Lab 8 Goals and Objectives:

Do not disrupt BHI broths: need to see surface growth. Do not shake FTM tubesMake all Gram stain smears from broth & plates early so they can dry! Read cultural characteristic results on BHI broths FIRST, then make slidesAfter 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 O2 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 shortTry to rule out some based on protease production or cultural characteristics!Save best streak for isolation plates (one each unknown) for use next class

Data to collect for Exercise 37 & 38Gram 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

Nutrient Gelatin StabInoculation method: stab with needleContains: beef extract, peptone, high gelatin concentration to gel (no

agar)Must be set on ice 5-10 min before readingDiscriminates organisms that can produce gelatinases to hydrolyze the

gelatin into amino acidsResults:

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 GelatinIndicators: noneBiochemical aspect: ability to produce gelatinases to

hydrolyze gelatin into amino acids

Data chart requires you fill in info regarding the assay: Media used: Nutrient GelatinIndicators: noneBiochemical aspect: ability to produce gelatinases to

hydrolyze gelatin into amino acidsEnzyme = gelatinaseSubstrate = gelatinProduct = 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.

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.

Lab 8 Goals and Objectives:

Do not disrupt BHI broths: need to see surface growth. Do not shake FTM tubesMake all Gram stain smears from broth & plates early so they can dry! Read cultural characteristic results on BHI broths FIRST, then make slidesAfter 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 O2 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 shortTry to rule out some based on protease production or cultural characteristics!Save best streak for isolation plates (one each unknown) for use next class