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&9 Goals and Objectives:

• Exercise 37: Morphological Study of Unknown BacteriumObserve results for unknown cultures and record all data:

– Use broth culture after reading cultural results in Ex. 38 (see below)– Use Gram stain to determine size, shape and arrangement of cells– Motility (from Motility agar and wet-mount)

• Exercise 38: Cultural Characteristics Observe results for unknown cultures and record all data:

– Amount of growth (from best plate)– Colony characteristics (from best plate)

• Color (from best plate)• Opacity (from best plate)• Form (from best plate)

– Amount of growth (from broth, mix completely)• Surface (from broth, NOT shaken!)• Subsurface (from broth, NOT shaken!)• Sediment (from broth)

– Temperature requirements (compare plates) no broth, • no spectrophotometer

– Oxygen requirements (from FTM)– Gelatin Protease production (from Gelatin stab) (Ice 10 min to read!)

• No type of growth in gelatin

Lab 8 Goals and Objectives:

Do not disrupt BHI broths: need to see surface growth. Do not shake FTM tubesMake all Gram stain smears 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

Data chart requires you fill in info regarding the assay: Media used: Indicators: Biochemical aspect: EnzymeSubstrate 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 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 acidsEnzyme = gelatinase Substrate = gelatin

Product = amino acids

Bacillus subtilis Enterobacter cloacae Escherichia coli Klebsiella pneumoniae

Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Shigella flexnari

Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes

1 2

5

9

3 4

6 7 8

10 11 12

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 Tail

Meow Woof

Bird Human Cat Dog

12 Possible Unknowns

Bacillus subtilis Enterobacter cloacae Escherichia coli Klebsiella pneumoniae

Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Shigella flexnari

Staphylococcus aureus Staphylococcus epidermidis Enterococcus faecalis Streptococcus pyogenes

1 2

5

9

3 4

6 7 8

10 11 12

12 Possible Unknowns

Gram Positive Gram Negative

Bacillus subtilisStaphylococcus aureusStaphylococcus epidermidisStreptococcus faecalisStreptococcus pyogenes

Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosaSalmonella typhimuriumShigella flexnari

12 Possible Unknowns

Gram Positive Gram Negative

Gelatinase + Gelatinase - Gelatinase + Gelatinase -

Bac

illu

s su

btil

is

Pse

udom

onas

aer

ugin

osa

Staphylococcus aureusStaphylococcus epidermidisStreptococcus faecalisStreptococcus pyogenes

Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Salmonella typhimuriumShigella flexnari