Culture media and methods

1. Presented by
Srinivas .T
Dept of cons & Endo
I MDS

2.  INTRODUCTION
 HISTORY
 CLASSIFICATION
 TYPES OF CULTURE MEDIA
 BIO CHEMICAL TESTS & REACTION
 CULTURE METHODS
 INDICATONS OF CULTURE METHODS
 TYPES OF CULTURE METHODS
 CONCLUSION
 REFERENCES

3.  Culture media is a liquid or solid substance
required to grow the organisms from infected
material to identify the causative agent.

4. The original media used by Louis pasteur (liquid med)
-Urine or Meat broth
-liquid medium = diffuse growth
-Solid medium = discrete colonies
Colony : macroscopically visible collection of millions of
bacteria originatimg from single bacterial cell.
Cooked cut potato by Robert koch (solid med)
Gelatin – not satisfactory
- becomes liquid med at 24 o C

5.  Water
 Electrolyte
 Peptone
 Meat extract
 Blood or Serum
 Agar

6.  Frau Hesse
 …. solid medium
 … seaweeds
 No nutritive value
 Not affected by growth of bacteria
 98 0C & 42 oC
 2 percent AGAR is employed in soild medium
 …. solidifying agent
 … powder form.

7. I Based on their consistency
a)solid medium
b)liquid medium
c)semi soild medium
II Based on the consistency /ingredients
a)simple medium
b)complex medium
c)synthetic or defined medium
d)Special media
III Based on oxygen requirement
a)aerobic media
b)anaerobic media

8. SOLID MEDIA
-CONTAINS 2% Agar
-Colony morphology , pigmentataion,hemolysis
can be appreciated.
E.g; Nutrient agar, blood agar
LIQUID MEDIA
- no agar
- For inoculum prep, blood culture, for the isolation
of pathogens from mixture
E.g; Nutrient broth
Semi solid medium
0.5% agar
E.g; Motility medium

10.  Enriched media
 Enrichment media
 Selective media
 Indicator media
 Differential media
 Sugar media
 Transport media
 Media for biochemical reactions

11. - they contain minimum ingreident that supports
growth of non- fastidious bacteria
E.g, Nutrient broth, nutrient agar
Nutrient broth consist of peptone, meat extract, NACL
Water
Nutrient broth +0.5% Glucose =Glucose broth
Nutrient broth +2% agar=Nutrient agar
Agar concentration reduced (0.2-0.5%)=semi solid med

12. I Media prepared from pure chemical subsatnces
II Exact composition is known
III Used for spl studies
eg., metabolic requirements
E.g, peptone water (1%peptone+ 0.5%NaCL in water)

13. I When basal media is added with additional nutrient
such as blood, serum or egg
E.g, blood agar: blood + nutrient agar
use for streptococus
chocolate agar: it is heated blood agar
use for Neisseria, H. inflenza
loffler’s serum slope: serum + nutrient agar
use for corneybacterium diptheria

15. I. It is a liquid media which increase the growth of
particular species or inhibits its competitors useful for
isolation of pathogen
E.g,, - Selenite F broth for isolation shigella
- Alkaline peptone water for vibro cholerae
- Tetrathionate broth for salmonella

16. I Enriched media & selective both are same principle but
they are solid media.
E.g,
- TCBS – V. cholerae
- LJ medium – M tubercolosis
- Wlison and Blair medium – S.typi
- Potassium tellurite medium –diptheria bacilli

19.  These media contains of indicator which changes
colour when a bacterium grows in them. S typhi grows
as black colour on wilson and blair medium.
 Eg
blood agar
mac conckey agar
christension urease medium

20.  This media differentiate between two groups of
bacteria by using an indicator.
 Eg Mac Conkey’s agar
 Distinguish between lactose fermenters & non lactose
fermenters
compostion: peptone
lactose
agar
Neutral red
Taurocholate

21.  lastose fermenters –pink colour
 non lactose fermenters – colour less colonies

