TITLE : BACTERIA COUNT

1.0 INTRODUCTION

2.0 LEARNING OUTCOMES

3.0 OBJECTIVE

4.0 THEORY

5.0 EQUIPMENT

6.0 PROCEDURES

7.0

RESULT AND CALCULATIONS

Plati
ng
Met
hod

Pou
r
Plat
e

Aver

Tota

Dilution

age

Colo

1/

1/1

1/1

ny /

10

00

000

1/104

1/105

1/106

bact
eria

Plate

/ mL

3.33

1.00

sel.
mL

47 x
106

Spre
ad
Plat
e

123.

3.50
sel.
mL

10

60 x
103

8.0

DATA ANALYSIS

1.

Show the calculation for each of the plating method and fill in the above table.

Calculation for Exact Total of Bacteria

For Pour Plate Method

Total bacteria
= (9.0 x 10) + (6 x 102) + (4 x 103) + (1 x 106)
= 1.0047 x 106

Average Colony / Plate ;

= 20
6
= 3.33 sel. mL

For Spread Plate Method

Total bacteria
= (10 x 10) + (5 x 102) + (3 x 103) + (2 x 104) + (1 x 105)
= 123.6 x 103

Average Colony / Plate ;

= 21
6
= 3.5 sel. mL

2.

Analyze the results by using the appropriate method. Explain your findings.
There are 2 methods that we apply for this experiment which is spread plate test
method and pour plate test method. The pour plate test method is very suitable for
sample which contains plenty of water while spread plate test method is just
suitable for 0.1 - 0.5ml of water content. The medium which contains nutrients is
a food which will help the growth of bacteria.

Based on our result, we can see the dead bacteria do form any colony. Some
bacteria will occur as single cells. Other species hang together in chains or clumps
of 2 or more bacteria. A piece of dirt with 10 bacteria on it will form a single
colony. For the pour plate 4 and pour plate 5, the result shows the bacteria form
colony. This is maybe because we are not scratching well on plate before we
placed the medium. For the spread plate 6, we predict that the bacteria colony
occur affected by environment and the degree of the dilution water also affect the
result.

3.

State the systematic bias error that could occur during this experiment.

Agar not truly hot till temperature 500C or more from the specific
temperature.

Every time dilution was made on samples, the pipette not been cleaning
properly with distilled water which can make the pollution to the next
samples.

Pouring agar which been pour into every plates not consistent that can course
spreading different bacteria in each sample bacteria.

There was an error while measured the weight of the nutrient media such as
the mix peptone, beef extract and the agar.

Some bacteria will remain on the spreader which causing the count to be too
small.

Some bacteria were unable to grow under the experiment conditions causing
the count to be too small.

4.

Usually, the result shows different reading for both methods. In some cases, both
methods produce the same result. Explain why the results are indistinguishable

The results show indistinguishable and different reading for both methods. These
are because of a few factors that occurs the differences such as:
i.

They require lengthy incubation for colonies to become visible.

ii.

Cell clumping can lead to an undercount of viable cells.

iii.

It is very easy to have too many or too few colonies on a plate to accurately
measure viable count.

iv.

Prevention of crowding often requires serial dilution.

v.

There is also have too few cells requires concentrating, e.g., by centrifugation
or filtration.

9.0

QUESTIONS

1.

Explain the meaning of phrases two times ten to the eight cells per mL in your
own convenient terminology.
Two times ten the eighth cells per ml = 2 x 108 cells/mL
It is a very convenient terminology used by all bacteriologists and molecular
biologist. This method of expressing numbers is called scientific notation. When
you examine a liquid culture of bacteria that has grown long enough you will
notice the culture is turbid. A culture that is just barely turbid contains nearly 100
million bacteria per mL.
Some bacteria are bigger than other species. Cultures of big bacteria are visibly
turbid at fewer cells per mL (at lower titer). 100000000 bacteria per mL is a
difficult number to write or comprehend. 100,000,000 is not much better. 108
means the same thing. 108 = 100,000,000 = 100 million. Notice that the 8 is the
number of zeros. 103 = 10 x 10 x 10 = 1000 = one thousand. 1 x 108 is the
scientific notation for 100 million. 2 x 108 is the scientific notation for 200
million.

