Bacterial Culture Media

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.
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).
 The food material or substances required for growing
microorganisms in vitro (outside the body) is called culture medium.
§ Reasons for bacterial culture:-
1- subsequent clinical diagnosis.
2- To study colony morphology and its identification.
3- Bacteria must be cultured in order to obtain antigens to develop
serological assay or for vaccines purpose.
4- Certain genetic studies and manipulations of the cells also need
that bacteria be cultured in vitro.

5- Culturing on solid media is another convenient way of separating

bacteria in mixture
• 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.
 Culture media

• Used to grow bacteria

• Can be used to-

 Enrich the numbers of bacteria.

 Select for certain bacteria and suppress others.

 Differentiate among different kinds of bacteria.

 Need for culture media

• It is usually essential to obtain a culture by

growing the organism in an artificial medium.

• If more than one species or type of organism are present each

requires to be carefully separated or isolated in pure culture .
 Basic requirements of culture media
• Nutrients :

 Energy source
-Phototrophs-light energy-Rhodospirillum rubrum
-Chemotrophs- chemical energy- E. Coli
 Carbon source:CO2, organic complounds
 Nitrogen source- nitrate, nitrite

• Mineral salts :

 Sulphates, phosphates, chlorides and carbonates of K,

Mg and Ca
 A suitable pH- 7.2- 7.4
 Bacterial 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

Factor x & v H.Influenzae

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 Common ingredients of culture media

• Water : Act as vehicle, pure or distilled water

. Peptone:
-Act as a buffer
-Hygroscopic in nature
- Partially digested protein obtained from meat, heart muscle, casein,
fibrine, soya meal etc
- Nitrogen source
- Protease, amino acids, inorganic salts and growth factors nicotinic
acid and riboflavin.
• Yeast extract :
- Prepare from cells of Backer yeast or Saccharomyces
- Contain amino acid, carbohydrates and growth factors
- Used as source of vitamin
Meat Extract:
- Prepare from fresh lean meat
- Contain gelatin, proteoses, amino acids, creatin , purine, mineral
salts, carbohydrates and Vit B
 Properties of Agar

- Golden yellow – Granular powder

- Used for preparing solid medium (solidifying agent)
- Obtained from different seaweeds algae.
- No nutritive value.
- Not affected by the growth of bacteria
- Resistance to action of all microorganism
- Melts at 95-98C and remain liquid up to 40-42C
- It gets solidified below 40C.
- 2% agar is employed in solid medium
- Easily available and economical
 Classification of culture media

Based on Based on presence of Based on nutritional

physical state molecular oxygen and factors
reducing substances

Liquid media Aerobic media Simple media

Semisolid media Anaerobic media Complex media

Solid media Synthetic media

Special media
 Special media
A. Enriched media

B. Enrichment media

C. Selective media

D. Differential media

E. Indicator media

F. Transport media

G. 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
Fastidious organisms: Only grow when specific nutrients are included in its diet.
Example:- Nutrient broth/agar
Peptone water
Composition:-
Lab-Lemco -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 of common
pathogenic bacteria.
2. It is used for subcultures of certain organisms.
Liquid media Nutrient broth Nutrient agar
Semisolid Solid
 Peptone water

• Type : basic liquid media

• Appearance : clear, colorless, watery, usually in test tube
• Composition :

Peptone 10 g
Sodium chloride, NaCl 5g
Water 1 litre

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 Uses Of Peptone Water:

 The media is used chiefly as the basis for

Carbohydrate fermentation media.

 Nutrient broths :

May contain a small amount of sugar derived from meat and it is

essential that the basal medium to which various carbohydrates are
added for fermentation tests should be free from natural sugars.
 Nutrient broths :

 It is also used to test the formation of indole.

 Culture of organisms for demonstration of motility.
 Complex media

Complex media have added ingredients for bringing out certain

properties or providing special nutrients required for growth of
the bacteria.

