Isolation of Bacteria

Into Pure Culture

Angelique Llorin
Noreen Lozano
Marielle Magpantay
Kristine Mendigorin
NTRODUCTON
A pure culture theoretically contains
a single bacterial species
- may be isolated by the use of
special media with specific chemical
or physical agents
Contamination means more than
one species is present in a culture
that is supposed to be pure
NTRODUCTON (cont.)
$impler methods for isolation of a
pure culture include:
(1) $pread plating on solid agar
medium with a glass spreader
(2) $treak plating with a loop, and
(3) Pour plating
NTRODUCTON (cont.)
Procedures require understanding of
the aseptic technique
8ep8i8 can be define as the
absence of infectious
microorganisms
Any technique designed to keep
unwanted microorganisms from
contaminating sterile materials
NTRODUCTON (cont.)
noculating loop8 or needle8
used to transfer microorganisms
Nichrome or platinum wire
$terilized between uses by flaming the
loop
Must be flamed before and after each
use
Inoculating loops/needles
NTRODUCTON (cont.)
!re8terilized pla8tic
loop8needle8
ADVANTAGE : they do not have to be
sterilized before use
DI$ADVANTAGE : they are single use
disposable items
Plastic loops and needles
NTRODUCTON (cont.)
!etri di8e8 (plate8 used to
culture microorganisms
$terile petri dish is filled with a solid
medium (agar) and the bacterial
culture is applied to the surface or
embedded within the medium
Petri plates
NTRODUCTON (cont.)
edia (8ing. medium
substance upon which a
microorganism is grown
Designed to mimic the environment in
which the organism naturally grows
Potato slices and the vitreous fluid of
cows eyes
Chemically defined or complex
noculate to implant microorganisms
or infectious material into a culture
media
NTRODUCTON (cont.)
A colony is visible mass of
microorganisms growing on a solid
medium
Is thought to have formed from
reproduction of a single cell so that all
the members of a colony are
descendant from that original cell
NTRODUCTON (cont.)
E. coli :
most studied and best understood
organism on the face of the globe
gramnegative rod shaped
bacterium
forms creamywhite colonies on
Nutrient Agar when incubated at
either 35C or room temp.
NTRODUCTON (cont.)
$. aureus :
grampositive spherical bacteria that
occur in microscopic clusters
resembling grapes
forms a fairly large yellow colony on
rich medium
often hemolytic on blood agar
$tapilococci are facultative anaerobes
that grow by aerobic respiration or by
fermentation that yields principally
lactic acid
NTRODUCTON (cont.)
. subtilis :
extremely common bacterium
rod shaped, grampositive
bacteria
NTRODUCTON (cont.)
Aerobic bacteria can grow freely in
oxygenrich environments
Anaerobic bacteria does not require
oxygen for growth
OBJECTVES
At the end of the activity, the
students are expected to:
1. Perform isolation techniques
2. Differentiate the microorganisms
isolated using spread plate and pour
plate techniques
3. Describe the distribution of colonies in
spread plate and pour plate
4. $tress the importance of pure culture
in microbiology
A method of isolating
individual bacterial strains
from a sample.
The goal is to obtain isolated
colonies on a large part of
the agar surface, so that
desired species can then be
brought into pure culture.
STREAK PLATNG
< Isolate genetically identical
bacteria, barring mutations,
without contamination of other
bacterial strains.
< Isolates aerobic organism
< $treak plates can be made
from a broth culture, an agar
slant or from an agar plate.
STREAK PLATNG
(CONT..)
STREAK PLATNG (CONT..)
noculating loop was heated until it glows red. t was slightly
cooled before touching the culture.
&sing aseptic technique
Mouth of the tube was flamed after removing the cap of the
test tube with mixed culture.
Loopful of the broth culture was picked out using the sterile
inoculating loop.
Mouth of the tube was flamed again. The cap was returned
to the tube.
STREAK PLATNG
(CONT..)
Using the T-method, the culture was spread over in the first
section. lways remember that it should always be in one direction
STREAK PLATNG (CONT..)
The inoculating loop was flamed again and allowed to cool
for few seconds.
Microorganism were streaked into the second section, passing the loop over
the initial streak area (1
st
section) a few times as the source of inoculum.
