If 6 variables (milk, probiotic capsule, yogurt, probiotic yogurt, kefir, and buttermilk) are compared, the probiotic capsule is the most beneficial to gastrointestinal and immune health because it retains the greatest abundance and variety of bacteria. Therefore, if the variables are grown on the same medium as oral bacteria (opportunistic pathogens), the oral bacteria’s growth will be inhibited, and the probiotic capsule will have the STRONGEST inhibition effect out of the 6 variables, due to the strength of its antimicrobial properties and abundance of probiotic species. 

 

Testable Questions

1. Which of 6 variables contains the most bacteria that are beneficial to gastrointestinal health? In other words, which variable is the “healthiest”?
2. What is the relationship between probiotic bacteria and oral bacteria when grown on the same medium? Do probiotics truly support immunity against pathogenic bacteria?
3. If the probiotic bacteria kill oral bacteria, which variable has the strongest inhibiting effect on the oral bacteria? In other words, which variable contains the probiotics that are most effective at supporting immunity?

Purpose: To determine the role probiotics play in supporting human health, specifically gastrointestinal function and immunity against opportunistic pathogens, and which of 6 variables, a dietary supplement and 5 dairy products, are the most effective in providing these benefits of probiotics.

 

My research focuses on…

*The differences between the 6 variables (pasteurized milk, buttermilk, probiotic yogurt, non-probiotic yogurt, probiotic pill, and kefir) to predict which would contain the most amount of probiotics 

*The types of probiotics that may be found in these variables, and how to identify them during the experiment

*Why probiotics are important to humans, and what occurs in the case of a lack or imbalance of them

*General bacteria found in human flora, some of which may be pathogenic

 

Common microorganisms beneficial to gastrointestinal health *These are bacteria that may be observed in samples of the variables

Bacteroidetes phylum: Gram-negative. Found in GI tract, metabolize fiber and polyphenols, extract energy from food

Firmicutes phylum: Gram-positive. Have similar functions to bacteroidetes, however, they make more efficient use of calories (extract more energy from food), so having more firmicutes than bacteroidetes in gut may lead to metabolic disorders (such as obesity, IBD, diabetes, etc)

Lactobacillus genus: Gram-positive, found in GI tract, mouth, skin, and dairy products. Most common bacteria in kefir. 

Bifidobacterium genus: Gram-positive, found in GI tract

Actinomyces genus: Gram-positive. Found in respiratory system, GI tract, on skin, and mouth

Pseudomonadota phylum: Gram-negative. Found in the GI tract. Can be beneficial, but some species can be infectious.
Saccharomyces genus: Gram-positive. A genus of yeast found in fermented foods such as kefir, sourdough, fruits, etc.

….in addition to many more. These are the most common.

 

Common oral and dermal microorganisms *These are bacteria that may be observed in the salival samples

Lactobacillus genus: Gram-positive, found in GI tract, mouth, skin, and dairy products. 

Streptococcus genus: Gram-positive cocci bacteria, found on skin and mouth (s. salivarius). May be opportunistic pathogens.

Granulicatella genus: Gram-positive cocci bacteria, found in mouth, GI tract, respiratory system

Prevotella genus: Gram-negative, primarily found in mouth, also in GI tract

Staphylococcus epidermis: Gram-positive species, cocci bacteria, opportunistic pathogen (can cause endocarditis, cellulitis, meningitis, and many more). Skin and nose

Staphylococcus aureus: Pathogenic, found on skin and in nose, can cause skin infections, sepsis, and respiratory illnesses 

Corynebacterium genus: Gram-positive, opportunistic pathogens, found in mouth and on skin. A common species is called corynebacterium diphtheriae, which causes the respiratory infection diphtheria

….in addition to many more. These are the most common.  

 

Microorganism Identification

Factors that will affect growth of a microorganism: temperature, the medium it is grown on, light, supplement of gasses (CO2, O2), potential of hydrogen, osmosis, and moisture of environment 

→this will affect what ends up growing on the agar plate, and what doesn’t. As the purpose of this experiment is to identify probiotics and oral bacteria, I have chosen selective and differential media that promote the growth of certain microorganisms if they are present in a sample.

Characteristics for identification: 

• Structure (shape, size, spores, capsules, flagella, uni. or multicellular organism)
  • Sphere-like / round shape: given the name “cocci”
  • Rod-like shape: given the name “bacilli”
  • Bacteria that come in chains: given the name “strepto”. Eg; streptococcus, a chain of sphere-like bacteria
  • Come in grape-like clusters: given the name “staphylo”. eg; staphylobacillus, clusters of rod-like bacteria
  • Come in pairs: given the name “diplo”. eg; diplococci, pairs of cocci
• Presence of mycolic acids
• Reaction to gram stains (positive or negative)
• Selective presence on certain media (what nutrients does this organism thrive off of?)
• Color and shape of colonies (flat or mucoid)
• Effect on the medium, such as in biochemical tests (eg; may indicate acidity, fermentation, or hemolysis)
  • Alpha (α) Hemolysis: Greenish discoloration around colonies caused by partial hemoglobin degradation.
  • Beta (β) Hemolysis: Clear, transparent zones around colonies due to complete lysis of red blood cells.
  • Gamma (γ) Hemolysis: No hemolysis or discoloration around colonies.

 

Strategies:

→Agar Plates: Petri dishes that contain nutrient agar as a medium for microbial growth. Agar is a substance that allows for growth of nutrients, which microorganisms feed off of, however agar itself is not digestible by these organisms. The media used in an agar plate can be selective or differential. Selective media will allow for the growth of one specific organism, while differential media allows for many types of similar bacteria to grow and tells them apart based on growth patterns (eg; varying species of a genus will change the medium’s color in different ways). A medium can be both selective and differential. 

 

→Gram Stains: A technique that can classify and identify bacteria based on their cell well. Cells are stained by being smeared with dyes such as methylene blue or safranin, and whether or not the cell wall absorbs these dyes classifies them as gram-positive (purple/blue) or gram-negative (pink/red). 

Gram-Positive Bacteria

• Thick peptidoglycan layer in cell walls retains and holds onto violet dye better. Appears purple or blue when stained
• Simpler cell wall
• Less resistant to antibiotics, making them easier to treat against. However, they can still be problematic to health (necrotizing fasciitis, sepsis, toxic shock syndrome, etc.)

Gram-Negative Bacteria

• Thin peptidoglycan layer
• Outer membrane makes cell wall more complex
• Complex membrane doesn’t allow violet dye to bind, and the thin peptidoglycan layer is not thick enough to trap dye. Appears pink or red when stained
• Outer membrane makes bacteria more resistant to antibiotics, so they are harder to treat

→Catalase test: Can determine if a bacteria contains catalase enzymes by placing drops of hydrogen peroxide onto a sample of bacteria. If bacteria has catalase enzyme, a positive reaction will take place and bubbles will form, as decomposition is occuring. H2O2 → O2 + H2O

→Phenol red broth, glucose, lactose and sucrose tests: Can identify a bacteria based on how well it ferments a carbohydrate in the broth and how it changes the color of the broth. Fermentation releases acids, causing it to turn a yellow color. 

→Mannitol Salt Agar test: Specifically supports the growth of Staphylococcus, as it is the only bacteria that can withstand the salinity of MSA and ferment it. Works the same way as the other fermentation tests.

