BACTER45P- Media Preparation

Diagnosis of Disease
Initial step in studying morphology and
identification
Obtain Antigen from developing serological
assays or vaccines
Genetic Studies
Reliable in estimating the number of viable
count
Separate and isolate bacteria in mixtures

The survival and growth of

microorganism depend on
available nutrients and
favorable growth
development

When culturing
bacteria, it is very
important to provide
similar environmental
and nutritional
conditions that exists
in its natural habitat

Artificial Culture Media

contains:
Water
Some of carbon energy
Source of nitrogen
Trace elements
Growth factors
Appropriate pH

Water
Peptone
Casein hydrolysate
Meat extract
Yeast extract
Malt extract

Consistency
Nutritional
component
Functional use

A.
B.
C.
D.

Liquid mediA
Solid Media
Semi Solid Media
Biphasic Media

Used to propagate large numbers of

microorganisms in fermentation studies and
various chemical tests
Suitable to grow bacteria when the numbers
in the inoculum is suspected to be low
Used when a large number of bacteria have
to be grown

Inoculating in the liquid medium also helps to

dilute any inhibitors of bacterial growth
Used to obtain viable count( dilution method)
Presence of more than one type of bacteria
can not be detected.

Example:

Supports growth of all types of organism

Resazurin- indicator of anaerobicity
O.075% agar prevent convection current
From carrying atmospheric oxygen through out
the broth
Thioglycolic Acid( Reducing Agent) create an
anaerobic environment deeper in the tube.Thus
allowing anaerobic bacteria to grow.

1.If only the top has growth, that means it is a strict aerobe because it is growing only in the
top part.
2. If the bottom has growth, it means its a strict anaerobe (aka obligate anaerobe) because it
can grow in the portion furthest away from the oxygen.
3. If there is growth throughout, but mostly at the top, it is a facultative bacteria since it
likes aerobic respiration best but can grow without it as well.
4. If there is only growth just below the very top, thats indicative of a microaerophile, because it
needs oxygen to live, but not a lot of oxygen, so its not going to grow closest to the top.
5. If there is growth throughout evenly diffused, thats indicative of an aerotolerant anaerobe as
the presence of oxygen doesnt affect them.

Any liquid media by the addition of certain

solidifying agents
Used:
1. Surface growth of microorganism(colony
appearance
2. Pure culture inoculations
3. Storage of culture
4. Biochemical reactions

1-3 % agar concentration to make the

medium solid
Agar is the most commonly used solidifying
agent.
Obtain from cell membranes of red

algae(Gelidium)
Long chains polysaccharide(70% agarose and 30%
agaropectin)
Melts @ 95c and soilidifies @ 42c

Example:

Agar of 0.2- 0.5% renders the medium semi

solid
Fairly soft
Useful in demonstrating bacterial motility
Motility is best observed at room
temperature
Example: SIM, Stuarts Amies, Cary Blair

Culture system comprises of both liquid and

solid medium in the same bottle
Besides agar, egg yolk and serum can be used
to soilidify culture media
Serum and egg(normally liquid) can be
rendered solid by coagulation using heat
Example: Loefflers Serum Slope, Lowenstein
Jensen Medium
Dorset egg Medium-solidified as well as
disinfected by a process of inspissation

Simple
Synthetic
Complex
Tissue

Can support non fastidious

bacteria
Non fastidious- able to grow
with minimal requirements
Peptone water and NA

Exact composition is known

Example: Davis and Mingioli Medium- Research
Purposes

Has ingredients whose exact components are

difficult to estimate
Contain at least 1 component which is not
chemically defined example: blood , serum,
yeast

For organism which cannot grow on cell free

media

Chick Embryo
Mc Coy cells (mouse cells)Chlamydia

Vero Cells-Derived from African

green Monkey Kidney Cells

A549 cells- Lung Carcinoma

Hela Cells- Cervical carcinoma
Hep2 Cells- Laryngeal Carcinoma

Basal Media
Enriched media
Enrichment media
Selective Media

Basically simple media that supports most

non fastidious organism
Example:peptone water, NA

contain nutrient supplement that supports

most non fastidious bacteria
Addition of extra nutrients in the form of
blood, serum, egg yolk, etc to basal medium
Used to grow nutrionally exacting (fastidiuos
bacteria)
Example: BAP, CAP, LSS

Liquid media that enhance the growth of

organism and serve to inhibit commensal in
the clinical specimen
Example: Selenite broth and tetrathionate
broth for Salmonella and Shigella
Alkaline peptone water- Vibrio

select for growth of a partial law organism.

