Jazer John T.

Lirazan BS Chemistry IV
Bureau of Fisheries and Aquatic Resources
Date: June 8, 2018

During our first day, we are first tour the laboratory and was introduce with the laboratory
staffs and analysts. We are then oriented on what is BFAR, what are the roles of BFAR the analysis
they make and the most important part is how to protect ourselves and our safety because that is
their main concern. We read their laboratory safety manual and Work instructions manual for us
to be knowledgeable of what are the instruments and analysis in the laboratory, and also to know
the SOP of their lab. The reading took us for almost the whole day because it’s so long. After we
have done reading the manual we were able to help perform their QC for their GFAAS because
they were detecting problems in their method so QC must be maintain in line with the ISO 17025
accreditation. Quality control, or QC for short, is a process by which entities review the quality of
all factors involved in production. ISO 17025 defines quality control as "A part of quality
management focused on fulfilling quality requirements”. We perform Standard addition in
GFAAS using 6 standards and 6 replicates. The method of standard addition is a type of
quantitative analysis approach often used in analytical chemistry whereby the standard is added
directly to the aliquots of analyzed sample. We help in the preparation of standards as well as in
the experiment proper. GFAAS is use to detect trace amounts of metals like lead and cadmium.
Graphite furnace atomic absorption spectroscopy (GFAAS) (also known as Electrothermal Atomic
Absorption Spectroscopy (ETAAS)) is a type of spectrometry that uses a graphite-coated furnace
to vaporize the sample. The analysis is very will take only hours because it is automatic but we
were not able to help interpret the result because its 5 pm already, so we just fixed our things and
go home because it’s raining.
Date: June 11, 2018
Today we were able to help in the calibration of their analytical balance, the action or
process of calibrating an instrument or experimental readings. "The measuring devices require
calibration" each of a set of graduations on an instrument. As an ISO accredited laboratory
analytical balance must be calibrated every week to insure its accuracy using calibrated weights
these weights are calibrated annually to the main office to insure its value. Weights are kept in a
box for them to insure its calibrated value. We then start our calibration we weighted 1, 10 and
100 g with each three trials, each weight has an indicated confidence interval for error to be
accounted. After weighing each weights we then check if it passes or not. The analytical balance
passes the calibration testing. After testing the calibration we helped in washing the laboratory
equipment for the microwave digester and was then dried using a microwave dryer set in a
particular temperature for 3 hrs. Microwave digestion is a common technique used by elemental
scientists to dissolve heavy metals in the presence of organic molecules prior to analysis by
inductively coupled plasma, atomic absorption, or atomic emission measurements. After that we
were given pipetting exercises we were task to pipette out 2 and 5 ml of water and was weighed
into the analytical balance to measure its accuracy and precision using standard deviation. It took
us a lot of time to achieve a 1% std. deviation but we are glad that we were able to practice a high
accuracy and precision pipetting skill. After lunch we received a sample from a private owner for
the examination of certain diseases in shrimps like WSSV (White spot syndrome virus) and
APHND/EMS (Acute hepatopancreatic necrosis diseases/Early mortality syndrome). In the
laboratory they PRC method to examine certain diseases present in the marine sample.
The analysis method is similar to conventional PRC in the way that the first principal component
is plotted against time, but differs from conventional PRC in terms of the precise definition of
explanatory variables and covariables. First step was Sample preparation and DNA extraction.
This includes sample preparation DTAB-CTAB DNA extraction procedure and DNA dissolution.
We help and first handedly experience the process from sample preparation to extraction itself.
The next step will be conducted on the other day due to lack of time. After the extraction process
we again did some practice for the histamine process, a pipetting exercise again. A simple and
rapid method for histamine analysis is now required all over the world. A simple and rapid method
for monitoring histamine levels in fish and fishery products is therefore needed to avoid delay in
the analysis in order to ensure safety of fish products. We were tasked to transfer a 2 ml of water
to another bottle every 20 mins, at first we find it hard but as we do 10 trials we were able to adjust
and done it successfully. After that we help cleaning in the lab we planned our next activities in
the laboratory and went home.

