Membrane filtration to detect total coliforms, and Escherichia coli

ENV H 433 LABORATORY EXERCISE 2 Membrane filtration to detect total coliforms, and Escherichia coli I. LABORATORY GOAL To determine concentrations of...
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ENV H 433 LABORATORY EXERCISE 2

Membrane filtration to detect total coliforms, and Escherichia coli I. LABORATORY GOAL To determine concentrations of total coliform, and E. coli in samples of surface water and drinking water using membrane filtration techniques with two different media. II. ASSIGNED READINGS Standard Methods (SM) 9222 Sections A and B (on class website) USEPA Method 1604 (on class websit) USEPA Method 1115 (on class websit) Difco™ & BBL™ Manual DBM for MI, and LTB (http://www.bd.com/ds/technicalCenter/inserts/difcoBblManual.asp) III.

LABORATORY SCHEDULE

Table 1. Tasks by day of experiment. Day 1 Tue 2

Task Dilute and filter samples, place a filter on mEndo-LES and MI plates Refrigerate mEndo-LES & MI plates Count samples on both media

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Interpreting results IV.

BACKGROUND

For some applications, membrane filtration (MF) may be preferable to the multiple tube fermentation technique. In this technique, samples are concentrated on a sterile filter, the filter is transferred to solid growth media, and resulting colonies are counted. While both MF and fermentation tube techniques rely on the ability of targeted organisms to grow under select conditions (i.e. media type and temperature), membrane filtration has the advantage that large sample volumes can be concentrated on a single filter for analysis. Note that sample interference can occur from either excessive solids or from non-targeted organisms, as these are also concentrated on the filter. Thus, primary wastewater samples and surface waters with high algae content should not be processed by MF. Two new media are introduced in this lab: mEndo-LES agar, and MI agar [Simultaneous Chromogenic-fluorogenic Detection and Enumeration of Total Coliforms and E. coli in drinking water by the membrane filter technique. Conforms with US EPA Approved Method 1604]. MI agar is named for the two specialized enzyme substrates that are included in its formulation: MUG and IBDG. MUG (4Methylumbelliferyl-β-D-galactopyranoside) was introduced in Lab 1, and is a fluorgen (i.e. fluoresces under UV light) triggered by the E. coli enzyme β-glucuronidase. Conversely, IBDG (indoxyl-β-Dglucuronide) is a chromogen (i.e. produces a blue color under ambient light) triggered by β-galactosidase enzyme found in all coliform. MI agar allows a single-step procedure for detection of both total coliform and E. coli.

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The second new media, mEndo-LES agar, contains both chemical inhibitors and color indicators. The inhibitors: sodium desoxycholate and sodium lauryl sulfate will limit the growth of Staphylococcus species. Acetaldehydes, produced by lactose-fermenting bacteria, react with sodium sulfate and fuchins to produce a red color. Further, aldehydes produced during rapid fermentation of lactose will cause the red colonies to have a metallic sheen. However, overgrown plates (i.e. too much inoculum) can suppress this metallic sheen. Thus, to limit the probability of false positives or negatives, following growth on the mEndo-LES agar, suspected coliform colonies can be confirmed by simultaneous inoculation into fermentation tubes containing lauryl tryptose broth (LTB) and brilliant green lactose bile (BGLB) broth – both of which were introduced last week.

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GENERAL COMMENTS Use aseptic technique. Quick reference

Sample Volumes - Process samples in the order listed! Lake Water Analysis  Filter 100 mL of Negative Control (Do This First!)  Next, add 10 mL of sterile phosphate buffered water with 1 mL of LW sample and filter (two plates for each media)  Next, filter 10 mL of the LW sample (two plates for each media)  Last, filter 100 mL of the Positive Control

