CULTURE MEDIA

GUIDED BY :
DR.P.KARUNAKAR PRESENTED BY :
DR.UMRANA FAIZUDDIN DR.M.RASAGNA
DR.ASHISH JAIN
CONTENTS
• Introduction
• Definition
• Types of media
Simple media
Complex media
Defined media
• Special media
Enriched media
Enrichment media
Selective media
Indicator media
Transport media
Sugar media
Anaerobic media
• Culture methods
• Endodontic microbiological assessment 
Introduction
Culturing of microorganisms from root canal
Microbial root canal sampling
Advantages
Disadvantages
Conclusion
references
• INTRODUCTION
DEFINITION 
• A growth medium or culture medium is a liquid or gel designed to
support the growth of microorganisms or cells or small plants like the
moss physcomitrella patens (Madigan M,Martinko J(2005).brock
biology of microorganisms (11th edition) prentice hall ).
 Microbial pathogens 

Growth ingredients 

Living systems    Artificial systems 

Animal host or cell in By gathering required

culture  conditions for growth
BASIC CONSTITUENTS 

• Water : source of hydrogen or oxygen .

• Electrolytes :      sodium   chloride   or other

electrolytes .
Peptone :
Partially digested
Complex mixture proteins 

obtained
 
• Meat extract :contains   protein  degradation products ,inorganic salts
,carbohydrates and  growth factors 

• Blood or serum:these are used for enriching culture

media 
 Agar :
Obtained
 

Long chain of polysaccharides 

Varying amounts of inorganic salts ,and,small amount of protein like
substances 
2-3% of agar acts as solidyifying agent 
It has no nutritive value 
CLASSIFICATION :
• ACCORDING TO  FORM OF CONSISTENCY OF MEDIA :
Solid media
Semi solid media
Liquid media
• ACCORDING TO THE CONSTITUENTS OF MEDIA :
Simple media
Complex media
Synthetic media
• SPECIAL MEDIA:
Enriched media
Enrichment media 
Selective media
Indicator or differential media
Sugar media
Transport media
• ACCORDING TO THE TYPE OF BACTERIAL GROWTH THEY SUPPORT :
Aerobic media 
Anaerobic media
• ACCORDING TO FUNCTIONS AND USE :
Basal media
Complex media
LIQUID MEDIA :
• Diffused growth
• No characteristics for identification
• It contains no agar
• Bacteria grow uniformly producing general turbidity 
• Example :  nutrient broth 
• Advantages: 
 Used for preparing bulk cultures of antigens or vaccines
• Disadvantages :
 Difficult to isolate different types of bacteria from mixed populations 
 SOLID MEDIA :
• It contains 2% agar
• Colony morphology ,pigmentation ,hemolysis can be appreciated.
• Example : nutrient agar,nutrient broth 
• SEMI SOLID MEDIA 
• It  contains 0.5% agar
• Such media are soft and are useful in demonstrating  bacterial motility .
• Example : motility media 
ACCORDING TO CONSTITUENTS OF MEDIA :

SIMPLE MEDIA :
• Simple media is the most common medium in routine diagnostic
laboratories.
• Example :
• Nutrient broth:peptone water ,meat extract ,sodium chloride ,water 
• Nutrient agar : nutrient broth +2-3% agar 
COMPLEX MEDIA :
• Media other than basal media 
• Added ingredients or special nutrients they bring out certain properties
required for the  growth of the bacteria under study.
SYNTHETIC OR DEFINED MEDIA :
• Media prepared from pure chemical substances and its exact composition
is known.
• Used for various special studies such as metabolic requirements .
• Example :
• Dubos medium
SEMISYNTHETIC OR SEMIDEFINED MEDIUM :
• Composition is approximately known
• Example :
• Simple peptone water medium
SELECTIVE MEDIUM :
• ENRICHED MEDIUM :
• Prepared to meet the nutritional requirements of more exacting bacteria
by the addition of substances such as serum ,blood  or egg to a basal
medium .
• Used for the cultivation of fastidious organisms .
• Example :
• Blood agar
• Chocolate agar
• Loefllers serum slope
ENRICHMENT MEDIA :

