Please please answer the right answers to the questions (1-32, skip #27 and 28 only). Read the lab exercise #10 to able to answer the questions.

D rinking water sources i nclude shallow wells, deep wells, moving surface waters (streams) and static surface waters (lakes, reservoirs, and cisterns). Regardless of its source, the water for human consumption must be free of human pathog ens , how ever it does not need to be sterile . The human colon harbors a wondr ous and varied population of microorganisms, none of which, whether aesthetically unpleasant or frankly pathogenic, should be found in domestic water. Domestic water that is contaminated with feces can harbor potentially pathogenic organisms such as Salmonella typhi, Shigella dystenteriae, Vibrio cholerae, Giardia lamblia, Entamoeba histolytica and several viruses (e.g .

polio and hepatitis). All the above organisms can be found in water contaminated by the feces of infected people or recovered carriers, and some, such as V. cholerae , can live and multiply i n water supplies. Deep g roundwater wells contai n water that has been filtered through many soil layers over tens of years , and if not mingled with surface water, this supply is likely pathog en free.

Shallow wells may easily be contaminated by surface r un- off. Surface waters may frequently contain heavy loads of microorganisms from defective sept ic tanks, leaking sewage systems, or the deliberate use of the water body for the disposal of domestic and industrial sewage from animal farms. Safe drinking water is difficult to find in many parts of the world. Many people are force d to use contaminated water. In addition, many of these places have very little available or affordable fuel to heat water to destroy pathogens or do not have the chemical means available, such as chlorine bl each, to tr eat the water . Boiling water to purify it is an energy inte nsive process . One alternative to treat drinking water is solar pasteurization. Pasteurization is a very useful tool, but there are some featur es of this process w e should review: • Pasteurization temperatures are below boiling. Temper atures of 63°C or 72°C are used. • Pasteurization does not sterilize. • Pasteurization does kill pathogens (this is the mai n reason we use it for purifying water or milk). • Pasteurization does not destroy endospor es. Two of the major success es of developed countries are their ability to isolate and Biology 15 10.2 distribute clean drinking water as w ell as to treat human waste before r eleasing it to the environment. The processes used to cl ean up water contaminated with human waste is know n as Wastewater Tre at ment. Given how limited our access is to clean water worldwide it is important to intr oduce the two types of wastewater: • Greywater – W ater fr om washing (clothing, bathing, dishes, etc); all water besides sewage water . • Sewage water – W ater contai ning fecal matter , also known as black water. This is what you flush dow n the toilet. In the U.S., we usually do not separate the two types of wastewater and both types are mixed and then treated at a wastewater treatment plant. As the number of clean water sources decreases and the cost of water incr eases, w e will likely see greywater being used in a more efficient manner i n the future. Gr eywater can be used to flush your toilet or irrigate your landscape. Greywater applied to the land is not only a source of water for growing plants, but it also helps recharge groundwater aquifers which are generally in a state of overdraft . Nevertheless, once was tewater enters a sewage line it is destined for the w astewater treatment plant w her e the main goals are to, one, reduce fecal -bor ne microbes and, two, reduce the amount of organic matter and other pollutants i n the water . If the o rganics ar e reduced, there will be no "food" for the pathogens & they will die. Wastewater T reatment S teps All wastewater treatment plants perform the following steps to improve the quality of the waste water so that it is safe to be r eleased to the environment without causing any negative impacts , Screening, Primary Treatment, Secondary Treatment, and Final Tr eatme nt.

Some larger facilities also perform a tertiary treatment (Figure 1 ).

Screening :

Large metal screens are used to filter out debris that often should not have been flushed down the drain in the first place. Things like a deceased pet, tampons, clothing e tc… Flushing these products down make life more difficult for wastewater engineers. How ever, cleaning these screens is much easi er than repairing a clogged pump. Primary Treatment: After scr een ing, the water is collected in a large basin where solids are allowed to settle, and floating debris is ski mmed off the surface.

