Quiz 2 Study Guide

Optimum Growth Temperature

Be able to list and define groups of bacteria based on preferred temperature range.

Psychrophiles: -5°C to 15°C

Psychrotroph: 20°C to 30°C

Mesophile: 25°C to 45°C

Thermophile: 45°C to 70°C

Hyperthermophile: 70°C or greater

Fermentative Patterns

Plate tests: know all reagents present in each type of plate and their function

Carbohydrate fermentation (Lactose)

Several selective and/or differential agars can be used to determine whether an organism is able to
ferment lactose.

Eosin Methylene Blue (EMB) Agar: Grows Gram Negative Bacteria, differentiates between groups of
Enteric Bacteria

 Contains peptone, lactose, sucrose, and the dyes eosin Y and Methylene blue.
 Selective because the dyes inhibit growth of Gram-positive bacteria.
 Differential media for bacteria that have enzymes to allow lactose fermentation; acid is
produced and the change in pH will cause the colony to be colored.

Hektoen Enteric (HE) Agar: Designed to specifically detect and differentiate between Salmonella and
Shigella from other Enteric Bacteria.

 Used for isolation and identification of Gram-negative bacteria that are found in feces.
 Ability to utilize lactose, sucrose or salicin
 The ability to reduce sodium thiosulfate to hydrogen sulfide gas (H2S)
 Bromothymol blue and acid fuchsin dyes are added as colored pH indicators that detect acid
production from the fermentation of sugars (differential characteristic).
 Bile salts are included to inhibit most Gram-positive bacteria (selective characteristic)

MacConkey (MAC) Agar: Gram Negative will grow, selects for Enteric bacteria

 Designed to grow Gram- negative bacteria and stain them for lactose fermentation.
  It contains bile salts, crystal violet dye (inhibits Gram-positive bacteria, selective characteristic),
neutral red dye (stains microbes fermenting lactose), lactose and peptone.
 Neutral red dye is an indicator that is colorless above a pH of 6.8 and red at a pH below 6.8.
 Acid accumulation from lactose fermentation turns the dye red.

Starch Utilization

Starch Agar:

 Used to test for the breakdown of starch by amylase.

 Medium contains beef extract and peptone to support growth, soluble starch and agar.
 Clearing in medium = starch hydrolysis
 No clearing in medium = No starch hydrolysis

Carbohydrate Fermentation and Gas Production – Broth

 The medium is a basal recipe in which a fermentable carbohydrate is added (i.e., glucose,
lactose etc.).
 Phenol Red Broth includes peptone and the pH indicator phenol red (PR) in the basal media.
 PR indicates whether or not fermentation is occurring.
 Yellow broth indicates fermentation with acid end products,
 Pink broth indicates Degradation of peptone; alkaline end products
 Red indicates no fermentation
 To test for gas production it is necessary to invert a Durham tube in the PR Broth. Bubble in
tube indicates gas production.

Enzyme Induction in E. coli

Understand the use of a positive and negative control

 Positive Regulation - Occurs when the DNA-binding form of a protein works to turn a gene on.
These proteins aid RNA polymerase in binding to the promoter region. CAP (Catabolite Activator
Protein) is the protein responsible for turning the Lac operon on, leading to gene expression.

 Negative Regulation - Occurs when the DNA-binding form of a protein works to turn a gene off.
These proteins work to inhibit the binding of RNA polymerase to the operon.

Know all reagents used and their function

Function and regulation of Lac operon and be able to predict if on/off based on culture media
  The Lac operon codes for proteins required to transport lactose into the cell and break it down
to glucose and galactose.
 The operon is activated in the presence of lactose (and low levels of glucose) and the β-
galactosidase enzyme is synthesized following the induction of the lac operon.

Draw and label a diauxic growth curve:

First glucose is use, followed by lactose.

Human Microbiome

Mannitol Salt Agar: Determines if salt tolerant or not and if organism can ferment mannitol.

Grows Staphylococci

 Selective and differential

 High salt concentration NaCl
 Grows halophilic bacteria, Selective.
 Contains a pH sensitive dye Phenol red, as well as the sugar alcohol mannitol.
 Bacteria that ferment mannitol (acid waste products) will grow and turn MSA yellow
 Bacteria that do not ferment mannitol will grow but MSA will remain pink

Lactic Acid Bacteria: know composition and use of YDC plates

Yeast Dextrose Calcium Carbonate Agar (YDC):

 Differential agar that determines whether or not the plated organism produces lactic acid
 CaCO3 in the YDC plates can be broken down to CO2 gas by lactic acid, producing a
clearing
 Clearing is the indicator for lactic acid positive bacteria.