GOLD

An Experiment With Culture

We are testing the effectiveness of disinfectants marketed as "environmentally friendly" as compared to conventional disinfectants.
Audrey Lo & Grace Berry
Grade 7

Hypothesis

If traditional disinfectants are used, then they will be more effective at killing bacteria than the eco-friendly products. Our reasons for this hypothesis are that we think traditional products are chemicals made specifically to disinfect. We assume that if the product is "green", the active ingredient is natural, and the bacteria may even have a substance to feed on.

 

Research

     Disinfecting is on everyone’s minds lately because of the pandemic. In any store you go to you will find whole aisles dedicated to disinfectants. The effects of chemicals on human health and the environment are also a concern lately. The current COVID-19 situation also adds worry that, since people are using more disinfectants, we are being exposed to a larger amount of these chemicals than ever. So, we should have a closer look at what we are using in our homes. Consumers might be influenced by brand names, costs, labeling, and the perception a product is “green” when shopping for disinfectants. However, even “eco-friendly” disinfectants are not completely “green” as they still kill organisms (bacteria). The consumer needs information on which cleaners work and which have the least negative effects. Although it would be nice to study how these disinfectants work on viruses like COVID-19, it is not possible to test viruses in our home or school labs so we decided to study bacteria instead. To prepare for our experiment, we researched what bacteria are our concern in the home, how a disinfectant is defined and tested, the types of disinfectants and how they act, their proper use, and lab techniques we could use. Additionally, we wanted to know about human health and environmental concerns, cost considerations, and other safety issues for the products we will test. For our experiment, we are planning to investigate whether products sold as eco-friendly disinfectants are as effective at killing bacteria as traditional products.

     Bacteria are prokaryotes. They have no nucleus or organelles attached to the membrane. They do have a cell membrane, ribosomes, cytoplasm, DNA (circular shape), and most have a cell wall. We need disinfectants to kill some types of potentially harmful bacteria before we get infected. The most common places in the house that people use disinfectants are the kitchen and bathrooms. In the kitchen, raw meats and food wastes can contaminate a variety of places. Raw vegetables can be a source of food poisoning because contaminated water may have been used to water or rinse the vegetables before being packaged for the grocery store. If cutting boards are not washed thoroughly enough, bacteria can be left behind. Sponges stay moist most of the time, which promotes bacterial growth. Food scraps that do not get cleaned up inside the sink can feed the bacteria already growing there. Toothbrush holders, floors, sinks, and faucets are additional surfaces that can allow bacteria and viruses to grow. Pet bowls and toys may also have harmful bacteria. Due to the pandemic, many people started to recognize the need to clean high-touch surfaces that get contaminated when everyone in the household touches them, such as light switches, doorknobs, and faucet handles. 

     People are sometimes confused about what "cleaners", "sanitizers", and "disinfectants" mean when they go to the store to pick a product. Cleaning refers to removing dirt, dust, or germs from a surface or object, not necessarily killing the germs. Sanitizing is lowering the number of germs on a surface or object to a safe level. A disinfectant is a substance applied to a surface or object that kills or inactivates germs (i.e. viruses, bacteria, fungi, protozoa) after being left for a period of time. In Canada, if a company wants to call a chemical a “disinfectant”, the manufacturer must provide data on safety, efficacy, and quality to the Natural and Non-Prescription Health Product Directorate (NNHPD), which is the part of Health Canada that evaluates all the applications. Because a disinfectant is considered a "drug", the rules for safety, efficacy, quality, and labels can be found in the Food and Drug Act. Once approved, the product will get a Drug Identification Number (DIN) and can be classified as a disinfectant. For safety testing, Health Canada requires data on how safe the product is when used as the label says. The company can submit other studies that have already been done instead of doing its own safety testing. Effectiveness is how well something completes a certain task in real world circumstances. Efficacy is how something completes a task to a certain degree in a controlled environment. To look at the efficacy, Health Canada is very strict about the testing methods that are used, depending on what the label’s claims are. There are different approved testing methods for various forms of disinfectants, such as sprays, wipes, and powders. The methods are also different if the label claims to kill bacteria, viruses, or fungi. Even different types of bacteria have specific testing methods. Data on quality should show that Good Manufacturing Practices set out by the Food and Drug Regulations are followed. The product will get a DIN when it has shown enough data to the NNHPD that the chemical can do what the label claims, such as "Kills 99.9% of Bacteria." The labels on the products need to include specifics like the brand name, active ingredient, how much of the active ingredient there is, a precaution part, and the company’s address. Although there may be information on toxicities, it is acceptable to only include some precautions and what to do if there are accidental exposures due to unsafe use. For example, bleach can damage aluminum surfaces. Also, if the chemical gets into the eye, the instruction is to rinse with water. Not all ingredients need to be listed either, so it is hard to know how other precautions consumers should take when using these products. Health Canada also recommends manufacturers to include information on potential acute oral toxicities, skin irritations, inhalation problems, and eye irritation, but it is up to the company what to include. Sometimes these details are in a Safety Data Sheet, but they are difficult for the consumers to access.

     Disinfectants can be divided into classes based on chemical characteristics and action. These classes are alcohols, acids, halogens, quaternary ammonium compounds, alkalis, phenols, peroxygens, biguanides, and aldehydes. When we went to the store to look at products labeled as “disinfectant”, we found that the main chemicals they contained were glycolic and citric acid (acids), sodium hypochlorite or bleach (halogen), quaternary ammonium compounds, sodium hydroxide (alkali), thymol (phenol), and hydrogen peroxide (peroxygen). We wanted to test disinfectants from different classes that are easily available at the store. So, we decided to use citric acid, sodium hypochlorite, a quaternary ammonium compound, sodium hydroxide, thymol, and hydrogen peroxide. There are several ways that disinfectants can kill both bacteria and viruses. One is to break open or weaken membranes, and the other is to disrupt the genetic materials inside so the bacteria cannot replicate, or the virus cannot insert its genetic materials into the host's. Additionally, they may block enzyme systems in microorganisms. The disinfectants which are considered to have the strongest effects on pathogens are peracetic acid, sodium hypochlorite, hydrogen peroxide, and aldehydes. For the most part, all disinfectants should have some effect on bacteria, but not always bacterial spores. Acids and alkalis have a limited effect on these spores and alcohols, quaternary ammonium compounds, phenols, and biguanides would do very little. Quaternary ammonium compounds have differences in parts of their chemical structure which affects how they work on different bacteria. Some may also not work that as against Pseudomonas bacteria. Based on this information, we would expect that all disinfectants should have some effect on kitchen bacteria as long as they are not in spore form.