Synthetic media
 These are prepared from pure chemicals and the exact
compositions of medium is very well known.
Example :- Dubo’s medium
 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.
Examples:-
Dorset’s Egg Medium.
 It is a creamy coloured opaque slope kept in screw
copped bottle
 Selective medium for isolation of Mycobacterium
tuberculosis.
 Composition: Hen’s egg, Nutrient broth
 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.
 Chocolate Agar
Also called Heated blood agar.
• Type : Enriched media.
• Appearance : Chocolate brown color.
Procedure:
Melt the desired amount of nutrient agar.
Cool it in a water – bath at 75º C .
Add 10 ml of sterile blood .
Allow the medium to remain at 75º C.
Mixing the blood and agar by gentle agitation from time to time until the
blood become chocolate brown in color, within about 10 min. Then
pour in plates.
 USES: Culture Of Neisseria , Haemophilus influenzae,
Pneumococcus
 Enrichment media

 In this media, it has a stimulating effect on the bacteria to be grown or

inhibits its competitors.

 This result in an absolute increase in the number of wanted

bacteria related to other bacteria.

 Example:- Selenite F broth

 It is enrichment medium for culture of Salmonella typhi and paratyphi

bacilli from stool sample

 Principle:-
 At neutral pH solution acid salinity has high toxicity to coli form group of
bacteria and not to most of the salmonella groups.
 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:-
 B i l e s a l t i n h i b i t t h e g r o w t h o f
n o r m a l c o m m e n s a l s ( u n w a n t e d
bacteria).

V i b r i o c h l o e r a e p r o d u c e a c i d b y
f e r m e n t a t i o n o f s u c r o s e w h i c h a c t s o n
b r o m o t h y m o l b l u e ( i n d i c a t o r ) p r o d u c i n g
yellow colonies.
 Differential media

• MacConkey agar is a culture medium designed to grow Gram-

negative bacteria. It is a useful medium for the cultivation of
enterobacteriacea.

• It shows lactose fermenter as red colonies while non-lactose

fermentor as white/pale colonies.

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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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• Gram-negative bacteria growing on the media are differentiated by
their ability to ferment the sugar lactose.

• Lactose fermenter cause the pH to drop and

is detected by neutral red, (red at pH's below 6.8.) which appear
as bright pink to red colonies on
the agar.

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 • Uses

Acting as a visual pH i n d i cato r, the a ga r

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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Description:
Lactose & non
lactose fermenters on
CLED medium.

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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 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

• Mueller-Hinton agar is an
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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 Choice of culture media

The choice of culture media to use in microbiology laboratories will

depend on:
• The major pathogens to be isolated, their growth requirements, and
the features by which they are recognized.

• Whether the specimens being cultured are from sterile sites or from
sites having a normal microbial flora. Although a selective medium is
usually more expensive than a non-selective one, it often avoids
subculturing, isolates a pathogen more quickly, and makes it easier
to differentiate and interpret bacterial growth.

• Cost, availability, and stability of different media in tropical countries.

• Training and experience of laboratory staff in preparing, using, and

quality controlling culture media
 How to prepare, sterilize, and test culture media

Use of dehydrated culture media

Dehydrated media is hygroscopic, i.e. it absorbs water. When
exposed to moisture, it rapidly becomes unfit for use.

A hard mass is formed which alters the chemical and microbiological

properties of the medium. This can be a serious problem for tropical
countries with humid climates.

Adequate precautions must be taken to prevent dehydrated culture

media deteriorating and having to be discarded before it is finished.
Such precautions include:

- Weighing the medium rapidly, and tightly capping the bottle as soon
as possible after removing the approximate amount

- Sealing the cap of the container with adhesive tape.

- Storing media in the coolest driest place available and always out of
sunlight
 Preparation of culture media

 Prepare media made from dehydrated products in as damp-free an

environment as possible.

 To prevent the risk of inhaling fine particles of dehydrated media,

wear a dust mask while handling dehydrated media.

 Once the ingredients are weighed, do not delay in making up the

medium. Follow exactly the manufacturer’s instructions.

