Propagating the microorganisms under
laboratory condition is known as cultivation. To accomplish this, one must know
the:
a. Food
nutrients required
b. Proper
environmental conditions
Nutritional requirements
1. Carbon
source
Autotrophs or
lithotrophs - require only CO2
for their carbon source.
Heterotrophs
or organotrophs – require and organic form of carbon
a. Saprophytes
– survive in dead organic matter.
b. Parasites
– require living organism for life
2. Nitrogen
source
Some uses
atmospheric nitrogen, some thrive on inorganic nitrogen compounds and others
derive their nitrogen from proteins or any naturally occurring organic nitrogen
compounds.
3. Minerals
In the form of
organic sulfur and free inorganic phosphates
4. Metallic
elements
Enzyme
activators like Mg, Fe, Na, K, Mn, Zn, Cu and Co.
5. Growth
factors like vitamins and amino acids
6. Water
Physical conditions
1. Temperature
requirements
On the basis
of temperature requirement, bacteria are classified as:
a. Psychrophiles
or cryophiles (cold–loving) – grow at 20oC or less
b. Mesophiles
(moderate–loving) – grow at 25oC–40oC
c. Thermophiles
(heat–loving) – grown between 45oC–60oC
Facultative
thermophiles or eurithermophiles – these are thermophilic bacteria that extends
into the mesophilic region
Stentothermophilic
– thermophilic bacteria that grows above 60oC.
2. Gaseous
requirements
The principal
gases that affect bacterial growth are oxygen and carbon dioxide. Based on oxygen
requirement, bacteria are divided into:
a. Aerobic
– grows in the presence of free oxygen
b. Anaerobic
– grows in the absence of free oxygen
c. Facultatively
anaerobic – grown in either absence or presence of free oxygen
d. Microaerophilic
– grows in the presence of minute quantities of free oxygen.
3. pH
or Hydrogen ion concentration
This is the
degree of acidity or alkalinity. For most bacteria, the optimum pH for growth
lies between 6.5 and 7.5. Some grow as low as pH 3 like the fungi and others
grow as high as pH 10.5 like the vibrios
4. Miscellaneous
physical requirement
a. Source
of energy
Photosynthetic
autotrophic – requires light as source of energy
b. Osmotic
or hydrostatic pressure
Osmophilic
bacteria – requiring high osmotic pressure
Others are
isolated from the deepest ocean trenches
c. High
concentration of salt
Halophilic
bacteria
THE CULTURE
MEDIA
A culture media is any material
containing essential nutrients for the growth and multiplication of bacteria.
Practically, all media are available commercially in powdered form.
Types of culture media
1. According
to physical state / consistency
a. Liquid
– contains no agar, no gelatin
e.g.
Thioglycollate, Trypticase Soy Broth
b. Solid
– with agar, gelatin or albumin
Agar – is a
polysaccharide derivative of seaweed, dissolves in water at boiling point and set
at 38oC. It is a solidifying agent with a concentration of 1.5 to
3%.
Gelatin – used
as a diagnostic medium and solidifies at 25oC and has 10 to 15%
concentration
Albumin –
coagulating factor of the agar
e.g. Blood
Agar Plate, Eosin Methylene Blue, TSI, LIA
c. Semi–solid
– contains 0.5 to 1% agar. It is used to demonstrate “swarming” growth of some
species of Proteus. Also used in motility test.
e.g. SIM
2. According
to application of function
a. Simple,
ordinary or basal – basic medium form which culture media are prepared and
supports the growth of many common bacteria
b. Enriched
– prepared so as to duplicate the natural environment of the desired bacteria
or fastidious heterotrophs that are difficult to grow.
e.g. Blood
Agar Plate, Chocolate Agar Plate, Loeffler’s serum agar
c. Differential
– differentiates two or more species of microorganisms by their colony
characteristics on the same medium due to the dye or indicator incorporated in
the medium
Eosin
Methylene Blue agar – eosin and methylene blue
Mannitol Salt agar – phenol
red
Simmon Citrate agar –
bromthymol blue
McConkey agar – neutral red
d. Selective
– allows the growth of some bacteria while preventing or suppressing the growth
of others because of inhibitory substances in the medium.
Inhibitory
substances Inhibits
Gentian Violet gram
positive bacteria
Sodium
Desoxycholate
Bile salts
Chloral
hydrate spreading
of Proteus
Alcohol
Sodium azide gram
negative bacteria
Potassium
tellurite
Penicillin /
Streptomycin bacterial
invaders or contaminants
Malachite
Green
e.g.
