PREPARATION OF CULTURE MEDIA,

INOCULATION AND IDENTIFICATION OF

BACTERIAL GROWTH CHARACTERISTICS
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.
 Culture media are artificially prepared media containing the
required nutrients used for propagation of microorganisms.
 Once the bacteria are grown we can:

1. Identify them either by presumptive lab diagnosis like Gram

stain or by definitive lab diagnosis like biochemical test

2. Test the antimicrobial sensitivity of the bacteria (drug

testing).
 Common ingredients of culture media

1. H2O : is essential for growth of microorganism 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.

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).

Eg. lean meat, heart meat, milk etc.

- It is the main source of nitrogen, some vitamins, carbohydrates,

nucleic acid fraction and minerals.
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

- PO42- - as a source of phosphor

- Nacl - is the common ingredient and source of

sodium and chloride

- Other trace elements include Mg, Mn, etc

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.

- Simple sugars includes glucose, maltose, sucrose, lactose, pentose,

hexose, galactose etc.
- The complex sugar include – polysaccharides, glycerol, manitol
etc.
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 .
• Is a good solidifying agent due to:
1. The protein is inert & undegradable by microorganism
2. Solid – gel transformation temperature is very high
i.e. it solidifies at 45oC and changed to gel > 45oC
3. It can form gel (solidify) at 1.5% w/v concentration and
4. It forms semi solid gel at 0.4 – 0.5% w/v concentration

Uses of agar
1. Solidify culture media
2. Provide calcium and organic ions for the bacteria
3. Used for adding heat sensitive ingredients such as blood and
serum while the agar is in gel phase at 45 – 50oC
4. The agar is very transparent so that growth of colonies is easily
visible.
B. Gelatin – is sometimes used as solidifying agent but it has many
disadvantages like:
 The protein which are found in gelatin are susceptible to
microbial digestion
– Solid to gel transformation is very low which is 24oC, so it will
become changed to gel at 24oC & can not be used for bacteria
which need incubation above 25oC.
– 15% w/v gelatin is required to solidify the culture.
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.
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
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.

3. For sub culturing pathogens from differential and selective

media prior to performing biochemical and serological
identification tests.
2. Enriched/Enrichment media
 These media are required for growth of organism with extra
nutritional requirements such as H. influenzae, 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.
 E.g: - Blood Agar (contain whole blood)
- Chocolate agar (contain lyzed blood)
• Enrichment media: this term is usually applied to fluid selective
media E.g. Tryptosoya broth, Selenite F broth
3. Selective media
• These are solid media which contain substances which inhibit the
growth of one organism to allow the growth of the other to be
more clearly demonstrated.
• The medium is made selective by incorporation of certain
inhibitory substances like bile salt, crystal violet, antibiotics, etc.
• E.g. Addition of crystal violet favors the growth of gram
negative bacteria and slows the growth of Gram positives.
• Selective medium is used when culturing a specimen from a site
having normal microbial flora to prevent unwanted contaminants
overgrowing a pathogen.
E.g.1. Thiosulphate citrate bile salt sucrose agar(TCBS) - is
alkaline medium and selective for V. cholera.
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 agar (SSA) - Selective for Salmonella

and Shigella species.
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.

E.g. Mac Ckonkey agar - contain neutral red as an indicator and

lactose as carbohydrate. Lactose fermenting bacteria will become
pink and other bacteria become colourless.
 Figure: differential media (Triple sugar iron/TSI agar media)

Figure: differential media (MacConkey agar media , pink colonies are

lactose fermenters)
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.
Example
1.Cary-Blair medium :– is used for preserving and transporting
enteric pathogens.
2.Amies transport medium:- is used for transportation of gonococci
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.
• 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.
N.B: The colonial appearances and changes in solid media made
by colonies may provide valuable identification information
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.

C. Fluid culture media

• Fluid media are most commonly used as enrichment when
organisms are likely to be few

E.g. blood culture

• Is also used for biochemical testing

E.g. Peptone water

 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 powder form or ready made standardized culture
media (in packs)
Use of dehydrated culture media (powder form)
• Usually ready made standardized dehydrated media should be used
(i.e. all the ingredients are incorporated to ensure good
performance and reproducibility)than buying the individual
chemical constitutes.
• Dehydrated media is hygroscopic (i.e. it absorbs water), so when
exposed to moisture, it rapidly becomes unfit for use. therefore to
avoid this:
• Weigh the medium rapidly
• Tightly cap the bottle as soon as possible
• Do not return small amounts of medium to the stock and
• Store the media in the cool, dry place.
Use of ready made standardized culture media (in packs)
• These media are weighed ,suspended ,sterilized and packed in
small volumes (usually 15ml)
• These packed media only require boiling the sachet (packet)
in 95oC till it is changed to gel and dispense in Petri dish
aseptically.
Major procedures during 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

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.
 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
2. Sterilization and sterility testing
• Sterilization is required to make the media free from any
Contaminant.

