MEDIA

Samantha Celena Triadi

1871152001
PPDS-1 Mikrobiologi Klinik
Fakultas Kedokteran Universitas Udayana/RSUP Sanglah
 Clinical Microbiologist purposes:
◦ Field of diagnostic microbiology
◦ Prevent infection
◦ Correct diagnosis and precise therapy (antibiotic)
◦ Regulation about infection prevention in hospital
 Process of growing microorganisms in culture
by taking bacteria from the infection site
 In vivo to In vitro
 Follow by observation in microscopy
 Need essential nutrients and appropriate
environmental conditions (the important part
of media)
• The food material or substances required
for growing microorganisms in vitro
(outside the body) is called culture
medium.
• This presentation consist of 2 parts :
culture media and quality control
 Louis Pasteur as the father of microbiology
used nutrient broth to grow microorganisms
 Koch as one of the pioneers of modern
microbiology used thin potato, which sliced
first by sterilized knife
 Agar is a polysaccharide extract from marine
algae. It melts at 84 celcius and solidifies at
38 celcius.
 Firstly, Koch used gelatin but it didn’t works,
because gelatin melts at 35 celcius and
bacteria were able to digest gelatin
 Divide into 2 type : fastidious (difficult to
grow) and nonfatidious
 Bacterial growth can be detected by eyes if
there at least 106 bacteria per mL of broth
 Location also define the type of bacteria
(aerob, anaerob, oxygen needed)
 Agarose is the most common solidifying
agent
I. Classification based on physical state

a) solid medium
b) semi solid medium
c) liquid medium
II. Classification based on the ingredients

a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
 Solid medium
agar is the most commonly used solidifying agent.

What is agar
• Golden –yellow granular powder
• Prepared from seaweeds.
• Not affected by the growth of thebacteria.
• Melts at 98oC & sets at 42oC
• Semi-solid media

Such media are soft and are useful in

demonstrating bacterial motility and
separating motile from non- motile strains .
• Liquid media
are sometimes referred as “ broth “.
bacteria grow uniformly producing general
turbidity eg. Nutrient broth
Simple media
- eg: Nutrient broth, N.agar
- NB consists of peptone, meat extract, NaCl,
- NB + 2% agar = Nutrient agar
• specially prepared media from pure chemical
substances for research purpose and
composition of every component is well known
• eg: peptone water –
1% peptone + 0.5% NaCl in water.
1. Enrichment non selective media
2. Selective (contain inhibitory agents such as dyes, bile
salt, alcohols, acids and antibiotics)
3. Differential (MacConkey agar : differentiates between
gram negative bacteria that can or cannot ferment the
sugar lactose)
4. Transport media
5. Anaerobic media
 These media are designed to support the
growth of most organisms without fastidious
growth requirements.
 Blood agar
 Chocolate agar
 Mueller-Hinton agar (for antibiotic
susceptibility testing of bacteria)
 Thioglycolate broth (anaerobic)
 Sabouraud dextrose agar (fungi).
Blood agar Chocolate agar
BA contains mammalian blood(usually contain red blood cells that have been
sheep or horse) typically at a lysed by
concentration of 5-10%, used to isolate slowly heating to 80 c .and it used for
growing fastidious bacteria, such as
fastidious organisms and detect
Haemophilus influenzae
hemolysis.
• The inhibitory substance is added to a solid media to
inhibit commensal or contaminating bacteria such as :

• Antibiotics
• Dyes
• Chemicals
• Alteration of pH
 Thayer Martin medium
selective for Neisseria gonorrhoeae
•It usually contains the following combination of
antibiotics:

•Vancomycin:
which is able to kill most Gram-positive organisms.
•Colistin,:
which is added to kill most Gram-negative organisms except Neisseria.
•Nystatin,:
which can kill most fungi
•Trimethoprim:
which inhibits Gram-negative organisms, especially swarming Proteus.
• Selective for gram negative bacteria
• The dye methylene blue in the medium inhibit the
growth of gram positive bacteria.
• Is used for isolation of Campylobacter jejuni from
fecal or rectal swab.
• Contain Bacteriological charcoal , Cefoperazone
and Amphotericin B.
• is solid medium used for
Mycobacterium tuberculosis.

• contain penicillin, nalidixic

acid and malachite green
to inhibit growth of gram
positive and gram negative
bacteria, in order to limit growth
to Mycobacteria species only.
• Differential media
• are designed in such a way that different bacteria can be
recognized on the basis of their colony color.