→Simmons Citrate Agar slant test: Bacteria that can utilize citrate as a carbon source produce citrate-permease, a membrane protein, to transport citrate into the cell. (Citrate is used by cells for energy and metabolic processes). These bacteria convert citrate into alkaline sodium carbonate, which raises the pH, and turns SCA agar blue. (This color change classifies organisms as citrate-positive.)

 

Characteristics of microorganisms predicted to be observed:

• Lactobacilli: Gram-positive, long, slender rods, non-spore forming, can occur in chains
• Bacteroidetes: gram-negative, rod-shaped, non-spore forming
• Firmicutes: gram-positive, can be spherical or rod-shaped, can be spore-forming and not
• Staphylococci: gram-positive, spherical, come in clusters, non-spore forming
• Bifidobacterium: gram-positive, rod structure and Y or V shaped, non-spore forming, come in clusters, chains, pairs, or individually
• Saccharomyces: gram-positive, spherical or egg shaped
• Streptococci: gram-positive, spherical, come in pairs or chains, non-spore forming

 

Yeasts VS Bacteria

Yeasts 

• Eukaryotic, making them complex organisms (organelles)
• Reproduce sexually and asexually
• Larger
• Fungi kingdom

Bacteria

• Prokaryotic, making them simpler organisms (no organelles)
• Reproduce asexually
• Smaller
• Bacteria kingdom

*Yeasts are A TYPE of fungi. Fungi can also be molds. Yeasts are unicellular, solitary cells that reproduce by budding.

What is the significance of probiotics, and what risks are humans posed with if there is a lack of probiotics in the body?

→Probiotics strengthen the immune system. Antimicrobial properties such as peptides and lactic acids. 

• By releasing toxic peptides called bacteriocins, probiotics kill harmful bacteria by inhibiting their growth and not allowing the pathogenic bacteria's cell to form. (Disrupts the functions of the cell membrane.)
• Lactic acids of probiotics lower the pH (therefore creating acidic conditions) and disrupting the functions of the pathogenic bacteria, killing it.

→Support general health. Probiotics promote digestion, metabolism, and nutrient intake. And, as mentioned above, strengthen the immune system. 

→ Dysbiosis is an imbalance between the microorganisms of the gut, caused by dietary changes, intake of antibiotics, and stress. This decrease in microbial diversity can lead to…

• Obesity
• Inflammatory Bowel Disease (IBD): overactive immune system begins attacking the healthy gut cells
• Cancers, including colorectal cancer: linked to alcohol, unhealthy eating, inactivity, etc.
• Bacterial gastroenteritis: bacterial infection caused by inflammation
• Irritable Bowel Syndrome (IBS): pain, gas, bloating, etc.
• Diabetes

 

→Streptococcal Infections cause skin and throat infections, group A streptococcus usually spread through skin injuries and transfer of oral fluids (coughing, sneezing, sharing food) and group B streptococcus through birth

 

GAS (strep. A)

• Found in skin and throat
• Mostly affects children
• Causes strep throat (swollen/itchy throat), necrotizing fasciitis, impetigo (skin infection), etc.

 

GBS (strep. B)

• Found in GI tract and vagina
• Mostly affects pregnant women, newborns, and people with chronic infections
• Causes UTIs, sepsis, pneumonia, meningitis, etc. 

 

Weight Loss Study from the European Journal of Nutrition

In this trail, a group of women was divided into 3 groups, and put on a diet plan. The control group received 2 servings a day of low-fat dairy products, women in the milk group received 4 servings of milk a day, and the kefir group received 4 servings of kefir a day. The result was that the milk and kefir groups had lost a significantly greater amount of weight after 8 weeks than the control group. There were no differences between the milk and kefir groups. 

It is seen that milk and kefir provide the same benefits in weight loss. This cannot be related to probiotic content, since milk does not contain probiotics, but kefir is rich in them. How did they both encourage weight loss? After some research, I found an answer to this question. 

• Calcium is present in milk, and calcium converts energy (calories) to heat instead of storing it as fat. The more dairy products consumed, the body knows that there is no need to store more fat. 
• Calcitriol is a form of Vitamin D that extracts calcium from dairy, and we know that calcium promotes weight loss through thermogenesis. If there is not enough calcium being consumed, the body creates more calcitriol, and calcitriol produces fat cells. If an individual consumes more calcium, the body will create less calcitriol, meaning it creates less fat.
• In other words, more calcium → less calcitriol → less fat.

*This was proved by a study led by Micheal Zemel, PhD.

Fibromyalgia and bacteria in the gut

Research done by McGill University, University of Montreal, and the Institute for Pain Medicine at Rambam Health Care Campus proved that chronic pain is related to the concentration of certain bacteria and bile acids in the GI system. Bile acids metabolize and absorb fat. Out of the women involved in the study, the healthier women had 5 times more alpha-muricholic acid (a type of bile acid) present in their gut. The study also involved mice, and the final result proved that the presence and absence of certain microorganisms determine the symptoms of fibromyalgia that an individual will experience. 

→The most effective way to prevent the negative outcomes of microbial imbalances is to eat healthy, and dairy products (as well as dietary supplements) are essential to maintaining a healthy gut microbiome. This leads us to the first testable question: “Which of 6 variables contains the most bacteria that are beneficial to gastrointestinal health?”

The 6 Variables

*To answer the first testable question (which is healthiest) I compared the positives and negatives of the variables.                                  

Variable Name	Pasteurized Milk (Control)	Buttermilk	Kefir	Probiotic Yogurt	Non-probiotic Yogurt	Probiotic
Description	Valley Pride Organic Milk, 2% milk fat	Dairyland Light Buttermilk, 1% milk fat	President’s Choice Plain Kefir, 1% milk fat	Activia Plain Probiotic Yogurt, 3.2% milk fat (No added flavors or sugar.)	President’s Choice Plain Greek Yogurt, 0% milk fat (No added flavors or sugar.)	Jamieson Probiotic 10 Billion Active Cells. Powder from the capsule was mixed with nutrient broth.
Positives	+High in calcium, vitamin D, B12, protein, zinc, etc +Nutrients, bone health, etc.	+Vitamins, calcium, protein, etc +Less fat/calories than pasteurized milk +Digestion, nutrients, bone health, etc. +Immune system support (probiotics kill “bad” bacteria)	+Digestion, nutrients, bone health, etc. +Vitamins, calcium, protein, etc +Immune system support (probiotics kill “bad” bacteria)	+Immune system support (probiotics kill “bad” bacteria) +Supports growth of blood cells +Vitamins, calcium, zinc, proteins, etc	+Digestion, nutrients, bone health, etc. +Vitamins, calcium, protein, etc +Less fats	+Offers a variety of microorganisms, and contains a high concentration in a small pill +Longer shelf life than food products +Zero calorie +Consistent intake of specific strains of microorganisms for every pill
Negatives	-No probiotics or bacterial cultures -Fats, cholesterol, and lipoproteins (weight gain, blockage of arteries)	-Lactose and probiotics cause gas, bloating, etc. -(Usually) has fewer probiotics than kefir	-Lactose and probiotics cause gas, bloating, etc. -May contain a little alcohol due to long fermentation	-Lipoproteins and saturated fats -Fewer probiotics than kefir -Gas, bloating, etc	-Contains bacterial cultures, however NO probiotics → not as much benefit to gut	-Doesn’t offer the additional nutrients that food does -Less sustainable (packaging, production) -Absorption not as strong, as pills lose potency and sometimes die due to acidity of stomach

*Kefir is classified as the dairy product with the most amount of probiotics due to several factors.

