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Plan of work.

Water quality and water purification.

Labteam 173:

Vincent Antonis
Vocational Education : Lab technician chemistry 4th level
ROC, Eindhoven
Mentor: Jack van Uytregt

Rob Luijten
Vocational Education : Lab technician biology 4th level
ROC, Eindhoven
Mentor: Jack van Uytregt

Aart Manders
Vocational Education : Lab technician chemistry 4th level
ROC, Eindhoven
Mentor: Jack van Uytregt

Plan of work V1.7 - Laboratory team 173. 1
Friday 19th April 2010.
Chapter X General Information.

X.1 Edukans
Edukans believes in a form of educations as the most durable form of
development. Education makes able-bodied and gives children skills they can use
their whole life. That’s the main reason Edukans works for more schools, school
desks, more schoolbooks and more teacher in developing countries.

Edukans also involves over 350.000 students in the Netherlands about education
in developing country’s whit actions like “Schoenmaatjes” and Going Global.
Edukans supports educations projects in Ethiopia, India, Kenia, Malawi, Uganda,
Albania, Peru.

X.2 Ex-change
Ex-change organizes internships in developing countries for students from the
Netherlands. Three times a year groups of Ex-chance students go to Malawi and
Kenia. They are going to work in groups, every group in their own specialty.
Diversify from hydraulic engineering to education, from IT to Tourism.

Together with students, schools and the locals the Dutch students work on
important basic facilities. The make dams and classrooms, give advice about
moneysaving and hygiene and the work on the quality of education. In the
internship they transfer knowledge and craftsmanship to the locals. This way Ex-
change provides an important contribute to the basic education in developing

Plan of work V1.7 - Laboratory team 173. 2
Friday 19th April 2010.
X.3 Malawi|
Information about Malawi
The Republic of Malawi is a landlocked country in southeast Africa that was
formerly known as Nyasaland. It is bordered by Zambia to the northwest, Tanzania
to the northeast and Mozambique, which surrounds it on the east, south and west.
The country is separated from Tanzania and Mozambique by Lake Malawi. The
name Malawi comes from the Maravi, one of the original Bantu tribes to inhabit the
Malawi was first settled during the 10th century and remained under native rule
until 1891 when it was colonized by the British, who ruled the country until 1964.
Upon gaining independence it became a single-party state under the presidency of
Hastings Banda, who remained president until 1994, when he was ousted from
power. Bingu Mutharika, elected in 2004 and again in 2009, is the current
president. Malawi has a democratic, multi-party government. Malawi has a small
military force that includes an army, a navy and an air wing. Malawi's foreign policy
is pro-Western and includes positive diplomatic relations with most countries and
participation in several international organizations.
Malawi is among the world's least developed and most densely populated
countries. The economy is heavily based in agriculture, with a largely rural
population. The Malawian government depends heavily on outside aid to meet
development needs, although this need (and the aid offered) has decreased since
2000. The Malawian government faces challenges in growing the economy,
improving education, health care and the environmental protection and becoming
financially independent. Malawi has several programs developed since 2005 that
focus on these issues, and the country's outlook appears to be improving, with
improvements in economic growth, education and healthcare seen in 2007 and
Malawi has a low life expectancy and high infant mortality. There is a high
prevalence of HIV/AIDS, which is a drain on the labor force and government
expenditures, and is expected to have a significant impact on gross domestic
product (GDP) by 2010. There is a diverse population of native peoples, Asians
and Europeans, with several languages spoken and an array of religious beliefs.
Although there was tribal conflict in the past, by 2008 it had diminished
considerably and the concept of a Malawian nationality had begun to form. Malawi
has a culture combining native and colonial aspects, including sports, art, dance
and music.

Capital: Lilongwe
Surface Area: 118.484 km2
Highest point: 3000 m (Mount Mulanie)
Lowest point: 473m (Lake Malawi)
Population: 9.840.747
Official languages: Engelish, Chichewa, Bantoetalen
Electricity voltages: 220-240 Volt
International land code: 265
Religion: Protestant, Roman Catholic, Muslim.
Currency: Kwacha, divided in 100 tambala.

Plan of work V1.7 - Laboratory team 173. 3
Friday 19th April 2010.
Chapter X Personal information and goals.

Rob Luijten: I always had a strong bond with travel and experiencing
something new. This internship will give me that
opportunity and will give me a chance to further increase
knowledge of the general population concerning the
management and quality of water.
Simply said this internship will develop me whilst I have a
chance to further help others.

Vincent Antonis: I traveled before and I would consider it to be a healthy
pastime/hobby for me.
Edukans provides me with a chance to travel and to help
in places where help is needed. I think this internship will
prove to be a challenge both mentally and physically and
it is a challenge I am more then willing to take.

Aart Manders: I’ve always wanted to travel around the world, see the
beautiful things this planet has to offer and meet people of
different cultures.
When I heard of Edukans Ex-change offering internships in
developing countries, I thought this would be a great
opportunity for me to do these things and combine it with a
chance to help the people of Malawi and even finish my own
I think I will learn a lot from this internship, not on chemistry
but more on communicational skills and on the capability of
improvising in a laboratory. Fact is that I already learned a lot
from just the preparations.

