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ENVIRONMENTAL MICROBIOLOGYLAB MANUAL
2013
Prepared by
Prof. Dr. Celal F. Gkay
Edited by
Prof. Dr. Filiz DilekTech. Kemal Demirta
Revised by
Dr. Robert W. MurdochRes. Asst. Firdes Yenilmez
Res. Asst. M. Selcen AkRes. Asst. Nilfer lgdr
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Lab Dates
No Date Experiment Teaching Assistants1 20-21.02.2013 Introduction S. Ak, F. Yenilmez, N. lgdr2 27-28.02.2013 1st Experiment Selcen Ak3 06-07.03.2013 2nd Experiment Firdes Yenilmez
4 13-14.03.2013 3rd
Experiment Nilfer lgdr5 20-21.03.2013 4th Experiment Selcen Ak6 27-28.03.2013 5th Experiment Firdes Yenilmez7 03-04.04.2013 6th Experiment Selcen Ak8 10-11.04.2013 7th Experiment Nilfer lgdr9 17-18.04.2013 8th Experiment Firdes Yenilmez10 24-25.04.2013 9th Experiment Nilfer lgdr11 15.05.2013 Final Exam S. Ak, F. Yenilmez, N. lgdr
Lab Technician: Mehmet Hamgl
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GENERAL FORMAT OF ENVE-202 LABORATORY REPORTS
1. IntroductionThe laboratory reports should be submitted the week following the completion of the experiment. It isdesired to have the reports clear, concise and informative rather than long, complicated and full ofnonsense knowledge.
2. General FormatThe following steps for format are intended to provide students assistance in reporting their Enve-202Laboratory Experiments.
1. PURPOSE (5 pt.) State the theoretical and rational basis for performing the experiment, including the
significance of the corresponding study. Describe the specific objectives of the experiment, (If any!). It is suggested that purpose
should not exceed 2-3 sentences and students use their own words and sentences.
2. PROCEDURE (5 pt.) Briefly and precisely, describe the methods, principles and the procedures involved inthe experiment conducted. (As a reminder to yourself) Please do not give the details of the experiment in this section.
3. THEORY (10 pt.) Give the theory of the experiment conducted, excluding unnecessary details, by
referring to the related references. It is suggested that the theory should not exceedthe 2-2.5 pages. (The longer the theory the more your TAs get bored)
In text, surname of the author and year of the publication should be written preciselyfor the reference used. e.g. (Reynolds, 1982) or (Muga and Reid, 1979). If thenames of the authors are more than three, use the surname of the first one. e.g.(Rogers et. al. 1984)
4. DATA ANALYSIS & CALCULATIONS (35 pt.) First give the data obtained from the experiment. It is advised to arrange the data in
tabular and/or figure forms as much as possible. Perform necessary calculations and summarize your findings in concise tables and
graphs. Show sample calculations. Number and title the tables and figures corresponding to the experiment in order to
facilitate the identification. Pay attention to all units employed and make sure the units of all outcomes are
written. Show the result of the experiment in this part.
5. DISCUSSION & CONCLUSION (35 pt.) Discuss the data gathered and the results obtained from the experiment in details,
referring to tables and figures and etc The intent is not to lead the reader through your interpretation of what happened but
why it did and what it means. (Try to think environmental engineering point of view.) The conclusion should necessarily summarize each outcome.
6. REFERENCES (5 pt.) Reference list should be given according to author surname in alphabetical order.
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If your reference is from web, then write the full address and date of web page. Reference should be written as follows: Surname of the author, initial of First name, Year. Name of Book, Publication
Company, Publication Edition (if any), Publication Place.
e.g. Bitton, G., 1994, Wastewater Microbiology, Wiley-Liss, Inc., New York.
3. Page and Typographical Format (5 pt.)Reports should be prepared via computers.Page Setup Margins:
Top 3.0 cm Bottom 2.0 cm Left 3.0 cm Right 2.5 cm Paper size A4
Paragraph Format and Spacing:
Line Spacing: Single Font: Times New Roman, 12 Indent: No indent Alignment: Justify Spacing between two paragraph and the title: 1 line
Cover Page:
Name of the Laboratory, (center) Name of the experiment, (center) Group number and members, (center)
Date (center at the bottom of the page)
General
Use passive voices as much as possible. Write the name of the figures below the figures (center) Write the name of the tables above the tables (center) Pages should be numbered without numbering cover page (center)
ENVE 202LABORATORY REPORT
EXPERIMENT-1
Submitted by:
Group 1Fadime KARA
Okan T. KOMESLIUmay G. ZKANFirdes YENILMEZ
03.03.2004
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TABLE OF CONTENTS
I. 1st WEEK
Media PreparationViable CountStreaking Plates to Obtain a Single ColonyContamination Experiment
II. 2nd WEEK
EnrichmentTurbidityDry WeightViable Counting by Spread Plate Method
III. 3rd WEEK
Isolation of anAzotobactersp. by Enrichment
Growth CurveEffect of Osmotic Pressure on Growth
IV. 4th WEEK
Isolation ofAzotobactersp.Effect of pH of Medium on GrowthEffect of Energy Source and Buffer on GrowthQualitative Demonstration of an Enzyme ActivityEnzymatic Hydrolysis of PolysaccharidesAcid Hydrolysis of Polysaccharides
V. 5th WEEK
Effect of Disinfectant on Bacterial GrowthEffect of Antibiotics on Bacterial GrowthEffect of UV Light on Bacterial GrowthMicroscopyStructures of Eukaryotic and Prokaryotic Microorganisms
VI. 6th WEEK
Sanitary MicrobiologyIndicator OrganismsTotal Coliform Enumeration in Potable Waters by Most Probable Number (MPN) MethodFlow Chart for Total Coliform MPN Test
Enumeration of Total Coliform Organisms in Sewage or Sewage Contaminated Water Samples byMPN with Two Tubes per Dilution SeriesDetermination of the Number of Total Coliform Organisms in a Water Sample by Membrane FilterMethodSterilization of Liquid by Passing through MFDetermination and Enumeration of Fecal StreptococciNotes on Sampling and Transportation of Water Samples
VII. 7th WEEK
Confirmation of Total Coliform Test by 9 Tube MPN ProcedureVerification of MF-Total Coliform TestDetermination of Fecal Coliforms in a Sewage-Contaminated Water Sample
Enumeration of Fecal Streptococci by MPN Method
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VIII. 8th WEEK
Chlorination of SewageTotal Residual ChlorineEffectiveness of DisinfectantsPreparation ofortho-toluidine ReagentPreparing the Chlorine Calibration CurveCompleted Test for Total Coliforms
IX. 9th WEEK
Gram StainBiochemical Identification of BacteriaMethyl Red TestVoger Prosheur TestIndole TestMotility TestCitrate UtilizationMulti Test Systems
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
1ST WEEK
Students will work in groups. A briefing on each day's practical will be given at start. Every group
will be provided with the following:
EMB plate
Nutrient agar plate
Escherichia coli (E. coli) culture suspension
Test tubes each containing 9 mL sterile distilled water
Sterile pipette
Test tubes
Distilled water
Media Preparation:Nutrient broth (NB) is a liquid and Nutrient agar (NA) is a solid media.
Both are general purpose rich media that support bacterial growth. In this weeks experiment, NA
will be used for bacterial counting ofE. coli and for a contamination experiment. The preparations
of these media are written in their prospectuses. For NA preparation, the required amount of
powdered NA is poured into the appropriate amount of distilled water. The media is autoclaved
for 20 min. at 121 oC and at high pressure in order to dissolve and sterilize the reagents.. The hot
liquid media is poured into the petri dishes (not more than 20 mL) and allowed to cool andsolidify. NB is identical to NA except that agar is excluded; thus the final media is liquid rather
than solid. NB is sterilized in flasks or tubes and allowed to cool at room temperature before use.
Sometimes, molten agar is poured into the test tubes and laid on bench so as to form sloping
surfaces. These are used for preparing stock cultures and are called slants or slopes (see the
drawing).
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What is sterilization?
Sterilization is needed for excluding unwanted or the contaminant organisms. The most common
method for sterilizing culture media is by heat. The best procedure is heat under pressure. Devices
for heating under pressure is called autoclaves. Autoclaving alone is not sufficient for excluding
contaminants however. The technique used for preventing contamination during the
manipulations of the cultures and sterile culture media is called aseptic technique. See Figure I
for the aseptic transfer as an example.