Methods of sterilization

A. Autoclaving –
• The majority of culture media are sterilized by being autoclaved.
• This ensures the destruction of bacterial endospores as well as
vegetative cells.
• It is important to sterilize a medium at the correct temperature and for
the correct length of time as instructed in the method of preparation.
• Under sterilization and over sterilization will affect the quality of the
culture media .
• N.B. Most culture media are sterilized at a pressure of 15 lb/in 2 and
temperature of 121oC for 15 minutes.

B. Steaming at 100oC
• It is used to sterilize media which contain ingredients that would be
inactivated at temperature over 100oC.

E.g. Cary-Blair transport media

• Steaming can be performed by placing media in boiling water for
certain time.
C. Filtration
• This provides a means of removing bacteria from fluids. It is used
mainly to sterilize additives that are heat sensitive and cannot be
autoclaved or heated at 100oC.

Eg. Serum and other solutions containing urea and certain

carbohydrates.
– Several different types of filters can be used including those made
from sintered glass or inert cellulose esters (membrane filters).
– They are available in a variety of pore sizes, and 0.22-0.45 µm
pore size is required for sterile filtration.
Sterility testing
• This step actually comes after the media is dispensed and ready for
use
• For sterile media in screw – cap tubes, bottles, or petridishes the
simplest way to test for contamination is to incubate the entire batch
or 3 – 5% of the batch at 35 – 37oC overnight.
• 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.
3. Addition of heat sensitive ingredients
• Refrigerated heat sensitive ingredients should be first warmed at
room temperature before added to a molten agar medium.
• Using an aseptic technique, the ingredient should be added when
the medium has cooled to 45oC and should be distributed
immediately unless further heating is required.
• Heat sensitive ingredients include serum, whole blood, egg yolk
etc.
4. 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 pH colour chart.
• 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

• The pH meter can also be used to measure Ph of a fluid or molten

medium.

NB. _The Ph of a dehydrated medium should not require adjustment

provided it has been prepared correctly using pure water and clean
equipment and it has not been over heated.
– The Ph of other media can be adjusted using 0.1 molar (0.1N)
NaOH and 0.1 M or (0.1N) Hcl.
5. Dispensing of culture media
• 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.
Sterilizing glass Petri dishes, tubes and other glass wares
• Culture media should be dispensed in sterile container s and
glass wares.
• Containers and glass wares are sterilized using dry heat (in dry
heat oven) and a temperature of 160oC held for 45 – 60 minutes
for effective sterilization.

NB: A cooling time is also necessary to enable the items in the

oven to cool slowly. The oven door must not be opened until
the temperature inside the oven has fallen to below 50oC. This
will avoid cracking the glass ware.
Dispensing agar media in petridish
1. Lay out the sterile Petri dishes on a level surface
2. Mix the medium gently by rotating the flask or bottle. Avoid
forming air bubbles.
3. Flam sterilize the neck of the flask or bottle and pour 12 – 15 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.
4. When the medium has gelled and cooled, stack the plates & tubes
and seal them in plastic bags to prevent loss of moisture and reduce
the risk of contamination.
5. Store at 2 – 8oC
Dispensing agar in culture tubes or bottles
• Sterilized agar media are dispensed aseptically half filled in a sterile screw cup bottles or
tubes and allowed to set in a slope or slant position to increase the surface area for aerobic
inoculation.

Figure. Agar media are dispensed aseptically half filled in a sterile

screw cup bottles or tubes and allowed to set in a slope or slant
position.
6. Quality control or performance test