• It is made with more species of bacteria and yeast
• It has a long fermentation time, allowing for more growth of probiotics
• The yeasts in kefir protect bacteria, ensuring more survival

(This will most likely be what I discover when I do my experiment - kefir sample will yield the most beneficial microorganisms.)

Extra notes;

*Professor Tim Spector (King’s College London) says that “having small amounts of kefir daily rather than a large amount once in a while is the best way to benefit from fermented foods.”

*Buttermilk is made from adding probiotics to the leftover milk from the butter-making process.

*Kefir is made when kefir grains and milk ferment together. Kefir grains are made of bacteria and yeasts

 

Notable Vocabulary

Polyphenols: organic compounds from plants that have more than one hydroxyl group, and are antioxidants

Fermentation: the process of glucose/carbohydrates being broken down by the enzymes of microorganisms into gases, acids, and ethanol (alcohol). Also called anaerobic respiration.

Enzymes: catalysts, are proteins that speed up metabolism and break down food

Anaerobically: without oxygen. Fermentation occurs when oxygen is not present.

Lactose: the sugar found in milk products

Bacterial phyla: major, broad taxonomic groups of bacteria that have distinct genetic features

Genus: more specific taxonomic group of related species of bacteria

Firmicutes/Bacteroidetes Ratio: ratio of the two most dominant bacteria in the gut

-Provide energy for the host through the fermentation of carbs and fiber to create short-chain fatty acids. Energy produced by SCFAs is used by the cells lining the GI tract

-A high F/B ratio may result in a variety of metabolic disorders. This is because firmicutes make a more efficient use of calories from food, which leads to obesity, as one example of health issues

-This ratio depends on testosterone/estrogen levels, diet, activity, and intake of medication

Short-chain fatty acids: group of fatty acids produced when bacteria in GI tract ferment carbs and fiber. SCFAs are absorbed by colonocytes, which line the large intestine. They enter the bloodstream, and assist in protein building and function of the immune system.

Colonocytes: cells that make up the epithelium 

Epithelium: thin layer of cells that line the hollow organs

Lipoprotein Lipase: enzyme that breaks down triglycerides which are stored as fat, into energy (this energy is absorbed by intestines)

Lipoproteins: transport cholesterol through bloodstream and may lead to blockage of arteries if there is an excess

Triglycerides: a type of fat found in blood

Fibromyalgia: a chronic pain and fatigue syndrome, mostly occurring in women due to hormonal changes

Cholesterol: waxy substance that assists in digestion and cell-building, however an excess develops fatty deposits in blood vessels

Bile acids: metabolize and absorb fat, found in liver and GI tract

Opportunistic pathogens: bacteria that usually do not cause harm, but if a hosts’ immune system is weakened they cause an infection

HDL: high-density lipoprotein is considered healthier due to the fact it transports cholesterol to the liver to break it down, removing it from the body

LDL: low-density lipoprotein is considered unhealthy because large amounts of it will result in the blockage of arteries, due to the fact it carries cholesterol

Spores: cells that protect the bacterium, most commonly produced by gram-positive bacteria. Spores have thick walls that protect against environmental conditions

Capsules: outermost layer of the cell, made mainly of polysaccharides. Not all bacteria have capsules, but those that do often cause diseases

Polysaccharides: chains of carbohydrates

Mycolic acids: Long-chain fatty acids found in some cell walls - help bacteria thrive and may cause diseases

Peptidoglycan: a polysaccharide that makes up the cell wall

Catalase enzyme: enzyme found in organisms that breaks down hydrogen peroxide into water and oxygen

Standard deviation: measurement that shows how far different points in a dataset are from the average of that set. If data points are further from average, there is higher deviation in that dataset. 

Hemolysis: the process of the destruction of red blood cells. (Can be observed on blood agar as green/brown color → to identify the bacteria present, such as streptococcus.)

Lysis: breakdown of a cell due to damage to its membrane

Serial dilution: a series of dilution where the dilution factor stays the same for each step

Strain: a subtype of bacteria that has unique features/characteristics from others. “Strain” can be used to refer to a bacteria that is unidentified.

Halotolerant: Ability of an organism to survive in extremely salty conditions

 

 

PART ONE - Preparation and growth of cultures 

Controlled: 

1. Pasteurized milk (does not contain probiotics)
2. Probiotic pill (not a natural source of probiotics like dairy) 

Manipulated / Independent: 

1. 5 dairy products
2. The oral bacterial culture (swab sample)
3. 6 types of agar that will be used to grow cultures
4. Dilution / difference in concentration of variables
5. The different time/conditions in which different types of agar will be grown for

Responding / Dependant: 

1. Growth or inhibition of oral bacteria in presence of probiotic and dairy products
2. The variety, abundance, and concentration of bacterial growth on agar plates depending on variable and selectivity of media.
3.  Hemolysis and / or discoloration of agar 

 

PART TWO - Well diffusion method and relationship between variables and oral bacteria

Controlled: 

1. Milk, saline solution, and liquid MRS agar (do not contain probiotics, meaning they should NOT inhibit oral bacterial growth)
2. Diameter of wells, which are 5mm
3. Mueller-Hinton and Mitis-Salivarius are the only mediums used for this experiment

Manipulated / Independent: 

1. The 5 probiotic-containing variables
2. The 3 types of agar from which strains are extracted
3. The concentration of independent variables → diluted with MRS broth
4. The amount of time the cultures will grow for and the conditions they will be grown in

Responding / Dependant: Growth or inhibition of oral bacteria around the well, measured by diameter. Differences in diameter indicate the strength / abundance of probiotics in a variable, affecting the relationship with oral bacteria.

 

PART THREE - Identification of strains 

Gram Stain and Biochemical Tests

Controlled: (Biochemical tests) Test tubes that do not contain culture / only contain medium

Manipulated / Independent: 

1. The amount of time the cultures will grow for and the conditions they will be grown in
2. The process of collection of bacterial smears diagnostic broths V.S. agar media
3. Different types of media (4 sugars, SCA agar, MCA broth, MSA broth)

Responding / Dependant: 

1. Cell’s retention of stain, positive or negative
2. Change in color of medium, indicating the bacteria’s ability to ferment and/or utilize citrate as carbon source
3. Presence or absence of bubbles, indicating if bacteria has a catalase enzyme or not

Materials

• Pre-prepared agar plates, with 8 types of media; Blood Agar, MCA (MacConkey agar), MSA (Mannitol Salt Agar), MSL (Mitis-Salivarius Agar, blue one), MRS agar (De Man-Rogosa-Sharpe Agar), Nutrient Agar, Mueller-Hinton agar, and Simmons Citrate Agar (SCA). 
  • Though “MSL” is not the correct abbreviation for Mitis-Salivarius Agar, it is the name I’ve given it to differentiate it from Mannitol Salt Agar.
• The 6 variables → 5 diary and a probiotic capsule
  • President’s Choice Plain Kefir, 1% M.F.
  • Dairyland Light Buttermilk, 1% M.F.
  • Valley Pride Organic Milk, 2% M.F.   (CONTROL, does not contain any bacteria)
  • Activia Plain Probiotic Yogurt, 3.2% M.F.
  • President’s Choice Plain Greek Yogurt, 0% M.F. (no probiotics added, therefore “non-probiotic yogurt”. Still DOES contain bacterial cultures.)
  • 1 pill of Jamieson Probiotic 10 Billion Active Cells mixed with 10mL of MRS broth (Each capsule contains 10 billion cells)

