WEN JIE U1520743G
Table of Contents. 1
Acknowledgement Page. 3
Assigned Tasks / Tests
1. General Routines. 5
2. Legionella. 5
3. Total Plate Count (TPC). 8
4. Total Coliform, Fecal Coliform and Escherichia Coli
(E. Coli). 9
5. Fecal Enterococcus. 12
6. Biochemical Oxygen Demand (BOD). 12
Annex A.. 18
The intern is assigned to the
microbiological laboratory in Admaterials
Technologies Pte Ltd. This report describes the main assigned tasks for the
intern in detail, where he aided the microbiological analyst in conducting microbiological
testing for environmental samples. The tests included quantitative and
qualitative analysis of samples for specific strains of bacteria such as
Legionella, Coliforms and Escherichia Coli (E. Coli). Another main test
includes the determination of the amount of Biochemical Oxygen Demand (BOD) consumed
by the microorganisms found in the sample.
I would like to express my heartfelt
appreciation to all those who have provided assistance to me during the 20-week
journey in Admaterials.
Special thanks are given to Admaterials for providing me with an opportunity to intern for 20 weeks at the company.
This has given me a valuable experience where I can be better prepared for any
future challenges faced in the industry.
Additionally, I would like to give a
special gratitude to Miss Chin Poh Yi for all the guidance and knowledge that
she has imparted throughout the journey.
The intern is assigned to the
microbiological lab in the Environmental (ENV) department. In the lab, the
intern was mainly involved in the microbiological testing of various water
samples. The method used for the testing of the bacteria species depended on
the requests of the customers. Additionally, the intern supported the lab in
the overall lab housekeeping, and the preparation of media and reagent used.
In this report, there will be a few
parts to each test that is conducted in the lab, namely, a brief introduction
to the bacteria tested and the reason why tests are conducted for the species.
Following that, the method number will be specified and the Standard Operating
Procedures (SOPs) will be briefly described. Any significant results,
confirmation testing, additional follow-ups
or quality assurance checks will be elaborated. The report is presented as such
to demonstrate the intern’s understanding of “What”, “Why” and “How” is he
conducting the tests on the samples for.
Additionally, this lab uses the Standard
Methods for the Examination of Water and Wastewater book that is published by the American Public Health Association
(APHA). The SOPs that is used in this lab is mainly devised from the APHA
methods. A British Standard (BS) method and an Australian/ New Zealand (AS/ NZ)
method is additionally used for Legionella testing. Thus, in-text citations
shall not be used for the APHA methods, when mentioned in the report. However,
it will be reflected in the References
& Appendices proper.
Tasks / Tests Conducted
During the first two weeks, the
intern was mainly involved in reading the SOPs, and the general routines that
the intern oversaw during the attachment. General routines included lab
housekeeping, media preparation and reagents storage. After learning the general
routines, the intern was involved in the testing procedures and also learnt
about the importance of proper paperwork
done for the result tabulation.
Legionella causes atypical pneumonia,
which is easily transmittable through water sources. The legionellae bacteria
can be easily found in freshwater environments globally, easily spreading and
infection a large number of people. The severity of pneumonia caused by Legionella can range from mild symptoms such as
coughing or fever to life-threatening effects or even fatality4.
There are also no vaccines for legionella. Therefore, water testing for
legionella is required and enforced by the organisations, such as Public
Utilities Board (PUB) and National Environment Agency (NEA), that overlooks
water safety and quality.
In the lab, the commonly requested
method for Legionella testing is the BS ISO
11731 method (BS 6068-4.12:1998), which is further broken down into two parts. The
lab is certified for other testing methods like the APHA method and the AS/ NZ method
(AS/ NZS 3896:2008).
ISO Part One
For BS ISO part one, sample
concentration is carried out based on the source of the water sample. If there
is an expected low legionella count in the water source, for example, potable
water, concentration is required. Instead, the concentration step is not
required for sources such as wastewater or surface water. Concentration can be
done using filtration or centrifugation. For the samples usually received by
the lab, a concentration factor of 10 is usually conducted using the filtration
method before testing proceeds. The sample is filtered, collecting the bacteria
on the membrane. The membrane is then resuspended in Page’s Saline to form the
sample concentrate. The concentration factor depends on the amount of sample
being filtered and how much Page’s Saline is used for resuspension.
