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1.

0 \INTRODUCTION

It is often important to know not only what types of bacteria are in a sample but also
how many of them are present. Food manufacturers are required by the FDA to monitor the
number and type of bacteria in their products. Dairies monitor the number of bacteria present
in milk after pasteurization. Water treatment plants monitor the effectiveness of their
sterilization process. Very few of the things we eat or drink are bacteria free. They merely
have greatly reduced numbers of harmless bacteria. Biotechnology firms closely regulate
bacterial growth as they manipulate these organisms to produce useful pharmaceutical
products. Beer and wine companies monitor the growth of yeast in their distilling process.
Clinical laboratories monitor the growth rate of bacteria from patients to determine their
antimicrobial sensitivity.

The problem is that often millions of bacteria are condensed in a small spot. For
counting bacteria, it is require preparing different dilutions of your original sample. For
example make 1:10, 1:100, 1:1000 and 1:10000 dilutions. We will then grow each dilution on
a different nutrient agar plate. When the bacteria grow, each bacterium will become a bacteria
colony that can be seen as a small spot on the petri dishes. By counting bacteria colonies, you
will know how many bacteria existed in the sample.

2.0 OBJECTIVE

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To measure the bacteriological quality of water sample by performing total plate count.

3.0 LEARNING OUTCOME


1. To be more proficient at dilutions.
2. To be more proficient at performing a standard plate count and determining bacterial
counts in a sample.

4.0 THEORY

Bacteria are remarkably adaptable to diverse environmental conditions: they are


found in the bodies of all living organisms and on all parts of the earthin land terrains
and ocean depths, in arctic ice and glaciers, in hot springs, and even in the stratosphere.
Our understanding of bacteria and their metabolic processes has been expanded by the
discovery of species that can live only deep below the earth's surface and by species that
thrive without sunlight or in the high temperature and pressure near hydrothermal vents
on the ocean floor. There are more bacteria, as separate individuals, than any other type of
organism; there can be as many as 2.5 billion of bacteria in one gram of fertile soil.

Many studies require the quantitative determination of bacterial populations. The


two most widely used methods for determining bacterial numbers are the standard, or
viable, plate count method and spectrophotometric (turbid-metric) analysis. Although the
two methods are somewhat similar in the results they yield, there are distinct differences.
For example, the standard plate count method is an indirect measurement of cell density
and reveals information related only to live bacteria. The spectrophotometric analysis is
based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive.

The standard plate count method consists of diluting a sample with sterile saline or
phosphate buffer diluent until the bacteria are diluted enough to be counted accurately.
Hence, the final plates in the series should have between 30 and 300 colonies. Fewer than
30 colonies are not acceptable for statistical reasons (too few may not be representative of
the sample), and more than 300 colonies on a plate are likely to produce colonies too
close to each other to be distinguished as distinct colony-forming units (CFUs). The
assumption is that each viable bacterial cell is separate from all others and will develop
into a single discrete colony (CFU). Thus, the number of colonies should give the number
of bacteria that can grow under the incubation conditions employed. A wide series of
dilutions (e.g., 10-4 to 10-10) is normally plated because the exact number of bacteria is

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usually unknown. Greater accuracy is achieved by plating duplicates or triplicates of each
dilution. Increased turbidity in a culture is another index of bacterial growth and cell
numbers (biomass). By using a spectrophotometer, the amount of transmitted light
decreases as the cell population increases. The transmitted light is converted to electrical
energy, and this is indicated on a galvanometer. The reading, called absorbance or optical
density, indirectly reflects the number of bacteria. This method is faster than the standard
plate count but it has limitation where sensitivity is restricted to bacterial suspensions of
107 cells or greater.

5.0 APPARATUS AND EQUIPMENT


a. Petri plate
b. Pipette

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c. Test tube
d. Glass rod
e. Bunsen burner
f. Incubator
g. Ethanol 95% @ methanol
h. Sterilize
i. Microscope
j. Bacteria medium: Peptone = 5g, Beef Extract = 3g, Agar = 15g, Distilled water =
600ml

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6.0 PROCEDURE

a) Procedures of preparing nutrient media

a. We mixed peptone, beef extract, agar and distilled water in 600mL beaker and
boiled it until the colour turned clear.
b. The agar was then cooled up to 45-50oC.
c. For the Spread Plate test, insert 40ml of nutrient media into the six petri plates.

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b) Dilution procedures

a. Removed 0.1mL from the bacteria sample and blow it into the 9.9mL of dilution
fluid by using clean, sterile, dry pipette.
b. Mixed thoroughly by blowing lots of bubbles with the pipette for a couple
seconds and discarded the pipette into the used jar for later cleaning.
c. We labelled tube 1 now contains 1/100 the concentration of bacteria in the
original sample because 0.1mL is 1/100 of 10mL. Next, we wiped the pipette
with Kleenex or toilet paper before inserting the pipette into tube 1 because
nearly 0.1mL of liquid may cling to the outside.
d. We used another clean, sterile, dry pipette remove 0.1mL from tube 1, wiped
pipette, blow contents of pipette into tube 2, and continued blowing bubbles for
a second or two for good mixing.
e. We repeated step 4 until we finished prepare tube 6.
f. All of sample tube we labelled with the dilution factor as to notice the bacteria
content in the tubes.