     Consumers want to pick products that are not a danger to their health. In Canada, when a company wants to call a new product a “disinfectant”, it must provide safety and toxicity data. Both the Environmental Protection Agency in the United States and Health Canada are similar in their approval process, but neither country requires the label on the product to list all the ingredients and precautions for consumers. Because disinfectants kill cells, none are 100% harmless. The Environment Working Group (EWG) in the U.S. is a non-government agency that researches health problems caused by many substances used in our daily lives. The way that disinfectants can harm humans can be either accidental or from being exposed to the chemicals repeatedly. The EWG found that there is a higher risk of birth defects in babies born to women who have cleaning jobs. Many new cases of asthma in people can be linked to workplaces that have regular exposure to cleaning supplies, such as health care workers, teachers, and custodial staff. Fragrances in some of the products can trigger breathing problems and headaches. Improper use of the disinfectant can result in skin and eye irritations, coughing, and burns. Accidental poisoning in children can also happen if the cleaners are not stored away safely. Sodium hypochlorite, or bleach, and chlorine are harmful to the mucous membranes in animals' respiratory and digestive systems. It is also corrosive so contact with the skin can cause swelling and blisters. It does not cause cancer by itself, but when it interacts with other substances, it can form other chemicals that can. In the U.S., bleach in a spray bottle is the most common cause of poisoning from household cleaners. Quaternary ammonium compounds can trigger asthma symptoms or cause skin and eye irritations. Sodium hydroxide is corrosive and can irritate the lungs if inhaled, cause skin reactions, and is toxic if swallowed. It can create dangerous chemicals if combined with other cleaners with bleach. Thymol comes from the thyme plant. It is also used as an herb in cooking. As a cleaning product, if it is not used according to the label’s directions, it can cause chemical burns, and skin and eye irritation. Accidental ingestion of a large amount can cause stomach irritation and maybe seizures or death. It has not been found to be a carcinogen. Citric Acid is low in toxicity, but if it is not used as carefully, it can cause irritation to the eyes and skin. If it is inhaled in a high concentration, it can cause sore throat and coughing. Hydrogen peroxide breaks down into water and oxygen quickly outside of the container, so it is not toxic to humans when used according to the label. Sometimes people will clean with several types of products that should not be mixed. For example, if hydrogen peroxide is accidentally mixed with vinegar, it forms a chemical called peracetic acid, which can produce toxic fumes. Exposure to hydrogen peroxide in higher concentrations or when not used properly, can cause irritation to the eyes, throat, airway, and skin. One study from the University of Saskatchewan found that mopping floors with commercial hydrogen peroxide cleans around 1% causes airborne levels of peroxide at the upper limit of what is recommended for long term exposure. This can be high enough to react chemically with other things in the air, making air quality poor. 

     In addition to concerns about health risks, consumers often want to use products that are less harmful to the environment. However, no disinfectant is officially “green” as the purpose of a disinfectant is to kill microorganisms. It is still possible for a disinfectant to be better than others for the environment. Consumers will have to look online for information about safe cleaner choices. In Canada, there is no current certification or division indicating whether a disinfectant is environmentally friendly or not. The EPA (in the USA) says that citric acid, quaternary ammonium compounds, sodium hydroxide, and hydrogen peroxide are the safest for the environment. Sodium hypochlorite and thymol are not rated. Packaging, addition of fragrances, and how a product is manufactured may also be important to consider. The active ingredient makes the biggest difference. Citric acid is made from fruit or by fermenting carbohydrates, and therefore a natural substance that breaks down quickly, which makes it environmentally friendly. Bleach is made by combining salt water, chlorine gas, and caustic soda in a chemical process. It breaks down relatively fast in the environment, transforming into sodium chloride (salt), oxygen, and water. Some breakdown products from bleach can combine with other substances in the environment to form toxic chlorinated compounds like chloroform and carbon tetrachloride. Quaternary ammonium compounds are made in a process that combines chemicals like ammonia, alcohols, and chlorides. They are fairly low toxicity but take a long time to break down in the environment so they might kill bacteria there or cause more resistant bacteria to grow. Some chemicals released when they breakdown during sewage treatment could form carcinogens. An electric current is run through salt water to produce sodium hydroxide. Sodium hydroxide falls apart quickly in the environment into sodium and hydroxide. It has low toxicity in small amounts but large amounts can increase the pH of a substance. Thymol is a naturally occurring disinfectant made from the leaves of the herb thyme. It is broken down quickly when exposed to sunlight but could persist longer if away from the sun. The usual process of creating hydrogen peroxide is to mix natural gas and oxygen together under high heat although it is possible to produce it by breaking down water with electricity. It separates very quickly into oxygen and water so has very minimal environmental effects. In conclusion, citric acid, sodium hydroxide, and thymol are the least toxic to produce. After use, citric acid, sodium hydroxide, thymol, and hydrogen peroxide have the least effects on the environment.

     There are also additional problems with some commonly used disinfectants. Firstly, some may create toxic substances if combined (bleach and acids, hydrogen peroxide and vinegar). Secondly, many have the potential to damage surfaces. For example, they might bleach, change the colour, or cause corrosive damage for delicate surfaces (sodium hypochlorite, hydrogen peroxide, and citric acid). When it comes to this issue, quaternary ammonium compounds and thymol are the safest to use. Thirdly, some disinfectants, especially quaternary ammonium compounds, can cause the growth of resistant bacteria. Disinfectants can bind to film or particles on surfaces or to cleaning cloths causing inactivation of the product being used. This problem mostly occurs with sodium hypochlorite, quaternary ammonium compounds, and hydrogen peroxide. Some disinfectants are also known to break down quickly once the bottle they are contained in is opened. Hydrogen peroxide has the shortest life. Others, like quaternary ammonium compounds, will probably last for more than 6 months. Lastly, cost can also be an issue. Most cleaners tested in our experiment were around $0.60-$0.89 per 100 ml. Sodium hypochlorite (Mr. Clean) was the least expensive at $0.38 per 100 ml and hydrogen peroxide (Natura Solutions) was the most expensive at $1.91 per 100 ml. Consumers should also consider these factors when deciding what disinfectant to buy.

