Download Lab 11

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***
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
Acid plus gas
Negative
Acid
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
_
MR-VP Medium: Voges-Proskauer Test
Contains: peptone and glucose
Additional reagents added: Barritt’s A (alpha napthol) and Barritt’s 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
_
+
_
Nitrate Reduction Broth
Contains: beef extract, peptone, KNO3 as nitrate source, durham
tube for gas collection
Additional reagents added: sulfanilic acid (reagent A), dimethylalpha-naphthylamine (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
Anaerobic respiration - Nitrate reduction
Electron acceptor
Products
NO3–
NO2–, N2 + H2O
SO4–
H2S + H2O
CO32 –
CH4 + H2O
NO3-  NO2nitrate
nitrite
NH4
ammonium
NO  N2O
nitric oxide
Nitrous oxide

N2
molecular nitrogen
sulfanilic acid (reagent A) + dimethylalpha-naphthylamine (reagent B)
1. A + B + nitrite = red
2. Zinc converts nitrate to nitrite
Nitrate to nitrite
add zinc to negative tubes
No reduction
Complete
reduction
Simmon’s 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
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-phenylenediamine (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)
Catalase Test
Discriminates aerobic organisms that produce catalase to degrade
hydrogen peroxide into water and oxygen
+
_
http://ftp.ccccd.edu/dcain/CCCCD%20Micro/Catalase.jpg
12 Possible Unknowns
Gram Negative
Gram Positive
Bacillus subtilis
Catalase +
Gelatinase -
Gelatinase +
Catalase -
Pseudomonas aeruginosa
Gelatinase +
Gelatinase -
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***
Fig. 40.1
Unknowns
Separate Plates!
broth
Control
broth
• 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
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
•
Literature Cited
– List here all source material cited in the laboratory report. Use proper CSE format for
scientific publications including listing the sources in this section in alphabetical order by
the first authors last name