Lab 11 Goals and Objectives:

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Lab 11 Goals and Objectives Exercise 39
Oxidation and Fermentation Tests Read results
some tubes will require additional reagents Do
controls first so you have something to compare
to! Exercise 40 Hydrolytic and Degradative
Reactions Set up according to Fig 40.1 except
both of your two unknowns well separated on
each type of plate and control on a different
plate (one control plate per pair) Save
streak plates of unknowns for use next class
2
Durham Sugar Tube Fermentation (Glucose, Lactose,
Mannitol) Contains single carbohydrate peptone
broth with durham tube for gas collection,
Phenol red pH indicator alkaline pH red,
acidic pH yellow Discriminates the ability
to ferment a single carbohydrate (glucose,
lactose, or mannitol) into acid products (e.g.
pyruvic acid) or acid plus gas Results Red
inert, negative for fermentation of specified
carbohydrate Yellow positive for
fermentation of carbohydrate to acid
products Yellow with bubble positive for
fermentation of carbohydrate to acid gas
Control Escherichia coli
3
Acid plus gas
Acid
Negative
4
MR-VP Medium Methyl Red Test Contains peptone,
glucose, and buffer (buffer will neutralize weak
acids so only strong stable acids will be
detected by methyl red) Additional reagents
added methyl red pH indicator acid pH red,
neutral or alkaline pH yellow Distinguishes
ability to catabolize glucose into stable mixed
acids (lactic, acetic, and formic acids) in the
mixed acid pathway Results Red positive for
mixed acid formation Yellow negative for
mixed acid formation
_

Control E.coli
5
MR-VP Medium Voges-Proskauer Test Contains
peptone and glucose Additional reagents added
Barritts A (alpha napthol) and Barritts B
(KOH) (will react with acetoin to produce a red
product, alone produce a copper colored
product) Distinguishes the ability to
catabolize glucose into the neutral end product
butanediol (the oxidized product is acetoin) in
the butylene glycol pathway Results Red
positive for acetoin and thus for 2,3-butanediol
production Yellow/Orange no acetoin,
negative for 2,3-butanediol production
_
_


Control Enterobacter cloacae
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Nitrate Reduction Broth Contains beef extract,
peptone, KNO3 as nitrate source, durham tube
for gas collection Additional reagents added
sulfanilic acid (reagent A), dimethyl-alpha-naphth
ylamine (reagent B), (together form a complex
with nitrite creating a red product), zinc
(reduces nitrate to nitrite allowing reaction
with reagent A and B) Discriminates organisms
that can produce nitrate reductases to utilize
nitrate as a final electron acceptor resulting
in the production of either nitrite (partial
reduction) or to NH4, N2O or N2 gas (complete
reduction). Results Red with reagents A and B
positive for nitrate to nitrite
reduction Clear with/wo gas
positive for complete reduction of nitrate
to nitrogen gas (or nongaseous N2O or NH4)
Red only after zinc negative for
nitrate reduction, negative for nitrate
reductases Controls Pseudomonas aeruginosa and
E. coli
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Anaerobic respiration - Nitrate reduction
Electron acceptor Products
NO3 NO2, N2 H2O
SO4 H2S H2O
CO32 CH4 H2O
NH4
ammonium
NO3- ? NO2-
NO ? N2O ? N2
nitrate
nitrite
molecular nitrogen
Nitrous oxide
nitric oxide
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sulfanilic acid (reagent A) dimethyl-alpha-napht
hylamine (reagent B)
1. A B nitrite red 2. Zinc converts
nitrate to nitrite
add zinc to negative tubes
Nitrate to nitrite
Complete reduction
No reduction
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Simmons Citrate Agar Contains citrate as sole
carbon source, ammonium salts as sole nitrogen
source, bromthymol blue pH indicator neutral pH
green, alkaline prussian blue Discriminates
organisms that can produce citrase to metabolize
citrate into oxaloacetate and pyruvate. These
organisms are forced to utilize ammonium salts
as the nitrogen source producing alkaline ammonia
waste. Results Prussian blue slant and or
butt positive for citrase
production Green negative for citrase
production
_

Citrate?Oxaloacetate ? Pyruvate ammonium
salt? Fermentation ?Alkaline pH
Control Enterobacter cloacae
10
Oxidase Test Discriminates organisms that can
produce cytochrome oxidase which catalyzes the
transfer of electrons from reduced cytochrome c
in the electron transport chain to molecular
oxygen. Test uses NNNN-tetramethyl-p-phenylenedi
amine (Oxidase Reagent) as an artificial
electron acceptor when oxidized it is colorless,
when reduced it turns purple
Look for color change on the bacteria, not on
the cotton swab! (The reagent will turn light
purple when exposed to oxygen in the air)
11
Catalase Test Discriminates aerobic organisms
that produce catalase to degrade hydrogen
peroxide into water and oxygen
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http//ftp.ccccd.edu/dcain/CCCCD20Micro/Catalase.
jpg
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12 Possible Unknowns
Gram Positive
Gram Negative
Gelatinase
Gelatinase -
Gelatinase
Gelatinase -
Catalase
Catalase -
Pseudomonas aeruginosa
Bacillus subtilis
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Exercise 40 Hydrolytic and Degradative
Reactions Set up according to Fig 40.1 except
both of your two unknowns well separated on
each type of plate and control on a different
plate (one control plate per pair) Save
streak plates of unknowns for use next class
14
Fig. 40.1
Unknowns
Separate Plates!
broth
Control
broth
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• Each pair needs
• 3 Starch plates
• 3 Skim milk plates
• 3 Spirit blue plates
• 5 Urea broths (replaces urea slant)
• 5 Phenylalanine slants
• 5 Tryptone broths
• One set of controls per pair using broth
  cultures
• Bacillus subtilis
• Staphylococcus aureus
• Escherichia coli
• Proteus vulgaris

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Title Identification of an Unknown Bacterial
Culture Laboratory Report

• Introduction ( Why the experiment is important? )
• State a hypothesis (an if then statement, may
  require multiple sentences) that is clear and
  appropriately addresses the purpose of this
  laboratory exercise.
• Materials and Methods (How each of the assay was
  performed?)
• Describe each essential assay separately in a
  separate paragraph.
• Media used to grow the organism and list any
  reagents or indicators
• Do not include the enzymatic reactions here save
  those for the Discussion
• Results (What are the results observed?)
• Include only the key assays, not every test that
  was performed. In cases where multiple assays
  were employed to determine one characteristic,
  only one type of assay need be presented.
• Discussion ( Discussion of the assays and
  interpretation of the results)
• Discuss only the assays for which you presented
  results in the table in the
• List these enzymes, substrates, products, and
  color change
• Conclusions (Interpretation of your collective
  results)
• Analyze the results and determine the
  identification of the unknown based on the
  collective results follow the dichotomous key.
• Approve or disapprove the hypothesis