These ar e often called settling tanks . The process of sedimentation is employed where larger solids drop to the bottom through the action of gravity. The solids are r emoved and either landfilled, composted, or pumped to an anaerobic digester (see bel ow) and t he liquid is pumped off to secondary treatment. Secondary treatment : This is w here the bulk of the treatment occurs.

The water arrives from primary treatment in another large basin that is equipped with an aeration device. Aeration can be achieved by pump ing air into the tank through a diffusion stone w hich makes many small bubbles , much like a fish tank aerator , or by large mixin g paddl es w hich act incr ease the surface area of the water driving more o xygen in to soluti on .

The goal of aeration is to increase the Biology 15 10.3 concentration of dissolved oxygen i n the water so that the microbes already found in the water can breakdown the organic m atter (waste) in the water as fast as possible . The microbes in wastewater tr eatment include a rich diversity of B acteria, Archaea and microbial Eukaryotes but are often collectively referred to as activated sludge . Secondary treatment typically has a non -aerated portion of the tank to allow the water to become anaerobic. This allows denitrifying bacteria to reduce the nitrate to nitrogen gas via denitrification. At the end of secondary treatment there is another settling process w her e any remaining solids are separated from the water, termed effluent at this point. Most of the solids that are settling at this sta g e are microbes that have eaten all the organic matter and once they run out of food they become less active and sink to the bottom through a proc ess called flocculation . A healthy wastewater microbial community is very effective at flocculating. An unhealthy wastewater microbial community will have trouble flocculating. When a wastewater engineer has trouble with flocculation it is typically because somethi ng came thr ough the plant that should not have, such as an unapproved chemical disposal. This is also why you ar e not supposed to fl ush chemicals and pr escription dr ugs down the toilet or drain. Final treatment Here the effluent has a dramatically reduced level of organic matter and nitrogen content and typically appears clear. Most wastewater treatment plants release their effluent to natural bodies of water such as the Sacramento River, in the case of the Chico W aste water Tr eatment Plant. To limit the number of potentially pathogenic organisms released into the environment the water is disin fected before it is release. The most common disinfection process is to chemically treat the water with bleach , an oxi dizing agent . Other chemical alter natives that some treatment plants use are chloramine or ozone .

Some plants will use UV light to disinfect the water. Other treatmen t steps: Treatment plants that are big enough and process enough was te will operate an anaerobic digester. These are large enclosed vessels that have a seeded community of microorganisms that specialize in the anaerobic breakdown of solid waste. These vessels are very effective at dramatically reducing the volume of solid waste. They also fos ter the growth of methanogenic a rchaea; microbes that produce methane (a major component of natural gas). T his gas can be harvested and burned to produce electricity or heat . In fact, Sierra Nevada B r ewery and the Chico Wastewater tr eat ment plant both have anaerobic digester s on site that breakdow n the solid waste further produci ng methane as a byproduct. The methane is burned, and the energy is use d to cr eate el ectricity and or heat kettles of beer in the brewing process .

L eftover solids at this point are typically either landfilled or applied to the land as a nutrient amendment. There is currently a lot of interest in using these biosolids to close the loop and use this waste product as a source of fertilizer. Checkout the Butte College Wastewater treatment video on Canvas. Biology 15 10.4 Figure 1 The wastewater treatment process begins by passing the water thr ough a ~6mm screen to remove debris that could damag e dow nstream components. The screened wastewater is homogenized to make the waste more bioavailable. Primary treatmen t allows the solids to settle throug h sedimen tation so that the cl earer liqui d can be siphoned off to the next step. In secondary treatmen t the water is aerated by mi xing or bubbling in air to encourage aerobic biodegradati on of the organic matter by native microorganisms . Portions of the tank are not aerated to encourage anaerobic respiration with nitrate (denitrification) to remove nitrate from the water. Once the microbial growth is complete, the biomass clumps together and falls to the bottom in a process called flocculation . The clean water is siphoned off the top and sent to fin al treatment where the water is disinfect ed with either chlorine, UV light or ozone before being rel eased to the environment.

