Culture media in periodontics

1. Gomathi.GD
PG I year
Culture media and culture methods

2. Introduction
 Culture media are required to grow the organisms
from infected material to identify the causative
agent.

3. INTRODUCTION
 Culture is the term given to microorganisms that
are cultivated in the lab for the purpose of
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 their growth.

4. History..
 History of culture media dates back to 19th
century.
 Louis Pasteur used urine medium and meat
medium for the isolation of bacteria.
 Robert Koch improvised the media by
adding cooked potato.
 Then they started to try gelatin as it is non
nutritive inert substance. However it was not
satisfactory as it liquefies at 24O C.

5. Constituents of culture media
 Water – source of hydrogen and oxygen.
 Electrolyte- sodium chloride or other electrolytes
 Peptone-complex mixture of partially digested
proteins
 Meat-contains protein degradation products,
inorganic salts,carbohydrates & growth factors
 Blood/serum-used for enriching culture media
 Agar-Obtained from seaweeds, melts at 98oC and
sets at 42oC . No nutritive value, but acts as
solidifying agent only.

6. Culture media
Based on
consistency
Solid media
Liquid media
semisolid media
Based on
constituents
Simple media
Complex media
Synthetic media
Special media
Based on
oxygen
requirement
Aerobic
media
Anaerobic
media
Classification:

7. Special media
 Enriched media
 Enrichment media
 Selective media
 Indicator media/Differential media
 Transport media
 Sugar media

8. Based on consistency
Solid media – contains 1.5% to 2% agar
• Colony morphology, pigmentation, haemolysis
can be appreciated.isolated pure bacteria can
be obtained
• Eg: Nutrient agar, Blood agar
Liquid media – no agar.
• For inoculum preparation.
• Eg: Nutrient broth
Semi solid medium – 0.5% agar.
• Eg: Motility medium

9. Based on ingredients
Simple medium
 Contains basic nutrients for the growth of
bacteria.
 E.g. Nutrient broth and Nutrient Agar
 Nutrient broth: peptone water, meat extract and
when glucose is added it becomes glucose broth.
 Nutrient Agar: Addition of 2-3%
agar to nutrient broth.
Used for the growth of non fastidious
bacteria

10. Complex medium
• Complex media are media that contain some
ingredients of unknown composition and/or
concentration.
• This type supplies amino acids, vitamins, growth
factors, and other nutrients
 E.g. Nutrient broth which contains meat extract.

11. Synthetic or defined medium
 Media prepared from pure chemical substance
and its exact composition and concentration is
known.
 Eg. Peptone water

12. Enriched medium
 Substances like blood, serum, egg are added to
the basal medium.
 Used to grow bacteria that are exacting in their
nutritional needs ie., fastidious bacteria
 Eg: Blood agar, Chocolate agar

13. Blood agar
 It is an enriched and differential medium
 The basal medium is mixed with 5% to 10%
sheep blood.
 Used to isolate streptococcus species which are
fastidious.
 Can also study haemolytic properties.

14. Types of haemolysis in blood agar
α hemolysis: zone of partial destruction of red blood cells
(RBCs) appears around the colony, by greenish to brownish
discoloration of the medium. Streptococcus
pneumoniae and many oral streptococci are α hemolytic.
β hemolysis: A clear, colorless zone appears around the
colonies, in which the RBCs have undergone complete
lysis. Streptococcus pyogenes, S. agalactiae, and several
other species of streptococci are β hemolytic.
(γ) hemolysis: No apparent hemolytic activity or
discoloration is produced .streptococcus fecalis.

15. Chocolate agar
 It is a variant of the blood agar plate.
 It contains red blood cells, which have been
lysed by heating very slowly to 56 °C.
 Chocolate agar is used for growing fastidious
bacteria, such as Haemophilus
influenzae,Neisseria
 These bacteria need growth factors, like NAD
and haematin, which are inside red blood
cells; thus lysis of the red blood cells is done.