• Performance test is used to check the quality of the culture media

prepared (whether it can be used to perform the intended test) using
control organisms.
• Whenever possible, the regional microbiology laboratory should
supply district laboratories with standardized tested media and when
this is not possible each district laboratory should set up its own
quality control of the media it prepared.
• A set of control organisms (stable stock strains) will need to be
obtained from the regional or central public health laboratory and the
viability of these organisms should be maintained with regular sub
culturing.
Control of nutrient agar, blood agar, chocolate agar
• Use appropriate control species. Inoculate slopes or quarter
plates of the medium to be tested with a 5 hour broth culture of
each control organisms.
• Depending on the species, incubate aerobically or in a carbon
dioxide enriched atmosphere at 35 – 37oC.
• After over night incubation, examine the cultures for the degree
of growth, size of colonies, and other characteristics such as
alpha or beta hemolysis.
• Record the result of each control species and compare with the
standard.
Control of a transport medium
• Immerse in the medium a swab of the specimen containing the
pathogen (s) to be preserved (E.g Urogential swab containing N.
gonorrhea in Amies transport medium)
• Leave the inoculated transport medium at room temperature for
the length of time the medium is intended to preserve the viability
of the pathogens it contains.
• After this time, inoculate the swab on an appropriate medium to
check for viability of the pathogen.
• NB. Similar control test is applied for selective, biochemical
testing media and other media
5. Storage of culture media
• Plates of culture media should be stored at 2 – 8oC, preferably in
sealed plastic bags to prevent loss of moisture.
• Antimicrobials in solution form should be stored at –20 OC.
• All types of prepared media should be stored in the dark.
• When in use, the media must be protected from direct light,
especially sun light.
• All culture media must be clearly labeled.
• Each batch of prepared medium should be given number and its
preparation date recorded.
 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
Depending on the characteristics and nutritional requirem ent,
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.
NB: 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.
3. Cost, availability, and stability of different media in tropical
countries.
4. Training and experience of laboratory staff in preparing, using, and
quality controlling culture media.
Inoculation of culture media

Inoculation: - is artificial seeding or introduction of micro

organisms on/in to culture media or animal body.
– 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.
E.g. Darkening of the medium
Any growth of micro organisms
• When inoculating culture media, an aseptic technique must be
used to prevent contamination of specimens and culture media,
and laboratory worker and the environment
Aseptic technique
• 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 necks of specimen bottles, culture bottles and tubes after
removing and before replacing caps or plugs.
• Use a safety cabinet when working with hazardous pathogens and
wear protective clothing.
Inoculating Techniques

NB: For sputum and stool samples since they have large number of
bacteria, prior to inoculation we have to dilute or homogenize the
samples.

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
Figure. Inoculation techniques.
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.

• 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. Fig. Inoculation of a deep (stab)
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.

Figure. … Inoculation of fluid media

Labeling of inoculated media
• 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.
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 at 25oC where as

many opportunistic and saprophytic mycobacteria grow at this
lower temperature.
• 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.
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.

Example- Streptococcus pyogens from throat swabs

• The hemolytic reactions of beta - hemolytic streptococci are also
more pronounced following anaerobic incubation.
• To create anaerobic environment a tightly closed container or jar is
required it is usually called Anaerobic jar.
•

Figure. Anaerobic jar

• There are several techniques for obtaining anaerobic conditions
1. Commercially produced sachets contain oxygen removing
chemicals
E.g. - Merck anaerocult anaerobic system
• The sachets contain a mixture of iron powder, citric acid and
sodium carbonate. Addition of a small volume of water
activates the chemical and oxygen is rapidly removed leaving
an aerobic conditions in the anaerobic jar. Some CO2 is also
produced.
• Palladium pellets are used as catalyst

Figure: Reactions in anaerobic jar

• Control of anaerocult system – is using anaerobic indicator strips
which will change colour in the absence of oxygen (anaerobic
condition).

2. Copper coated steel wool to remove oxygen

Steel wool + acidified copper sulphate solution – this will coat the
iron and rapidly absorb oxygen when put in a sealed plastic bag.

3. Use of reducing agents in culture media

E.g. Thioglycollate broth which is used mainly to culture

anaerobes in blood , contains the reducing agent sodium
theioglycolate and the indicator methlene blue to show that the
medium is reduce.
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.

Figure. Culturing in carbon Dioxide

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.
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.
E.g. - V. cholera in TCBS agar appear Yellow
- Corny bacterium diphteriae in Tellurite agar appear black.

Figure:TCBS Agar - Vibrio chol.

Changes which may occur in the medium when bacteria are
cultured on solid agar.
• These include hemolytic reactions, pigment production, color changes
surrounding carbohydrate fermenting colonies, and blackening due to
hydrogen sulphide production.

* An example of pigment forming organism is pseudomonas

aeroginosa which produces a yellow – green color in media such as
blood agar and MacConkey agar.

* An examples of organism that produces a color change is Vibrio

chorera which is sucrose fermenting, giving a yellow color in TCBS
agar.
• Blacking due to hydrogen sulphide production is seen with many
Salmonella cultured in kligler iron agar(KIA).
• Hemolytic reaction in blood agar is seen with beta hemolytic
Streptococci (complete hemolysis) and alpha hemolytic
pneumococci(partial hemolysis)

Figure: Beta hemolysis Figure: Alpha hemolysis

NB. Morphological appearance of colonies on blood agar can vary
depending on the species of blood used. E.g. Horse, sheep, or
goat blood.
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.

NB. For isolating organism from sterile sites use culture media
which is non selective, and enriched.
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’.
• 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.
Questions?

68 01/16/24