During the incubation, the bacteria
multiplies and produce colonies.
Each of the colony are composed of million
of cells derived from a single parent
bacterium.
Isolated colonies from the last streaked
(third section) represents well isolated
strains.
In ensuring strains purity, streak plating
procedure is repeated few more times using
an isolated colony as the source of starting
bacteria.
STREAK PLATNG
(CONT..)
STREAK PLATNG
(CONT..)
Using the T-method, the culture was spread over in the first
section. lways remember that it should always be in one direction
STREAK PLATNG
(CONT..)
Using the sterile cooled loop, the microorganism were streaked on the
third section, overlapped with the second section only.
Flame the loop again and incubate the plate in an
inverted position at 37C for 48 hours.
During the incubation, the bacteria
multiplies and produce colonies.
Each of the colony are composed of million
of cells derived from a single parent
bacterium.
Isolated colonies from the last streaked
(third section) represents well isolated
strains.
In ensuring strains purity, streak plating
procedure is repeated few more times using
an isolated colony as the source of starting
bacteria.
STREAK PLATNG
(CONT..)
STREAK PLATNG
(CONT..)
solated
colony
Advantage and Disadvantage:
A. $treak Plate
- Advantage:
- For isolation of bacterial cultures.
- Has distinct separate colonies.
- Disadvantages
- Higher probability of contamination prior
to isolation
2. $pread Plating Method
1. Pipet 0.1 ml of diluted sample into an agar plate.
2. Flame sterilize the L shape
stirring rod and cool.
3. $pread the sample evenly
over the surface of agar.
4. Incubate the plate in an inverted manner.
SPREAD PLATE
Note:
- $preading technique also a surface colonies.
- $pread plates are generally used for plate
counts or when your dealing with a larger
liquid volume. The bacteria are then
distributed evenly over the surface by a special
streaking technique. After colonies are grown,
they are counted and the number of bacteria in
the original sample calculated.
Advantage and Disadvantage:
. $pread Plate
- Advantages:
- It is generally used for plate counts or
when your dealing with a larger liquid
volume.
- Disadvantages:
- More microbes
- Presence of more colony forming units.
3. Pour Plating Method
1. Flame sterilize the pipet.
2. Pipet 1ml of the inoculum
And pour into the plate.
3. Pour the contents of the culture
Medium into the plate. Mix well
And allow to solidify.
4. Incubate the plate in an inverted manner.
A pour plate is an alternative method for
using agar plates to obtain isolated
colonies. Pour plates are used when it is
necessary to know the number of
organisms present per unit volume of
specimen or other sample.
When a specific aliquot is placed in the
Petri dish, a count of the colonies that grow
after incubation reveals their concentration
in the original sample.
W Pour plates are used commonly in the
bacteriologic examination of milk, or could
also be used to determine whether
sufficient bacterial numbers are present in
urine samples to signify the patient has a
urinary tract infection.
POUR PLATE
W %e pour plate tecnique can be u8ed to
determine te number of microbe8mL or
microbe8gram in a 8pecimen. t a8 te
advantage of not requiring previou8ly prepared
plate8 and i8 often u8ed to a88ay bacterial
contamination of food8tuff8.
W We can identify weter bacteria i8 an
anaerobe aerobe or a facultative aerobe. n a
pour plate bacteria will trap in te agar and te
anaerobe and facultative anaerobe will grow
witin te media.
Advantages of Pour Plating
the reduced growth rate of
obligate aerobes in the depth of
the agar
the danger of killing heat
sensitive organisms with hot
agar. These objections cannot
be raised against the streak
plate method.
i8advantage8 of !our !lating
- Disadvantages:
- Picking subsurface colonies would
interrupt other colonies by digging out of
the agar.
- Reference:
1. $pread Plate Method.<
http://www.studentsguide.in/microbiology/microbiologytools
techniques/spreadplatemethod.html> Last Accessed 10 Aug
2011.
2. Microbiology techniques. Retrieved August 5 2011 from
http://www.edu.biology/cellweb/techniques/microspread.com
3. Microbiology. Oxygen. Retrieved August 5 2011 from
http://www.inst.dept.edu/index=module