The bi o solids collected in primary and secondary treatment are either landfilled or subjected to tertia ry treatment, also called an anaerobic digestor , w here the bi osolids are broken dow n further throug h anaerobic metabolism. A bypr oduct of anaerobic digestors is methane production by methanogenic archaea. This methane (aka natural gas) can be collected, purified, and used as a source of heat and or electricity in the facility. Biology 15 10.5 Septic Tank s Septic tanks are a wastewater treatment strategy employed i n rural residential areas (Figure 2). These are areas that have low density residential populations so there are not enough people to merit the construction and maintenance of sew er lines and a wastewater tr eatment plant. Instead, each home manages their ow n waste onsite with a septic tank. A septic tank is a large tank (~750- 1,250 gallons) that is buried in the ground. All wastewater from a single -family home is collected i n the tank. In the tank the same sedimentation process described above is occurring. Solids settle to the bot tom and liq uids remain on top. The sealed tank encourages the growth of anaerobic bacteria which degrades the solids and dissolved organic matter (waste). Near the top of the tank the liqui d layer flow s out of the tank into the surrounding ground (soil) thr ough a bra nching network of porous pipe . There the septic effl uent will leach throug h the ground in the leech field . All the while, native microorganisms that came with the waste will be bio degrading the organic matter in the tank and in the surrounding ground (l eac h field). At some poi nt the amount of solids will accumulate too much for the tank and they will need to be pumped out although if the tank is sized properly this should be a rare practice . The pumped solids will be either landfilled or added to a wastewat er treatment plant. Figure 2 The Home Septic System collects all the wastewater in the home in a large tank which is where a significant amount of the waste is broken down through microbial biodegradation. Solids will settle and accumulate at the bottom of the tank w hile the liquids will flow out of the top of the tank into a por ous branching drainage system known as a l eech field. The leach fi eld needs to be sized appr opriately for the size of the home so that the soil microbes can absorb and breakdow n the remaining excess nutrients in the septic tank effluent. Biology 15 10.6 Water Qu ality Testing The mai n questions with respect to water quality are :

1. How do w e know treated wastewater is safe to be rel eased into the environment? 2. How do we know it is safe to recreate in a natural body of water? 3. How do w e know if a water source is safe to drink? These ar e no doubt questi ons you have had at some point in your life. In this lab we will learn two tests that are routinely used to test water quality : C olifor ms testing and Biochemical Oxygen Demand (BOD) testing. A descripti on of each follows: Coliform te sting The overall goal of coliform testing is to detect fecal contamination based on the presence of an indicator organism , which are organisms used to detect a certain feature of a sample or an environment. For exampl e, certain algae occur only in very pur e natural water; their presence indicates that the water is clean. C oliforms are i ndicator organisms that are used to i ndic ate the pr esence of contaminated water . They are non -pathog enic bacteria whose presence indicates fecal contamination and, therefo re, the possible presence of pathogenic organisms. C oliforms are Gram negative, facultative , non -spore for ming rod - shaped bacteria, capable of fermenti ng lactose with the producti on of carbon dioxide gas as a byproduct . It is not practica l to attempt to detect pathog ens in a water supply; they are too varied, may be pr esent i n very low concentrations, and the tests required for the detection of some are long and expensive.

Ther efore, indicator organisms are used instead.

H owever, indicator organisms do not always correlate with the pr esence of true pathogens. Some water samples that test positive for coliform bacteria carry pathogens, but this is not true 100% of the ti me.

Nevertheless, we always err on the side of caution when coliforms ar e detected and assume that true pathogens are also present. E. coli is generally considered a fecal coliform. Although it can survive in the environment for some time, it can only flourish and grow in animal G.I. tracts. Total coliform counts include not only coliforms of fecal origin, but also non- fecal coliforms which may have originated from other sources, such as r otting vegetation and would include organisms such as Enterobacter . We will learn two ways to identify fecal and non- fecal coliforms in a wa ter sample in this lab Purple Lactose Broth Test This medium is used to determine if an organism can produce gas w hile fermenti ng lactose, the hallmark indicator of a coliform. T his br oth medi um contains lactose, a dur ham tube, and the pH indicator bromocresol purple (differential ingredi ent) and there is no strong selective ingredi ent . If coliforms are present, they will ferment the lactose which will produce acids that decrease the pH turning the media from purpl e to yel low. They will also produce carbon dioxide gas, w hich will be trapped i n the durham tube. This test cannot Biology 15 10.7 differentiate between fecal and non -fecal coliforms (Figure 3 ). Figure 3 The purple lactose broth illustrating the thr ee types of results . (a) Coliforms ar e present due to the lactose fermentation (yellow color ) and gas bubble trapped in the durham tube . (b) C oliforms ar e not pr esent due to the lactose fermentation (yellow color) and no gas bubbl e trapped in the durham tube . (c) Coliforms are not present due to no lactose fer mentation (pur ple color) and no gas bubble trapped in the durham tube .

