Aseptic and Sterilization Techniques Broth to Agar Slant
 Insert the loop into the broth tube and withdraw a
Culture transfer technique
 used when preparing and maintaining stock culture, and
when carrying out a number of microbiological test
procedures.
Procedures include:
 Holding the tubes and sterilizing the inoculating loop.
 The transfer process
 Capping the tubes and re-sterilizing the loop
Holding the tubes and sterilizing the inoculating loop
 If you are right-handed, hold the stock culture tube
(from which you will make the transfer) and the tube to
be inoculated in the palm of your left hand.
 If your are left handed, the tubes should be in your right
hand.
How did we sterilized the inoculating loop in the
experiment?
 The inoculating loop is not sterile.
 To sterilize it, hold the handle of the instrument in your
free hand like you would in a pen or pencil.
 Place the loop in the hottest portion of the Bunsen
burner flame, which is the top blue flame.
 In a few seconds, the entire loop will turn red hot. Move
the rest of the wire rapidly through the flame.
 Once sterilized, do not place the loop in the lab bench.
Hold it in your hand and allow to cool for 20 seconds
The Transfer Process
 The caps or closures of the two tubes are removed
by grasping the cap of the left tube between your
little and ring finger; the cap of the right tube
between the ring and middle finger.
 Now remove the caps and briefly flame the necks of
the tubes in the Bunsen burner flame.
loopful of liquid.
 You should see a watery film on the loop.
 The broth-containing loop is immediately inserted
into the agar slant tube.
 Place the loop flat on the agar surface and move the
loop rapidly in a zigzag motion gently across the agar
surface from bottom to top.
 Remove the loop.
Agar Slant to Broth tube
 Inert the loop into the agar slant and touch the
loop on the agar slant surface where the
bacteria are growing.
 Remove just a small sample of bacteria.
 The loop is immediately inserted into the broth
tube.
 Shake the loop gently to dislodge the bacteria
and suspend them in the broth.
 Withdraw the loop from the broth tube.
Capping Tubes and Re-sterilizing the Loop
 Following the transfer, the necks of the tubes
are briefly re-flamed and the caps placed back
on the respective tubes from which they were
removed.
 The loop is again flamed to incinerate any
remaining bacteria still on the loop.
 Place the tubes in the test tube rack or
appropriate tube holder.

Sterilization (Autoclaving)
 Sterilization is a process whereby all forms
of microbial life, including bacterial spores,
are killed.
 Sterilization maybe accomplished by
physical or chemical means.
 Physical methods of sterilization include
the following:
 Incineration
 Moist heat
 Dry heat
 Filtration
 Ionizing (gamma) radiation
 An autoclave is essentially a large pressure
cooker.
 Moist heat in the form of saturated steam
under 1 atmosphere (15psi or pounds per
square inch) of pressure causes the irreversible
denaturation of enzymes and structural
proteins.
 Items such as media, liquids and instruments
are usually autoclaved for 15 min at 121 °C.
 Infectious medical waste is often sterilized at
132 °C for 30-60 minutes to allow penetration
of the steam throughout the waste and the
displacement of air trapped inside the
autoclave bag.
Definition of Terms
 By definition, there are no degrees of
sterilization—it is an all-or-nothing process.
Chemical or physical methods may be used to
accomplish this form of microbial destruction.
 The word sterile is a term that is relevant to the
method used. For example, a solution that has
been filtered through a certain pore-size filter
(>0.22 µm) is often referred to as sterile.
 Disinfection refers to a process that eliminates
a defined scope of microorganisms, including
some spores.
 Physical or chemical methods may be used, but
most disinfectants are chemical agents applied
to inanimate objects.
 A substance applied to the skin for the purpose
of eliminating or reducing the number of
bacteria present is referred to as an antiseptic.
 Antiseptics do not kill spores.
Culture media
Plain agar plate
Ingredients:
5.2 g Agar powder
100 mL Distilled water
Equipment:
Lab thermometer
Glass stir rod
Sterile Petri dish
Beaker/flask
Autoclave
Plain agar preparation Nutrient broth agar preparation
1. Obtain 100 mL of deionized water/distilled 1. Heat 10 ml of water to boiling
water in a grad. cyl 2. Add the dry ingredient
2. Weigh 5.2 g of agar powder 3. Stir constantly until dissolution
3. Add the powder to the flask 4. Slowly add the remaining 90 mL to the flask
4. Pour 75mL of deionized/distilled water 5. autoclave for 15 min@ 15 psi 121C
5. Stir for 25 minutes then add the remaining 6. repeat steps for “pouring” and “storage”
25 mL of H2O
6. Stir the solution until visible clumps have
been broken down Other culture media
7. Cover the flask with aluminum foil
8. Autoclave for 15 minutes at 121C 15psi
9. Take it out from the autoclave while stirring
10. allow to cool until 45C