Date: June 13, 2018

Today we again calibrated the analytical balance same method was done during the last
calibration a standard weight was weighed for three times the mean was then computed to see if it
varies from its designated confidence interval. We saw a variation on the weight so internal
calibration of the analytical balance was done. Internal calibration allows the weighing scale or
balance to calibrate itself, often automatically without needing manual input from its users or
calibration weight sets. There are various degrees and technologies used to ensure precision that
change depending on model, make and price range. Some balances even allow you to set
calibration at specific times or intervals (for example, during lunch or in the morning before people
come to work, or even every couple of hours). They also allow for the scale or balance to be
calibrated with a single button press. These balances have built-in calibration weights, often motor
driven. Then we continued the analysis for WSSV (White spot syndrome virus) and APHND/EMS
(Acute hepatopancreatic necrosis diseases/Early mortality syndrome). The sample were submerge
in a 95% alcohol for preservation and avoid deterioration and was stored in a refrigerator. After
the samples was removed from the refrigerator a kit for this analysis was also prepared. A problem
was encountered in the laboratory that one of their refrigerator was turned off due to the electrical
shortage which affects the AVR and results to shut down. The kits inside the ref was also task to
analyze especially the enzymes present to see if it was destroyed during the incident. With that
matter we did two analysis with the samples given by the private sector using a new kit and a kit
from the refrigerator. The new kit was done by the laboratory analyst and the old kit was done by
us fist handedly “happy accident”. I was task to dilute the EBT buffer solution from 10x to 1 times
so I did from that buffer solution helps in the formation of gel in the electrophoresis process. Using
the prepared buffer solution I single handedly prepared a gel for the electrophoresis process which
involves weighing 1 g of gel powder then dilute to 150 ml buffer then heat at high temp for 3 mins
remove then heat again for a min. let it settle then pour into the plate. Going back to the analysis
we did the amplification reaction protocol which amplifies the DNA and RNA present in the
sample it involves reagent preparation, reaction condition, and reaction procedure. The laboratory
analyst was first to do the experiment to show us how it’s done following the lab manual and using
the new kit for the analysis, then we do the same on what he did using the old kit which suspect to
be destroyed because of the incident. In the sample preparation process during this step cleanliness
and organization must be observe since we are dealing ul amount of solution a careful and keen
experimentation was done. When everything was prepared it was dyed using a dye from the kit
with a 1 ul of dye to 5 ul of solution ratio. The solution was then injected into the gel for
electrophoresis. Gel electrophoresis is a method for separation and analysis of macromolecules
and their fragments, based on their size and charge. After the electrophoresis it was then submerge
into a bromine solution and then water and was then analyzed. The analysis of WSSV and
APHND/EMS is very similar the only variation is base in their amplification process. Results was
then examined by the laboratory analyst and gladly the enzyme from the refrigerator incident was
not damage at all so basically we really did the analysis first handedly and successfully. We ate
lunch after everything. After lunch we were tasked to weigh the samples from a private sector for
Chloramphenicol analysis. Chloramphenicol is an antibiotic useful for the treatment of a number
of bacterial infections. This includes as an eye ointment to treat conjunctivitis. This is needed for
the export of these following samples. Sample weighing was not that easy because sample were
squishy it almost took us 1.5 hrs to weigh 10 samples with each 2 replicates. This analysis uses the
method of LC MSMS. Liquid chromatography–mass spectrometry is an analytical chemistry
technique that combines the physical separation capabilities of liquid chromatography with the
mass analysis capabilities of mass spectrometry. The laboratory manual was followed during the
process the laboratory analyst has a huge trust on us and he let us do the experiment just following
the lab manual he first demonstrate the spiked samples then we do the rest. Spiking was done to
increase the accuracy of the data. First was the addition of solution from the lab manual, then it
was then centrifuge to separate the layers. The upper layer was remove due to the presence of ethyl
acetate and hexane was then added to dissolve it. It was centrifuged to separate the layers and after
that the lower layer was then gathered and filtered using a nylon microfilter ready for the analysis.
Analysis of the LC MS was done by the lab analysis and was set overnight to run. We were not
able to help him further because it was already 6 pm. We cleaned the lab and washed and go home.
Today was very productive since we were able to really conduct the experiment by ourselves not
just to watch but to actually do it.

Date: June 14, 2018

The analysis for chloramphenicol was done using LC-MS or liquid chromatography mass
spectrometry is an exceedingly sensitive and specific analytical technique that can precisely
determine the identities and concentration of compounds within the sample. After the analysis the
sample containers were washed, the washing process includes separation of the caps and the bottles
the the solution inside the bottle are then discarded into a chemical bottle. The bottles are then
rinse with tap water individually, after that it will be rinse again with ultra pure water. Ultra pure
water refers to water with high purity that has been made as close as possible to H2O by integrating
all the elemental technologies for water. The purity of water is upgraded to an ultra high level by
removing not only solid substance and salts but also gas dissolved in water. Then it is rinsed with
acetonitrile, but before that it was vacuum filtered to remove other contaminants since LC-MS
column is very sensitive. Acetonitrile is the simplest organic nitrile and is used in LC MS. After
that it was then oven dried to dry all volatile substances. The washing took all day because the
process was very tedious because it was performed carefully to avoid contaminants.

