PRACTICAL 9: TECHNIQUE IN MICROBIOLOGY

A. VIABLE COUNT OF BACTERIA CULTURE

INTRODUCTION
It is a common practise to determine microbial counts for both liquid and solid specimen
suspension of bacteria culture in nutrient broth all the way to soil and burger meat. Most
specimen have high enough numbers of microorganism that the specimen must serially
diluted to quantitate effectively.

OBJECTIVES
i. To make a serial dilution of bacteria culture.
ii. To transfer the bacteria to petri plates.
iii. To compare the number of bacteria presents in petri plate.

MATERIALS
i. 24 hours E.coli culture
ii. Nutrient agar plates
iii. Mac conkey agar plates
iv. Nutrients broth
v. Sterile graduated (14ml) plastic tubes
vi. Test tube racks
vii. 1ml micropipettes
viii. 200ul micropipettes
ix. Blue tips
x. Yellow tips
xi. 70% alcohol
xii. Bunsen burner
xiii. Glass spreader
xiv. Inoculating loop
xv. Vortex
METHODS
i. Five sterile raduated test tube was labelled as Y1 to Y5.
ii. The base of each sterile nutrient agar and Mc conkey agar plates was labelled with P1
to P5.
iii. 9ml of sterile nutrient broth was transferred to each of five test tubes.
iv. 1ml of bacteria culture was transferred using a sterile 1ml micropipette to Y1. This
gives 10X dilution.
v. Y1 tubes was shaken gently to ensure thorough mixing.
vi. Using fresh tip, 1ml was transferred from tube Y1 to the next test tube Y2 and steps 4
and 5 was repeated until the last tube Y1.
vii. Using fresh tip, 100ul was transferred from tube Y1 using 200ul micropipettes to the
sterile plate labelled PI (X10-1), the lid was lifted to the minimal amounts.
viii. The glass spreader was dipped in 70% alcohol, the alcohol was allowed to drip off and
the spreader was hold vertically in Bunsen Burner flame.
ix. The spreader was cooled and the diluted bacteria culture was spreaded over the
spreader of the plate.
x. The spreader was re-steriled.
xi. Step 7 to 10 was repeated for tube Y2 (X10-2) until the last tube Y5 (X10-5).
xii. The lid of the plates was taped down to avoid the risk of pathogens being spread.
xiii. The 5 plates was incubated upside down ot 37C for 24 hours.
xiv. The plate for bacterial growth was examined and counted the number of individual
colonies on each plate the next day.
xv. The colony counter was used to estimated the total number of bacteria on the plate
in CFU/ml.
 B. INOCULATING OF BACTERIA CULTURE
INTRODUCTION
The human body had billion of bacteria which constitutes he normal flora fighting against
the invading pathogens. It is tedious to isolate a particular type of bacteria from a clinical
sample. The streak plate method is a rapid qualitative isolation method. The techniques is
commonly used for isolation of discrete colonies initially require that the number of
organism in the inoculum be reduced. It is a essentially a dilution technique that involves
spreading a loopful of culture over the surface of an agar plates. Although many types of
procedure are performed, the four ways of quadrants streak is mostly done.

OBJECTIVES
i. To inoculate bacteria from bacteria culture by using streaking method.
ii. To determine the presence of bacteria in petri plates.

MATERIALS
i. 24 hours E.coli culture
ii. 24 hours Serratia sp. culture
iii. Nutrient agar plates
iv. Inoculating loop
v. Bunsen burner
vi. Incubator

METHODS
i. The inoculating loop were placed over the Bunsen burner until the loop is red hot.
ii. The loop were allowed to cool down and then dipped into the sample of bacterial
culture.
iii. The lid of the sterile agar plate were slightly lifted and the contents of the inoculating
loop was spread over the surface of the agar plate by using zig-zag motion.
iv. The lid of the plate was closed.
v. At the base of the plate were labelled with permanent marker.
vi. The steps were repeated for the second specimen.
vii. The two plates were placed in an incubator upside down to prevent condensation falling
onto cultures.
viii. The plates were observed after 24 hours of incubation.
DISCUSSION
Normally it is very difficult to determine the actual number of microorganisms in a
population. Viable plate count is the method being used most frequently in enumeration of
bacterial population. We can either use pour plate method or spread plate method for
viable plate count. Colony forming unit (CFU) is introduced in viable plate count for the
enumeration. Viable plate count has its limitations such as CFU only accounts the visible
colony in enumeration, take some time for the visible colonies to grow and too many
colonies could cause error in the count. To overcome the over crowded problem, dilution is
required.
To perform a serial dilution, a small amount of a well-mixed solution is transferred
into a new tubes and additional water is added to dilute the original solution. The diluted
sample is then used as the base solution to make an additional dilution. From the results
obtained, it shows that Petri plates P1 and P2 contain mass number of colonies that are too
much too count. In plates P3, the number of colonies is 236, P4 is 160 and P5 is 60 colonies.
To count the Colony-Forming Unit/ml or (CFU/ml), formula of [number of colonies x dilution
factor / amount plated] were used and number of bacteria present in each plates were able
to be compared of. To simply put the result obtained, the number of colonies decreasing in
each dilution of every test tubes.
Streak plate technique is used for the isolation into pure culture of the organisms
(mostly bacteria), from mixed population. The inoculum is streaked over the agar surface in
such a way that it thins out the bacteria. Some individual bacterial cells are separated and
well spaced from each other. As the original sample is diluted by streaking it over successive
quadrants, the number of organisms decreases. Usually by the third or fourth quadrant only
a few organisms are transferred which will give discrete colony forming units (CFUs).
In this experiment, only two type of bacteria being used which is E.coli and Serratia sp. For
both of the bacterial culture, the inoculum is dipped and streaked over the agar surface in
such a way that it thins out the bacteria. The plate is incubated at proper temperature
overnight so that the cells can grow and multiply to give isolated colonies. Based on the results
obtained, it shows that both of the specimen produced a positive results which matched with
the proposed theory. Specimen E.coli yields a TMTC on first quadrant, 180 colony on second
quadrant, 93 colony on third quadrant and TFTC on fourth quadrant. While for the specimen
Serratia sp. yields a TMTC on first quadrant, 162 colony on second quadrant, 74 colony on
third quadrant and TFTC on fourth quadrant. The terms TMTC can be referred as Too
Many To Count as the colony appeared in mass number that it is not easy to count. The terms
TFTC can be explained as Too Few To Count as the colony appeared in small quantity
which is below 30.
CONCLUSION
As a conclusion, in viable counts of bacteria culture experiment, we are able to make a
serial dilution of bacteria culture. The bacteria is then transferred to petri plates and
incubated for 24 hours. The next day, we compared the number of bacteria presents in the
petri plates.
As in streak plate method, we are able to inoculate the bacteria culture by using streak plate
method. The presence of bacteria in the petri plates was determined.

REFERENCES
i. Van Soestbergen, A. A., & Lee, C. H. (1969). Pour plates or streak plates?. Applied
microbiology, 18(6), 1092.
ii. Gilchrist, J. E., Campbell, J. E., Donnelly, C. B., Peeler, J. T., & Delaney, J. M. (1973).
Spiral plate method for bacterial determination. Applied microbiology, 25(2), 244-
252.
iii. Wright, D. J., Chapman, P. A., & Siddons, C. A. (1994). Immunomagnetic separation
as a sensitive method for isolating Escherichia coli O157 from food
samples. Epidemiology & Infection, 113(1), 31-39.