LAB1 :

STERILIZATION
LA Dalia Hesham, TA Hoda Raafat, TA Omnia Bassam, TA Nada Nasser,
TA Marina Nashaat, TA Hesham Fady, TA Zeyad Hamdy, TA Nour Ahmed,
TA Nada Sherif, TA Amr Walid, TA Youssef Maged, TA Maya Hany &
TA Marwan Tarek
Sterilization
Any process that effectively kills or
eliminates moving agents (such as fungi,
bacteria, viruses, spore forms, etc.) from
a surface, equipment, article of food or
medication, or biological culture medium.
 Sterilization Methods
1- Heat
2- Chemicals
3- Radiation
4- Filtration
5- High pressure
 1. Heat Sterilization

1.1.Moist heat sterilization

2.1.Dry heat sterilization

1.1. Moist Heat
Sterilization
◦ The most useful approach is autoclaving, in which items are sterilized by
exposure to steam at 121°C and 15 bars of pressure for 15 minutes or
longer, depending on the nature of the item.

◦ Under these conditions, microorganisms, even endospores, will not

survive longer than about 12 to 13 minutes.

◦ This method is rapid and dependable.

• *Endospores: consists of the bacterium's DNA and part of its cytoplasm,

surrounded by a very tough outer coating & can survive without nutrients.

• In moist heat, the microbial proteins undergo denaturation, a process in

which the three-dimensional form of the protein reverts to a two-
dimensional form, and the protein breaks down.
 1.2. Dry Heat
Sterilization
◦Dry glassware such as pipettes and petri plates
must be sterilized.
◦Steam tends to etch glassware and also leaves it
damp.
◦Therefore, such items are generally dry-heat
sterilized.
◦The glassware is placed in an electric oven set to
operate between 160° and 170°C.
◦Since dry heat is not as effective as wet heat, the
glassware must be kept at this temperature for
about 2 hours or longer.
 1.2. Dry Heat
Sterilization
◦ Dry heat kills microorganisms by reacting with
and oxidizing their proteins. Dry heat can be
used in incineration devices, such as the
Bunsen burner or the hot-air oven.
3. Chemical
Sterilization
1-Ethylene oxide
2-Oxidizing agents such as ozone.
3-Hydrogen peroxide.
4-Peracetic acid.
5-Aldehydes such as glutaraldehyde
6- Silver ions
 4. Silver Ions
Silver compounds show a toxic effect on some
bacteria, viruses, algae and fungi

Typical for heavy metals like lead or mercury, but

without the high toxicity to humans that are
normally associated with these other metals. Its
germicidal effects kill many microbial organisms in
vitro, but testing and standardization of silver
products is yet difficult.
 5. Radiation
Sterilization
1-Electron beams

2-X-rays

3-gamma rays

4. UV rays
6. Sterile filtration
-This method is commonly used for sensitive
pharmaceuticals and solutions in biological
research.

-A filter with pore size 0.2 µm will effectively

remove bacteria. If viruses must also be
removed, a much smaller pore size around 20
nm is needed. Solutions filter slowly through
membranes with smaller pore diameters
 Types of Microbes

Archea Viruse
Fungi
s

Bacteri Protist
a a
Types of
Media
 1- Non-synthetic
medium
1. Chemically undefined ingredient:
– Non-synthetic medium contains at least one
component that is neither purified nor completely
characterized nor even completely consistent.

– Nutrient broth, for example, is derived from

cultures of yeasts.
• Often these are partially digested proteins from
various organism sources.
 2- Synthetic media

Synthetic media is a type of chemically defined media and is

produced from pure chemical substances. A defined media refers to
a medium having a known concentration of ingredients, like
sugar (glucose) and nitrogen source.
 3-Selective medium
– Selective medium is designed to suppress the
growth of some microorganisms while allowing
the growth of others (i.e., they select for certain
microbes).
– Solid medium is employed with selective medium
so that individual colonies may be isolated.
• Examples of selective media include:
– Mannitol salts agar (selects against on-skin
flora) = inhibit
– MacConkey agar (selects against gram-positives)
– Eosin-methylene blue agar (selects against
gram-positives)
– Phenylehyl alcohol agar (selects against gram-
negatives)
 McConkey’s Agar
 Selective for: gram-
negative bacteria
 Growth of gram-positive
bacteria (e.g.:
Staphylococcus aureus
in the image below) is
inhibited by the crystal
violet dye and bile salts
in the media
4- Differential
medium
– Differential media allow the growth of more
than one microorganism of interest but with
morphologically distinguishable colonies.