Date: June 18, 2018

Today we washed again some bottles for the LC-MS process using acetonitrile then after
that we were able to solve for the chloramphenicol content of the analyzed samples. We solved for
theoretical yield which the volume used multiplied by the concentration all over the sample spike
weight. This is the expected amount of yield collected. We also solve for the actal yield by
multiplying the concentration of the calculated with the dilution factor over the spike weight. This
is the actual yield of your samples. We also solved percent recovery by subtracting the actual
minus sample concentration all over theoretical multiplied by 100. Final concentration is also
solved using concentration sample multiplied by dilution factor over sample weight then divided
by the percent recovery. We are then given a quick lecture about the concepts of LC-MS and how
to interpret its data. Then its lunch time. After lunch we were able to help in the sample preparation
for the analysis of histamine in fish samples like sardines. As a part of immune response to foreign
pathogens, histamine is produced by basophils and by mast cells found in nearby connective tissue.
Histamine increases the permeability of the capilliaries to white blood cells and some proteins, to
allow then to engage pathogens in the infected tissue. First we separate the meat of the fish with
the bones then grind it with the blender to homogenize the samples. The sample are then labeled
based on their names. After that we help cleaning in the laboratory then go home.
Date: June 19, 2018
Today we prepared the sample containers for LC-MS method by washing the sample with
ultrapure water then it was then placed in a separate container for it to be ready for analysis. Shrimp
samples from a private sector has arrived to be analyzed for white spot and APHND/EMS since
we already have an experience with this method we were task to do the sample preparation all by
ourselves. First the shrimp were separated from the water using a strainer then it was put in a tube
using a forceps. We were not able to help with the analysis since the samples that are given are so
small and it’s very dangerous to do some error since the analyst will not be able to run the test
again. After the lunch break we again clean the bottles for LC-MC instrument using the ultrapure
water we were able to use the machine and we were taught how does it Ball-valves, Butterfly-
valves, needle-valves, Floating-valves, gate-valves, Forged-valves, Check-valves, Y-Strainers,
Joints. Material in Brass, Stainless Steel of Cast Iron. Are the basic parts of the machine. Since we
will be going for a field the next day for red tide analysis at isla gigantes we were taught how too
use the machine and we were able to calibrate the machine base on its pH and DO for water
analysis. We just follow the manual that is with the machine for calibration. Some concepts are
also discussed for us to have an idea what will happen for the next day, pH and Do are analyzed
since in theory it at an ideal pH and Do phytoplanktons are able to reproduce more which elevates
the risk for red tide. They only do a positive and negative results since the method for the complete
red tide analysis is not yet available it is the mouse bio assay. The mouse bioassay is a functional
assay that detects biologically active toxin. The assay requires a three part approach: toxin
screening, toxin titer, and finally toxin neutralization using monovalent antitoxins. The process
requires two days of analysis at each step. When it reads positive the regional office will forward
the sample to the main office for further analysis. We were also able to do moisture determination
of sardine samples, first sardines are cut into small pieces and was homogenized and the sample
was then put in a moisture teller then we were able to determine the exact weight and moisture of
the sample. After all the orientation we then help in cleaning the laboratory and went home after.
Date: June 20, 2018
Today was our field work we arrived 4:30 in the morning at the assembly place ceres
terminal for the first trip going to isla gigantes, the trip is very long it took us almost 5 hours
including the boat ride. When we arrived at where will be staying we had our lunch. After eating
our lunch we prepared the tools for the sample collection; 3 transparent bottles for water sampling,
a plankton net and 6 small battles for plankton collection and a container for the collection of
shellfish. After we prepared everything we went into the boat to head at the sampling site. At first
the waves were bearable but as we approach the deep sea the weaves are getting larger we will
collect sample in 3 barangay here in the island to check if there is a presence of red tide, or harmful
algal bloom. Red tide is a common name for a worldwide phenomenon known as an algal bloom
when it is caused by species of dinoflagellates and other organisms. When we arrive at our first
destination we first collect water sample we get a transparent bottle then submerge it into the water
so that it will be filled and no air bubbles must appear inside the container because it might affect
the dissolve oxygen content of the water. Then we collect plankton samples using a plankton net.
We use the horizontal method in collecting the sample. We first submerge a 5 meter long rope
with the net into the water then after that the net was raised at a rate of .25 m per second when the
net was fully raised it was then put in a small reagent bottle and was labelled. It was done again
using a 10 meter depth of water same process was done, it was then again put in a bottle and was
labelled. After then we first allow the fisher men to get samples of shell fish for us at the deep sea,
we gather bukol-bukol and scallops because these kinds of shellfish are abundant throughout the
year and date data can be compared because of that. When it was collected it was put in a container
and was then labelled. We then head for our second and third destination and same process was
done. During the last destination our supervisor allow us to do the sampling all by ourselves for us
