TITLE REPORT

NAME STUDENT(S)

AFFILIATION, BACHELOR PROGRAM, COURSE

SUPERVISORS
PLACE AND YEAR,DATE
(WHEN THE EXPERIMENT TOOK PLACE)
The report is written on the basis of the laboratory journal. What did you do, what did you
find? Commonly used to report experimental research in many scientific disciplines is the
following structure:

1
SUMMARY
Short summary of the experiment (in a few sentences, max. 200 words): what did you research
and what are the most important results.

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CONTENT
SUMMARY 2
CONTENT 3
1. INTRODUCTION 5
2. THEORETICAL BACKGROUND 6
2.1. Paragraph 1 Error! Bookmark not defined.
2.2. Paragraph 2 Error! Bookmark not defined.
3. MATERIALS AND METHODS 7
3.1. Paragraph 1 Error! Bookmark not defined.
3.2. Paragraph 2 Error! Bookmark not defined.
4. RESULTS 10
4.1. Paragraph 1 Error! Bookmark not defined.
4.2. Paragraph 2 Error! Bookmark not defined.
5. DISCUSSION 11
5.1. Paragraph 1 Error! Bookmark not defined.
5.2. Paragraph 2 Error! Bookmark not defined.
6. CONCLUSION 11
6.1. Paragraph 1 Error! Bookmark not defined.
6.2. Paragraph 2 Error! Bookmark not defined.
REFERENCES 14

APPENDICES (OPTIONAL) 15
APPENDIX I: TITLE 15
APPENDIX II: TITLE 15

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1. INTRODUCTION
Bacterial identification has significant importance in many different industries including the
pharmaceutical industry, when considering microbial contamination.
Microbial contamination refers to the accidental introduction of an infectious material, such as bacteria,
yeast, fungi or virus, to the production line (Donaldson, 2003). By inspecting for microbial contamination
in products, it ensures quality control as there won't be any damage, namely in the way of chemical
degradation, which can lead to the loss of efficacy as well as toxic side effects and medicine spoilage, all
of which can be detrimental to a patient's health. The identification and characterization of bacteria is a
vital component in microbiology in both the scientific research and clinical settings because they provide
valuable information regarding the microbial ecology of a specific environment, the metabolic
capabilities of a bacteria, their pathogenicity or lack of, etc. This is more evident in the clinical field, in
which the quick identification of bacteria allows physicians to save lives by: 1) providing them with the
information required to make a more accurate diagnosis; 2) prescribing an appropriate treatment; 3)
assessing the effectiveness of a treatment.

There are some few microorganisms (bacteria) found in pool water such as Crypto, E-coli and Giardia
which are usually spread in pools where chlorine and pH levels are too low. Microorganisms’ presence in
pool water can cause illness. An experiment using the Gram stain and membrane filtration method was
used to isolate and determined the type and species of microorganisms found in the sample of a pool
water.

This experiment has proven to be effective as it helped identified the presence of bacteria in the
sample of the pool water (gram stain method) and membrane filtration helped with the isolation of the
bacteria.