Figure I : Aseptic transfer
1. Viable Count: You will be provided with a liquid overnight culture ofE. coli. In order to find
how many bacterial cells there are in one mL of this suspension you must perform Viable
Counting which can be done by eitherSpread Plate Method orPour Plate Method. Dilute this
suspension 1/10 (10-1) by transferring 1 mL of the suspension into a tube containing 9 mL sterile
water (as in Figure III). Shake this tube by flicking the base with your thumb to obtain thorough
mixing. After mixing, transfer 1 mL from the 10 -1 dilution tube (that you just prepared) to a new
tube of 9 mL sterile water to obtain a 1/100 or 10-2 dilution. Apply the same procedure three more
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times to obtain 10-5 dilution. Then transfer 0.1 or 0.2 mL from 10-3, 10-4 and 10-5 dilution tubes to
the surface of agar plate (petri dishes with approximately 20 mL nutrient agar as described in the
first part) with sterile pipette and spread over it with sterile glass spreader (L baguette)
homogenously. After proper spreading, put them in the 35 oC incubator. Count the number of
colonies produced on each plate after 24-48 h and calculate the number of cells/mL, considering
only plates containing 100-200 colonies (if more than 200 colonies are observed, then do more
dilution). Report the number of colonies you found in each plate and number of organisms you
calculate in theE. coli culture.
No. of organisms = No. of colonies x Dilution factor in 1 mL
This method is called Spread Plate Method. In the pour late method, inoculations are done
into empty petri dishes and then nutrient agar is poured over each of them by providing well-
mixing. These methods and procedure for viable count are shown in Figure II and III respectively.
Figure II : Two methods for performing a viable count (plate count)
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Figure III: Procedure for viable count using serial dilutions of the sample
2. Streaking Plates to Obtain Single Colony: Streak a nutrient agar plate provided with a loop full
ofE. coli suspension. See the demonstration for correct streaking. See also the drawing for
streaking pattern. Put streaked plate into 35 oC incubator and observe after 24-48 h. Also streak an
Eosin-methylene blue (EMB) agar plate with a loop full of wastewater. EMB-agar is a selective
medium which supports only enteric bacteria, termed "Coliforms" collectively. These enteric
bacteria inhabit the intestinal tract of man and are discharged along with feces into receiving
waters. Therefore sewage contains them in great numbers. Without eosin and methylene blue,
other bacteria would also grow on these plates. EMB media stops the growth of other microbes
and also causes coliform organisms to appear shiny and metallic,. Place inoculated plates into a 35
oC incubator and observe after 24-48 h.
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3. Contamination Experiment: Every group is provided with one NA plate. Some of the groupswill open the lid of the plates by exposing its contents to air for different time periods; 5, 10, 15
min. One other group will draw a single line with the thumb on the agar surface without tearing
the surface, and another group will put a piece of hair on the agar surface. Then put all the plates
into the 35 oC incubator for 24-48 h and observe any growth on the plates. Discuss the
contaminations by exchanging the results.
PS Write the followings on the bottom of petri dishes for all experiments;
- Group numbers,
- Experiment name or number,
- Other necessary information (depending on the experiment).
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
2ND WEEK
Work in groups. Every group is provided with the followings:
125 mL mineral salts medium + ethanol (0.1 M) in 500 mL conical flask
Soil sample (humid)
Sterile pipettes (1, 5, 10 mL)
NA plate
E. coli overnight culture
Test tubes containing 9 mL sterile water
Test tubes
0.45 filter paper which has been brought to constant weight
Fresh nutrient broth
Ready-weighed aluminium foil moulds
1. Observe all the plates from the previous week.
2. Enrichment: By changing the contents of a culture medium you can enrich and isolate those
organisms which are best suited to your culture environment. This is called enrichment
technique as you know from the class. During enrichment, the most successful organism under
these conditions outgrows all the rest and predominates. Today you will start enriching for anAzotobacter sp.and isolate in pure culture in the coming weeks. As you knowAzotobacteris an
aerobic heterotroph requiring organics for both carbon and energy. However, Azotobacterhas the
added advantage over other aerobic heterotrophic organisms; it can fix atmosphereic nitrogen.
Therefore, you do not need to include any nitrogen source into your mineral salts medium. This
will exclude most other microorganisms.
Preparation of Mineral Salts Medium: Mineral salts medium (which is already done for you)
contains the following per liter: 2 gr. K2HPO4; 0.05 gr. FeSO4 .7H2O; 0.02 gr. MgSO4.7 H2O;
0.02 gr. CaCl2. Also add 0.1 M ethanol as carbon and energy source to this medium and adjust
pH to 7.0. Inoculate this medium with about 1 g of soil provided. Cover the flasks and put them
into 30 oC, shaking incubator. (To be continued next week)
3. You are provided with an overnight culture ofE. coli. You will do:
1-Turbidity Calibration,
2-Dry Weight,
3-ViableCounting,
with this culture.
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Turbidity: As you may have noticed, un-inoculated liquid cultures are crystal-clear whereas
inoculated ones become turbid or cloudy due to the bacterial growth. There is a linear relationship
between the number of cells and optical density (O.D.). We shall verify this now. Take a 5 mL
sample ofE. coli culture and put it into a test tube and mark this tube (e.g. as 5/5). Also do a series
of 5 different dilutions ofE. coli culture (4/5, 3/5, 2/5, 1/5, 2/4) with distilled water. For example,
to dilute 3/5 you must put 2 mL distilled water and 3 mL E. coli culture and so on. Measure the
extinction of these dilutions in the spectrophotometer at 550 nm wavelength against blank.
Dry weight (by membrane filter): Take 10 mL ofE. coli suspension and filter it through a
membrane filter, which has been brought to constant weight, by vacuum. Membrane filter disks re
made of cellulose acetate and their pore size is 0.45, smaller than the size of a bacterium.
Therefore the bacteria in suspension will be retained on the surface of paper. Then, in order to
determine dry weight of cells, put your filter paper into the drying oven at 105 oC. After one hour,
cool it in the desiccator, weigh the paper, and work out dry-weight of cells in 10 mL ofE. coli
suspension. After you calculate dry-weight of cells/5 mL of suspension, calculate dry-weight of
cells in g/mL for each dilution (4/5, 3/5, 2/5, 1/5, 2/4).
Viable counting by spread-plate method: Aseptically dilute yourE. coli suspension 10-1, 10-2,
10-3, 10-4, 10-5, ............ 10-11 times with 9 mL sterile water provided in test tubes (use 1 mL sterile
pipette), as in the first week experiment. Be very careful not to contaminate anything or your
results will be wrong and you may have to do this experiments all over again next week. Take 0.1
or 0.2 mL from the last 3 dilutions (10-9, 10-10, 10-11), put them on to nutrient agar plates and
spread them homogenously with a spreader provided. Put your plates into a 37 oC incubator.
Count the colonies formed after 24-48 h and work out the number of viable cells that were in your
1 mLE. coli suspension.
4. Take a discrete colony showing diagnostic metallic shine on EMB agar and streak it on a nutrient
agar plate that you prepared in the first weeks 3rd experiment. Put the plate in a 37 oC incubator
for 24-48 h. Write a report on what you did today.
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
3RD WEEK
Work in pairs. Each student will be provided with the following:
Agar plates, each containing mineral salts medium + ethanol (1.5 % agar but
no any nitrogen source)
500 mL conical flasks containing 150 mL NB
Glucose agar containing 0.5 % NaCl
" " " 10 % NaCl
" " " 25 % NaCl
" " " 0.5 % Sucrose
" " " 15 % Sucrose
" " " 30 % Sucrose
" " " 60 % Sucrose
Fungi, yeast, andE. coli cultures
Note: Glucose agar is prepared by adding 0.5 % glucose into nutrient agar.
1. Isolation of an Azotobacter sp. by enrichment: In order to isolate Azotobactersp. which you
have enriched using mineral salts medium + ethanol in the previous week (see medium
composition in 2nd week's lab. manual), you have to transfer on a solid medium which is mineral
agar. The provided agar plates (where agar is used for solidification) contain exactly the sameingredients which were used for the enrichment of cultures in shaking flasks. Take a loop full of
medium from flasks and streak the provided two plates in the usual manner to obtain isolated
colonies. Put these in a 30 oC incubator for 1 week (to be continued next week).
2. Growth curve: You are provided with two 500 mL flasks containing 150 mL sterile fresh NB..
Inoculate both flasks withE. coli culture. Incubate one at 37 oC, without shaking, and the other
with shaking at 37 oC. Every few hours for two days, pour about 3 mL into spectrophotometer
cuvettes from these cultures and do an optical density reading with the spectrophotometer at 550
nm (for both flasks) against a blank. After two days plot your results on a graph paper; turbidity
versus time. The moment you did inoculation is the time "zero". Also plot your results on a
semi-logarithmic paper: "Turbidity" being on the logarithmic axis, "time" on the normal axis.