Image 1: strains of probiotic found in one capsule of Jamieson Probiotic

Image 2: Probiotic content of the variable "probiotic yogurt"

Image 3: The 5 dairy products (probiotic variable not included here)

Image 4: Dairy products in sterile containers for transportation to lab

   

           

                   

• Hydrogen peroxide (H2O2)
• Saline solution 
• Nutrient broth 
• Mechanical pipette and sterile pipette tips
• Sterile containers for product storage
• Bunsen burner
• Glass test tubes + test tube rack
• Anaerobic jar and sachet 
• Glass cell spreader and inoculating loop
• Sterile cotton swabs
• Plastic or glass microscope slides
• Wax pencil
• 95% ethanol, crystal violet dye, Gram’s iodine solution, safranin, and water
• Lab coat, sterile gloves, safety goggles
• Autoclave
• 6.5% NaCl (salt) nutrient broth, 15% NaCl broth

Instructions

*I did not brush my teeth the morning of experiment in order to preserve as many oral bacteria as possible

*Product storage (for transportation to lab): use freshly bought products, pour into separate sterile cups, filling them about halfway. Close the lid immediately after pouring the product in to prevent contamination. 

 

PART ONE - Preparation and growth of cultures

1. Prepare probiotic by cutting open a capsule and mixing it with 10mL of nutrient broth in a test tube.
2. Pipette 1mL of each of the 6 variables (products) into test tubes, using a mechanical pipette.
3. Swab one side of the mouth with 3 sterile cotton swabs, and repeat on the other side of the mouth. Swab around gums, tongue, and top of mouth. (Total of 6 swabs) Avoid touching other surfaces with the swab. Using one swab for each tube, mix it into a test tube containing variables (products). Let test tubes sit for 1 hour. 
4. After letting the 6 variables (introduced to oral swab samples) sit for 1 hour, perform serial dilution in test tubes.
   1. Dilute the 1mL of variable with 9mL of nutrient broth (1:10 ratio / 10^-10) 
   2. Repeat several more times to create samples down to ratio 10^-7 to provide a clear view of colonies when grown. (Otherwise the growth will be too concentrated). 
5. *(36 plates) On 6 different media (blood agar, MCA, MSA, nutrient agar, MRS, MSL)
   1. Pipette samples of the diluted 6 variables onto 6 types of agar plates and use a glass cell spreader to spread it over the plate. 
   2. Sterilize the spreader with ethanol and then Bunsen burner between each sample. 
   3. Change pipette tip between each sample
6. Swab both sides of the mouth again, around the gums, tongue, and top of mouth. Avoid touching other surfaces with the swab. After sampling, immediately place the swab into a 1 mL sterile nutrient broth. This suspension will preserve microbial viability. Mix the swab content in the broth.
7. Using new swabs, soak them in the suspension; then swab diagnostic plates (MSA, MCA, Blood Agar, MSL), rolling the swab on one corner of the plate. Using the sterile loop, perform the streak plate technique:
   1. Spread bacterial sample from the corner where the bacterial sample was rolled to the next corner. 
   2. Sterilize the inoculating loop with a Bunsen burner before repeating; streak from second corner to third, sterilize, and steak from third corner to fourth. 

→This technique is performed in order to isolate individual colonies by diluting the sample.

8. Place all MSL agar plates into an anaerobic jar, and place an anaerobic sachet into the jar. Close the jar and place it into the autoclave for 48 hours at 35℃. Do not place the other agar plates in an anaerobic jar, but straight into the autoclave for 24 hours 35℃. 

→ MSL plates were grown in anaerobic jar because it is meant for the growth of anaerobic organisms. MSL is selective for streptococcus which grows anaerobically (without oxygen). The rest of the plates grow bacteria that grow aerobically (with oxygen) or either way.

9. After incubation, isolate individual colonies and streak them on fresh plates. Again, incubate for 24 hours, and 48 hours for MSL, at 35℃. Incubate MSL in anaerobic conditions. These strains will be identified later on.

 

PART TWO - Well diffusion method 

1. Using an inoculating loop, gently touch a sample of the strain colony from MCA (made MCA5) and the three strain colonies from MSA (made MSA6, MSA7, and MSA8), and transfer these bacteria onto the plates of Mueller-Hinton agar plates(2 plates of each strain.) 
2. Using an inoculating loop, gently touch a sample of the two strain colonies on the MSL plate and isolate them onto two separate MSL plates (to make MSL1 and MSL2). 
3. Collect these samples one by one, and remember to sterilize the inoculating loop before each sample collection. When isolating the oral bacteria, make sure to collect a sample from an individual colony that grew apart from a cluster in order to avoid picking up multiple different strains to isolate individually. 
4. Streak the isolated pure cultures over the Mueller-Hinton and MSL plates. These plates will now grow the unidentified strains of oral bacteria. 
5. Using a sterile borer, cut 4 wells, each 5 mm in diameter, into each agar plate. 
6. Dilute 1mL of each independent variable (fresh from container) with 9mL of MRS broth (assist growth of lactobacillus) to create a concentration of 1:10. Do not dilute the controlled variables. (Milk, saline, MRS broth).
7. Pipette 0.1mL of each newly made solution (of variable and broth) into a well on the two separate plates of the same culture. (8 variables, 6 strains, 2 “trials” / plates  per strain = 24 plates, 96 wells)
8. Allow the Mueller-Hinton plates to sit in the autoclave for 24 hours at 35℃, and the MSL plates for 48 hours at 35℃.