Plan of work V1.7 - Laboratory team 173. 4
Friday 19th April 2010.
Chapter X Inventory.

X.1 Knowledge transfer.
A transfer of knowledge will occur between several contacts and or connections
laboratory team 173 will either have from previous laboratory teams or from new
contacts made whilst stationed in Africa.

These contacts might include;
Local African students, teachers, local residents, the previous laboratory team, the
next laboratory team and us.

This transfer of knowledge concerns several basic points of water research and
water purification methods.
These points are:

- How to set-up and maintain a laboratory for analysis on water on both
chemical and microbiological level.
- How to analyze and test water on chemical and microbiological
- How to theoretically create and possible practically create and test a
water purification method.
- How to develop a long term program on water quality testing and water
- How to pass on general knowledge about water quality and healthy
water management.
- How to (safely) involve the school community.

More has to be known about potential health risks and water sources these risks
might come from.
To achieve greater knowledge 2 surveys might be conducted.
Doctors stationed at hospitals can further increase efficiency by providing extra
input over the general health.
The Robert Laws School and the neighboring hospital might be willing to provide
additional contacts which would most probably be of great help.

Plan of work V1.7 - Laboratory team 173. 5
Friday 19th April 2010.
We are the second laboratory team that will be stationed in Malawi.
Because of this fact there are several points we must address to eventually be able
to progress with the work left to us by the previous laboratory team.
This will be done by evaluating and re-evaluating results and recommendations left
for us by the previous laboratory team.

Point 1. Setting up an inventory list with equipment that is either
Inventory needed or available.
This list should only contain items that can be provided by
either companies or individuals from a laboratory team.
(Optional equipment will also be included in the inventory)
If an inventory list is already available this list will be

Point 2. The chemical and microbiological quality of water from
Experimental several water sources will be assessed. Either a
work purification method will be developed, thoroughly tested
and reevaluated or a purification method created by a
previous laboratory team will be defined. A long term
purification program will also be developed and/or be

Point 3. Conclusions will be drawn from the results of laboratory
Conclusion team 173 and laboratory team 138 combined. From
these conclusions recommendations towards a new
laboratory team will be made. This will be done to
guarantee improvement will be made by the next
laboratory team if they would chose to further our

Plan of work V1.7 - Laboratory team 173. 6
Friday 19th April 2010.
Point 4. Advice & Recommendations
First point of order would be setting up an inventory.
- A assessment has to be made about the quantity, quality and
availability of water sources and its water.
In chapter (XXX) several methods of harvest rainwater are described.
In chapter (XXX) several analysis methods concerning the quality of
water are described.
With that taken into account laboratory team 173 will be able to
determine and analyze water sources and its water in multiple ways
and be able to do so in multiple situations.

- To determine if people undergo health risks by drinking water from a
water source or to determine if people have a below average general
health. Laboratory team 173 will compare data found to data provided
by the world health organization.
The residents and/or locals who make use of the water sources will be
asked to take part in a survey if at all possible.

- Laboratory team 173 will analyze water sources of farmers, households
and schools. Laboratory team 173 will also make a summary of the
different water sources target groups use to get water.
To grant further insight locals can fill out a form where they get their
water, how they transport it, contain it, and store their water.
Laboratory team 173 will also inquire how long they walk from the
sources back home. If they have other means of transport are available
these too will be included in the inquiry.

- Laboratory team 173 shall investigate the environment near water
sources and the general area surrounding it as well.
To achieve this, the surrounding area and the sampling process will be
photographically recorded. This will have to be done at times locals
would normally use the water source to ensure whether or not live
stock is also using the water source or is somewhere in the near

- Laboratory team 173 should also think about which water purification
method is most useful at a certain location. For example a location
with electricity available will have access to more purification methods
in comparison with places that do not have access to electricity.
Laboratory team 173 should also get a clear indication of how many
people use the water source. For example a water source based in a
school would have access to different purification techniques in
comparison with a general household.
A comparison will be made between different water purification
processes and the maintenance needed for the aforementioned

Plan of work V1.7 - Laboratory team 173. 7
Friday 19th April 2010.
It should be kept in mind that locals might be analphabetic.
Because of this we need to approach one of our contacts so that he/she will be
able to act as an interpreter so laboratory team 173 should be able interact with the
residents and/or hospital staff thus being able to attain information that could be of
use in any one of the surveys.
It should also be kept in mind that several of aforementioned points are impossible
to put into practice whilst still in The Netherlands.
Additional info and/or input will all be obtained in Africa and will be added to the
report to provide additional insight somewhere on a later stage in the internship.

Three steps for research on water quality and purification:
Water quality research and purification research follows a general concept best
described in 3 steps.
Each step divided further into points of interest and points that should be taken into
consideration when developing or opting for a certain water purification method
and/or process.

