II.

Biological Techniques, Procedures and Methods

•1. Preparation of Reagents

•2. Aseptic Technique
•3. Preparation of Microbiological Media
•4. Sterilization and Disinfection
1. Preparation of Reagents

•Reagent
is a substance or compound
added to a system to cause a
chemical reaction or added to test if
a reaction occurs.
 Preparation of Reagents
• Three basic rules to obey when taking chemicals from a bottle:

• 1. use a clean spoon

• 2. always add acid to base
• 3. wear gloves and mask if necessary.
Note:
all reagents are made with double distilled water unless
otherwise noted.
Preparation of Reagents

•Stock Solutions
* relatively small values are commonly
used ( less than a mL) which creates
problems for making solutions from scratch
every time it is needed.
Preparation of Reagents

Stock Solutions
* to get around the problem of weighing small
amount, buffers and other solutions at concentrations
10 times ( 10X) or greater than needed are being made.
Simply add the appropriate volume to the
recation being set up, to get the desired final
concentration.
Preparation of Reagents
• Stock Solutions
For example:
Restriction enzyme buffers are usually made
as a 10X stock. The working concentration is
usually 1X.
It means that regularly calculating the dilution
factors is needed.
Preparation of Reagents
Some Practice Problems
1) 1 M Tris
dissolve 121.1 g of Tris - HCl in 800 mL of H 2O. Adjust
the pH to the desired value by adding concentrated HCl.
desired pH amount of conc. HCl
7.4 70mL
7.6 60 mL
8.0 42 mL
Check pH using a Tris compatible electrode, and adjust.
Bring volume to 1 L. Dispense in 100 mL aliquots and autoclave
for 15 min at 15 psi on liquid cycle.
more examples
more examples
more examples
 Guidelines in making Reagents, buffers
• 1. Use highest grade of reagents wherever possible.
• 2. Prepare solutions with double distilled or distilled
water.
• 3. Autoclave solutions whenever possible.
Solutions that cannot withstand autoclaving should
be sterilized using 0.2 mm filter.
4. pH meter should carefully be checked using freshly
prepared standard solutions of pH before using it for
 Guidelines in making Reagents, buffers
• 5. Label all containers with name of solution,
percentage or concentration, date.
• 6. Use plastic containers for highly basic solutions like
1(M) NaOH, since glass will be corroded by bases.
• 7. Store all solutions in cold or 4oC, wherever possible.
• 8. Prepare concentrated stock solutions to make up a
range of dilutions, eg. 1(M) Trio. This saves time.
Percentage of Solution
- the exact concentration of the solute in 100 mL
of solvent.
a. % by mass
mass/volume = m/v = g/mL
b. % by volume
volume/volume = mL/mL
ex. 1% v/v acetic acid
= 1 mL Acetic acid / 100 mL solution
 Dilution of Solution ( % by volume)

- lowering the concentration

( %age available)(unknown volume)=(%age required)(volume wanted)
C1V1 = C2V2
example:
• How will you make a 1000 mL of 70% V/V ethanol
from 95% (V/V) ethanol?
Sample problem

•When 10% v/v is required to

prepare a 100 mL HCl and 36%
v/v is available . How do you
prepare the solution?
Molar Solutions
Molarity ( M) mol/L
- mole or molecular
- one in which the molecular wieght of
the solute is dissolved in one litre
( 1000 mL) of the solvent.
Molar Solutions
• * Molecular weight or Molar mass
- the sum of the atomic weights
ex: 1M NaCl
Atomic mass: Na=1 x23= 23
Cl = 1 x 35=35
58 g
1 M NaCl means, 58 g NaCl in 1L of distilled
water.
Molar Solutions

• How will you prepare 100 mL of 1M NaCl?

2. Aseptic Technique
• using practices and procedures to prevent
contamination from pathogens.
* pathogens
- disease-causing bacteria
> applying the strictest rules to minimize the risk
of infection.
 Aseptic Technique
• asepsis(aseptic)
• - free from pathogenic microorgsnisms
• surgical asepsis = “sterile technique”

- used in surgery
medical asepsis = “clean technique”
- involves procedures to reduce the
number and transmission of pathogens
3. Preparation of Microbiological Media
• Microbiological Media
( bacterial growth media)
- a growth medium / nutrient solutions
used in laboratories to grow bacteria.
* the survival and growth of microorganisms
depend on available and a favourable growth
growth environment.
3. Preparation of Microbiological Media
• example of a culture media: AGAR
 4. Sterilization and Disinfection
decontamination processes:
disinfection - the process of eliminating or
reducing harmful microorganisms from
inanimate objects and surfaces.
sterilization - the process of killing all
microorganisms
4. Sterilization and Disinfection

• 3 levels of disinfection:
• a) high-level disinfection ( HLD)
- kills all vegetative microorganisms,
mycobacteria, lipid and nonlipid viruses,
fungal spores, and some bacterial spores.
4. Sterilization and Disinfection

• 3 levels of disinfection:
• b) intermediate-level disinfection ( ILD)
- kills mycobacteria, most viruses, and
bacteria, and is registered by the
Environmental Protection Agency (EPA) as
a “ tuberculocide”
4. Sterilization and Disinfection

• 3 levels of disinfection:
• c) low-level disinfection ( LLD)
kills some viruses and bacteria
5. Preparation of Biological Specimen

•specimen
5. Preparation of Biological Specimen
• Preparation Options:
1. Whole-mounts
where an entire organism or structure is small
enough or thin enough to be placed directly onto
microscope slide
2. “Squash” preparations
where cells are intentionally squashed or
crushed onto a slide to reveal their contents.
5. Preparation of Biological Specimen

Preparation Options:
3. Smears
where the specimen consists of cells suspended in fluid.
Ex. blood, semen, cerebro-spinal fluid, or a culture of microorganisms
4. Sections
where specimens are supported in some way so that very thin slices
can be cut from them, mounted from slides, and stained.
prepared using a “ microtome”