Calculate the time required for the culture to double itself (doubling time), that is the time taken
for optical density (O.D.) to double in the logarithmic (sloping) portion of the graph (e.g. on the
logarithmic portion, time interval between 0.5 and 1.0). Using your calibration curve that you
have obtained in the second week experiment, plot growth curve (on a normal paper or semi-log
paper) dry weight (DW) of cells versus time.
Compare the two growth curves and comment on the results. (Typical growth curve is shown in
Figure I).
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Figure I: Typical Growth Curve
3. Effect of osmotic pressure on growth: When a bacterial cell is placed in a medium, an osmotic
pressure proportional to the concentration of dissolved solvents in the medium is exerted across
the semipermeable membrane that surrounds the cell. If inside the cell is more concentrated
(hypertonic) relative to outside, water will tend to diffuse into the cell (Fig. II). If bacteria did not
have a rigid cell wall, they would burst due to the excess water entering the cell. The cell wall
prevents this to happen. If the opposite is true, that is outside more concentrated (hypertonic) than
inside the cell (hypotonic), water will flow out; bacteria have no mechanism to stop this.However, yeast and fungi, unlike many bacteria, can withstand high osmotic pressures.
This exercise is intended to show how microbial species differ in their response to osmotic
pressure (in the extreme conditions). You are provided with plates of glucose agar (0.5 % glucose
in 1 L nutrient agar) containing 0.5 %, 10 %, 25 % NaCl and four plates of glucose agar
containing 0.5 %, 15 %, 30 %, 60 % sucrose.
Label each plate appropriately and divide every plate into three equal parts with your glass-pens
as shown in Figure III. Inoculate one segment of each plate with E. coli and the other two
remaining segments with yeast and a fungal culture provided, respectively (with a small loop full
of culture). Inoculation will be carried out by streaking in a small Z pattern as shown in Figure
III. Write the names of the inoculated organisms on the proper segments of the petri dishes.
Incubate at 30 oC and observe the growth after 2 days. Discuss the results of different
concentrations of NaCl and sucrose. Report your findings.
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Figure II
Fun i Yeast
Bacteria
Figure III
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
4TH WEEK
Students will work in groups. Every group will be provided with the following:
NA Plate
Glucose-agar plate (pH 3, 5, 7, 9)
Tubes containing (10 mL);
a. 1 % tryptone + 1 % yeast extract
b. 1 % tryptone + 1 % yeast extract + 1 % glucose
c. 1 % tryptone + 1 % yeast extract + 1 % glucose + 0.5 % K2HPO4
d. 1 % tryptone + 1 % yeast extract +0.1 % glucose + 0.5 % K2HPO4
pH paper
Iodine solution (0.1 N in 3 % KI) Iodine solution (0.005N in 3%KI)
Sodium chloride (1 %) Glass rods
1 % glucose 10 % glucose
Test tubes 25 % (v/w) H2SO4
0.1 M Phosphate buffer pH 0.7 20 % (v/w) KOH
1 % NaCl
Benedict's Reagent
0.1 M Phosphate buffer including 5 % starchE. coli, Yeast, Fungus
1. Isolation ofAzotobacter sp.: Streak nutrient agar plate with a discrete colony that you will pick
from solid medium (mineral salts agar containing ethanol but no N-source) so as to purify the
culture.
2. Effect of pH of medium on growth: Most organisms have an optimum pH for growth, although
they will grow over a fairly wide range of pH. Generally microorganisms grow best at the pH of
their natural habitat. The pH effect will be shown in this experiment.
You are provided with four glucose-agar plates (see 3rd week manual for media composition) of
different pH values (pH 3, 5, 7 and 9). Draw three equal segments on each plate (as you did in the
previous experiment) and inoculate every segment with the cultures ofE. coli, Saccharomyces
cerevisiae (baker's yeast) and a fungus provided respectively. Incubate plates at 30 oC. Observe
any growth after 24-48 h and record your results in your reports.
3. Effect of energy source and buffer on growth: As microorganisms utilize their substrates, they
tend to change the pH of the surrounding medium due to the accumulation of organic acid end-
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products. Low pH also inhibits growth of bacterium. You are provided with anE. coli culture and
four tubes of liquid medium containing:
a. 1 % tryptone + 1 % yeast extract,
b. 1 % tryptone + 1 % yeast extract + 1 % glucose
c. 1 % tryptone + 1 % yeast extract + 1 % glucose + 0.5 % K2HPO4
d. 1 % tryptone + 1 % yeast extract + 0.1 % glucose + 0.5 % K2HPO4
Put a drop on the strips of pH papers in order to find the initial pH of these media. Inoculate all the
tubes withE. coli. Incubate for 2 days; observe growth in tubes with naked eye. Report the tube in
which maximum growth is observed. Check pH of media once again using pH paper and report
your results. Explain the effect of buffer by considering the difference between initial and final pH
values.
4. Qualitative demonstration of enzyme activity: The first step in the microbial breakdown of fats,
polysaccharides and proteins is hydrolysis into monomers (constituent parts). Enzymatic splitting
of such polymeric compounds does not require presence of oxygen, therefore may be undertaken
both in the presence and absence of molecular oxygen. These polymers are rather big and cannot
get inside the bacterial cell; therefore microbes produce extracellular enzymes to achieve
splitting outside of the cells.
In this practical, hydrolysis of starch (a polysaccharide) into its smaller constituents will be carried
out both enzymatically and by acid-hydrolysis. The course of reaction will be followed in two
ways. Firstly, by observing starch depletion with iodine and secondly by observing production of
reducing sugars with Benedict's reagent. Free aldehyde and ketones have a reducing property on
Benedict's Reagent whereas polysaccharides have not since polysaccharides have fewer free
aldehyde and ketone groups, principally located on the terminal ends. A fragmentation of
polysaccharide molecule produces more and more reducing ends by enzymatic or acidic
hydrolysis. These reducing ends may be detected with Benedicts Reagent.
a. Enzymatic hydrolysis of polysaccharides: Amylase, present in saliva, hydrolyses 1.4
linked D-glucose units. The hydrolysis of amylase and production of polysaccharides by somebacteria in a random manner gives maltose, a disaccharide, as the final product. Amylase attacks
starch (and also to some other polysaccharides) as a substrate. Hydrolysis of starch to maltose
proceeds via various dextrins. Starch and higher dextrins give a blue colour with iodine, the
intermediate dextrins give a reddish-brown colour, while the lower dextrins and maltose give no
reaction with iodine. The action of the amylase can be followed by observing the time taken to
reach the achromic point. The time at which the reaction mixture no longer gives color with
iodine solution is called Achromic Point.
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Collect a sample of saliva and dilute it to 1/20 with distilled water (different dilutions may also be
done). Divide the diluted sample into 2 parts. Boil the first one for 2 min and incubate the other
one at 37 oC. Meanwhile prepare 3 tubes as shown in table below. Add the required amount of
boiled and incubated saliva samples immediately into these tubes. While doing that, one student
from each group should prepare 3 series drops of dilute iodine solution on the tile. At time zero
and at intervals of 1 min., take 1 drop of reaction mixture from all tubes (Test, Control 1 and
Control 2) and mix with a drop of iodine on the tile. Glass rods used must be thoroughly cleaned
between each test. The time taken when reaction mixture no longer gives a colour with the iodine
is the achromic point. Continue dropping the reaction mixture on to the iodine solution until the
achromic point is reached. If the achromic point is not reached in 40 min. then "zero" activity is
observed. If this point is reached in less than 4 min dilute your saliva sample.
Report your findings .......... min taken for 1 mL of saliva to hydrolyse per gram of starch to
completion and ...... g starch hydrolysed x mL-1 saliva x min-1. This is how activity of an enzymeis reported.
Test
(1 mL saliva)
Control 1
(1 mL boiled saliva)
Control 2
(No saliva)
0.5 % starch 5 mL 5 mL 5 mL
0.1 M phosphate buffer
pH 6.7
2 mL 2 mL 3 mL
1 % NaCl 1 mL 1 mL 1 mL
The achromic point can also be determined by Benedicts Reagent if required. For this detection,
take 5 drops from each 3 tubes (test, control 1, control 2) at the beginning and at the end of iodine
test and put them into 3 tubes each containing 2 mL Benedicts reagent. Compare the results of
Benedicts Reagent tests which are carried out at the beginning and at the end of the iodine test.
You will see the difference.
