Media preparation and sterilization,

Aseptic technique & pure culture concept

 Introduction
❖In natural environments, microorganisms usually exist as
mixed populations. However, if we are to study, characterize,
and identify microorganisms, we must have the organisms in
the form of a pure culture.
❖A pure culture is one in which all organisms are
descendants of the same organism. Techniques for obtaining
pure cultures from a mixed population will be described in
this Lab.
 Aseptic technique.
❖ In working with
microorganisms, we must
have a method of
transferring growing
organisms from a pure
culture to a sterile
medium without
introducing any unwanted
outside contaminants.
❖For the most part, bacterial physiology only can
be studied in pure cultures.
❖The best way to obtain a pure culture is to start
with a single bacterial cell.
❖This cell then divides quickly, and may produce
millions of cells within 24 hours.
❖A single unwanted contaminant cell can do the
same thing in an otherwise pure culture, making
the culture useless.
For this reason, and to protect
against disease, strict sterile
procedures must be used.
 Media preparation & sterilization
❖ In working with microorganisms we must also have a sterile
nutrient-containing-medium in which to grow the organisms.
Anything in or on which we grow a microorganism is termed
a medium.
❖ Medium (media, plural): a nutrient blend used to
support microbial growth.
❖ Re-hydrate powder according to manufacturer’s instructions.
❖ Micro-organisms are grown and sub-cultured on different
types of chemical media; either chemically defined or
synthetic or complex or non-synthetic.
❖ A medium is sterilized (living organisms removed) before usage
in the lab with different sterilization methods include:
➢ Autoclaving, Dry-heat, Filtration, UV exposure, Ethylene
oxide.
Note: plastic Petri dishes are supplied in already sterilized packs; packs of
sterile plastic pipettes are also available but cost may be a consideration.
▪ Culture media and solutions: Autoclave/pressure cooker.
▪ Glass spreaders and metal forceps: Flaming in alcohol.

❖Autoclave:
The principle of sterilization in an autoclave is that steam under
pressure is used to produce a temperature of 121ºC which if held for
15 minutes all microorganisms including bacterial endospores will be
destroyed.
❖Sterilization of equipment and materials
• Wire loop: Heat to redness in Bunsen burner flame.
• Empty glassware and glass (not plastic!) pipettes and
Petri dishes: Either, hot air oven, wrapped in either grease
proof paper or aluminum and held at 160-180ºC for 2 hours,
allowing additional time for items to come to temperature (and
cool down!).
❖ A sterile medium is one which is free of all life forms. It is usually sterilized
by heating it to a temperature at which all contaminating microorganisms are
destroyed.
❖ Colony: is the smallest bacterial unit that can be seen with the naked eye.
❖ Culture: Is part of specimen grown in cultural media.
❖ Culture media: is a medium(liquid or solid) that contains nutrients to grow
bacteria in vitro. Because sometimes we cannot identify with microscopical
examination directly, and sometimes we do culture for antibiotics sensitivity
testing.
❖ Prosperities of Media:
1. Media has to support the growth of bacteria, should be
nutritive(contains the required amount of nutrients).
2. With suitable pH (neutral to slightly alkaline 7.3-7.4).
3. Suitable temperature.
4. Suitable atmosphere (bacteria grow at 37c).
Agar
• Frau Hesse
• Used for preparing solid medium
• Obtained from seaweeds.
• No nutritive value
• Not affected by the growth of the bacteria.
• Melts at 98oC & sets at 42oC
• 2% agar is employed in solid medium
Types of Culture Media
I. Based on their consistency
a) solid medium
b) liquid medium
c) semi solid medium
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
 Types of Culture Media
Special media
• Enriched media
• Enrichment media
• Selective media
• Indicator media
• Differential media
• Sugar media
• Transport media
• Media for biochemical reactions
III.Based on Oxygen requirement
- Aerobic media
- Anaerobic media
Types of Culture Media
• Solid media – contains 2% agar
• Colony morphology, pigmentation, hemolysis can be
appreciated.
• Eg: Nutrient agar, Blood agar
• Liquid media – no agar.
• For inoculum preparation, Blood culture, for the isolation
of pathogens from a mixture.
• Eg: Nutrient broth
• Semi solid medium – 0.5% agar.
• Eg: Motility medium
 Types of Culture Media
Liquid Media
1) Broth tube: are tubes containing a liquid
medium. A typical nutrient containing broth
medium such as Trypticase Soy broth ,
nutrient broth. After incubation, growth
(development of many cells from a few
cells) may be observed as one or a
combination of three forms:
a) Pellicle: A mass of organisms is floating on top of
the broth.
b) Turbidity: The organisms appear as a general
cloudiness throughout the broth .
c) Sediment: A mass of organisms appears as a
deposit at the bottom of the tube.
 Types of Culture Media
Solid Media
1) Slant tubes: are tubes containing a nutrient
medium plus a solidifying agent, agar-agar. The
medium has been allowed to solidify at an angle
in order to get a flat inoculating surface.
2) Stab tubes (deeps): are tubes of hardened agar
medium which are inoculated by "stabbing" the
inoculum into the agar.
3) Agar plates: are sterile petri plates that are
aseptically filled with a melted sterile agar
medium and allowed to solidify. Plates are much
less confining than slants and stabs and are
commonly used in the culturing, separating, and
counting of microorganisms.
Types of Culture Media