Technical effectiveness.
- The ability to purify water.
- Removing and/or inactivating pathogens.

This point needs to be assessed in order to be sure if a certain technique could be
effective at a certain location.
If a technique proves to be ineffective thus not being able to clear step one the
chosen purification method will not be able to be used and thus be taken out of

Consumer acceptance.
- Availability of the material required for a specific purification method
- Availability of the knowledge required for a purification method
- Material and/or Educational costs.
- Taste and clarity of treated water.
- Safety of treated water

Plan of work V1.7 - Laboratory team 173. 8
Friday 19th April 2010.
Achieving widespread sustained use of aforementioned purification methods.

In order for this project to achieve maximum efficiency and to grant a nonobjective
view about the water quality in a certain area,
samples must be taken and analyzed.

To be able to grant insight on the water quality over a general area one must work
statistically correct.
This requires sampling:

- At different places
- In Different seasons
- At different depths of submersion in a water source
- At different water sources
- Before raining
- After raining
- Over a longer period of time
- Multiple analysis from each source

Target groups.
Target groups we are going to concentrate our research upon are:
- The children at the different schools in Embangweni.
- Local residents near our place of stay.
- Educated laboratory technicians and/or Doctors near our place of stay
and/or work

Laboratory team 173 will have to make an inventory provided by the input given to
us by the previous laboratory team.
This will then be adjusted to accommodate our needs for either more or for less
material and/or reagents. Laboratory team 173 will need several different reagents
and materials.
Below is a summary of what laboratory team 173 believes is needed to set up a
working laboratory.

Plan of work V1.7 - Laboratory team 173. 9
Friday 19th April 2010.
Chemical laboratory.
Essential material and/or features for a laboratorium.
- Water (Preferably running)
- Electricity
- Gas (optional)
- Tables (hard surface)
- Stone floor (even floor)
- (Open) window or a different form of ventilation
- Storage closet
- Fire proof storage (optional)
- Lights
- Lab coat
- Safety goggles
- Refrigerator

Chemical Laboratory.
- Glassware (Beakers/conical flasks)
- Weighing equipment
- photo spectrometer and cells
- heating(cooking)plates
- Demineralized water
- Glass Pipettes
- Glass Burettes
- pH/Ec meter
- Jerry cans
- Sample flasks

Biological laboratory.
- Mathematical grade calculator
- High pressure autoclave
- Incubator (37 °C)
- Microscope
- Centrifuge
- Standard weighing balance
- 2x Bunsen burner
- Tripod
- Stir wire
- Pipette
- Oses
- Demineralized water
- Saline
- Spectrophotometer
- Gram staining reagents
- Oxidase reagents
- Catalase reagents
- Short colored row (TSI, Indol, Urease)
- Glass conical flasks
- Glass measuring jar
- Glass petri dishes
- Specific agars (PCA, EMB, SS)

Plan of work V1.7 - Laboratory team 173. 10
Friday 19th April 2010.
Material can be purchased from Lab Enterprises Limited
POBOX 51712, Limbe, Malawi
Tel +2651875293
Fax +265 1876611

Unit pack Product Price (MK)
3 Erlenmeyer flask/conical flask MK ??
3 Volumetric flask MK ??
1 Thermometer MK ??
500 Petri dish MK ??
50 Plastic sample bottles MK ??
1 Test tube ith s/cup glass 150* 15mm MK 1,600
1 Alcohol 99vol% MK ??
3 Oses Mk ??
500g Plate Count Agar MK 19,380
500g Eosin Methylene Blue Agar MK 19,750
500g Salmonella Sghighella Agar MK 18,850
Colloidal silver MK ??
2,5l Kovac’s oxidase enzym MK 7,900
100ml H2O2 MK 18,312
1 roll Aluminium foil MK 4,800
500g Cotton wool MK 650

To be certain an e-mail will be sent to the Robert Laws School to give us a
summary of material and reagents already present.
This will be done as an indication for how much material and/or reagents laboratory
team 173 will have to bring themselves.

Water is going to be tested at several different locations with several variables.
The previous laboratory team started with 4 different locations.
Laboratory team 173 will also visit multiple locations as well and analyze water
samples taken from his or her respective source.
If proven necessary laboratory team 173 will locate and analyze other water
sources as well.
This will be done in conjunction with the company and/or schools laboratory team
173 will be coupled with and/or work together with.

Plan of work V1.7 - Laboratory team 173. 11
Friday 19th April 2010.
Chapter X Analysis.

The world Health Organization creates, manages and tries to enforce international
norms stating the acceptable quality of water.
These norms are formed in guidelines that should be used as an instruction for the
standard setting.
These guidelines if put to use will result in drinkable water and will not pose any
threats to either your health or those of others.
The current water quality in most of the water sources in Malawi does not hold up
against these guidelines.
The water from these water sources could or could not contain a multitude of;
Bacteria, parasites, chemical pollution or organic compounds which could possibly
be of harm.

