culture media, Techniques &

Identification

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 CULTURE AND THE MEDIUM

• CULTURE :
Is the term given to microorganisms that are
cultivated in the lab for the purpose of identifying
and studying them.

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• MEDIUM:
Is the term given to the combination of ingredients
that will support the growth and cultivation of
microorganisms by providing all the essential
nutrients required for the growth (i.e., multiplication)
in order to cultivate these microorganisms in large
number to study them.

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• Microbiological culture: which are used for
growing microorganisms, such as bacteria or
yeast.

• The most common growth media for

microorganisms are nutrient broths and agar
plates

• Specialized media are sometimes required for

microorganism and cell culture growth.
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Culture techniques are designed to promote the
growth and identify required bacteria, while
restricting the growth of the other bacteria in the
sample.

• In the laboratory, bacteria are usually grown using

solid growth media such as agar plates or liquid
media such as broth.

• Solid, agar-based media can be used to identify

colonial characteristics (shape, size, elevation, margin
type) and to isolate pure cultures of a bacterial strain

• liquid growth media are used when measurement of

growth or large volumes of cells are required.
Growth in stirred liquid media occurs as an even cell
suspension, making the cultures easy to divide and
Transfer

• isolating single bacteria from liquid media is difficult.

• The use of selective media (media with specific nutrients
added or deficient, or with antibiotics added) can help
identify specific organism.

• Most laboratory techniques for growing bacteria use

high levels of nutrients to produce large amounts of
cells cheaply and quickly.
• However, in natural environments nutrients are limited,
meaning that bacteria cannot continue to reproduce
indefinitely
Cultural characteristics
Basic conditions for cultivation
• Optimum environmental moisture. It is possible to
cultivate bacteria in liquid media or solid media with a
gelling agent (agar) binding about 90% of water.
• Optimum temperature for cultivation of bacteria of
medical importance is about 370C. Saprophytic bacteria
are able to grow at lower temperatures.
• Optimum pH of culture media is usually 7.2-7.4
Lactobacillus spp need acid pH and vibrio cholera needs
alkaline pH for the growth.
• Optimum constituents of bacteriological culture media.
• All culture media share several common constituents
necessary to enable bacteria to grow in vitro.
Colony morphology
• Form - the basic shape of the colony
ex: circular, filamentous etc.
• Size – The diameter of the colony.
• Elevation - This describes the side view of a colony.
Turn the Petri dish on end.
• Margin/border - magnified shape of the edge of the
colony
• Surface - colony appearance
ex: smooth, glistening, rough, wrinkled or dull.
• Opacity - ex transparent (clear), opaque, translucent
(like looking through frosted glass), etc.
• Colour (pigmentation) ex: white, buff, red, purple, etc.
Effect of media
• different types of media, which contain different
nutrients can affect the cultural characteristics of
bacteria.
• Some types of media are much more nutritive and
will encourage hearty growth. Some types of media
may restrict growth.
• Colonial morphology may also be affected by the
temperature at which the bacteria is incubated. Some
bacteria grow better at body temperature and grow
weakly at room temperature, or vice versa.
• Some bacteria express certain characteristics, such as
the formation of pigment, more strongly at some
temperatures than at others.
 BASIC REQUIREMENTS OF CULTURE
MEDIA
• NUTRIENTS :

 Energy source
 Carbon source
 Nitrogen source

• MINERAL SALTS :

 Sulphates, phosphates, chlorides and carbonates of K, Mg

and Ca
 A suitable pH- 7.2- 7.4
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 GROWTH FACTORS 
ARGININE E.COLI