0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL

Test tubes contain 9.9 mL dilution fluid


Test tubes contain 9.0 mL dilution fluid
water
sample
1.0 mL
Water 1.0 mL
sample 1.0 mL
1/10 1/100 1/103 1/104 1/105 1/106
1.0 mL
1.0 mL
1.0 mL
1/106
1/104
1/105
1/103
water
sample

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c) Spread plate test method
a. Removed 0.1mL of diluted sample by using clean, sterile, dry pipette into six
different petri plates contain sterile agar.
b. The petri plate closed.
c. The petri plates are placed inside the incubator for 18-24 hours with a
temperature of 37oC.

d) Pour plate test method

a. Removed 0.1mL of diluted sample by using clean, sterile, dry pipette into six
different petri plates.
b. The agar poured into the plates and we waited until agar to solidify.
c. The petri plate closed.
d. The petri plates are placed inside the incubator for 18-24 hours with a
temperature of 37oC.

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e) Method of counting bacteria

a. After being incubate for 1 day, the petri plates are took out.
b. Placed the petri plate on the counting chamber.
c. The bacteria colonies on the culture being counted.

7.0 RESULTS AND DATA ANALYSIS

Average Total
Plating Dilution
Colony / Bacteria/
Method 1/10 1/100 1/1000 1/104 1/105 1/106
Plate mL
Pour 1.48 x
10 23 17 8 5 4 1
Plate 106

Spread 0.14 x
7 18 13 6 3 1 0
Plate 106

Calculation for Average Colony /Plate and Total Bacteria


Pour Plate Method
Average colony / plate = (23+17+8+5+4+1) / 6

= 9.67 10

Total Bacteria = (23 x 101) + (17 x 102) + (8 x 103) + (5 x 104) + (4 x 105) + (1 x 106)
= 1.48 x 106 colony /ml
Spread Plate Method

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Average colony / plate = (18+13+6+3+1+0) / 6

= 6.83 7

Total Bacteria = (18 x 101) + (13 x 102) + (6 x 103) + (3 x 104) + (1 x 105) + (0 x 106)
= 0.14 x 106 colony /ml

Analyse the results by using the appropriate method. Explain your findings.

In the experiment, we used the standard plate count method to count the number of bacteria.
This method consists of diluting a sample with sterile saline or phosphate buffer diluents until
the bacteria are diluted enough to count accurately. The final plate in the series that we should
have is between 30 and 300 colonies.

However, we did not get the final result that follow the series due to some errors throughout
the experiments such as human error, random error and environmental error.

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

i. Over-heated nutrient media

ii. Invisibility of spores.


iii. Movement of spores.
iv. Inaccuracy of counting the spores in a chamber and personal bias in counting.
v. Sampling error due to distribution of spores in the counting chamber.
vi. Temperature of agar
vii. Equipment such as pipette not cleaned properly after each dilution

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

Bacteria growing on a pour plate have slightly less available oxygen and are confined by the
agar so they tend to grow smaller than those on a pour plate. Spread plates are used to isolate
single bacteria, while pour plates are used to enumerate bacteria.
However, if the results are indistinguishable, we can conclude that the agar was not
prepared properly according to the specified steps. Besides, the temperature of agar do affect
the results of the experiment as well. The pouring and spreading of nutrient in the petri plate

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were not poured or well-spread hence affected every sample. Thereby, the results may not be
as accurate.

8.0 QUESTIONS

1. Explain the meaning of a phrase two times ten to the eight cells per mL in your own
convenient terminology.
Terminology is used by all bacteriologists and molecular biologist universally. This
method of expressing numbers is called scientific notation. Some bacteria are bigger than
other species. Cultures of big bacteria are visibly turbid at fewer cells per mL (at lower
titer). As we know, 108 equals to 100,000,000 = 100 million. Notice that the 8 is the
number of zeros. 103 = 10 x 10 x 10 = 1000 = one thousand. Thus, 1 x 10 8 is the scientific
notation for 100 million. If there are enough bacteria in a liquid culture to make the
culture barely cloudy, counting the cells commonly reveals nearly one hundred million
bacteria per milliliter (1 x 108 cells/mL). In short, meaning of phrases two times ten to
the eight cells per mL is one hundred million bacteria per milliliter (1 x 108 cells/mL).

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

TNTC is the acronym for too numerous to count because it is exceed beyond its ideal
range. The ideal range for colony counts differ for various kinds of bacteria. Usually the
number of colonies of 300 and above per plate are considered as TNTC.

Number of colonies 100


Colonies / 100 mL = Volume of sample filtered

3. Design an experiment to compare the bacteria counts in different water samples (tap
water, lake water, swimming pool water and rain barrel water). Explain the difference
of bacteria count for each type of water sample?

Tap water no bacteria will be detected as the water supply had been filtered.