To grant the laboratory team a further insight on the current level of water quality
the water will be tested with both several chemical and biological tests.
Eventually these results will be used as a guideline and as a reference for
upcoming laboratory teams.

X.1 Microbiological Analysis.

Plate count agar (PCA)
Plate count agar is a non-selective microbiological growth medium used to observe
the total of living bacterial growth.
Being non-selective plate means that all types of micro-organisms will be able to
Several dilutions of a water sample will be made and preferably a dilution
containing between 30 and 300 colony forming units will be used to determine the
rough amount of living bacteria in the initial suspension.
This is done by smearing out 10 µl of the sample on the plate count agar plate and
incubating said plate overnight at 37°C.

Eosin and Methylene blue agar (EMB)
Eosin and methylene blue agar is a selective and differential agar normally used
for the isolation of gram-negative enteric bacteria.
Said agar contains both eosin Y and methylene blue which acts as a inhibitor for
the growth of gram positive bacteria.
Two types of colonies can be formed on this agar plate:
Aerogene-type colonies; Often seen as a dark center surrounded by a wide, light
coloured ridge.
This shows us that a organism is not capable of fermenting lactose (Klebsiella and

Coli-types of bacteria will give a very distinctive metallic green shine.
This is due the metachromatic properties of the dyes used in the agar plate.
These organisms are also capable of fermenting lactose.
Those organisms are known as E.coli and several strains of Citrobacter

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Friday 19th April 2010.
Salmonella Shigella agar (SS)
Salmonella Shigella Agar is a selective and differential media normally used for the
isolation of pathogenic enteric bacteria, mainly those belonging to the genus
Salmonella. This medium contains a high concentration brilliant green and bile
salts. This medium is not recommended for the primary isolation of Shigella.

Clear streak.
A clear streak is used to separate different colonies present on a given agar plate.
This is done to isolate ‘suspicious’ colonies which might be determined by using
several identification tests.
Several inferences might occur whilst using a clear streak the most common one is
overgrowth meaning that a incorrect dilution is smeared on a the agar plate
resulting in massive growth all across the agar plate inhibiting the possibility of
isolating ‘suspicious’ bacteria.

An added technique can be used to ensure the isolation of colonies.
A single colony is smeared out over a new agar plate.
That said the colony will have more room to spreading itself over a greater area.
Loose colonies from these plates enlarge the chance of only using one type of
bacteria for the identification tests.

Gram Staining.
Gram staining is a staining method used to differentiate bacterial species into two
large groups.
These groups are known as Gram-negative and Gram-positive bacteria.
The staining method is based on the current physical properties of cell walls and
on the chemical properties of the reagents used.

Gram-negative bacteria are stained as a light purple (nigh pink) colour.
Gram-positive bacteria are stained as a deep (dark) purple.
(test results rely on the physical properties of cell walls so results may vary
between living and/or dead cells)

The basic morphology of the bacteria present might also be seen.
The only differentiation able to be made in this stage is between rods and cocci.

Oxidase is an enzyme that plays an important part in the electron transport chain
during aerobe respiration. An oxidation or reduction reaction takes place which
involves the molecular oxygen (O2) as the electron acceptor. In this reaction,
oxygen is reduced to water (H2O) or to hydrogen peroxide (H2O2). The oxidation
test makes a difference between the species Neisseria (pathogen) and
Pseudomonas who are oxidase positive (blue) and the Enterobacteriaceae who
are oxidase negative. (colorless)

Plan of work V1.7 - Laboratory team 173. 13
Friday 19th April 2010.
During aerobe respiration micro-organisms produce hydrogen peroxide (H2O2)
accumulation of said substance can be fatal to micro-organisms. Some organism
can produce catalase what can break down H2O2.

To test if a micro-organism has access to catalase one can use the catalase test.
A single drop of H2O2 is introduced to the micro-organism.
If the sample reacts to the H2O2 in the form of bubbles the micro-organism will be
positive for catalase.
If no bubbles show the test will be considered negative and the micro-organism will
be determined to not have access to catalase.

Short colored Row.
Triple-sugar-iron. (TSI)
Triple-sugar-iron is a test used to help determine if a microbe has the ability to
make acid or acid and gas out of carbohydrates, or if it can form H2S out of sulphur
concerning amino acids
The medium contains lactose, glucose and sucrose
Some bacteria ferment the glucose but do not ferment the lactose or sucrose.
These organisms use the protein at the aerobic surface of the slant as a carbon
source, the pH increases and the top of the slant becomes red. The black is due to
hydrogen sulfide production. Substantial hydrogen sulfide production makes it
difficult to determine color in the butt of the slant.
Some organisms only ferment the glucose but do not produce hydrogen sulfide
(note gas production shown as a bubble in the TSI tube). Some ferments all three
sugars, the pH goes down and the slant is yellow.

Indole production test.
This test is used to determine if the bacteria can breakdown the amino acid
tryptophan into indole. The presence of indole is detected with Kovac’s reagent
which will show a perfect red ring on top of the fluid.

