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Environmental Sampling_ Dilution

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					Environmental Sampling, Dilutions & Population Counts

This is a multi-purpose and continuing experiment. It will be performed in laboratory groups and will begin with each group sampling a specified environmental site (or processing a sample). In addition to site sampling techniques and population counts, this experiment will provide an introduction to differential and selective media, and practice of aseptic technique. Also it will provide you with the starting materials for, production of “pure cultures”, introduce you to the wonders of storage of cultures (short term/long term), and provide your group unknowns for identification in future labs. To be succinct this will be a long term experiment, don’t mess up, don’t loose your experimental results. You will also get plenty of chances to practice and refine your pipetting skills from Cell Biology. As techniques and results will differ somewhat, it is necessary that each and every group exchange methods and results. Population or viability counts are methods used to estimate the number of reproducing organisms (concentration or density) from a sample/site. It is an extremely important piece of information for many applications within microbiology. Is it safe to eat, drink or to come into contact with (Tacos to beech closings)? Your analysis will use a common counting method employing a series of dilution to reduce the number of bacteria in the original sample so that it may be plated and representative numbers determined. Colonies form on plates as each bacterium produces “clones” around itself so that one colony is equal to 1 bacterium (or colony forming unit) in the original culture. Frequently one may be interested in identifying specific microorganisms [Salmonella, Escherichia coli (E. coli), etc.] instead of total bacterial numbers, in which case selective media (inhibits the growth of other microorganisms) and or differential media (provides for a differential phenotype between microorganisms). Plates having 30 to 300 colonies after incubation are considered “countable”. Counts above 300 CFU have a higher probability of having a single colony being derived from 2 cells instead of one. At concentrations less than 30 CFU slight variability’s in distribution within a sample could pose problems. To ensure that a countable plate

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is produced, a sample is plated from several of the dilution tubes within the series, usually in replicate to ensure precision and validity. HOMEWORK DETERMINE THE SELECTIVE AND DIFFERENTIAL CHARACTERISTICS OF MANITOL SALT AGAR and MACCONKEY AGAR. WILL NEED IT FOR PART 2. ● Task: Each group will perform each of the following tasks as assigned by their lab instructor; HOWEVER EVERYONE will be responsible for understanding principals and application of the tasks. A. Sampling a liquid volume (Is the water safe to drink or swim in?): Usually investigations are looking at E. coli as an indicator organism and volumes are really large. B. Testing soil for the presence of bacteria. A key aspect of all of these measures is that it will yield a result in colony forming units per unit (volume, or mass). ● Procedure:

Activity A 1. For analysis of bacterial counts present in water samples, collect six dilution blanks (three sterile aliquots of 99 mls [A blanks] and three of 99.9 ml aliquots [B blanks]). 2. Collect water analysis plates (50 mm dia. with media absorbent pad. Label base of the plates (the portion of the plate with etched Company name) with sample designation, dilution volume (three plates with 1 ml & 3 with 100μl) date, initials of group, section #, table #. 3. Collect 6 tubes of Millipore ColiBlue 24 Media, apply to water analysis plates (one tube per plate). 4. Prepare dilution blanks: Invert water sample ten times to mix. Using aseptic technique, rapidly transfer 1m of the water 2

sample to each of the 99 ml water blanks. Transfer 100μl of water sample to your 99.9 ml blanks. DO NOT GET BLANKS MIXED UP, DOUBLE DILUTE OR FAILE TO ADD A SAMPLE. 5. Take samples and labeled plates with media to the vacuum manifold station. Will have vacuum manifold, sterile filters in filter holders, alcohol, forceps, sterile water, sterile graduated cylinder. The process is as followed: in an aseptic manner (don’t handle surfaces that will come into contact with microbes, alcohol and flame forceps as needed) press three filter assemblies onto black stoppers on vacuum manifold. Turn on vacuum. Mix one group of diluted samples (100 μl, B samples) by inversion. Pour one bottle into each of the three filter holders. Holder. WHEN THE INITIAL VOLUME HAS PASSED THROUGH THE FILTER. Fill each blank with 100 ml of sterile water (Wash) mix and pour into the corresponding filter holder. 6. When the was volume has passed through the filter, turn off the vacuum, take off the top o the filter holder and using flamed forceps, transfer the filters to the labeled plates (filter grid side up). Incubate lid up as directed by your lab instructor. Empty the vacuum manifold. 7. Repeat procedure with dilution A set. 8. Congratulations, you are not through yet, empty the vacuum manifold. Follow lab instructor’s instructions; wash filter holders assemble with filter, package and autoclave. (Introduction to autoclaving, a truly valuable technique.)

