29-Feb-20

Dr. Rania Abdel Halim

Lecturer of clinical Pathology
Ain Shams University

Growth Media
 Bacteria and other microbes have
particular requirements for growth.

 In order to successfully grow bacteria

in lab, we must provide an environment
suitable for growth.

Growth media are used to

cultivate microbial growth.

 Media = mixtures of nutrients that the microbes need to live.

Also provides a surface and the necessary moisture and PH to
support microbial growth.

From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Streak plate of E. coli, T. Port

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A microbiological culture, is a method of

multiplying microbial organisms by letting them
reproduce in predetermined culture media under
controlled laboratory conditions.

Colony – macroscopically visible collection of

millions of bacteria originating from a single
bacterial cell.

Purpose:
Microbial cultures are used to determine the type
of organism, its abundance in the sample being
tested, or both.

How is media made?

 When lab personnel make media
they measure out a quantity of
dry powdered nutrient media, add
water and check the pH.

 They pour the media into bottles,

cap it and autoclave.

 The autoclave exposes the media

to high temperature (121°C) and
pressure (15 psi) for 20 minutes.

 Once the media is autoclaved it is

considered sterile (all life forms
killed).

From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Autoclave, Astell Scientific; Pressure cooker, Rama

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Preparation
Wash hands & wear gloves

Sterilize all equipments

Pour D/W in glassware (required amount)

Add powder ingredients (required amount)

Heat to dissolve completely

Autoclave

Dispense the medium into tubes ,bottles &plates

Store at required temperature

TS
Y

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Labeling Plates

All Petri plates for should be labeled and stored in the following manner:

1. Make certain that all plates are labeled on the bottom half (i.e.
the portion of the dish plate that contains the media).

2. Include the following:

a. Name of patient
b. Date
c. Type of specimen

3. All plates are incubated in the "upside down"

position (to minimize contamination &
to keep culture & agar properly hydrated)

“Upside down” means that the ½ of the Petri plate with

media faces up. The empty ½ of the Petri plate is down.

Images: Positive & negative differential reaction

From the Virtual Microbiology Classroom on ScienceProfOnline.com on Mannitol Salt Agar, T. Port

Oct 07 Dr Ekta, Microbiology, GMCA

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Oct 07 Dr Ekta, Microbiology, GMCA

Atmospheric requirements

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Classification of bacteria according to atmospheric requirements

Category Requirement Examples

Obligate aerobe 15%-21% O2 Mycobacteria, Pseudomonas

Acenitobacter, Vibro, fungi.

Microaerophile Reduced concentrations Campylobacter spp., Legionella,

of O2 (6%) Helicobacter, atypical
Mycobacteria.

Facultative Multiplies equally well Enterobacteriaceae, most

anaerobe in the presence or staphylococci, streptococci
absence of O2
Obligate anaerobe Strict anaerobic Most Bacteriodes spp., many
environment Clostridium, Eubacterium,
(<0.5%O2) Fusobacteri spp.,
peptostreptococcus spp.,
Porphyromonas spp., most strains
of Veillonella parvula
Capnophile 5%-10% CO2 Some anaerobes, Neisseria,
Haemophillus spp, Brucella

Gaspak
 Commercially available disposable envelope.
 Contains chemicals which generate H2 and CO2 on
addition of water.
 Cold catalyst – in the envelope
 Indicator is used – reduced methylene blue.
 Colourless – anaerobically
 Blue colour – on exposure to oxygen

Oct 07 Dr Ekta, Microbiology, GMCA

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Anaerobic Culture Methods

 Anaerobic jar

Figure 6.5

Anaerobic Culture Methods

 Anaerobic
chamber

Figure 6.6

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Capnophiles Require High CO2

 Candle jar

 CO2-pak

Figure 6.7

Culture Media
Indications/ Need for culture -
 Isolate bacteria in pure cultures.

 Demonstrate their properties.

 Obtain sufficient growth for preparation of

antigens & for other tests.

 Typing bacterial isolates.

 Antibiotic sensitivity.

 Estimate viable counts.

 Maintain stock cultures.

