Appendix B W24 Laboratory Formal Experiment (Enumeration of Probiotics in yogurt) FINAL

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B1 Appendix B BLG144 LABORATORY FORMAL EXPERIMENT Microscopic Analysis and Enumeration of Probiotic Bacteria in Yogurt Yogurt is a staple food in several cultures, originating from countries in Western Asia and the Middle East. The word yogurt is believed to be derived from the Turkish word “yoğurmak,” which means to thicken, coagulate, or curdle. [1] Yogurt is made when heated milk is combined with bacteria, specifically the probiotic microbes Lactobacillus bulgaricus (Fig B1.1) and Streptococcus thermophilus (Fig. B1.2), and left to incubate for several hours at a warm temperature (110-115°F). The bacteria convert the sugar in milk, called lactose, to lactic acid, which thickens the milk and develops its distinctive tart flavor. Additional types of lactobacilli ( Lactobacillus acidophilus or casei ) an/or bifidobacteria ( Bifidobacterium bifidus ) may be added to provide extra probiotics as well as give yogurt its distinct flavour and texture. Today, yogurt can be found in a variety of forms — including plain, but often with added fruit or sweeteners. Thickeners and stabilizers such as gelatin and pectins may also be added for a thicker texture and richer taste. Probiotics are living microbes that benefit your health which is why they are often referred to as the “good bacteria”. Eating yogurt can increase the number of probiotics in your diet, and thus in your natural microflora. It is beneficial to have more “good bacteria” in your system since they enhance your health, as well as keep the harmful “bad bacteria” at bay. Other examples of foods and drinks that contain probiotic microbes include sauerkraut, kimchi, pickles, kefir and kombucha. Probiotics can also be taken as supplementals in forms of pills or capsules. Does your yogurt really have probiotics? The Food and Drug Administration (FDA) requires all yogurt manufacturers to use probiotic bacteria called Streptococcus thermophilus and Lactobacillus bulgaricus . These two organisms are the necessary probiotics to ferment milk and turn it into yogurt. Since probiotics are used in the fermentation process, one must assume that there is a high number of probiotic bacteria in yogurt, but that is not always the case. According to the FDA, a product that specifies to contain “live and active cultures” (a LAC Seal) on its label must have at least 100 million living bacteria or colony forming units per gram (CFU/g) when it was made. CFUs or colony forming units/gram, is the number of living bacteria per gram. This is an indication of how many live bacteria are present in the yogurt at the time of manufacture. Yogurt typically contains at least 1,000,000, or 10 6 CFUs, which is the minimum needed to display a “Live and Active Cultures” (LAC) seal on the packaging. This minimum number is the standard provided because it is expected that some bacteria will be destroyed when passing through the digestive tract and exposed to stomach acid.

B2 Figure B1.1 Micrograph of a Gram-stain of Lactobacillus sp Figure B1.2 Micrograph of a Gram-stain of Streptococcus thermophilus How much probiotic is really in yogurt? In this year’s BLG144 Lab Formal Experiment, since you will be introduced to the study of microorganisms, we will take a closer look at probiotic bacteria. We will be analyzing a variety of yogurts available in our Canadian grocery stores. Only plain yogurt with similar milk fat content will be used in the experiment to keep other variables, such as sugar and fat content, to minimal affecting factors in the bacterial count. Yogurt probiotics are lactic acid-producing bacteria (LAB). Tomato juice agar and MRS Agar has been used for culturing yogurt to analyze lactic acid bacteria (LAB). De Man – Rogosa – Sharpe agar, often abbreviated to MRS , is a selective culture medium designed to favour the luxuriant growth of Lactobacilli and will be used for this lab study.

