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ABSTRACT

There are several different methods of getting a pure culture from a mixed culture.
The two most frequently used methods involve making a streak plate, spread plate and pour
plate. These three methods are the methods that we were using during the experiment A.
During streak plate, we need to streak the bacteria over the surface of the molten agar in a
Petri dish and this procedure was use to obtain a single isolated pure colonies of bacteria. The
bacteria than we are using were Bacillus sp. and E.colli. For spread plate, we were using a L-
shaped bend rod on the molten agar to spread the bacteria over the surface. The last method
was pour plates. We need to release some into surface of the Petri dish and pour the medium
into the dish. Make sure the medium covers the entire plate. For experiment B, Serial of
dilution take place by using water and broth culture. The bacteria culture undergo 4 times of
serial dilution and each mixture were being examined for the calculation the number of
colony present.
After we had done the experiment, the result showed that, only E. colli has growth
and negative for Bacillus sp. but for spread plate both bacteria shows positive growth and
negative growth for pour plate. For experiment B, the Bacillus sp. shows a positive growth
for spread and pour plate but negative growth for E. colli. Thus, the spread plate is better for
Bacillus sp. than the other while streak plate is better for E. colli. For experiment B, pour
plate is better for Bacillus sp. Then, we calculate the CFU from range 30 to 300 colonies.

INTRODUCTION
Microorganisms are very diverse and any medium contains many different kinds of
them. To obtain a pure culture, a microorganism must be isolated from the others, because a
pure culture consists of microorganisms of the same species. Also, without pure cultures, it is
almost impossible to determine the characteristics of an organism that are of the most general
interest, such as nutritional requirements, responses to environment,methabolic products or
pathogenicity. Also to ensure that only the correct bacteria to be grown in the medium is
grown and there is no contamination and to prevent incorrect identification of bacteria as the
medium is contaminated, hence, pure cultures must be used.
In streaking technique, microorganisms are streaked on an agar plate by using
inoculating loop so that a single cell will be obtained near the end of the streak while in
spreading technique, microorganisms are spread all over the surface of the agar plate by using
a sterile glass rod so that a single layer of cells will form (Fankhauser, 2005).

OBJECTIVES
1. Isolated and identified discrete colonies from mixed microbial colonies using streak,
spread and pour plate methods.
2. Demonstrate the diverse methods in determination of the number of cells in culture.
3. Calculate quantitatively the number of viable cells via continuous cell dilution, colony
counter, spectrophotometer and colony forming unit (CFU).
THEORY
A pure culture is derived from a mixed culture that is the one containing by many
species. It is done by transferring a small sample into new, sterile growth medium in such a
manner as to disperse the individual cells across the medium surface or by thinning the
sample manyfold before inoculating the new medium. In streak plate, if more than one shape
or colour of colony on the streak lines is evident, this indicates a culture contains more than
one type or species of bacteria. This technique is used to check the purify of cultures that are
being maintained over a long period (Fankhauser, 2005).
In spread plate, the bacteria is spread to the plate to dilute the amount of bacteria in
each section of the plate continuously because the streaking technique gradually dilutes the
amount of bacteria in each quadrant of the plate. So, the last quadrant should have small
isolated colonies that can be easily studied (Smith, 2013).
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. The microbe may have the ability to grow provided that it has enough
oxygen to grow. But it only get oxygen from the molten agar. (Smith, 2013).
In experiment B, after the bacteria is diluted from full strength stock into sterile
distilled water and continuous into 3 other tubes, the growth of bacteria will become less
heavily grow in the last tube and heavily grow in the first tube because in contain the most
bacteria and after several dilutions been conducted, the concentration is decreased. Then the
CFU ( colony-forming unit) is calculated. CFU is used to find the number of bacteria in the
original solution. The number of colonies is valid from range 30 to 300 colonies.
PROCEDURE
A) Isolation of microorganism via streak plate, spread plate and pour plate
techniques.