Oct 07 Dr Ekta, Microbiology, GMCA

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Classification of Media
 Based on the consistency :
Solid media
Semisolid media
Liquid media

Oct 07 Dr Ekta, Microbiology, GMCA

Solid media - contains 2% agar

 Colony morphology, pigmentation, hemolysis can be
appreciated.
 e.g.: Nutrient agar, Blood agar

Liquid media - no agar.

 For inoculum preparation, Blood culture, for the isolation
of pathogens from a mixture.
 e.g.: Nutrient broth

Semi solid medium - 0.5% agar.

 e.g.: Motility medium (MIO).

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Classification
 Based on nutritional requirements:

a ) Simple e.g peptone water

b ) Complex e.g blood agar

c ) Synthetic e.g Davis /mingioli medium

Classification
 Based on the function or property:
- Enriched media
- Enrichment broth
- Selective media
- Indicator media
- Differential media
- Composite media
- Transport media

Oct 07 Dr Ekta, Microbiology, GMCA

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Simple Media

• These are simple media contains only basic substances

&minerals.

• Support those organisms that don't require special

nutrient.

• Often used in the preparation of enriched media.

• Also used for sub culturing of pathogens

Oct 07 Dr Ekta, Microbiology, GMCA

Simple Media

 Also called Basal medium

 Most commonly used in routine labs.
e.g. Nutrient broth, NA

Oct 07 Dr Ekta, Microbiology, GMCA

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Enriched Media

 Blood, serum or egg added to the basal medium.

 To grow bacteria which need exact nutritional
requirements.
e.g. BA

Oct 07 Dr Ekta, Microbiology, GMCA

Blood agar
Most specimens received in a clinical microbiology lab
are plated onto Blood agar. It is an enriched medium
that will grow even fastidious bacteria.

Also contains 5% sheep blood.

This media is not selective. It is enriched and

differential:

Certain bacteria produce enzymes that act on red cells

to produce either:

* Beta hemolysis: Enzymes lyse the blood cells

completely, producing a clear area around the colony.

* Alpha hemolysis: Incomplete hemolysis produces a

greenish discoloration around the colony.

* Gamma hemolysis: No effect on the red cells.

Images: Beta-hemolysis, Alpha-hemolysis and

From the Virtual Microbiology Classroom on ScienceProfOnline.com a sterile plate of Blood Agar, T. Port

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Chocolate agar

is a non-selective, enriched growth medium. It is a variant of the

blood agar plate. It contains red blood cells, which have been
lysed by heating very slowly to 56 °C. Chocolate agar is used for
growing fastidioud respiratory bacteria, such as Haemophylis
influenzae

 These bacteria need growth factors, like

NAD and hematin, which are inside red
blood cells; thus, a prerequisite to
growth is lysis of the red blood cells.
The agar is named for the color and
contains no actual Chocholate

Enrichment Broth (media)

 For mixed cultures or materials containing more

than one bacterium.

 Contains substances which stimulates wanted

bacteria & inhibits unwanted bacteria.

e.g. Selenite broth (enrichment broth for

isolation of salmonella & sheigella in stool
specimens)

Oct 07 Dr Ekta, Microbiology, GMCA

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Selective Media

 These are solid media contains inhibitory

substances i.e bile salts ,dyes, antibiotics etc.

 Inhibit the growth of one organism to allow the

other one
 e.g. TCBS agar for Vibrio cholerae.

Oct 07 Dr Ekta, Microbiology, GMCA

Thiosulfate-citrate-bile salts-sucrose agar or

TCBS agar
TCBS Agar is highly selective for the
isolation of V. cholerae and V.
parahaemolyticus as well as other vibrios.

Inhibition of gram-positive bacteria is

achieved by the incorporation of bile
salts.

The alkaline pH of the medium enhances

the recovery of V. cholerae. Thymol blue
and bromthymol blue are included as
indicators of pH changes.

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XLD agar
Selective growth media used in the isolation of
Salmonella and Shigella species from clinical samples
and from food.
It has a pH of approximately 7.4, leaving it with a
bright pink or red appearance due to the indicator
phenol red.