• Examples of differential media include:

– Mannitol salts agar (mannitol fermentation =

yellow)
– Blood agar (various kinds of hemolysis)

– MacConkey agar
 McConkey’s Agar
Differential for: lactose fermentation
Neutral red pH indicator turns red in the presence of acid by-
products of lactose fermentation
Gram Negative Enterobacteria E. aerogenes Proteus vulgaris
bacteria Escherichia coli & Salmonella
and Enterobacter typhimurium
aerogenes
Ferment Produces pink to red Produces pink to red __________
colonies often with a colonies on
lactose
reddish bile McConkey's agar
Media precipitate
surrounding colonies
on McConkey's agar
Do not ferment Grow on McConkey's
agar, but do not
lactose
ferment lactose
Media (media appears
yellow to light pink in
color & colonies are
colorless)
 Forms of media
1- Agar media (Solid)

2- Broth Media (liquid)

2 1
Media Preparation
◦Agar is a gelling agent extracted from red
seaweed.
◦Nutrient agar is a commonly used food
medium for microbial cultures (especially
bacteria).
◦Nutrient agar contains:
◦Beef extract (provides carbohydrates,
nitrogen, vitamins, salts)
◦Peptone (source of nitrogen)
◦Agar (a carbohydrate used as a solidifying
agent)
◦Distilled water (an agent for distributing
food materials to growing colonies of micro-
organisms)
Procedures
• Suspend 28 g of nutrient agar powder in 1 liter of
distilled water using conical flask (2000ml) or add 7
gram per 250 ml of dist. Water using 500 ml conical
flask.
• Bring to the boil to dissolve completely. Using autoclave
at 121oC and 15 bar for 15 minutes.
• Once the program is done the autoclave lid will be
opened when the temperature decreased to 90oC.
• Pour it into a sterile plates carefully in aseptic
conditions .
• Leave the plates for ten minutes till the agar solidified.
• The plates now prepared for culturing.
Recipe: Agar + Distilled
Water = Yield
Yield Distilled Agar
Water
50 Plates 1000 ml 28 g
25 Plates 500 ml 14 g
10 Plates 250 ml 7g
CULTURE OF SIMPLE
MICROFLORA
 Aseptic Technique
Asepsis means free from sepsis [a toxic
condition resulting from the presence
of microorganisms.]

◦Importance of Aseptic technique:

1. Prevent culture contamination.

2. Protects against infection with
microorganism being handled.

Always work near the flame

 Aseptic transfer

◦A transfer may involve the transport of

organisms from an isolated colony on a
plate/tube of medium to another plate/tube
for various types of tests.
◦Aseptic transfer of a culture from one
culture vessel to another is only successful
if no contaminating microorganisms are
introduced in the process.
 Conditions
1.Work Area Disinfection: The work area is first
treated with a disinfectant to kill any
microorganisms that may be present. This step
destroys vegetative cells and viruses; endospores,
however, are not destroyed!
2.Loops and Needles: The transport of organisms
will be performed with an inoculating loop or
needle. To sterilize the loop or needle prior to
picking up the organisms, heat must be applied
with a Bunsen burner flame, rendering them
glowing red-hot.
Conditions
3. Culture Tube Flaming: Before inserting the
cooled loop or needle into a tube of culture, the
tube cap is removed and the mouth of the
culture tube flamed. Once the organisms have
been removed from the tube, the tube mouth
must be flamed again before returning the cap to
the tube.
4. Liquid Medium Inoculation: If a tube of
liquid medium is to be inoculated, the tube
mouth must be flamed before inserting the loop
into the tube. To disperse the organisms on the
loop, the loop should be twisted back and forth
in the medium.
 Conditions
5. Final Flaming: Once the inoculation is
completed, the loop or needle is removed from the
tube, flamed as before, and returned to a
receptacle. These tools should never be placed on
the tabletop. The inoculated tube is also flamed
before placing the cap on the tube.
6. Petri Plate Inoculation: To inoculate a Petri
plate, no heat is applied to the plate and a loop is
used for the transfer. When streaking the surface
of the medium, the cover should be held diagonally
over the plate bottom to prevent air contamination
of the medium.
Conditions

7. Final Disinfection: When all work is

finished, the work area is treated with
disinfectant to ensure that any
microorganisms deposited during any of the
procedures are eliminated.