Lab 8&9 Goals and Objectives:
• Exercise 37: Morphological Study of Unknown Bacterium
•
Exercise 38: Cultural Characteristics
•
•
•
Discuss observations of the 12 possible organisms
Discuss how to rule some out
Start working on a dichotomous key of logic for solving all 12 unknowns
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 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
Opacity
Transfer to Data Chart
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
Data chart requires you fill in info regarding the assay:
Media used:
Indicators:
Biochemical aspect:
Enzyme
Substrate
Product
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.
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
Enzyme = gelatinase
Substrate = gelatin
Product = amino acids
1
2
Bacillus subtilis
5
3
Proteus vulgaris
9
7
Pseudomonas aeruginosa
10
Staphylococcus aureus
Escherichia coli
Enterobacter cloacae
6
4
8
Salmonella typhimurium
11
Staphylococcus epidermidis
Klebsiella pneumoniae
Shigella flexnari
12
Enterococcus faecalis
Streptococcus pyogenes
For next lab:
Fill in information you have collected thus far (Gelatinase,
Gram stain result, size of cells, shape and arrangement of
cells) in the data charts for each unknown.
Read over formal lab report directions in packet.
State a hypothesis
“If the collective results of a profile of key biochemical assays
are compared to the known results for limited subset of
bacterial species, then a specific unknown culture from that
subset of bacterial species can be correctly identified based on
the results obtained from the biochemical assays.”
The Dichotomous Key
-a visible map of logic flow
-divides a group of possible things into smaller and smaller
groupings until it leads to the identification of just one
-tracing the pathway backwards shows all the characteristics
that collectively are unique to only that one
-assesses only one characteristic per branch
-lines all the answers up across the bottom of the key
Example: explain with the least number of characteristics how
to tell the difference between an old lady and her three pets:
sort a human, a cat, a dog and a bird. Construct a
Dichotomous key to ascertain the identity of each from this
small group:
Animals in the house
External covering:
feathers
External covering:
hair
No tail
Bird
Human
Tail
Meow
Woof
Cat
Dog
12 Possible Unknowns
1
2
Bacillus subtilis
5
3
Proteus vulgaris
9
7
Pseudomonas aeruginosa
10
Staphylococcus aureus
Escherichia coli
Enterobacter cloacae
6
4
8
Salmonella typhimurium
11
Staphylococcus epidermidis
Klebsiella pneumoniae
Shigella flexnari
12
Enterococcus faecalis
Streptococcus pyogenes
12 Possible Unknowns
Gram Positive
Bacillus subtilis
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus faecalis
Streptococcus pyogenes
Gram Negative
Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae
Proteus vulgaris
Pseudomonas aeruginosa
Salmonella typhimurium
Shigella flexnari
12 Possible Unknowns
Gram Negative
Gram Positive
Gelatinase +
Gelatinase -
Gelatinase +
Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae
Proteus vulgaris
Salmonella typhimurium
Shigella flexnari
Pseudomonas aeruginosa
Staphylococcus aureus
Staphylococcus epidermidis
Streptococcus faecalis
Streptococcus pyogenes
Bacillus subtilis
Gelatinase -