- addition of certain inhibitory agents that
dont affect the pathogen.
Various approaches to make the medium
selective include the addition of antibiotics,
dyes, chemicals, alteration of ph, or a
combination of these.

Inhibitors for Gram + bacteria

1. Dyes : Gentian violet; crystal violet
2. Bile Salts: Sodium desoxychocolate

Inhibitors for Gram Negative Bacteria

1. Na azide
2. Potassium tellurite

- Different bacteria can be recognized on the

basis of their colony color.
- various approaches include , incorporation
of dyes, metabollic substrates so that those
bacteria that utilize them appeara as
differently colored colonies.
E.g. MAC, TCBS

Clinical specimen must be transported to the

laboratory immediately after collection to
prevent overgrowth of contaminating
organisms or commensals
Semi soilid media
Prevent drying(dessication) of specimen
Maintain the pathogen commensal ratio
Inhibit overgrowth of unwanted bacteria

Charcoal is added to
neutralize inhibitory factors
and to absorb fatty acids
which are toxic to some
bacteria.

Anaerobic bacteria need special media for

growth because they need low oxygen
content and reduced oxidation reduction
potential and extra nutrients
Boiling of media serves to expel any dissolved
oxygen
1% glucose, 0.1% thioglycollate, 0.1%
ascorbic acid, 0.05% cystein, reed hot iron
flings make the medium reduced

- a process of rendering a medium or material

free of all forms of life.

3 methods:
1. autoclaving 121c @ 15psi for 15 min
2.dry heat 160-170 for 2 hrs
-glasswares **
3. Bacteriologic filter -more than 121 c
- >0.22um

Ex. Scintered glass

Seitz asbestos pad filter
Cellulose or polycarbonate base *
Pls see safety consideration/precaution page 54.

1. refrigerator to avoid dehydration

2.agar plates are air tight
3.tube media with fitted capped is store at
room temp
4. agar/plate should be warmed before use

1. wear your mask,lab gown and gloves

2.disinfect the laboratory table top
3.prepare the plates,flask and tubes ( sterile)
4. prepare the media powder, distilled water
etc.
5. follow aseptic technique from preparation
to dispensing.
6.autoclave the media and store properly.

Before autoclaving ,cover each tube with

cotton
Place tubes in large beaker and cover with
foil.

BAP
* Add 100 ml of distilled water per flask
+4ml blood
Good for 5 plates of 20ml/plate
MRVP
(Broth) *add 75 ml distilled water and heat.
Dispense 5 ml/tube. Autoclave

SIM
(butt) * add 75ml distilled water and heat.
Dispense 5ml/tube. Autoclave.
LIA
(butt slant) * add 100 ml distilled water and
heat.
Dispense 7 ml /tube. Autoclave

TSI
(butt slant) * add 100ml distilled water and
heat.
Dispense 7ml/tube.Autoclave
SCI
(slant) * add 75ml distilled water and heat.
Dispense 5ml/tube. Autoclave

Common bacteriology media

BACTERIOLOGICAL MEDIA
BACTER45P

LOWENSTEIN JENSEN
MEDIUM
For Mycobacterium
tuberculosis
Inhibitor: MALACHITE GREEN

PHENYLETHYL ALCOHOL
For Gram positive bacteria; inhibits
gram negative bactera

MUELLER TELLURITE
For Corynebacterium
diphtheriae
Inhibitor: Potassium
tellurite

MAC CONKEY AGAR

For negative bacteria; inhibits gram
positive bacteria

 Thayer Martin
Vancomycin- inhibit gram positive bacteria
Colistin- inhibit gram negative bacteria except Neisseria
Nystatin- fungi inhibitor

