ENVIRONMENTAL MICROBIOLOGY LAB MANUAL

2013

Prepared by Prof. Dr. Celal F. Gkay

Edited by Prof. Dr. Filiz Dilek Tech. Kemal Demirta Revised by Dr. Robert W. Murdoch Res. Asst. Firdes Yenilmez Res. Asst. M. Selcen Ak Res. Asst. Nilfer lgdr

Lab Dates No 1 2 3 4 5 6 7 8 9 10 11 Date 20-21.02.2013 27-28.02.2013 06-07.03.2013 13-14.03.2013 20-21.03.2013 27-28.03.2013 03-04.04.2013 10-11.04.2013 17-18.04.2013 24-25.04.2013 15.05.2013 Experiment Introduction 1st Experiment 2nd Experiment 3rd Experiment 4th Experiment 5th Experiment 6th Experiment 7th Experiment 8th Experiment 9th Experiment Final Exam Teaching Assistants S. Ak, F. Yenilmez, N. lgdr Selcen Ak Firdes Yenilmez Nilfer lgdr Selcen Ak Firdes Yenilmez Selcen Ak Nilfer lgdr Firdes Yenilmez Nilfer lgdr S. Ak, F. Yenilmez, N. lgdr

Lab Technician: Mehmet Hamgl

GENERAL FORMAT OF ENVE-202 LABORATORY REPORTS 1. Introduction

The laboratory reports should be submitted the week following the completion of the experiment. It is desired to have the reports clear, concise and informative rather than long, complicated and full of nonsense knowledge. 2. General Format

The following steps for format are intended to provide students assistance in reporting their Enve-202 Laboratory 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. PROCEDURE (5 pt.) Briefly and precisely, describe the methods, principles and the procedures involved in the 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 exceed the 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 precisely for the reference used. e.g. (Reynolds, 1982) or (Muga and Reid, 1979). If the names of the authors are more than three, use the surname of the first one. e.g. (Rogers et. al. 1984)

2.

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.

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
ENVE 202 LABORATORY REPORT EXPERIMENT-1

Cover 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) Name of the Laboratory, (center) Name of the experiment, (center) Group number and members, (center) Date (center at the bottom of the page)

Submitted by: Group 1 Fadime KARA Okan T. KOMESLI Umay G. ZKAN Firdes YENILMEZ 03.03.2004

TABLE OF CONTENTS I. 1st WEEK Media Preparation Viable Count Streaking Plates to Obtain a Single Colony Contamination Experiment II. 2nd WEEK Enrichment Turbidity Dry Weight Viable Counting by Spread Plate Method III. 3rd WEEK Isolation of an Azotobacter sp. by Enrichment Growth Curve Effect of Osmotic Pressure on Growth IV. 4th WEEK Isolation of Azotobacter sp. Effect of pH of Medium on Growth Effect of Energy Source and Buffer on Growth Qualitative Demonstration of an Enzyme Activity Enzymatic Hydrolysis of Polysaccharides Acid Hydrolysis of Polysaccharides V. 5th WEEK Effect of Disinfectant on Bacterial Growth Effect of Antibiotics on Bacterial Growth Effect of UV Light on Bacterial Growth Microscopy Structures of Eukaryotic and Prokaryotic Microorganisms VI. 6th WEEK Sanitary Microbiology Indicator Organisms Total Coliform Enumeration in Potable Waters by Most Probable Number (MPN) Method Flow Chart for Total Coliform MPN Test Enumeration of Total Coliform Organisms in Sewage or Sewage Contaminated Water Samples by MPN with Two Tubes per Dilution Series Determination of the Number of Total Coliform Organisms in a Water Sample by Membrane Filter Method Sterilization of Liquid by Passing through MF Determination and Enumeration of Fecal Streptococci Notes on Sampling and Transportation of Water Samples VII. 7th WEEK Confirmation of Total Coliform Test by 9 Tube MPN Procedure Verification of MF-Total Coliform Test Determination of Fecal Coliforms in a Sewage-Contaminated Water Sample Enumeration of Fecal Streptococci by MPN Method

VIII. 8th WEEK Chlorination of Sewage Total Residual Chlorine Effectiveness of Disinfectants Preparation of ortho-toluidine Reagent Preparing the Chlorine Calibration Curve Completed Test for Total Coliforms IX. 9th WEEK Gram Stain Biochemical Identification of Bacteria Methyl Red Test Voger Prosheur Test Indole Test Motility Test Citrate Utilization Multi Test Systems

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 of E. 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 and solidify. 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).

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 of E. 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 either Spread Plate Method or Pour 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

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 the E. 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 wellmixing. 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)

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 of E. 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
o

C incubator and observe after 24-48 h.

3.