2.

What the meaning of TNTC and the significant amount due to the TNTC?
Give the formula for determining bacteria count.

In the determination of microorganisms by a technique in which individual

viable units are determined, such as by plate count assay of bacteria or by plaque
count assay of viruses, with insufficiently diluted samples an overgrowth or dense
formation of colonies is noted which is conventionally reported as TNTC
(too numerous to count) that the total number of bacterial colonies exceeds 200 on
a 47-mm diameter membrane filter used for coli form detection.. The significant
are to count the number of bacteria colonies that appear on each of the plates that

has between 30 and 200 colonies where any plate which has more than 200
colonies is designated as "too numerous to count" (TNTC).

Formula :

Colony Forming Unit = C.F.U. The calculation is performed, thus :

C.F.U/mL original sample = {C.F.U/plate} x {1/mL aliquot plated} x {dilution

factor}

3.

Design the experiment for comparing the bacteria counts of water sample (tap
water, lake water, swimming pool water and rain-barrel water). Explain the
different of bacteria count for each kind of water sample?

Comparing Bacteria Counts of Water Samples. Suggested water samples: tap

water, well water, rainfall caught in a sterile vessel, rain barrel water, ditch water,
pond water, river water, lake water, ocean water, swimming pool water. Sterile
tools must used at all stages analysis.
Materials Needed:

Sterile collection container (sterile bottle or test tube) with water sample

Sterile pipetts or sterile transfer pipetts (dropper pipetts)

Sterile petri dish

Coliscan Easygel (Micrology Laboratories)

Incubator set

Procedures:
1.

Put a drop of water of well water on a sterile plate of TGY or other agar.

2.

Spread it uniformly over the surface of the agar with any non-absorbent
sterile tool. A glass rod bent into an L-shape is ideal. The bottom of a
teaspoon will also work.

3.

Incubate the plate at 30C or room temperature (r.t.).

4.

Examine the plate as often as you like. At room temperature, it will probably
take a day or two for single cells to grow into colonies large enough for you
to see. Different species of bacteria grow at different speeds and some
species will take many days.

5.

Count the colonies. There are about 18 drops per mL, the size of drop
depends on the orifice. Small tips make small drops and it can take 30+ drops
to make one milliliter.

6.

Comparing Bacteria Counts of Water Samples.

City tap water are used in the above experiment probably got no bacteria. If used
lake water in the above experiment probably got many bacteria. If used water
from a swimming pool, you probably got many bacteria per drop and it is likely
the plate was covered and could not be counted.

4.

In many experiment there are 2 types of control used which are positive and
negative control. Due to this experiment what is the suitable control? How the
control will effect to your findings?

Positive control

Inoculate 1 mL of suspension into each of 2 petri dishes. Add about 20 mL of

cooled molten YEA to the plates at the same time as the pour plate method is
carried out on test samples. Incubate one control plate with the test plates at
37C and the other plate with the test plates at22C for the appropriate times.

Negative control (blank)

Sterility checks are to be performed for each bottle of agar. Aseptically pour
about 20 mL of molten agar, cooled to 45C, into 2 Petri dishes. This should
be done at the same time as the test samples are inoculated. Incubate the
control plates with the test plates at 37C and 22C for the appropriate times.

10.0

DISCUSSION

11.0

CONCLUSIONS

12.0

REFERENCES

1.

Master, Gelbert M (1998) Introduction to Environmental Engineering and

Science New Terzey : Prentice Hall

2.

Hammer, MarkJ. (2001)Water and Waste water Technology Frouth Edition New
Terzey: Prentice Hall

3.

Davis Cornwell (1998) Introduction to Environmental Engineering New York:Mc

Graw Hill