CHAPTER VIII

PREPARATION OF CULTURE MEDIA,

INOCULATION AND IDENTIFICATION
OF BACTERIAL GROWTH
CHARACTERISTICS

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Learning objectives
At the end of this chapter, the student will be able :
1. List the common ingredients of culture media.
2. List the different types of culture media.
3. Discuss the different steps while preparing culture media.
4. Describe the storage methods for culture media.
5. Discuss the inoculation and aseptic techniques during
inoculation of culture media.
6. List the different inoculation techniques.
7. Discuss the culturing of anaerobes.
8. Perform quality control on culture media.
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Culture media are artificially prepared media
containing the required nutrients used for
propagation of micro- organisms or living tissue
cells.

Uses
• Isolation and identification of micro-organisms

• Performing anti-microbial sensitivity tests

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 Common ingredients of culture media
1. H2O : is essential for growth of micro-organism and
80% of bacterial cell is H2O.

• It must be free of mineral salts which can inhibit

bacterial growth.
• Either distilled or deionised water should be used.

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2. Peptone: contains water soluble products obtained from
break down of animal or plant protein (contains free
amino acids, peptides, and proteoses (large sized
peptides).

Example: lean meat, heart meat, milk etc.

It is the main source of nitrogen, some vitamins,

carbohydrates, and minerals.

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3. Meat extract : this provides amino acids, minerals, and
essential growth factors .

4. Yeast extract : is a very good growth stimulant.

It provides amino acids, water soluble vitamins (vit.B),

inorganic salts, and etc.

5. Mineral salts : are essential for growth and for

metabolism of living organism.

e.g SO4……………….. as a source of sulphur

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6. Carbohydrates : simple and complex sugars are used:
• As energy and carbon source.

• For differentiation of bacteria.

Example:

- Sucrose in TCBS agar differentiates vibrio species

- Lactose in MacConkey agar differentiates enterobacteria

etc.

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• PO24 ……………..a source of phosphors

• Nacl ………………source of sodium and chloride

• Other trace elements include Mg, Mn, etc.

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7. Solidifying agents: are agents which are used to solidify the
culture media.

A. Agar : is a commonly used solidifying agent of culture media.

• Is a mixture of two poly saccharide - agarose (70 -75%) &
agaropectin (20- 25%).
• It is produced from sea weeds (Rhodophyceae) which form the
agarophyte group of marine algae .

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B. Gelatin : is sometimes used as solidifying agent but it has
many disadvantages such as :
• The protein which are found in gelatin are susceptible to

microbial digestion
• 15% w/v gelatin is required to solidify the culture

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Agar Is a good solidifying agent due to:

1. The protein is inert & undegradable by micro-organism

2. It can form a firm gel (solidify) at 1.5% w/v concentration.

3. It forms semi solid gel at 0.4 - 0.5% w/v concentration. It is

mainly used to prepare transport media such as Amies
medium and stock culture.

4. Agar also provides micro- organisms with calcium and other

organic ions.

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8. Accessory growth factors
• Include vit B (thiamine, niacin, and
biotin).These provide the necessary condition
for growth
• 9. Other ingredients include, blood, serum,
egg yolk etc depending on the organisms need.

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Types of culture media

The main types of culture media are:

1. Basic media

2. Enriched or enrichment media

3. Selective media

4. Indicator (differential) media

5. Transport media

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1. Basic Media

These are simple media that will support the growth of

micro organisms that do not have special nutritional
requirements.

Example - Nutrient Agar

- Nutrient broth

Purposes of basic media

1. Are used in preparation of enriched media.

2. Are used to maintain stock culture.

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2. Enriched
 These media are required for growth of organism with extra
nutritional requirements such as H. influenza, Neisseria spp,
and some streptococcus species.
 An enriched medium increases the number of a pathogen by
containing all the necessary ingredients to promote its growth.
 The media can be enriched with whole blood, lyzed blood,
serum, vitamins, and other growth factors.

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Example: - Blood Agar (contain whole blood)

- Chocolate agar (contain lyzed blood)

3. Enrichment media
• Fluid medium that increases the number of a pathogen by
containing enrichments and/ or substances that discourage
multiplication of unwanted bacteria.

Example:

- Selenite Faecal (F) broth is used as an enrichment

medium for Salmonellae in faeces or urine prior to subculture.

