Transcript Lab8

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