LAB 7 – Biochemical and Serological Assays Information and Worksheet(1)

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Arkansas Tech University *

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Biology

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Apr 3, 2024

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LAB 7 – BIOCHEMICAL & SEROLOGICAL ASSAYS Part 1 (18 Total Pts) Nutrition on the cellular level requires the breakdown of large macromolecules. This breakdown is facilitated by enzymes in a series of oxidation reactions called metabolic pathways. The activity of these metabolic pathways can be viewed macroscopically. Microorganisms oxidize carbohydrates to produce energy in a process known as carbohydrate catabolism . Lipids and proteins may also be catabolized, but today’s lab will focus on carbohydrate catabolism because it is the primary source of energy for microorganisms. Through the inoculation of B. subtilis, E. coli, and S. epidermidis on starch agar, and in lactose broth, you will be able to see which organisms hydrolyze starch and ferment lactose. This lab will have 3 parts. Please carefully read the directions for each part. Part 1 Starch Agar – Starch Hydrolysis Test Procedure 1. Obtain a starch agar plate and label the bottom in the following manner (remember initials and section number): 2. Using an inoculation loop, you will inoculate each labeled area as shown above. 3. In 2-4 days, check the plate for growth. 4. Flood the plate with Gram’s iodine and wait for 2 minutes. Areas of starch hydrolysis will appear clear while un-hydrolyzed starch will remain blue-black. 5. Record your results in Table 1. ( 3pts ): Table 1. Data for the observation of growth and starch hydrolysis Starch Hydrolysis Organism Growth (+ or - ) Starch Hydrolysis (+ or - ) Bacillus subtilis Staphylococcus epidermidis Escherichia coli B. cereus E. coli S. marcescens + + + + - - permease cloesptyre mylasenydre a 3-4 drops E starch iodine Stuck in : hydrolysis Coils makes it blue-black Belle demon e

Part 2 – Lactose Broth Fermentation Procedure for the inoculation of any liquid medium 1. After sterilizing an inoculation loop, obtain a small sample of the target organism. 2. Remove the lid from the broth tube and gently stir the loop in the broth close to the glass side of the container. 3. Remove the inoculation loop and replace the cap. Gently flick the tube to mix the organism into the broth. Procedure: Lactose Broth Fermentation Test 1. Obtain 4 tubes of lactose broth. Make sure that each tube contains a Durham tube. The Durham tube will catch gas produced by bacteria that ferment lactic acid. What is the fourth tube for? 2. Label and inoculate E. coli into one tube, B. subtilis into the second, and S. epidermidis into the third. Be sure to label them well. The incubator will be very crowded. 3. Incubate the tubes for 24 hours. 4. Recording your observations: If you need to make a comparison between tubes, the volume of the Durham tube is approximately 0.5 ml. You may estimate the volume to make a comparison, otherwise just enter a “+” for the presence of gas and “ - “ for the absence of gas. Even if you see a bubble the size of the head of a pin, that is a positive. Table 2. Data table for the observation regarding the presence of gas and/or color change as an indicator of lactose fermentation (3pts) Lactose Fermentation Organism Gas Present (+ or - ) Color Change (+ or - ) Escherichia coli Bacillus subtilis Staphylococcus epidermidis E. coli B. cereus S. marcescens + - - + - - ⊥ > control bubble indicates CO2 Carbonic acid H20 + CO2 > CH203 e > ·

Questions (13 Total points for this section) 1. What interaction occurs between the iodine and starch molecules that causes a blue black color? In the context of your answer to the first question, explain what happened in the area of hydrolysis? **NOTE** Just saying that starch turns blue-black in the presence of iodine does not answer the question. (3pts) 2. Only one of your organisms should produce CO 2 in lactose broth. Which organism ferments lactose? What enzyme is responsible for fermenting lactose? **NOTE** (2pts) 3. Write a summary about Part 1 or Part 2 of this lab? (DON ’ T write a paragraph about both of them…just choose one. In your summary include: what you did, the results you expected, the results you observed. If you did not observe the expected results, supply a plausible explanation. (8 pts) Iodine sticks to the coils of starch molecules which gives starch a blue-black color and indicates growth. When hydrolysis occurs via amylase, there is a break where the coils are; therefore, iodine cannot stick to the coils and give starch a blue-black appearance. E. coli is the organism that produced carbon dioxide in the lactose broth. Lactase is the enzyme responsible for fermenting lactose. For part 2 of this lab we inoculated 3 test tubes with E. coli, B. cereus and S. , marcescens. We also left one tube without an organism for our control. In this experiment all 4 test tubes contained lactose both and a Durham tube. A air bubble of carbon dioxide and a color change due to changes in pH indicated fermentation. For this experiment, I predicted E. coli would ferment while B. cereus and S. marcescens would not because E. coli is gram negative while B. cereus and S. marcescens are gam positive. The results were as predicted: E. coli indicated fermentation.

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