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DIFFERENT TYPES OF CULTURE MEDIA
For a culture medium to be successful in growingthe pathogen sought it must provide all essential
nutrients, ions, and moisture, maintain the correctpH and osmotic pressure, and neutralize any toxic
materials produced. It is also essential to incubatethe inoculated medium in the correct atmosphere,
atthe optimum temperature and for an adequateperiod.
The main types of culture media are:
Ɣ Basic
Ɣ Enriched
Ɣ Selective
Ɣ Indicator
Ɣ Transport
Ɣ Identification
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c : These are simple media such as nutrient agar and nutrient broth that will support
the growth of microorganismsthat do not have special nutritional requirements.They are often used
in the preparation of enriched media, tomaintain stock cultures of control strains of bacteria, and for
sub culturing pathogens from differential or selective mediaprior to performing biochemical and
serological identificationtests.
 c  : Enriched media are required for the growthof organisms with exacting
growth requirements such asï
 ,

species, and some species. Basic
media may be enriched with whole or lyzedblood, serum, peptones, yeast extract, vitamins and
othergrowth factors. An enriched medium increases the numbers ofa pathogen by containing all the
necessary ingredients topromote its growth. Such a medium is often used for specimens
collected from sites which are normally sterile to ensurethe rapid multiplication of a pathogen which
may be presentonly in small numbers.
|
|  | : This term is usually applied to fluid selectivemedia which contain
substances that inhibit the growth ofunwanted organisms, e.g. Rappaport-Vassiliadis broth which
is often used as an enrichment medium for  serovars in faeces.



c  : These are solid media which contain substances(e.g. bile salts or other
chemicals, dyes, antibiotics)which inhibit the growth of one organism to allow the growthof another
to be more clearly demonstrated. A selectivemedium is used when culturing a specimen from a site
havinga normal microbial flora to prevent unwanted contaminantsovergrowing a pathogen. Media
made selective by incorporatingantibiotics are usually expensive
  c 
c  c These are media to which dyesor other substances
are added to differentiate microorganisms.Many differential media distinguish betweenbacteria by
incorporating an indicator which changes colourwhen acid is produced following fermentation of a
specificcarbohydrate e.g. MacConkey agar.
: Many media used to isolate pathogens are both
selectiveand enrichment or both selective and differential.
 
 c cThese are mostly semisolid media thatcontain ingredients to prevent the
overgrowth of commensalsand ensure the survival of aerobic and anaerobic pathogenswhen
specimens cannot be cultured immediately after collection.Their use is particularly important when
transportingmicrobiological specimens from health centres to the microbiology laboratory or
specimens. Examples of transport media includeCary-Blair medium for preserving enteric
pathogens andAmies transport medium for ensuring the viability of gonococci.
  c cThese include media to which substratesor chemicals are added to
help identify bacteria isolated onprimary cultures. Examples include peptone water sugars,urea
broth, and Kligler iron agar. Organisms are mainly identifiedby a change in the colour of the
medium and or theproduction of gas. Organisms used to inoculate identificationmedia must be first
isolated in pure culture.

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Culture media can be classified by consistency as:
Ɣ Solid
Ɣ Semi-solid
Ɣ Fluid
„




|| 
Media are solidified by incorporating a gelling agentsuch as agar or gelatin.
# c
Agar (polysaccharide extract obtained from seaweed) iscommonly used to solidify culture media
because of its highgelling strength, its setting temperature of 32±39 C and
melting temperature of 90±95 C. Most agars used in bacteriologicalwork produce a firm gel at an
agar concentrationof 1.5% w/v. The low gelling temperature allows heat-sensitivenutrients such as
whole blood to be added safely at 45±50 C. At a concentration of 0.4±0.5% w/v, agar is added
to transport media such as Amies medium to give a semisolidgel.Solid media are used mainly in
petri dishes as platecultures. Also in bottles or tubes as stab (deeps) orslope cultures. The
inoculation of plates, slopes anddeeps is described later in this subunit. The purpose
of culturing on solid medium is principally to isolatediscrete colonies of each organism present in
thespecimen. This will enable pure cultures to beproduced for identification and sensitivity testing.
The colonial appearances and changes in the mediamade by colonies may provide valuable
identificationinformation.
„|„




|| 
This form of culture medium is prepared by addinga small amount of agar (0.4±0.5% w/v) to a
fluidmedium. Semi-solid media are used mainly astransport media, and for motility and
biochemicaltests.






|| 
Fluid media are most commonly used as enrichmentwhere organisms are likely to be few e.g. blood
culture. Some organisms produce a surface growthon the medium in which they are growing e.g.
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 when growing in alkaline peptonewater. Fluid media may also be used for
biochemicaltesting e.g. peptone water sugars or the use ofmedia containing tryptophan to detect
indole productionby some enterobacteria.
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Of the many media available, Müeller-Hinton agar is considered to be the best for routine
susceptibility testing of nonfastidious bacteria for the following reasons:
* It shows acceptable batch-to-batch reproducibility for susceptibility testing.
* It is low in sulphonamide, trimethoprim, and tetracycline inhibitors.
* It gives satisfactory growth of most nonfastidious pathogens.