Contamination Experiment: Every group is provided with one NA plate. Some of the groups will 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 an Azotobacter sp. and isolate in pure culture in the coming weeks. As you know Azotobacter is an aerobic heterotroph requiring organics for both carbon and energy. However, Azotobacter has 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 of E. coli. You will do: 1-Turbidity Calibration, 2-Dry Weight, 3-Viable Counting, 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 of E. 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 of E. 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 of E. 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 of E. 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 your E. 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 mL E. 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, and E. 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 Azotobacter sp. 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 same ingredients 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 with E. 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

Fungi

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. b. c. d. 1 % tryptone + 1 % yeast extract 1 % tryptone + 1 % yeast extract + 1 % glucose 1 % tryptone + 1 % yeast extract + 1 % glucose + 0.5 % K2HPO4 1 % tryptone + 1 % yeast extract +0.1 % glucose + 0.5 % K2HPO4

pH paper Iodine solution (0.1 N in 3 % KI) Sodium chloride (1 %) 1 % glucose Test tubes 0.1 M Phosphate buffer pH 0.7 1 % NaCl Benedict's Reagent 0.1 M Phosphate buffer including 5 % starch E. coli, Yeast, Fungus Iodine solution (0.005N in 3%KI) Glass rods 10 % glucose 25 % (v/w) H2SO4 20 % (v/w) KOH

1.

Isolation of Azotobacter 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 of E. 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 an E. coli culture and four tubes of liquid medium containing:

a. b. c. d.

1 % tryptone + 1 % yeast extract, 1 % tryptone + 1 % yeast extract + 1 % glucose 1 % tryptone + 1 % yeast extract + 1 % glucose + 0.5 % K2HPO4 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 with E. 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 some bacteria 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 enzyme is reported.

Test (1 mL saliva) 0.5 % starch 0.1 M phosphate buffer pH 6.7 1 % NaCl 1 mL 5 mL 2 mL

Control 1 (1 mL boiled saliva) 5 mL 2 mL

Control 2 (No saliva) 5 mL 3 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. Cu2O + H2O + O-2

2 Cu(OH)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 of E. coli, fungus, yeast Microscopes Ocular micrometer Stage micrometer Prepared slides Test tubes with 9 mL sterile distilled water

1.

Pick a discrete colony of Azotobacter sp. from NA-plate aseptically and streak onto two NAslants provided. This ends isolation of an Azotobacter sp. from soil.

2.

Effect of disinfectant on bacterial growth: Place 0.2 mL of E. 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
o

C 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 of E. coli. A lawn culture of E. 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 adjustment screw; 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 to distinguish 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 cedaroil, 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 with reference 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. b. Blue green algae: Draw a unicellular and filamentous blue - green algae. 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 of Sacchromyces 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.

22

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 Agar Tap 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 nearly absent 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

23

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) and Klebsiella. Of this group, only Escherichia is a true indicator of fecal contamination, implying possible presence of pathogens. The rest of the genera, though giving similar reactions as Escherichia 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. 2. 3.

Both acid production and gas bubble formation => "Positive" Acid production but no gas formation => Negative No acid production but gas formation => Suspicious => Wait for another 24 hr. and if the same conditions are observed => Negative

24

Example:

10

10

10

0.1

0.1

0.1

+ 3

+ 2

If you look 3 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 and confirmed tests are undertaken on routine basis, and completed test is confined more or less to doubtful or special cases (see the flowchart).

25

FOR COLIFORMS Sample

Lauryl Tryptose Broth (or MacConkey or Lactose Broth) 35 0C, 24 h

Gas (+) PRESUMPTIVE TEST Gas (+)

Gas (-) Reincubate 24 h

Gas (-) Negative Test

Brilliant Green (BGB) or Lactose Bile Broth 35 0C, 24 h

Gas (+)

Gas (-)

Reincubate 24 h CONFIRMED TEST Gas (+) Negative Test Gas (-)

Eosin Methylene Blue or Endo Agar 24 h, 35 0C (If typical colonies exist) COMPLETED TEST Nutrient Agar Slant 24 h, 35 0C Lauryl Tryptose Broth 24-48 h, 35 0C

Gram (+) Spore formers NEGATIVE

Gram (-) Nonspore formers COLIFORM PRESENT

Gas (+) COLIFORM PRESENT

Gas (-) NEGATIVE

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 108 - 1014 coliforms/100

26

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 than once. 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 2 (+, +) 10-4 2 (+, +) 10-5 1 (+,-) 10-6 0 (-,-) 10-8 0 (-,-)

Significant No:

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. 10-3 0 10-4 1 10-5 0 10-6 0 10-7 0

Dilution

Significant No:

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. 10-3 : 2 10-4 2 10-5 1 10-6 1 (1+1) 2 2 1 2 0 10-7 1

Dilution

Result out of 2 tubes Significant No:

27

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 Case Case Case 1 2 3 6.0 0.5 20.0 Dilution 10-4 10
-3 -4

95 % Confidence 9 100 - 400 000 76 3 300

MPN/mL 60.000 5.00 200 000

10

30 000 - 1 300 000

This experiment will not continue next week since the confirmation stage is the same as for experiment 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 a pre-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

28

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 runoff from farmlands.