• In a mixed cultures or materials containing  more than one bacterium,bacteria

to be isolated is often overgrown by unwanted bacterium.
• In such situations,certain stimulating or inhibitory substances are incorporated
in the medium which facilitates the growth of wanted bacterium and inhibits
the growth of unwanted bacterium.
• Examples :
• Selenite f broth 
• Tetrathionate broth
• Alkaline peptone water 
SELECTIVE MEDIA :

• It is a solid medium
• Here the inhibitory substances are added to inhibit the growth of
unwanted bacteria
• Examples :
• Deoxycholate citrate agar (DCA),xylose lysin deoxycholate agar for
samonella and shigella 
• Thiosulfate citrate bile salt sucrose agar (tcbs) for vibrio cholerea 
• Lowenstein jensen medium 
• Thayer martin medium
INDICATOR MEDIA 
• Certain indicator substances are added into the medium which
changes color when bacteria grows on the medium
• Example :
• Wilson and blair medium for salmonella typhi 
• Mc leods medium(potassium tellurite agar) for corynebacterium
diphtheriae 
SUGAR MEDIA
• It is used to determine the ability of an organism to ferment a specific
carbohydrate  present in the medium.
• It contains 1% sugar in peptone water along with  durham's tube is
kept inverted to detect gas production and with andrade's indicator 
• Acid production is detected by color change 
TRANSPORT MEDIA :
• Media used for transporting clinical specimens containing delicate
organisms  
• Bacteria remain viable without multiplication
• Examples :
• Stuarts medium-gonococci
• Pikes medium-streptococci
• Cary –blair medium-vibrio cholerae
• Buffered glycerol saline –shigella
 ANAEROBIC  MEDIUM :
• Media used for the cultivation of anaerobes
• Examples :
• Robertson's cooked  meat medium
• Thyoglycolate broth
STERILISATION OF CULTURE MEDIA :

• Autoclaving at 121 degrees for 15 minutes

• Media which cannot be autoclaved –selenite f broth and tetrathionate
broth are sterilised by  steaming at 100 degrees
• LJ medium for mycobacterium tuberculosis and loefflers serum slope for
corynebacterium diphtheriae are sterilised by inspissation  using
inspissator at 80-85 degrees
INDICATIONS FOR CULTURE :
• Isolate bacteria in pure cultures from clinical specimens and
their  identification by various methods 
• Determination of antibiotic sensitivity
• Prepare antigens for serodiagnosis of infective diseases
• Maintain stock cultures
CULTURE METHODS :
• AEROBIC CULTURE METHODS :
Streak culture
Lawn culture
Stroke culture
Stab culture
Pour- plate culture
Liquid culture
• ANAEROBIC CULTURE METHOD
STREAK CULTURE (SURFACE PLATING ) :
• It is routinely employed for isolation of bacteria in pure cultures from
clinical specimens 
LAWN CULTURE :

• Uniform surface growth of bacterium

• Mainly used for 
• Antibiotic sensitivity  testing(disc method )
• Preparation of bacterial antigens and vaccines
• For bacteriophage typing
STROKE CULTURE :
• Done in tubes containing agar slopes 
• Used for :
• Pure growth of bacterium
• For slide agglutination and other biochemical tests 
 STAB CULTURE :
• It is performed by a straight wire,charged with culture material ,by
puncturing deep inside the agar
• It is used to :
• Demonstrate gelatin liquefaction
• Oxygen requirement of the bacteria
• To maintain stock culture for preservation of bacterium
POUR PLATE TECHINQUE :

• Tubes –15ml of agar medium –melted-cooled in water bath 45-50 degrees.

