Professional Documents
Culture Documents
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
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
countries.
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.
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.
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
Education.
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.
This transfer of knowledge concerns several basic points of water research and
water purification methods.
These points are:
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.
- 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
process.
Technical effectiveness.
- The ability to purify water.
- Removing and/or inactivating pathogens.
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
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
Material.
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.
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)
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.
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.
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.
Oxidase.
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)
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.
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).
pH
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.
Conductivity
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.
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
indicator.
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
nitrite.
So it is important to understand that values of nitrate, nitrite and ammonium can be
closely related.
Phosphate (PO43-)
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.
Monday.
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.
Tuesday.
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
Monday.
Wednesday.
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.
Thursday.
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.
Friday.
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.
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
bacteria.
If there is any change in growth what so ever extra steps should be made to
ensure the consistency of water quality.
Conclusion
This all will be conducted on our buddies, colleagues, teachers present and if
possible locals as well.
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.
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.
Method:
Preparation: (0,5 liter)
Method:
Preparation: (0,5 liter)
Method:
Preparation: (0,5 liter)
Method:
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)
Method:
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.
Method:
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
Oxidase:
Materials:
Bacterial colonies on the clear streaks
Bunsen burner
Inoculation loops
Filter paper
Kovac’s-oxidase reagent
Method:
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.
Catalase:
Materials:
Colony from a clear streak
Bunsen burner
Inoculation loop
Glass slides
3% H2O2
Method:
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 (-)
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
appears.
Note the color of the urease tube.
Note the color of the citrate tube.
Note the interpretation of the TSI tube
TSI
Materials:
Bacteria culture from the clear streak.
Triple Iron Sugar agar.
Distilled water.
Tubes and caps.
Inoculation needle.
Method:
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.
Method:
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.
Urease
Materials:
Bacteria culture from clear streak.
Urea agar.
Urea 40%.
Tubes.
Inoculation loop.
Method:
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.