Preparation of Benedicts Reagent: Dissolve 172 gr. sodium citrate and 100 gr. Na2CO3 in
about 800 mL of warm water. Filter through a filter paper into a 100 mL measuring cylinder and
make up to 850 mL with distilled water. Meanwhile dissolve 17.3 gr. of CuSO4 in about 100 mL
of water and make up to 150 mL Pour the first solution into a 2 lt. beaker and slowly add the
CuSO4 solution with stirring.
Copper salts in alkaline medium will form Cu(OH)2. In the presence of reducing substances,
cupric ions (+2) will be reduced to cuprous (+1) oxide and precipitate.
2 Cu(OH)2 Cu2O + H2O + O-2
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If organics with reducing groups and hydroxyl groups are present, cuprous ions (+1) will form
stable soluble complexes with rust-brown colors. There is also a quantitative Benedicts Reagent
with which one can determine the concentration of reducing sugars colorimetrically with
reference to a calibration curve.
b. Acid hydrolysis of polysaccharides: Starch may be hydrolysed chemically just as in the
enzymatic process. However, due to the absence of suitable enzymatic catalysts, the reaction only
proceeds in the presence of strong acids and by the aid of net head (energy) input.
In order to see this, you may take 5 mL of starch solution and add 1 mL 25% sulphuric acid, boil
for 15 min. Test with Benedicts Reagent and Iodine before and after boiling.
CAUTION: Neutralize test solution with 20% KOH, before testing with Benedicts Reagent.
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
5TH WEEK
Work in groups. Every group will be provided with the following:
NA Plates
NA slant
Paper discs
Concentrated culture ofE. coli, fungus, yeast
Microscopes
Ocular micrometer
Stage micrometer
Prepared slides
Test tubes with 9 mL sterile distilled water
1. Pick a discrete colony ofAzotobacter sp. from NA-plate aseptically and streak onto two NA-
slants provided. This ends isolation of anAzotobactersp. from soil.
2. Effect of disinfectant on bacterial growth: Place 0.2 mL ofE. coli culture on NA-plates
provided and spread these on the agar surface so as to obtain a lawn (dense) culture. Use a sterile
(dipped into alcohol and flamed) spreader to do this. Put 1 mL of concentrated disinfectant(Zefirol) into a test tube containing 9 mL sterile water using a sterile pipette and mix thoroughly.
Transfer 1 mL from 1/10 dilution into another 9 mL sterile distilled water to obtain 1/100 dilution.
Carry on two more times like this to obtain 1/1000 and 1/10000 dilutions respectively. Then soak
two paper disks in each dilution tube. Transfer these disks onto the lawn (dense) cultures prepared
and mark the proper dilutions on the lid of petri dishes. Place petri dishes upside-down in the 35
oC incubator. Check growth after 24-48 h. Note the maximum dilution giving no growth under
and around disinfectant-disks.
3. Effect of antibiotics on bacterial growth: Place antibiotic-impregnated (absorbed) discs
provided on one of the inoculated plates prepared in the section 2. The particular antibiotic with
which each paper disc is impregnated with is indicated on small discs. Incubate plates at 35 oC.
Observe any growth after 24-48 h. Comment on result.
4. Effect of UV light on bacterial growth: The lethal effect of UV light on bacterial growth will be
shown by using a lawn culture ofE. coli. A lawn culture ofE. coli, prepared in the second section,
will be exposed to UV light for various periods (15, 20, 25, 30 min). Half of the lawn will be
shielded by a cardboard during irradiation. Each group will expose for different periods. After
exposure to UV, put plates in the 35 oC incubator and observe any growth after 24-48 h. Record
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your result along with other groups findings as well. Comment on the period of exposure that is
needed to kill all bacterial cells by drawing the graph of number of cells vs. time.
5. Microscope: A microscope is used to magnify small objects such as microorganisms. It is made
of a few essential parts of which all will be demonstrated on the adjacent figure. Coarse focusing
is done by the coarse focus knob. Fine focusing is done with the fine adjustment screw. A thin
specimen is placed on a glass slide (lam) and covered with a coverslip (lamel) and placed under
the objective on the stage and secured with clips. The lowest powered objective (the shortest)
ought to be chosen to start with, so as to eliminate the danger of breaking the slide and the
objective. Once the specimen is focused, high-powered objective should be selected without
altering the focal setting. A final adjustment is done with the fine adjustment screw for maximum
focus.
CAUTION: Never lower the high-power objective on to the slide with the course adjustmentscrew; if adjustment is required, do it very carefully using the fine adjustment screw turning it not
more than once downwards. Otherwise you will break the slide and possibly the objective too.
When using the low-power objective, you must also dim the sub-stage condenser by adjusting the
aperture of its iris diaphragm with the lever and vice versa for high-power objectives. The level of
sub-stage condenser should be adjusted for optimum light (especially for opacity) conditions with
the adjustment screw. For high-power objectives the sub-stage condenser should be raised (close
to the specimen).
The magnification of a microscope is the product of eye-piece magnification and objective
magnification.
Total magnification = Eye-piece magnification x Objective magnification
e.g. Total magnification = 10 x 40 = 400
The power of the objective and eye-piece is always written on them (x 10, x 3.5 etc.). Another
useful thing to know about microscope is resolution, which is the ability of an objective todistinguish two adjacent points. The better the receiving power of an objective, the smaller the
distance would be between any two points. The resolving power is a function of the wavelength
of the light used and the numerical aperture of the objective:
Resolving power = Wavelength / Numerical aperture x 2
Since the refractive index of air is less than that of glass, light rays will be refracted away from the
objective. This phenomenon may be overcome by placing a drop of immersion oil, such as cedar-
oil, which has a refractive index equal to that of glass (that is 1.00) between the specimen and the
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objective. Very-high powered objectives (95-100x) can only be operated by using immersion oil.
These objectives are called "immersion objectives" and are signified by the word "Oil" on them.
Due to their small size, bacteria can be observed with immersion objectives only (with a few
exceptions), whereas protozoa and algae may be observed with dry-objectives.
Microscope measurement: The assessment of microscopic-particle size distribution is often
required for the design of filters etc. by sanitary engineers. One of the few methods available for
the purpose is microscopic determination. This is possibly the easiest and most reliable one.
Particle size distribution assessment may be done either by direct counting or by taking a picture
of the image and counting the particles of different size groupings on the photograph. The former
is tedious and less reliable. Whichever way is chosen, the microscope ought to be calibrated with
a stage micrometer.
You will be provided with a stage micrometer and a graduated eye piece. Calibrate the piece withreference to the stage micrometer (Try to fix 2 lines in stage micrometer with 2 lines in graduated
eye piece and record how many lines in graduated eye piece correspond to how many millimetres
in micrometer). Do this for every objective and then measure a particulate specimen that will be
provided by each of four objectives (See Fig.1).
Figure I: Standardization of the ocular micrometer
6. Structures of eukaryotic and prokaryotic microorganisms: Each group will be provided with
slides of the following microscopic organisms:
a. Blue green algae: Draw a unicellular and filamentous blue - green algae.
b. Eukaryotic algae: Draw at least one of the following; green algae (filamentous or
colonial), Chrysophyte (desmid), diatom, dinoflagellate.
c. Protozoa: You will be provided with a culture of protozoa. Try to identify protozoa in it.
Draw one ciliate
d. Fungi: Examine fungal suspension provided on your slides. Draw different parts such as
hyphae, mycelium, sporangium, conidia spores (asexual spores) and sexual spores if you
can identify.
e. Yeasts: Examine and draw the cells ofSacchromyces cerevisae (baker's yeast). Notice
budding phenomenon.
f. Bacteria: You will be provided with gram stained preparates of bacteria. Examine gram
(+) and gram (-) rods, cocci.
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
6TH WEEK
Work in groups. Each group will be provided with the following material:
Single strength MacConkey broth (with Durham vials)
Double strength MacConkey broth (with Durham vials)
Sterile pipet (1, 5, 10 mL)
0.45 m filter paper
Vacuum pump
M-Endo broth
Glass spreader
Tubes with 9 mL sterile distilled water
Molten M-Enterococcus AgarTap water
Disposable plastic Petri dishes
SANITARY MICROBIOLOGY
Indicator organisms: Cholera, typhoid fever, bacillary dysentery and amoebic dysentery are
classic water-borne diseases. Cholera is absent from developed regions of the world and nearlyabsent in our country. Also typhoid fever, bacillary dysentery and amoebic dysentery have been
reduced to low levels. These diseases are transmitted by food and water. Control of these intestinal
infections became feasible when it was realized that they are transmitted by sewage-contaminated
water in late 19th century. Since then water-borne epidemics are greatly controlled by monitoring
such waters with bacteriological techniques. An apparent absence of pathogenic organisms does not
indicate that water is safe for human consumption. When pathogens are present in mixed
populations, relatively complicated procedures are required for their detection, but these procedures
are not suitable for routine use. It is fortunate that when pathogens are present they are scarce.
