Lab Exam 2 Study Guide
The lab exam will be half short answer questions, half practical questions. It will be
worth a total of 50 points. The practical questions will consist of examples of lab cultures
we grew and observed in our lab exercises as well as microscope specimens prepared
with stains. You will be asked to look at them and answer two or three questions based on
each culture or specimen. Below are the general concepts I want you to focus on. It is up
to you to know these are. Bold terms are especially important.
Ex. 3-10
Know the steps in the Spore Stain and their purpose. Malachite green, heat, water,
safranin.
Ex. 3-8
As above, understand Acid Fast Staining. This helps identify mycobacteria. Carbol
fuchsin, heat, acid alcohol, methylene blue.
For the rest of the exercises you need to understand the principle of the test and how to
read the results. Know what selective and differential media are. A good way to study
for this is to create a chart that includes the media, important ingredients, purpose,
important reactions, and expected results. Here is an example:
Test
MR-VP
Citrate
Ingredients
Purpose/Enzyme
Reaction
Results
Glucose,
methyl red
(add later)
Glucose,
Barritt’s A +
B (add later)
Citrate,
bromphenol
blue
Detect glucose
fermentation to acid
byproducts
Detect fermentation
to NON-acid
byproducts
Detects enzyme
citrase and citrase
permease
Red ring on top +
Yellowish -
None
Acids change methyl
red pH indicator to
red
Acetoin reacts with
Barritt’s to form a red
color
CO2 released forms
sodium citrate which
reacts with
bromphenol blue
2H2O2  2H2O + O2.
Bubbles from O2
Detects catalase.
Differentiates Staph
from Streps.
Inhibits nonhalophiles Hypertonicity. Staphs
(selects for Staph.)
resists osmotic
pressure
Detect mannitol
Acids change phenol
fermentation to acids red to yellow
Catalase
NaCl
MSA
Mannitol,
phenol red
Red ring on top +
Yellowish Blue media +
Green -
Bubbles formed +
No bubbles Growth +
No growth Yellow media +
Red media -
Exs. 4-4, 4-5, 5-3
MSA – ingredients are mannitol, phenol red, NaCl. Selects for halophiles. Yellow is
positive for mannitol fermentation.
MacConkey – ingredients are lactose, neutral red, crystal violet, bile salts. Inhibits
Gram+. Red/pink is positive for lactose fermentation.
Phenol Red Broths – ingredients sugar (lactose, glucose, or sucrose), phenol red,
durham tube. Growth means sugar utilization. Fermentation to acid is yellow,
fermentation to gas gives air gap in durham tube.
Ex. 5-5, 5-6
Catalase turns hydrogen peroxide to water and oxygen. Bubbles are positive for this
reaction. Staphylcoccus are positive while Streptococcus are negative.
Oxidase is an electron transport chain enzyme. Oxistrips have an artificial substrate
(TPPD) that turns purple when oxidase oxidizes it. Aerobic organisms are positive while
enterics are negative.
Exs. 5-12, 5-14, 5-15, 5-17
Understand the concept of an exoenzyme. Amylase breaks down starch. Plate is read by
flooding with iodine to see zone of clearing. Casease breaks down casein and lipase
breaks down lipids. These plates are directly read by seeing the zone of clearing.
Gelatinase breaks down gelatin. Tubes are chilled before reading. Liquifaction is
positive for this test.
Ex. 5-4, 5-7
MR-VP – main ingredient is glucose tubes. Methyl red is added to detect acid
byproducts. Red ring means positive. Barritt’s reagents A and B are added to detect
non-acid byproducts (acetoin). Red ring means positive.
Nitrate Reduction – main ingredient is nitrate in tubes. NO3- (nitrate)  NO2- (nitrite)
 N2 (nitrogen gas) or NH3 (ammonia). Add reagents A and B, if red then you had
nitrite. If colorless, add zinc (turns nitrate to nitrite). Now if red, then you had nitrate. If
still colorless, then you had nitrogen gas or ammonia.
Ex. 5-8, 5-13, 5-20
Citrate – ingredients are citrate, bromthymol blue. Citrase breaks down citrate. Blue is
positive for alkaline product.
Urea – ingredients are urea, phenol red. Urease breaks down urea. Hot pink is positive
for the alkaline product ammonia.
SIM – ingredients are ferrous sulfate, tryptophan, agar deep. Sulfur – if hydrogen
sulfide gas (H2S) is produced, it combines with ferrous sulfate to give black precipitate.
Indole – produced if tryptophan is metabolized. Add Kovac’s reagent. Positive is a red
ring. Motility – cloudiness away from stab line is positive.