Different Techniques in the

Inoculation of BacteriA

GROUP 2:
BAUTISTA, GALDONEZ, LAPID, TAMONDONG, VENTURA

Nutrient agar plates
Tubed media (Slants, Butts, Butt-slants)
Nutrient broth
Stock broth culture
Inoculating needle/loop
Bunsen burner/ alcohol lamp

Clean an area of bench, and
wipe it with disinfectant.
Flame a wire loop, bringing
it all to red heat, and when
it is cold enough, fish out a
loopful of the specimen or
culture.
Hold the petri dish in your
left hand partially opened
covering the agar plate.
Place the drop of culture on
one side of the agar medium
away from you. Streak the
culture on the agar surface
back and forth, edge to edge
in parallel lines moving
towards you. (NOTE Make
the streak lines close to
each other and inoculate
gently to avoid gauging the
agar)
After the entire surface of
the agar medium has been
streaked, cover completely
agar culture and label.
A. INOCULATION OF AGAR PLATES
(SIMPLE STREAK METHOD)
Hold the tube lightly with the
left hand and loop or wire
(previously heated) with the
right hand.
With the little finger of the
right hand, pull out the
cotton plug with a slightly
rotary motion.
Heat the mouth of the test
tube and proceed with the
inoculation. (NOTE: Never put
the cotton plug or screw cap
on the table or elsewhere)
Slant the tube slightly and
rub the loop with the
inoculum on the side of the
tube at a spot lower than the
level of the medium if the
tube is an erect position.
If the inoculum stick to the
loop and it could not be
dislodged by rubbing on the
inside of the tube, then,
twiriling of the handle of the
loop between the thumb and
forefinger may dislodge the
inoculum.
B. INOCULATION OF LIQUID MEDIA
(NUTRIENT BROTH)
Hol d the tube l i ghtly wi th the
l ef t hand and the l oop or
strai ght wi re (j ust previ ously
heated) wi th the ri ght hand.
Wi th the l i ttl e fi nger of the
ri ght hand, pul l out the cotton
pl ug wi th a sl i ghtl y rotary
moti on.
Heat the mouth of the test tube
and proceed wi th the
i nocul ation. (NOTE: Never put
the cotton pl ug on the tabl e or
el se where)
Starti ng from the butt end of
the sl ant, draw the strai ght
wi re or l oop over the surface i n
a strai ght l i ne towards the end
of the sl ant.
Starti ng agai n from the butt
end, trace a zi gzag course
from si de to si de, at the same
ti me sl owl y drawing the wi re or
l oop towards the end of the
sl ant.
Heat the mouth of the tube
once more and pl ace the cotton
pl ug.
Heat the strai ght wi re or l oop.

C. INOCULATION OF SLANTED MEDIA
(AGAR SLANT)
Follow the procedure 1-5 in the inoculation
procedure of Agar Slant. Then make a stab from
the center of the slant, down to the bottom of the
tube. Withdraw the straight wire along the same line
of inoculation.
Heat the mouth of the test tube, replace the cotton
plug and heat wire as in the above procedure.
D. INOCULATION OF SLANTED MEDIA
(AGAR SLANT WITH BUTT)
Using the loop, take a drop of the liquid culture
medium (broth) provided and spread it carefully in a
line across the surface of the agar as shown. With
the same loop, a second, third and fourth line may
be drawn parallel to the first. Close the lid of the
petri dish immediately.
Sterilize the loop in the flame once again, and allow
it to cool.
Turn the petri dish so that the end of the previous
lines can be the start of the next ones.
E. MULTIPLE STREAKING METHOD:
Take a cooled loop and make 2 or 3 strokes as
before. Close the lid of the petri dish immediately.
Repeat 2,3,4 until there is no more space around the
edge (4 or 5 times), then finish off with a single
zigzag streak across the middle.
Seal and label the petri dish with the culture
reference and your name and the date. Place it in an
inverted position in the incubator at an appropriate
temperature.
E. MULTIPLE STREAKING METHOD:
Agar Plate Culture- Upside Down Position
Slant Agar Tubes- Upright Position On A Rack
37 Degrees C, For 18-24 Hours.
F. INCUBATION OF THE BACTERIAL
CULTURE
Inoculation Techniques Inoculating device
used
Incubation period Results
1. Inoculation of agar
plates (Simple
Streak Method)
Inoculating loop 18-24 hours (37
degrees C)
Formation of distinct
pigmented colonies
1. Inoculation of
liquid media
(Nutrient Broth)
Inoculating loop 18-24 hours (37
degrees C)
Turbidity
1. Inoculation of
slanted media
(Agar Slant)
Inoculating loop 18-24 hours (37
degrees C)
Formation of distinct
pigmented colonies
1. Inoculation of
Slanted Media
(Agar Slant with
Butt)
Inoculating needle 18-24 hours (37
degrees C)
Formation of distinct
pigmented colonies
1. Inoculation of Butt
Media
Inoculating needle 18-24 hours (37
degrees C)
Formation of distinct
pigmented colonies
1. Multiple Streaking
Method
Inoculating loop 18-24 hours (37
degrees C)
Formation of distinct
pigmented colonies
III. EXPERIMENT RESULTS
a. Interpretation of results