Urease test.
Some bacteria can degrade urea into ammonia, water and CO2 by the action of the
urease enzym. The reaction occurs as follows: (NH2)2CO + H2O → CO2 + 2NH3
The production of ammonia leads to alkalinity in the medium and will cause the
medium to turn dark pink because the pH is >8,1 (the indicator used is phenol red).

Plan of work V1.7 - Laboratory team 173. 14
Friday 19th April 2010.
X.2 Checmical Analysis.

The indicator for acidity or alkalinity is known as the pH value. A pH value of 7
means a substance is neutral. The lower the pH value is indicates acidity, and a
higher pH value is a sign of alkalinity. The pH value determines whether water is
hard or soft. The pH of pure water is 7. In general, water with a pH lower than 7 is
considered acidic, and water with a pH greater than 7 is considered basic. The
normal range values for pH in surface water systems is 6.5 to 8.5 and for
groundwater systems 6 to 8.5. Water with a low pH (< 6.5) could be acidic, soft,
and corrosive. This could indicate the presence of toxins in the drinking water and
these are associated with health risks.
Water with a high pH (> 8.5) could indicate that the water is hard. Hard water does
not pose a health risk, but can cause other problems. These problems include an
alkali taste to the water, formation of deposits on dishes.

The electrical conductivity in a solution can be measured with a conductivity meter.
The conductivity of water is of interest because it gives an indication for the values
of anions and cat ions in water. The higher the value of the conductivity, the more
ions are present in the water sample. Regular tap water in the western world has
conductivity of about 500µS/cm, higher values can indicate poor quality of water.

Ammonium (NH4+)
The presence of ammonium in water is an important indicator for fecal pollution.
A lot of the organic wastes which could be present in drinking water contain
proteins and amino acids. These compounds contain nitrogen and can be broken
down to smaller chemical compounds by bacteria and enzymes.8 The first
compound that it is broken down to is ammonium. Ammonium is a nutrient for
plants, but if there’s too much ammonium present in the water or there are not
enough plants to consume the ammonium, bacteria will process this. The presence
of the ammonium cationin drinking water could lead to the formation of nitrite which
could also indicate the presence of ammonium-oxidizing bacteria.

Plan of work V1.7 - Laboratory team 173. 15
Friday 19th April 2010.
Nitrate (NO3-)
Nitrate is a wide spread contaminant of ground and surface waters worldwide.
Nitrate is a molecule made up of nitrogen and oxygen. It is formed when nitrogen
from ammonium or other sources, combines with oxygen in water.6 Nitrate is a
natural compound of plants and is found in vegetables at varying levels depending
on the amount of fertilizer applied and on other growing conditions.
The accumulation of nitrate in the environment results mainly from7:
- Water or soil from agricultural areas which have been overly exposed to
nitrogen-fertilizers drain into other waters.
- Waste from human and animal sewage entering the waters
These pollutants are eventually deposited into bodies of water, such as lakes,
rivers, wetlands, coastal waters, and underground sources of drinking water.
When nitrate is taken in by eating food and drinking water, nitrate is converted in
the digestive system to nitrite, which then combines with haemoglobin and
decreases the ability of the blood to carry oxygen. Infants are more susceptible to
nitrate toxicity than older children or adults.
According to the World Health Organization, most adults ingest 20-70 milligrams of
nitrate- nitrogen per day with most of this coming from foods like lettuce, celery,
beets, and spinach. When foods containing nitrate are eaten as part of a balanced
diet the nitrate exposure is not thought to be harmful.

Nitrite (NO2-)
Nitrate does not normally cause health problems unless it is reduced to nitrite.
Nitrite is generally formed by the action of bacteria on ammonium and organic
nitrogen. The presence of nitrite does not always signify pollution, although, in
combination with ammonium and nitrate, the presence of nitrite is a pollution
Nitrate is more stable than nitrite. This means nitrite easily changes into nitrate in
groundwater and the results of a nitrate plus nitrite test are almost always
predominantly nitrate. But as said in the explanation about nitrate, when it is
consumed, it can be converted into nitrite again which poses health risks.
Testing labs report nitrate plus nitrite results as nitrogen present as nitrate and
So it is important to understand that values of nitrate, nitrite and ammonium can be
closely related.

Phosphate (PO43-)

The addition of large values of phosphates to waterways accelerates algae and
plant growth in natural waters. This lowers the oxygen level in the water body thus
causing the death of oxygen needing organisms. Large amounts algae can form
and can completely cover small lakes. As a result, water can become polluted from
decaying organic matter.10 Sources of phosphate are similar to that of nitrate and
include human sewage, agricultural waters used for crops, sewage from animal
feeding locations, pulp and paper industry, vegetable and fruit processing,
chemical and fertilizer manufacturing.