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Lake water most probably, many bacteria detected varies on the location and
environment.
Swimming pool water - many bacteria per drop and it is likely the plate was covered
and could not be counted.
Rain barrel water - Some contamination is inevitable. Bird droppings will be washed
off the roof into the barrel. Bacteria and moss grow on cedar and asphalt roofs. Rain
barrel water is not potable for these reasons. Plant debris that accumulates in the
bottom of your rain barrel can support microbial growth

The different of each type of bacteria count for each type of water sample are due to
the differences of solvent, rates of oxygen and other chemical substances in the water sample,
rates of biochemical oxygen demand, and biological oxygen demand. The presence of
bacteria was affected by those water quality parameters, such as physical, chemical and
biological parameters.

4. In many experiments there are 2 types of control used which are positive and negative
control. Based on this experiment what is the suitable control? How will the control
affect your findings?
From the results obtained, it is shown and proven that positive control is the suitable
control to be used.
Positive control: a positive control should give the desired outcome of the
experiment, provided that all the reagents and equipment are functioning properly. For
example, if your experiment results in the ability of bacteria to grow on a petri plate
containing antibiotic, your positive control will be bacteria that are known to carry the
appropriate drug resistance marker. Even if none of your experimental bacteria grow,
as long as there is growth of the positive control you know that growth was possible.
Negative control: a negative control should be designed to not give the desired
outcome of the experiment. In the example above, bacteria which do not carry a drug
resistance marker should not be able to grow on a petri plate containing antibiotic. If
growth is observed, it is a red flag that something is wrong with the experiment.

If the positive control does not produce the expected result, there may be something
wrong with the experimental procedure, and the experiment is repeated. For difficult or

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complicated experiments, the result from the positive control can also help in comparison
to previous experimental results.

9.0 DISCUSSION

The extent of bacterial activity in a given sample in a definite set of conditions


mainly depends on the total number of bacteria present in it irrespective of their species.
Therefore, it is very often required to find out the total number of bacteria present in
samples of food, water, soil, air and tissue during their microbiological analysis. This total
number of bacteria includes both living and dead bacteria. Dead bacteria cannot grow
and reproduce.

It is only the living bacteria (viable bacteria), which can grow and multiply resulting in
specific bacterial activity. Therefore, it is very often required to enumerate the viable
bacteria cells in different samples.

The spread plate relies on bacteria growing a colony on a nutrient medium so that
the colony becomes visible to the naked eye and the number of colonies on a plate can be
counted. Selective media can be used to restrict the growth of non-target bacteria.
Whereas, the pour plate method is used when the analysis is looking for bacterial species
that grow poorly in air, for example water samples.

The colony becomes visible to the naked eye and the number of colonies on a plate
can be counted. To be effective, the dilution of the original sample must be arranged so
that on average between 30 and 300 colonies of the target bacterium are grown. Fewer
than 30 colonies makes the interpretation statistically unsound and greater than 300
colonies often results in overlapping colonies and imprecision in the count. To ensure that
an appropriate number of colonies will be generated several dilutions are normally
cultured. The laboratory procedure involves making serial dilutions of the sample (1:10,
1:100, 1:1000 etc.) in sterile water and cultivating these on nutrient agar in a dish that is
sealed and incubated.

As the serial dilution takes place in terms of 10 times, mathematically it is obvious


that no two dilutions can have colonies between 30 and 300. For example, if 10 -3 has 50

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colonies, 10-2 should have 500 (i.e. >300) and 10 -4 should have 5 (i.e. <30) colonies.
However, this does not occur in reality, as bacteria do not occur as a homogenous
solution; rather occur as a suspension in the diluents. If there are two dilutions having
countable colonies (between 30 and 300) first calculate the number of colony forming
units/gm or ml using each dilution. If one value is more than double of the other, report
the lower value. If not, take the average of the two values and report that value.

10.0 RECOMMENDATION
a) Make sure gloves are worn throughout the whole experiment to prevent
contamination of bacteria.
b) During the process of dilution, make sure no air bubbles in the pipette.
c) Make sure the pipette is washed with distilled water to avoid the bacteria from
shifting.
d) The sampling of bacteria should be left 24 hours to have its optimum reading of
nutrient absorption and so as of colony counts.
e) All petri plates and test tubes must be sterilised in the autoclave before using.

11.0 CONCLUSION
The objective of the experiment is to determine the total bacteria cell and yeast cell
inside the sample using total viable count is achieved. Regarding the result obtained, the
total pour plate is 1.46x106 colony /ml, while for the spread plate is 0.14x106 colony /ml.
Besides, the average value for pour plate is 10 colonies/plate and the spread plate is 7
colonies/plate. The observation concludes that the average of pour plate is much higher
than the spread plate.

REFERENCES
1. Hammer, MarkJ. (2001)Water and Waste water Technology Forth Edition New
Terzey: Prentice Hall
2. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice
Hall. Upper Saddle River, New Jersey. pp. 140-144.

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3. Aziz, A. et al. (2014). BFC 32403 Environmental Engineering, Faculty of Civil and
Environmental Engineering. First Edition

APPENDIX

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