Basic technique in Microbiology

( 102)

MITU AFRIN
BGIB
Class-3
 Q-2020-Answer
• 3.a) What is culture media? Mention the ingredients
used for culture media. M-3
• Culture media is a gel or liquid that contains nutrients
and is used to grow bacteria or microorganisms. They
are also termed growth media.
• Different cell types are grown in various types of
medium. Nutrient broths and agar plates are the most
typical growth media for microorganisms.
• Some microorganisms or bacteria need special media
for their growth.
• Significance – Culture media is used in order to identify
the causative agent from infected material.
Common ingredients of culture media:
• Water: essential for bacterial growth, use deionized or distilled water.
• Peptone: from hydrolised animal or plant protein, it provides nitrogen and
amino acid.
• Meat extract: provides amino acid, vitamins, mineral salts (phosphate and
sulphate).
• Yeast extract: used to stimulate the growth of bacteria.
• Mineral salts: traces of magnesium, potassium, iron and calcium which are
essential for bacterial enzyme activity.

• Carbohydrates: to provide bacteria with energy and carbon source.

• Agar: inert polysaccharide from sea weed or marine algae, it is solidifying

agent with concentration of 1-2%, dissolves at 90-100 °C, solidify at 45 °C.
 3.b)Write down the types and criteria
of culture media. M-5
• Types of Culture Media
• The culture media are classified in many different ways:
• Based on the physical state
– Liquid media
– Solid media
– Semisolid media
• Based on the presence or absence of oxygen
– Anaerobic media
– Aerobic media
• Based on nutritional factors
– Simple media
– Synthetic media
– Complex media
– Special media
 Culture media
• Simple culture media includes nutrient broth.
One percent meat extract and peptone water
makes up such broth.
• Nutrient broth becomes glucose broth when
glucose is added to it. Likewise, it becomes
nutrient agar when 2-3% agar is added.
• For purposes of diagnosis, this is the simplest and
most common media used in laboratories.
• A semisolid medium that permits the propagation
of motile bacteria can be produced if the
concentration of agar is decreased. Examples –
peptone water, nutrient agar and glucose broth.
 Culture media
• Complex Media
• Other than simple media, all forms of media are termed complex
media. Complex media contain additional components for bringing out
specific qualities or providing the unique nutrients needed for the
bacterium’s growth. Here, the source of the amino acids contains a
variety of chemicals whose precise composition is unknown, thus it is
an undefinable media. Examples – MacConkey agar medium and
chocolate agar.

• Synthetic Media
• A synthetic medium is a defined medium. A defined medium
(chemically defined medium) is a medium in which there is no yeast,
plant or animal tissue present, and all the chemicals employed are
known. These are made from only pure ingredients whose exact
composition is known. These are employed in specialised
investigations like those on metabolic needs. Example – Dubo’s culture
medium with tween 80.
 Culture media
• Special Media
• Special media are of seven different types:
• Enriched media – It is created when a basic medium is supplemented with
nutrients like eggs, blood or serum. For example, a blood agar medium is used for
the growth of bacteria like Streptococcus which specifically requires blood for its
proliferation.

• Selective media – Selective media contain components that prevent the

growth of all but a small number of bacterial species and make it easier to isolate a
specific species. When mixed bacterial flora is anticipated in specimens, these
media are utilised to isolate specific bacteria from those specimens. For example,
bile salt acts as a selective agent in BSA or bile salt agar. While preventing the
growth of other intestinal organisms, it favours the growth of Vibrio cholerae.

• Differential media – It is a term used to describe a medium that has

components that aid in identifying the various properties of bacteria. Peptone,
agar, lactose, neutral red and sodium taurocholate are all ingredients in
MacConkey’s medium. Here, the colonies made by lactose fermenters are pink,
but those made by non-lactose fermenters are pale or colourless.
 Culture media
• Enrichment media – This media contains several ingredients that either
stimulate the bacteria being grown or suppress their competitors. Examples – Alkaline
peptone water and tetrathionate broth.

• Transport media – These are employed when dealing with delicate organisms
that might not make it through the transit period or might become covered with non-
pathogenic germs. Special media are developed for the transportation of such bacteria
to laboratories and these are known as transport media. Example – Stuart’s transport
medium.

• Indicator media – When bacteria multiply in these media containing an indicator,

they tend to change their colour. MacConkey’s medium is also an example of an
indicator medium. Another classic example is the black colonies of Salmonella
typhi that develop on sulphite-containing Wilson and Blair media.

• Sugar media – It contains 1% sugar, which can be any fermentable substance like
glucose, mannitol, sucrose and lactose. The generation of acid following the
fermentation of sugar transforms the medium into pink due to the presence of an
indicator. Also to show that gas is produced, Durham’s tube is kept inverted inside the
sugar tube and gas bubbles are observed.
 3.c) Describe the preparation technique of
nutrient agar and nutrient broth media. M-7
• Nutrient agar and nutrient
broth) from Oxoid share
almost the same medium
composition.
• The main difference Example of nutrient agar in a petri dish
between them is that
nutrient agar contains a
solidifying agent, agar
powder that causes the
medium to solidify in
room temperature,
whereas nutrient broth
remains in liquid form. Example of nutrient broth in a culture bottle
 Nutrient Agar
• Nutrient agar contains nutrients that suitable
to subculture a wide range of microorganisms
and makes it an excellent agar media to check
on the purity before any biochemical or
serological test.
• Besides, the addition of agar solidifies nutrient
agar, which makes it suitable for the
cultivation of microorganisms. You can also
add up to 10% blood or other biological fluids
that meet your experimental purpose.
 Preparation of nutrient agar
• Suspend 28g of nutrient agar powder (CM0003B) in 1L of distilled
water.
• Mix and dissolve them completely.
• Sterilize by autoclaving at 121°C for 15 minutes.
• Pour the liquid into the petri dish and wait for the medium to
solidify. Be sure that you are preparing the agar in the clean
environment to prevent any contamination.
• Once the agar solidifies, the agar is ready to use.
• ** Here’s a tip for you to ensure that your prepared agar is
sterile. You can incubate your freshly prepared agar in an
incubator for a few days. If there aren’t any microorganisms grow
on the agar, you are safe to use it, else, prepared again!
• Storage condition and shelf life for nutrient agar
• You have to store the dehydrated medium at 10-30°C and use
before the expiry date on the label. Once you prepare the
nutrient agar in the petri dish, store them at 2-8°C.
 Nutrient Broth

• Basically, the nutrient broth is the nutrient

agar that lack of the solidifying agent, agar
powder. They remain in liquid form at room
temperature and are usually used to maintain
the stocks of microorganisms. In general, they
are used to grow fastidious organisms. Also,
you can enrich your nutrient broth with blood,
serum, sugars… etc for special purposes.
 Preparation of nutrient agar
• Add 13g of nutrient broth powder (CM0001B) in 1L of
distilled water.
• Mix and dissolve them completely.
• Pour them into the final containers (eg. conical flask)
• Sterilize by autoclaving at 121°C for 15 minutes.
• What are the storage condition and shelf life for
nutrient broth?
• You have to store the dehydrated medium at 10-30°C
and use before the expiry date on the label. Once you
prepare the nutrient broth medium, store them at
below 25°C.