     People often want to kill viruses too when considering disinfectants. COVID-19 is a big concern now. It would have also been good to look at the effects of disinfectants on viruses but, in this experiment, we could only test household bacteria as very specialized equipment is required. Unlike bacteria which are cells, viruses are particles of DNA or RNA inside a protein shell called a "capsid". Most viruses also have a fatty membrane that surrounds the capsid. This membrane helps the virus get into host cells as it fuses with the cell membrane. If a virus is enveloped, it can easily be destroyed by disinfectants. The reason is because all the cleaner must do is break down the usually delicate envelope. All types of coronaviruses are enveloped viruses. Strong protein covers on viruses, however, make them a lot harder to kill. An example of a virus that is very hard to kill is Norovirus, which causes a serious gastrointestinal illness. The disinfectants we tested would most likely have some effect on enveloped viruses. When used against viruses, alcohols, acids, aldehydes, alkalis, halogens, and peroxygens are the most effective. Sodium hydroxide is the only cleaner we used that has not been rated as effective for COVID-19 by the EPA. Out of the products used in our tests, sodium hypochlorite is the only one which will work well against non-enveloped viruses like Norovirus. Hydrogen peroxide will have only a partial effect and the rest, no effect at all. Based on our research, all our disinfectants except possibly sodium hydroxide could be used to kill COVID-19 besides working for bacteria but they may not all work for tougher viruses. Governments consider research evidence to decide which disinfectants to certify for bacteria and viruses.

     Although disinfectants are useful when killing harmful microorganisms, it is important to know how to use them correctly. Using too much disinfectant can be damaging as well. Scientists think that exposure to microorganisms when people are young helps reduce the chance of developing allergies later so a "too clean" house may actually be harmful! Some bacteria are good. So, disinfectants should be used only in places that are likely to have pathogen growth such as the kitchen, bathroom, and pet areas and on items used in public such as cellphones and keys. Before using a cleaner, consumers should read and understand the directions (including what surfaces to avoid using it on and time needed for action). They should also clean the surface they plan to disinfect first to remove any film of particles and reduce overall bacteria count, use good ventilation, and keep the cleaner somewhere away from children and pets. As the kitchen is an area where a disinfectant is warranted, we plan to test the products on cultures from the sink. Additionally, we will plan to mimic how a product would be applied to a surface in the home when used properly. 

     There are several testing methods that are approved by Health Canada for disinfectant companies to run tests to get results. The Association of Official Agricultural Chemists (AOAC) Spray Method is approved for testing spray disinfectants. In this procedure, bacterial culture is spread over glass slides, sprayed with the test disinfectant, and treated over the contact time on the label. The slides are then placed in a broth medium to incubate, and the amount of growth is measured after. This would be a good contender for our experiment because it simulates real life.  Another AOAC test is the Use-Dilution test, where the glass slides with bacteria are soaked with a large amount of test disinfectant for a certain contact time and then incubated. This would not be a good test for the sprays, because it would be unrealistic to try to soak surfaces in a home with a huge amount of disinfectant. For example, it would be impossible to soak light switches and faucets handles in disinfectant. Our school mentor showed us different ways to grow bacteria, such as agar and broth, and count the bacteria. To apply bacteria for incubation, a person could use the cotton swab to swab a surface, then gently wipe the cotton tip back and forth on an agar plate once or turn it 45 degrees three times while swabbing after every turn. Because the two experimenters cannot work together due to social distancing, we needed a method that allows us to carry out the experiment separately but be consistent with each other. Although the spray test is the best way to carry out this experiment, our homes may have different humidity levels and slight temperature differences. The sprayed disinfectants may evaporate at different rates in each of our homes and at different times of the day. The Use-Dilution method is not as close to real life situations, but we can control the amount of exposure to our test disinfectants better. Therefore, we will combine the two methods. The chemist at the store where we get our test tubes from told us to incubate no more than about 24 hours because we shouldn't grow too much bacteria at home or it will be harmful to us, so we picked 16 hours. We will be using tryptic soy broth because it is useful to grow and store many types of bacteria, and it is easier for us to combine our cultures together, then divide it for each of us to use, so we would have the same amount of bacteria. Since we are using broth, we are going to use a spectrophotometer to measure the turbidity of the broth to show how much bacteria are in our test samples. Our spectrophotometer (SpectroVis by Vernier) has a rectangular space for a cuvette, with one side of the space containing a light and the other side containing a sensor to measure turbidity. A "Blank" sample is measured first to calibrate the machine. In our experiment, the broth medium that is used will be the blank so that all the readings of the test samples after will ignore the colour and density of the broth and not count it as part of the turbidity. The reading of the blank sample should be zero or very close. An optical density (OD) of 600nm wavelength will be used. The wavelengths of 420 to 600nm is usually used when measuring bacteria. OD600 is a common absorbance number used to measure bacterial content, because it is safely above the spectrum of UV light, which can kill bacteria. The bacteria are not actually absorbing any light, it is scattering light, and that is what is being measured. OD600 is also in the range of orange and yellow light and creates minimal interference with the orange and yellow broth. The more bacterial "biomass" there is in the cuvette, the more light will be scattered, and the measurement will be higher. The less absorbance, the clearer the solution is, and in our case, since we are measuring bacteria amounts, the less bacteria in the solution. We will pick three "conventional" disinfectants and compare their effectiveness with three "environmentally friendly" ones. The common brands are older and the brand names are recognizable. The three conventional cleaners contain active ingredients sodium hypochlorite, quaternary ammonium, and sodium hydroxide. The three "green" disinfectants are ones usually found in organic grocery stores. These products have the active ingredients thymol, citric acid, and hydrogen peroxide. With a variety of products available, we narrowed it down to disinfectants that all have a DIN number, and they all claim to at least kill over 99% of bacteria.