• 1ml of inoculum+molted agar –poured into petri dishes –allowed to set .
• Estimate the viable bacterial count in suspension
LIQUID CULTURE
• Test tubes are inoculated by touching with a charged loop or by adding the
inoculum with pipettes or syringes.
• Uses are :
• Blood culture and sterility tests.
• Preffered for specimens containing antibiotics.
• When large yields of bacteria are needed
• Disadvantage :
• Does not provide pure culture from mixed inocula
ANAEROBIC CULTURE METHODS :
• Production of a vaccum
• Displacement of oxygen with other gases
• Absorption of oxygen by chemical or biological methods
• By using reducing agents
DESSICATOR :
• In dessicator some oxygen is left .
• Not suitable for fluid culture
DISPLACEMENT OF OXYGEN WITH OTHER GASES :

CANDLE JAR : 
• Inoculated plates are kept
• Burning candle use up  all the oxygen
• But a little oxygen is left
• But the presence of co2 stimulates the growth of bacterium
DISPLACEMENT OF OXYGEN BY CHEMICAL OR
BIOLOGICAL METHOD : 
• Buchner first introduced alkaline  pyragallol for anaerobiosis which absorbs
oxygen.
• Pyragollic acid added to a solution of sodium hydroxide in a large test tube
placed inside an air tight jar provides anaerobiosis.
• MIXTURE OF POWERDED CHROMIUM AND SULFURIC ACID : 
• Rosenthal method –mixture of chromium and sulfuric acid 
• Or white yellow phoshorus 
• To produce anaerobiosis.
GASPAK :
• Method of choice for preparing anaerobic  jars.
• Commerically available as disposable envelope ,containing chemicals which
generate hydrogen and co2 when water is added .
• Reduced methylene blue is used as a indicator ,remains colorless when
anaerobically but turns blue on exposure to oxygen.
• DISADVANTAGE :
• Major disadvantage is that the plates have to be removed  from the jar  to
be examined  and this of course ,exposes the colonies to oxygen.
• In order to avoid that ,a suitable holding system should be used.
BIOLOGICAL METHOD :
• Absorption of oxygen by incubation with aerobic bacteria,germinating
seeds or chopped vegetables.
• BY DISPLACEMENT AND COMBUSTION OF OXYGEN :
• Mcintosh and  fildes anaerobic jar 
• Most reliable and widely used anaerobic method.
• Complete anaerobiosis
• Consists of a metal jar or  glass jar  with a metal lid which can be clamped
air tight.
• When electrified palladinised asbestos heating acts as a catalyst for
combination of hydrogen with residual oxygen which produces complete
anaerobiasis.
ENDODONTIC  MICROBIOLOGICAL ASSESSMENT 
• HISTORY :
INTRODUCTION
  W.D.miller  
 Authored a book called ''microogranisms of human mouth''.
 First researcher to identify bacteria in the diseased pulp.
 onderdonk recommended the use of bacteriological test to determine the
presence of infection in a root canal.
 Kakehashi et al proved bacteria was responsible for pulpal and periapical
infection.
 Moller and his associates illustrated the importance of  bacteria in the 
developmental of pulpal and periradicular diseases.
MICROFLORA OF PULPAL SPACE :
GRAM POSITIVE BACTERIA :
• Streptococci :
Alpha hemolytic streptococci – streptococcus viridans(5% of microflora)
9% -non hemolytic streptococci
8%-streptococcus salivarius
• Staphylococcus
GRAM NEGATIVE BACTERIA :
• Lactobacillus
• Enterococccus faecalis
• porphyromonas
• pnemococcus
•     Nesseria
•    bacillus subtlis
•    Sarcinae
MISCENALLOUS MICROORGANISMS :
• Candida 
• Actinomyces
• nocardia
 REASONS FOR CULTURING OF ROOT CANAL 

To isolate microbial
To asssess the bacteriological flora for antibiotic
status of the root canal sensitivity and
system before obturation and resistance profile in
determine the efficacy of cases of persistent
debridement procedure infections