Agar pouring
11. Obtain a petridish that will hold the agar
12. using your thumb and index finger, hold the
top cover of the petridish while middle
finger is assisting. Pinky and ring finger will
be the one holding the bottom.
13. Pass the petridished through the heat 3
times for sterilization
14. Pour the prepared agar into the dish until all
the surface is covered.

Plate storage
15. Rotate the plates upside-down to prevent
moisture from condensing in the agar surface
16. stack em' up!
17. If you are planning to use it later, store the plates
in a plastic sealed packets to prevent the
moisture from escaping the plate

Techniques
 The base of the plate must be covered, agar must 1. Blood agar
not touch the lid of the plate and the surface Ingredients:
must be smooth with no bubbles. 37.5g blood agar powder/agar powder
 To fasten and ensure dissolution, heat the agar 1L distilled water
mixture into boiling before subjecting it to
autoclave

Nutrient agar
Ingredients:
0.5% peptone or
0.3% beef extract or
1.5% agar or
0.5% NaCl Blood agar plate preparation
1. Follow step 1 to 9 of plain agar preparation
2. Cool the solution at 50C to 55C
3. Aseptically add 5% to 7% sterile defibrinated
horse/sheep blood and selective supplements as
required
4. Pour into plate by following step 12 to 15 of “agar
pouring”
5. Store accordingly by following step 16 to 18 of
“plate storage”
 Hemolytic pattern of blood agar
1. α-Haemolysis
 Formation of greenish to brownish halo
around the colony e.g., streptococcus
gardonii and streptococcus pneumoniae.
2. β-Haemolysis
 Virtual complete hemolysis of blood cells
thereby giving rise to a distinct clearing
effect around growth in the colony e.g.,
Staphylococcus aureus and Streptococcus
pyogenes.
3. Nonhemolytic Pattern.
 No change occurs in the medium e.g.,
Staphylococcus epidermidis and
Staphylococcus saprophyticus.
2. Chocolate agar
 To prepare chocolate blood agar, just hold the
temp at 75C to 80C until the blood becomes
chocolate brown in color
 Specifically made from ‘pre-heated blood’. e.g.,
Haemophilus influenzae and Neisseria
gonorrhoeae
Enrichment media required appropriate nutrients for
growth while differential media uses chemical
indicators
1. . MacConkey agar
 Inhibits growth of Gram-positive bacteria and thus is
selective for Gram-negative bacteria.
The MacConkey agar medium serves both differential* and
selective, because it predominantly contains lactose and
neutral red dye whereby the particular lactose-fermenting
colonies distinctly appear pink to red in color, and are
distinguished from the ‘colonies of non-fermentors quite
easily.
2. Thayer-Martin agar and Martin-Lewis agar
 chocolate agars containing extra nutrients plus several
antimicrobial agents
 are selective for N. gonorrhoeae.
3. Mannitol Salt Agar (MSA)
 Only salt-tolerant (haloduric) bacteria can grow
 e.g. Staphyloccoci.
 differentiation of the ‘pathogenic staphylococci’
from the ‘nonpathogenic staphylococci’
differentiation of the ‘pathogenic staphylococci’ from the
‘nonpathogenic staphylococci’ due to the fact that the former
augments fermentation of mannitol to yield ‘acid’ whereas the
latter fails to do so
MSA medium also serves as both differential and selective.
Mannitol salt agar is used to screen for S. aureus; not only will S.
aureus grow on MSA, but it turns the originally pink medium to
yellow because of its ability to ferment mannitol
 Differential media
Permits the differentiation of organisms that grow on the
medium

Salmonella-shigella agar

Reducing media
contains reducing agents which is used specifically for the
cultivation of Anaerobes.

 Isolate both Salmonella and Shigella species

 its ‘bile salt mixture’ inhibits the growth of
several cardinal groups of coliforms
 give rise to almost colorless colonies by virtue
of the fact that they are not capable of
fermenting lactose

Materials:

Chocolate agar
To prepare chocolate blood agar, just hold the temp at
75C to 80C until the blood becomes chocolate brown in
color