Notes for B.Sc nursing students for little help...

1. Jayesh Soni
Associate professor
Venkteshwar College of Nursing
Udaipur

2. Culture media
A culture media is a special medium used in
microbiological laboratories to grow different kinds
of microorganisms. A growth or a culture medium is
composed of different nutrients that are essential for
microbial growth.
Composition of culture media

3. Types of Culture Media
Classification based on physical state
Classification based on the ingredients

4. Classification based on physical state
• Solid medium-
 Solid medium contains agar at a concentration of 1.5-2.0% .
 Solid medium has physical structure and allows bacteria to grow in
physically informative or useful ways (e.g. as colonies or in streaks).
 Solid medium is useful for isolating bacteria or for determining the colony
characteristics of the isolate.
• Semisolid medium-
 They are prepared with agar at concentrations of 0.5% or less.
 They have soft custard like consistency and are useful for the cultivation
of micro-aerophilic bacteria or for determination of bacterial motility
• Liquid (Broth) medium-
 These media contains specific amounts of nutrients but don’t have gelatin
or agar.
 Broth medium serves various purposes such as propagation of large
number of organisms, fermentation studies, and various other tests. e.g.
sugar fermentation tests.

6. Classification based on the ingredients
• Simple medium-
 Nutrient broth and nutrient agar are considered as basal /
simple medium. These media are generally used for the
primary isolation of microorganisms.
 It contains Peptone water, meat extract 1% and glucose 0.5%
• Undefined/Complex Medium-
 An undefined medium also known as a basal or complex
medium contains carbon source such as glucose, water,
various salts, amino acids and nitrogen (e.g., beef, yeast
extract)
 This is an undefined medium because the amino-acid source
contains a variety of compounds with the exact composition
being unknown.

7. Cont…
• Defined Medium:
A defined medium/synthetic medium is a medium in which all
the chemicals used are known and no yeast, animal, or plant
tissue is present.
• Special Media:
a) Enriched media
b) Enrichment media
c) Selective media
d) Differential media
e) Indicator media
f) Transport media
g) Dehydrated media

8. a) Enriched media-
It contain the nutrients required to support the growth of a wide
variety of organisms
They are commonly used to grow as many different types of
microbes as are present in the specimen.
E.g.- Blood agar which is nutritionally-rich, whole blood
supplements the basic nutrients.
- Chocolate agar is enriched with heat-treated blood (40-
45°C), which turns brown and gives the medium that
chocolate color.
Cont…

9. b) Enrichment media-
 Enrichment media promotes the growth of a particular
organism by providing it with the essential nutrients and
rarely contains certain inhibitory substance to prevent the
growth of normal competitors.
E.g.- Selenite F broth favors the growth of Salmonella also
prevents the growth of normal competitors like E. coli. E. coli
does not die in the medium but they do not flourish like
Salmonella does.
So the conclusion is that- “Enriched media selects for a certain
group of microbes while an enrichment media selects for one
microbe.”
Cont…

10. c) Selective media-
 Selective media are used to isolate a particular strain of
microorganisms
 Selective media use specific growth characteristics of a
microorganism to selectively grow that M.O. in the growth
medium where mixed bacterial flora are expected
E.g.- Deoxycholate citrate agar (DCA) act as selective agent
for enteric bacilli
d) Differential media-
 Differential media are used to identify and differentiate a
closely-related group of microorganisms.
 Differential media allow the characterization of several M.O.
based on the growth patterns of them.
E.g.- MacConkey’s agar is differential for lactose fermentation
Cont…

11. Cont…

12. e) Indicator media-
 A type of bacteriological medium which may contain a fermen
table sugar plus a pH indicator that gives a color change.
 It is used to identify bacteria on the basis of a characteristic
biochemical reaction.
E.g.- MacConkey’s agar is differential as well as indicator
medium. Lactose fermenters- pink colonies,
Non Lactose fermenters- colourless colonies
f) Dehydrated media-
 D.M. are simply reconstituted in distilled water and sterilized
before use. With D.M. the process of media making has
become simpler & its quality more uniform
Cont…