I. Streak Plate:
1) The cap of the bottle containing the inoculums was loosened.
2) The inoculation loop was hold in right hand.
3) The loop was flame and allowed to cool.
4) The test tube containing the inoculums was lifted with left hand.
5) The cap of the tube was removed with the little finger of right hand.
6) The neck of the test tube was flamed.
7) The loop was inserted into the culture broth and withdraws. The loop was holding as
still as possible at all the times.
8) Flame the neck of the test tube again.
9) The cap of the test tube was replaced using the little finger of right hand. Then, the
test tube was placed on the bench.
10) The lid of Petri dish containing the solid medium was lifted partially.
11) The charged loop was holding parallel to the surface of the agar. The inoculum was
smeared backwards and forwards across a small area of the medium.
12) The loop and Petri dish was removed and closed respectively.
13) The loop was flamed again allowed to cool.
14) The dish was turned through 90 anticlockwise.
15) With cooled loop, the plate was streaked from across the surface of the agar in three
or four parallel lines.
16) The loop and Petri dish was removed and closed respectively.
17) The loop was flamed again and allowed to cool. The dish was turned through 90oC
anticlockwise again and streak across the surface of the agar in three or four parallel lines.
18) Steps 14 until 17 were repeated for the last time.
19) The plate was tapped closely and incubates the plate in an inverted position.

II. Spread Plate
1) 0.1mL of bacterial suspension was pipette onto the middle of the agar surface.
2) The spreader was lifted out of the alcohol, and passed the spreader rapidly through a
flame.
3) The spreader was placed on the surface of agar and use is to push and spread the
liquid all over the surface.
4) At 5 to 10 seconds spreading finished, the spreader was lifted away and replaced the
cover of the plate.
5) The spreader was put back in the alcohol.

III. Pour Plate
1) The lid of the Petri dish was lifted slightly with right hand and inserted the pipette into
the Petri dish.
2) The required volume of inoculums was released gently onto the centre of the dish.
The lid was replaced.
3) The pipette was putted into a discard pot.
4) The medium was poured into the dish.
a) A bottle of sterile molten agar was collected from the water bath.
b) The bottle was hold with right hand. The cap was removed with the little f
inger of left hand.
c) The neck of the bottle was flamed.
d) The lid of the Petri dish was lifted with the left hand and poured the sterile
molten agar into the Petri dish. The lid was replaced.
e) The neck of the bottle was flamed and the cap was replaced.
f) The dish was moved gently to mix the culture medium thoroughly.
g) The plate was allowed to solidify.
h) The plate was taped closely and incubates in an inverted position.

B. Determination number of the cell.
Preparation of Dilution:
1) 4 bottles of agar deeps, liquefied and tempered were obtained.
2) The bacteria culture was obtained in broth and 4 sterile 0.1% peptone water,
containing 9mL each.
3) 1mL was transferred using a sterile pipette from original culture into the first tube of
9mL of peptone water to make a 1: 10 dilution.
4) 1mL was transferred from tube 1 to the second tube containing 9mL of peptone water
to make 1: 100 dilution.
5) 1mL was transferred from tube 2 to the third tube containing 9mL of peptone water to
make 1: 1000 dilution.
6) 0.1mL was transferred from tube 3 to the fourth tube containing 9.9mL of peptone
water to make 1: 100,000 dilution.
7) The inoculums was cultured using spread plate and pour plate method.
8) The cell was observed after 24 hours using colony counter and the CFUs number was
calculated for each dilution used.
APPARATUS
1. Test tubes.
2. Inoculating loop.
3. Bunsen burner.
4. Petri dish.
5. Tape.
6. Incubator machine.
7. Sterile chamber.
8. L shape bend rod.
9. Pipette
10. Parafilm tape.
11. Plate counter.
MATERIALS
1. Alcohol.
2. Bacteria (s.p Bacillus and s.p E.colli)
3. Distilled water.
4. Molten agar.
RESULT AND CALCULATION
A. Isolation of microorganism via streak plate, spread plate and pour plate
techniques.
Streak Plate Spread Plate Pour Plate
Bacillus sp.
Growth
Negative
Positive single
colony
Negative
E. colli
Growth
Positive Heavy
growth
Positive 2 colonies Negative


B. Determination number of the cell.

1) Spread plate
1.1) Bacillus sp.
Dilution Ml of dilution
plated
Number of colonies CFU/ml