Salmonella–Shigella agar, Petragnanni medium, Mannitol Salt agar,
Saboraud’s agar
3. According
to composition
a. Synthetic
or chemically defined – pure chemicals which substitute the requirement derived
from natural sources.
e.g. Glucose –
inorganic salts – for E. coli
b. Non–synthetic
– exact composition is not known.
e.g.
Commercially prepared media
c. Tissue
culture – made of living cells
e.g. HeLa cell
lines
4. According
to form or distribution
a. Plated
medium – placed on a petri dish
b. Tubed
medium – placed on a test tube
Preparation of culture media
1. Weighing
of different ingredients and placing appropriate container
2. Dissolving
the material in proper amount of distilled water
3. Titration
– adjusting to right pH
4. Sterilization
5. Distribution
on sterile petri dishes
·
For tubed method – distribute
before sterilize
·
Inside refrigerator – normal
position (cover is on top)
·
Inside incubator – inverted
position, to prevent evaporation
Aerobic cultivation of large quantities
1. Use
of suitable containers such as Kolle flask, Roux bottle and Fernbach flask.
2. Constant
shaking of liquid medium to introduce oxygen.
3. Forcing
air through the medium by pressure or suction.
Anaerobic cultivation
1. Addition
of reducing agent such as sodium thioglycollate
2. Mechanical
removal of oxygen from an enclosed vessel, by pumping the air out and replacing
it with nitrogen, hydrogen or H2–CO2 mixture (GasPak).
3. Sealing
tubes of agar with a layer of petrolatum, oil or paraffin
4. Chemical
reaction within an enclosed vessel containing the inoculated medium to combine
free O2 into a compound by combustion:
a. Burning
of a candle – Candle Jar method
b. Use
of Brewer anaerobic jar
5. Chemical
reaction using a mixture of 40% pyrogallic acid and NaOH
6. Use
of enriched agar in deep tubes
Containers
with bacteria are placed in an incubator, an apparatus in which constant degree
of temperature (37oC) is maintained for purpose of growing cultures
of bacteria.
Control of culture media
1. Follow
exact directions of the manufacturer regarding methods of preparation and
sterilization (overheating is the most frequent source of error).
2. Sealing
and dating properly all dehydrated media
3. Check
the pH of the final product
4. Freshly
prepared media are preferable for anaerobic bacteriology
Quality
control program is best carried out by performance testing for the desired
reaction with a number of stock cultures of known microorganisms of known
stability.
Stock culture
media may be safely stored for months if care is taken to retain moisture.
Transport media
1. Amies
transport medium with charcoal – maintenance of fastidious organisms in
original specimen
2. Carry
and Blair – for feces
3. Stuart’s
THE PROCESS OF
INOCULATION
Inoculation is a process of implanting
microbes or infectious material into a culture media. Cultures are growth of
microorganisms on nutrient medium while culture means to grow the
microorganisms on such medium.
Types of cultures:
1. Pure
cultures – when microorganism in a culture medium is all of the same species.
2. Mixed
cultures – when there are two or more different species of microorganism
growing in a culture medium.
3. Contaminated
cultures – when the cultures accidentally contain more than one species of
microorganisms.
4. Stock
culture – is a pure culture of microorganisms used as a source of supply for
research, industry or student use.
Manner of inoculating a culture
medium:
A culture medium is inoculated by
streaking or swabbing gently a previously sterilized swab or platinum loop or
needle containing such as microbes, sputum, urine, blood or pus.
a. Petri
dish containing the culture medium – by streaking using either:
(1) Simple
streaking
(2) Radial
method
(3) Clock
method
b. Butt
culture medium – by stabbing
Using a
previously sterilized needle, stab the butt medium at the center. The portal of
entry of the needle should be the same as the portal of exit.
c. Butt–slant
culture medium – stab and streak
Using a
previously sterilized needle, the center of the butt portion is stabbed,
withdraw the needle and streak in a “zigzag” motion up to the upper portion of
the slant.
d. Slant
culture medium – streak
This is
inoculated by streaking the surface of the slant with a previously sterilized
loop with a “zigzag” motion up to the upper portion of the slant.
e. Liquid
medium – incline the tube at an angle and rub the material against the tube
wall.