AIM
obtain single isolated pure
colonies.
If more than one shape or
colour of colony on the
streak lines is evident, -a
culture contains more
than one type or species
of bacterium or yeast.
check the purity of
cultures that are being
maintained over a long
period of time
regular sampling and
streaking will show any
contamination by other
microbes.

A. SIMPLE STREAK METHOD:
TURBIDITY (cloudiness)
INDICATION
number of organisms
present

DEGREE OF TURBIDITY
VARIES WITH:
Organisms
Conditions
phase of growth
use of turbidity as a form of
enumeration requires previous
standardization.

B. INOCULATION OF LIQUID MEDIA
(NUTRIENT BROTH)
Non-motile bacteria-only grow
where they were inoculated.
Motile bacteria- grow along
the stab and will also swim
out away from the stabbed
area.

Negative Result: growth in a
distinct zone directly along
the stab.
Positive Result: diffuse
(cloudy growth), especially at
the top and bottom of the
stab.
C. INOCULATION OF SLANTED MEDIA
(AGAR SLANT)
THE DEEP STAB
tube filled with a solid medium containing agar
Inoculating needle
presence of
Agar- which slows the diffusion of oxygen from the air
process of autoclaving
bacteria itself- which tend to deplete the oxygen
oxygen gradient is set up in the tube such that high
concentrations of oxygen may be found at the top and low if any
amounts at the bottom.
presence and location of growth in the tube then
indicates the oxygen requirements for the organism.

D. INOCULATION OF SLANTED MEDIA
(AGAR SLANT WITH BUTT)
determining a bacteria in a clinical sample.
When the bacteria is streaked and isolated,
the causative agent of a bacterial disease can
be identified.

E. MULTIPLE STREAKING METHOD
24 to 36 hours
to allow the bacteria to reproduce
the end of incubation there should be enough
bacteria to form visible colonies in the areas
touched by the inoculation loop.
single bacterial or fungal species can be identified
based on their morphological (size/shape/colour)
differences, and then sub-cultured to a new media
plate to yield a pure culture for further analysis.
INCUBATION:
We did not get the correct results for Simple and
Multiple Streaking Method because the agar in the
Petri dish was contaminated. There is a fungi/molds
present in our results.
For our Liquid Media, we got the correct results
because theres the presence of turbidity in our broth
culture.
We also got correct results for the Inoculation of
Slanted Media (Butt, Butt Slant and Slant) theres a
presence of cloudy zigzag (Butt Slant and Slant) and
a straight (Butt) growth in the culture media.
b. EXPECTATIONS:
1. Why should the loop wire be heated
over the burner before and after use?
Loop and wire should be heated over the
burner so that it will be sterilized and to
ensure that there is no contamination. Thus,
preventing bacterial growth on it.


2. Why should you let the inoculating loop
or needle cool first before using it?
Because when you use the inoculating
loop/needle while it is hot, the
microorganisms in it will die. This is to
prevent the chances of killing the bacteria.


3. Why is aseptic bacteriologic technique
so important in the isolation of pure
culture?
So that no impurities and no other bacteria
can live on the bacteria culture. It ensures
that you wont contaminate the culture.



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