PART THREE - Recording Data / Identification of strains 

1. After growing the first 36 plates (part one), record observations. 
   1. How much growth, physical appearance, count colonies and cells (calculate colony formula units)
   2. For the variables that were grown in more than one concentration, record the data of the concentration that shows the clearer growth of colonies (the more diluted concentration).
   3. If there is growth on a SELECTIVE media, identify which type of bacteria is present
2. Well diffusion: after growing the various strains on Mueller-Hinton agar for 24 hours at 35℃ and MSL agar for 48 hours at 35℃, record the diameter of inhibition (of oral bacterial growth), calculate the average, and standard deviation of inhibition between each 2 wells (trials) for every variable.
3. Collection of bacterial smears from the diagnostic media that was incubated (MSA, MCA, Blood Agar, MSL):
   1. Draw a dime-sized circle in the middle of your microscope slide with a wax pencil. This will help locate your sample when you have finished staining the bacteria. Turn the slide over before continuing.
   2. Label the slide with a wax pencil. Add one drop of tap water to the center of the slide - this will be in the center of the circle.
   3. Sterilize an inoculating loop with a Bunsen burner flame, allowing it to cool down, and use it to transfer a very small part of a single colony from an agar plate into the tap water. If the amount of culture on the loop is easily visible you have taken too much.
   4. Make a suspension of the culture in the tap water on the slide and thoroughly but gently spread it evenly over an oval area of up to 2 cm length. The objective here is to spread out the cells so that they can be viewed individually under the microscope.
   5. Sterilize the loop and set it aside.
   6. Allow the slide to air-dry COMPLETELY, then pass the glass slide 3-5 times at a moderate pace over the flame. This will cause the cells to adhere to the slide, and to accept the stain more easily. Allow the slide to cool before staining.
4. Collection bacterial smears from diagnostic broths: Making a bacterial smear from broth differs from the above only in that no water is added to the slide. However, since the broth cultures are fairly dilute, gently shake the tube to mix the culture and use several loopfuls of culture.
   1. Draw a dime-sized circle in the middle of your microscope slide with the wax pencil. This will help you locate your sample when you have finished staining the bacteria. Turn the slide over before continuing.
   2. Label the slide with a wax pencil
   3. Sterilize an inoculating loop with a Bunsen burner flame, allowing it to cool it down, and then mix loop inside the culture. Then transfer a few loopfuls of culture to the slide.
   4. Sterilize the loop and set it aside.
   5. Allow the slide to air-dry COMPLETELY, then pass the glass slide 3-5 times at a moderate pace over the flame. This will cause the cells to adhere to the slide, and to accept the stain more easily. Allow the slide to cool before staining.
5. Perform the gram stain technique on the glass slides containing bacterial smears:
   1. Place the slide on the staining rack in the sink. Cover the bacterial smear with crystal violet solution and allow it to sit for 60 seconds. Wash the slide in a gentle stream of tap water for 2 - 3 seconds. Gently tap the slide to remove excess water.
   2. Cover the bacterial smear with Gram’s iodine solution, and allow it to sit for 60 seconds. Wash with tap water as above. The iodine serves to increase the interaction between cell wall and crystal violet so that the cell takes up more stain.
   3. With the slide held at a 45° angle over the sink, slowly drip 95% ethanol over the slide until the ethanol drips are colorless. This step removes the crystal violet from Gram-negative bacteria, decolorizing them.
   4. Wash the slide with water.
   5. Cover the bacterial smear with Safranin solution and allow it to sit for 60 seconds.
   6. Wash the slide with water. Gently blot (do NOT rub) the slide dry with paper towel.
   7. Observe the results under oil immersion (to magnify the image). Determine shape of cells and results of the Gram’s stain. Cover slips are not used for this procedure.
   8. Gram-positive species will have retained the crystal violet stain, and will appear blue or purple. Gram-negative species will have lost the violet stain when treated with alcohol, but will have been stained red or pink by the Safranin.

 

Identification of the Streptococcus species: Mitis-Salivarius Agar (MSL) is a selective and differential medium used to isolate and differentiate species of streptococci from the oral cavity, particularly Streptococcus mitis, S. salivarius, and S. mutans. Trypan blue and crystal violet in MSL

inhibit Gram-negative bacteria and some Gram-positive bacteria. Tellurite (1% solution) inhibits most non-streptococcal species, particularly Staphylococcus and Micrococcus. 

• S. mutans produces irregular, blue to dark blue, granular colonies (rough, textured surface)
• S. salivarius produces large, light blue, gumdrop-shaped, mucoid colonies due to sucrose fermentation. 
• S. mitis produces small, flat, blue colonies. 
• Some Enterococcus species produce small dark blue/black colonies.

 

1. Two strains were isolated from the MSL plate: “MSL 1” and “MSL 2.” Both cultures were stained with Gram staining and analyzed under a microscope. (These strains were used for well-diffusion tests).
2. Colonies were tested for catalase. The catalase test distinguishes between catalase-positive (Staphylococcus species) and catalase-negative bacteria (Streptococcus and Enterococcus species). If bacteria has catalase enzyme (enzyme found in organisms that breaks down hydrogen peroxide), a positive reaction will take place and bubbles will form, as decomposition is occuring. H2O2 → O2 + H2O. Drip hydrogen peroxide onto the bacterial smear to perform catalase test, and observe for bubbles.
3. To further differentiate Streptococcus from Enterococcus, isolated strains were grown in 6.5% NaCl broth. Growth after 72 hours was determined by turbidity. Enterococcus can tolerate high salt concentrations (halotolerant), while Streptococcus species cannot grow in 6.5% sodium chloride.
4. Isolated strains were grown on Blood Agar. Alpha-hemolytic streptococci (streptococci that cause partial hemoglobin degradation) including S. mitis and S. salivarius produce hydrogen peroxide, which bleaches the heme iron (iron containing part of hemoglobin, found in animal blood/meat) in red blood cells, resulting in a greenish zone around the bacterial colonies on blood agar.

 

Identification of the Staphylococcus and Micrococcus Species: Mannitol salt agar (MSA) is a selective and differential medium used to isolate and identify Staphylococcus aureus. As its name suggests, mannitol salt agar (MSA) contains 1% mannitol (sugar), 7.5% salt, and agar as a solidifying agent. A selective property of MSA is the high salt concentration. 7.5% NaCl inhibits the growth of most bacteria, making the medium selective for halotolerant organisms. 

• Most non-halotolerant bacteria are unable to grow on MSA. 
• Staphylococcus and Micrococcus are the most common organisms to grow on MSA 

A differential property of MSA is color change, relating to mannitol and the pH indicator phenol red. 

• Organisms that ferment mannitol will produce acid (lowers the pH and turns the medium yellow). S. aureus is one example of a mannitol-fermenting organism.
• Organisms that do not ferment mannitol will leave the medium red or pink. 
• Staphylococcus epidermidis and Micrococcus luteus are halotolerant and can grow on MSA. However, they do not ferment mannitol, so the medium remains red/pink if they are present.

 

1. On an MSA plate, three strains with different types of colonies were isolated: white, bright yellow, pale yellow. The medium changed color indicating mannitol consumption, but because the culture was mixed, a conclusion could not be drawn out on which isolated strain was fermenting the mannitol. To clarify this, the strains were isolated into “MSA 6”, “MSA 7”, and “MSA 8”, and were incubated on MSA individually for 24 hours at 35℃. (These strains were later transferred to Mueller-Hinton agar plates for well-diffusion test.) 
2. Additionally, the isolated strains were incubated in Phenol Red (PR) Sugar Fermentation broth with durham tubes (to detect the production of gas by microorganisms). The fermentation of two sugars, mannitol and glucose, was tested. 

*S. aureus actively ferments mannitol + glucose, and changes media to bright yellow due to acid production / lowered pH. It also produces gas (CO₂ or H₂) which was captured in the Durham tube. 

*S. epidermidis and M. luteus are poor fermenters, meaning they make little to no change to the media (neutral pH).

3. Blood Agar was used to identify hemolytic Staphylococcus. Beta hemolytic microorganisms such as S. aureus make the red blood cells clear (complete degradation), while non-hemolytic microorganisms such as S. epidermidis and M. luteus leave the media unchanged. Individual cultures were grown on Blood Agar for 24 hours at 35℃. 
4. To distinguish between S. epidermidis and M. luteus, the isolated strains were grown in 15% NaCl nutrient broth. Micrococcus luteus cannot tolerate such high salinity. After 24 hours at 35℃, growth was determined by turbidity.
5.  The isolates were gram stained, and morphology was analyzed.