     We are used to seeing our parents use different types of cleaners and disinfectants around the house. In the past year, we have seen a huge increase in disinfectant use around our homes due to the pandemic. We are also hearing more people begin to discuss the health and environmental effects of using so many disinfectants. There are so many brands of disinfectants; how do people know which to choose? We hope that our experiment will help us find out if there is a difference in the effectiveness of the conventional disinfectants and their "greener" counterparts. It would be nice to be able to use a "greener" product but we want something that works. Disinfectants are an essential in the modern home, due to the need to ensure the house is clean and the family will not get sick. We can not use too much disinfectant at a time to ensure the bacteria do not develop resistance. Some bacteria are good. The approval process for disinfectants is very strict, but it is not considered necessary to include all the health or environmental hazards on the labels for buyers to see. Fortunately, many reliable websites can give more background on a diverse number of disinfectants to assist consumers in choosing the right product for them, such as Health Canada, the Environmental Working Group, and the Safer Choices site. We have selected 3 disinfectants that are considered more eco-friendly (thymol, citric acid, and hydrogen peroxide) and plan to investigate whether they are as effective as 3 more traditional products (sodium hypochlorite, sodium hydroxide, and a quaternary ammonium compound). In addition, our test disinfectants come from different classes of chemicals available at the stores. Our disinfectants will be our manipulated variables, and how effective they are in killing bacteria will be our responding variable, as measured in the amount of turbidity with a spectrophotometer, as optical density. Our procedure will reflect how we can implement our experiments in two different homes due to the inability for the two experimenters to get together, and the uncertainty of the school remaining open.


 

 

Variables

Manipulated variable:

The disinfectant used to treat the bacteria-coated surface - sodium hypochlorite, sodium hydroxide, quaternary ammonium chloride, citric acid, thymol, hydrogen peroxide.

 

Responding variable:

The number of bacteria (kitchen sink) on the surface that are killed by the different disinfectants.

 

Controlled variables:

Equipment and materials - tubes, slides, spectrophotometers, swabs, broth

General type of incubator and temperature range during incubation

Location of experiments - used same locations each time (2 home work areas)

Worksurfaces - methods for disinfection/sterilization, templates for slides

Source of bacteria - kitchen sinks, combined into a shared master culture

Source and type of broth used

Amount of broth used in each falcon tube

Methods of sanitizing and disinfecting - time for exposure of bacteria to test disinfectant

Amount of bacterial broth on each slide - used micropipette to apply standard amount

Amount of disinfectant used on slides - used standard size of paper dipped in disinfectant

Surface where bacteria and disinfectants applied - sterilized glass slides

Techniques for handling slides and tubes to minimize contamination - flamed forceps

 

Procedure

Materials:

For each experimenter:

  • 2 sterile cotton swabs
  • 1x1mL vial of sterile normal saline
  • BBQ lighter
  • 250mL Tryptic Soy broth (need 10ml/tube x 20 tubes plus a bit extra for blank calibrating cuvettes). Keep in refrigerator at 4-8 degrees Celsius before use.
  • 19 Falcon plastic 50mL sterile test tubes with caps
  • Marker - for labelling
  • Test tube holder big enough for 19 test tubes (styrofoam block) 
  • Homemade incubator – (GE 40w 330 lumen appliance light bulb, extension cord, light bulb socket with plug, Taylor Indoor/Outdoor Wired thermometer, styrofoam cooler box with lid (1.34 - 1.5cubic feet), duct tape, scissors, ruler)
  • 18 glass slides
  • Pyrex baking pan - for sterilizing slides
  • Baking parchment paper - for sterilizing slides
  • Paper templates, each with five rectangles representing the glass slides, with a 1cm x 2cm portion drawn at one end of each slide pictures
  • Plastic paper protectors - one for each template
  • 2 tin oven liner trays (for a sterilizable work surface)
  • Adjustable micropipette (0.1-10uL)
  • Sterile tips (at least 4) for micropipette
  • Three different test disinfectants (30 ml each)
    • Audrey
      • Method Antibac Disinfecting All-Purpose Cleaner (5.0% citric acid)
      • Seventh Generation Disinfecting Bathroom Cleaner (0.05% thymol)
      • Natura Solutions All-In-One (1.25% Hydrogen Peroxide)
    • Grace
      • Fantastik All-Purpose Cleaner with Bleach (3.0% sodium hypochlorite)
      • Mr. Clean Multi-Surface Disinfectant (0.34% sodium hydroxide)
      • Lysol All-Purpose (Lemon) Cleaner (0.08% alkyl (67% C12, 25% C14, 7% C16, 1% C8-C10-C18) dimethyl benzyl ammonium chlorides, 0.02% alkyl (50% C14, 40% C12, 10% C16) dimethyl benzyl ammonium chlorides)
  • 4 x 125 mL sterilized mason jars
  • No. 4 coffee filters - cut into 12 pieces each measuring 2.5x4cm
  • Scissors - to cut filter pieces
  • Metal forceps - for moving filter papers and slides
  • SpectroVis Plus spectrophotometer (Vernier) - borrowed from school lab
  • LabQuest2 (Vernier) with connecting cable for spectrophotometer - borrowed from school lab
  • 18 Cuvettes for measuring absorbance of samples
  • Containers for safe disposal of materials
  • 1 bottle isopropyl alcohol (minimum 70%) - for sterilizing work surfaces and equipment
  • Alcohol hand sanitizer
  • Home oven - for dry sterilization of slides and metal trays
  • 1 pair sterile gloves - optional
  • Paper and pen/pencil - for recording observations and results
  • Camera - for photographing procedure and results

 

                     Traditional disinfectants tested                      Eco-friendly disinfectants tested

 

                                                               Home incubators

 

                                       Home “labs" with materials and equipment used

 

                      Micropipette - set to draw up 5uL             Spectrophotometer, analyzer, cuvettes
 

Procedure:

 

Main Procedure

We first made an incubator

Procedure

  1. Connect lightbulb to an extension cord in order to reach power source
  2. Dangle the sensor of the thermometer halfway down one side of the cooler 
  3. Tape the thermometer wire down with duct tape a few inches above the temperature sensor
  4. Plug in and turn on lightbulb and thermometer
  5. Use popsicle sticks to lift the lid slightly open
  6. Adjust the opening of the lid to reach the temperature wanted (aimed for 32-35 degrees Celsius as is similar to what is used in lab bacteria incubators)

 