- Tryptosoya broth supports the growth fastidious organisms such
as Neisseriae and Pnemococci.
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4. Selective media
• These are media which contain substances that prevent or slow
down growth of micro- organisms other than pathogen for which
the media are intended.
• The medium is made selective by incorporation of certain
substances like bile salt, crystal violet, antibiotics, etc.
• Selective medium is used when culturing a specimen form a site
having normal microbial flora to prevent unwanted contaminants
overgrowing a pathogen.
Example:. 1.Thiosulphate citrate bile salt sucrose agar(TCBS) - is
alkaline medium and selective for V. cholera.
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2. Xylose Lysine Deoxy Cholate (XLD) agar - selective
for Salmonella and Shigella.

3. Modified New York City (MNYC) medium - Selective

for Neisseria gonorrhoeae.

4. Butzler medium - Selective for Campylobacter species.

5. Salmonella and Shigella (SS) agar - Selective for

Salmonella and Shigella species.
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5. Differential (Indicator) media
• These are media to which dyes or other substances are added
to differentiate micro-organisms.
• Many differential media distinguish between bacteria by
incorporating an indicator which changes colour when acid is
produced following fermentation of a specific carbohydrate.
Example : Mac Ckonkey agar - contain neutral red as an
indicator and lactose as carbohydrate. Lactose fermenting
bacteria will become pink and other bacteria become
colourless.
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6. Transport media
• These are mostly semisolid media that contain ingredients to
prevent the overgrowth of commensals and ensure the survival
of aerobic and anaerobic pathogens when specimens cannot be
cultured immediately after collection.
• Their use is particularly important when transporting
microbiological specimens form health centres to the district
microbiological laboratory or regional public health laboratory.

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Example

1.Cary-Blair medium :– is used for preserving and

transporting enteric pathogens.

2.Amies transport medium:- is used for transportation

of gonococci.

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Culture media can be classified by their consistency (form) as:
• Solid media
• Semi-solid media and
• Fluid media

A. Solid media
• Are solidified by agar

• Are used mainly in Petri dishes as plate cultures, in bottles or

tubes as stab (deeps) or slope cultures.

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• The purpose of culturing on solid medium is principally to isolate discrete
colonies of each organism present in the specimen. this will enable pure
cultures to be produced for identification and sensitivity testing.

B. Semi solid media

• Are culture media prepared by adding small amount of agar (0.4 - 0.5%
W/V) to a fluid medium.
• Semi solid media are used mainly as transport media, and for

motility and biochemical tests.

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C. Fluid culture media

Is used for biochemical testing as enrichment media

and blood Culture. e.g. Peptone water.

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Preparation of culture media
 For successful isolation of pathogens, culture media
should be

prepared carefully following manufacturers instruction.

 Most culture media are available commercially in
ready-made dehydrated form

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Preparation of culture media

1. Weighing and dissolving of culture media

2. Sterilization and sterility testing

3. Addition of heat sensitive ingredients

4. pH testing of culture media

5. Dispensing of the culture media

6. Quality assurance of culture media

7. Storage of culture media

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1. Weighing and dissolving of culture media
• Use ingredients suitable for microbiological purpose

• Use clean glass ware, plastic or stainless steel equipment

• Use distilled water.

• Weigh accurately using a balance.

• Wear a facemask and glove while weighing and dissolving

toxic chemicals.
• Do not delay in making up the medium after weighing.
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2. pH testing
• 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 papers or a pH meter.
• A fluid medium can be tested by dipping a narrow range pH
paper in to a sample of the medium when it is at room
temperature and comparing the colour of the paper against the
p colour chart.
H
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• An agar medium can be tested by pouring a sample of
the molten medium in to a small beaker or Petri dish
and when it has solidified, laying a narrow range pH.
paper on it’s surface.
• The colour of the paper is then compared against the
pH colour chart

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• Add powdered ingredients to distilled water and mix by rotating
or stirring the flask.
• Stir while heating if heating is required to dissolve the medium.

• Autoclave the medium when the ingredients are dissolved.

• Over heating may result in:

- alteration of the nutritive value

- alteration of pH and
- alteration of gelling property
• Always autoclave media at the correct temperature and for the
time specified

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3. Sterilization and sterility testing
Sterilization is required to make the media free from any
Contaminant.

Methods of sterilization

A. Autoclaving - is used to sterilize most agar and fluid culture

media.

B. Steaming at 100oC - It is used to sterilize media containing

ingredients that would be inactivated at temperature over 100
O C and re-melt previously bottled sterile agar media.
C. Filtration - is used to sterilize additives that are heat-sensitive
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and can not be autoclaved.
4. Dispensing of culture media
• Media should be dispensed in a clean draught free room.

• Most fluid media, unlike solid media, are first dispensed into
screw capped bottles or tubes, and then sterilized by
autoclaving.
• Media should be dispensed in a clean draught-free room using

aseptic technique and sterile container.