Inoculating loop
or needle should be flame–sterilized before and after using it. The neck of the
tube or bottle of culture should be flame–sterilized before and after re –
capping or replacing the cap.
In incubation,
petri dishes containing the inoculated agar should be placed inside the
incubator in an inverted position to prevent the accumulation of moisture in
the cover to drop into the medium and destroy colonies.
Methods employed in obtaining a
pure culture:
1. Streak
plate method – surface streaking
The routine procedure for isolating bacteria in a pure culture.
2. Pour
plate technique
The dilution
(thinning) of the specimen in tubes of liquid (cooled) agar medium. The streak
plate and pour–plate techniques can be made more effective for isolating
specific kinds of bacteria by using selective or differential media.
3. Enrichment
– culture technique
It provides a specially
designed cultural environment which will favor the growth of the particular
type of bacteria being sought but will be unsuitable for the growth of other
types.
4. Serial
dilution technique
Series of
dilutions in tubes of an appropriate medium containing decreasing number of
bacteria.
5. Single–cell
isolation technique
This technique
uses a micromanipulator to pick out a single organism from a hanging drop
preparation.
Pure cultures
should be maintained and preserved by:
a. Periodic
transfer to fresh media
b. Overlaying
cultures with mineral oil
c. Lyophilization
– drying in a frozen state
d. Storage
at a very low temperature
Cultural characteristics
One of the major features of bacteria
is their appearance following growth on various media. Such commonplace
characteristics as the color, abundance of growth and even the odor of the
culture provide useful clues for identification.
1.
Agar plate
colonies
a. Size
– colonies range in size from extremely small (pinpoint) to large colonies.
Many form a colony of a limited size regardless of the period of incubation.
Others, like Pseudomonas and Proteus spread across the entire agar surface.
b. Margin
or edge – it may be evenly circular or it may show irregularities as rounded
projections, irregular notches, threadlike or rootlike projections.
c. Elevation
– colonies may either be flat or raised.
d. Chromogenesis
or pigmentation – colonies may be colored or not.
e. Optical
features – maybe opaque, translucent or opalescent
2.
Growth on agar
slant
a. Amount
– scanty, moderate or abundant
b. Margin
or edge of growth – similar to agar plat
c. Consistency
of mass growth – butyrous or butterlike consistency, easily removed with
transfer needle; viscous or stingly; dry and brittle.
d. Chromogenesis
or pigmentation – similar to that described for colonies.
3.
Growth on
nutrient broth
a. Amount
– scanty, abundant or moderate
b. Distribution
or growth – evenly turbid; growth confirmed to surface of broth as a scum or
film (pellicle); or growth accumulated as sediment, which may be granular or
viscous.
c. Odor
– may be putrid, fruity or aromatic or negligible
4.
Growth in
gelatin stabs
a. Growth
(no liquefaction) along line of inoculation – growth may be confined to zone of
inoculation streak or may exhibit varying degrees of spreading away from this
streak.
b. Liquefaction
of gelation – may start evenly from top or various designs of liquefied medium
(funnel–like) may occur.
Chromogenesis is one of the most
striking cultural characteristics. Not all bacterial species have this
distinctive feature. Not all bacterial species have these distinctive features.
In some, the pigment is retained within the cell and the mass of bacterial
cells is colored; others the pigment is excreted and colors the medium.
The intensity of the pigment is
influenced by the comparison of the medium and the conditions of incubation.
Pigment production is best observed from growth on solid media.
Enrichment broth:
1. Alkaline
peptone water Vibrio
cholera
2. Selenite
F broth Salmonella
and Shigella
3. Tetrathionate
broth Salmonella
except S. typhi
4. Gram
negative broth Salmonella
and Shigella in feces
Ringer’s
solution – for dissolving calcium alginate swab
CULTURE MEDIA PURPOSE
Blood
Agar Plate cultivation
of fastidious organisms;
determination
of hemolytic reactions
Brain
Heart Infusion Agar cultivating
the pneumococcus for the liquid
bile
solubility test
Brilliant
Green Agar highly
selective for the isolation of Salmonella
Brucella–Vit
K1 Blood Agar for the isolation
and subculture of anaerobes
Campy–blood
agar selective for the isolation of Campylobacter
Buffered
Charcoal Yeast Extract selective for
Legionella sp.
CDC
anaerobic agar isolation
of anaerobic organisms
Cefsulodin–Irgasan–
Novobiocin
Agar selective for
Yersinia sp.