Activity B 1. For the “solid sample”, weigh 100 milligrams of the soil sample (record actual weight you will determine CFU/ gram). Label three MSA plates and three of the other designated selective/differential media (as indicated by your lab instructor) with sample designation, plating volume (100 μl or 10 μl ), date, initials of group, section #, table #. Label one 5 ml sterile snap cap tube with group initials. 2. Transfer the soil sample to the sterile tube, add 1 ml of sterile water, vortex for 5 minutes and then allow the soil to briefly 3

settle (30-60 seconds). While this vortexing frenzy is going on, transfer 900μ of sterile water to a second sterile tube (your dilution blank). 3. Transfer 100μ of the supernatant to the 900 μ of diluent. Mix well. (With memories of Cell Biology, What is the dilution factor?) 4. From your diluted sample, spread plate volumes of 100 μl and 10 μl on the labeled plates [NOTE will have to first add 50 μl of sterile media in a single drop to the center of the plates for the 10 μl platings. Then add the 10 μl volumes to the drops (carrier volume)]. 5. As demonstrated by your lab instructor spread-plates are done as described below: a. Dip the end of a spread bar into alcohol and sterilize by quickly passing the bar through a flame to ignite the alcohol. b. After allowing the bar to cool, quickly and evenly distribute the sample over the agar surface of plate E. c. Re-sterilize the spread bar and repeat the process for the remaining plates. d. Place inoculated plates into plate holder (can) and incubate overnight. and incubate overnight at room temperature, 37° C, and 45°C 6. Clean up and lab area. 7. AFTERWARD STORE AT 4º - 10º C.

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Cautions  When plating from a dilution series using the same pipette or tip, samples must be taken from the LEAST concentrated tube to the MOST concentrated tube. The spread bar should be tipped down and away from hands to prevent contact between alcohol and skin. Pass the spread bar through the flame to ignite the alcohol. DO NOT LET THE SPREAD BAR REMAIN IN THE FLAME AS THE GLASS WILL MELT.

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● Results: (second laboratory period) Each group will tooth pick ten colonies/ student (40 if you have 4 students in your group) onto a gridded Flurocult plate and incubate overnight for future use. GET YOU CFU NUMBERS FIRST AND EXCHANGE WITH THE OTHER LAB GROUPS.

Activity A 1. For ColiBlue plates determine the number of red colonies and blue colonies at a countable dilution (the dilution may differ for the two different phenotype colonies). Determine CFU of β-glactosidase + colonies and CFU of β-glactosidase negative colonies per 100 ml of water sample (deciliter). EVIL HIGHER MATH. 2. Also toothpick your (10/student) lactose positive colonies to a Flurocult plate. Incubate at 37º C overnight. Activity B 1. Which platings (media/temp) gave countable colonies? How many phenotypes did you obtain? Determine CFU per gram of soil for each phenotype. 2. For MSA plates determine the number of red colonies and yellow colonies at a countable dilution (the dilution may differ for the two different phenotype colonies). Determine CFU of

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mannitol fermenting and non mannitol fermenting colonies per 1 cm2. Also determine total salt tolerant CFU/ 1 cm2. 3. For MAC plates determine the number of pink/red colonies and colorless/transparent colonies at a countable dilution (the dilution may differ for the two different phenotype colonies). Determine CFU of lactose fermenting and non lactose fermenting colonies per 1 cm2. Also determine total bile salt/ crystal violet tolerant CFU/ 1 cm2. 4. Our next step is to start the process of isolating different phenotypes based on colony morphology. Selection of colonies should be distributed as evenly as possible among the plates that showed growth. Toothpick 12 Mannitol fermenting colonies to an MSA plate. Also toothpick your (12/student) lactose positive colonies and 12 lactose negative colonies to a Flurocult plate. Incubate at 37º C overnight. You will have recorded phenotype and incubation temperature with grid numbers when patching (single phenotype/temp. per row would be a wise option: Incubate at 37º C overnight.

Calculations: Questions: 1. When and why is it acceptable to use the same pipette or tip when taking samples from several tubes within a dilution series?

2. You add a 100 μl sample to 900 μl diluent. What is the dilution factor? What is the concentration (CFU/ml) of the original stock if you determine that there are 93 CFU/ml in the diluted tube? 3. You add 0.001 ml to 0.099 ml. What is the dilution factor? What are the CFU/ml of the original stock if you determine that there are 45 CFU/ml in the diluted tube? 4. Define the differential and selective characteristics of the media used. (a bit of work but it will be worth it.) 5. Did incubation temperature impact colony counts? If there was a difference, what do you think was the cause of this difference? Would extended incubation (48 hrs) produce results? 6

Practice Dilution Problem:

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A Stock 99 mls

B A 99 mls

C 9.0 mls

D 4.5 mls 0.2 ml

10µl

Plate B 30 colonies

Plate A 150 colonies Tube A DF FDF ______ ______ Tube B ______ ______ Tube C ______ ______ Tube D ______ ______

What is the concentration of tube C? What is the concentration of the stock as calculated from plate B? What is the concentration of the stock as calculated from tube C?

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