CULTURE MEDIA

PowerPoint Presentation by Frances Rowena Mercado, MAED General Science

Culture Media
 A liquid or gel designed to support the growth of
microorganisms or cells.
 Culture medium- nutrients prepared for microbial
growth
 Inoculation- introduction of microbes into
medium
 Culture/Colony- microbes growing in/on culture
medium
In the history…

 Robert Koch- described his culture techniques

in 1881.
 Fanny/Frau Hesse- suggested the use of agar.
 Richard Julius Petri- invented the glass Petri
dishes.
 Joseph Lister- the first person to obtain a pre
culture of bacterium (Streptococcus lactis) in a
liquid medium.
Classification of Culture Media Based on
Whether the Exact Contents are Known

 Chemically defined media- exact

chemical composition is known
 Complex media- exact contents are not
known, from extracts and digests of
yeasts, meat, or plants
Liquid and Solid Media
 Liquid media- or broths are contained in tubes,
referred to as tubed media.
 Solid media- prepared by adding agar to liquid
media and then poured into test tubes or Petri
dishes, where the media solidifies.
 Agar plate - one grown on a medium, usually agar or
gelatin, on a Petri dish
 Agar slant - one made on a slanting surface of a
solidified medium in a tube, the tube being tilted to
provide a greater surface area for growth.
 Agar butt/deep - one in which a tube of solid medium
is inoculated by a needle thrust deep into the
contents.
Bacterial Media

 Selective
 Differential
 Enriched
Selective Medium

 Has added inhibitors that discourage the

growth of certain organisms without inhibiting
growth of the organism being sought.
 Solid medium is employed with selective
medium so that individual colonies may be
isolated.
Examples:
 MacConkey agar- screen for S. aureus and is selective
for Gram (-) bacteria.
 Phenylethyl alcohol agar (PEA) and colistin-nalidixic
acid agar (CNA)- inhibit growth of Gram (-) bacteria.
 Thayer-Martin agar and Martin-Lewis agar- selective
for N. gonorrhoeae.
 Mannitol salt agar (MSA)- only for salt-tolerant
(haloduric) bacteria
 Eosin methylene blue agar (EMB) – selective against
gram-positives
MacConkey agar E. coli on EMB
Differential Medium

 Permits the differentiation of organisms

that grow on the medium.
 Reveals the presence of 2 or more similar
microorganisms by differences in the
appearance of their colonies.
Examples
 MacConkey agar- used to differentiate various
Gram (-) bacilli that are isolated from fecal
spcimens.
 Gram (-) bacteria are able to ferment lactose
produces pink colonies, those are unable to ferment
lactose produce colorless colonies.
 Differentiates between LF and NLF Gram (-) bacteria.
 Mannitol salt agar- used to screen for S.
aureus, pink to yellow.
 Centrimide agar - used for the differentiation of
strains of Pseudomonas spp.
P. aeruginosa on centrimide agar Two different species of
Staphylococcus growing on
mannitol salt agar (MSA).
Enriched Medium

 Broth or solid medium containing rich

supply of special nutrients that promotes
the growth of fastidious organisms.
 Prepared by adding extra nutrients to a
medium called nutrient agar.
Blood Agar Types
 Blood agar plates (BAP)
 Contains mammalian blood, typically at a
concentration of 5–10%
 Used to isolate fastidious organisms and detect
hemolytic activity (Neisseria and Streptococcus).
 Chocolate agar (CHOC)
 blood cells have been lysed by heating the cells
to 56 °C
 used for growing fastidious (fussy) respiratory
bacteria, such as Haemophilus influenzae.
BAP CHOC
Remember…
 Various categories of media are not mutually
exclusive.
 Ex: blood agar is enriched and differential
 MacConkey agar and MSA are selective and
differential
 PEA and CNA are enriched and selective
 Thayer-Martin and Martin-Lewis are highly enriched
and highly selective
 Thioglycollate broth (THIO) is a liquid medium that
supports the growth of all categories of bacteria.
Examples

 Hektoen enteric agar (HEA) - selective and

differential agar primarily used to recover
Salmonella and Shigella from patient specimens
 Salmonella-Shigella agar (SS) – selective and
differential for Salmonella and Shigella
 Bile Esculin Agar (BEA) is a selective
differential agar used to isolate and identify
members of the genus Enterococcus
Fungal Media
 Sabouraud agar
 Sabouraud agar is used to culture fungi and
has a low pH that inhibits the growth of most
bacteria; also contains the antibiotic
gentamicin to specifically inhibit the growth of
Gram-negative bacteria.
 Hay infusion agar
 Specific for the culturing of slime molds
(though not technically fungi).
 Potato dextrose agar
 PDA is used to culture of certain types of fungi.
Preparation of CM
 Nutrient Broth
 Dissolve 8 g of NB powder in 1000 ml of distilled
water in an Erlenmeyer flask. Mix and heat over the
magnetic stirrer until the medium becomes clear or
transparent.
 Dispense 8 ml of the medium into sterile test tubes.
 Immediately stopper the tubes completely.
 Sterilize in the autoclave at 15 psi, 121 C for 15
mins.
Preparation of CM