1:10
0.1 2192 TNTC

1:100
0.1 2600 TNTC

1:1000
0.1 832 TNTC

1:100,000
0.1 32 3.2 x



Calculation of colony using CFU formula.
Colony per plate= 32
Dilution factor = 1:1 x


Volume of dilution added to plate = 0.1ml
32 x

= 32 x

CFU
32 x

/ 0.1 = 3.2 x

CFU/ml

1.2) E. colli
Dilution
Ml of dilution
plated
Number of colonies CFU/ml

1:10
0.1 Ngative -

1:100
0.1 Negative -

1:1000
0.1 2 TFTC

1:100,000
0.1 Negative -

2) Pour plate
2.1) Bacillus sp.
Dilution Ml of dilution
plated
Number of colonies CFU/ml

1:10
0.1 8892 TNTC

1:100
0.1 4680 TNTC

1:1000
0.1 3276 TNTC

1:100,000
0.1 39 3.9 x



Calculation of colony using CFU formula.
Colony per plate= 39
Dilution factor = 1:1 x


Volume of dilution added to plate = 0.1ml
39 x

= 39 x

CFU
39 x

/ 0.1 = 3.9 x

CFU/ml
2.2) E. colli
Dilution
Ml of dilution
plated
Number of colonies CFU/ml

1:10
0.1 Negative TFTC

1:100
0.1 Negative TFTC

1:1000
0.1 Negative TFTC

1:100,000
0.1 Negative TFTC


DISCUSSION
From the result for experiment A, it shows that only in E. colli has positive heavy
growth while negative growth for Bacillus sp. in streak plate. While both bacteria shows
positive growth for spread plate and negative growth for both bacteria in pour plate.
According to the theory the pour plate will have the least growth because the bacteria only
get the oxygen from the molten agar. But in this experiment its shows negative result.
Perhaps the bacteria needs a longer time to growth because it has a long lag phase or the
bacteria has died because did not have enough oxygen to grow.
Based on the result for experiment B, the mixture from 1 : 100000 shows a positive
result for Bacillus sp. The number of colonies is 32 and using CFU formula we calculated the
number of CFU/mL in the original sample thus, we get 3.2 x

CFU/mL. This is for the


spread plate and same goes for the pour plate. The mixture from 1: 100000 shows a positive
result that is 3.9 x

CFU/mL. But the dilution for 1:10, 1:100 and 1:100 for both spread
and pour plate shows a negative result because the number of the colonies for each plate is
more than 300. So, these will be classified as too numerous to count (TNFC). However, this
is correct according to the theory. It state that the lowest concentration will have the less
growth of bacteria and the highest concentration will have the highest number of growth. But
the number of colonies is too many that we cannot count by using the CFU formula.
But for E. colli , it shows negative result. This is because the bacteria need a longer
time to grow due to a longer lag phase and for pour plate, perhaps the bacteria has died
because it did not get enough oxygen to gorw. Because for pour plate it only gets oxygen
from the molten agar. Supposedly, the bacteria shows a positive growth for spread plate
because it has enough oxygen. But throughout the experiment, it shows that the aceptic
technique is correctly done because none of the plates has been contaminated by others
microorganisms.
CONCLUSION
As the conclusion, for experiment A, streak method is the best method to inoculum
the E.colli because it shows a positive heavy growth than the other method. But in
experiment B, both spread and pour method can be used to inoculum Bacillus sp. because it
shows a heavy growth. The CFU can be calculated from the original number of colonies can
be calculated from the 1 : 100000 tubes and we gets 3.2 x

CFU/mL and 3.9 x


CFU/mL for Bacillus sp. The aseptic technique also has been done correctly because there
are no contamination in all the plates after incubate for 24 hours.
RECOMMENDATION
1) The apparatus should be sterile properly to avoid contaminations.
2) Increase the concentration of E.colli so that it will grow faster.
3) Shake the plate lightly after pour the medium into the plate that contain the bacteria to
avoid the bacteria die.
References
Fankhauser, D. B. (2005, Jun 23). POUR PLATE TECHNIQUE FOR BACTERIAL ENUMERATION. Retrieved
from http://biology.clc.uc.edu/
Smith, S. (2013). Encyclopedia Britannica. Retrieved from Encyclopdia Britannica, Inc. :
http://corporate.britannica.com/