IMV iC Biochemical Testing for Coliforms The standard biochemical tests learned in the Gram -negative rods lab may also be used to identify and distinguish between fecal coliform (E. coli ) and non- fecal colifor ms ( Enterobacter spp. ). These two organisms will give different responses to the IMViC test s, or Indol e, Methyl Red, V oges -Proskaur, and the Citrate tes t. The distinguishing responses of the tw o species are shown in the following table. Bacterium Indole Methyl Red Voges P ro skau r Citrate Escherichia coli + + – – Enterobacter – – + + Petrifilm by 3M This commercially available medium is used to demonstrate the presence of fecal and non - fecal coliforms by detecting lactose fermentation with CO 2 gas production. The growth medium on the Petrifilm is Vi olet Red Bile Lactose (VRBL) media that is contained on a sheet of paper covered by a clear plastic sheet. VRBL agar contains peptone, lactose, BCIG, bile salts, pH indicators (neutral red and crystal violet) a redox indicator (tetrazolium) and a gelling agent (similar to agar). 1.0 mL of sample water is add ed to the surface of the film and then sealed in (cover ed) with the clear plastic sheet and allowed to incubate at 37 ℃ for 24 hr. Organisms capable of fermenting lactose will produce acids causing the pH to dr op produci ng red colored colonies. Figure 4 (a) The 3M Petrifilm and (b) an incubated petrifilm showing the fecal coliforms (blue with gas bubbl e) and non -fecal coliforms (red with gas bubble). Biology 15 10.8 Most E. coli (about 97%) produce the enzyme beta -glucuronidase which will cause BCIG to turn blue. Since the media is cover ed with a plastic sheet any gas p roducti on is trapped betw een the plastic and agar media causi ng a CO 2 bubble(s) to form around the colony. All other coloni es red or other color w/o gas are non- coliforms and therefore are not counted .

The test allows you to detect fecal coliforms, (E. col i) blue /pur ple colonies with a CO 2 gas bubble, and non- fecal coliforms (such as Enterobacter ) – r ed col onies with a CO 2 gas bubble. The differential Ingredients are the pH indicator (neutral red) and BCIG and the s elective Ingredients are the c rystal viole t and bile salts w hich i nhibit the growth of Gram - positive bacteria. The Petrifilm is effectively a viable plate count adapted to a differential and sel ective media so that just coliform bacteria and fecal indicator bacteria can be enumerated.

Depending o n the water sample , undiluted or serially diluted samples can be used. Each colony is assumed to have arisen from a single cell. Results are typically reported as total and fecal coliforms/100 mL so the number of coliforms /mL needs to be multiplied by 100.