Plan of work V1.7 - Laboratory team 173. 16
Friday 19th April 2010.
Sulphate (SO42-)
A sulphur cycle exists which includes atmospheric sulphur dioxide (SO2), sulphate
ions (SO22-) and sulphides (S-). Sulphides, especially hydrogen sulphide (H2S), are
soluble in water and are toxic to both humans and fish.
Sulphate is a substance that is naturally present in drinking water. High values of
sulphate can cause diarrhea. Sulphides are produced by bacteria under conditions
where there is a lack of oxygen (anaerobic). Because of their foul "rotten egg"
smell they are avoided by both fish and humans. Sulphides formed as a result of
coal mining or other mineral extraction and from industrial sources, can be oxidized
to form sulphates, which are less toxic.
Problems caused by sulphates are most often related to their ability to form strong
acids which changes the pH. Sulphate ions also are involved in reactions which
affect solubility of metals and other substances.

Quality control
This will be done to make sure our results are reliable.

Control samples
For the positive control we will use H2O obtained from a water bottle purchased for
drinking. For the determination of every chemical component a control sample will
be made, to make sure the spectrophotometer does its work properly. As long as
our calculations and the values given by the spectrophotometer differ no more than
10%, the determination will be reliable.

Plan of work V1.7 - Laboratory team 173. 17
Friday 19th April 2010.
Chapter X Weekly Schedule.

On Mondays the collection of water samples from different sources will take place.
As of yet it’s uncertain if the same or a different source will be tested per week. It
may be possible that the laboratory team might have to travel for extended periods
of time and over great lengths of distance.
If this is the case it might be possible for the laboratory team to depart on a Sunday
and arrive back on Monday with the water sample.
This can, will and must only be done in consultation with our hosts and under
guidance by either a ‘buddy’ or one of the trusted locals/colleagues.

Once the laboratory team has returned at the laboratory the water sample will be
divided in two bottles.
These bottles will contain all the sample needed by both the biological part of the
team and the chemical part of the team.

Biologists will plate the water on three culture medium plates (PCA-, EMB- and SS
medium), which will stay overnight at 37°C as mentioned before.

The plates are checked for bacteria.
If there are bacteria colonies present, clear streaks are made if possible and
incubated overnight at 37°C.
If overgrowth occurs the laboratory team will be forced to repeat the steps made on

A gram staining will be made to identify bacteria found on plates from Tuesday.
This will and must be done BEFORE other tests will be used.
Clear streaks are checked and suspicious micro organisms are put through a
series of tests to possibly identify said bacteria.
These tests would be aforementioned short coloured row, catalase and oxidase.

If needed the results of tests from Wednesday are checked.
Conclusions will be made about said micro-organisms and these will be added to
the inventory.

On Friday preparations will be made for new culture medium plates.
These plates might be infected during the weekend.
So it might be possible that work has to be done on Saturday and Sunday as well.
Fridays through Sundays updates for our bi-monthly letter might be added as well.

Plan of work V1.7 - Laboratory team 173. 18
Friday 19th April 2010.
Water transport.
Water will be transported in tubes filled to the top so that little to no air is polluting
and/or degrading the water sample.

This will be tested on the water sample by added a optional extra test.
Water will be collected in a source near the direct vicinity of our laboratory.
These samples will be tested and several intervals in time.
These intervals will be 8, 16, 24, and 48 hours from the time of sampling.
The water samples will be stored in several conditions as well.
One will be placed in a refrigerator the other two will be placed at room
temperature and the direct sunlight.

This test will show us if time and/or storage conditions will be of influence on said
If there is any change in growth what so ever extra steps should be made to
ensure the consistency of water quality.

Sample in the Sample at Sample in Tested for
refrigerator room the sun
8 hours PCA, CFUs/ml,
pH, conductivity
16 hours PCA, CFUs/ml,
pH, conductivity
24 hours PCA, CFUs/ml,
pH, conductivity
48 hours PCA, CFUs/ml,
pH, conductivity


Plan of work V1.7 - Laboratory team 173. 19
Friday 19th April 2010.
Community awareness program.
A program will be set up whilst in Holland and executed whilst stationed in Africa.
The idea behind this entire program/project is to create some form of awareness
concerning water quality and management.

This all will be conducted on our buddies, colleagues, teachers present and if
possible locals as well.

Possibilities are vastly determined by the materials provided by either the
laboratory team.
Or by the materials available in Africa.
Currently the idea’s opted for stand as followed;

Provide the people means to understand the current situation of the water and the
management of water by giving ‘lectures’ and if possible(and if chosen for) provide
them with graphic imagery concerning the subject at hand.
This can either be done by images created beforehand (either photographed or
hand drawn) or by actually showing them ‘parts’ of the laboratory.

This might be achieved by ‘’schooling’’ the people of interest either during school
hours or as a extracurricular activity after school. (weekends might be opted for this
as well)
With each passing lesson depth can be added and more complex details of water
and its current situation might be addressed.

As it stands the following subjects will be addressed;
- What is water ? (different kinds of waters, pollutions, compounds)
- Why do you need water ? (self sustainment, food and drinks,
agricultural tool)
- What happends when it goes wrong ? (water contamination and illness)
- What should/can we do ? (tips about water management and improving
water quality)

Of course it should stand as noted that one might not be able to address a child
the same was as he/she would be able to address a teacher.