Non-legionellae bacteria interfere
with the growth of legionellae bacteria when cultivated on culture media. Thus,
pre-treatment procedures such as acid or heat treatment are applied to kill off
these non-legionellae bacteria. For the acid treatment, the sample is exposed
to an acid buffer (pH 2.2) for 5 minutes. For the heat treatment, the sample is
exposed to heat via incubation in a 50oC water bath for 30 minutes. Following
the treatments, the treated sample will be inoculated onto the culture media
using the spread plate method. Thus, the final units for the results will then
be in cfu/ mL, following the procedure
where a concentration factor of 10 is used. The units “cfu” stands for colony forming units, where small isolated clusters
of bacterial growth are found on the culture plates and can be visibly counted.
After plating the samples onto the selective culture media, it is then
incubated for up to 10 days at 36oC.
ISO Part Two
For the BS ISO part two, it is used
to test for large volumes of sample. In this method, up to 1 L of the sample can be filtered and the final units for
the results are in cfu/ # mL, where # is the amount of sample
being filtered. The sample is only subjected to acid treatment, as opposed to
part one. Following the acid treatment, the membrane filter is then placed on
top of the culture media for incubation up to 10 days at 36oC.
Confirmation tests are conducted to ensure that the colonies found
on the culture media after incubation is
legionellae bacteria. This is done by using 2 types of Buffered Charcoal Yeast
Extract (BCYE) agar, one containing growth supplement with L-cysteine (BCYE+),
and one containing growth supplement without L-cysteine (BCYE-). The L-cysteine
is crucial for the growth of legionellae bacteria, without it, legionellae
bacteria are unable to grow. Therefore,
positive results are obtained when there is growth on BCYE+ and there is no
growth on BCYE-, confirming that the suspected colony is Legionella.
2.3. Confirmation Testing Procedures
Upon customers’ requests, further
tests such as latex bead agglutination, indirect immunofluorescent antibody
assay, and enzyme-linked immunosorbent assay, can be done to identify the
serogroup of the legionellae-like species. In the lab, the latex bead agglutination
kit is used to identify if the particular colony belongs to serogroup 1 or 2 –
14 of Legionella pneumophila, or if they are of other legionellae species. In the
latex kit, there are three key reagents that test
for the classification of Legionella
species. To test for Legionella pneumophila serogroup 1, a test reagent that contains blue latex particles
sensitised with specific rabbit antibody is used as it is selectively reactive
only to the serogroup 1 antigen. For serogroup 2 – 14, the test reagent uses a
different rabbit antibody that is reactive only to serogroup 2 – 14 antigens.
For other Legionella species, the test
reagent uses another different rabbit antibody that is reactive to other Legionella species. The confirmed legionella
colony is first suspended in a suspension buffer
before the three test reagents are separately added to the legionella
suspension buffer. A positive result occurs when the blue test reagent turns colourless,
with the appearance of blue beads in the solution. A negative result is
observed when the solution remains blue5.
Government health agencies focus on Legionella pneumophila
serogroup 1 as it is the most virulent strain amongst other Legionella species. Majority of the infections caused
by Legionella species is a result of Legionella pneumophila
serogroup 1. For the rest of the Legionella
species, they are considered non-pathogenic until symptoms of disease surface6.
Therefore, identification of Legionella
colonies is an important procedure.
Total Plate Count (TPC)
In this method, the total plate count
is done to estimate the number of heterotrophic bacteria in the water body. Heterotrophs are a group of
microorganisms found in all sources of water. Examples of heterotrophs include
bacteria, moulds and yeast. They use organic carbon sources to grow.
There are limitations to the TPC
method, making this method not reliable in determining whether the water is
safe or has potential adverse human health effects. The results only show the amount of culturable organisms present in
the water as not all bacteria or disease-causing organisms known as pathogens
are able to grow on the culture media used in this method. Additionally, there
are no indications as to whether the grown colonies belong to harmful organisms7.