Sugar fermentation lowers the pH and the phenol red

indicator registers this by changing to yellow

XLD agar
Salmonella species: red colonies, some with
black centers. The agar itself will turn red due
to the presence of Salmonella type colonies.

Shigella species: red colonies.

Coliforms: yellow to orange colonies.

Pseudomonas aeruginosa pink, flat, rough

colonies. This type of colony can be easily
mistaken for Salmonella due to the colour
similarities.

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Examples of selective media:

• MacConkey agar for Gram-negative bacteria

• Lowenstein Jensen's agar for M . bacterium.

• TCBS for vibrio ( selective and differential

medium for Vibrio cholera and other Vibrio spp.)

• XLD for salmonella & shigella.

• Modified New York City (selective and

differential medium for N. gonorrhea)

 mannitol salt agar (MSA) which is selective for Gram-

positive bacteria and differential for mannitol
fermentaion

 Skirrow ҆s medium (selective & enriched medium for

Campylobacter spp. )

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Indicator media

 Dyes , indicator or other

substances are added to
differentiated organisms i.e
Neutral red , phenol red , eosin , or
methylene blue, bromothymol blue.

 Change in the colour of indicator

with the growth of bacteria.
e.g. Wilson Blair media
for Salmonella typhi.

Oct 07 Dr Ekta, Microbiology, GMCA

Differential Media

 To bring out differing characteristics of

bacteria.
e.g. MacConkey’s agar

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MacConkey's (MAC)

MacConkey’s media is both selective & differential.

1. Selective because it only grows Gram-negative

bacteria. Inhibits the growth of Gram-positive
bacteria.

2. Differential because neutral red (pH-sensitive

dye) and lactose (type of sugar) have been added
to media.

- Bacteria that use lactose for food (lactose fermenters),

produce acidic
metabolites that trigger the pH sensitive dye to turn pink.

- So lactose fermenting bacteria will grow in bright pink

colonies while
non-lactose fermenters will be colorless and clear.

Enteric bacteria are the most frequently encountered bacteria

isolated from many types of clinical specimens. They are most
commonly lactose fermenters.
Image: McConkey’s growing Salmonella on the
From the Virtual Microbiology Classroom on ScienceProfOnline.com left, and E. coli on the right, T. Port

CLED agar
CLED agar (cysteine lactose electrolyte deficient
medium) 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. Cysteine promotes
the formation of cysteine-dependent dwarf
colonies.

Lactose fermenters produce yellow colonies on

CLED agar; non-lactose fermenters appear blue. It
has a pH of approximately 7.3. Bromthymol blue is
used as a pH indicator

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Composite Media

 For identification of
isolates
e.g. Triple Sugar Iron

Oct 07 Dr Ekta, Microbiology, GMCA

Transport Media
•These are semi-solid media.
Used for the temporary storage of
microorganisms in the specimen.

•Delicate organisms may not survive the

time taken for transporting the
specimen without a transport media.

•Transport media contain ingredients

preventing overgrowth of commensals.

Examples:

a) Thioglycollate broth for strict

anaerobes .
b) Cary-blair medium for enteric
pathogen.
c) Amies medium for gonococci.

Oct 07 Dr Ekta, Microbiology, GMCA

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Anaerobic media

 These media are used to grow anaerobic organisms.

 e.g.: Robertson’s cooked meat medium, Thioglycolate medium.

Mueller-Hinton agar

Müller-Hinton agar is an microbiological growth medium

that is commonly used for antibiotic susceptibility testing.

It is also used to isolate and maintain Neisseria and

Moraxella species.

Five percent sheep blood may also be added when

susceptibility testing is done on Streptococcus species. This
type is also commonly used for susceptibility testing of
Campylobacter.

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Antibiotic susceptibility testing by disc

diffusion method on Mueller-Hinton agar

Oct 07 Dr Ekta, Microbiology, GMCA

Oct 07 Dr Ekta, Microbiology, GMCA

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AST and Emerging Resistance

Beta-lactamases

Gas capture blood culture bottles

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Bactec automated system for blood culture

Signal blood culture bottles used in

BACTEC

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Semi quantitative urine

culture Method

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