Chocolate
Agar cultivation
of Haemophilus and Neisseria
Columbia
Colistin–Nalidixic
Acid
agar (CAN) selective for
isolation of gram positive cocci
Cycloserine–cefoxitin
fructose selective for Clostridium
difficile
Agar
(CCFA)
Cystine–lactose
electrolyte isolation
and enumeration of bacteria in
Deficient
Agar (CLED) urine
Cytine
Tellurite Blood Agar isolation of
C. diptheriae
Decarboxylase
test media for
differentiating members of the
Enterobacteriacea
Desoxycholate
Agar for
the isolation of gram negative enteric
Bacilli
and the differentiation of lactose–
fermenting
and non–lactose fermenting
species
DNA
agar for
testing S. aureus for thermostable nuclease
Dextrose
Ascitic fluid
Semisolid
agar for spinal fluid
cultures
Ellinghausen,
McCullough,
Johnson
and Harris (EMJH) cultivation
of leptospires
medium
Fetal
Bovine Serum agar with isolation of
H. ducreyi
Vancomycin
Gelatin
medium (dilute) differentiation
of Nocardia and Streptomyces
Hektoen
Enteric Agar (HEA) for isolation
and differentiation of gram
negative
enteric pathogens; coliforms are
salmon
to orange in color; salmonella and
shigella
are bluish green
Hemin
solution as
a supplement for recovery of Hemophilus
species
from respiratory culture
Horse
blood–bacitracin agar as a selective
medium for Hemophilus species
Human
Blood Bilayer (HBB) selective and
differential isolation of
Gardnerella
vaginalis
Kanamycin–Vancomycin
for primary inoculation of
clinical specimens
Blood
Agar (KVBA) for
selective isolation of anaerobes, particularly
Bacteroides
Kelly’s
medium non–selective
modified for the isolation of
B.
burgdorferi & other spirochetes
Lombard–Dowell
agar isolation of
anaerobic organisms
Lysine
Iron Agar for
determining whether members of the
Family
Enterobacteriacea can decarboxylate
or
deaminate lysine
MacConkey
agar Inhibitory
for gram positive bacteria and
differential
for enteric bacteria rather than
selective
McBride
medium cultivation
of Listeria
Middlebrook
7H10 agar isolation of and
antimicrobial susceptibility
testing
of Mycobacteria
New
York City (NYC) agar selective
for Neisseria gonorrhea and
M.
hominis
Methylene
Blue milk identification
of enterococci
Milk
media – Litmus milk determination
of litmus milk reactions of
Clostridium
Mueller
Hinton Agar Useful
for the disk agar diffusion method of
testing
for antimicrobial susceptibility testing
Oxidative–Fermentative
(O–F) differentiation of Pseudomonas,
Alcaligenes,
Basal
medium Acinetobacter
from Enterobacteriacea
Peptic
Digest Agar a
good medium for H. influenza
Phenylethanol
Agar (PEA) isolation of gram
positive cocci and inhibition
of
gram negative bacilli particularly Proteus
Schaedler
Agar Nonselective
medium for the recovery of
anaerobes
and aerobes
Scott’s
modified Castaneda For blood
cultures
Media
and Thioglycollate broth
Seller’s
differential agar for
differentiating and identifying
Nonfermentative
gram (–) bacilli that produce
an
alkaline reaction on TSI agar; useful in
differentiating
Pseudomonas aeruginosa,
Acinetobacter
calcoaceticus and Alcaligenes
fecalis
Sodium
bicarbonate supplement
for anaerobes
Sodium
chloride broth selective
for enterococci and salt tolerant
organisms
Streptococcal
selective agar selective for
Streptococcus pyogenes
Thioglycollate
medium recovery of
anaerobes, aerobes,
without
indicator microaerophilic
& fastidious organisms
Tinsdale
agar isolation
of C. diphtheria
Todd–Hewitt
broth for growing streptococci for serologic
identification
Trypticase
soy agar Excellent blood agar base and can be
used
For
the isolation and maintenance of all
organisms
Trypticase
soy broth supports
growth of pneumococci and
Streptococci
and Brucella
Tween
80–albumin broth cultivation
of spirochetes
Xylose Lysine
Desoxycholate isolation &
differentiation of Salmonella
(XLD) agar &
Shigella agar from other enteric bacilli
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