GLUTATHIONE GONOCOCCI
CHOLESTEROL MYCOPLASMA

ARYL SULPHATE, AMIDE ATYPICAL MYCOBACTERIA

GLYCEROL MYCOPLASMA HOMINIS

SULFONAMIDES RIKETTSIA

TRYPTOPHAN SALMONELLA TYPHI

L-CYSTEINE LISTERIA MONOCYTOGENS

SODIUM CHLORIDE VIBRIO PARAHAEMOLYTICUS
10/17/22 12
FACTOR X & V H.INFLUENZAE
 Culturing Microorganisms
• To grow microorganism of choice in the laboratory
• There are two basic culture techniques used in
microbiology:
1. Liquid culture: bacteria, algae, and some fungi can be reared
in culture tubes (test tubes) in a liquid medium.
 Liquid medium is best when you want to rapidly increase
the concentration of the organism or when you want to
grow the cells.
 Culturing Microorganisms
• There are two basic culture techniques used in microbiology:
2. Culture Plates: Liquid medium is solidified using agar
and poured as a thin layer in the bottom of a culture dish
(also sometimes called petri plate)
 Culture plates are used when you want to test (1)
antibiotic sensitivity, (2) purified culture (3) isolate
individual colonies from samples.
Colony – macroscopically visible collection of millions of
bacteria originating from a single bacterial cell.
 Forms Of Culture Media
1) Broth tube: are tubes containing a liquid medium. After
incubation, growth (development of many cells from a few cells)
may be observed as one or a combination of three forms:
Patterns of growth
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.
2) 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.

 Slanting the surface of the agar gives the bacteria a

greater surface area on which to grow in a test tube.
2) 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.
 CLASSIFICATION
BASED ON
PRESENCE OF BASED ON
BASED ON
MOLECULAR NUTRITIONAL
PHYSICAL STATE
OXYGEN AND FACTORS
REDUCING
SUBSTANCES
LIQUID MEDIA AEROBIC MEDIA SIMPLE MEDIA

SEMISOLID MEDIA ANAEROBIC MEDIA COMPLEX MEDIA

SOLID MEDIA SYNTHETIC MEDIA

SPECIAL MEDIA
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SPECIAL MEDIA

ENRICHED MEDIA
ENRICHMENT MEDIA
SELECTIVE MEDIA
DIFFERENTIAL MEDIA

INDICATOR MEDIA

TRANSPORT MEDIA

SUGAR MEDIA
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 SIMPLE MEDIA
Simple or basal media are culture media which contain the minimum
adequate nutrition for non fastidious organisms

Example:- Nutrient broth/agar

Peptone water
Composition:-
Meat extract-10gm
peptone-10gm
NaCl- 5gm
Distilled water-1000ml
- When 2-3% agar is added ,then we have it as nutrient agar.
- For semisolid media – agar concentration is 0.2-0.4%
Uses:-
1. This is basis of most of the media used in the study at common
pathogenic bacteria.
2. It is used for subcultures of certain organisms.
 NUTRIENT AGAR

NUTRIENT BROTH
 COMPLEX MEDIA
Complex media have added ingredients for bringing out certain properties
for bringing out certain properties or providing special nutrients
required for growth of the bacterium in question.
SYNTHETIC MEDIA
 These are prepared from pure chemicals and the exact compositions of
medium is very well known.
Example :- Dubo’s medium (routine cultivation of tubercle bacilli)

SEMIDEFINED MEDIA
 In these media the exact chemical composition of the constituents is not
known because substances like meat and peptone are used.

 Most of the culture media used for routine diagnostic work are
semidefined culture media.
 SPECIAL MEDIUM
ENRICHED MEDIA

 When basal medium is added with some nutrients such as blood,

serum or egg is called enriched media.
 They are used to grow bacteria which are more exacting in their
nutritional needs.

Example:-
CHOCOLATE AGAR
Also called Heated blood agar
 Type: Enriched
 Purpose: Cultivation of fastidious organisms such as Neisseria or
Haemophilus sp.
 Interpretation: Some organisms grow on Chocolate that do not grow
on standard media
 BLOOD AGAR

• TYPE : Enriched media.

• APPEARANCE : Red color.
• COMPOSITION :
Sterile Nutrient agar + Defibrinated sheep blood

USES :
 Routine culture
 Widely used in medical bacteriology
 It is also an indicator medium showing the haemolytic
properties of bacteria such as Streptococcus pyogenes.