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2. THEORETICAL BACKGROUND
The goal of this experiment is to isolate and identify two different bacteria based on their
morphological, physiological and metabolic characteristics, to this end, several biochemical tests will be
performed to correctly identify the unknown bacteria down to the genus and species level.
Nowadays, the identification of unknown bacteria by using biochemical and/or physiological tests is
considered for the most part obsolete because it is a process that can be both labor intensive and time
consuming (days to weeks). Additionally, the test results are never guaranteed to be completely.
accurate since bacteria mutate constantly to adapt to their environments, thus they can occasionally
express genes that change a key biochemical or morphological characteristic, rendering this technique
inadequate (Rosselló, Amann 2001). Even so, in the clinical setting, in which speed is paramount,
biochemical tests (coupled with other immunological techniques) are often the preferred way to quickly
assess whether a known specific bacterium is present or absent in a sample.
By identifying bacteria, sterilization of products can take place in the hope of removing pathogens
and reducing the risk of contamination. Contamination can occur at any stage of production; however,
companies regularly assess the purity of a product on a frequently timed schedule to ensure an infested
product is not shipped.
These routine tests are performed on the raw materials as well as the manufactured medicines. By
sourcing the microbe's identity, the growth and metabolism can be investigated with the aim of finding
an appropriate way to prevent it from growing any further and elimination; this is usually by recognizing
the antibiotic which can destroy the cell membrane or the function of the pathogen. This can be done by
isolating the strain of microbe and exposing it to various antibiotics and observing and recording the
different sensitivities; a popular method involving this is the disk diffusion method. Additionally, this
process can reveal which strains of bacteria are resistant to certain sterilization techniques and
antibiotics. This is an important field of research in microbiology as antibiotic resistance is a common
clinical issue that is faced today and is of growing concern in the pharmaceutical industry due to the fact
that, every year, routinely used antibiotics are becoming redundant in the face of bacterial resistance
(Toy, 2008).
There are some few microorganisms (bacteria) found in pool water such as crypto, E. coli and
giardia are spread in public pools where chlorine and pH levels are too low. Pools are full of germs that
can make you sick. Some of the common issues you can get from swimming in a lake or pool are
diarrhea, skin rushes, respiratory illness and swimmer’s ear. People typically contract one of these
illnesses when the accidentally ingest contaminated water. So, it is very important to do this experiment
and to find measures to control it.
Filtration is the process in which solid particles in a liquid or gaseous fluid are removed by the use
of a filter medium that permits the fluid to pass through but retains the solid particles. In this
experiment, a specific type of filtration technique is used called “membrane filtration”. Membrane
filtration is a technique for testing water samples. In this procedure, water is drawn through a special
porous membrane designed to trap microorganisms larger than 0.45µm. In this experiment, a filter
paper is used as the trap. Afterward, the filter is applied to the surface of Endo agar plates and
incubated for 24 hours.
Microorganisms’ growth characteristics are important to know, so we can predict or control their
growth under particular conditions. In the medical field, this diagnostic is very important to identify a
pathogen isolated from a patient. Food industry helps identifying a microbial containment responsible
for food spoilage. In this experiment, the gram stain procedure is used to gather information about the
contents of a species’ cell wall. A purity streak is used in this experiment, to help identify microbe. In this
technique, a loopful of culture is spread on an agar plate to get individual cells far apart enough from
each other. The streaking method gradually dilutes the inoculum such that the bacterial cells can be
counted as colony forming units (CFUs).

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3. MATERIALS AND METHODS
3.1. Materials and Equipment
3.1.1. List Of Materials and equipment used for this experiment;
Bunsen burner Glass filter holder Microscope
Lighter 0,45 µm cellulose acetate filter,
47 mm
Sterile pair of tweezers Sterile 250 mL Erlenmeyer flask Immersion oil
Buchner filtration flask Black tile Object glass with opaque edge
Water jet pump Inoculation loop set

3.1.2. List Of Chemicals, solutions and samples used for this experiment;
chemical manufacturer City Country of provenience
Sterile demi water Reymerink Ankeveen North Holland
Standard Plate Count K.F. Wannee (plate Vlissingen Zeeland
Agar (SPC), 4X count)
Pseudomonas Agar K.F. Wannee (plate Vlissingen Zeeland
Base (PAB), 3X count)
70% ethanol aqueous WMM chemie Oosterwolde Friesland
solution
Gram staining *Found in any chemical - -
solutions shop
Crystal violet:
Lugol (iodine solution):
Ethanol:
Fuchsine:

Container with Can be made by self or - -

swimming pool water get a sample from any
containing sodium pool water available
thiosulphate
*Refer to protocol CU20626 Pool Chemistry and CU20626 Pool Chemistry.

3.2. Experimental Procedure:

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To determine the type of bacteria, present in the sample pool water, standardized experiments were
performed in a laboratory setting in a specially constructed filtration setup and an inoculated agar plate.

Figure 1.1. Figure 1.2.