General colony counting rules (applies to both media types). 1. Count all plates with < 200 colonies. Plates having 20-80 colonies are considered preferable. 2. Report plates with >200 colonies as "too numerous to count" (TNTC). 3. As you count each colony mark it with a black sharpie on the bottom of the petri plate. 4. You may have to carefully remove the lids from the plates to clearly see the colonies on the filter surface. mEndo-LES agar  pink, blue, white, or colorless – noncoliform, do not count (mark as · )  red with greenish-gold metallic sheen – score as total coliform colonies “typical” (mark as +) The sheen may cover the entire colony, may only be in the center or may appear only around the edges.  dark red, mucoid, or nucleated without sheen – score as “atypical” colonies (mark as ◦) MI agar – appears blue or indigo under ambient light. Count plates under long wavelength (366 nm) ultraviolet light as follows: (NOTE: To prevent burns, you must have on your lab coat, gloves, and full-face shield before turning on UV lamp. Room lights should be off.)  blue/green fluorescent colonies or with fluorescent edges – E. coli  blue/white, non fluorescent – total coliforms, but not E.coli The sum of these two categories (fluorescent colonies and blue, non-fluorescent colonies) are reported as total coliform.

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MATERIALS Membrane Filtration (Day 1) 1. Lake Water or Drinking Water samples (Note: Drinking water sample contains sodium thiosulfate to quench any chlorine residual in water – See standard method 9060.A.2). (1 liter of each sample per group) 2. Disposable sterile membrane filtration apparatus - consists of membrane filter holder and vacuum flask (2 per group). 3. Membrane filters (sterile, individually wrapped)- mixed cellulose esters, 47-mm-diameter, 0.45-µm-pore-size, gridded ("up" side has grid pattern) (6 per group) 4. Forceps (sterile, individually wrapped) - for holding and transferring filters (2 per group) 5. 60-mm-diameter Petri dishes with media: mEndo-LES agar for total coliforms (6 per group) and MI agar (6 per group) 6. 10-mL serological pipettes (sterile, individually wrapped, 2 per group), and 10-mL pipette pump (green) or pipette bulb 7. 1-ml serological pipettes (sterile, individually wrapped, 2 per group), and 10-mL pipette pump (blue) or pipette bulb 8. Air incubator – set at 35 ºC for all agar plates 9. 200 mL Nalgene bottle containing Positive Control (1 per group) 10. 500 mL bottle containing Negative Control (sterile phosphate buffer solution) Note: Use sterile phosphate buffer for washing down

sides of filter holder. (1 per group) PROCEDURE

Mon Day 1 1. Label plates on the back side (group number and sample identification including date). Note: For each group, there will be one plate each of the negative and positive controls, and two plates each of two different dilutions of lake water sample. 2. Set up a vacuum flask and the lower section (base) of a filter unit. Be sure to handle the base using aseptic technique and avoid touching the porous surface on which the membranes are placed. 3. Use a sterile forceps to aseptically place a membrane filter, grid side up, on the filter base. Align and attach the upper, funnel section of the filter unit to the base. 4. Using a sterile graduated cylinder, add the designated sample volume, and record the precise volume (see quick reference above) 5. After each filter, rinse the interior walls of the funnel by dispensing about 10 ml of sterile phosphate buffer into the filter funnel containing the membrane filter while the vacuum is on. 6. After filtration, turn off the vacuum. 4

7. Carefully remove the funnel from the base and rest upside down on a clean paper towel. 8. Use a sterile forceps to aseptically remove the filter, and apply it, grid side up, to the surface of a 60-mm-diameter Petri dish containing the agar media (mEndo-LES and MI agars). The filter is applied to the agar by "rolling" it onto the surface. Avoid trapping air bubbles under the filter.

9. Repeat steps 2 through 8, paying careful attention to the order that samples should be processed, as outlined in the quick reference box. 10. Incubate all plates inverted in a 35ºC air incubator Tue TA refrigerate plates Wed Day 2 Remove plates from the refrigerator. Calculate coliform and E. coli densities according to Standard Method 9222.B.6. 1. Determine the plates that are suitable for counting (preferably between 20 to 80 colonies). 2. Compare results between the two sets of tubes. A confirmed coliform will have both growth (turbidity) and gas in both LTB and BGLB broth. 3. For verified coliform counts, adjust the initial count based upon the positive verification percentage and report as “verified coliform count/100 mL.” Percentage verified coliforms =

number of verified coliform  100 total number of coliform colonies subjected to verification