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Müeller-Hinton agar preparation includes the following steps.
1.c Müeller-Hinton agar should be prepared from a commercially available dehydrated base
according to the manufacturer's instructions.
Example 36g dissolved in 1litre of water, the dissolved agar should be boiled to dissolved
completely
2. Immediately after autoclaving, allow it to cool in a 45 to 50uC water bath.
3. Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed petri dishes
on a level, horizontal surface to give a uniform depth of approximately 4 mm. This
corresponds to 60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml
for plates with a diameter of 100 mm.
4. The agar medium should be allowed to cool to room temperature and, unless the plate is
used the same day, stored in a refrigerator (2 to 8uC).

5. Plates should be used within seven days after preparation unless adequate precautions, such
as wrapping in plastic, have been taken to minimize drying of the agar.
6. A representative sample of each batch of plates should be examined for sterility by
incubating at 30 to 35uC for 24 hours or longer.
NUTRIENT AGAR
This is a general purpose media which support the growth of large variety of microbes particularly
chemoheterotrophs
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c
Nutrient agar preparation includes the following steps.
1.c nutrientagar should be prepared from a commercially available dehydrated base according to
the manufacturer's instructions.
2.c Example 28g dissolved in 1litre of water, the dissolved agar should be boiled to dissolved
completely

2. Immediately after autoclaving, allow it to cool in a 45 to 50uC water bath.

3. Pour the freshly prepared and cooled medium into glass or plastic, flat-bottomed petri dishes
on a level, horizontal surface to give a uniform depth of approximately 4 mm. This
corresponds to 60 to 70 ml of medium for plates with diameters of 150 mm and 25 to 30 ml
for plates with a diameter of 100 mm.
4. The agar medium should be allowed to cool to room temperature and, unless the plate is
used the same day, stored in a refrigerator (2 to 8uC).

5. Plates should be used within seven days after preparation unless adequate precautions, such
as wrapping in plastic, have been taken to minimize drying of the agar.
6. A representative sample of each batch of plates should be examined for sterility by
incubating at 30 to 35uC for 24 hours or longer.
  c%
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s/no Name of media Description Direction for use
1 Nutrient broth Liquid medium for the dissolve 13g in 1liter of
isolation of large variety distilled water and
of microorganism e.g. autoclave at 121 c for
bacteria 15 minutes
2 Saborauddextroux Liquid medium for the dissolve 30g in 1litre of
broth(SDB) isolation of fungi distilled water and
autoclave at 121 for 15
minutes
3 macConkey agar For isolationand 48.8g dissolve in 1litre of
differentiation of enteric distilled water and
bacteria,also support the autoclave at 121 for 15
growth of staphylococci minutes
and enterococci
4 Saborauddextroux agar Semi solid medium for Dispense 62g in1litre of
the isolation of fungi water,allow to soak for10
minute,boil to dissolve
completely and sterilized
by autoclave
5 mannitol salt agar for detection of 111g dissolve in 1litre of
pathogenic staphylococci distilled water,boil to
in food and other dissolve completely and
materials sterilize with autoclave
+c Salmonella, shigella Differential, selective 63g in1litre of distilled
agar medium recommended for water bring to boil with
the isolation of salmonella frequent agitation to
and shigella from dissolve agar
stool,food and clinical completely,do not
materials autoclave
,c EMB(Eosine methylene Differential medium for 57.5g in 1liter distilled
blue) the isolation and water boil to dissolve
enumeration of coliform completely and sterize
organism with autoclave
-c Tryptic soy agar Precultivation medium 40g in 1 litre of distilled
for enumeration of E.coli water
.c Others
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Information provided by microscopically techniques can often provide rapid presumptive
diagnosis of an infectione.g pulmonary tuberculosis using the Ziehl-Neelsen staining technique.
Other techniques can help to identify a pathogen, e.g. the Gram staining technique can indicate
whether an organism is Gram positive or Gram negative, a coccus or bacillus. Such information
is particularlyr

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1 Beta ±glucoromidase To identify |

2 Bile solubility To differentiate  A heavy inoculum of the test
from other alpha- haemolytic organism is emulsified in
streptococci physiological
saline and the bile salt
sodium deoxycholate is
added.
This dissolves S. pneumoniae
as shown by a clearing of the
turbidity
within 10±15 minutes.
Viridans and other
streptococci
are not dissolved and
therefore there is no clearing
of the turbidity.