3.3. Filtration Process

The filter holder of Figure 1.1.is sprayed with 70% ethanol, the excess of 70% ethanol is rinsed with demi
water (a sterile Pasteur pipette is used to rinse the walls). The water tap is opened to let the water
pump start creating a low-pressure zone in the Buchner funnel. Some sterile demi water was poured on
the filter to wash the filter. The sample of pool water is poured into the setup and with the help of 0,45
μm cellulose acetate filter, 47 mm (this is used for sample 2 and sample 3), and 100 ml sterile Demin
water (this is used as the Blanc sample.) onto the filter and the filtered sample (filtrate) stays on it. This
was filtered completely. The pool water sample is collected in a 10-liter jar with a tap. Collect the sterile
200 ml pool water in a sterile Erlenmeyer flask. *100 ml of the pool water is used for each sample.
Three samples of petri dishes are used, and each marked with a specific name Blanc (sample 1),
Plate count agar (sample 2), Pseudomonas agar base (sample 3). The filtration was made thrice and the
filtrate on the cellulose acetate filter was placed upside down on each petri dish (each sample).
Incubate these plates for at least 48 to 72 hours at 37°C (or for a longer period at room temperature)
and place them in the fridge afterwards.

3.4. Gram stain & purity streak

Purity streak:
Four agar plates each containing the samples Standard Plate Count Agar (SPC) and Pseudomonas Agar
Base (PAB), two plates for each Figure 1.2.. An inoculation loop is sterilized with 70% ethanol aqueous
solution and a Bunsen burner by soaking the loop in the ethanol solution and with the help of the
Bunsen burner, start burning the end of the loop to the tip and allow to cool down for a minute before
inoculation.
Two colonies with a different appearance from the PAB plate used for the filtration setup is inoculated
on the new PAB plate and two colonies with a different appearance you take from the SPC plate used
for the filtration setup is inoculated on the new SPC plate. The plates are incubated overnight in the
incubator at 37 °C.
Gram stain:
An Object glass with opaque edge is placed on a black tile and a drop of sterile demi water is dropped
onto the glass. A sterile inoculation loop is used to pick a colony from the previously used PAB and SPC

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plate containing the sample of the pool water and transfer a fragment of the colony to the drop of
water on your slide and use the inoculation loop to mix and spread it over the surface of the slide until it
covers an area approximately 1,5 cm in diameter. Take for each colony a fresh inoculation loop. Leave
the drop to dry at room temperature or dry the drop by waving the slide back and forth high over the
flame of the burner. Fixate the bacteria by passing the slide - using a wooden peg - slowly through the
flame three times. Once you have done this, your slide will be microbiologically safe. It is now ready for
storage or immediate staining.
The actual Gram staining (differential staining) * refer to protocol CU20626 Pool Chemistry.
1. Stain for 1 minute using crystal violet by placing the slide in a small container with crystal violet
solution.
2. Rinse the slide with water making sure that the waste is collected in the waste container
intended for this purpose.
3. Stain for one minute using lugol (= iodine solution) by placing the slide in a small container with
lugol; do not now rinse the slide!
4. Discolor for 1 minute with ethanol by placing the slide in a small container with 96% ethanol.
5. Rinse the slide with water making sure that the waste is collected in the container intended for
this purpose.
6. Stain for 2 minutes using fuchsine by placing the slide in a small container with fuchsine
solution.
7. Rinse the slide with water making sure that the waste is collected in the container intended for
this purpose.
8. Dry the slide by dabbing it with absorbent paper. NB. Do not wipe, since by doing this you will
wipe the bacteria off the slide!

Using the 100X objective of the microscope* refer to protocol CU20626 Pool Chemistry.
● Place the slide on the microscope table and turn the revolver until you have objective 10x in front of
you (condenser as high as possible, diaphragm closed).
● Look for a suitable part of the slide and focus according to the usual manner.
● Now place a drop of immersion oil on the slide under the light beam.
● Carefully turn now until you have the 100x objective in front of you, the objective will touch the drop
of oil without any further intervention.
● Now open the diaphragm that is located under the condenser completely.
● Look at the slide through the eyepieces and whilst you continue to look through the eyepieces, focus
using only the micrometer screw.
● If you are going to study more slides using the 100x objective, there is no need to clean the objective
yet.
When you have finished using the microscope
● Turn the microscope table as far down as possible so that you can easily access the 100x objective.
● Wipe the objective using a piece of lens paper, whereby you keep using a clean bit of the paper. The
lens is clean when there is no more oil to be wiped off.