Plan of work V1.7 - Laboratory team 173. 20
Friday 19th April 2010.
Protocol 1 – Preparing media for plates
PCA (Plate count agar):
PCA agar powder.
Distilled water.
Erlenmeyer flask. (1000 ml)
Bunsen burner.
Autoclave (121°C).
Sterilized Petri-dishes.

Preparation: (0,5 liter)

Suspend 8,75 g in 0,5 liter distilled water.
Dissolve and heat to boil.
Sterilize by autoclaving at 121°C for 15 minutes.
Cool to about 50°C and mix well.
Pour the agar into the petri plates and fill them until 0,5 cm.

EMB (Eosin and Methylene blue agar):
EMB agar powder.
Distilled water.
Erlenmeyer flask. (1000 ml)
Bunsen burner.
Autoclave (121°C).
Sterilized Petri-dishes.

Preparation: (0,5 liter)

Suspend 18,75 g in 0,5 liter distilled water.
Dissolve and heat to boil.
Sterilize by autoclaving at 121°C for 15 minutes.
Cool to about 50°C and mix well in order to oxidize the methylene blue.
Pour the agar into the petri plates and fill them with 0,5 cm.

Reading the result:
Escherichia coli : Good growth, green metallic sheen.
Klebsiella pneumoniae : Good growth, purple colonies, no sheen.
Shigella flexneri : Good growth, transparent colonies (lactose negative)

Plan of work V1.7 - Laboratory team 173. 21
Friday 19th April 2010.
SS (Salmonella Shigella agar):
SS agar powder.
Distilled water.
Erlenmeyer flask. (1000 ml)
Bunsen burner.
Sterilized Petri-dishes.

Preparation: (0,5 liter)

Suspend 31,5 g in 0,5 liter distilled water.
Dissolve and heat to boil.
Do NOT autoclave or overheat because it may destroy the selectivity of the
Cool to about 50°C and mix well.
Pour the agar into the petri plates and fill them with 0,5 cm.

Reading the result:
Salmonella typhi : Colorless colonies, black centers. (H2S positive).
Shigella flexneri : Colorless colonies. (H2S negative)
Escherichia coli : Pink colonies.

Protocol 2 – Counting colonies (CFU/ml)
Plate with bacteria colonies.

Count the total number of colonies on each plate.
If half plates are used multiply the amount by two.
When there are a lot of colonies divide the plate in to quarters, count one quarter
and multiply the amount by four.
When there are more than 300 colonies it can be noted as >300
Calculate the CFU/ml for the dilutions that contain between 30 and 300 colonies.
The following formula can be used:
N x D x V = CFU/ml
N = the number of colonies
D = Dilution factor
V = Volume factor (30µl = 33,3)

Reading the result:
Once the CFU/ml has been determined write the results in the lab journal.

Plan of work V1.7 - Laboratory team 173. 22
Friday 19th April 2010.
Protocol 3 – Clear streak
Bacteria colonies.
Bunsen burner.
Inoculation loops.
PCA, EMB and SS plates.

Take a sterile inoculation loop and tip the selected colony
Make four to five lines on the selected plate. (1)
Sterilize the inoculation loop and make four to five lines
through the grid. (2)
Repeat step three, to make lines through the second
grid. (3,4)
Sterilize the inoculation loop and incubate for 24 hours.
The EMB and SS must be incubated at 37 °C and the PCA at room temperature.

Reading the result:
The clear streak has worked if the colonies are of the same species bacteria. The
colonies must also be separated from each other making it possible to tip only one
colony at a time

Plan of work V1.7 - Laboratory team 173. 23
Friday 19th April 2010.
Protocol 4 - Gram Staining
1 bacteria colony.
Physiological salt.
Crystal violet.
Ethanol (95%, 70%)
Microscopy slides.
Inoculation loop.
Bunsen burner.
Marker pen or pencil.
Immersion oil.
Absorption paper.

Clean object glasses .
Place a drop of physiological salt on the microscope slide.
Take one bacteria from the plate and smear in the physiological salt using an
sterile, cooled inoculation loop.
Leave the smears to air dry and then heat fix in the usual manner by sweeping the
slide three times through the flame of the Bunsen burner.
Drop 3-4 drops of crystal violet on the smear for 1 minute.
Rinse the dye off with demi-water.
Drop 3-4 drops of lugol on the smear for 1 minute.
The lugol can be tapped off the side of the slide.
Drop 3-4 drops of ethanol 95% on the smear for 30 seconds.
Remove the ethanol 95% by rinsing with 70% ethanol
Rinse with demi-water.
Drop 3-4 drops of safranin on the smear for 1 minute.
Rinse with demi-water.
Leave to air dry.
Once dry place under microscope to read the results.
Focus the microscope with the 5, 10 and 40 magnification.
Before using the 100 magnification place one drop of immersion oil on the smear
and focus.
The bacteria group can now be determined

Reading the result:
Bacteria with a purple colour can be seen as gram positive bacteria. Bacteria with
a pink colour can be seen as gram negative bacteria.
Further the shape of the bacteria is important. The two main shapes are rods and
cocs. Rods can be long and thin bacteria. Cocs are a more circular shape.