• Stack the plates after the medium has gelled or cooled.
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Dispensing agar media in petridish
• Lay out the sterile Petri dishes on a level surface.

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

• Flame sterilize the neck of flask or bottle.

• Pour 15 ml of medium in each Petri dish.

• Store the plates in a refrigerator.

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 Figure----. Agar media are dispensed aseptically half filled in
a sterile screw up bottles or tubes and allowed to set in a slope
or slant position to increase the surface area for aerobic
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5. Storage of culture media
• Dehydrated culture media and dry ingredients should be stored
at an even temperature in a cool dry place away from direct
light.
• Plates of culture media, and additives like serum, blood and
antimicrobials in solid form require storage at 2-8 OC.
• Antimicrobials in solution form should be stored at -20 OC.
• All culture media and additives should be labelled with the

name and date of preparation.

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6. Culturing of bacteria

Materials for Culturing Bacteria

Basic materials used for culturing bacteria.

• Culture media

• Petri dishes

• Test tubes

• Inoculating loops, straight wire loops

• Bunsen burner

• Incubator

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Depending on the characteristics and nutritional requirement,
bacteria can be inoculated on different culture media hence
the choice of culture media for inoculation of samples
depends on:

1.The major pathogens to be isolated, their growth

requirements, and the features by which they are
recognized.

2. Whether the specimens being cultured are from sterile sites

or from having normal microbial flora.

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• Although a selective medium is usually more expensive than
a non selective one, it often avoids sub culturing , 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.

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Inoculation of culture media
• Inoculation: - is artificial seeding or introduction of
micro organisms on/in to culture media or animal
body.
• When inoculating culture media, an aseptic technique
must be used to prevent contamination of specimens
and culture media, and laboratory worker and the
environment
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• Immediately before inoculating a culture medium, check the
medium for visual contamination or any change in its
appearance medium for visual contamination or any change
in its appearance which may indicate deterioration of the
medium.

Example. Darkening of the medium and any growth of micro

organisms.

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 The aim of inoculating media (solid) is to get isolated single colonies
which are derivatives of single bacterium. (i.e. pure colony).
 Up on inoculating the sample successively its distribution will
become decreased and at last end up in isolated colonies.
 The technique used to inoculate media in Petri dishes (plate) must
provide single colonies for identification
 A pathogen must be isolated in pure culture before it can be

identified and tested for antimicrobial sensitivity.

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• The inoculation of media in Petri dishes is referred to as ‘plating
out’ or ‘looping out’.
• It is not necessary to use whole plates of media for every
specimen. Considerable savings can be made by using half or
even a third of a plate, but the area of medium used must be
sufficient to give separate colonies.
• Before inoculating a plate of culture medium, the surface of the
medium must be dried, other wise single colonies will not be
formed.
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Aseptic technique during inoculation of culture media
• Decontaminate the workbench before and after the work of
the day.
• Use facemask and gloves during handling highly infectious
specimens.
• Flame sterilize wire loops, straight wires, and metal forceps
before and after use.
• Flame the neck of specimen and culture bottles, and tubes
after removing and before replacing caps and plugs.

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 Sterilizing the
inoculating loop Inoculating
with flame the fluid
media with
sterilized
loop

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• 7. Inoculating Techniques

1.Using a sterile loop or swab of the specimen, apply

the inoculation to a small area of the plate.

2. Flame sterilize the loop, when cool or using a second

sterile loop, spread the inoculation systematically.
This will ensure single colony growth

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 Figure …… Inoculation techniques.
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Inoculation of Slopes

To inoculate slopes (such as Dorset egg medium or TSI) use a

sterile straight wire to streak the inoculation down the center of

the slope and then spread the inoculation in a zigzag pattern as

shown in the figure.

Figure. … Inoculation of Slopes.

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To inoculate a slope and butt medium such as kligler iron agar
(KIA), use a sterile straight wire to stab in to the butt first and
then use the same wire to inoculate the slop in a zigzag
pattern.

Inoculation of Stab Media (deeps)

Use a sterile straight wire to inoculate a stab medium. Stab

through the centre of the medium taking care to with draw the

wire along the line of inoculation with out making further stab
lines.

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 Figure. … Inoculation of a deep (stab)
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Inoculation of fluid media
• Broths and other fluid media are inoculated using a sterile
wire loop, straight wire, or pasture pipette depending on
whether the inoculums is a colony, a fluid culture or a
specimen.
• When using a wire loop, hold the bottle or tube at an angel
and rub the loop against the side of the container below the
level of the fluid.

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 Figure. … Inoculation of fluid media.
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Using grease pencil or marker, label inoculated media with the
date and the patient’s number always label the base of the
culture plate not lids.