Sjogren et al. Reported that complete periapical healing occurred in 94% of

the cases that yielded a negative culture.whereas the samples were positive
prior to root sampling,the success rate of treatment was just 68%
SPECIMEN COLLECTION :
• Specimen collection in involved root canal or periradicular region should be
under strict aseptic conditions with out any error .
• FUNDAMENTAL CONSIDERATION IN SPECIMEN COLLECTION 
1. Specimen  collection should be from the actual infection site with the
minimal contamination.
2. Optimal timing :culturing should be done before and after cleaning and
shaping  of the root canal system.
3. Sufficient quantity :adequate quantity of specimen is required to grow the
microbes .
4. Appropriate collection devices ,specimen containers and culture media.
MICROBIAL ROOT CANAL SAMPLING :
• Based on moller's work, to perform microbiological  root canal sampling
several steps were followed :
• ASPETIC SAMPLE FROM ROOT CANAL :
Remove all the hard &soft  tissue deposits from the root
               surface 

Isolate the tooth with rubber dam

 Surface of the tooth and surrounding field must be
disinfected with 30% hydrogen peroxide and 10% iodine
tincture 

Solution was inactivated with 5% sodium thiosulfate in

order to avoid any interference with the bacteriological
sampling

Acess to the canal is made with sterile burs and manual

irrigation with sterile solution was used
 Canal was rinsed with sterile saline to remove the
remanants of filling material and debris and to
moisten the canal before sampling 

Working length  of canal was determined 

radiographically  

Sterile paper point was then placed in the full length of

the canal and retained in that position for 60 seconds for
microbial sampling 

Paper point was immediately placed in a transport

medium containing  3ml of sterile reduced transport
fluid
Then submitted to the microbiology laboratory
within 15 minutes

The maxium time between sample collection

and laboratory processing was 4 hrs 

Dispered by sonication or by vortex mixer 

Dispersed on to various types of agar media
and cultured under aerobic or anaerobic
enviroment 

After a suitable period of

incubation,individual colonies are
subcultured and identified on the basis of
multiple aspects including colony,cellular
morphology ,gram staining pattern,o2
tolerance, metabolic end product
analysis,biochemical reactions
 SAMPLING THROUGH ABSCESS
Flucantant abscess is palpated,most dependent
part of swelling is determined

Surface of mucosa –iodophor or alcohol

Penetrated with 16-20 gauge needle ,aspiration of

exudates

Aspirate is then injected into anaerobic transport

medium
SAMPLING THROUGH SINUS
Orifice of sinus ,surrounding mucous
membrane dried with cotton rolls
cleansed with 5-10% h202

Cleansed area inactivated by 5%

thiosulphate for 8-10 sec

Paper points introduced 10 sec transferred 

to culture medium incubated for 10-14
days 
CULTURE REVERSAL :
• Grossman found that 2%  of the cultures were negative after 48 hours of
incubation ,but they turned positive when incubated for 10 days.
• It is advisable to allow  more than 48 hours between taking culture and
filling the root canal,preferable for 96 hours or more hours.
 Lower the
accuracy of
the MRS
The higher (microbial
the number root canal
of culture sampling )
reversals 
WHY PERFORMANCE OF MRS NOT PARTICULARLY
HIGH ?
• False negative :
It is posssible that bacteria may be in the root canals from the  very
beginning , these bacteria repopulate after the first MRS shows negative
result .
A carryover effect of antibacterial irrigants or medicaments can inhibit the
bacterial growth during cultivation process.

• FALSE POSITIVE :
Failure in sterilization of operating field and instruments.
Rubber dam leaks.
Use of unsterile paper points and cotton tube plugs.
Air or hand contamination during collection or transport.
BACTERIAL STATUS MAY CHANGE BETWEEN
APPOINTMENTS FOR INCIDENTAL REASONS UNRELATED
TO THE STUDY ITSELF 
• INCIDENTAL FALSE NEGATIVE :
• Bacteria are low in number in number at the intitial sampling,at the second
visit they might be retreived simply by chance.
• MRS  measured bacterial status ,but results are incorrect .

• INCIDENTAL FALSE  POSITIVE :

• Bacteria can re-enter the root canal system between visits.
• Anarochesis.
METHODS OF ISOLATING PURE CULTURE :
• Surface plating.
• Pre treatment of specimens with appropriate bactericidal substances
which destroy the unwanted bacteria.
• Obligate aerobes and anaerobes may be separated by cultivation under
aerobic or  anaerobic  conditions.
• Separation of bacteria with different temperature optima can be effected
by incubation at different temperatures.
• Bacteria of differing sizes may be separated by the use of selective filters.
REASONS FOR BACTERIAL UNCULTURABILITY :

•  Lack of essential nutrients or growth factors in the artificial medium.

