Transcript Culture media

Bacterial Cultivation
(Culturing)
Function of
Clinical Microbiology Lab
• Participation in management of patients
with infectious diseases by processing of
clinical specimens:
1- Microscopy.
2- Cultivation & isolation of pathogens.
3- Identification of isolates: macroscopy,
microscopy, biochemical testing & serology.
4- Antimicrobial susceptibility testing (AST)
of isolates.
Bacterial Cultivation
Definition & Purposes
• Collection of bacteria-containing clinical specimens from
patient’s infection site (i.e. in vivo) & growing these
bacteria in artificial Lab bacteriological culture media (i.e.
in vitro) → isolation of dispersed (individual), significant
(↑ number), pure bacterial colonies → macroscopic
examination of colony characteristics on primary culture
media → early preliminary information regarding bacteria
isolated → subsequent laboratory confirmatory
procedures → definitive characterization & identification
and Antimicrobial Susceptibility Testing (AST) → interim
& final reporting to the clinician.
Bacterial Cultivation
Growth Requirements
• The appropriate growth requirements for in
vitro bacterial cultivation include:
1- Nutritional needs in culture media.
2- Environmental/ incubation conditions.
Growth Requirements
1- Nutritional needs
• Water: used to dissolve
materials to be transported
across the cytoplasmic
membrane.
• Source of carbon (glucose):
required for the construction of
all organic molecules.
• Source of nitrogen: obtained
either inorganic or organic
source.
• Source of minerals.
• Buffer System.
Growth Requirements
Nutritional needs
• Nutrients as blood, milk, yeast extract & beef
extract are incorporated into culture media.
• Pathogenic bacteria are classified into:
Fastidious (needs are relatively complex, with
exceptional media components) vs Nonfastidious (needs are relatively basic &
straightforward).
• various types of culture media are used in
Microbiology lab according to different needs of
pathogenic bacteria.
Growth Requirements
2- Environmental/ Incubation conditions
• Four environmental (incubation) conditions to
support the growth of clinically relevant bacteria
include:
 Oxygen & carbon dioxide (CO2) availability.
 Temperature.
 pH.
 Moisture content of medium & atmosphere.
Oxygen & Carbon dioxide (CO2) availability
Oxygen (O2)
• Most clinically relevant bacteria are either:
• Aerobic: use oxygen as a terminal electron
acceptor & grow well in room air.
• Facultatively anaerobic: being able to grow in
presence (i.e. aerobically) or absence (i.e.
anaerobically) of oxygen.
• Strictly anaerobic: are unable to use oxygen as an
electron acceptor.
Oxygen (O2)
• some bacteria, as Pseudomonas spp., members
of Neisseriaceae family, Brucella spp.,
Bordetella spp., & Francisella spp., are strictly
aerobic & cannot grow in absence of oxygen.
• Other aerobic bacteria require only low levels of
oxygen & are referred to as being
microaerophilic, or microaerobic.
Carbon dioxide (CO2)
• Capnophilic organisms grow best with higher
CO2 concentrations (i.e. 5-10% CO2) than is
provided in room air.
• For some bacteria, 5-10% CO2 concentration is
essential for successful initial cultivation from
clinical specimens.
Temperature
• most medically important bacterial pathogens
generally multiply best at temperatures similar to
those of internal human host tissues & organs (i.e.
37°C) → incubators with temperatures 35°-37°C are
used for cultivation.
• Incubation at certain temperatures can be used as
an enrichment procedure for enhanced recovery of
certain organisms:
 Campylobacter jejuni grows at 42°C, whereas many
other pathogens & non-pathogens cannot.
 Cold enrichment for Listeria monocytogenes &
Yersinia enterocolitica at 0°C, however they grow
best at temperatures between 20° & 40° C.
pH
• Most clinically relevant bacteria prefer a near
neutral pH range (6.5-7.5).
Moisture
• Loss of water from media can be deleterious to bacterial
growth in two ways:
 less water is available for essential bacterial metabolic
pathways .
 relative increase in solute concentration of the media.
• Measures to ensure that appropriate moisture levels are
maintained throughout the incubation period include:
 sealing agar plates to trap moisture.
 using humidified incubators.
Culture media
• Culture medium or
growth medium is a
liquid or gel designed
to support the growth
of bacteria.
Culture Media
Physical Status
1- Liquid (Broth): contains NO agar.
2- Solid (Agar): contains 1.5% agar.
(N.B. Biphasic medium contains liquid &
solid parts e.g. Brucella Castaneda
medium).
3- Semisolid: contains 0.5% agar e.g.
Motility medium.
Culture Media
Broth media
• nutrients are dissolved in
water.