13. g) Transport media-
 Transport media used in the cases of delicate organism,
which may not survive the time taken for transit or may
overgrown.
 Temporary storage of specimens being transported to the
laboratory for cultivation
 Maintain the viability of all organisms in the specimen
without altering their concentration
 It Lack of carbon, nitrogen, and organic growth factors so
as to prevent microbial multiplication
E.g.-Thioglycolate broth & Stuart transport medium
Cont…

14. • Streak culture (Surface planting):-
Streak plate technique is used for the isolation into pure culture
of the organisms (mostly bacteria) from mixed population.
Procedure:-
 Sterilize the inoculating loop in the Bunsen burner by putting
the loop into the flame until it is red hot. Allow it to cool.
 Pick an isolated colony from the agar plate culture and spread
it over the first quadrant (approximately 1/4 of the plate)
 Streak the inoculating loop very gently over a quarter of the
plate using a back and forth motion
 Flame the loop again and allow it to cool. extend the streaks
into the second quarter of the plate
CULTUER METHODS

15. Cont…
 Flame the loop again and allow it to
cool. extend the streaks into the
third quarter of the plate.
 Flame the loop again and allow it to
cool. extend the streaks into the
center fourth of the plate.
 Flame your loop once more.
Result-
 Streaked plate are incubated
at 37°C for 24 hours.
 Colonies grown in the plate All
colonies should have the same
general appearance.
 If there is more than one type of
colony, each type should be
streaked again on a separate plate
to obtain a pure culture.

17. Lawn culture:-
This type of culture provide a uniform surface growth of the
bacteria
Uses-
 For bacteriophage typing
 Antibiotic sensitive testing
 In the preparation of bacterial antigen and vaccines
Preparation-
 Lawn culture are prepared by flooding the surface of the plate
with a liquid culture or suspension of the bacterium
 Culture plate may inoculated by a sterile swab soaked in liquid
culture
Result:
 Incubated 37 degree overnight to obtain bacterial colonies.

18. Anaerobic Culture method
• Anaerobes are bacteria that can live only in the absence of
oxygen.
• Anaerobic bacterial cultures are performed to identify
bacteria that grow only in the absence of oxygen and
which may cause human infection.
• Anaerobic infections result in such serious consequences
as amputation, organ failure, sepsis, meningitis, & death.
• Culture is required to correctly identify anaerobic
pathogens and institute effective antibiotic treatment.
• Anaerobiosis obtained by- “McIntosh & Filde’s anaerobic
jar” which is most reliable and widely used method.

19. McIntosh & Filde’s anaerobic jar
About the equipment -
 McIntosh and Fildes’ jar consists of a 8x5 inch (20x12.5 cm)
jar of stout glass or metal with a tight fitting metal lid.
 The lid can be clamped airtight with a screw and is fitted with
two tubes with taps, one for introduction of gas inside
(inlet)and the other as outlet for vacuum valve. The lid also
contains two terminals that can be connected to an electric
supply.
 A capsule containing 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.
 Nowadays, catalyst active at room temperature is also
available.

20. Procedure -
 Keep the inoculated culture plates inside the jar along with an
indicator.
 Screw tight the lid
 Close the inlet tube and connect outlet tube to a vacuum pump (
at least three quarters of the air of the jar can be removed).
 Note the pressure on a vacuum gauze and when the pressure is
reduced to 100 mm Hg (i.e., 600 mm below atmospheric),
tightly close the outlet tap.
 Connect the inlet tap is to a hydrogen supply and then open it.
Hydrogen is passed through a small wash bottle.
 Bring the reduced pressure up to 760 mm Hg (i.e., atmospheric)
by monitoring on the vacuum gauze as 0.

21.  Switch on the electric
terminals for heating the
palladinised crystal (When
room temperature catalyst is
used heating is not required).
– The catalyst helps the
combination of hydrogen
and residual oxygen to
form water. This process is
allowed to continue for 20
minutes.
 Incubate the McIntosh and
Filde‘s jar in an incubator at
37°C for 48 hours.