Total coliforms are the sum of the fecal and non- fecal coliforms. Equations used to perfor m these calculations are shown below: (# blue colonies w/ gas) Fecal Coliforms = (100mL) 100mL (1 mL)(TDF) (# red colonies w/ gas) Non-Fecal coliforms = (100mL) 100mL (1 mL)(TDF) ( ) () Non-Fecal Coliforms Fecal Coliforms Total Coliforms = + 100 mL 100 mL Example 3M Petr ifilm Calculations After incubating a Petrifilm with a water sample that was diluted by a factor of 10 you find; 10 are r ed with a gas bubble (non -fecal) and 4 are blue with a gas bubble (fecal). Without accounting for diluti on factor, w e have the followi ng per mL: Non -Fecal Coliforms = 10/mL Fecal Coliforms = 4 /mL Total Coliforms = 10 + 4 = 14/mL Next, we need to consider the dilution factor and report the value per 100 mL. We only tested 1 mL so w e will need to multi ply by 100. -1 (4) 4,000 Fecal Coliforms = (100mL) = 100 mL (1 mL)(10 ) -1 (10) 10,000 Non-Fecal Coliforms = (100mL) = 100 mL (1 mL)(10 ) ( ) ( ) 10,000 4,000 14,000 Total Coliforms = = += == 100 mL 100 mL 100 mL = Note,= in your calculations you do not dilut e your water samples so =ther e is no need to divide by the dilution fac tor.=According to the= Environmental Protection Agency (EPA),=4,000 fecal coliforms/100 mL =is above the= permissible level to drink or swim in. The= 14,000 total=coliforms/100 mL also exceeds the permissible levels to drink or swim in. T he= recommended permissible Coliform levels for drinking water or recreational waters ( ie.

swimming) are shown in the table below. = Biology 15 10.9 Permissible Total Coliform / 100 mL Permissible Fecal Coliform / 100 mL Drinking Water 0 0 Recreational Water <1000 <200 Biochemical Oxygen Demand Biochemical Oxygen Demand (BOD) is a test used to deter mine th e amount of bioavailable organic matter in water. I t is ac tually somewhat complicated to measure the actual concentration of organic matter in water because there are so many differ ent types of organic molecules that make up organic matter which require more elaborate analytical equipment and numerous standards to calibrate it . A simpl er approach is to incubate a water sample in an airtight container with a fixed amount of oxygen and measure the chang e in oxygen before and after a 5 -day incubation. Over time, microorganisms already in the water sample (or added if testing a very clean sample) will break dow n the organic matter using the oxygen at the same time through aerobic respiration. The change in dissolved oxyg en (DO) concentration, is direct ly proportional to the amount of organic matter in the initial sample that can be respired. If ther e is a lot of organic matter in a water sample, the bacteria will use that organic matter as a food source for respiration, which uses oxygen. If there is ve ry little organic matter in the water, very little respiration will take place and little or no oxygen will be used up. Water polluted with feces or decomposing organic matter will have a high BOD such as the wastewater flowing into a sewage tr eatment plan t. Water that is very clean will have a very low BOD such as water in a high mountain lake or stream or water released (effluent) from a sewag e treatment plant. In summary, the BOD test uses the depleti on of O 2 via respiration as a reflection of the initial amount of organic matter (food) present . The test is routinely used by the technicians w ho monitor the perfor mance of a wastewater treatment plant.

It is easy to perform and gives a good coarse metric on the total organic matter load in the waste water. The BOD test is not as well suited to environmental water quality testing w here organic loads tend to be much l ower and this test is not as sensitive to low amounts of organic matter. Biochemical Oxygen Demand Calculation s BOD is a measure of change in dissolved oxygen concentration (DO) between an initial and 5-day incubation t ime point and is reported as mg/L oxygen. ( ) if DO - DO BOD = Dilution Factor D O i = Initial Dissolved Oxygen in mg/L D O f = Final Dissolved Oxyg en in mg/L Consider the following example BOD calculation problem. Y ou dilute 2.5 mL of an unknow n water sample into 2 47.5 mL of diluent water and measure the initial dissolved oxygen concentration to be 8.0 mg/L. After 5 days you measure the final dissolved oxygen t o be 4.0 mg/L. First determine the dilution factor using the IDF equati on. Biology 15 10.10 volume transferred IDF = volume transferred + initial volume in t ube 2 2.5 mL 2.5 mL 1 = = = 0.01 = 10 2.5 mL + 247.5 mL 250 mL 100 −= Note: If there is no diluti on then the dilution factor is equal to 1 . 250 mL 1 or 1 00% of the sample 250 mL = = Now that we have the= dilution factor, w e can subtract the DO values and divide by the= dilution factor to solve for BOD in this= example. = ( ) 2 -2 8 - 4 mg BOD = = 400= O L 10 = 400 mg/L is a typical value for raw sewage.