However, people still request for this test to assess if the water sample
contains high culturable organisms count, and will usually pair up with an
additional test for coliform or E. Coli.
According to APHA 9215 method, TPC
can be done using the pour plate, spread plate or the filtration method. The
filtration method will be further discussed in Point 4, where the main testing
method used is filtration. In this lab, filtration method is seldom used for
The pour plate method uses molten
agar kept at 45oC, or it will solidify prematurely. A small amount of sample, up to 1 mL, is
pipetted onto an empty petri dish first, before pouring the molten agar into
the dish. Once the molten agar is poured into the dish, swirling of petri dish
is done to ensure uniformity of the mixture. As the molten agar has to be kept
at a temperature, higher than the temperature of the water sample, the bacteria
found in the sample may be exposed to heat shock, even if it was for a short
period of time. Heat shock can cause inaccurate results. Additionally, the
bacterial growth is usually found inside the agar itself since they were mixed
in molten agar.
The spread plate method uses
pre-plated solidified media. Instead, a small amount of sample is pipetted onto
the media, then spread evenly on the surface till it dries, meaning that the
sample has been fully absorbed by the media. This method will not cause any
possible heat shock to the bacteria and the colonies will form on the surface
of the agar instead. This makes it easy to transfer the colony from the agar to
another culture media using a culture loop. This is done as required if any
further steps are required. Once the sample has been inoculated onto the
culture media, it is incubated based on the media used, water source and the
required specifications. Usually, the lab receives inoculated potable water
sample where it is incubated at 35oC for 2 days. There are no
confirmation tests as this method tests for generic heterotrophic bacteria
Total Coliform, Fecal Coliform and Escherichia
Coli (E. Coli)
Coliform bacteria are commonly tested
as a measure of the quality of water. An increase in the levels of coliform
usually indicates that there is a
possible failure in water treatment or possibly damaged pipelines resulting in
possible contaminations from unwanted sources. Tests for coliform bacteria is
usually requested as coliform bacteria can be used as a pre-indicator of the amount of pathogens present in the water
sample. Some harmful effects of these
pathogens include diarrhoea and vomiting.
Coliform bacteria are defined as
rod-shaped, gram-negative, and non-spore forming bacteria that can ferment
lactose when incubated at 35oC to produce acid or gas7.
The faecal coliform is a subset of total
coliform, and E. Coli is a subset of faecal
coliform. Fecal coliform is usually found in faecal
matter. The test methods used in this lab for the Coliform groups are the same,
which is either the IDEXX method and the membrane filtration method via APHA
4.1. IDEXX Method
The IDEXX method gives a quantified bacterial count of 100 mL samples
using the IDEXX reagent, known as Colilert. This mixture of reagent and sample
is then poured into a tray before it is
sealed and incubated for 1 day at 35oC. After incubation, the number
of yellow wells is counted and compared against the Most Probable Number (MPN)
table provided together with the reagent and trays. It is then placed under
Ultra Violet (UV) light to check for fluorescing.
The number of yellow wells reflects the amount of total coliform, and the
number of wells that fluoresce reflects the amount of E. Coli found in the
sample. For the same sample, it is possible to have different MPN counts for
total coliform, and for E. Coli. This method is simple to execute, and results
can be obtained quickly, however it is expensive, and the obtained results are
only an approximation. Additionally, there
are no confirmation tests for the IDEXX method.
4.2. Filtration Method
In the filtration method, a large
volume of sample (100 mL) is filtered and the filter membrane is placed onto
the agar surface while ensuring that
there are no air bubbles trapped in between. Air bubbles prevent surface contact between the media and
the filter membrane, inhibiting colony growth at the affected area as there is
no contact with the nutrients on the culture media. The agar is then incubated.