22.  Media containing any fermentable substance.
 E.g glucose, arabionse, lactose, starch e.t.c,
 Media consist of one % of sugar

23.  This is used for transport the specimen. In this media
bacteria not multiply only remains viable when delay
transporting the specimen from site of collection to
laboratory
 Eg
-stuart medium: non nutrient soft agar containing
a reducing agent
-bufferd glycerol saline: enteric bacilli
-Amies media - Neisseria

25.  these media are used to grow anaerobic organisms
 EG: Robertson cooked meat medium,
Thioglycolate medium

26.  Culture methods employed depend on the purpose for
which they are intended.
 The indications for culture are:
 To isolate bacteria in pure cultures.
 To demostrate their properties.
 For bacteriophage & bacteriocin susceptibility.
 To determine sensitivity to antibiotics.
 To estimate viable counts.
 Maintain stock cultures.

27.  Streak culture
 Lawn culture
 Stroke culture
 Stab culture
 Pour plate culture
 Liquid culture
 Anaerobic culture methods

28. Intro
 It is most comman inoculaton method used for
bacterial isolation in pure culture(solid media)
Requirement
-culture plate
-platinum or nichorme wire
-specimem
-solid media

29.  Procedure
-A platinum or nichrome wire loop of 2-4mm internal
diameter is used
-the loop is first sterilized in the bunsen flame by
making it red hot and cooled by touching an un
inoculating
-then a loop full of specimen is smeared onto the
surface of a dried plate near the peripheral area.
this is named as primary inoculum.

30.  From the primary inoculum it is sperad thinly over the
final series of streaks.
 Incubate at 37 0C overnight
 Use – for isolation of bacteria in pure culture.

34.  Also called as carpet culture
Requirement:
-specimen
-sterile cotton swab
-petri plate
Procedure:
-lawn culture is obtained by flooding the surface of
plate with a liquid culture or suspension of bacterium

35. - Alternatively the culture plate may inoculated by a
sterile swab soaked in liquid bacterial culture
- plate is incubated at 37 degree C overnight to obtain
bacterial colonies

37.  Store culture is made in tube containing agar Slant/slope
 Requirement
-tube containing agar slope
-zig zag pattren wire loop
-specimen
Procedure
- (nutrient agar slope) liquid culture is poured in to the
tubes
-tube are placed in the slant position
-after solidfication a slant is ready for inoculation
-specimen is inoculate streaking the straight wire in zig zag
fashion

38.  Uses
-provides a pure growth of bacterium for slide
agglutination and other diagnostic test.

39.  Prepared by puncturing … or stabbing the semi solid agar
Requirement:
 gelatin or glucose agar
 straight charged wire.
 test tube and specimen
Procedure:
 Performed by a straight charged wire with culture material
by puncturing deep inside agar.
Uses:
 Demonstration of gelatin liquefaction.
 Oxygen requirements of the bacterium under study.
 Maintenance of stock cultures.

41. Requirement:-
 Molten agar
 water bath
 Petri dish
 Specimen
Procedure:
 Tube Contain 15 ml of agar medium are melted and kept to Cool
in water bath at 45-50°C
 The inoculum to be tested in diluted in serial dilution.
 Add 1 ml of diluted Sample in petri dish and Pour melted agar
mix well and allow to Solidify.
 Plate are incubated at 37°C for overnight.
 Colonies will be seen throughout the depth of the medium and
can be counted

43.  Liquid Culture is used for Culture of specimen Such as blood or
body fluid.
Requirment:-
 Specimen
 Liquid media
 Pipette / Syringe
 Screw-Capped bottle or flask.
Procedure:-
 Inoculated by adding the Specimen directly in to the liquid
medium with the help of Syringe or Pipette.
 Bacterial growth is detected by observing turbidity in the
medium and some aerobic bacteria form Surface pellicles.
Uses
 blood, body fluid Culture, water analysis.