Representative =BOD values=for several different types of water= ar e show n in the table= below . = Water Sample BOD (mg/L) Pristine river < 1 Moderately polluted river 2 – 8 Raw untreated sewage 200 – 600 Treated sewage (effluent) < 20 Summary of Coliforms a nd BOD 1. Specific types of p athogens may enter water supplies sporadically. Most of them do not survive l ong, and they require complicated pr ocedur es to detect. 2. When testing for individual pathogens, the ir number might be so low that they are missed in laboratory analyses. 3.

If present even in small numbers, pathogens can cause disease, even epidemics. 4. We don't r outinely check water for specific pathogens because of their s poradic presence , short survival times, l ikelihood of missing them even if present , difficulty of testing often requiring long incubati on times, assuming we find them at all . 5. We routinely check drinking water for total coliforms (normal fl ora of ani mal G.I.

tracts). If found to be pr esent, we know the water is contaminated with fecal contamination. We assume the possibility of pathogens i n the water. Sometimes, further tests are done for fecal coliforms (E. coli) if the total coliform count is high. If we want to absolutely i dentify the coliforms found in water, we do the IMViC tests or other nucleic acid -bas ed testi ng .

6. To ensure safety, municipal drinking water is chlorinated or filtered to kill or remove microbes. In developing countries, since water is not treated in this way, pasteurization may be a practical alternative .

7. Most treated wastew ater is r eleased to a natural water body ( such as a river or the ocean). To ensure these natural water systems are not contaminated, t reated wastewater is also routinely checked with the Biochemical Oxyg en Demand (BOD) test to make sure it does not have to o much organic matter in it. Once the organic matter is removed, micro bes do not survive in it well, especially when a low concentration disinfectant is also used before the water is rel eased. Biology 15 10.11 Protocols – Part I – Coliforms Objectives: Students will learn how to test a water sample for coliforms to determine whether wastewater treatment was effective and whether it is safe to drink or recreat e in.

This is an at -home lab where students will be collecting water samp les from around the region and sharing your results with you group on Canvas. Butte College Provided Materials • 1.5 mL Sterile tube • 1 3M Petrifilm (stored in refrigerator) • 1 plastic sterile pipette Materials provided by the students (or already given in a previous lab) • Access to a water sample in the region • Ziplock bag • Sturdy shoes • DIY tube rack • Multi -purpose disinfectant • Tupperware box for i ncubation and secondary containment • Bleach to destroy Petrifilm cultures after incubati on 1. Determine w hich l ocation you will sample from the list below . a. Big Chico Cre ek at one -mile po ol in Lower Bidwell Park. b. Big Chico Creek at five -mile in Middle Bidwell Park. c. Bear Hole – Upper Bidwell Park d. Horseshoe lake in Upper Bidwell Park .

e. Butte Creek at the Honey Run Road Bridge .

f. Sacramento River (Scotty’s Landing or your choice) g. Magalia Reservoir h.

Feather River (your choice) i. Feather River – Ther malito forebay j. Feather River – Ther malit o afterbay 2. Sample collection : When visi ting your sampling location, ear enclosed shoes and long pants and sleeves. Riverbanks ar e often slippery with uneven footing and could have poison oak growing. Tell someone you trust where you are goin g to sample water befor e you leave and when you will be back. If possible, bring a buddy with so that someone is with you in case somethi ng goes wrong. Once at your site using your 1.5 mL tube to collect water from a representative location within the site . Fill the tube above the 1.0 mL line. Label a Zi plock bag with the sample location, sample date and sampl e ti me and put the water sampl e in the bag. Proceed immediately home to complete the next steps. The longer you wait the more l ikely the microbes in the tube will die. 3. Once back home, use your multipurpose disinfectant to disinfect your work surface. 4. Bring one Petri -film out of the refrigerator being careful not to open the film or damage it. It is somewhat fragile and does not like to be squeezed. Hold it from just the white paper board border. 5. Label the Petrifilm by writing directly on top of the Petrifilm in the bottom area with your name, date, “Petrifilm” and sample locati on. 6. Use the plastic pipette to remove the excess water from the sampl e so you are left with 1 mL i n the tube using the Biology 15 10.12 graduations on the side of the tube as your guide. Discard the excess water down the drain or in the trash.