                                                          Home incubators
 

Day 1:

Procedure

  1. Turn on incubator lightbulb to preheat to 32-35 degrees Celsius
  2. Label falcon tubes "Master Broth 1", "Master Broth 2", and "Control"
  3. Write date of test on tubes
  4. Put falcon tubes into test tube holder
  5. Pour 5 ml of broth into each tube and place lid over top to reduce dust contamination
  6. Open saline

Control:

  1. Swirl 1 sterile swab in saline about 6 times
  2. Open tube labeled "Control"
  3. Swirl swab in tube about 6 times and take out
  4. Cap loosely so that if a person lifted the tube by the cap it would not drop but it shakes
  5. Write time on tube
  6. Put tube into small test tube holder

Master Broths:

  1. Swirl 1 sterile swab in saline about 6 times
  2. Thoroughly swab the dirty inside rim of the kitchen sink
  3. Open tube labeled "Master Broth 1"
  4. Swirl swab in tube about 6 times and take out
  5. Cap loosely so that if a person lifted the tube by the cap it would not drop but it shakes
  6. Write time on tube
  7. Put tube into the test tube holder
  8. Repeat with tube labeled "Master Broth 2"

 

                                                     Swabbing sinks and culturing
 

To incubate:

  1. Lower the test tubes in the test tube holder into incubator
  2. Record temperature of incubator after placing test tubes in
  3. Make sure the temperature is about 33 degrees Celsius, or 32-35 degrees Celsius
  4. Wait about 16 hours for bacteria to grow
  5. Check temperature multiple times before going to bed to make sure temperature is stable

 

     Prepared cultures                                              Incubating

 

Day 2:

In Day 2, we sterilized the glass slides by baking them in the oven before using them to for our experiment. And we wiped down our aluminum pans and materials with alcohol to minimize contamination.

Sterilizing Slides by Baking

Procedure

  1. Put parchment paper in pan or glass dish
  2. Put glass slides in a single layer on top of parchment paper
  3. Put pan on middle rack of oven
  4. Turn oven to 325 degrees Fahrenheit on convection bake
  5. When oven has finished preheating, set timer for 2 hours
  6. When timer is up, turn oven off but leave closed for one hour so the glass doesn't crack with the sudden temperature change

                        Baking slides

 

Cleaning the Work Surface and Materials

Procedure

  1. Spray work surface with bleach solution
  2. Wait 5 minutes
  3. Wipe down
  4. Use isopropyl alcohol to wipe down both sides of the aluminum liners 
  5. Let the liners dry

 Prepared slides and templates

 

Procedure for Taking Absorbance for the Control and Master Broth from Day 1

  1. After 16 hours of incubation, take out the test tubes labeled "Control", "Master Broth 1" and Master Broth 2"
  2. Since the experiments are being done separately, the bacterial culture broths will be shared
    1. Each experimenter has 2x5ml Master Broths to mix together (Total of four tubes of 5ml)
    2. All four tubes are mixed together to get 20ml of broth with bacteria
    3. Then the 20ml is divided into 2 tubes of 10ml, one tube for each experimenter to use
  3. Turn on and calibrate spectrophotometer (see instructions at the end below)
  4. Record absorbance of the Control and Master Broth
  5. Put down aluminum liners on the work surface
  6. Label the four spots on each paper template:
    1. Untreated Control
    2. Disinfectant 1, 2, and 3
  7. Put the paper templates inside plastic protectors
  8. Spray plastic protectors with isopropyl alcohol on both sides and wipe down
  9. Put templates on top of aluminum liners
  10. Label the jars 
    1. Disinfectant 1, 2, and 3
  11. Assign the numbers to each brand of test disinfectants
  12. Take the jars and pour each halfway full with the assigned test disinfectant
  13. Close the lids of the jars for about 20 minutes for any foam to settle
  14. Remove the cap from fresh tryptic soy broth
  15. Pour 10ml of fresh broth into each labeled test tube
    1. Controls 1, 2, and 3 - put a clean glass slide in each of these tubes right away because they are just controls with no bacteria or disinfectant exposure
    2. Untreated controls 1, 2, and 3
    3. Tests # 1.1, 1.2, 1.3, 2.1, 2.2, 2.3, 3.1, 3.2, 3.3
      1. The first number is the Trial #, the second number is the number assigned to the corresponding disinfectant 
      2. For example, Test #2.3 will be the second trial, disinfectant number 3
    4. There should be 15 test tubes
    5. Keep the tubes capped when not in use for the trial
  16. Put the rest of the sterilized slides on top of templates according to the outline of the template (4 slides per template)
  17. Use scissors to cut coffee filter paper into nine pieces, measuring about 2.5 x 4 cm each
  18. Place three filter papers into each of the three jars to soak for at least one minute

 

                                     Lab set-up

To do each trial on one template:

  1. Push the micropipette end into a new sterile tip so it's secure
  2. Gently shake the Master Broth so it is cloudy throughout
  3. Dip the micropipette into the broth and withdraw 5.0µl amount
  4. Eject the liquid onto the slide, inside the small rectangle shown on the template
  5. Use the micropipette tip to distribute the liquid over this small rectangular area
  6. Repeat Steps 3 to 6 with the other three slides on the template
  7. Let the liquid culture on the slide dry for about 4 to 5 minutes
  8. Open the lids of the jars of disinfectants
  9. Pick up a piece of disinfectant-soaked filter paper from the first jar with clean forceps
  10. Gently shake off the drips
  11. Place the filter paper on top of the part smeared with bacteria (the small rectangle) of the slide, making sure the filter paper covers the entire part where the bacteria is
  12. Repeat Steps 10 to 13 for the other two test disinfectants and corresponding slides

(The Untreated Control will only have bacteria on it, but no exposure to disinfectants)

  1. Set the timer for 5 minutes
  2. After 5 minutes, clean the forceps with BBQ lighter in between, remove each filter paper
  3. Let the slides dry, about 5 to 10 minutes
  4. Put each slide into the corresponding labeled test tubes
  5. Cap the tubes loosely
  6. Repeat Steps 1 to 19 with the other two Trials on the two other templates

 

       Adding tip to micropipette       Drawing up 5uL of culture      Putting bacteria on slides