However, this complicates the problem of their detection. At present it would be impossible to
monitor waters for certain pathagens such as viral hepatitis (a very important pathogen), since their
cultivation outside of a human host has not been achieved yet.
The procedures adopted for testing potability rely on the detection of bacteria which are native to
the intestines of healthy humans and other warm-blooded animals. They indicate the presence of
feces and are called the indicator bacteria. When fecal matter is present, so may be intestinal
pathogens, and thus the water is suspect. According to US EPA (Environmental Protection
Agency) and TSE (Trk Standartlar Enstits), water containing 1 coliform organism or less per
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100 mL is considered to be safe bacteriologically. These indicator organisms also serve an
important design and operation parameter for waste treatment and water supply plants.
An ideal organism to indicate fecal pollution would always be present in feces in large numbers.
They should be absent in unpolluted environments, should persist in the environment somewhat
longer than pathogens, and should be detected easily among other microorganisms. No ideal
indicator is known, but two bacterial groups, coliforms and fecal streptococci, satisfy the
requirements to a high degree.
1. Total coliform enumeration in potable waters by Most-Probable-Number (MPN) method
(with 3 tubes method): The coliform or total coliform group includes all the aerobic and
facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose
in 24-48 h at 35 oC 0.5. The definition includes the genera; Escherichia, Citrobacter,
Enterobacter (Aerobacter) andKlebsiella. Of this group, only Escherichia is a true indicator offecal contamination, implying possible presence of pathogens. The rest of the genera, though
giving similar reactions asEscherichia in MPN test procedure (interfering with MPN results), are
not indicative of fecal contamination as they do not normally inhabit intestinal track of man and
animals. A test capable of distinguishing fecal coliforms (Escherichia) from non-fecal coliforms
does has been devised. However, standard procedure to assess bacteriological quality of potable
waters is to count total coliform only. Fecal-specific coliform counts are usually made only in
doubtful conditions or for special studies.
This week, you will start with the following test; inoculate three 10 mL, single strength, Lauryl
Tryptose (LT) broth or MacConkey broth tubes, whichever is available, with 1 mL of water
sample provided and another three 10 mL single strength LT broths (or equivalent) with 0.1 mL
of water sample. Lastly, inoculate 3 double strength, 10 mL LT broths (or equivalent) with 10 mL
samples. Place all tubes into the 35 oC incubator. Look for gas bubble formation and color change
after 48 h. Score those tubes as;
1. Both acid production and gas bubble formation => "Positive"
2. Acid production but no gas formation => Negative3. No acid production but gas formation => Suspicious => Wait for another 24
hr. and if the same conditions are observed => Negative
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Example:
10 10 10 1 1 1 0.1 0.1 0.1
+ + + + + + - --
3 2 1
If you look3 2 1 from the table, you will read;
MPN / 100 mL => 150
Lower Limit => 30
Upper Limit => 440
The upper and lower limit is given with 95% confidence limit.
Flow chart for the total coliform MPN test: As a standard procedure, only presumptive andconfirmed tests are undertaken on routine basis, and completed test is confined more or less todoubtful or special cases (see the flowchart).
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FOR COLIFORMS
Sample
Lauryl Tryptose Broth (or MacConkey or Lactose Broth)35 0C, 24 h
Gas (+) Gas (-)
PRESUMPTIVE Reincubate 24 hTEST
Gas (+) Gas (-)Negative Test
Brilliant Green (BGB) or Lactose Bile Broth35 0C, 24 h
Gas (+) Gas (-)
Reincubate 24 hCONFIRMEDTEST
Gas (+) Gas (-)Negative Test
Eosin Methylene Blue or Endo Agar24 h, 35 0C
(If typical colonies exist)COMPLETEDTEST
Nutrient Agar Slant Lauryl Tryptose Broth24 h, 35 0C 24-48 h, 35 0C
Gram (+) Gram (-) Gas (+) Gas (-)Spore formers Nonspore formers COLIFORM NEGATIVENEGATIVE COLIFORM PRESENT
PRESENT
The above multi tube method is especially suitable for potable waters as number of organisms that
are likely to be found is such media will be low. For example, if you examine MPN score-charts,
3x3 tube combination will score in the order of 103 organisms/100 mL up most. There are also
other combinations available but none has a maximum as high as 3x3 combination. However, in
sewage analysis, the order of magnitude of organisms is perhaps around 10 8 - 1014 coliforms/100
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mL In order to count that many organisms, sewage samples should be diluted a sufficient number
of times so that they will fall within the multi-tube range, that is, below 1100. For every dilution, a
set of multi-tubes must be inoculated, resulting in enormous number of tubes at the end. For
example, for counting organisms in the range of 108, at least 8 set of multi tubes must be prepared;
if it does not fall within the range, more trials must be made. Considering that 9 tubes used for
each dilution, total of 9 x 8 = 72 tubes will be involved in determination. MPN method with two
tubes per dilution series may be used for sewage analysis. Around 10 sets of two-tubes can be
used for counting organisms in the range of 108.
2. Enumeration of total coliform organisms in sewage or sewage contaminated water samples
by MPN with two tubes per dilution series: You are provided with a series of tubes containing
9 mL lauryl tryptose broth and inverted durham tubes. Set your tubes which contain broth in two
series. Inoculate two tubes with 1 mL of sewage sample (10 -1 dilution). Transfer from each of the
two tubes into new tubes having 9 mL medium each (10
-2
). Never use the same pipet more thanonce. After each inoculation, change your pipet. Continue like this until you reach 10 -8 dilution.
Inoculate all tubes in the 35 oC incubator for 24-48 h. Score acid and gas producing tubes as
positive. Consult MPN charts (Table 2). When using these charts, the situations that you may face
are:
Case 1: Select the highest dilution having all positives and take the next two consecutive dilutions.
Dilution : 10-3 10-4 10-5 10-6 10-8
Significant No: 2 (+, +) 2 (+, +) 1 (+,-) 0 (-,-) 0 (-,-)
Case 2: If the result of the entire test is similar to the following example, the three tubes should be taken
so as to throw the positive result in the middle dilution.
Dilution : 10-3 10-4 10-5 10-6 10-7
Significant No: 0 1 0 0 0
Case 3: When a positive occurs in a dilution higher than the three tubes chosen according to case 1, this
positive should be included in the result of the higher dilution.
Dilution : 10-3 10-4 10-5 10-6 10-7
Result out of : 2 2 1 1 1
2 tubes (1+1)
Significant No: 2 2 1 2 0
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When the proper significant numbers have been selected, using the distributed table, determine the MPN of
organisms present in 1 mL of original sample. In the significant number column, find the set of 3 numbers
which corresponds to the results, as shown in cases 1-3 and note the "probable number" of organisms in the
opposite right hand column. Multiply this MPN value by the dilution of the first of the three positive tubes
selected. The calculation of MPN of organisms per 1 mL follows the below examples for cases 1, 2, and 3.
Example:
Least
MPN/mL Dilution 95 % Confidence MPN/mL
Case 1 6.0 10-4 9 100 - 400 000 60.000
Case 2 0.5 10-3 76 - 3 300 5.00
Case 3 20.0 10-4 30 000 - 1 300 000 200 000
This experiment will not continue next week since the confirmation stage is the same as forexperiment 1. From all positive tubes confirmatory media, brilliant green bile broth or equivalent
is inoculated and if gas production and acid production still persists experiment confirmed. Report
your results and MPN of organisms/mL in sample. Give also confidence limits.
3. Determination of the number of total coliform organisms in a water sample by membrane
filter method: This method, through very costly, is very much straight forward, quick and
versatile. Membrane filter discs you are provided which are made of cellulose acetate and their
pore size is 0.45 m 0.05 m, smaller than the size of any known bacteria. When liquid samples
are filtered with these, bacteria are retained on the surface (viruses however are not retained).
When the filter placed on a pad impregnated with special media, organisms grow over the surface
and form colonies. After incubation, we can count the number of colonies formed and determined
bacterial count in the sample. When applying this method to sewage samples, sample must be
diluted sufficiently so as to yield 20-80 colonies/disc. The primary disadvantage of this method is
the membrane filter discs cost.