45.  Anaerobic bacteria grow only in the absence of oxygen
 Anaerobiosis can be established by various methods.
 Methods:
- Production of vacuum
- Displacement of oxygen with other gases
-Reduction of medium
-by reducing agents
- anaerobic chamber

46. Production of a Vacuum
 Cultivation in vacuum was attempted by incubating
cultures in a vacuum desiccator, but it proved to be
unsatisfactory. This method is not in use now.
Displacement of Oxygen
 Displacement of oxygen by inert gases like hydrogen,
nitrogen, carbon dioxide or helium is sometimes employed.
Oxygen can never be removed completely by this method.
By Displacement and Combustion of Oxygen
 Anaerobiosis obtained by "McIntosh and Filde's" anaerobic
jar is the most reliable and widely used method.

47.  Procedure
 McIntosh and Filde's anaerobic jar consists of a stout glass or
metal jar with a metal lid which can be clamped air -tight with a
screw The lid is fifted with two tubes with taps, one acting as
inlet for introduction of gas and the other as the outlet.
 The lid also contains two terminals which can be connected to an
electrical supply. A catalyst (alumina pellets coated with
palladium) is suspended under the lid by stout wires which are
connected with the terminals to heat the catalyst for its activity.

49.  Culture plates inoculated with specimens are placed
inside the anaerobic jar with an indicator. The lid is
clamped tight.
 The outlet tube is connected to a vacuum pump to
remove the air inside the jar while inlet tube is closed.
 The outlet tube is then closed and hydrogen gas is
passed through inlet tube till reduced atmospheric
pressure is brought to normal atmospheric pressure (
760 mm Hg) which is monitored on the vacuum gauze
as zero.

50.  Electric terminals are switched on. The catalyst helps
to combine hydrogen and residual oxygen to form
water
 Reduce methylene blue generally used as indicator for
anaerobiosis in jar. it remains colourless in anaerobic
conditions, but turns blue on exposure to oxygen

51.  By reducing agents
oxygen in culture media can be prepared by various
agents such as glucose, thioglycollate, cooked meat
pieces, cysteine and ascorbic acid.

52.  They provide additional information for the
identification of bactreium
 The tests include
 -oxidase test
-indole test
-Citrate utilization
-Urease test

53.  Detects the presence of an enzyme “oxidase”
 Postive test ….purple colour
 Oxidase positive – Pseudomonas, vibro,Neisseriae
 Oxidase negative-salmonella, shigella

54.  INDOLE TEST
 Used to detect indole production by the organism.
 After overnight incubation, a few drops of indole
reagent(Kovac's reagent)is added.
 Positive test is indicated by a pink ring.
Negative indole test- yellow ring –
 Indole positive- E.coli
 Indole negative- Klebsiella,Salmonella.

55.  CITRATE UTILIZATION
 Done in Simmon's Citrate medium.
 Contains Sodium citrate and bromothymol blue as the
indicator.
 Citrate positive-blue colour
 Citrate negative- green colour
 Positive- Klebsiella
 Negative- E.coli

56.  UREASE TEST
 Done in Christensen's urease medium.
 This test is used to detect organisms that produce
urease.
 Urease positive- pink colour
 Urease negative- yellow colour
 Positive- Proteus, Klebsiella
 Negative- E.coli, Salmonella

58.  A sterile paper point (#25) was introduced into the full
length of the canal (as determined by a preoperative
radiograph) and kept in position for 30 seconds. The
paper point then was removed, and nine parallel lines
were slowly drawn on the surface of a anaerobe 5%
Sheep Blood agar plate with a chip of the paper point.
The plate and an anaerobic gas-producing pouch were
immediately set in an anaerobic jar. The jar was then
incubated at 37◦C for 3 days. The numbers and
thickness of lines made by bacterial colonies were
checked at each treatment procedure

59.  If no bacterial lines or colonies were detected on the
plates, then the decision was made to proceed to root
canal filling

61.  Ananthanarayan’s – textbook of micro biology
 Bhatia’s – textbook of microbiology
 C.P Baveja – textbook of microbiology