    Flaming forceps     Picking up disinfectant-dipped paper     Applying to bacteria on slide

 

Second Incubation:

  1. Place the test tube holder with all 15 test tubes in the incubator preheated to about 33 degrees Celsius (range 32-35 degrees Celsius)
  2. Incubate for 16 hours total

 

                                              Slides in tubes, incubating 

Day 3:

Procedure

  1. After 16 hours of incubation, take out the test tube holder with the 15 tubes
  2. Calibrate the spectrophotometer if not already done
  3. Gently shake the first test tube in case bacteria settled on the bottom
  4. Pour some liquid into a cuvette to about three quarters full, so when you put the cuvette into the slot in the spectrometer, the liquid comes up above the rim
  5. Record the absorbance
  6. Repeat the Steps 3 to 5 for the rest of the test tubes and cuvettes

 

                                                Cultures to be analyzed 

 

 Using the Spectrophotometer to Measure Turbidity

  1. Calibrating the Spectrometer (Spectro Vis attached to the LabQuest2, both from Vernier)
    1. Click on SENSOR menu at the top
    2. Click CALIBRATE
    3. Click USB Spectrometer
    4. Let it warm up
    5. When the meter asks for a blank cuvette, use fresh tryptic soy broth as a BLANK
      1. NOTE: the cuvettes have two opposite clear sides and two ridged sides. Only take the cuvette by the ridged sides because the meter reads the amount of light going through the clear sides
      2. Make sure the liquid comes up above the rim in the slot
    6. Press "Finish Calibration"
    7. Press "OK"
    8. The red box shows the absorbance reading, which should be very close to 0 for the BLANK
  2. Remaining set-up
    1. Select Time-based mode
      1. Tap on Mode box on the right 
      2. Press "Time-based" on the drop-down menu
      3. Press "OK"
    2. Change wavelength to 600nm
      1. Tap on the red box at the middle of the screen
      2. Press "Change Wavelength"
      3. Enter "600 in the box
      4. Press "OK"
    3. Double-check calibration
      1. Put the BLANK back into the slot and make sure absorbance is still 0. 
      2. Repeat calibration steps if absorbance is not 0
    4. Measure Absorbance of the samples
      1. Put the cuvette with sample in the slot
      2. Record absorbance shown in the red box
      3. Before each set of samples from a different trial, put the BLANK back into the slot to make sure absorbance for the BLANK is still 0

                                        

 

Observations

Traditional Disinfectants:

 

Trial 1 (January 10):

 

Quantitative Results

Spectrophotometer Absorbance Readings

Trial

Clean Control

Untreated

Control

Test Disinfectant 1

(sodium hypochlorite)

Test Disinfectant 2

(sodium hydroxide)

Test Disinfectant 3

(quat. ammonium chloride)

1

0.028

0.660

0.022

0.028

0.029

2

0.025

0.724

0.021

0.416

0.030

3

0.021

0.787

0.020

0.023

0.026

 

Qualitative Results

Tube

Observations

Clean Control

All 3 tubes clear, smelled like original broth

Inoculated Control

All 3 tubes very cloudy, no big particles, smelled bad

Test disinfectant 1

All 3 tubes clear, smelled like original broth.

Test disinfectant 2

Tubes 1, 3 clear, smelled like original broth. Tube 2 slightly cloudy and smelled bad.

Test disinfectant 3

All tubes clear, smelled like original broth.

 

Trial 2 (January 17):

 

Quantitative Results

Spectrophotometer Absorbance Readings

Trial

Clean Control

Untreated

Control

Test Disinfectant 1

(sodium hypochlorite)

Test Disinfectant 2

(sodium hydroxide)

Test Disinfectant 3

(quat. ammonium chloride)

1

-0.003

0.726

0.000

-0.006

-0.003

2

0.064

0.745

0.000

-0.005

0.000

3

-0.010

0.830

-0.005

-0.003

-0.007

 

Qualitative Results

Tube

Observations

Clean Control

All smelled like original broth. Tubes 1 and 3 were very clear, tube 2 was very slightly cloudy.

Inoculated Control

All 3 tubes very cloudy, some particle in tubes 1 and 3, smelled very bad.

Test disinfectant 1

All 3 tubes clear, smelled like original broth.

Test disinfectant 2

All 3 tubes clear, smelled like original broth.

Test disinfectant 3

All 3 tubes clear, smelled like original broth.

 

 

Eco-Friendly Disinfectants:

 

Trial 1 (January 24):

 

Quantitative Results

Spectrophotometer Absorbance Readings

Trial

Clean Control

Untreated

Control

Test Disinfectant 1

(thymol)

Test Disinfectant 2

(citric acid)

Test Disinfectant 3

(hydrogen peroxide)

1

-0.004

0.784

0.002

0.006

-0.020

2

-0.005

0.695

0.000

0.550

0.004

3

0.000

0.684

0.004

-0.004

-0.010

 

Qualitative Results

Tube

Observations

Clean Control

All 3 tubes clear, smelled like original broth.

Inoculated Control

All 3 tubes very cloudy and very smelly but had no particles.

Test disinfectant 1

All 3 tubes clear, smelled like original broth.

Test disinfectant 2

Tubes 1 and 3 were clear, smelled like original broth. Tube 2 was cloudy with a slight odour.

Test disinfectant 3

All 3 tubes clear, smelled like original broth.

 

 

 

Trial 2 (January 24):

 

Quantitative Results

Spectrophotometer Absorbance Readings

Trial

Clean Control

Untreated

Control

Test Disinfectant 1

(thymol)

Test Disinfectant 2

(citric acid)            

Test Disinfectant 3

(hydrogen peroxide)                     

1

-0.005

0.651

-0.001

-0.003

-0.002

2

0.000

0.714

-0.003

-0.002

-0.001

3

-0.001

0.630

-0.001

-0.006

-0.007

 

Qualitative Results

Type of tube

Observations

General

Yellow color, a little bit of condensation on the side of each tube

Clean control

The clean controls were all very clear, can see through the tubes to the other side, smelled a little bit musty

Inoculated control 

The inoculated controls were all very muddy looking and very smelly, cloudy, kind of really light pastel yellow

Test Experiment 1

All the tubes were very clear, actually looked slightly clearer than the control, not much smell

