Micro Lab Exam 2 Flashcards
What are coliforms? Characteristics?
Members of the family Enterobacteriaceae
- Enterobacter, Klebsiella, Citrobacter, Escherichia
-Gram negative
-Non-spore forming rods
-Lactose fermenting (w/ acid and gas production)
-Part of normal enteric (gut) flora
-Class of bacteria found in the environment and feces of warm blood animals
What are non-coliform enterics?
Members of the family Enterobacteriaceae that…
-Do NOT ferment lactose (or ferment very slowly)
-Part of enteric flora or true pathogens
What are coliforms used for with water?
Bacterial indicator of the sanitary quality of food and water. Drinking water must be free of disease-causing organisms called pathogens.
To actually test water for specific harmful viruses, protozoa, and bacteria is very time-consuming and expensive. Testing water for specific organisms is limited to investigating specific waterborne disease outbreaks.
What are the two main reasons coliform bacteria are used as water quality indicators?
-Coliforms may be associated with the source of pathogens contaminating water
-The analysis of drinking water for coliforms is relatively simple, economical, and efficient
Multiple Tube Fermentation Method
How do we measure coliforms?
Standardized test used to estimate the total number of coliforms in a sample
It involves inoculating multiple series of broth tubes that also contain Durham tubes
After incubation the tubes are observed for bacterial growth and gas production
The number of tubes that are positive are cross referenced to a standardized table that will indicate the most probable number of coliforms per 100 mL of sample (MPN).
Soil is a storehouse for microorganisms. What are the three groups of microorganisms.
-Typical bacteria
-Actinomycetes: important organic matter decomposers and humus producers
-Fungi: some are plant and animal pathogens. Most are harmless saprophytes that decompose dead or dying organic matter.
Glycerol Yeast Entract Agar (GYEA)
Media used for this exercise are designed to isolate specific groups of
Designed for actinomycetes
Not enough nutritive value for typical bacteria or fungi
Tryptic Soy Agar (TSA)
Designed for a broad range of bacteria but not fungi
Media used for this exercise are designed to isolate specific groups of
Sabouraud Dextrose Agar (SDA)
Designed for** fungi** but will also support bacteria
Ampicillin/Streptomycin added to discourage bacterial growth
Media used for this exercise are designed to isolate specific groups of
What are antimicrobials?
Term used to describe ALL microbial agents, both natural and synthetic, used to treat bacterial infections
-Antibiotics
-Bactericidal vs. Bacteriostatic
Antibiotics
Natural antimicrobial agents produced by microorganisms
Example: penicillin, which is produced by the mold Penicillium notatum
Bactericidal vs. Bacteriostatic
Bactericidal: kills the microbe
Bacteriostatic: stops the growth but does not kill the microbe
What’s the Kirby-Bauer test, or disk diffusion test? Procedure?
Standard tool for measuring the effectiveness of antimicrobials against
pathogenic microorganisms
Procedure:
-A plate is inoculated to form a “lawn” of bacteria
-Disks coated with antimicrobials are placed on a plate
-Plates are incubated to allow growth of the bacteria and time for the agent to
diffuse
-If the organism is susceptible, a clear zone, where growth is inhibited, will appear around the disk
–**Size of the “zone of inhibition” **depends upon the sensitivity of the bacteria to the
specific microbial agent
Brilliant Green Lactose Bile Broth
What is it made of? Purpose?
**Selective and Differential Medium **
Contains bile and brilliant green dye which both inhibit the growth of Gram (+) organisms (selective)
Contains lactose as the main carbon source for fermentation, only organisms that can ferment lactose will grow (differential)
Whats a postive and negative test of the Brilliant Green Lactose Bile Broth?
A positive result is indicated by
turbidity (cloudiness) in the broth
and a gas bubble in the durham
tube
A negative results is indiacted by turbidity (Cloudiness) and no gas
How do you calculate Orginal cell density (CFU/g)
= (# of colonies/(vol*dilution))
*(100ml/10g)
The effect of temp of microbial growth
Each species classified by its cardinal temps.
Minimum, Maximum, Optimum
–Temperature at which an organism shows the highest growth rate
Thermophiles
adapct to temps >45 C
Obligate Thermophiles
will not grow at tempures <40C
Faciltative thermophiles
will grown below 40C
Extreme thermophiles
grow best above 80C
What is the order of all the -philes?
(Lowest)
Psychromphiles (-5-15C)
Psythchrotrophs (-2-35C)
Mesophiles (15-45C)
Thermophiles (41-80C)
Hyperthermophiles (65-105C)
The Effect of pH on icrobial Growth
what is pH?