 

Identification of the Escherichia coli Species: MacConkey Agar is a selective and differential medium commonly used for the isolation and identification of Gram-negative enteric (intestinal) bacteria, including Escherichia coli. MCA contains bile salts and crystal violet, which inhibit the growth of Gram-positive bacteria, allowing for the selective growth of Gram-negative bacteria like E. coli. MCA also contains lactose as a fermentable sugar and neutral red as a pH indicator. 

• Lactose fermenters (such as E. coli, Enterobacter and Klebsiella) produce acidic byproducts, turning the colonies from pink to red. 
• Non-lactose fermenters (such as Proteus, Salmonella and Shigella) remain colorless or pale because they do not produce acid from lactose fermentation.

1. Strain MCA5 which was isolated on MCA demonstrates strong lactose fermentation, proved by the development of dark pink colonies. These type of colonies can be produced by E. coli, Enterobacter and Klebsiella. Klebsiella is excluded because it produces mucoid, gum-drop shaped pink colonies, while this isolate produced flat dry pink colonies, meaning it could not be Klebsiella. 
2. To prove fermentation capacity of the isolate, it was incubated in in Phenol Red (PR) Sugar Fermentation broth with Durham tubes. The fermentation of four sugars was tested: mannitol, glucose, lactose and sucrose. All tests were positive for fermentation and gas production, which supports the identification of the isolate as E. coli or Enterobacter.
3. To further distinguish the microorganism, inoculate a Simmons Citrate Agar slant (A test-tube filled with liquid agar that is left to solidify at an angle to create a slanted surface for bacterial growth) with a loopful of the strain and incubate at 35°C for 48 hours. (If an organism can utilize citrate as its carbon source, the medium will change from green to blue because of the alkaline byproducts produced.) Simmons Citrate Agar contains sodium citrate as the only carbon source and ammonium phosphate as the only nitrogen source. 

*Bacteria that can utilize citrate as a carbon source produce citrate-permease, a membrane protein, to transport citrate into the cell. (Citrate is used by cells for energy and metabolic processes). These bacteria convert citrate into alkaline sodium carbonate, which raises the pH → makes SCA agar blue. Alkaline means having a pH above 7 (basic).

*The medium contains bromothymol blue, a pH indicator that changes color based on alkalinity (water’s ability to neutralize acids). 

4. Additionally, the cultures were tested on Blood Agar for hemolysis.

 

GENERAL PROCEDURES

1. Wash hands before/after every procedure
2. Wear a lab coat, close-toed shoes, and goggles, especially if working with a Bunsen burner. Tie back long hair
3. Before and after conducting every experiment, wipe bench tops with a disinfectant solution 
4. Do NOT place contaminated instruments, such as inoculating loops, needles, and pipettes, on bench tops. Loops and needles should be sterilized by incineration
5. On completion of the laboratory session, place all cultures and materials in the disposal area
6. Always practice aseptic techniques and careful handling of microbial cultures to prevent contamination of cultures
7. Wear gloves when working with dyes and other solutions
8. Handle all cultures with extreme care. Never open petri dishes or culture tubes outside of a controlled environment and minimize exposure to the air
9. Label Clearly: Always label all petri dishes, test tubes, and other containers clearly with the microorganism and medium
10. Dispose Properly: Dispose of all biohazardous waste, including cultures, gloves, and pipettes, in designated biohazard containers. Never dispose of these materials in regular trash bins.
11. Retract objects from Bunsen burner immediately after sterilization
12. Turn off gas (for Bunsen burner) before leaving the lab

PART ONE - Bacterial Growth on Plate

*CFU is calculated by multiplying the amount of colonies on a plate by the dilution factor to obtain the number of cells grown on the plate.

*Of the plates that were left to grow in autoclave, most showed successful growth of colonies. Some of the plates had growth that was too concentrated, meaning it was not possible to count the individual colonies and cells using CFU factor. I created a scale (ranging from 0 - 3) to describe the observed growth due to the fact that using colony formula units was not applicable to every agar plate.

*3- Lots of growth, covering most of the plate, 0- No growth

Blood Agar Plates (all had dilution factor 10^-2) → no CFU measurement, scale only. Forgot to count colonies.

Milk

• Growth scale 3
• Evidence of hemolysis; swamp-green spots
• Change in red color, darker undertones. 2 sizes of white colonies

Yogurt

• Growth scale 2
• Evidence of hemolysis; swamp-green spots
• Very small white colonies growing in string-like structure

Kefir

• Growth scale 1
• Evidence of hemolysis; very slight hint of swamp-green
• Extremely small white colonies, scattered throughout

Probiotic Yogurt

• Growth scale 3
• Evidence of hemolysis; swamp-green spots
• Change in red color, darker undertones. Extremely small white colonies, scattered throughout

Probiotic

• Growth scale 2
• Small green and white colonies, widespread

Buttermilk

• Growth scale 3
• Small green and white colonies, a bit clustered

 

MacConkey Agar Plates (all had dilution factor 10^-1)

Milk

• Growth scale 3
• Plate changed from orange-red to raspberry pink color
• Large and small red colonies, some growing in streaks and some in clumps
• 1200 colonies / 12,000 cells (1.2 x 10⁴ CFU)

Yogurt

• Growth scale 1
• 4 very large, pink colonies 
• 4 colonies / 40 cells (4 x 10¹ CFU)

Kefir

• Growth scale 1
• 1 very large, pink colony
• 1 colony / 10 cells (1 x 10¹ CFU)

Probiotic Yogurt

• Growth scale 2
• 2 very large, pink colonies
• 2 colonies / 20 cells (2 x 10¹ CFU)

Probiotic

• Growth scale 0

Buttermilk

• Growth scale 1
• 3 very large, pink colonies
• 3 colonies / 30 cells (3 x 10¹ CFU)

 

Mannitol Salt Agar Plates (all had dilution factor 10^-1)

Milk

• Growth scale 3
• Many small yellow colonies
• 1800 colonies / 18,000 cells (1.8 x 10⁴ CFU)

Yogurt

• Growth scale 3
• Three visible types of colonies: very small white, small yellow, and large yellow
• 1200 colonies / 12,000 cells (1.2 x 10⁴ CFU)

Kefir

• Growth scale 2
• Yellow large colonies and white large colonies
• 84 colonies / 840 cells (8.4 x 10² CFU)

Probiotic Yogurt

• Growth scale 3
• Yellow colonies grew in worm-like streaks
• Rosy pink discoloration in one corner of agar plate
• 920 colonies / 9200 cells (9.2 x 10³ CFU)

Probiotic

• Growth scale 0
• Plate completely changed from yellow to orange-pink color

Buttermilk

• Growth scale 3
• Some yellow colonies growing in worm-like streaks, some grown in clusters or individually
• 720 colonies / 7200 cells (7.2 x 10³ CFU)

 

Nutrient Agar Plates 

Milk (dilution factor 10^-7)

• Growth scale 3
• Many extremely small white colonies, and 7 very large yellow colonies
• 700 colonies / 7,000,000,000 cells (7 x 10⁹ CFU)

Yogurt (dilution factor 10^-7)

• Growth scale 0

Kefir (dilution factor 10^-7)

• Growth scale 1
• Very small white colonies, a few larger white colonies
• 80 colonies / 800,000,000 cells (8 x 10⁸ CFU)

Probiotic Yogurt (dilution factor 10^-7)