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4. RESULTS
4.1. Observations and Results
4.1.1. Filtration Process
After Incubation, it was seen that, some colonies of bacteria appear on the filter paper. The table
below shows the number of colonies found on each agar plate.
SAMPLES NUM.OF COLONIES NUM OF BACTERIA PER LITER
Blanc 130 13
Plate count agar (SPC) or E. 258 25.8
coli
Pseudomonas agar base (PAB) 660 66
or Pseudomonas aeruginosa
Table .1

4.1.2. Calculations
!"#$%&'" 012∗2.0
CFU (number of bacteria) = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 ()*+,% ). #+*$+&% /*"$% = 0.2∗02^67=13.0 L

4.2. Gram stain & purity streak

It was seen after incubation that; the PAB plates have different colonies of bacteria fused together
forming a single colony on the purity streak agar so as the SPC plates/ Blanc (Figures 2.3, 2.4 and 2.5).
The stain microscope glass was viewed under the microscope and the bacteria of the Pseudomonas agar
base (PAB) (Figure 2.1) was found to appear pinkish- purplish which was concluded that this is a gram-
negative bacteria given that they are unable to retain the primary stain (crystal violet). The Plate count
agar (SPC) was found to appear pinkish- purplish which was concluded that this is a gram- negative
bacteria (Figure 2.2).

Figure 2.1 (400x microscopic view of Pseudomonas agar base (PAB)) Figure 2.3 (bacteria growth on pool water” sample 2”) Figure 2.4 (bacteria growth on pool water” sample 1”)

Figure 2.2 (400x microscopic view of Plate count agar (SPC)) Figure 2.5 (bacteria growth on sterile demi water” sample Blanc”)

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5. DISCUSSION
The TVC (Total viable count ) of the pool water examined and the isolation of pathogenic bacteria
indicates the need for increased monitoring. Microbial contaminants in recreational water are mostly
controlled by disinfection. Improper maintenance of public and semi-public facilities frequently fails to
protect the public against chlorine-sensitive pathogens (Yoder et al., 2004). From table .1 the results
show the number of colonies of bacteria present in each agar plate count and also the number of
bacteria present in a liter of swimming pool water. The Dutch law requires 100 CFU of E. coli per liter of
swimming pool water. The results were calculated according to the range and estimated using CFU
values.

6. CONCLUSION
In this study, some microorganisms were identified from the pool water. Effective management of
swimming pools and proper control of the physical, chemical and microbiological property of
water pools can produce the healthy recreational activity. According to the Dutch law, 1 liter of
swimming pool water must contain 100cfUs of E. coli. And from the results (Table .1) if can be
seen that, the number of bacteria per liter (CFU) of Plate count agar (SPC) or E. coli is
approximately 25.8 liters which is below the Dutch law recommendation. The isolation of
pathogenic bacteria E. coli and Pseudomonas aeruginosa from this study is an indication of poor
bather hygiene and poor compliance of the standards. Therefore, there is a need to increase
monitoring of the recreational facilities, increased bather hygiene education, as well as pool staff
education and improved pool circulation. Further studies should be carried out on other microbial
contaminants apart from bacteria that contaminate swimming pool water and also on pool
architecture and bather population that contribute to poor compliance of the standards.

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REFERENCES
Cite any resources or publications you used. Did you consult a paper that somehow related to
the project? Give credit. References are needed for all facts.

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APPENDICES (OPTIONAL)
An appendix contains information that is non-essential to the understanding of the report but
may present information that further clarifies the results section.

Appendix headings: Each appendix should be identified by a Roman numeral in sequence, e.g.,
Appendix I, Appendix II, etc. Each appendix should contain different material, such as raw data.

Figures and Tables are often found in an appendix. These should be numbered in a separate
sequence from those found in the body of the paper. So, the first Figure in the appendix would
be Figure 1, the first Table would be Table 1, and so forth. Also, here you may include the
number of the paragraph: Figure I-1, Figure I-2, etc.

APPENDIX I: TITLE

APPENDIX II: TITLE

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