Test Experiment 2

All the tubes were very clear, actually looked slightly clearer than the control, not much smell

Test Experiment 3

All the tubes were very clear, actually looked slightly clearer than the control, not much smell

 

 

 

 

 

Analysis

Table 1:

To show the effectiveness of different disinfectants on bacterial growth as measured in optical density

Trial:

1

2

3

4

5

6

Average

Clean Control 1

0.03

0.03

0.02

0

0.06

-0.01

0.02

Untreated Control 1

0.66

0.72

0.79

0.73

0.75

0.83

0.75

Sodium Hypochlorite

(Disinfectant 1)

0.02

0.02

0.02

-0.01

-0.01

0

0.01

Sodium Hydroxide

(Disinfectant 2)

0.03

0.42

0.02

-0.01

-0.01

0

0.01

Quaternary Ammonium Chloride

(Disinfectant 3)

0.03

0.03

0.03

0

0

-0.01

0.02

Clean Control 2

0

-0.01

0

-0.01

0

0

0

*Untreated Control 2

0.78

0.70

0.68

0.65

0.71

0.63

0.69

Thymol

(*Disinfectant 1)

0

0

0

0

0

0

0

Citric Acid

(*Disinfectant 2)

0

0

-0.01

0.01

0.55

0

0.00

Hydrogen Peroxide

(*Disinfectant 3)

0

0

-0.01

-0.2

0

-0.01

-0.04

*There are two sets of Untreated Controls and Disinfectants 1, 2, 3 because the two experimenters were testing a different set of disinfectants separately.

 

Figure 1:

Averaged optical density (bacterial material) with different disinfectants

 

Table 2:

Average percentage of bacteria killed by different disinfectants

Disinfectant

Average % killed

Sodium Hypochlorite

98.89

Sodium Hydroxide

99.20

Quaternary Ammonium Chloride

98.22

Thymol

100

Citric Acid

100

Hydrogen Peroxide

105.31

 

Figure 2:

Graphing the averaged percentage of bacteria killed by different disinfectants

 

Our experiment studied the relationship between the main active ingredient in disinfectants and the number of bacteria killed by the tested disinfectants. We tested this by incubating a master broth, smearing sterile glass slides with the mixed master broth, and applying disinfectant to the slides using a coffee filter. We then put the slides into clean broth and the tubes holding the slides into our homemade incubators. After the broths had incubated for sixteen hours, we measured the turbidity of the broths with optical density of 600 nanometers. The numbers that showed in the spectrophotometer have three decimal points; however, the last digit fluctuates, so it was decided to round to the second decimal point. Sometimes the measurement showed a negative number. It could be due to the third digit fluctuating or that the dried disinfectant on the glass slide then diluting the broth slightly when it dissolved again during the incubation process. There could be other minor ingredients in the solutions that are not listed. These can also mix with the broth and affect the scattering of light. These reasons could have caused the hydrogen peroxide percentage of bacteria killed to come out to 105.31% ([0.69-(-0.04)] / 0.69 x 100). From these results, it means that all the disinfectants are equally as effective in killing germs from the rim of the kitchen sink. The results from six trials are shown. As a comparison in our tables and graphs, the measurements for the Untreated Control are included. In Table 1, Because the Master Broths were mixed then split into two for each of the experimenters to use as their controlled bacteria source, measurements of Untreated Controls from both experimenters are used to calculate the average for Table 1. The measured OD for the Untreated Control for one experiment was 0.75 who tested the conventional disinfectants. The OD was 0.69 for the Untreated Control for the experimenter who tested the "eco-friendly" options, giving the average of 0.72 in the graph in Figure 1. All our turbidity measurements for the disinfectants are close to zero. Figure 1 shows how much bacteria material is left after being treated with disinfectants compared to the slide that was coated with bacteria but untreated. Table 2 is another way of showing the effectiveness of the disinfectants in killing bacteria. It is calculated as the percentage of bacteria killed. Each percentage is calculated by subtracting the OD of each treated result from the untreated measurement, then divided by untreated, multiplied by one hundred:

                                                    (untreated-treated)/untreated x 100

 

 

 

Conclusion

     We discovered that all six of the disinfectants we tested worked equally well and killed all bacteria we cultured from our kitchen sinks when we used an application technique that mimics what people would do at home. A five-minute exposure time was adequate and is likely close how long a cleaner would stay wet on a surface in normal use at home. Our hypothesis was incorrect as we initially thought that our eco-friendly disinfectants (thymol, citric acid, and hydrogen peroxide) would not be as effective as the traditional products (sodium hypochlorite, sodium hydroxide, and a quaternary ammonium compound). Knowing this information, consumers should feel comfortable that any of these disinfectants are effective for their kitchens and can then consider other factors when picking a product. Some other things to consider are the human health and environment impacts when they are produced and when they are used. While most of the cleaners are similarly priced, “green” cleaners can sometimes be more expensive. Also, consumers should keep in mind that certain chemicals can create toxic products when accidentally mixed with other chemicals in the home, cause damage to delicate surfaces, and may be inactivated under some conditions. Not all disinfectants will be effective for all types of pathogens. It turns out that all our study disinfectants except sodium hydroxide are rated as being effective for COVID too. If very resistant viruses (like Norovirus) or microorganisms are a concern, sodium hypochlorite is the most likely to be effective for the widest variety of pathogens. However, we do not think bleach is needed for the most common home cleaning situations. It is especially important that consumers know where to find information on the disinfectants they are planning to use, including learning how to read labels and product ingredients. Unfortunately, the labels do not always contain all the information needed on safety and toxicity so consumers also must look at on-line resources too. After considering the results of our experiment and our research on the safety profiles, costs, and other concerns, we have chosen to use citric acid, thymol, or hydrogen peroxide in our own homes.