• Used 1- for growth of pure
batch cultures e.g. Nutrient
broth & 2- as Enrichment broth
e.g. alkaline peptone water for
Vibrio cholera.
• Growth is indicated by change
in appearance from clear to
turbid (i.e. cloudy).
• The more bacterial growth, the
greater the broth’s turbidity.
• At least 106 bacteria/mL of
broth are needed for turbidity
to be detected with unaided
human eye.
Culture Media
Solid media
• made by adding solidifying agent to nutrients &
water.
• Agarose:
polysaccharide
extracted
from
seaweeds, most commonly used solidifying
agent, No nutritive value, NOT affected by
growth of bacteria, & melts at ≥95°C but resolidifies at <50°C → allows for heating to
extremely high temperature required for
sterilization, cooling to 55-60°C for distribution
into petri dishes and tubes, & on further cooling,
forming stable solid gel (Agar).
Culture Media
Solid media
• Three forms:
1- Agar plate (petri dishes): provide large surface
area for culture. Incubated in inverted position
(agar-compartment upwards & dish-cover
downwards).
2- Agar Slant (+ butt): e.g. Triple sugar iron.
3- Agar Deep: used for storage & study of
gaseous requirements of bacteria.
Culture Media
Agar plates
Nutrient agar
Blood agar
Preparation of Culture Media
General
• Broth & agar media are prepared by
reconstituting (dissolving) specified amount of
powder in water (distilled or deionized).
• Boiling is often required to dissolve the powder.
• Most media require sterilization to allow only
growth of bacteria from patient specimens &
prevent growth of contaminants from water or
powdered media.
Preparation of Culture Media
Additives
• Other sterile supplements as sheep blood or specific
vitamins, nutrients, or antibiotics, should be added
when molten agar has cooled, just before
distribution to plates.
• Delicate media components that cannot withstand
steam sterilization by autoclaving (e.g. serum,
certain carbohydrate solutions, certain antibiotics, &
other heat-labile substances) can be sterilized by
membrane filtration:
 Passage of solutions through membrane filters with
pores 0.2-0.45μm in diameter.
 This will effectively remove most bacterial & fungal
contaminants but NOT viruses.
Preparation of Culture Media
Agar Plate Medium
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Weight dehydrated media.
Dissolve in distilled H2O to 1L volume.
Adjust pH to 7.0
Boil media then distribute in bottles or large flasks
covered with plastic screw caps or aluminum foil
respectively.
• Sterilize for 15 minutes using autoclave or pressure
cooker at 121°C under 15 Ibs.
• Cool to 50°C, & pour molten agar into individual petri
plates.
Preparation of Culture Media
Tube Medium
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Weight dehydrated media
Dissolve in distilled H2O to 1L volume.
Adjust pH to 7.0
Boil media then distribute in individual test tubes.
Sterilize for 15 minutes using autoclave or pressure
cooker at 121°C under 15 Ibs.
• Cool to 50°C, & pour into individual tubes:
 For slant agar, place tubes in slant position until cool.
 For broth media or deep agar, place tubes in upright
position until cool.
1- Assemble all chemicals in
work area before you begin
2- Accurately weigh each of the dry
ingredients in your culture media
3- Add each dry culture medium
ingredient to the culture flask
4- Add distilled (or
deionized) water to make
the correct volume. Heat
& stir (agar will burn if
it is NOT stirred) until
all of the ingredients go
into solution. When the
media boils, it is ready
for sterilization.
5- Media sterilization
6- Sterilize for 15
minutes using the wet
cycle (autoclave) or at
maximum pressure in a
pressure cooker.
Remember to cover the
top of the flask or jar
with aluminum foil to
prevent contamination
when the media cools.
N.B. When using a pressure
cooker, don’t over fill the cooker!
7- Line your sterile petri
plates along the edge of the
table. Transfer hot media to
small sterile container &
pour 15-20 ml of culture
media into each petri plate.
The petri plate lid should be
open slightly, but NOT
completely open as this
increases contamination.
The Petri Porter
12-Apr-17
NM Kaplan
35
Culture Media
Types
• Four general categories of
according to their function & use:
 Supportive.
 Enrichment.
 Selective.
 Differential.
media
Culture Media
Supportive
• contain nutrients that • Nutrient agar plate
support growth of
most non-fastidious
organisms
without
giving any particular
organism a growth
advantage.
• Examples: Nutrient
agar & Nutrient broth.
Culture Media
Enrichment
• Contain nutritive substances like blood, serum, or egg →
grow most bacteria e.g. Blood agar & Chocolate agar.
• Some contain specific nutrients → grow particular
bacterial pathogens that may be present alone or with
other bacterial species in patient specimen e.g. Buffered
charcoal-yeast extract (BCYE) agar that provides Lcysteine & other nutrients required for growth of
Legionella
pneumophila
(causative
agent
of
legionnaires’ disease).