Incubation of inoculated media

• Inoculated media should be incubated as soon as possible. A
delay in incubation can affect the viability of pathogens.

• Micro organisms require incubation at the temperature,

humidity & gaseous atmosphere most suited for their

metabolism.
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Temperature of Incubation
• The temperature at which micro-organisms grow best is
referred to as its optimum temperature.
• The temperature selected for routine culturing is 35 -37oC,
however, most microbiologists recommend 35oC.
• Temperature of growth is also used in the differentiation of
mycobacterium species. E.g. No growth is produced by

M. tuberculosis and H. ulcerans at 25oC where as many

opportunistic and saprophytic mycobacteria grow at this lower

temperature.
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• Some exception is that yersinia enterocolitica grows
best at 20 -28oC which helps to identify the species.
• Humidity -growth atmosphere which is too dry can
affect the growth and viability of many pathogens.

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Culturing of Anaerobes
• An anaerobic atmosphere is essential for the growth
of strict anaerobes such as clostridium species,
bacteroides, and anaerobic streptococci.
• Anaerobic incubation also helps to differentiate
pathogens and isolate facultative anaerobes from
specimen containing commensals.

E example- Streptococcus pyogens from throat swabs

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• The hemolytic reactions of beta - hemolytic streptococci
are also more pronounced following anaerobic incubation.

Figure. … Anaerobic jar

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Control of anaerocult system – is using anaerobic indicator strips

which will change colour in the absence of oxygen (anaerobic

condition).

Figure: Reactions in anaerobic jar

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Culturing in carbon Dioxide
• Carbon dioxide enriched atmosphere is used for the
growth of N. gonorrhea,N. Maningitidis, Brucella species,
and streptococcus pneumonia.
• The simplest and cheapest way of providing a carbon
dioxide enriched atmosphere is to enclose the inoculated
plates in an air tight jar with a lighted candle. As the
candle burns, the oxygen content is reduced releasing a
carbon dioxide content of 3-5% by the time the candle
extinguished.
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Figure. … Culturing in carbon Dioxide
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Appearance of Bacterial colonies on solid Media

Bacterial colonies should be examined in a good light and a low

power magnifying lens can help to see morphological details.

1. When viewed form above: colonies may appear round,

irregular crenated, or branching.

They may be transparent or opaque and their surface may be

smooth or rough, dull or shiny. Eg. The colonies of
pneumococci have a ringed appearance.

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2. When viewed form the side: Colonies may appear flat or
raised in varying degrees some times with beveled edges or
with a central elevation or depression.

3. When touched with wire loop: Some colonies are soft and
easily emulsified such as S.aureus. Where as others are
difficult to break up such as S. pyogens.

4. The colour of colonies: this also helps to identify bacteria,

especially when using differential media containing indicators.

Example- V. cholera in TCBS agar appear Yellow

- Corny bacterium diphteriae in Tellurite agar appear black.

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Reporting culture results

The manner of reporting culture depends on whether the

specimen is either from a sterile site or from site with a

normal microbial flora.

Sites normally Sterile: Identify and report all bacteria

isolated up to their genera. And if helpful identify the
actual species using bio chemical tests. Sterile sites
include blood, bone marrow, CSF, pleural and
peritoneal fluids.
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Sites Having a Microbial Flora:
 Interpretation requires patients’ clinical data to judge whether
an isolate is a pathogen which causes the patients illness.
 The laboratory report should indicate those organisms for
which isolation technique has been performed. For example
when the pathogen have been isolated from feacal specimen
cultured on selective medium like SSA (salmonella shigela
agar).

The report should state as ‘no salmonella or shigella organism

isolated’.
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8. Quality control

• Inoculate quarter plates of the medium with a five hours broth

culture for each control organism.
• Use a straight wire to inoculate and wire loop to spread the
inoculums.
• Depending on the species, incubate aerobically, CO2
enriched atmosphere and anaerobically at 35-37 OC for 24hours.
• Examine for the degree of growth, morphology and other
characteristics of microbial colonies.
• Record the result of each control species and compare to
your standard reading

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Performance testing

Inoculate a known and representative organism on a

batch of prepared culture media and evaluate for
growth.

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• Sites with normal flora include faeces (stool),
sputum, skin, throat and nose swabs,
• vagainal, cervical, and urethral swabs.

• NB: For isolating organism from sites with

normal flora selective media are much
• better than general media.
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 Sterility testing

1. Contamination by micro organism capable of over

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

2. The simplest way to test for contamination is to

incubate the prepared sample media at 35-37 OC for
24 hours. Turbidity in fluid media and microbial
growth in solid media confirm contamination.

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