16. Loeffler’s serum slope
 Serum is added for enriching the medium.This is
used for isolation of corneybacterium diphtheriae.

17. Enrichment medium
 Liquid media used to isolate pathogens from a
mixed culture.
 Media is incorporated with inhibitory substances
to suppress the unwanted organism.
 Eg:
 Selenite F Broth – for the isolation of
Salmonella
 Alkaline Peptone Water – for Vibrio
cholerae

18. Selective medium
 The inhibitory substance is added to solid media.
Thus inhibits certain bacteria and allows the growth
of wanted bacteria.
Eg:
 Mac Conkey’s medium for gram negative bacteria
 TCBS (Thiosulphate Citrate Bile Salts Sucrose
Agar)– for V.cholerae

19. Differential medium/Indicator medium
 A media which has substances incorporated in it
enabling it to distinguish between bacteria.
 These media contain an indicator which changes its
colour when a bacterium grows in them.
 Eg: Mac Conkey’s medium
 Distinguish between lactose fermenters & non
lactose fermenters.

20. MacConkey agar
 MacConkey agar is a culture medium designed
to grow Gram-negative bacteria and gives pink
colour colonies for lactose fermenting bacteria.
 It contains bile salts (to inhibit most Gram-
positive bacteria) crystal violet dye (which also
inhibits certain Gram-positive bacteria), neutral
red dye (which give pink colour on fermenting
lactose), lactose and peptone.

21. Depending on oxygen requirement
Anaerobic medium:
 These are used for cultivation of anaerobic
bacteria
 Example :Robertson's cooked meat
broth,thioglycollate broth.
 Robertson replaced brain tissue with beef heart
tissue for cultivating and classifying anaerobic
bacilli.
 Dextrose, yeast extract, haemin and vitamin K are
added to enhance the growth of anaerobic
microorganisms.

22. Robertson cooked meat medium
(RCM)
 Amino acids and other nutrients are supplied
by the muscle protein in the heart tissue
granules.
 Growth of spore-forming and non-spore-
forming obligate anaerobes is supported by
this medium.
 Cooked Meat Medium is also useful as an
enrichment broth for cultivating organisms
from a very small inoculum.
 Clostridium tetani is normally grown in this
medium.

23. Transport medium
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• These are used for the temporary storage of specimens
being transported to the laboratory for cultivation.
• Allow organisms to survive
• Non-nutritive - does not allow organisms to proliferate
E.g
• Stuart transport medium-a non-nutrient soft agar gel
• containing a reducing agent to prevent oxidation, and
charcoal to neutralise certain bacterial inhibitors-for
gonococci.
 Buffered glycerol saline for enteric bacilli.

24. Sugar media
 Media containing any fermentable sugar.
 Media consists of nutrient broth,peptone
water,bromocresol
purple,sugar(glucose,sucrose,lactose,starch,malt
ose)
 To determine which sugar is utilized by bacteria.
 Type of sugar contained in test tube is known by
the color of cotton plug of test tube, which are
 .1. glucose – green
 2. sucrose- white
 3. Lactose- red
 4. Maltose- Blank

25. Sterilization of culture media
 Sterilization is a process by which surface of the
article is made free from all microorganisms
including vegetative and spore forms.
Sterilization of culture media is best carried
out in a steam autoclave at temperature
between 121°C for 15 minutes at 15lbs
pressure to make sure all pathogens are killed.