Take a 100 mL of "potable" water sample and filter it through asterile membrane filter (MF) that
has been placed in a special pre-sterilized filter holder assembly. Place a sterile absorbent pad in apre-sterilized plastic petri dish and pour 1.8-2.0 mL M-Endo broth (or alternatively 5-6 mL
Melted M-Endo Agar without pad) onto the pad. Place the membrane filter over the nutrient pad,
close the lid of the petri dish and put it into the 35 0C incubator for 24 2 h (do not invert the
dishes). Count the resulting colonies that show a metallic sheen and report the number of total
coliform organisms/100 mL
This a single-step MF method can be used where there are not many background organisms and
where there is no toxicity problem which may suppress coliform organisms. Otherwise a two-step
MF method is used. In the first step, the filter retaining the microorganisms is placed on an
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absorbent pad saturated with lauryl tryptose broth. After incubation for 2 h at 35 oC, the filter is
transferred to an absorbent pad saturated with M-Endo broth or an equivalent and incubated for
20-22 h. at 35oC. The two-step method is especially used for chlorinated secondary or tertiary
sewage effluent and for industrial waste effluents. Statistically speaking it is claimed that the
MPN method is more reliable than the MF method, especially where there is high solids content
in the sample or toxic conditions are present (e.g. in industrial waste effluent).
Sterilization of liquid by passing through membrane filters: Compounds which are heat-labile
in aqueous solutions may be sterilized by passing them through a membrane filter. For example;
many carbohydrates (e.g. glucose, lactose, etc.) and many other organics are chemically altered
upon heating thus ought to be sterilized in this way.
4. Determination and enumeration of fecal streptococci: The term fecal streptococci will be
used to describe the streptococci which indicate the sanitary quality of water and wastewater.Fecal streptococci data verify fecal pollution and may provide additional information concerning
how recent the pollution occurred and what its probable origin might be. As mentioned in the
notes of the first experiment, in the total coliform test, non-fecal organisms (Aerobacter,
Klebsiella etc.) are also resolved along with the fecal ones. However fecal streptococci are
exclusively fecal in origin, therefore they definitively indicate fecal pollution by warm-blooded
animals (including man) and are superior to total coliform data in this respect. Moreover, their
survival in the environment is far shorter than the coliforms hence they indicate recent fecal
pollution. See the adjacent list of the organisms.
Further identification of streptococcal types in the sample by biochemical characterization gives
useful information leading to the source of fecal contamination. For example, S. bovis and S.
equinus are host-specific and are associated with the fecal excrement from a non-human source.
High numbers of these are associated with pollution from meat processing, dairy wastes and run-
off from farmlands.
Fecal streptococci
S. facealis
S. facealis
sub.sp.liquefaciens Group D (human source)
Enterococcus sub.sp.zymogenes
Group S. faecium
S. bovis
S. equinus Group Q (non-human source)
S. avium
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There are three different procedures to differentiate and enumerate fecal streptococci. These are
MPN, MF and direct pour plate methods. MF and pour plate methods are superior to MPN. Pour
plate methods are superior to MPN method where there is too much solids content in the sample.
You are provided with molten M-Enterococcus Agar. Dilute a sewage sample down to 10 -5 and
transfer 0.1 or 0.2 mL onto each of 3 agar surfaces starting from the 10 -3 dilution (10-3, 10-4, 10-5
respectively) and spread the samples over the surface of the plates (Spread plate method).
Incubate them at 35 oC for 24-48 h. Count typical colonies in plates showing 30-300 colonies and
report your results as number of organisms/100 mL. This so called presumptive test is often
used. If confirmation is necessary, then additional tests have to be carried out (see future
experiments). If sample is expected to be relatively clean, then obviously a method such-as MPN
or MF involving larger sample inoculum should be chosen (e.g. for MF, 100-200 mL sample may
be used) to increase the sensitivity.
In MF technique, sample (roughly 100 mL) is filtered and filters are placed on KF-Agar (orequivalent) just as it is done in the coliform test.
NOTES ON SAMPLING AND TRANSPORTATION OF WATER SAMPLES
Water samples are taken in pre-sterilized containers. Typically, 250 mL bottles are used for this.
The neck of the bottle and inner surface of the stoppers should not be handled at all. If flame is
available, then the mouth of the sample bottle must be flamed before unstoppering and stoppering.
If water sampling is being done from a tap then the tap must be opened and allowed to run for at
least 4-5 minutes. Before taking the sample, flame the mouth of the tap as well. After filling the
250 mL water bottle, make sure that there is an air space over the liquid. Normally a space
corresponding to 1/4 of the bottle volume is adequate. If water to be sampled is chlorinated, put
0.1 mL of 10 % sodium thiosulfate (Na2S2O3) solution per 125 mL of sample volume. This is
enough to neutralize 15 mg/l free chlorine. Add thiosulfate solution before you sterilise bottles in
an autoclave at 121 oC, 15 min.
All samples must be kept on ice or at 1-4 oC in a refrigerator and must be analysed as soon as
possible. Maximum allowable time lapse between sapling and analysis is 6 h. This period may beextended to 30 h at most in special conditions but reliability of results must be checked.
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TABLE I
Number of positive tubes out of 95 % Confidence Limit
Three 10-mLtube
Three 1-mLtube
Three 0.1-mLtube
MPN per 100 mL Lower Upper
000000000000
0000111111111
1111111222
000011112222
3333000011112
2223333000
012301230123
0123012301230
1230123012
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Table I continued
Number of positive tubes out of MPN per 100mL
95 % Confidence Limits
Three 10 mL
tubes
Three 1 mL
Tubes
Three 0.1 mL
Tubes
Lower Upper
222222222222
23333333333333333
011112222333
30000111122223333
301230123012
30123012301230123
26152027342128354229364453233964954375120160931502102902404601100
>1100
2.8
3.5
3.56.9
7.11430
153035
3671150460
44
47
120130
210230380
380440470
130024004800
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TABLE II
MPN of Organisms With Two Tube Per Dilution
Significant Number MPN (/1 mL) Confidence Limits (95 %)000001010011020100101110111120121
200201210211212220221222
0.00.50.50.90.90.61.21.32.02.03.0
2.55.06.0
13.020.025.070.0110
.076 - 3.3
.076 - 3.3.14 - 6.0.14 - 6.0.091 - 4.0.18 - 7.9.20 - 8.6.20 - 13.30 - 13.45 - 20
.38 - 16
.76 - 33
.91 - 407.0 - 863.0 - 1303.8 - 16011 - 460
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
7TH WEEK
Work in groups. Each group will be provided with the following materials:
Brilliant Green Bile Broth (BGB), single strength, with Durham vials
MacConkey broth tubes with Durham vials
Tubes of EC Medium with inverted Durham vials
Azide Dextrose Broth (Single Strength)
Azide Dextrose Broth (double broth)
Sewage Sample
Sterile pipet (10 and 1mL)
1. (Continued from last week) Confirmation of total coliform test by 9 tube MPN procedure:
In order to confirm that gas and acid production in tubes from the presumptive test is really due to
coliform organisms and nothing else, transfer a drop or a loop full of media from tubes showing
gas and acid (therefore positive) (from MacConkey broth) in the presumptive test into
corresponding BGB tubes. Incubate inoculated tubes for 24-48 h at 35 oC. Observe gas (Durham
tubes must be at least half full with gas bubble) and acid production (green colour of tubes will
turn yellowish-green) in these and score them as positive. Go through your 9 tube MPN charts
and find the corresponding combination and work out the MPN of organisms per 100 mL
Standard procedure ends here and you can report your findings in your report. The TurkishStandard method TSE 266 follows the same procedure with one exception, that is 3 tubes rather
than 9 tubes are used and consequently it is slightly less sensitive than ours and the maximum
number of organisms that can be counted is 240 rather than 1.100 as in our case.
For full description of the procedure, see 6th week's sheet.
Verification of MF-Total Coliform test: Colonies showing metallic sheen on m-Endo medium
has already verified. Test ends there. However, verification of representative numbers of colonies
may be required in evidence for gathering or for quality control procedure. You should follow the
procedure below when verification of a MF-Total coliform test is required.
Pick one typical colony showing metallic sheen and one non-typical colony without metallic
sheen (in fact for quality control 10 typical and 10 non-typical colonies are used) and inoculate
them into 2 lauryl tryptose (or MacConkey) broths respectively. Put them into the 35 oC incubator
for 24-48 h. If typical colonies do not give positive results in further experiments and/or non-
typical ones do give positive results, than your MF method is doubtful. See if your m-Endo
medium has gone useless (It may be outdated or stored wrong etc.). Inoculation of BGB broths
from those tubes showing gas and acid should be done after 1 week.
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2. Determination of fecal coliforms in a sewage-contaminated water sample: As has already
discussed, the total coliform test does not distinguish between coliform organisms of fecal origin
and non-fecal origin. Although all drinking water standards are based upon total coliform counts,
better and more accurate information regarding origin of coliforms may be obtained by the fecal
coliform test. Flow diagram of this procedure is given in Figure I.