B3 Table B1.1 A Variety of Yogurt Brand Samples Used in the Experiment Brand Description LAC Seal Price Price/100 g Dahi Plain, live cultures 3.25% mf yes $2.87/750 g $ 0.38 ACTIVIA Plain, 3.25% mf yes $6.99/650 g $1.07 SKOTIDAKIS Strained, probiotic yes /500 g Farm Boy Organic probiotic, 4.3% mf yes /750 g Astro BioBest Plain probiotic, 3.25 % mf yes /750 g Yoplait The objectives of this experiment are to determine the following: 1. What types of probiotics are found in the yogurt we eat? Does this differ from one brand to another? 2. What are the three most prominent type of probiotics found in your sample of yogurt? 3. What is the number of probiotics found in yogurt? Does this differ from one brand to another? 4. Do the yogurt samples with the LAC seal contain the minimum number of CFU/g of probiotics as required by the FDA? 5. Is the price of the yogurt sample justified by the number of probiotics? Ie Are there more probiotics found in the more expensive brand yogurt compared to the cheaper brands?

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B4 Experiment B1.1 Microscopic Examination of Probiotics in a Yogurt Sample A microscopic analysis of the yogurt sample will be performed to determine the types of probiotic microorganisms found in the sample, as well as determine the three most prominent types that are seen in the sample. A heat-fixed slide of the yogurt sample will be prepared in order to do a Gram-stain of the sample. The Gram-staining procedure will help in the identifying the Lactobacillus , Streptococcus and Bifidobacteria . All the probiotics in yogurt have a Gram-positive cell wall structure. The Gram-stain technique (Fig. B1.4) Is a differential staining method to differentiate between two main types of bacteria, based on their cell wall structures. At the end of staining process, the Gram- Positive cell walled bacteria will appear PURPLE , while the Gram- Negative bacteria will appear PINK. Lactobacillus sp. are Gram-positive bacillus, and will appear as purple bacillus or rod shaped bacterial cells. Streptococcus sp. are also Gram-positive, and will appear as purple cocci or spheres in the cellular arrangement of chains. The Bifidobacteria are Gram-positive as well, and will appear as purple branched rod-shaped bacteria in random cellular arrangement. A. Preparation of Heat-fixed smear Materials: (per group of 4) • Glass microscope slides • Dilute yogurt sample • Inculating loop • Bunsen burner/Incinerator Procedure: 1. Set up your Bunsen burner or incinerator as demonstrated by your TA. 2. Using your inoculating loop, place a loopful of water on a glass slide. (Fig B1.3) 3. Sterilize your inoculating loop by placing it in the flame or incinerator until it glows red hot. Remove from heat and let cool. 4. Using your sterile inoculating loop, take a loopful of your yogurt sample. 5. Touch the loop with yogurt sample into the drop of water. Swirl the yogurt around with the loop into the drop of water and spread the drop into a thinner smear. The thinner the you spread the drop of water, the faster it will air dry. 6. Place a coverslip on top. Remove excess solution around the coverslip with a paper towel or tissue. 7. View in the compound microscope at 4 x or 10 x initially, before moving to higher magnification. Bacteria will appear small even at the highest magnification.

B5 (data:image/jpeg;base64) Figure B1.3 Preparation of a Heat-fixed Smear B. Gram-staining of Probiotic Smear Materials: (per group of 4) • Heat-fixed smear (prepared as above) • Gram-stain set up • Microscope • Camera/Phone • Bibulous paper (blotting paper) • Lens paper • Lens cleaner/swab • Immersion Oil Procedure: 1. Take the slide of the heat-fixed smear and place it in the slide holder with crystal violet. Let it sit in the stain for 1-2 minute . (Fig. B1.4) 2. Remove slide and place over the waste beaker. Using the water bottle rinse the slide with water until all the stain is removed. 3. Dip the slide in the mordant iodine stain holder and let it sit for 1-2 minute . 4. Remove slide and rinse as in Step 2. 5. While holding the slide over the waste beaker, decolorize the slide for 10-20 seconds using ethanol bottle to apply on the slide. 6. Rinse slide over the waste beaker with water as in Step 2.