 Use completely clean glassware, and the container in which the

medium is prepared should have a capacity of at least twice the
volume of the medium being prepared.

 Use distilled water or Deionized water to dissolve the media.

 Add the powdered or granular ingredients to the water and stir to
dissolve.

 Heat the medium where required to completely dissolved.

 Autoclave a medium only when the ingredients are completely

dissolved.

 Dispense medium in bottles or tubes in amounts convenient for

use.
Checking the pH of a culture medium:
The pH of most culture media is near neutral. An exception is alkaline
peptone water.
The simplest way of testing the pH of a culture medium is to use
narrow range pH papers or a pH meter.

Sterilizing culture media:

The following methods are used to sterilize culture media:
– Autoclaving
– Steaming at 100 C
– Filtration
How to dispense culture media:

 Media should be dispensed in a clean draught-free room.

 Most fluid media are dispensed into screw capped bottles or tubes,
and then sterilized by autoclaving.

 Sterile media must be dispensed into sterile petri dishes, tubes or

bottles using an aseptic technique.
Dispensing sterile media into petri dishes:

 Lay out the sterile petri dishes on a level surface.

 Mix the medium gently by rotating the flask or bottle.

 Avoid forming air bubbles.

 Flame sterilize the neck of the flask or bottle and pour 15–20 ml of
medium into each dish (90–100 mm diameter).
 If air bubbles enter while pouring, rapidly flame the surface of the
medium before gelling occurs.

 Rotate the dish on the surface of the bench to ensure an even

layer of agar.

 When the medium has gelled and cooled, stack the plates and seal
them in plastic bags to prevent loss of moisture and reduce the risk
of contamination.

 Do not leave the plates exposed to bright light especially sunlight.

 Store at 2–8 C
Note:

 Agar plates should be of an even depth (not less than 4 mm) and
of a firm gel.

 The surface of the medium should be smooth and free from

bubbles.
Sterility testing:

 For ‘sterile’ media in screw-cap tubes or bottles, the simplest way

to test for contamination is to incubate 5% of the batch at 35–37 C
overnight.

 Contamination by microorganisms capable of overnight growth, will

be shown by a turbidity in a fluid medium and growth on or in a
solid medium.

 Media in petri dishes are best examined for contamination

immediately before use.
Performance testing:

A set of control organisms (stable stock strains) will need to inoculate

on respective media .

Sources of well-characterized stable strains of control bacteria are;

 National Collection of Type Cultures (NCTC)
 American Type Culture Collection (ATCC)
 Centers for Disease Control (CDC) Bacterial Diseases Division,
Atlanta , USA etc
Control of nutrient agar, blood agar, chocolate agar:

 After overnight incubation, examine the cultures for the degree of

growth, size of colonies, and other characteristics such as alpha-
or beta-haemolysis.
 Record the results of each control species and compare with the
results of previous performance tests.

Control of a differential medium:

 After overnight incubation, examine the cultures for the differential

characteristics of the medium.
 Record the results of each control species and compare with the
results of previous tests.
Control of a transport medium:

 Immerse in the medium a swab of the specimen containing the

pathogen(s) to be preserved.

 Leave the inoculated transport medium at room temperature

(protected from direct light) for the length of time the medium is
intended to preserve the viability of the pathogen(s) it contains.

 After this time, inoculate the swab on an appropriate medium to

check for viability of the pathogen.
Long-term preservation of control strains:

 To reduce the risk of contamination and changes in the growth

characteristics of control strains, stock cultures should not be sub
cultured more than is necessary.
 Several subcultures should be prepared at one time.
Labelling and storage of culture media:

• Plates of culture media should be stored at 2–8 C, preferably in

sealed plastic bags.

• Most media in screw-cap tubes or bottles can be stored at room

temperature (20–28 C).

• Prepared media should be stored in the dark. When in use, the

media must be protected from direct light, especially from sun light.

• All culture media must be clearly labelled with identification and

date of preparation.