CULTURE MEDIA

&
CULTURE
METHODS
 Culture and Medium

Culture is the term given to microorganisms that are

cultivated in the lab for the purpose of identifying and
studying them.

Culture media can be defined as artificially supplied

nutrients for the growth of microorganism in the
laboratory.
Basic requirements of culture media
 Nutrients:
- Energy source
- Carbon source
- Nitrogen source
 Mineral salts – Sulphate, phosphates, chlorides &
carbonates of K, Mg & Ca.
 A suitable pH – 7.2 – 7.4
 Accessory growth factors:
- Tryptophan for Salmonella typhi
- X & V factors for H. influenzae
 Classification of Culture media
A. Based on the consistency:
 Liquid media-
1. Peptone water
2. Nutrient broth
 Semisolid media -
1. 5% Nutrient agar stabs
 Solid media-
1. Blood agar
2. Nutrient agar
3. MacConkey agar
4. Chocolate agar Sea weed Agar
 Classification of Culture media
B. According to characteristics of the media:
I. Simple or Basal media:
1) Peptone water
2) Nutrient broth
3) Nutrient agar
Simple media support the growth of the organisms that
do not need special nutrient requirements.
Used in preparation of enriched media.
 Classification of Culture media
II. Complex culture media:
1) Enriched media
2) Selective media
3) Indicator media
4) Indicator media
C. Transport media Complex medium

D. Identification media

E. Based on oxygen requirement-

I. Aerobic Media
II. Anaerobic Media
 Enriched media
 Addition of extra nutrients in the form blood, serum, egg
yolk, etc. to basal medium makes them enriched media.
 Stimulate growth of desired bacterium and inhibit growth of
unwanted bacterium
 Media is incorporated with inhibitory substances to
suppress the unwanted organism, thus increase in numbers
of desired bacteria.
 Examples of Enriched media: Chocolate agar, Blood
agar(Nutrient agar + 5 to 10% sheep blood)
.
 Culture procedure
Inoculation of the media on 1st day
 Immediately before inoculation, media should be
checked for visual contamination.
 Aseptic techniques must be applied to avoid
contamination.
 Flame sterilize wire loops, straight wires & metal
forceps before & after use.
 Flame the necks of specimen bottles, culture bottles
& tubes after removing and placing caps.
 Cont.
 Before inoculating, the surface of the medium must
be dried otherwise single colony will not form.
 To do this, the lid should be removed and incubate
the medium at 350-370C for 30-40 minutes.
 A sterile loop containing specimen or swab of the
specimen is used as inoculum and applied to a small
area of the plate-‘’the well’’.
 Flame sterilization of the loop should be done.
Inoculum should be spread as shown in figure
 
Inoculation of slopes:
To inoculate a slope and butt such as Kligler iron agar, use a
sterile straight wire to stab into the butt first and then use
the same wire to streak the slope in a zigzag pattern.

Inoculation of fluid media:

Broth and other fluid media are inoculated using a sterile
wire loop, straight wire or Pasteur pipette depending on
whether the inoculum is colony, a fluid culture or a
specimen.

When using a wire to subculture colonies, hold the bottle or

tube at an angle and rub the loop against the side of the
container below the level of the fluid.
Inculcation of slopes
 Incubation of the inoculated media:
 Inoculcated media should be incubated as soon as
possible.

 The temperature selected for routine culturing is 350-

370C

 The length of time of incubation depends on how long

an organism takes to develop the cultural characteristics
by which it is recognized.

 Usually in our laboratories we do it overnight.

 Procedures on 2nd day:

Observation of primary plate:

a)Colony morphology:
1) Size of the colony-pin head/pin point, small, moderate,
large diameter of the colony.
2) Pigment production:-
 Non-pigmented white, creamy, etc.
 Pigmented-yellow, gray, red, pink, greenish blue,etc.
3) Appearance-Raised/depressed/flat, opaque/transperant,
shiny, dull.
4) Zone of haemolysis-present/absent, if present size of the
zone.
5) Texture-mucoid/rough, dry/moist
6) In MacConkey agar-Pale/Pink colour colony
 Subculture: Is done to isolate pure colony. 2-3
isolated single colony are taken from primary
plate and inoculated in the media, incubted
overnight and examined in the next morning (3rd
day) for pure growth.

 Gram stain-Gram positive/negative organism

detection

 Wet film:
 pus cell-present/absent
 Motility of the organism-motile/immotile
Drug sensitivity-
 Methods of antimicrobial sensitivity testing-
1) Diffusion method
2) Dilution method
3) Diffusion & Dilution

 Disk Diffusion Method: To standardize the inoculum

density McFarland standard is used.

 Within 15 minutes after adjusting the turbidity of the

inoculum suspension, a sterile cotton swab is dipped
into it, excess inoculum is removed and the dried
surface of the Mueller –Hinton media is inoculated by
streaking the swab over the entire sterile agar surface.
Muller Hinton Agar
for
Antibiotic Testing
 This procedure is repeated by streaking two more
times, rotating the plate approximately 600 each
time to ensure an even distribution of inoculum.

 As the final step, the rim of agar is swabbed.

 The antibiotic disk must be pressed down to ensure

complete contact with the agar surface.

 No more than 10 disks should be placed on one

plate.
 Disks should not be closer than 24 mm from center
to center.

 Because some of the drugs diffuses almost

instantaneously, a disk should not be relocated once
it has come into contact with agar surface.

 Within 15 minutes after the disks are applied the

plates are inverted and placed in an incubator which
is set at 350C .

 Quality control in antibiotic susceptibility testing is

done by reference strains named ATCC.
Results on the 3rd day:

 Examination of the
subculture
 Examination of the
drug sensitivity
 Examination of
biochemical tests.
 Reporting of culture