B6 7. Finally, dip the slide in the counterstain safranin for 30s-1 minute . 8. Remove slide from the stain holder, and place above the waste beaker for a final rinsing. 9. Rinse the slide untll all the stain has been washed off. 10. Blot slide dry between sheets of bibulous paper. DO NOT RUB! Only pat dry. 11. Once the slide is dry. View under the microscope using oil immersion objective lens at 1000x total magnification as final magnification. 12. See Appendix 1: Microscopy Skills Workshop for proper use and care of microscope. 13. Record your observations by drawing or taking a picture. Estimate which type of bacterial cell morphologies are most prominent and their possible identities. 14. Please remove ALL oil from the oil immersion lens after use using lens paper and lens cleaner. 15. Slides can be thrown out after use in the BIOHAZARD pail. 16. Slides can be saved to be viewed the following if more time for observations is needed in the class slide holder case. (https://microbiologie-clinique.com/img/gram-stain-procedure.jpg) Figure B1.4 The Gram-stain Technique Table B1.2 Microscopy Analysis and The Three Most Prominent Cellular Morphology of Probiotics in Yogurt Order of Numbers Gram Rxn Cellular Morphology/Arrangements Possible Bacteria Percentage Estimate 1 2 3

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B7 Experiment B1.1 Enumeration of Probiotics in a Yogurt Sample An 11 g sample of yogurt was weighed, and added to bottle of 99 mL dilution buffer. This 10 -1 diluted yogurt sample will be aliquoted to 5 mL per sample tube. This sample tube will have a dilution of 10 -1 and will be used for the preparation of the dilution series for the enumeration of the probiotic organisms in a yogurt sample. Since the density of the diluent is about 1 g/mL, we can say that the 11 g of yogurt in 99.0 (g or mL) of diluent is a 10 -1 dilution. Dilution Math: Sample weight = 11 g yogurt______ = 11 g____ = 1 g__ = 10 -1 Total weight (11 g + 99 g diluent) 110 g (mL) 10 mL Materials: (per group of 4) • 1 – 5 mL sample of yogurt in (10 -1 g/mL Dilution) • 5 – 9.0 mL sterile dilution tubes • 5- 1 mL pipettes • Pipette pump/aid • P100 Micropipettor • Sterile yellow tips (100 uL) • 8 MRS agar plates • Sterile glass balls • Beaker with ethanol f(or used contaminated glass balls) Procedure: A. Preparation of Dilution Series (10 -2 to 10 -6 ) (TA DEMO) 1. Take a sample of yogurt (10 -1 g/mL dilution) from the side bench. 2. Using a 1 mL pipette and pipette pump, aseptically transfer 1.0 mL of your 10 -1 yogurt sample in a 9.0 mL dilution tube. Pipette the liquid up and down several times to ensure it is thoroughly mixed. This will be a 1/10 dilution of your 10 -1 sample to make a 10 -2 dilution. 3. Using a fresh 1 mL pipette and dilution tube, repeat Step 2 to prepare another 1/10 dilution for a final dilution of 10 -3 . 4. Repeat Steps 2-3 until you have completed a dilution series from 10 -2 to 10 -6 . 5. Once the dilution series are prepared, they are ready for plating. (Fig B1.5)