Different agar bases are used for different coliform groups. The Endo agar is
used for total coliform testing, the Membrane Fecal Coliform (m-FC) agar is
used for faecal coliform testing, and the
Dual-chromogen agar (mColiBlue24) is used for total coliform and E. Coli
testing. The red colonies on the mColiBlue24 agar are counted as total coliform, and E. Coli colonies are
differentiated by blue or purple colonies. This is due to a reaction caused by a
enzyme on 5-bromo-4-chloro-3-indolyl- ?-D-glucuronide (BCIG), thus producing the
blue or purple colonies. For total coliform and E. Coli, they are incubated at
35oC for 1 day, and for the fecal
coliform, it is incubated at 44.5oC for 1 day.
4.3. Confirmation Testing Procedures
There are confirmation tests for the membrane
filtration method. For coliform testing, suspected colony growth on the agar is
transferred to Lauryl Tryptose Broth (LTB) and Brilliant Green Bile Broth
(BGBB) with a Durham Tube inside. After incubation at 35oC for 2
days, gas present in the Durham Tube represents a positive result, which
indicates that total coliform is confirmed to be present in the sample. A
second confirmation test is to inoculate the suspected colony growth into (EC)
broth and incubate at 44.5oC for 2 days to check for the presence of
thermotolerant coliform. Gas present in the Durham Tube indicates a positive
The detection of faecal enterococcus in water is an important
indicator to determine faecal pollution. It
causes urinary tract, wound, and soft tissue infections. It is highly
infectious as the mode of transmission can be through water, food, or even
through human-to-human interactions8. Additionally, it is able to
survive in harsh conditions for up to 4 months.
The testing for faecal enterococcus is similar to the membrane
filtration steps for total coliform. According to APHA 9230 method, the agar
used is Membrane Enterococcus Agar (m-EA). It is incubated at 35oC
for 2 days. Any enterococci colonies that surface
on the agar is pink or red in colour.
5.1. Confirmation Testing Procedures
There are multiple confirmation tests
required for faecal enterococcus. The
suspected colonies will be inoculated in other different media cultures for
confirmation. In summary,the growth of catalase-negative, gram-positive cocci on Bile Esculin Agar (BEA)
at 35oC for 2 days, Brain Heart Infusion (BHI) broth with 6.5% NaCl
at 35oC for 2 days, and BHI broth at 45oC for 2 days or
BHI agar at 10oC for 2 days is all required in order to confirm that
the colony belongs to the Enterococcus genus.
Biochemical Oxygen Demand (BOD)
This is an empirical test where the
relative oxygen requirements of the water sample are determined. Usually, this
is done for wastewater, effluents, and polluted waters. The theory behind this
test determines the amount of dissolved
oxygen (DO) consumed by aerobic biological organisms found in the water sample
over a period of 5 days. Consumed DO can be a result of biochemical degradation
of organic material, oxidation of inorganic materials such as sulphides and
ferrous iron, or oxidation of reduced nitrogen. However, an inhibitor is
usually used to prevent oxidation of reduced nitrogen as it is categorized
under nitrogenous demand instead. In general, the higher the consumed DO, the
higher the amount of microbes in the
water sample, this in turns indicates that the water is increasingly polluted9.
In this lab, the APHA 5150 B method
is used for BOD testing. For this test, the commercially
prepared nutrient concentrate is used to provide nutrients and seed to
provide microorganisms for the sample. A calibration curve is set up for each
batch of BOD sample to ensure the accuracy of the obtained results. To obtain
the curve, the setup of 4 bottles containing 15 mL, 20 mL, 25 mL, and 30 mL of
seed suspension is added to nutrient water and then incubated. The curve
obtained will give an insight as to how much DO consumed
is attributed to the amount of seed added to the sample.
The general SOP is as follows. Firstly, the nutrient is added
to water, which is then agitated vigorously via shaking for 30 minutes. The
purpose of agitation is to aerate the water with dissolved oxygen. The seed is
then added to 500 mL of the nutrient water, stirred for 30 minutes using a
magnetic bar stirrer. The dilution factors for the sample that is to be
performed is determined based on the Chemical Oxygen Demand (COD) results
obtained from the chemical lab, referring to Annex A. 2 mL of seeded nutrient
water is added to every bottle containing sample volume. The bottles are sealed
using parafilm before it is placed in the
incubator for 5 days at 20oC. The DO of the samples is measured before and after incubation.