 Nutrient Agar
 Dissolve 28 g of NA powder in 1000 l of
distilled water in an Erlenmeyer flask. Mix and
heat over the magnetic stirrer until the
medium becomes clear or transparent.
 Sterilize in the autoclave at 15 psi, 121 C for
15 mins.
Dispensing the CM
 NA plate
 Lay out several sterile Petri dishes on the table with
the cover partially open and dispense the medium
inside and inoculating hood.
 Dispense the NA medium (30 ml) and allow 5-10
minutes to elapse before completely covering the
dish to avoid contamination.
 When the medium is solidified, label the NA plate.
Wrap and label the plate.
 NA slant
 Dispense 8 ml of the medium into sterile test
tubes using pipette or syringe.
 Cover the tube partially with sterile cotton plug
or screw cap and allow the agar medium to
harden in an incline position on an agar slant
rack.
 Stopper the tube completely when the
medium has solidified. Label the upper portion
of the test tube slant.
 Sterilize in the autoclave at 15 psi, 121 C for
15 mins.
 NA deep or butt
 Dispense 8 ml of the medium into sterile test
tubes using pipette or syringe.
 Cover the tube partially with sterile cotton plug
or screw cap and allow the agar medium to
harden in an upright position on a test tube
rack.
 Stopper the tube completely when the
medium has solidified. Label the upper portion
of the test tube slant.
 Sterilize in the autoclave at 15 psi, 121 C for
15 mins.
 Inoculation of Culture Media

 Inoculation- adding a portion of the

specimen to the medium.
 Involves the use of sterile inoculating loop
to apply a portion of the specimen to the
surface of the medium; a process
commonly referred to as “streaking”.
 Materials
 petri dishes
 test tubes
 bunsen burners/alcohol lamps
 wire inoculating loops
 bottles of staining reagents
 incubators
 Importance of Using
“Sterile Technique”
 Necessary to exclude all microorganisms from
a particular area, so that area will be sterile.
 Media should remain sterile before inoculation.
 Contaminants- unwanted microorganisms
 Contaminated- if the sample contains
contaminants
 Streaking the Agar Plate:
Simple Streak
 Flame sterilize the inoculating loop then let it cool for a
few seconds, afterwards fish out a loopful of specimen
from pure culture.
 Hold the sterile Petri dish with cover by your left hand,
partially open the agar plate near the flame of the
alcohol lamp.
 Place a loopful of specimen on one side of the agar
medium away from you and streak the culture back
and forth from edge to edge of the plate,
 When the entire medium has been streaked, flame
sterilized the cover of the plate completely.
 Label the Petri dish then incubate for 24-48 hours at 37
C.
Streaking the Agar Plate
 Inoculating the Agar Slant
 Fish out a loopful of bacterial culture using a
flamed sterilized loop.
 Hold the agar tube with the left hand and screw
cap should be held by the small finger. Never lay
the screw cap of the test bacterial specimen
anywhere.
 Pass the mouth of the test tube through the
alcohol lamp’s flame. Flame immediately and
inoculate the agar surface by moving the
inoculating loop from the bottom to the top of the
agar slant or in a snake-like motion.
 Inoculating the Agar Slant
 Withdraw the inoculating loop and
immediately flame-sterilize it. Pass the
mouth of the agar tube on the alcohol
lamp’s flame.
 Sterilize the screw cap and cover the agar
tube.
 Label the agar slant tube and place it in
the test tube rack, then incubate.
 Inoculating the NB
 Fish out a loopful of bacterial culture using a flamed
sterilized loop.
 Hold the agar tube with the left hand and screw cap
should be held by the small finger. Never lay the
screw cap of the test bacterial specimen anywhere.
 Inoculate the medium from top to bottom. Assume
equal distribution of inoculums by suspending the
inoculating loop into the broth, then shake.
 Flame sterilize the mouth of the test tubes before
and after inoculation.
 Sterilize the screw caps, cover, and label the broth
culture, then incubate.
 Inoculating the NA butt
 Fish out a loopful of bacterial culture using a flamed
sterilized loop.
 Hold the agar tube with the left hand and screw cap
should be held by the small finger. Never lay the screw
cap of the test bacterial specimen anywhere.
 Inoculate the inoculum into the agar butt by stabbing the
inoculating needle into the agar without touching the sides
of the test tubes at the middle and halfway of the agar.
 Flame sterilize the mouth of the test tubes before and after
inoculation.
 Sterilize the screw caps, cover, and label the broth culture,
then incubate.
FINISHED!