• Growth scale 1
• 6, medium-sized white colonies
• 6 colonies / 60,000,000 cells (6 x 10⁷ CFU)

Probiotic (dilution factor 10^-5)

• Growth scale 1
• 6 small white colonies
• 6 colonies / 600,000 cells (6 x 10⁵ CFU)

Buttermilk (dilution factor 10^-5)

• Growth scale 2
• Large, bright yellow colonies
• 47 colonies / 4,700,000 cells (4.7 x 10⁶ CFU)

 

De Man-Rogosa-Sharpe Agar Plates

Milk (dilution factor 10^-5)

• Growth scale 0

Yogurt (dilution factor 10^-5)

• Growth scale 2
• Individually growing white colonies and some growing in worm-like streaks
• 80 colonies / 8,000,000 cells (8 x 10⁶ CFU)

Kefir (dilution factor 10^-5)

• Growth scale 3
• Large white colonies and smaller white colonies
• 560 colonies / 56,000,000 (5.6 x 10⁷ CFU)

Probiotic Yogurt (dilution factor 10^-5)

• Growth scale 3
• Off-white colonies growing in worm-like streaks, look like splatters
• 280 colonies / 28,000,000 (2.8 x 10⁷ CFU)

Probiotic (dilution factor 10^-7)

• Growth scale 3
• Large off-white colonies, as well as very small ones growing in worm-like streaks
• 280 cells / 2,800,000,000 cells (2.8 x 10⁹ CFU)

Buttermilk

• Growth scale 3
• All dilutions had much too concentrated growth to count. Looked like waves or spread butter.

 

Mitis-Salivarius Agar Plates

Milk (dilution factor 10^-7)

• Growth scale 3
• 2 shades of blue (dark and light) colonies, some grew as circles and some as streaks
• 320 colonies / 3,200,000,000 cells (3.2 x 10⁹ CFU)

 

Yogurt (dilution factor 10^-7)

• Growth scale 3
• Near-black color larger colonies and smaller dark blue ones
• 240 colonies / 2,400,000,000 cells (2.4 x 10⁹ CFU)

Kefir (dilution factor 10^-7)

• Growth scale 2
• Small white colonies
• 120 colonies / 1,200,000,000 cells (1.2 x 10⁹ CFU)

Probiotic Yogurt (dilution factor 10^-7)

• Growth scale 3
• Very large, dark blue colonies. Some are more faint / faded color
• 82 colonies / 820,000,000 cells (8.2 x10⁸ CFU)

Probiotic (dilution factor 10^-5)

• Growth scale 1
• 9 Large, blue colonies
• 9 colonies / 900,000 cells (9 x 10⁵ CFU)

Buttermilk (dilution factor 10^-7)

• Growth scale 3
• Some extremely small dark blue colonies and lighter large colonies
• Growth too concentrated to count colonies

Visual Growth Scale Chart

Visual Growth Scale Graph (same data as above)

Bacterial Growth Measuremeant (Colony Formula Units) Forgot to count blood agar.

*THIS IS THE MORE ACCURATE / SCIENTIFIC DATA

Further Observations 

• Though varying from medium to medium, milk was usually the variable that showed the largest amount of bacterial growth, and probiotic showed the least (inhibition). Keep in mind that the media in which milk variable grew the most and probiotic grew the least are the media selective for species of oral bacteria.
• MRS is selective for lactobacilli (most common probiotic), and in the charts we see milk displaying zero growth on MRS, and probiotic displaying the most.
• Discoloration on MSA plates
• Evidence of hemolysis on blood agar plates. Hemoglobin in blood cells is being degraded. 

  • Note; 

  • Alpha (α) Hemolysis: Greenish discoloration around colonies caused by partial hemoglobin degradation.
  • Beta (β) Hemolysis: Clear, transparent zones around colonies due to complete lysis of red blood cells.
  • Gamma (γ) Hemolysis: No hemolysis or discoloration around colonies.

PART TWO - Well diffusion method and relationship between variables and oral bacteria 

*Because it is not possible to give a diameter to negative inhibition (promoted growth) of bacteria, I used a scale of 0 to -3 to describe the concentration of promoted bacterial growth around the control wells. (Milk, MRS media, and saline.) 0 means normal amount of growth around the well (no inhibition or promotion) and -3 means lots of promotion of bacterial growth.   

Plate 1 - Top right will always be probiotic, bottom right kefir, top left buttermilk, bottom left probiotic yogurt

Plate 2 - Top right will always be yogurt, bottom right milk, top left MRS, bottom left saline

On the culture MSA8, the independent variable that inhibited bacterial growth the most was probiotic, and the least was probiotic yogurt. The controls did not show any inhibition.

 

On the culture MSA7, the independent variable that inhibited bacterial growth the most was the probiotic, and the least was probiotic yogurt. The controls did not show any inhibition. 

On the culture MSA6, the independent variable that inhibited bacterial growth the most was probiotic, and the least was probiotic yogurt. The controls promoted bacterial growth around their wells, and the milk well showed the most promotion of growth.

On the culture MCA5, the independent variable that inhibited bacterial growth the most was the probiotic, and the least was probiotic yogurt. The controls did not show any inhibition. 

On the culture MSL1, the independent variable that inhibited bacterial growth the most was kefir, and the least was probiotic yogurt. The controls milk and saline promoted bacterial growth around their wells.  No image available of MSL1 plate.  (Milk should display negative inhibition on graph as well - this is an error)

On the culture MSL2, the independent variable that inhibited bacterial growth the most was kefir, and the least was probiotic yogurt. The controls milk and saline promoted bacterial growth around their wells. No image available of MSL2 plate.

 

PART THREE - Identification of strains

Streptococcus Identification Test Results

MSL1 (Strep. Salivarius) and MSL2 (Strep. mitis) growing on an MSL plate.

 

Staphylococcus and Micrococcus Identification Test Results

“Tetrads” - groups of four   -   “PR positive” means it changed to yellow. - "15% NaCl positive means growth occured."

MSA6 (Staph. aureus), MSA8 (Staph. epidermidis), and MSA7 (Micrococcus luteus) growing on an MSA plate.

A - Mannitol, B - Glucose, C - MSA broth (control)

MSA6: the mannitol and glucose turned yellow, indicating that Staph. aureus was fermenting them

MSA 8: the mannitol and glucose did not change color, indicating that Staph. epidermidis did not ferment them

MSA7: the mannitol and glucose did not change color, indicating that Micrococcus luteus did not ferment them

 

Escherichia coli Identification Test Results

• Blood Agar hemolysis test: After 24-hour incubation, the isolate revealed α-hemolysis, detected by production a greenish or brownish discoloration around bacterial colonies. The color change is due to the production of biliverdin, a byproduct caused by the incomplete breakdown of hemoglobin. This test confirmed the presence of E.coli.

MCA 5 (E. Coli) growing on an MCA plate.

A- Mannitol, B - Glucose, C - Lactose, D - Sucrose, E - MCA broth (control). E does not contain any bacterial growth.

The growth of MCA5 (E. coli) on phenol red broth, containing 4 different sugars. Positive fermentation of each sugar.

 

The growth of MCA5 (E. coli) in the Simmons Citrate Agar slant test. No color change was observed. The medium remained green, which means citrate was not utilized. This result indicates the presence of E. coli and excludes Klebsiella and Enterobacter which are citrate-positive and turn the medium blue. No difference between the slant growing MCA5 and the control (second tube, SCA agar).