26. Identification OF
BACTERIA
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27. Identification of bacterial species
27
o Staining
o Growth in solid media: Morphology of bacterial
colony.
o Growth in liquid media
o Hanging drop preparation - motility
o Biochemical tests

28. GRAM STAINING:
 Place slide with heat fixed smear on staining tray.
 Gently flood smear with crystal violet and let
stand for 1 minute.
 Tilt the slide slightly and gently rinse with tap
water or distilled water using a wash bottle.
 Gently flood the smear with Gram's iodine and let
stand for 1 minute.
 G+ve organisms are
Streptococci,Staphylococci,corneybacterium,clost
ridium,$
bacillus
 G –ve organisms are
Salmonella, Shigella, Enterobacteriaceae, Pseud
omonas,Moraxella, Helicobacter, Stenotrophomo

30. Ziehl-neelsen (ZN) staining
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 Differentiates AFB from non-AFB
 Eg. Mycobacterium tuberculosis and M. leprae.
Both are acid fast in nature.
Procedure
1.Place the slide on the slide rack over the metal
staining tray  concentrated carbolfuchsin and
boil to steam for 5 min  rinse very well*
2.Cover with the acid-alcohol solution  2 minutes
 rinse
3.Flood with methylene blue  let stand 1 minute
 rinse
4.Blot dry  observe

31. Acid Fast Stain
 Carbolfushin at high concentration+boiling 
penetrate the lipid cell wall  reach the
cytoplasm
 Cytoplasm once stained resist decolorization 
acid-alcohol can not dissolve the cell wall and
penetrate beneath it
  hence the name acid fast
 Bacteria that do not have mycolic acid are readily
decolorized by the acid-alcohol

32. Biochemical tests
Catalase test
• Catalase is an enzyme that converts hydrogen
peroxide into water and oxygen.
• The presence of catalase can be easily detected
by the slide method.
• A drop of 3% hydrogen peroxide is put on a slide
and the bacteria is emulsified in the drop.
• The presence of bubbles is evidence of the
production of oxygen.

33. Uses of catalase test
 To differentiate streptococcus(catalase
negative) from staphylococcus(catalase
positive).
 To differntiate aerobic and anaerobic bacteria
 Used for idetification of M.tuberculosis
 Used to differntiate aerotolerant strains of
clostridium( C –ve)from bacillus species(C +ve)
 Can be an aid in identification of
enterobacteriaceae

34. Hanging drop preparation-
motility test
 In this method a drop of culture is placed on a
coverslip that is encircled with petroleum jelly. The
coverslip and drop are then inverted over the well of a
depression slide. The drop hangs from the coverslip,
and the petroleum jelly forms a seal that prevents
evaporation. This preparation gives good views of
microbial motility.
 Uses: Presumptive diagnosis of Vibrio Cholerae

35. Oxidase test
The identification of some bacteria is aided by
detecting their ability to produce the enzyme
cytochrome c oxidase
This enzyme, in the presence of atmospheric oxygen,
oxidizes the colourless substrate tetramethyl-p-
phenylenediamine dihydrochloride to form a dark-
purple compound(iodophenols).
Oxidiase positive organisms are Neisseria , vibrio
cholerae, pseudomonas, camphylobacter,
helicobacter/haemophilus.
Oxidiase negative organisms are all species of
enterobacteriaceae.

36. IMViC Test
 Indole, Methyl Red, Voges-Proskauer, Citrate
(IMViC) Tests:
 The following four tests comprise a series of important
determinations that are collectively called the IMViC
series of reactions
 The IMViC series of reactions allows for the
differentiation of the various members of
Enterobacteriaceae.
Principle
Certain microorganisms can metabolize
tryptophan that leads to the formation of
indole,presence of which is detected by addition
of Kovac's reagent

37. IMViC: Indole test
Method:
 Inoculate tryptone water with the test microorganism
 Incubate at 37°C for 24 hours
 After incubation, add 1 ml Kovac’s reagent, shake the
tube gently and read immediately .
Methyl Red test Voges-Proskauer test
Red: +ve MR (E. coli) Pink: +ve VP
(Klebsiella

38. Sugar fermentation test
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 An anaerobic process during which
carbohydrates are broken down for energy
production.
 The types of carbohydrates which are fermented
by a specific bacteria can serve as a diagnostic
tool for the identification of that bacteria( Glucose-
enterobacteriaceae,lactose- E.coli,mannitol-
salmonella)
 The test organism is inoculated into a broth
containing the test sugar and
incubated.Production of gas bubble is detected
with a Durham tube.
 Positive – Yellow (acidic)