Fecal streptococci

S. facealis S. facealis sub.sp.liquefaciens Enterococcus Group sub.sp.zymogenes S. faecium S. bovis S. equinus S. avium Group Q (non-human source) Group D (human source)

29

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 (or equivalent) 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 be extended to 30 h at most in special conditions but reliability of results must be checked.

30

TABLE I
Number of positive tubes out of Three 10-mL tube 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 Three 1-mL tube 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 Three 0.1-mL tube 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 MPN per 100 mL <3 3 6 9 3 6.1 9.2 12 6.2 9.3 12 16 9.4 13 16 19 3.6 7.2 11 15 7.3 11 15 19 11 15 20 24 16 20 24 29 9.1 14 20 95 % Confidence Limit Lower Upper 0 9

0.085

13

0.085 0.87

20 21

0.88

23

2.7

36

1.0 2.7

36 37

31

Table I continued Number of positive tubes out of Three 10 mL tubes 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Three 1 mL Tubes 0 1 1 1 1 2 2 2 2 3 3 3 3 0 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 Three 0.1 mL Tubes 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 26 15 20 27 34 21 28 35 42 29 36 44 53 23 39 64 95 43 75 120 160 93 150 210 290 240 460 1100 >1100 MPN per 100 mL 95 % Confidence Limits Lower Upper

2.8

44

3.5

47

3.5 6.9

120 130

7.1 14 30 15 30 35 36 71 150 460

210 230 380 380 440 470 1300 2400 4800

32

TABLE II
MPN of Organisms With Two Tube Per Dilution Significant Number 000 001 010 011 020 100 101 110 111 120 121 200 201 210 211 212 220 221 222 MPN (/1 mL) 0.0 0.5 0.5 0.9 0.9 0.6 1.2 1.3 2.0 2.0 3.0 2.5 5.0 6.0 13.0 20.0 25.0 70.0 110 Confidence Limits (95 %)

.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 - 40 7.0 - 86 3.0 - 130 3.8 - 160 11 - 460

33

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 Turkish Standard 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 nontypical 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.

34

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 of E. coli (fecal coli) organisms per 100 mL of sample. Record your results.

The relationship of fecal coliform to fecal streptococcus density may provide information on the potential 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 Man Duck Sheep Chicken Pig Cow FC / FS RATIO 4.4 0.6 0.4 0.4 0.4 0.2

4.

Enumeration of fecal streptococci by MPN method (with 3 tubes method): Enumeration of fecal streptococci by the direct pour plate method using M-Enterococcus Agar does not usually necessitate 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.

35

FOR FECAL COLIFORMS Sample

Lauryl Tryptose Broth or MacConkey Broth 35 oC, 24 h

Gas, acid (+)

Gas (-)

PRESUMPTIVE TEST

Reincubate 24 h

Gas (+)

Gas (-) Negative Test

Elevated Temperature Test. EC Medium 44.5 oC, 24 h

Gas (+) CONFIRMED TEST

Gas (-) Negative Test

Fecal Coliforms Present

Calculate Fecal Coliform MPN

36

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+ + 2ClNa+ + OCl- + H2O

NaOCl + 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;

37

NH3 + HOCl NH3 + 2HOCl NH3 + 3HOCl

NH2Cl + H2O mono-chloramine NHCl2 + 2H2O di-chloramine 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 of o-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 38

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 following empirical 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 log C = 1/n log K - 1/n log tp

............. (1)

Plot of log 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).

39

Immediately proceed with the next step and transfer, with a pipette, 5 mL from each chlorinebeaker and control beaker into corresponding empty test tubes.

Put 3-5 drops of o-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

40

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 of ortho-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 of o-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 % KOH Kovacs reagent

1.

Gram Stain: As discussed earlier in the class, bacteria may be classified broadly as grampositive 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 nontypical 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 oC for 48 h. Add 0.6 mL -naphthol reagent and 0.2 mL 40% KOH solution to 1 mL of 48 h incubated old MRVP medium. Shake vigorously for 10 seconds and allow the mixture to stand 24 h. A pink colour indicates a positive test result. 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 g p-dimethylaminobenzaldehyde in 75 mL amylalcohol, add 25 mL concentrated HCl.

c.

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. Nonmotile 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 E. coli Citrobacter Aerobacter Aerogenes Klebsiella Pseudomonas Aeromonas 3.

Indole Test + +,+

MR Test + + -

VP Test + + +

Citrate Test + + + + +

Motility Test + + + + +

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 Salmonella S. typhi Citrobacter Shigella Aerobacter E. coli

G: high amount of gas g: small amount of gas

Slant K K K N or K A A

Buff A, g A A, g A A, g A, g

H2S +1 to +4 +1 +1 to +3

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.

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