 

 

PART ONE - Bacterial Growth on Plates

• Evidence of lactobacillus: 
  • The MRS medium is selective for lactobacilli (most common probiotic), and the data displayed that, of the six variables, the milk plate of MRS grew nothing, while the probiotic pill grew the most. This supports the hypothesis, and proves that the probiotic pill contains the greatest number of probiotic species. It also proves that pasteurized milk is completely sterile.

 

• Evidence of antimicrobial properties: 
  • On MSA, MCA, and MSL, probiotic plates showed the least growth. These media are selective for Staphylococcus, various gram-negative bacteria, and Streptococcus, respectively. These are all bacteria which are commonly found in the oral cavity.  
  • Since the selective media for oral bacteria showed the least growth on the probiotic plates, this shows that the probiotic was the variable with strongest inhibition of opportunistic pathogens, proving that the capsule contained the greatest amount of probiotics out of the six variables. 
  • The data proves the hypothesis correct. When probiotics are grown on the same medium as oral bacteria, the antimicrobial properties of probiotics result in the inhibition of the oral bacterial growth. In other words, probiotics DO support the immune health of a human. 

 

• The discoloration on MSA agar plates, a selective media for Staphylococcus and Micrococcaceae, means that acidity was released. The color change is evidence of these microorganisms fermenting the mannitol.
• The hemolysis on blood agar plates is evidence of growth of Staphylococcus and Streptococcus.

 

PART TWO - Well diffusion method and relationship between variables and oral bacteria 

• Evidence of antimicrobial properties: the 5 independent variables, which contain probiotics, inhibited growth around their wells, creating a clear ring of no visible colonies. 
• On every plate, each containing different strains of bacteria, there was a consistent pattern: of the 5 independent variables, the probiotic and kefir had the strongest inhibition effect, and the probiotic yogurt had the weakest. Probiotic, however, showed stronger inhibition than kefir most of the time.
  • The reason for probiotic yogurt to have the weakest inhibition effect is probably due to the fact it only contained one strain of probiotic, being Bifidobacterium lactis
• An unusual and unexpected relationship occurred on MSA6, MSL1, and MSL2 plates: the controlled variables (milk, saline, and MRS broth, all three of which do NOT contain bacterial culture) promoted bacterial growth. Perhaps the strains growing on these plates prefer to grow on milk, saline, and MRS broth as media. 
• Otherwise, on the other plates, the controls did not inhibit bacterial growth, as predicted.

 

PART THREE - Identification of strains

• 6 observed organisms extracted from the oral cavity:
  • Streptococcus salivarius (culture MSL 1)
  • Streptococcus mitis (culture MSL 2)
  • Staphylococcus aureus (culture MSA 6) 
  • Micrococcus luteus (culture MSA 7)
  • Staphylococcus epidermidis (culture MSA 8) 
  • Escherichia coli (culture MCA 5)

1. Of 6 dietary products (milk, kefir, yogurt, probiotic yogurt, buttermilk, and a probiotic pill) the probiotic pill contains the greatest abundance of probiotic bacteria. The most prominent species of bacteria in these products was lactobacillus, and a singular pill offers the largest supply of lactobacilli per 1mL. The probiotic, 4 out of 6 times, inhibited growth better than any of the other variables on the selective media agar plates and the Mueller-Hinton + MSL agar during the well diffusion test. The probiotic also grew the most lactobacillus on the MRS plate than any other variable. In relation to probiotic content, the probiotic pill can be classified as the “healthiest” or most beneficial product, because of the abundant growth of lactobacillus it yields, and the strong inhibiting effect of opportunistic pathogens. This is due to the lactic acids and peptides called bacteriocins that probiotics release, destroying the cell membrane of opportunistic pathogens, and killing them.

 

2. Pasteurized milk contains ZERO bacterial culture, and has proven itself to be a perfect medium for bacterial growth (once introduced to the milk.) Along with saline and MRS broth, it appeared to attract the growth of oral bacteria on the MSA6, MSL1, and MSL2 plates during the well diffusion test.

 

3. The oral bacteria that were identified were Streptococcus salivarius, Streptococcus mitis, Staphylococcus aureus, Micrococcus luteus, Staphylococcus epidermidis, and Escherichia coli. The relationship that occurred between these bacteria and the probiotics with the 6 product variables prove that probiotics offer immunity benefits. When grown on the same medium,  growth of these oral bacteria / opportunistic pathogens was inhibited. (Killed by probiotics)

My hypothesis was proven CORRECT. The probiotic capsule was proven to be the most beneficial variable to gastrointestinal and immune health because it retains the greatest abundance and variety of bacteria. The probiotic capsule had the STRONGEST inhibiting effect on the oral bacteria's growth, due to the strength of its antimicrobial properties, and abundance and variety of probiotic species.

The results of this study give insight on how different dietary products benefit humans and which ones offer the more of these benefits. Using the data of this experiment, it can be drawn away that a probiotic pill, packaged by humans, offers the most variety of probiotic strains and the best protection of immune health. This can be used in making dietary choices - if looking for the product with most effective gastrointestinal benefits, a probiotic capsule would be the suitable choice. However, my research provides insight on the benefits of the other variables / foodstuffs - such as the nutrients and natural supplements found within them that probiotic pills do not offer.

 

This study also identified the bacterial species that reside within the oral cavity. It can be applied as a stepping stone to medical research, such as identifying which opportunistic pathogens cause infections and illnesses, how probiotics can be used as antimicrobials against them, and which probiotics have the strongest effect against the opportunistic pathogens.

 

 

1. Contamination of cultures (eg; air currents, opening petri dish lid too much, misplacing sterile equipment onto countertop or touching other surfaces). 
2. Inaccuracies in measurement → serial dilution and dilution factors 
3. Calibration issues (setting mechanical pipette to incorrect measurement)
4. Only conducting one trial of parts one and three of the experiment. If multiple trials were conducted there would be higher accuracy in data collected. Only one trial was possible for these parts of the experiment due to time and resources required. 
5. Human estimation of bacterial growth of cultures on agar plates which CFU could not be counted

 

Errors that occurred in experiment:

1. Forgetting to count the CFU of Blood Agar plates, forgetting to take pictures of gram stains underneath the microscope, and of MSL plates in well diffusion method. 
2. Not enough dilution of certain variables, making counting the bacterial growth difficult / impossible. These plates were not given a colony formula unit measurement, hindering the accuracy of data.

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• Everything You Need to Ace Biology in One Big Fat Notebook by Matthew Brown

I would like to thank Victoria Herbst, for her support and dedication in helping me throughout this project. Truly, it would not have been possible without her. She has helped me pursue my passion of science and challenged my sense of what I believed impossible. My heartfelt gratitude goes out to her for teaching me so much throughout this experience, and being the very beacon of knowledge. I would also like to thank my mother, who constantly persuaded me to continue when I felt like I couldn't, and whose knowledge of microbiology inspired me to dedicate this project to the topic. I cannot express how grateful I am for her patience and support. My last acknowledgement goes out to Mr. Nayak, my science fair coordinator, for his reliability and dedication throughout the process. Thank you for always answering my questions, and supporting me through the process of participating in this fair.