3 Catalase test To differentiate staphylococci Catalase acts as a catalyst in

from streptococci the breakdown of hydrogen
peroxide to oxygen and
water. An organism is tested
for
catalase production by
bringing it into contact with
hydrogen
peroxide. Bubbles of oxygen
are released if the organism is
a
catalase producer. The
culture should not be more
than 24
hours old.
4 Citrate utilization To differentiate enterobacteria This test is one of several
techniques used occasionally
to assist in the identification
of enterobacteria.
The test is based on the
ability of an organism to use
citrate as its only source of
carbon

5 Coagulase test To identify S.aureus Coagulase causes plasma to

clot by converting fibrinogen
to
fibrin. Two types of
coagulase are produced by
most strains of

:
_ Free coagulase which
converts fibrinogen to fibrin
by activating
a coagulase-reacting factor
present in plasma. Free
coagulase is detected by
clotting in the tube test.
_ Bound coagulase (clumping
factor) which converts
fibrinogen directly to fibrin
without requiring a
coagulasereacting
factor. It can be detected by
the clumping of
bacterial cells in the rapid
 slide test.
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The principles of determining the effectivity of a noxious agent to a bacterium were well
enumerated by Rideal ,Walker and others at the turn of the century, the discovery of antibiotics
made these tests(or their modification)too cumbersome for the large numbers of tests necessary
to be put up as a routine. The ditch plate method of agar diffusion used by Alexander Fleming
was the forerunner of a variety of agar diffusion methods devised by workers in this field .The
Oxford group used these methods initially to assay the antibiotic contained in blood by allowing
the antibiotics to diffuse out of reservoirs in the medium in containers placed on the surface.

With the introduction of a variety of antimicrobials it became necessary to perform the

antimicrobial susceptibility test as a routine. For this, the antimicrobial contained in a reservoir
was allowed to diffuse out into the medium and interact in a plate freshly seeded with the test
organisms. Even now a variety of antimicrobial containing reservoirs are used but the
antimicrobial impregnated absorbent paper disc is by far the commonest type used. The disc
diffusion method of AST is the most practical method and is still the method of choice for the
average laboratory. Automation may force the method out of the diagnostic laboratory but in
this country as well as in the smaller laboratories of even advanced countries, it will certainly be
the most commonly carried out microbiological test for many years to come. It is, therefore,
imperative that microbiologists understand the principles of the test well and keep updating the
information as and when necessary. All techniques involve either diffusion of antimicrobial
agent in agar or dilution of antibiotic in agar or broth. Even automated techniques are variations
of the above methods

 !"c )c ! "1c
 *!1!2c!)cc

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The pH of each batch of Meller-Hinton agar should be checked when the medium is prepared.
The exact method used will depend largely on the type of equipment available in the laboratory. The
agar medium should have a pH between 7.2 and 7.4 at room temperature after gelling. If the pH is
too low, certain drugs will appear to lose potency (e.g., aminoglycosides, quinolones, and
macrolides), while other agents may appear to have excessive activity (e.g., tetracyclines). If the pH
is too high, the opposite effects can be expected. The pH can be checked by one of the following
means:
* Macerate a sufficient amount of agar to submerge the tip of a pH electrode.
* Allow a small amount of agar to solidify around the tip of a pH electrode in a beaker or cup.
* Use a properly calibrated surface electrode.

!"c
If, just before use, excess surface moisture is present, the plates should be placed in an incubator
(35uC) or a laminar flow hood at room temperature with lids ajar until excess surface moisture is
lost by evaporation (usually 10 to 30 minutes). The surface should be moist, but no droplets of
moisture should be apparent on the surface of the medium or on the petri dish covers when the
plates are inoculated.

 !cc/2c"c/2c

Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of
sulfonamides and trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all,
which may result in false-resistance reports. Meller-Hinton agar that is as low in thymidine
content as possible should be used. To evaluate a new lot of Müeller-Hinton agar°
|

   ATCC 29212, or alternatively, |
   ATCC 33186, should be tested with
trimethoprim/sulfamethoxazole disks. Satisfactory media will provide essentially clear, distinct
zones of inhibition 20 mm or greater in diameter. Unsatisfactory media will produce no zone of
inhibition, growth within the zone, or a zone of less than 20 mm.

 !cc"!cc4!c!c

Variation in divalent cations, principally magnesium and calcium, will affect results of
aminoglycoside and tetracycline tests with m
 strains. Excessive cation content will
reduce zone sizes, whereas low cation content may result in unacceptably large zones of inhibition.
Excess zinc ions may reduce zone sizes of carbapenems. Performance tests with each lot of
Meller-Hinton agar must conform to the control limits.
!)c!"c!/!cc!c)"5c! !"2c
Only aerobic or facultative bacteria that grow well on unsupplementedMüeller-Hinton agar should
be tested on that medium. Certain fastidious bacteria such as ï  spp °




 ,
 , and viridans and ß-haemolytic streptococci do not grow
sufficiently on unsupplementedMüeller-Hinton agar. These organisms require supplements or
different media to grow, and they should be tested on the media described in separate sections.
c





 
 




 


Antimicrobial susceptibility testing methods are divided into types based on the principle applied in
each system. They include:
cc c c c c c c c
Stokes method
Kirby-Bauer method
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Minimum Inhibitory Concentration
i)c Broth dilution
ii)c ii)Agar Dilution

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E-Test method
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