to experience it first handedly, we did it successfully. After collection of sample we then head back
to the shore for sample preservation and analysis of water. Using a multiparameter tester we
analyze waters pH, temperature, dissolved oxygen, and many other things. Things like these will
analyse if the water is suitable for algal bloom. The plankton was preserved by adding formalin in
the collected samples it was then sealed well and labeled. For the shell fish the fishermen was
tasked to remove the inside from the shell, then it was put in a sealable container and let the water
lessen for some time. After then it was put in an ice box for to be preserved. We finished early in
our task for the day so we asked our teacher if we can tour the place then he was kind to let us do
some sightseeing around the place. We then go back for dinner.
Date: June 21, 2018
We wake up early today and we then we clean up everything and arrange out tools that we
used during the sampling process, we eat breakfast and bought ice for the shellfish sample for it to
be preserved, we then head back to Iloilo, we traveled to estancia then back to Iloilo.
Date: June 22, 2018
Rest day, we were allowed to rest for a day since the trip was very exhausting.
Date: June 25, 2018
Today we were tasked to read the manuals about LC-MSMS because we will be helping
for the method verification of their analysis using the quetchers method. We learned the basic parts
of the LC MS triple quadrupole. A triple quadrupole mass spectrometer (TQMS), is a tandem mass
spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass-
resolving) radio frequency (RF)–only quadrupole between them to act as a cell for collision-
induced dissociation. This configuration is often abbreviated QqQ, here Q1q2Q3. In the manual
we learned how does the machine operates and the software use during its operation. I also scanned
through the analysis done using LS MS. Triple quadrupole analyzers are often used when higher
sensitivity and specificity is required. They may also be used to generate additional fragmentation
data from ions of interest. The instruments are rugged, offer 0.1 m/z resolution, exhibit linear
response, and resulting MS/MS spectra can be helpful in structural identification. We are also
given the tasked to search about different related literatures regarding the quetchers method for us
to have an overview how it is done. After lunch we were tasked to filter acetonitrile for the final
washing of the bottles to be used in the LC MS, the bottles are then thoroughly washed then dried
in the oven to let it dry. We then observe the analysis using the shellfish, the samples are first
grinder to be homogenized then HCl was dropped into it, the pH was then checked to have a range
of 3-4 when pH is lower in the expected range add NaOH when pH is higher than the expected
range add NaOH, after that it was heated and the stirred after heating it was then cooled pH was
check to the same range and was transfer in a test tube to be centrifuge after that the separated
layers are then used to and dropped in a testing kit to see if its positive or negative, the results are
based on standard reference, it is same in the pregnancy test but is opposite, if it shows one line it
is positive and If it shows to lines it is negative. We are also tasked to prepare a 95% ethanol for
the sampling and farm evaluation in negros. We are then invited to attend the talk about method
uncertainty. Measurement uncertainty is a property of measurement result, not of the method, i.e.
of the testing/measurement process. Assuming that the testing/measurement process is the chief
source of uncertainty, it is possible to estimate measurement uncertainty on the grounds of its
performance characteristics. In this talk it was discussed the ISO recommendation to evaluate
method uncertainties, its concepts and to analyze it and how to account for the uncertainties when
conductinhg and interpreting results of the analysis. Sir Robert the head of the lab asked us random
questions and thankfully I answered mine correctly. After the talk was done we went home.
Date: June 26, 2018
Today we helped in labelling the newly arrived reagents, consisting of name, date
purchased, control number, expiration date, remarks and etc. this is to keep track of the reagents
that will be used in the future experiment. Sir did also asked us for our assignment and different
update. QuEChERS is a solid phase extraction method for detection of pesticide residues in food.
The name is a portmanteau word formed from "quick, easy, cheap, effective, rugged, and safe".
We then help and observed in the analysis of plankton present in the samples we collected in isla
gigantes. Plankton are the diverse collection of organisms that live in large bodies of water and are
unable to swim against a current. They provide a crucial source of food to many large aquatic
organisms, such as fish and whales. To identify the dominant species of plankton present in the
sample we used a compound light microscope, the term light refers to the method by
which lighttransmits the image to your eye. Modern compound light microscopes, under optimal
conditions, can magnify an object from 1000X to 2000X (times) the specimens’ original diameter.
The term red tide is also sometimes used to describe harmful algal blooms. This type of bloom
is caused by another species of dinoflagellate known as Alexandrium fundyense. Using the
microscope we try to examine if there are species of planktons that causes redtide in the samples.
Then a small amount of sample is dropped in a glass slide for examination. Dominants species of
planktons present in the samples are then listed. Since the results in the shellfish analysis is positive
theoretically there is a plankton that causes redtide present in the water. The analysis and
examination took us a long time because it must be examined keenly and carefully because
planktons are like transparent species that is hard to find. The results are not yet finished and its
almost 5 pm so we just clean the lab and will continue the analysis the day after.