B8 Figure B1.5 Preparation of a Dilution Series and Spread Plating Inoculation B. Spread Plating your samples using Copacabana Method (TA DEMO) 1. Take a stack of 8 MRS agar plates and label the plates with the letter of your sample ie “A to F”” followed by the dilution. 2. The dilutions 10 -3 to 10 -6 will be used for the spread plating in replicates. 3. For the sample “A” , label 2 plates each with A10 -3 , A10 -4 , A10 -5 , A10 -6 . 4. Take and sign out a micropipettor from the rack at the side or front bench. 5. Set the volume of micropipettor to 100 uL and load with a fresh tip. 6. Starting with the most dilute (A10 -6 ), aseptically inoculate 100 uL of the sample into the agar plate labelled A10 -6 . Repeat for the replicate plate. Note: you can use the same tip for all the inoculations as long as you are going from most dilute(least bacteria) to least dilute (most bacteria). 7. Inoculate the next dilution (A10 -5 ), into the replicate plates labelled A10 -5 as in step 7. 8. Repeat steps 7-8 for the rest of the dilutions to be plated (10 -6 to 10 -3 ) 9. Once all the samples have been aliquoted into the agar plates, take a tube of sterile glass beads. 10. Add by shaking 4-5 glass beads in each of the 8 MRS agar plates. Close lids and stack 4 plates at a time. Move the stacked plates from side to side on the bench, rotate 1/4 turn, move from side to side again, repeating this motion 4 times until you have gone all the way around so that the glass beads can roll around the entire agar surface and spread the bacteria evenly around the whole plate. 11. Remove glass beads into a waste beaker with ethanol. 12. Once all the plates have been spread plated, put them in the plate rack provided. 13. The plates will be incubated at 37 o C for 3-4 days or at 37 o C (optimal temp). 5 - 9.0 mL Dilution Tubes 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 yogurt sample 1.0 mL 1.0 mL 1.0 mL 1.0 mL 1.0 mL 100 uL. 100 uL 100 uL 100 uL 100 uL 8 MRS Agar Plates

B9 Next Week After incubation, only one plate in the series should give a countable range of counts between 30-200 colonies. The rest of the plates will fall in the TMTC (too many to count >300 colonies) or TFTC (too few to count <30 colonies). Table B1.3 Results of Enumeration Count of Probiotics Dilution Number of colonies Average CFU g/mL 10 -3 10 -4 10 -5 10 -6 Questions to consider when writing your report: 1. What would your null and alternate hypotheses be for this experiment? 2. What are some of the limitations of this experiment? 3. What are other variables, that may play a factor in the types or numbers of probiotics in yogurt, that are not considered in this experiment? Eg type of milk (cow vs sheep); initial starter cultures 4. Is there a control in this experiment? If not, what can be used as a control group for this experiment? 5. What kind of statistical analysis should be used to compare if there are any significant differences in the number of probiotics in the various yogurt samples? 6. How would you change this experiment to improve the outcome or account for other variables?

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B10 Want to Make Yogurt home? Here is a simple recipe and method to use. If you want to try making strained yogurt at home, it’s not as difficult as you might think: 1. Heat 1/2 gallon of milk (may be skim, 2%, or whole). Pour the milk into a 3- quart saucepan and set over medium heat. Warm the milk until almost boiling when small bubbles appear; stir the milk periodically to prevent scorching. 2. Cool the milk to a warm temperature of about 110°-115° F and transfer to a glass or ceramic bowl. A higher temperature can destroy the bacteria, while a cooler temperature can prevent fermentation. 3. Whisk into the milk ½ cup plain yogurt with live cultures or a yogurt starter package. Cover the bowl with a lid or clean plate. Wrap the bowl in a towel. 4. Heat oven to warm setting for 1-2 minutes and turn on oven light. Turn off the oven (leave light on) and place yogurt into oven. Let sit for at least 4 hours but may be left overnight. The oven temperature should be about 110°F. 5. The yogurt is then ready to use. If you prefer this thinner consistency, refrigerate for a few hours before eating. If you wish a thicker Greek-style yogurt, strain the yogurt. Place a fine mesh sieve over a large bowl and line with a coffee filter or cheesecloth. Pour the yogurt into the sieve and refrigerate for up to several hours, which will strain out the liquid whey and excess water. 6. The yogurt may be stored in the refrigerator for about 7 days References 1. Fisberg, M., and Machado, R. History of yogurt and current patterns of consumption. Nutr Rev . (2015) 73 (suppl_1): 4-7. 2. Retrieved from internet Jan 17 th , 2024 https://www.hsph.harvard.edu/nutritionsource/food-features/yogurt 3. Retrieved from internet Jan 17 th , 2024 https://zoe.com/learn/best-yogurt-for-probiotics

Appendix B W24 Laboratory Formal Experiment (Enumeration of Probiotics in yogurt) FINAL

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