS preparation.

• Dissolve each constituent separately, adding

CaCl2 at last.
• Make up to 1L
• Adjust pH to 6.5
 • Sterilize the solution by autoclaving or
filtration.
• Note:- With autoclaving, the pH must be
kept below 6.5 to prevent calcium and
magnesium phosphate from precipitating.
• If glucose is included, the solution should be
filtered to avoid caramelization of glucose.

• With autoclaving, marks the level of the liquid

before autoclaving. Store the solution at room
temperature and if evaporation has occurred,
make up to mark with sterile ultrapure water
before use.

Do you know?
Ringer's solution is one of the first laboratory
solution of salts (BSS) developed by physiologist
Sidney Ringer (1882). Using this he successfully
kept frog heart beating after dissection and removal
from the body.
Simple Growth Media

• What is a simple/basal media?

• The term ‘basal medium’ refers to the medium that is
used at the beginning of a culture. It should contain
all essential nutrients that are required to support and
promote culture growth. A robust and productive
basal medium is a prerequisite of any medium
development effort and essential for successful feed
medium development and overall cell culture process
development.
• It contains 29 components, including 13 amino acids,
8 vitamins, 6 ionic species, glucose, and serum
protein.

• Types of media

• What are some of the types of Eagle's Media?

• Basal Medium Eagle (BME)
• Minimum Essential Medium (MEM)
• Dulbecco's Modified MEM (DMEM)

• What is 'Minimal Essential Medium' MEM?

• It was developed by Harry Eagle based on the
concept of minimally required nutrient categories for
mammalian cells.
• The MEM medium contains 29 components,
including 13 amino acids, 8 vitamins, 6 ionic species,
glucose, and serum protein with appropriate pH,
 buffering capability, and osmolality.
• It contains twice the amount of amino acids found in
Basal Medium Eagle and twice the HCO3− and CO2
concentrations to achieve better buffering.
• MEM contains balanced salt solution and sodium
pyruvate.
• It is formulated with a reduced sodium bicarbonate
concentration (1500 mg/l) for use with 5% CO2.
• Eagle’s MEM contains only the water-soluble
vitamins (the B group, choline, folic acid, inositol,
and nicotinamide, excluding biotin)

• What are the features of Dulbecco's Modified

MEM?
• DMEM (Dulbecco’s Modified Eagle Medium) is the
most broadly suitable medium for many adherent cell
phenotypes among defined media for cell and tissue
culture.
• It contains approximately four times as much of the
vitamins and twofold amino acids present in the
original formula and two to four times as much
glucose.
• Non essential amino acids which can be
biosynthesized by cells were eliminated.
• Additionally, it contains iron and phenol red.
• DMEM is further divide into high-glucose type
(4500g/L glucose) and low-glucose type (1000g/L
glucose). High-glucose DMEM is suitable for some
tumor cells with faster growth speed and difficult
attachment, as it is beneficial to retain and grow in
one place.
• DMEM is a basal medium and contains no proteins
or growth promoting agents. Therefore, it requires
supplementation to be a “complete” medium. It is
most commonly supplemented with 5-10% Fetal
Bovine Serum (FBS).

• Which buffer system is used for DMEM?

DMEM utilizes a sodium bicarbonate buffer system
(3.7 g/L) and therefore requires artificial levels of CO2
to maintain the required pH. Powdered media is
formulated without sodium bicarbonate because it tends
 to gas off in the powdered state. Powdered media
requires the addition of 3.7 g/L of sodium bicarbonate
upon dissolving it in water.

• Describe the media primarily formulated for

suspension cultures.
• RPMI-1640 was developed at Roswell Park
Memorial Institute (RPMI) in Buffalo, New York.
RPMI-1640 is a modification of McCoy’s 5A and
was developed for the long-term culture of peripheral
blood lymphocytes.
• It uses a bicarbonate buffering system and differs
from the most mammalian cell culture media in its
typical pH 8 formulation.
• It is characterized by low levels of calcium and
magnesium and high levels of phosphate.
• RPMI 1640 in particular has quite widespread use,
often for attached cells, despite being designed for
suspension culture and lacking calcium. Cells may be
grown as monolayers.
• As a medium for suspension cultures; for example, of
white blood cells, lymphocytes, and hybridomas are
cultured.

• What is Ham's F-12?

• Ham's nutrient mixtures were originally developed to
support the clonal outgrowth of Chinese hamster
ovary (CHO) cells.
• The original F-10 was modified numerously to more
complex flormulations such as F-12 that are suitable
for serum free prepration.
• The supplementation of serum depends upon the cell
lines being cultured.
• Being a rich media, F-12 may prove helpful in failed
cloning practices where the initial media might have
been inadequate.
• What is mixed media?
• Some cell lines do not grow adequately in the
medium available, therefore two or more media are
combined in defined ratios to remediate that.
 • DMEM/F-12 -constitutes a 50:50 mixture of
component-rich Ham’s F12 medium and the nutrient-
rich DMEM medium and compatible with the
requirements of a variety of cell types, it is most
often used as a basal medium for serum-free culture.
• RPMI 1640/DMEM/F-12 (RDF)-Developed for the
serum-free culture of hybridomas, it is a mixture of
RPMI 1640, DMEM, and Ham’s F-12 at 2:1:1 and is
typically used as a serum-free medium with added
insulin, transferrin, ethanolamine, and selenite.

• Which medium is used for transportation?

• Leibovitz L-15 medium
• It contains a higher concentration of sodium pyruvate
(550 mg/L) but lacks NaHCO3 and does not require
CO2 in the gas phase.
• The inclusion of pyruvate in the medium enables
cells to increase their endogenous production of CO2,
making them independent of exogenous CO2, as well
as HCO3−.
• Buffering is achieved via the relatively high amino
acid concentrations.

• Cell lines and recommended Media

• What is the difference between DMEM and

MEM

DMEM MEM
DMEM is a modified type of
basal medium, with increased
amino acids and vitamins
concentration. This also includes
some more substitutions which
increase the nutrients content of
the media.
Amino acid concentration has
increased up to twofold in DMEM
medium.
Vitamin concentration has
increased up to fourfold in
DMEM.
Glucose concentration has been
increased up to 4500 mg/L in
DMEM.
Components like ferric nitrate,
sodium pyruvate are present in
DMEM.
EMEM is one of the first types of
animal cell culture media
developed by Harry Eagle. It is a
simple, basal media with the
minimum amounts of nutrient
compositions.
Minimal concentration of amino
acids is used in EMEM.
Minimal concentration of
vitamins is used in EMEM.
Glucose concentration is 1000
mg/L in EMEM.
MEM contains the minimal
amount of nutrients.