4. Estimate the 95% confidence intervals for the MF data using Table 9222.II, Standard Method 9222B 5. Share your results with the other groups, and participate in class discussion. Estimating densities when dilutions are not ideal: 6. Number of colonies is too high at all dilutions: estimate the number of colonies by counts from plates having >80 but 200 colonies as "too numerous to count" (TNTC). 3. As you count each colony mark it with a black sharpie on the bottom of the petri plate. 4. You may have to carefully remove the lids from the plates to clearly see the colonies on the filter surface. mEndo-LES agar  pink, blue, white, or colorless – noncoliform, do not count (mark as · )  red with greenish-gold metallic sheen – score as total coliform colonies “typical” (mark as +) The sheen may cover the entire colony, may only be in the center or may appear only around the edges.  dark red, mucoid, or nucleated without sheen – score as “atypical” colonies (mark as ◦) MI agar – appears blue or indigo under ambient light. Count plates under long wavelength (366 nm) ultraviolet light as follows: (NOTE: To prevent burns, you must have on your lab coat, gloves, and full-face shield before turning on UV lamp. Room lights should be off.)  blue/green fluorescent escent colonies or with fluorescent edges – E. coli  blue/white, non fluorescent – total coliforms, but not E.coli The sum of these two categories (fluorescent colonies and blue, non-fluorescent colonies) are reported as total coliform. Reading Results 5. Remove plates from the air incubator. 6. Count colonies according the to strategy in the quick reference boxes (next page). 7. After counting, place the mEndo-LES plates into sealable, Zip-Lock bags and store at 4ºC until next class period. Two step Procedure Part II–Confirmation of Total Coliforms and E. coli from mEndo-LES Plates Day 3 1. Remove plates from refrigerator and allow to warm to room temperature. 2. Label tubes. 3. Using a sterile inoculation loop, touch a “typical” colony and inoculate a tube of LTB. 4. Using a new inoculation loop, touch the same colony and inoculate a tube of BGLB broth. 5. Repeat for up to five typical colonies from each replicate plate (10 total). 6. If present, repeat inoculation of LTB and BGLB broth tubes for up to five “atypical” colonies from each replicate plate (10 total). 7. Repeat this for each sample if necessary. 8. Incubate tubes at 35oC. 9. After inoculating the LTB and BGLB tubes, transfer membranes having countable colonies on m-EndoLES agar to nutrient agar with MUG (NA-MUG) plates. 10. Record the time that incubation was started. 11. Incubate the NA-MUG plates 4 hours at 35oC. Wait 4 hours – continue with single step procedure on MI agar 9

Single step Procedure: Membrane Filtration Follow Day 1 procedure steps 1 to 10, substituting the MI agar plates. Day 3 (plus 4 hours) Reading Results 1. Read membrane filters on NA-MUG according to the quick reference below: Day 4

NA-MUG agar –Count plates under long wavelength (366 nm) ultraviolet light as follows: (NOTE: To prevent burns, you must have on your lab coat, gloves, and full-face shield before turning on UV lamp. Room lights should be off.)  blue fluorescent around the edge – E. coli Note that non-E. coli coliforms may produce a metallic sheen on the mEndo-LES agar, but do not fluoresce on NA-MUG.

1. Gently shake the rack of tubes back and forth several times to release gas in positive tubes. 2. Record the presence of growth (turbidity or cloudiness) and gas (look in the small inverted tube) for all tubes. 3. Read MI agar plates following the quick reference box on pg 4. Day 5 Calculate coliform and E. coli densities according to Standard Method 9222.B.6. 8. Determine the plates that are suitable for counting (preferably between 20 to 80 colonies). 9. Compare results between the two sets of tubes. A confirmed coliform will have both growth (turbidity) and gas in both LTB and BGLB broth. 10. For verified coliform counts, adjust the initial count based upon the positive verification percentage and report as “verified coliform count/100 mL.” Percentage verified coliforms =

number of verified coliform  100 total number of coliform colonies subjected to verification

Estimating densities when dilutions are not ideal: 1. Number of colonies is too high at all dilutions: estimate the number of colonies by counts from plates having >80 but