• Over feeding conditions.
• Toxicity of the culture medium itself, which can inhibit bacterial growth.
• Production of substances inhibitory to the target microorganisms by other
species present in a mixed consortium.
• Metabolic dependence on other species for growth.
• Bacterial dormancy :bacteria unable to divide or form colonies on agar plates.

  
CULTURE MEDIA FOR FUNGI

POTATO DEXTROSE AGAR

SABOURAUD'S DEXTROSE AGAR CORN MEAL AGAR
CELL CULTURE FOR VIRUSES :
• Three types of cell culture :
• Primary cell culture
• Secondary or diploid cell strains
• Continuous or established cell line
CULTURAL CHARACTERISTICS OF CERTAIN BACTERIA :

ENTEROCOCCUS FAECALIS :
CULTURAL  CHARACTERISTICS OF CERTAIN MICROFLORA

 STREPTOCOCCI :
• It has been isolated in large  proportions of  cases with primary endodontic
infections.
                            
Oral streptococci mutans

salivarius

oralis

angiosus
 •   CANDIDA ALBICANS         
• Candida albicans is the predominant yeast
identified directly from the infected root canals.
• EDTA is one of the regularly used chelating agents
and is found to have significant antifungal activity.

• ACTINOMYCES :
• In the root canals of retreated tooth ,A.israelii  is
most commonly found .
• A.meyeri  has been isolated from teeth with
persistent periapical infections.
ADVANTAGES OF CULTURE MEDIA :
• Broad –range nature ,identification of unexpected species.
• Allow quantification of all major viable cultivable microorganisms in
samples.
• Allow determination of antimicrobial susceptibilities of isolates.
• Physiologic  studies are possible.
• Pathogenicity studies are possible.
• Widely available.
LIMITATIONS OF CULTURE MEDIA :
• Impossibility of culturing a large number of extant bacterial species.
• Not all viable bacteria can be recovered.
• Once isolated,bacteria require identification using a number of techinques.
• Misidentification of strains with ambiguous or  aberrant phenotypic
behavior .
• Low sensitivity.
• Strict dependence on the mode of sample transport.
• Samples require immediate processing.
• Costly,time consuming and laborious ,as for cultivation of anaerobes
• Specificity is dependent on the experience of microbiologist.
• Extensive expertise and specialized equipment needed to isolate
anaerobes.
• Takes several days to weeks to identify most anaerobes.
CONCLUSION :
• Rationale of endodontic treatment lies in removal of nidus of infection as
described by FISH 1939.
• And hence,it necessitates to study the microbiology of root canal in detail
so as to successfully deal with persistent infection for better healing.
• Currently practiced,intracanal sampling techinques suffer from deficiencies
that limit their predictive value,that does not indicate end to the culture
media.
• It emphasizes the need for more detailed clinical studies,as well as
refinement of techniques for microbial sampling of canals.
• Bacterial culturing is not an end in itself ,but its main purpose clinically is
to serve as a predictor of healing.
REFERENCES :
• Textbook of microbiology-anathnarayana &paniker (9 th edition)
• Endodontics –ingle and bakland(6 th edition)
•  Pathways of pulp -Stephen cohen(11 th edition)
• Textbook of endodontics –nisha garg (3 rd edition)
• Endodontic practice –grossman(12 th edition)
• How Useful Is Root Canal Culturing in Predicting Treatment Outcome?
 (Chankhrit Sathorn, DClinDent, Peter Parashos, PhD, and Harold H. Messer,PhD)
• INVESTIGATION OF MICROORGANISMS IN INFECTED DENTAL ROOT CANALS (E.
Ercan1, M. Dalli2, İ. Yavuz3, T. Özekinci4)
• ROOT CANAL MICROFLORA(Marina George, Romana Ivančaková)