Modified Thayer Martin

Thayer Martin plus Trimethoprim lactate which prevent the
swarming of Proteus

Martin Lewis

Vancomycin
Colistin
Trimethoprim lactate
Anisomycin- inhibit the growth of fungi

New York City Agar

Vancomycin+ Colistin+ Trimethoprim lactate and Amphotericin B
that inihibit the growth of fungi

Medium: Mannitol Salt Agar( Selective and Differential)

Inhibitor: 7.5 % NACl
CHO: Mannitol
pH indicator: Phenol Red (Acid= Yellow; Alkaline= Red)
Mannitol Fermenter- Yellow colony due to
production of acid. Example: S. aureus
Non Mannitol Fermenter- Pink- S. epidermidis and
S. saprophyticus

Medium: TCBS( Thiosulfate Citrate Bile Salts)

CHO: Sucrose
pH indicator: Bromthymol blue
Acid: Yellow; Alkaline: Yellow
Sucrose fermenter- Vibrio cholerae; Vibrio alginolyticus
Non sucrose fermenter- Vibrio parahemolyticus- green
colony

Selective/ Differential Media for Gram negative enteric

bacilli
Inhibitor: Crystal Violet and Bile Salts
CHO: Lactose
Lactose Fermenter: Pink Colonies; presence of lactose
permease and beta galactosidase to degrade lactose
Late Lactose Fermenter: only beta galactosidase present
Non Lactose Fermenter: Colorless Colony; no enzyme
present to degrade
the lactose

Rapid Lactose
Fermenters
Escherichia
Klebsiella
Enterobacter

Late Lactose
fermenter
S. arizoane
S. sonnei
Serratia
Hafnia
Yersinia

Non Lactose
Fermenter
Salmonella
Shigella except S.
sonnei
Proteus
Providencia
Morganella
Edwardshiella
Erwinia-plant
pathogen

Eosin Methylene Blue

Selective and differential Media

CHO: Lactose
Lactose fermenter: Pink to Purple
Colonies
Non Lactose fermenter: Colorless
Colony
E.coli- Green Metallic Sheen
Klebsiella- Pink Mucoid colonies
Enterobacter- Pink colonies with dark
center FISH EYE

Purpose: Blood agar is used to determine the ability of an

organism to hemolyze erythrocytes (RBCs).
Medical Applications The ability to produce hemolysis is
associated with virulence. Many pathogenic species of
Streptococcus, Staphylococcus and Clostridium demonstrate
hemolysis.
Streptococcus pyogenes, which causes streptococcal sore
throat, scarlet fever, rheumatic fever, and other diseases, is (
hemolytic.
S. pneumoniae, which causes pneumonia and pneumococcal
meningitis, is a hemolytic.
Most strains of Staphylococcus aureus, the causative agent of
boils, toxic shock syndrome, food poisoning and other
diseases, are hemolytic.
Clostridium botulinum and C. tetani are hemolytic and cause
botulism and tetanus, respectively.

Principle Exotoxins that cause destruction of RBCs (hemolysis) are

called hemolysins.
There are three categories of hemolysis.
hemolysis is the complete destruction of RBCs and hemoglobin and
results in a clearing around the growth on a blood agar plate.
Partial destruction of RBCs and hemoglobin ( hemolysis) produces a
greenish discoloration of the blood agar plate.
In hemolysis, there is no destruction of RBCs and hemoglobin, so
there is no change in the medium.
Alpha-prime-hemolysis () A small zone of complete hydrolysis
that is surrounded by an area of partial hemolysis

recommended for use in the isolation and cultivation of

fastidious organisms.
CHOC is an enriched medium supplemented with
cofactor, which provides NAD to facilitate the growth of
Haemophilus influenzae, Neisseria gonorrhoeae and
Neisseria meningitidis.
Heated sheep blood is added to give the medium its
chocolate appearance.
This medium is prepared, stored and dispensed under
oxygen-free conditions to prevent the formation of
oxidized products prior to use.

What is
essential is
invisible to
the naked
eye