7. Using the same pipette, transfer all of the 1 mL sample ont o the center of the Petrifilm (underneath the plastic film).

Dispense slowly so as not to overshoot your target. Slowly cover the sampl e with the plastic film and then watch it spread to all edges of the circle. It usually does not need help. Resist the ur ge to fix air bubbles or areas w here the liquid did not reach. Poking with your fing er or pipette usually only makes it worse. 8. Dispose of the pipette and tube in the trash. Since nothing was cultur ed/enriched yet w e do not consider this biohazardous waste. 9. Place the Petrifilm in your Tupperware container and label it with “Biohazard – Do Not Disturb”. Allow the Petri- film to incubate for 2 days before inter preting. 10. Interpretation : Using the lab manual as a guide, interpret the colonies on your Petrifilm and calculate the number of Fecal Coliforms /100 mL and Total Coliforms /100 mL . Since there was no dilution perfor med there is no need to divide by a diluti on factor as shown in the example calcu lation. Enter your results into this online form . Use the entire class dataset to answer questions in the lab report quiz. 11. Take a picture of your Petr ifilm in a w ell-lit area without direct spot lighting or flash.

Crop the image around the P etrifilm. You will be uploading the image into a discussion on Canvas. 12.

After you have completed your interpretations you can dispose of the P etrifilm. While, wearing gloves and eye protection, carefully open the Petrifilm while it is still in the Tupperware container. Pour 2% diluted bleach over the surface of the film where there are coloni es. If you ran out of diluted bleach, consult the instructions in L ab 4 to prepare more 2% bleach. C over the container and put someplace out of the way , like in a garage for 6 -12 hours to disinfect the cultures. Once complete pour the excess down the drain chased with a gener ous amount of running water. Place the petri - film in a Zi ploc bag and dispose of i n the trash Protocols – Part II – BOD Objectives: Students will learn how to test a water sample for BOD to determine whether wastewater treatment was effective and whether it is safe to drink or recreate in.

This is not possibl e to perform at home, but you will answer questions on the procedure, calculations, and interpretations in the lab report quiz on canvas .

Vernier is the company that manufactures the dissolved oxygen probe that w e use in the lab.

They have a short video on calibrating the measuring DO with their DO probe. We do not focus on calibration of the probe so feel free to ski p to the 7 minute mark to watch how the DO measurement is taken . 1. Depen ding on the sample used for the BOD determination a dilution may be required. Add your sample to your dilution bottle and then add freshly aerated (swirl vigorously for 30 seconds) Biology 15 10.13 dilution water to the shoulder of the bottle . Dilution water is non -sterile water with inorganic salts so as not to put the organisms under hypotonic stress. 2. Insert the DO pro be no more than halfway into your BOD sample so as not to cause it to overflow as the probe will displace water . Gently stir the bottle with the probe while waiting for the reading to stabilize. Record the DO as soon as the value has stabilized for 10 seconds. 3. Optional: For water sampl es that are believed to be very “cl ean” a small amount of “seed bacteria” are added after taking this DO readi ng to ensure adequate microbial respiration can take place during the incubation . 4. Rinse the probe over the rinse beaker and place it back in the original flas k.

5. Add more BOD dilution water or if you had no dilution add more sampl e so that it is ½ up the neck of the bottle .

6. Take the BOD bottle to the tray on the back bench, then cap the BOD bottle fir mly; water should overflow and there should be no air bubbl es insi de . Fill the trough above the glass stopper with a DI water rinse bottle to further bl ock the ability of oxygen from getting into the bottle . 7. Incubate your BOD bottle at room temperature for ~5 days in the dark . 8. Da y 2 : After the 5 -7 days of i ncubation, repeat step 2 above to record the final DO value . 9. Calculate the BOD value using the instructions given in this manual