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 Enrichment Media

• Enrichment media refers to the liquid media that

inhibits the growth of unwanted bacteria.
• Enrichment media allow the growth of a particular
type of microorganism in the medium.
• Enrichment media are liquid media
• The examples of enrichment
media are Selenite F broth,
tetrathionate broth, alkaline
peptone water (APW), etc.
 SELECTIVE MEDIA
 It is a medium in which certain substances are present which
inhibit all other bacteria except the desired bacteria.
 It encourages the growth of particular species from a mixed
inoculum.

Example:- TCBS
-It is light green translucent medium kept in petridish
-It is selective medium for Vibrio cholera
-Principle:-
Bile salt inhibit the growth of normal
commensals (unwanted bacteria).

 Vibrio chloerae produce acid by fermentation

of sucrose which acts on bromothymol blue
(indicator) producing yellow colonies.
Mannitol Salt Agar
Type: Selective and differential
Purpose: Selects for Staphylococci, which grow at high salt
concentrations; differentiates Staphylococcus aureus from other
Staphylococci
Interpretation: Staphylococcus aureus is yellow (ferments
mannitol), other staphylococci are white
 DIFFERENTIAL MEDIA
• Differential media contain compounds that allow groups of
microorganisms to be visually distinguished by the
appearance of the colony or the surrounding media,

• usually based on some biochemical difference between the

two groups.

• Blood agar is one type of differential medium, allowing

bacteria to be distinguished by the type of hemolysis
produced.

• Some differential media are also selective, for example,

standard enteric agars such as MacConkey and EMB agars,
which are selective for gram-negative coliforms and can
differentiate lactose-fermenting and non-lactose-fermenting
bacteria. 30
 MAC CONKEY AGAR

• MacConkey agar is a culture medium

designed to grow Gram-negative bacteria.

• It is a useful medium for the cultivation of

enterobacteriacea.

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MacConkey agar
showing both
lactose and non-
lactose fermenting
colonies.

Lactose fermenting
colonies are pink
whereas non-
lactose fermenting
ones are colourless
or appear same as
the medium.
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• It contains Bile salts to inhibit non-intestinal
bacteria and most Gram-positive bacteria,
except Enterococcus and some species
of Staphylococcus i.e. Staphylococcus aureus.

• Neutral red dye : which stains microbes

fermenting lactose.

• Crystal violet dye : which also inhibits certain

Gram-positive bacteria).

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• Uses
• Acting as a visual pH indicator, the agar
distinguishes those Gram-negative bacteria
that can ferment the sugar lactose (Lac+) from
those that cannot (Lac-).
This medium is also known as an
• "indicator medium"
• "low selective medium".
• Absence of electrolytes serves to inhibit
swarming by Proteus species

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 Lac+
• By utilizing the lactose available in the
medium, Lac+ bacteria such as
 Escherichia coli
 Enterobacter spp.
 Klebsiella spp.

will produce acid, which lowers the pH of the

agar below 6.8 and results in the appearance
of red/pink colonies
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 CLED
(Cystine Lactose Electrolyte Deficient medium)

• It is a valuable non-inhibitory growth medium

used in the isolation and differentiation of
urinary organisms.

• Being electrolyte deficient, it prevents the

swarming of Proteus species

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 INDICATOR MEDIUM

 These media contain an indicator which changes colour when

bacteria grow on them.

 Example:- Wilson and Blair medium

 For isolation of Salmonella typhi and S. paratyphi

 They appear as black colonies

 Principle:- The black colour of colonies is due to the ability of

these organisms to reduce bismuth sulphite to sulphide in the
presence of glucose coliforms are inhibited by brilliant green
and bismuth sullphite
 TRANSPORT MEDIUM
These are used for the temporary
storage of specimens being transported to
the laboratory for cultivation.

Such media ideally maintain the viability

of all organisms in the specimen without
altering their concentration.

Transport media typically contain only

buffers and salt.