Inoculate EC broth tubes from the sewage sample or from all the corresponding tubes showing
gas and acid production in the presumptive total coliform test (Lauryl tryptose or MacConkey
broths) of last week. Incubate inoculated tubes at 44.5 0.2 oC for 24 h (a drop or a loop full of
inoculum is sufficient). Observe gas production and score all tubes as (+) or (-). Refer to your 9
tube MPN charts to calculate MPN ofE. coli (fecal coli) organisms per 100 mL of sample.
Record your results.
The relationship of fecal coliform to fecalstreptococcus density may provide information on thepotential source of contamination. Estimated per capita contributions of indicator bacteria for
animals were used to develop FC/FS ratios. When FC and FS are examined in the same sample at
the same time, the adjacent list is used for the ratio of FC/FS. From the data, it can be reasoned
that ratios greater than 4:1 (more realistically 2:1) are indicative of pollution from human
domestic wastes (body wastes). Ratios less than 0.7 suggest that contamination is originating from
livestock and poultry wastes or from storm run-off.
ORIGIN FC / FS RATIO
Man 4.4
Duck 0.6
Sheep 0.4
Chicken 0.4
Pig 0.4
Cow 0.2
4. Enumeration of fecal streptococci by MPN method (with 3 tubes method): Enumeration offecal streptococci by the direct pour plate method using M-Enterococcus Agar does not usuallynecessitate further verification. However, for quality assurance, further verification may be
necessary by observing the catalase reaction and growth in azide dextrose broth. For MPN
procedure, inoculate 3 single strength azide dextrose broth tubes with 10 mL water inoculum, 3
single strength tubes with 1 mL and the other three with 0.1 mL water inoculum. Incubate these
for 24-48 h at 35 oC. Score positive for tubes with growth and negative for those without those
without growth. Turbidity in the tubes indicates growth. Consult the MPN chart and estimate
MPN of fecal streptococci per 100 mL of sample. Further confirmation may be done by streaking
KF, PSE or M-Enterrococcus Agar plates from each positive tube and observing typical colonies
appearing on these plates.
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FOR FECAL COLIFORMS
Sample
Lauryl Tryptose Broth or MacConkey Broth
35 oC, 24 h
Gas, acid Gas(+) (-)
PRESUMPTIVETEST Reincubate
24 h
Gas Gas(+) (-)
Negative Test
Elevated TemperatureTest. EC Medium44.5 oC, 24 h
Gas (+) Gas (-)Negative Test
CONFIRMEDTEST
Fecal ColiformsPresent
CalculateFecal Coliform
MPN
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
8TH WEEK
Work in pairs. Each pair will be provided with the following material.
OCl- stock solution
Sewage sample
Endo agar plates
Tubes containing 9 mL dist. water (sterile)
o-toluidine reagent
Test tubes
4 Beakers (250-500 mL)
I. Chlorination of sewage: This will be a review of information given to you in Environmental
Chemistry course. It is here intended to refresh your memories. Whether you apply gaseous
chlorine (Cl2) or combined chlorine (OCl-), you always end up with OCl- at above pH 4,
according to the following equilibrium:
Cl2 + H2O HOCl + H+ + 2Cl-
NaOCl + H2O Na+ + OCl- + H2O
This establishes an equilibrium with hydrogen ions as follows:
OCl- + H+ HOCl
The amount of OCl- and HOCl in the solution depends upon pH. Below pH 5, there is 100%
HOCl and above pH 10 100% OCl-. It is found that HOCl is much more powerful as a
disinfectant than any other Cl0 species. Therefore, the most effective chlorination for disinfection
is between pH 4-5.
Chlorination is applied for the purpose of disinfection. Disinfection is a process in which
pathogenic (disease producing) organisms are destroyed or otherwise inactivated. Chlorine and
several derivative compounds are by far the most widely used chemical disinfectants in the world.
Chlorine and hypochlorous acid reacts with a wide variety of substances including ammonia;
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NH3 + HOCl NH2Cl + H2O mono-chloramine
NH3 + 2HOCl NHCl2 + 2H2O di-chloramine
NH3 + 3HOCl NCl3 + 3H2O tri-chloramine
to give mono-, di-, tri-chloramines. Mono-and di-chloramines have significant disinfectant
properties therefore they are of interest. Some reducing organics (unsaturated, double covalent
bonded, reducing compounds i.e. R-SH etc.), though having no disinfectant properties, and
inorganics (i.e. Fe, Mn, NO2 etc.) react with Cl2 thus depleting available chlorine for disinfection.
The total of chlorine species listed above is termed COMBINED RESIDUAL CHLORINE.
Reaction of chlorine is given in the adjacent figure. All the applied Cl will initially combine as
discussed above (O-A), and only after A, BREAK POINT, is reached will FREE RESIDUAL
CHLORINE (Cl2, HOCl, OCI-) will predominate.
Total Residual Chlorine (Free residual + Combined residual): Ortho-toluidine is a dye
compound which is oxidized in acid solution by chlorine and chloramines and other oxidizing
compounds to produce a yellow product. Chloramines react slowly with o-toluidine (3-5 min at
20 oC) whereas free-residuals react instantly (within 5 sec). Therefore, color formation measured
after 5 min will correspond to total residual chlorine. However if you add a reducing agent such as
sodium arsenite, 5 sec after addition ofo-toluidine, this will reduce chloramines instantaneously,
stopping further reaction with o-toluidine. Color produced in this case will be principally due to
free residual chlorine.
In this experiment you will determine total residual chlorine only. Disinfection action of chlorine
(or any other disinfectant) is proportional to the concentration applied and the period of contact.The CHLORINE DEMAND of a water sample is the amount of chlorine that must be applied to
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leave desired free, combined or total residual chlorine or that amount which is necessary to kill all
the bacteria of interest (total coliforms in this case). Measurement of chlorine demand can be
readily made by treating a series of water samples in question with known but varying dosages of
Cl2 or OCl-. After the desired contact period, determination of residual chlorine or viable bacteria
in the samples will demonstrate the dosage which satisfies the the chlorine demand.
Effectiveness of Disinfectants: Factors determining effectiveness of a particular disinfectant are:
1. Type of disinfectant
2. Contact time
3. Concentration of disinfectant
4. Temperature
Effect of temperature on disinfectant effectiveness can best be expressed by the followingempirical equation:
Cn tp = K where;
C is the disinfectant concentration, tp is the contact time required for a given percentage of kill
(e.g. 99 % kill); n and K are constants. The above equation can be linearized as:
n log C + log tp = log K ............. (1)
log C = 1/n log K - 1/n log tp
Plot oflog C versus log tp will yield a straight line with a slope of-1/n.
Important: The following 1st and 2nd experiments will be conducted simultaneously. That is,
when one student is doing 1st experiment, a second student should do the 2nd experiment.
1st Experiment: You are provided with a known OCl- stock solution. The exact concentration of
chlorine in this solution is written on the bottle. From this solution, pipet enough into each of four
50 mL sewage samples in beakers to obtain 2, 4, 6, 8 mg/L chlorine concentrations in the flasks.
Do not put any chlorine solution into the fifth sample as this will be the control beaker without
any chlorine. Wait for 10 to 30 min for contact.
At the end of contact period, immediately transfer 1 mL from each beaker containing a different
amount of chlorine into 9 mL sterile distilled water supplied for you (to avoid lengthening of the
contact period you should immediately do this step. Otherwise your contact time will be altered
and this will effect calculations).
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Immediately proceed with the next step and transfer, with a pipette, 5 mL from each chlorine-
beaker and control beaker into corresponding empty test tubes.
Put 3-5 drops ofo-toluidine reagent into each test tube and mix with hand. Note that when you
add o-toluidine if a strong reddish-yellow colour is produced that means your chlorine
concentration in the tube is too high to read in the spectrophotometer and you must further dilute
your sample (chlorine - beaker) to obtain the desired o-toluidine colour which should be a shade
of yellow.
Wait for 5 min and read O.D. at 435 or 490 nm. against reagent blank. Reagent blank is the tube
corresponding to the beaker without any chlorine. Refer to the calibration curve and work out total
residual chlorine in each flask.
2nd Experiment: Transfer 1 mL from each of the beakers containing 0 (control), 2, 4, 6, 8 mg/l
chlorine prepared in the 1st experiment into test tubes containing 9 mL distilled water. Mix well.
You should further dilute these tubes according to the below table:
0 mg/l : 10-1 , 10-2 , 10-3 , 10-4 , 10-5 , 10-6 , 10-7
2 mg/l : 10-1 , 10-2 , 10-3 , 10-4 , 10-5
4 mg/l : 10-1 , 10-2 , 10-3 , 10-4
6 mg/l : 10-1 , 10-2 , 10-3
8 mg/l : 10-1 , 10-2
Take care to dilute these using sterile pipettes.
