Lab 6: Bacterial Enumeration Flashcards
What is the primary stain of gram stains?
Crystal violet
What is the primary stain step of acid fast stains?
Carbol fuchsin flood smear (paper towel)
What is the primary stain step of endospore staining?
Malachite green flood smear (paper towel)
What is the mordant step of a gram stain?
Iodine
What is the mordant step of an acid fast stain?
Heat carbol fuchsin to 90-95 degrees C (steaming) for 3.5 minutes, then rinse with water
What is the mordant step of an endospore stain?
Heat malachite green to 90-95 degrees C (steaming), rinse with water
What is the decolorization step of a gram stain?
95% alcohol
What is the decolorization step of an acid fast stain?
Add alcohol dropwise for 20-30 seconds, rinse with water
What is the decolorization step of an endospore stain?
There isn’t one
What is the counterstain step of a gram stain?
Safranin
What is the counterstain step of an acid fast stain?
Methylene blue for 1 minute
What is the counterstain step of an endospore stain?
Safranin for 1 minute, rinse with water
What are the steps of an endospore?
1) Primary stain: Malachite green flood smear (paper towel)
2) Mordant: Heat at 90-95C (steaming), keeping paper towel wet, for 3.5 minutes. Rinse with water.
3) Counterstain: Safranin for 1 minute, rinse with water
What are the steps to an acid fast stain?
1) Primary stain: Carbol fuschin (paper towel) flood smear
2) Mordant: Heat to 90-95C for 3.5 minutes, rinse with water
3) Decolorization: Add alcohol dropwise 20-30 seconds, rinse with water
4) Counterstain: Methylene blue for 1 minute
What are the acid fast colors?
Acid fast positive = purple/ red
Acid fast negative = blue
How do you make bacteria make spores?
Leave them in an incubator for a week or more
Would you ever acid-fast stain a species that gram-stains well?
No
How do you calculate stepwise dilution factor? Give an example
1 divided by the volume of dilution, then convert to a decimal.
-Ex: 1: 1ml + 99ml equals 1:100, then 1/100 = 0.01, which = 10^-2
1) How do you calculate total dilution factor? Give an example
2) What is the typical initial total dilution factor of the culture?
1) Stepwise dilution factor + previous total dilution factor
-Ex: 10^-2 x 10^-4 = 10^-6 (adding not multiplying)
2) Typically 10^0 for the culture
In the 1890s, we learned the causative agent of what two things? What was the causative agent? Why wasn’t this very beneficial at first?
1) Typhoid fever and cholera
2) Causative agent was mammalian feces
3) Monitoring those agents was time consuming
What technique was created to monitor for potentially contaminated water?
Monitoring fecal contamination with an indicator species called coliforms
What do coliforms do?
1) Indicate fecal contamination
2) Lactose fermentation (usually within 24 hours)
Define lactose fermentation
The production of acid + gas
Historically, the issue of enumerating bacteria arose due to what?
Public health concerns for clean, uncontaminated water, which led to attempts to monitor water quality
How is water quality counted?
Using MPN tests with phenol red lactose fermentation
What are other ways to count bacteria? (3)
1) Serial dilutions
2) Viable plate counts
3) Hemocytometer (graph thing)
When does phenol red turn yellow?
At pH greater than or equal to 6
What does an MPN test do, and what does MPN stand for?
Tests water quality; stands for ‘most probable number’
What are the 3 parts of an MPN test?
1) Presumptive: data generated
2) Confirmed: data verified
3) Complete: published and acted upon (recommendations, policy, etc)
What is the formula to calculate the number of bacteria in the original culture (cells/mL)?
Number of colonies divided by total dilution factor
True or false: The number of colonies on a plate is the minimum number of cells originally put on that plate
True
The statistically valid range of colony counts depends on what?
The size of the plate
What range is considered a reliable colony count?
30-300
How