• Enrichment broth: used to enhance the growth of
particular bacterial pathogen from mixture of organisms
e.g. selenite F broth (Salmonella species) & alkaline
peptone water (Vibrio cholera).
Blood agar
Chocolate agar
Growth of Legionella pneumophila
on BCYE enrichment agar
Culture Media
Selective
• contain one or more agents that inhibit the
growth of all unwanted organisms except those
being sought.
• Inhibitory agents used include dyes, bile salts,
alcohols, acids, & antibiotics.
• Example: MacConkey agar & SalmonellaShigella agar.
Culture Media
Differential
• contain an indicator as pH indicator that allows
colonies of one bacterial species to exhibit
certain metabolic or culture characteristics that
can be used to distinguish them from other
bacteria growing on the same agar plate.
• Examples:
 MacConkey agar that differentiates between
lactose-fermenter (LF) & non lactose-fermenter
(non LF) Gram-negative bacteria.
 Blood agar & different hemolytic patterns.
Culture Media
Many with more than one function
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Selective & differential:
MacConkey agar: Gram negative bacilli as LF vs. NLF.
Mannitol Salt agar (MSA): Staphylococci.
Hektoen
enteric
(HE)
agar
&
Xylose-lysinedesoxycholate (XLD) agar.
• Enrichment & differential as sheep blood agar which
contains general nutrients & 5-10% sheep blood →
different hemolytic patterns of Streptococcus species
colonies.
MacConkey agar
• Selective for Gramnegative bacilli due to bile
salts & crystal violet that
prevent growth of most
Gram-positive bacteria.
• Differential due to
lactose (sugar) & pH
indicator neutral red:
• Lactose fermenter; acidic
pH; deep purple colonies.
• Non-lactose fermenter;
alkaline pH; colorless
colonies.
Mannitol Salt Agar
• Selective for staphylococci
due to high salt (NaCl)
concentration (7.5%).
• Differential due to mannitol
(sugar) & pH indicator
phenol red:
• Mannitol fermenter; acidic
pH; yellow colonies; S
aureus.
• Mannitol nonfermenter; red
colonies; alkaline pH; S
epidermidis & S
saprophyticus.
Different hemolytic patterns on
Sheep Blood agar
1- Beta hemolysis:
complete lysis of RBCs →
colorless zone surrounding
the colony.
2- Alpha hemolysis: zone
of partial hemolysis
surrounding the colony,
often accompanied by
greenish discoloration of
agar.
3- Gamma reaction: NO
hemolysis or discoloration of
agar surrounding the
colony.
Bacterial Cultivation
Tools
• Cotton swabs.
• Plastic pipettes.
• Loops & wires/ needles:
1- Plastic.
2- Metallic: made of platinum or
Ni-chrome; sterilized by Flaming =
incineration of all life forms, which
begins from handle towards the
tip to prevent bacteria from
forming aerosols (= water droplets
of live bacteria sprayed into air).
Bacterial Cultivation
Tools
Bacterial Cultivation
Culturing Methods
in Broth medium
• by direct inoculation of
clinical specimen using
cotton swab or plastic
pipette.
on Agar plate
1- Streak-plate method:
(commonest & routinelyused).
2- Pour-plate method.
3- Spread-plate method.
(2 & 3 uncommonly used).
1- Streak-plate method
1- direct inoculation of one loop-full of the
specimen onto small peripheral area of the surface
of well dried agar plate.
2- Streaking in a standard pattern to distribute
inoculum thinly in a series of parallel lines in
different segments of the plate using loop (with
flaming between different series) → even
spreading over the entire surface.
3- Following incubation, separated colonies are
obtained over the last series of streaks.
Inoculation & Streaking with flaming
Semi-quantitative culturing using calibrated loop streaking technique =
inoculation of measured amount of liquid specimen as urine for
enumeration of bacterial colonies (colony-forming units; CFUs).
Streaking Pattern
Well-isolated dispersed
separated colonies
Contamination
2- Pour-plate method
3- Spread-plate Method
Bacterial Cultivation
Subculturing
• Transfer of microorganisms from one
media to another.
• Purposes:
1- Isolation of pure culture from mixed one.
2- Performance of microbiological test
procedures.
3- Preparation & maintenance of stock
culture.
Isolation of pure culture from
mixed one
Picking Colonies
General Aseptic Precautions
1. Sterilize the bench working area before & after use.
2. Sterilize metallic inoculation loop or needle before &
after use.
3. Work quickly & never place the tube’s plug on bench.
4. Flame the lips of the tube before & after work.
5. Never leave the tube open any longer than the time
needed to transfer the culture.
6. The container must be covered to prevent entrance of
microorganisms.