Transfer 0.1 or 0.2 mL to the center of each of petri plates containing Endo agar from the last two
dilutions of each series. Using sterile L-shaped glass baguettes spread the droplets on the plates to
obtain a homogeneous layer.
Incubate plates for 24-48 h in 35 oC incubator. After incubation, count the number of colonies
formed and calculate no. of cells in the corresponding chlorine and control beakers.
Calculations:
1. Work out percentage kill for each chlorine beaker.
% kill = 100 - (No. of viable cells remaining / No. of viable cells initially present) * 100
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For each chlorine concentration, plot percent kill versus initial chlorine concentration in the
sewage sample.
2. Using the percent kill and contact times, calculate specific kill rate (k) for different chlorine
concentrations, from:
N = N0 e -kt
Given that:
N : plate count after contact time in chlorine-beakers,
N0 : plate count for control beaker having no chlorine is N0,
t : contact time with chlorine
Calculate specific kill rates (k) for each initial chlorine concentration.
Having calculated k values, calculate contact time, tp, for 99.9% kill at each initial chlorine
concentration.
Plot log C (initial chlorine concentration) versus log tp (contact time for 99.9 % kill) and calculate
constants K and n in eq. 1.
Note that the concentration of chlorine initially present in the sample (applied dosage)corresponding to 99.9 % kill is the chlorine demand.
3. Plot total residual chlorine versus percent kill. This curve is useful for practical monitoring of
waters
Technical:
Preparation ofortho-toluidine reagent: Dissolve 1.35 g ortho-toluidine dihydrochloride in 500
mL dist. water. Add a mixture of 350 mL dist. water and 150 mL concentrated HCl with stirring.
Store in a dark bottle.
Preparing the chlorine calibration curve: Dilute the stock solution with dist. water to give 2, 4,
6, 8, 10 mg/L chlorine (if necessary). Transfer 10 mL from each of these dilutions into test tubes.
Add 3-5 droplets ofo-toluidine reagent into each tube. Mix well. Wait for 5 min. Read O.D. at
435 or 490 nm against the reagent blank. Draw the calibration curve, concentration versus O.D.
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II. Completed test for total coliforms: Spread a loop full of medium taken from a Brilliant Green
Bile broth tube showing gas and acid in the confirmed test onto an Endo agar plate (ideally the
same for each positive BGB should be done). Incubate at 35 oC for 24-48 h. Observe colonies
with metallic sheen at the end of 24-48 h.
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ENVIRONMENTAL MICROBIOLOGY PRACTICAL
9TH WEEK
Work in groups. Each group will be provided with the following materials:
Set of gram stain (dyes: safranin, lugol, crystal violet)
Motility test (10 mL in test tube)
Methyl red test (MRVP Medium) (10 mL in test tube)
Voger-Prosheur test (MRVP Medium) (10 mL in test tube)
Simmons' citrate agar slants
Tryptone broth (10 mL in test tube)
Kligler iron agar or triple sugar iron agar slants
-naphthol
40 % KOHKovacs reagent
1. Gram Stain: As discussed earlier in the class, bacteria may be classified broadly as gram-positive and gram-negative based on their gram reaction. Gram staining also constitutes the final
step (completed test) of total coliform analysis. NA slants, inoculated with typical colonies from
Endo agar, will be used for the gram staining experiment. It is essential that the culture to be used
is a growing one (has not reached the final stationary phase), otherwise false results may be
obtained.
Procedure: Place a small drop of distilled water on a clean slide. Transfer a loop full of culture
from surface of an NA slant or from the previous colony into the drop on the slide using a sterile
loop. Spread inoculum on the slide. Let it dry in air. When dried, fix material on the slide by
passing it over a Bunsen flame as if cutting the flame across. Make sure that the surface of the
smear is pointing up and away from the fire. It is customary to run two controls for quality control
for a known gram-positive organism and a gram-negative organism.
1. Flood the fixed-smear with ammonium oxalate-crystal violet stain (or crystal violet
only) for 1 min.2. Wash the slide in a gentle stream of tap water
3. Flood it with Lugol's iodine solution for 1 min.
4. Decolorize with acetone/alcohol mixture by adding it dropwise on the titled slide until
no more blue-colour leaching occurs or for 30 seconds.
5. Flood the smear with safranin counter stain for 30 seconds.
6. Wash with tap water and dry slowly
7. Observe under microscope after putting a drop of immersion oil on the slide and
immersing the immersion objective (x100) into the oil. Choose an area on the smear
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where the cells are not dense, it is often misleading to observe an aggregate of organisms
rather than individual cells to determine the gram colour.
Gram-positive organisms will retain the crystal violet stain and are blue in colour. Gram-negative
cells are decolorized firstly and then counterstained with safranin, therefore they will appear pink,
reddish. A representation of staining cells for microscopic observation is shown in Figure 1.
Figure 1: Staining cells for microscopic observation
2. Biochemical Identification of Bacteria: In order to identify and characterize an unknown
organism, a series of biochemical tests are carried out. Some of the very common tests are
discussed below. Always do each test in duplicate, one for a typical colony and one for a non-
typical colony.
a. Methyl Red Test: Inoculate two buffered glucose-broth tubes (MRVP media) with a typical and
a non-typical colony from Endo medium respectively. Incubate for 5 days at 35 oC. Add a few
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drops of methyl red indicator. A distinct red colour indicates positive test. If in doubt, repeat the
test.
b. Voger-Prosheur Test: Inoculate two MRVP medium tubes just as discussed above. Incubate at
35
o
C for 48 h. Add 0.6 mL -naphthol reagent and 0.2 mL 40% KOH solution to 1 mL of 48 hincubated old MRVP medium. Shake vigorously for 10 seconds and allow the mixture to stand 2-
4 h. A pink colour indicates a positive test result.
c. Indole Test: Inoculate two tryptone broth tubes (1% tryptone) as usual and incubate at 35 oC for
24 h. Add 0.2-0.3 mL Kovac's test reagent and shake. Allow mixture to stand for 10 min. A red
colour in the amyl alcohol layer on top indicates a positive test, whereas the original colour of the
reagent shows negative. Kovac's Reagent: Dissolve 5 gp-dimethylaminobenzaldehyde in 75 mL
amylalcohol, add 25 mL concentrated HCl.
d. Motility Test: Stab-inoculate the centre of the tube of motility test medium to at least half depth.
Motility test medium is very similar to nutrient agar but contains half as much (4 g/l) agar which
results in a much softer medium. This permits motile organisms to diffuse into the medium. Non-
motile organisms grow only along the line of inoculation. Motile organisms grow outward from
the line and spread throughout the medium producing a cloudy appearance. Examples are shown
below.
Non-motile organism Motile organism
e. Citrate Utilization: Lightly inoculate a pure culture into a tube of Simmon's citrate agar using a
needle to stab, then streak the surface of the medium. Incubate 48 h, at 35 oC. Examine tube for
growth and colour change. A distinct blue colour with growth indicates positive test. Discuss your
results by using the following table.
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Bacterium IndoleTest
MRTest
VPTest
CitrateTest
MotilityTest
E. coli + + - - +Citrobacter - + - + +
Aerobacter
Aerogenes
- - + + +
Klebsiella +,- - + + -Pseudomonas - - - + +Aeromonas + - + + +
3. Multi Test Systems (Reaction with Triple Sugar Iron Agar): You are provided with duplicate
tubes of TSI slants. Inoculate these with a typical and a non-typical colony respectively which are
taken from Endo agar. Inoculate by stabbing the buff and streaking the slant. Incubate at 35 oC for
18-24 h. Read and record reactions. Colour of slant or buff is yellow for an acid reaction
(abbreviated as A) or red for alkaline reaction (K) and no colour change for neutral reaction (N).
Bubbles in the medium indicate gas production. Abbreviate abundant gas bubbles with G and
small amount of gas bubbles with g. H2S production is evidenced by blackening of the medium.
Grades of blackening are given empirically between +1 and +4.
Typical Reactions:
COLIFORMS Slant Buff H2S
Salmonella K A, g +1 to +4S. typhi K A +1Citrobacter K A, g +1 to +3Shigella N or K A
Aerobacter A A, gE. coli A A, g
G: high amount of gas
g: small amount of gas
H2S: blackening of the medium (grades of blackening; +1 to +4)
A: acid production
K: alkaline reaction
N: no colour change
TSI Medium is only presumptive evidence hence must be supported with additional biochemical
tests.