Application

     Unnecessary overuse of disinfectants can result in excess chemicals in the environment which can be damaging to humans and theoretically the environment. This may also make bacteria more resistant to disinfectants and antibiotics. More "natural" products may be more expensive, but in the long run, damaging our environment and physical health cost more. One crucial aspect of disinfectants is that they help prevent disease. This is especially important in schools, medical facilities, restaurants, and various public places because they are most subject to infection spread. This makes it difficult for these places to trust environmentally friendly products because the products may be unknown to them. Most medical facilities stick with more trusted conventional brands, but if they test more environmentally friendly kinds, they will find that they are all equally effective and they may be more open to other brands. The pandemic has brought more attention to cleaning and disinfecting, resulting in higher use of these products, and the importance of understanding the pros and cons of each are vital in our current situation and beyond. Interest levels have shifted to the environment recently, and people wonder whether it is worth the cost to buy the "greener" disinfectants. Eco-friendly products are just as effective as conventional disinfectants and may be less harmful to the human body than processed disinfectants. Schools are, as mentioned earlier, one of the areas of high contact. Many students and teachers are exposed to these disinfectants. Some chemicals have been proven to cause developmental problems and disruption to the hormone systems of the body, which is problematic for students who spend the majority of their time in these schools. Pregnant teachers are also exposed to these fumes, which increase the probability of birth defects. Some ingredients in disinfectants can also trigger or worsen asthma, allergies, skin problems, and sensitivity. Hospitals and senior's homes are two of the largest disinfectant users, where people are already suffering from illnesses. The ability for these organizations to trust and use a less toxic product can decrease extra workload from the health care system. Consumers need to inform their government representatives to make regulations stricter and make information more accessible, like having more safety information on the packaging instead just making the labels look like the product is "reliable" or more "green." Currently, choosing a new chemical to trust is difficult due to the pandemic, and it is easier to choose a disinfectant that they have been using for years rather than a new disinfectant. Effectiveness of a disinfectant is also affected by how it is applied. It is most likely best to just follow package instructions. Consumers need to be better educated in how to make good disinfectant choices and which choices are the least toxic. Buyers also need to do their research on factors like costs, advantages, disadvantages, and medical issues. 

 

Sources Of Error

     During our experiment, we had some potential sources of error including issues with accidental contamination, spectrophotometer use, and aspects of applying the disinfectants and culturing. Although we tried our hardest to avoid accidental contamination, it was possible we did have some. We sterilized the forceps and tops of the tubes and broth bottles with a flame, baked the slides and trays, and treated the templates with isopropyl alcohol but they could have become contaminated despite our best efforts. It was difficult to keep a firm grip on the slides with forceps while moving them and some did drop back onto the templates. It is unlikely that the broth or slides were not sterile to start as the control group did not grow much. We could have accidentally touched the tube edges and slides during the procedure. Secondly, we had a few problems with the spectrophotometer. It did need careful calibration. In one trial, we noted in retrospect that the spectrophotometer was reading 0.025 too high when supposedly zeroed suggesting all our readings were off by this amount although it was not enough to alter the averages. In later trials, we paid extra close attention to recalibrating often to reduce this risk. Also, one hydrogen peroxide treated slide culture had an absorbance reading that was an unusually significant negative number. As a result, it gave a calculated 105% average success rate in killing bacteria for the hydrogen peroxide group. A negative absorbance reading could have arisen by an error in calibration or if chemical left over from the dried disinfectant got in the broth and caused refracting of the light or fluorescence. It is also possible to introduce error in the absorbance readings by not filling the cuvette enough so the light does not shine through the broth, getting fingerprints on the cuvette, or placing the cuvette into the chamber in the wrong orientation. However, we were very careful and are quite sure that we did not have these issues with cuvettes. Using absorbance to "count" bacteria only gives an estimate of the number of bacteria in the broth. Larger bacteria will give a higher absorbance than smaller ones even if there are the same number. Although we let the residual disinfectant on the slides dry before placing the slides into the tubes, it was possible some active chemical was still present that continued to kill bacteria in the broth. Researchers working on testing disinfectants do sometimes add neutralizers to stop further action of the disinfectants in culture but we did not have access to these agents. When applying the disinfectant-dipped papers, we could have wiped off bacteria or washed them under the slides. However, we felt that dipping the slides into disinfectant could wash off bacteria too and using a spray technique would have been difficult to do uniformly. Culturing was another part of the procedure where error could have occurred. When making our master culture, we could have also grown fungi which may not have responded the same way as bacteria when exposed to disinfectants. In the incubator, some parts might have been warmer or cooler than others as the tubes varied in distance from the light bulb. We also did not agitate the test tubes so there is a chance that the bacteria did not get sufficient oxygen.  As we did do an initial trial and still got good growth up to 32 hours (the absorbance continued to increase) without agitation, we feel that the bacteria likely had adequate conditions over the 16 hours of culture. Since we needed to do our experiment in two different labs (homes), it was hard to use absolutely identical techniques although we discussed how we were going to carry out the tests before and used a written procedure for both. It is indeed a challenge to work with bacteria in our home labs.

     If we could have done more in this experiment, we would have liked to test different disinfectants than what we used (like glycolic acid and other types of quats). We could also have tried longer and shorter exposure times (by varying the amount of time the papers coated in disinfectant stayed on the slides). It would have been nice to test different dilutions of the disinfectants as well to see if we could tell which are more effective (whichever works the best at the greatest dilution) or use a different testing method. Different testing methods include adding a disinfectant to the liquid culture then plating on agar to see which produced the lowest colony count. Another option would be to do the lawn on an agar plate and measure the zone of inhibition around the filter paper dipped in disinfectant. We could also have tried doing the experiment using a known bacterial culture. This was quite hard though as we were not able to use pathogenic bacteria in our tests. The last thing we could have done differently was creating our master culture from swabs taken in other areas in our homes which might have given us a different mix of bacteria (like the bathroom, shower, faucet, or toothbrush holder, or refrigerator) to see if our results would also be the same for more areas. Bacteria are fascinating to study as there are so may ideas that could be tested.

 


 

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Acknowledgement

We would like to acknowledge the following people and organizations for all the help with our project:

Beatriz Garcia-Diaz, B.Sc., Ph.D., Senior School Lab Coordinator, Webber Academy - advice, supplies and equipment

Danielle Grelowski, B.Sc., B.Ed., Junior High Science Teacher, Webber Academy - advice

Dalynn Biologicals - supplies and advice on bacterial culturing

Doug McFadyen, HypoIndustries Ltd. - clarification of Health Canada regulations

Steven Sawchuk, Ph.D, M.Sc., B.Sc. - for explanation of optical density

Our parents - advice and purchasing of supplies and equipment, helping with recording for the presentation