For assurance of the accuracy of the
obtained results, three quality controls are in place. Firstly, the DO of the blank
nutrient water is measured and then incubated. There is a requirement for the
DO of the blank nutrient water to drop by less than 0.2 mg/ L to ensure that
the quality of water used was acceptable, validating the results. Secondly, there
is a minimum residual DO and DO depletion requirement stated by the method. For
the results to be valid, the minimum residual DO to be ? 1.0 mg/ L and the minimum DO depletion to be ? 2.0 mg/ L for the results obtained for the DO of the bottles
with sample water. This validates the results as there is sufficient residual
DO, ensuring that the DO uptake by the sample was not restricted by
insufficient DO in the water and that
there is a meaningful measure of oxygen consumed. Thirdly, one bottle
containing 2 mL of seed, 3 mL of GGA and nutrient water for the remainder is
incubated as well. The final DO is measure and the BOD is calculated. The BOD
value should be 198 ± 30.5 mg/ L.
This checks for the effectiveness of the seed to ensure the accuracy of the
obtained results. Consistent high BOD values for this check can indicate the
following: too much seed suspension used, contaminated dilution water, or
nitrification. Consistent low BOD values for this check can indicate the
following: poor seed quality, too little seed suspension, or the presence of
toxic materials in the water. Checking on the water source and seed source is
required to correct this issue.
The quality controls for BOD is
extremely crucial in ensuring the validity of the obtained results. It is easy
for the BOD results to be invalidated due to any of the stated reasons above,
thus the emphasis on the quality control.
During the internship attachment, the
intern has learnt more about how a commercial lab works. Through the
attachment, the intern has gained more knowledge in the significance of water
testing and the possible implications if water quality is not ensured.
Additionally, the intern learnt to perform various sample analysis methods for
water testing. The intern has demonstrated understanding as to why certain
procedures are performed and has executed
these procedures during the tests for the specified bacteria. Last but not
least, the intern has gained some valuable industrial job experience, which can
better prepare him for the challenges ahead.
1 BS EN ISO 11731:2017, Water quality.
Detection and enumeration of Legionella. Britain: International Organization
for Standard; 2017
2 AS/ NZS 3896:2017, Waters –
Examination for Legionella spp. Including Legionella pneumophila. Australia: Australia Standards; 2017
3 APHA, Standard Methods for the
Examination of Water and Wastewater, 23rd ed. Washington, DC, New York:
American Public Health Association; 2017
4 “Legionella | About the Disease
| Legionnaires | CDC”, Cdc.gov, 2017. Online. Available:
https://www.cdc.gov/legionella/about/index.html. Accessed: 21- Dec- 2017.
5 DR0800, Legionella Latex Test | Oxoid
– Product Detail”, Oxoid.com, 2017. Online. Available:
Accessed: 21- Dec- 2017.
6 Q. Zhang, H. Zhou, R. Chen, T. Qin,
H. Ren, B. Liu, X. Ding, D. Sha and W. Zhou, “Legionnaires’ Disease Caused
byLegionella pneumophilaSerogroups 5 and 10, China”, Emerging
Infectious Diseases, vol. 20, no. 7, 2014.
7 J. Bartram, J. Cotruvo, M. Exner, C.
Fricker and A. Glasmacher, Heterotrophic Plate Counts and Drinking-water
Safety – The Significance of HPCs for Water Quality and Human Health. Padstow,
Cornwall, UK: TJ International (Ltd), 2003.
8 A. Kau, S. Martin, W. Lyon, E. Hayes,
M. Caparon and S. Hultgren, “Enterococcus faecalis
Tropism for the Kidneys in the Urinary Tract of C57BL/6J Mice”, Infection
and Immunity, vol. 73, no. 4, pp. 2461-2468, 2005.
9 “The Importance of Biochemical
Oxygen Demand (BOD) in the Water Analysis Sector – May 01 2015 – Tintometer
GmbH – Environmental Science News Articles – Envirotech
Online”, Envirotech-online.com, 2015. Online. Available:
Accessed: 21- Dec- 2017.
recommended based on BOD value as estimated from 60% of COD