Simple media / basal media

• Eg: Nutrient Broth, Nutrient Agar
• NB consists of peptone, meat extract, NaCl,
• NB + 2% agar = Nutrient agar
Types of Culture Media
Complex media
• Media other than basal media.
• They have added ingredients.
• Provide special nutrients
Synthetic or defined media
• Media prepared from pure chemical substances and
its exact composition is known
• Eg: peptone water – 1% peptone + 0.5% NaCl in
water
 Types of Culture Media
Enriched media
• Substances like blood, serum,
egg are added to the basal
medium.
• Used to grow bacteria that
are specific in their nutritional
needs.
• Eg: Blood agar, Chocolate agar
Types of Culture Media
Enrichment media
• Liquid media used to isolate pathogens
from a mixed culture.
• Media is incorporated with inhibitory
substances to suppress the unwanted
organism.
• Eg:
• Selenite F Broth – for the isolation of
Salmonella, Shigella
• Alkaline Peptone Water – for Vibrio
cholerae
Types of Culture Media

Differential media
• Distinguishes microorganisms
growing together by differences in
their cultural characteristic
• Eg:
• Mac Conkey’s medium
• Lactose fermenter – Pink
• Non-Lactose fermenter -
White
Types of Culture Media
Selective media
• The inhibitory substance is added to a solid media.
• Gram (+) – gentian violet, Na desoxycholate, bile salts
• Gram (-) – potassium tellurite, Na azide
• Eg:
• Mac Conkey’s medium for gram negative bacteria
• TCBS – for V. cholerae
• LJ medium – M. tuberculosis
• Wilson and Blair medium – S. typhi
• Potassium tellurite medium – Diphtheria bacilli
• XLD Agar – Salmonella and Shigella
Types of Culture Media
Selective media

Mac Conkey’s medium TCBS

Types of Culture Media
Selective media

Potassium Tellurite media LJ media

Types of Culture Media

Specific media
• Specifically prepared to support the specific growth of
microorganism
• Eg:
• Thayer-Martine Agar - Neisseria
• Petragagni medium – M. tuberculosis
• MacBride Agar – L. monocytogenes
Types of Culture Media

Indicator media
These media contain an indicator
which changes its colour when a
bacterium grows in them.
Eg:
• Blood agar
• Mac Conkey’s medium
• Christensen’s urease medium
Types of Culture Media
Transport media
• Media used for transporting the
samples.
• Delicate organisms may not survive
the time taken for transporting the
specimen without a transport media.
Eg:
• Stuart’s medium – non nutrient
soft agar gel containing a reducing
agent
• Buffered glycerol saline – enteric
bacilli
Types of Culture Media

Anaerobic media
• These media are used to
grow anaerobic
organisms.
• Eg: Robertson’s cooked
meat medium,
Thioglycolate medium.
 Culture Methods
Culture methods employed depend on the purpose for which they
are intended.
The indications for culture are:
1. To isolate bacteria in pure cultures.
2. To demonstrate their properties.
3. To obtain sufficient growth for the preparation of antigens and
for other tests.
4. For bacteriophage & bacteriocin susceptibility.
5. To determine sensitivity to antibiotics.
6. To estimate viable counts.
7. Maintain stock cultures.