1. Lab 8 Goals and Objectives: Do not disrupt BHI broths: need to see surface growth. Do not shake FTM tubes Make all Gram stain smears from broth & plates early so they can dry! Read cultural characteristic results on BHI broths FIRST, then make slides After heat fix, smears can be stored to stain later: make at least 5 smears of each today but only stain one pair (save some in case things don’t go well!) Collect all data and add to charts for your unknowns: Exercises 37 and 38 1. FTM tubes for O 2 requirements (Ex. 38 pg. 267) 2. BHI broth for growth in liquid characteristics (Ex. 38 pg. 266-267) then Gram stain (Ex. 37 pg. 260) and get size, shape, and arrangement. 3. Gelatin stab culture for gelatin hydrolysis ability (Ex. 38 pg. 267) Ice for 10min before reading! Put on ice now so you don’t forget! 4. Three BHI plates each unknown grown at 25°C, 30°C, 37°C for optimal temp (Ex 37&38): compare colony size. Use best plate of colonies for colony characteristics (Ex. 38 pg. 267-268) 5. Motility test media stab (Ex. 37 pg. 262) Observe characteristics of all 12 potential unknowns in cultures supplied by PA: see these today while they are fresh, you can stain next lab if time is short Try to rule out some based on protease production or cultural characteristics! Save best streak for isolation plates (one each unknown) for use next class

2. Data to collect for Exercise 37 & 38 Gram result, size, shape, arrangement Transfer to Data Chart Motility Transfer to Data Chart Amount of growth Color Opacity Form Surface (broth) Subsurface (broth) Sediment (broth) Growth (broth) Temperature Transfer to Data Chart Oxygen requirements (FTM) Transfer to Data Chart Gelatin Transfer to Data Chart Colonies

3. Nutrient Gelatin Stab Inoculation method: stab with needle Contains: beef extract, peptone, high gelatin concentration to gel (no agar) Must be set on ice 5-10 min before reading Discriminates organisms that can produce gelatinases to hydrolyze the gelatin into amino acids Results: liquid = gelatin hydrolysis, positive for gelatinase production solid = negative for gelatinase production Data chart requires you fill in info regarding the assay: Media used: Nutrient Gelatin Indicators: none Biochemical aspect: ability to produce gelatinases to hydrolyze gelatin into amino acids

4. Data chart requires you fill in info regarding the assay: Media used: Nutrient Gelatin Indicators: none Biochemical aspect: ability to produce gelatinases to hydrolyze gelatin into amino acids Enzyme = gelatinase Substrate = gelatin Product = amino acids Make sure you understand what the assays are for!!! We are testing for particular biochemical reactions. The reactions performed by living cells involve enzymes. The enzymes act on a particular starting material called the substrate and enzymatically alter it (cause a reaction) that results in a particular ending material called the product.

5. For next lab: Fill in information you have collected thus far (Gelatinase, Gram stain result, size of cells, shape and arrangement of cells, motility, oxygen requirements) in the data charts for each unknown. Read over formal lab report directions in packet. We will go to the library to get information about resources to help you find sources for your formal paper.

6. Lab 8 Goals and Objectives: Do not disrupt BHI broths: need to see surface growth. Do not shake FTM tubes Make all Gram stain smears from broth & plates early so they can dry! Read cultural characteristic results on BHI broths FIRST, then make slides After heat fix, smears can be stored to stain later: make at least 5 smears of each today but only stain one pair (save some in case things don’t go well!) Collect all data and add to charts for your unknowns: Exercises 37 and 38 1. FTM tubes for O 2 requirements (Ex. 38 pg. 267) 2. BHI broth for growth in liquid characteristics (Ex. 38 pg. 266-267) then Gram stain (Ex. 37 pg. 260) and get size, shape, and arrangement. 3. Gelatin stab culture for gelatin hydrolysis ability (Ex. 38 pg. 267) Ice for 10min before reading! Put on ice now so you don’t forget! 4. Three BHI plates each unknown grown at 25°C, 30°C, 37°C for optimal temp (Ex 37&38): compare colony size. Use best plate of colonies for colony characteristics (Ex. 38 pg. 267-268) 5. Motility test media stab (Ex. 37 pg. 262) Observe characteristics of all 12 potential unknowns in cultures supplied by PA: see these today while they are fresh, you can stain next lab if time is short Try to rule out some based on protease production or cultural characteristics! Save best streak for isolation plates (one each unknown) for use next class