Plan of work V1.7 - Laboratory team 173. 24
Friday 19th April 2010.
Protocol 5 – Biological tests

Bacterial colonies on the clear streaks
Bunsen burner
Inoculation loops
Filter paper
Kovac’s-oxidase reagent

Sterilize the inoculation loops
Take a colony of the plate with a sterile inoculation loop.
Smear the colony on the filter paper.
Put on a droplet of Kovac’s-oxidase reagent on the filter paper.
Wait for 5 to 10 seconds and observe the colour.

Reading the result:
With a positive reaction with the oxidase reagent the color changes to blue. With a
reaction the color changes to pink or there is no color change.

Colony from a clear streak
Bunsen burner
Inoculation loop
Glass slides
3% H2O2

Place a drop of 3% H2O2 on the slide.
Place a colony on a sterile inoculation loop from the clear streak.
Smear the colony on the droplet.
Observe the reaction.
Reading the result:
- Positive reaction: formation of O2 bubbles (+)
- Negative reaction: absence of O2 bubbles (-)

Plan of work V1.7 - Laboratory team 173. 25
Friday 19th April 2010.
The short colored row  TSI, Indol, Urease and Citrate test.

Day 1
Describe the bacterial colony.
Make a gram staining to control the cleanness of the culture and to make sure your
dealing with a gram negative rod.
Suspend ± 3 colonies in 5 ml PBS.
With a dropper ± 3 drops of the fluid are dropped on the agars. (make sure the fluid
is spread evenly on the agar)
The TSI agar also has to undergo a depth twinge. (do this one at last, or else the
agar gets stuck in your dropper)
Make a clear streak on normal culture medium.
Put overnight at 37°C.

Day 2
Control the clear streak, if this is not one bacteria the test is not accurate.
Indole test: drop some drops of Kovac’s reagent in the tube. Positive: a red ring
Note the color of the urease tube.
Note the color of the citrate tube.
Note the interpretation of the TSI tube

Bacteria culture from the clear streak.
Triple Iron Sugar agar.
Distilled water.
Tubes and caps.
Inoculation needle.

Inoculate each bacteria culture into its appropriate labeled tube by using the streak
and stab method.
Stick the needle about 3 cm in the butt of the medium and use the same needle to
inoculate the slant by moving the needle sideward’s from the bottom to the top.
Always use one tube as a negative control by not inoculate.
Incubate for 18-24 hours at 37°C.

Reading the result:
Red: does not ferment either lactose or sucrose
Yellow: ferments lactose and/or sucrose
Red: no fermentation, the bacterium is an obligate aerobe
Yellow: some fermentation has occurred, acid has been produced, it is a facultative
Gas formed: Seen as cracks in the agar, bubbles, or the entire slant may be
pushed out of the tube. (Caution: these gassy fermenters may have bacteria close
to the opening.)
Black: H2S has been produced.

Plan of work V1.7 - Laboratory team 173. 26
Friday 19th April 2010.
Bacteria culture from clear streak.
Tryptone water.
Distilled water.
Kovac’s reagent.
Inoculation loop.

Inoculate each bacteria culture into its appropriate labelled tube
Mix the colony in the indole.
Inoculate the tubes at 37°C for 24-48 hours.
After inoculation remove the caps and add 5 drops of Kovac’s reagent with a
pipette. Shake the tubes gently and observe the colour.

Reading the result:
If the Kovac’s reagent turns red than this indicates a positive test (+)
If the Kovac’s reagent stays green/yellow than this indicates a negative test (-)
With a negative test, make sure that the bacteria has grown. If not, the result is not

Bacteria culture from clear streak.
Urea agar.
Urea 40%.
Inoculation loop.

Inoculate each bacteria culture into its appropriate labelled tube by using the streak
and stab method. (Streak and stab method: Insert the loop into the medium to
approximately one-fourth of its depth. If testing motility, use an inoculating needle
and stab it in the centre of the agar tube to the bottom. Draw the needle out
carefully, keeping it straight.)
Inoculate the tubes at 37°C for 8-48 hours depending on the speed of the hydrolyse
of urease.

Reading the result:
If the media has growth on it and it’s turned purple/pink this is a positive (+) result.
If the media has no growth and is the media still is the same colour as before this is
a negative (-) result.
If there is no colour change and no growth after 48 hours. This counts as a
negative result. With a negative test, make sure that the bacteria has grown. If not,
the result is not reliable.

Plan of work V1.7 - Laboratory team 173. 27
Friday 19th April 2010.
Appendix 3 • Identification scheme

Plan of work V1.7 - Laboratory team 173. 28
Friday 19th April 2010.
Plan of work V1.7 - Laboratory team 173. 29
Friday 19th April 2010.