Conventional means of expressing the [H+] in solution
–[H+] increases as the pH decreases
Logarithmic scale with a range of 0-14
pH= -log[H+]
What pH do acidophiles live in?
live below pH 5.5 (acidic pH)
What pH do Neutroiphiles live in?
prefer pH between 5.5 and 8.5
What pH do Alkaliphiles live in?
live above pH 8.5 (alkaline or basic pH)
Regardless of their habitat, bacterial maintain a ______
near-neutral internal enviornment
Why are pH changes lethal to a cell?
pH changes outside an organism’s range may destroy membrane potential and denature vital enzymes
Culturing in* vitro*
What can destory an enzymes integrity?
Acids from carbohydrate fermentation and alkaline products from protein metabolism are enough to destroy enzyme integrity
Buffers are added to growth media maintain equilibrium
Aerobic and aneorobic
organisms ability or inability to live inthe presence of oxygen. Aerobic in presence of oxygen. Anaorobic takes palce in absence of oxygen.
Thioglycollate
used to determine if an organism is aerobic or anaerobic by observing its growth patterns in the media
Classification of organisms
Obligate (strict) aerobes
organisms that require oxygen for respiration. Will grow at the top of the media where oxygen is most plentiful
facultative anaerobes
grow in the presence or absence of oxygen. when an oxygen gradient exisits, these organisms will grow throughout the gradient. Will grow throughout the medium (making it all cloudy) but will appear denser at the top.
obligate (strict) anaerobes
organisms for which even small amounts of oxygen are lethal. only grows in the bottom part of the media where there is no oxygen (the top blue part is clear and indicates no bacterial growth)
aerotolerants
organisms that don’t require oxygen and are not adversely affected by it. Will grow uniformly throughout the medium
microaerophiles
survive only in environments containing lower than atmospheric levels of oxygen
How is oxygentiated area indicated in the test tube?
the blue line of media at the top
Fluid Thioglycollate Medium
Appropriate for a broad variety of aerobic and anaerobic microorganisms
Oxygen removed during autoclaving will
diffuse back into the medium as the tubes
cool to room temperature
–Small amount of agar included slows oxygen diffusion
–Produces a gradient of oxygen concentrations from fully aerobic at the top to anaerobic at the bottom
Anaerobic jar
plastic jar to create anaerobic, microaerophilic or CO2-rich conditions
components:
-chemical gas generator packet with NaBH4 and NaHCO3, palladium as a catalyst
-paper indicator strip saturated with methylene blue (O2 indicator)
Eosin Methylene Blue Agar
Selective and differential medium
Inhibits growth of Gram (+) organisms
Differentiate enterics based on fermentation
What are the different colors for fermentation on the Eosin Methylene Blue Agar?
Non-lactose fermenters = normal colored colonies
Vigorous Lactose fermenters = dark purple to black colonies
Less Aggressive Lactose fermenters = pink to dark purple colonies
How does the Eosin Methylene Blue Agar differentiates different coloforms based on color?
E.coli = dark colonies with a metallic green sheen
Enterobacter/Klebsiella= pink to dark purple colonies
Hektoen Eneric Agar
Selective and differential medium
Inhibits growth of Gram (+)
organisms
Enterics that ferment 1 or more
carbohydrates turn yellow or
salmon colored
Hydrogen sulfide reduction
produces black precipitate
How does Hektoen Enteric Agar isolate Salmonella and Shigella from other enterics by color?
Salmonella = black colonies
Shigella = green colonies
Other coliforms = yellow or salmon
colored colonies
Bacterial Transformation
The process by which competent bacterial cells pick up DNA from the environment and make use of the genes it carries
Bacterial Transformation
Operons
Operons are closely linked genes that help to regulate protein synthesis
Each includes a promoter and structural genes that encode for enzymes
Arabinose operon
Promoter (Pbad)
Three structural genes (araB, araA, araD) - Code for enzymes used to digest arabinose
araC is a binding protein that attaches to the promoter – “on/off switch”
What happens if arabinose is present?
If arabinose is present to be digested, it binds to araC , changes its shape, and then enables RNA polymerase attach and transcribe the genes that come after the promoter. The organism can then produce
enzymes that digest the arabinose.
What happens if arabinose is not present?
If arabinose is not present there is nothing to bind to araC to shape its shape. Therefore RNA polymerase cannot attach and the enzymes won’t be produced.
Green Fluorescent Protein (GFP)
Introduce Green Fluorescent Protein (GFP) (protein responsible for bioluminescence in jellyfish) into competent E. coli cells
–GFP is carried on a pGLO plasmid (vector used to introduce the GFP gene into recipient E. coli cells)
–Plasmid also contains antibiotic resistance gene (confers resistance to
ampicillin) to differentiate between cells that were successfully
transformed from those that were not
Ability of the cells to fluoresce (i.e. glow green) will provide visual
evidence of transformation and subsequent gene expression