39. Citrate utilization test
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Simmon’s Citrate Agar contains sodium citrate
(carbon source), ammonium ion (nitrogen source),
& pH indicator -bromothymol blue. Medium- green
colour.
Reading Results:
+ve result is blue and –ve result remains green
 Klebsiella pneumoniae,Enterobacter species ,
Citrobacter freundii,Salmonella other than Typhi and
Paratyphi A,Serratia marcescens Proteus mirabilis are
the positive organisms
 Escherichia coli,Shigella spp,Salmonella Typhi and

40. Urease Production
• Inoculate Christensen’s urease medium with
inoculating loop.
• This test is done to determine a bacteria’s ability to
hydrolyze
urea to ammonia using the enzyme urease.
Urease test positive bacterias are Proteus,nocardia,
cryptococcus neoformans, helicobacter pylori.
E.coli is negative organism.

41. Phenylalanine deaminase Test
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Phenylalanine deaminase medium tests the ability of
an organism to produce phenyl pyruvic acid with the
enzyme deaminase.
After incubation of the culture in phenylalanine
medium, 10% ferric chloride is added to the media; if
phenylpyruvic acid is produced, it will react with the
ferric chloride and turn dark green.
If the medium remains a straw colour, the organism is
negative for phenylalanine deaminase production.
Proteus sp., Morganella sp., Providenica are +ve
organisms
Enterobacteriaceae species are negative

42. Triple Sugar Iron (TSI) Agar
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 TSI contains Glucose Lactose ,Sucrose,Phenol
red,ferrous sulphate.
 Principle
To determine the ability of an organism to attack a
specific carbohydrate incorporated into a basal
growth medium,
with or without the production of gas, along with the
determination of possible hydrogen sulphide production.
♦ Phenol red indicates pH change
♦ Ferric compound indicates H2S gas production

43. triple Sugar Iron (TSI) Agar
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♦ .
♦ Reactions are:
♦ No growth- red color
♦ Growth with no acid
♦ Acid production in the butt only- only glucose fermented-
yellow/red
♦ Acid production in both butt and slant- ferments all sugar
yellow
♦ Acid production in the bottom and H2S gas formation
(black)
♦ K-alkaline A- acidic

44. Name of
organism
Slant Butt Gas H2S
Escherich
ia,
Klebsiella,
Enterobac
teria
Acid (A) Acid (A) Pos (+) Neg (-)
Shigella,
Serratia
Alkaline
(K)
Acid (A) Neg (-) Neg (- )
Salmonell
a, Proteus
Alkaline
(K)
Acid (A) Pos (+) Pos (+)
Pseudom
onas
Alkaline
(K)
Alkaline
(K)
Neg (-) Neg (-)

45. Culture methods
 Culture methods employed depends on the
purpose for which they are intended.
 The indications for culture are:
 To isolate bacteria in pure culture.
To demonstrate their properties.
To determine antibiotic sensitivity
To estimate viable counts
Maintain stock cultures.
To obtain sufficient growth for the preparation of
antigens and for other tests

46. Types
 Streak culture
 Lawn culture
 Stroke culture
 Stab culture
 Pour plate method

47. Streak culture
 Used for isolation of bacteria in pure culture from
clinical specimens
 Platinum wire or nichrome wire is used
 One loopful of the specimen is transferred onto
the surface of a well dried plate.
 Spread over a small are at the periphery
 The innoculum is distributed thinly over plate by
streaking it with a loop in series of parallel lines in
different segments of the plate.