The lack of carbon, nitrogen, and

organic growth factors prevents microbial
multiplication.

Transport media used in the isolation of

anaerobes must be free of molecular STUART TRANSPORT BROTH
oxygen.
 CHARACTERISTICS OF TRANSPORT MEDIA:
• It should be non-toxic
• It should not promote or inhibit the bacterial growth
• It should be easy to carry and transport

Examples:
1. Buffered glycerol saline transport medium
2. Cary and Blair medium

Cary and Blair medium

 ANAEROBIC MEDIUM
These media are used to grow anaerobic organisms.
Examples:-
 Thioglycollate broth
 Robertsons Cooked Meat Medium
Mueller-Hinton agar

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• Mueller-Hinton agar is a microbiological
growth medium that is commonly used for
antibiotic susceptibility testing.

• Originally formulated for isolation of Neisseria

species.

• It is also used to isolate and maintain

Neisseria and Moraxella species.

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10/17/22 47
 General protocol for growing bacteria

• Pouring media “plates”

• “Streaking” bacteria on media plates

• “Picking” a “colony” from the plate

• “Inoculating” a liquid culture

Everything needs to be done under sterile conditions!!
 Culturing Microbes
The Five “I’s
Innoculation: Producing a pure culture
Isolation: Colony on media, one kind of microbe,
pure culture
Incubation: growing microbes under proper
conditions
Inspection: Observation of characteristics (data)
Identification: use of data, correaltion, to ID
organism to exact species
 Microbiology – Chapter 3
Culturing Microbes
The Five “I’s
Innoculation: Producing a pure culture
Introduce bacteria into a growth medium using “aseptic technique” to
prevent contamination. Tools: Bunsen burner, loop. Needle, etc.
 Microbiology – Chapter 3
Innoculation: Producing a pure culture
Introduce bacteria into a growth medium using “aseptic technique” to
prevent contamination. Tools: Bunsen burner, loop. Needle, etc.
 Microbiology – Chapter 3
Isolation: Colony on media, one kind of
microbe, pure culture: isolation on general
and special “differential media”
General growth media: NA, TSA
Differential: Mac, EMB, SS
These have dyes, salts, inhibiting
agents
 Microbiology – Chapter 3
Isolation: Colony on media, one kind of
microbe, pure culture
 Microbiology – Chapter 3
Isolation: Colony on media, one kind of
microbe, pure culture – Streak Plates
 Microbiology – Chapter 3
Isolation: Colony on media, one kind of
microbe, pure culture. Many colonies? Use
a needle, pick one, and redo streak plate
 Microbiology – Chapter 3
Differential: Mac, EMB, SS
These have dyes, salts, inhibiting
agents : see differences on plates
 Microbiology – Chapter 3
• Blood agar : rich with nutrients, can see a
difference, thus differential; much more
later
 Microbiology – Chapter 3
• Incubation: Allow organisms to grow under the optimal
conditions
• Temperature, with or without oxygen etc
 Microbiology – Chapter 3
• Incubation: Allow organisms to grow under the optimal
conditions
• Temperature, with or without oxygen etc
• Candle jar reduces oxygen
 Microbiology – Chapter 3
• Inspection: Observation, description
• Colony Morphology, Microscopic examination (grams
stain)
• Systematic recording of “DATA”
 Microbiology – Chapter 3
• Microscopic study: Gram + bacilli, Gram -
bacilli
 Microbiology – Chapter 3
• Microscopic study: Acid fast, and capsule
 Microbiology – Chapter 3
• Identification: Correlating data from all observations to
ID organism to species
• Resources: flow charts, Bergey’s manual etc.
• Ex. Gram – bacilli, ferments lactose, green sheen on
EMB: E.coli
 Microbiology – Chapter 3
• Identification: Correlating data from all observations to
ID organism to species
• Gram + cocci, grape like clusters, golden yellow colonies, catalase
+, coagulase +, resistant to Methicillin (MRSA)
• Staphylococcus aureus