• Dyes and metabolic substrates are incorporated so that

those bacteria that utilize them appear as differently
colored colonies.

Examples:
• MacConkey agar
• CLED agar
• TCBS agar
• XLD agar
MacConkey medium
•Distinguish between lactose fermenters & non
lactose fermenters.
•Lactose fermenters – Pink colonies
•Non lactose fermenters – colorless colonies
 Xylose Lysine DeoxycholateAgar(XLD)
• Used for the recovery of Salmonella andShigella
species.
Cysteine Lactose Electrolyte
Deficient Agar(CLED)
• For cultivation of pathogen from urine specimen ,
inhibit swarming of proteus sp.

CLED,serratia
Thiosulfate-citrate-bile salts-
sucrose agar(TCBS)
• highly selective for the isolation of V. cholerae
and V. parahaemolyticus

Yellow coloured (sucrose fermenting) colonies

of Vibrio cholerae on TCBSagar.
 Media used for transporting the samples.
 Delicate organisms may not survive the time
taken for transporting the specimen without a
transport media.
 Eg:
– Stuart’s medium
– Buffered glycerol saline
• These media are used to grow anaerobic organisms.

Eg:
• Robertson’s cooked meat medium.

• Thioglycolate broth medium.

 Ph meter
 Beaker glass, erlenmeyer
 NaOh for neutralize the media
 Media
 Scale
 Waterbath
 Safety Cabinet
 Autoclave
 Steam Indicator tape
 Scissors
 Sterilize the area with desinfectant
 Close doors
 Weigh the ingredients or dehydrated powders on
a balance
 Prepare the aquabides or distilled water and
check the pH by pH meter
 Mix and recheck the pH (optimally 6,5-7,5)
 Sterilize the medium in autoclave by cover it with
alluminium foil for 15 minutes in 121 celcius
temperature
 Wait until lukewarm
 Dispense approximately 20 ml into sterile plates
 Store these plates on incubator for 24 hours
 Media sterilization : use autoclave for 15
minutes and 121 celcius – until reach 50
celcius – distribute into petri plates each 20-
25 ml (except for T.C.B.S Cholera Medium
and S.S Agar)
 Four most critical environmental factors to
consider include oxygen, carbondioxide
availability, temperature, pH, moisture
content of medium and atmosphere
 Why QC culture media?
◦ A regulatory expectation
◦ Variations with manufacturing
 Including sterilisation
◦ Variations with the quality of raw materials
◦ Transport conditions
◦ Storage conditions
◦ Different applications of media in different
environments
 Physical parameters (Visually)
◦ Solid media: excessive bubbles or pits, unequal
filling of plates (uniform levelling), cracked medium
in plate, gel strength and freezing or crystallization
◦ Liquid media: Clarity of broth
◦ All media: pH value of the medium (pre- and post-
sterilisation)
 Storage conditions are important
◦ Routine storage
◦ When and how expiry time is assessed
 Different storage conditions affect:
◦ Drying out (due to different humidity levels, which
can affect the water activity of solid media)
◦ Chemo-oxidation (due to physical factors like heat)
◦ Photo-oxidation (from sunlight)
 Test on media sterility (Sterility Test)
◦ A small number of uninoculated items are
incubated alongside the test items for the same
period of time.
◦ No growth in these items is often part of a
successful test release.
 Mengambil sejumlah 5 %volume dari tiap
wadah media yang dibuat.
 Media diinkubasi selama 1-2 hari pada suhu
35° C.
 Apabila terdapat pertumbuhan lebih dari 2
koloni mikroorganisme/cawan petri atau
lebih, hal itu menandakan seluruh media dari
wadah tersebut tidak dapat digunakan.
 Uji spesifitas dengan penanaman
mikroorganisme kontrol positif dan control
negatif. Mikroorganisme kontrol kualitas
(strain kuman) adalah mikroorganisme
spesifik yang seharusnya tumbuh pada media
tertentu. Mikroorganisme tersebut memiliki
ciri morfologi, biokimia, serologi yang dapat
diuji dan mampu menunjukkan stabilitas
reproduksi yang tetap ketika ditempatkan
pada kondisi yang sesuai.
 Important due to the variations of
manufacturing
◦ As a routine
◦ When the supplier changes or moves premises
◦ To follow up customer complaints
◦ Check tests conducted by supplier:
 Growth promotion
 pH
 Gel-strength
 Clarity and colour
 THANK YOU