48. Lawn culture:
 Provides a uniform surface
growth of the bacterium
 Uses –
 Anti biotic sensitivity testing
 In the preparation of bacterial antigens and vaccines
 Lawn cultures are prepared by flooding the
surface of the plate with a liquid suspension of
the bacterium

49. STROKE CULTURE
 Stroke culture is made in tubes containing agar
slope/slant
Uses
Provides a pure growth of bacteria for slide
agglutination

50. STAB CULTURE
 Prepared by puncturing a suitable medium
Uses:
• demonstration of gelatin liquifaction
• Oxygen reqirement of the bacteria under study

51. POUR PLATE CULTURE
Agar is melted and cooled at 45 c
One ml of the inoculum is added to the molten agar
Allow it to set and incubate at 37C
Uses“:
Gives estimate of viable bacterial count
Quantitive urine culture

52. Culture of yeast
 Culturing yeast is simple and rapid with a doubling
time in rich medium of approximately 90 minutes.
 Cells divide mitotically by forming a bud,which is
subsequently pinched off to form a daughter cell.
 Yeast can be grown in either liquid medium or on
the surface of a solid agar plate
 Yeast cells grow more rapidly in the
presence of rich medium that contains
reagents such as yeast extract.

53.  Yeast cells will grow on a minimal medium
containing dextrose as a carbon source and salts
that supply nitrogen, phosphorus and trace
metals.
 Media that are commonly used for fungal culture
are Sabouraud dextrose, malt extract and brain
heart infusion medium.
 To prevent contamination of the medium by
bacteria, chloramphenicol is used,but prevents
the growth
of Actinomyces, which other wise grows well on
Sabouraud dextrose agar.

54. Identification of viruses

55.  Virus must be grown in living cells as they cant be
grown in culture media .
 They must have living cells to support their
replication.
 Live animals have been used for some virus.
 Embryonated eggs can serve as a substitute for
some virus.

56. PLAQUE METHOD
• Virus, bacteria and agar mixed, plated and
incubated.
• After replication the virus lyses the bacteria
forming plaques or clear zones.
• Each plaque is assumed to come from a singe
viral particle
TISSUE CULTURE
Organ culture
Cell culture

57.  Viruses can be identified by the following
methods:
 Serological methods
 Western blot
 Cytopathic effects
 Diagnostic inclusion bodies are associated with
rabies virus, measles virus , vaccinia virus,
smallpox virus , herpes virus , adenoviruses.
 Molecular methods includes PCR and RFLP’s

58. Periodontal Pathogens

59. Bacteria Typ
e
Cultu
re
media
used
Appearence in the
culture plate
Description
S.mitis G
+vC
occi
e
Blood
agar
Clear halo surrounding
the colony
V.Parvula G-
veC
occi
Blood
Agar
transprant colony
through haemolytic
activity
A.Viscous G
+ve
Rod
Blood
agar
Tiny white spherical
colonies
L. bacillus G –
veR
od
Rogos
a agar
Seasame seeds pattern

60. Bacteria Type Culture
media used
Appearence
in the culture
plate
Description
S.gardonii G +ve Cocci Blood Agar Clear halo
surrounding
the colony
F. nucleatum G –ve Rod CVE agar Round
opaque
colony
P.gingvalis G- ve Rod Blood agar Biege to
brown
coloured
colony
AAC G-ve Rod TSBV agar
plate
Small circular
dome shaped
colonies

61. Bacteria Type Culture media
used
Appearence in
the culture plate
Description
Tannerella
forsythia
Anaerob
ic
G-ve
rod
Blood agar Smooth
white colony
with faded
edge
Camphylobact
er rectus
G-ve
rod
Hammond
plate
Smooth
opaque
black round
colonies
Eubacterium
nodatum
G+ve
rod
Blood agar Depends on
its
substrate.its
growth is
vey slow
S.sorbinus G+ve
cocci
Tycsb agar Colony with
white halo

62. conclusion
 Though other many laboratory methods are used
for diagnosing the disease and the underlying
microorganism involved
 Culture medium method is still used in
identification of bacteria, antibiotic sensitivity.
 Periodontal pathogens are identified by culturing
to provide proper treatment