Lab Practical 2 Flashcards
Why do you want to count microbes only from a plate with 30-300 colonies?
Only numbers between 30-300 are considered statistically valid. If greater then 300, there is a probability of overcrowding and inhibits cell growth. Less then 30 involves sample error
Why are population counts important?
Allows one to see which organisms are alive and growing
What are the advantages of the standard plate count?
Easy to use
Can be used for a number of different organisms
Universally used
What are the disadvantages of standard plate count?
Can get artificial numbers, both dead and alive organisms are counted
Obligate (strict) aerobes
Require oxygen for growth because they carry out respiratory metabolism in which oxygen functions as a terminal electron acceptor.
Microaerophiles
Bacteria prefer to grow in oxygen concentrations of 5-10% rather then 20%
Example: helicobacter pylori
Facultative aerobes
Bacteria can grow either aerobic or anaerobic depending on culture conditions
Example: e. Coli
Aerotolerant anerobes
Can grow in the presence of oxygen and are not usually harmed by its presence in the environment. Metabolism doesn’t require oxygen but involves fermentation
Obligate (strict) anaerobes
Bacteria cannot tolerate oxygen and must be cultivated under conditions where oxygen is removed
Example: clostridium
Superoxide dismutase
Which converts superoxide to oxygen and hydrogen peroxide
Peroxidase
Detoxifying enzyme
Fluid thioglycollate medium (FTM)
Rich liquid that supports the growth of both aerobic and anaerobic bacteria. Contains a dye called resazurin
Resazurin
An indicator in the FTM for the presence of oxygen. The dye becomes pink if oxygen is present. The medium will become pink at the top and colorless in the middle and the bottom
Brewers anaerobic agar
Is a solid medium for culturing anaerobic bacteria in a Petri dish.
GasPak anaerobic jar
Provides an oxygen free incubation environment for the Petri dishes of anaerobic agar.. Hydrogen is generated in the jar which removes the oxygen by forming water. Palladium pellets catalyze the reaction at room temperature
In a purple Petri dish,Purple indicates?
Oxygen is present
In a Petri dish, if it is clear it indicates?
Oxygen is not present
Aerobic growth in a test tube
Located at the top of the tube
Microaerophillic growth in a test tube
Located in the middle of the test tube
Facultative growth in a test tube
Located throughout the entire tube
Anaerobic growth in a test tube
Located in the bottom of the test tube
Cultivation and identification of anaerobes temp and for how long
32 degrees C
24-48 hours
Effects on temperature on growth lab temp and for how long
One incubator 37°C Another incubator 55°C Refrigerator 10°C Room temperature 22°C 24-48 hours
Psychrophile
Cold loving
Will grow in temperatures -5°C to 20°C
Not infectious to humans
Responsible for spoiling food and frozen foods
Mesophiles
Middle loving
Grow in temperatures of 20°C to 50°C
Optimum temperatures for human pathogens are around 37°C
Thermophiles
Heat loving
Will grow in temperatures 50°C to 80°C
Found in natural hot springs
Hyperthermophiles
Extreme heat
Will grow in temperatures above 80°C
Halo tolerant
Bacteria that can tolerate moderate concentrations of salt up to 10%
Halophilis
Bacteria that require high levels of salt. 15-30% to live and grow
Losmophiles
Bacteria that will grow in environments in high concentrations of sugar
Sugar fermentation experiment temperature and length of incubation
37°C
48 hours
Ferment manitol
Durham tube (upside down tube within a tube): indicates the presence of gas that was produced as a result of fermentation
Contains a phenol red pH indicator
**begins red
If no color change: no fermentation occurred
Color change to yellow: fermentation occurred
MR-VP (methyl-red vogues proskauer) test
Broth
Results: 2 tubes, the original and one with the 1ml of broth
In the original tube: add methyl red indicator, will turn red if pH dropped below 5
(Red= mixed fermentation)
Tube with 1ml broth: add VP solution A and VP
If it turns pink/red alcohol fermentation occurred
**always a +MR/-VP or -MR/+VP result
Catalase production experiment
37°C for 48hours
Use an agar plate
Drop hydrogen peroxide on plate if bubbling occurs then it is positive for catalase
Citrate utilization experiment
Uses Simmons agar slant (starts dark green)
If a positive change occurs it will change to a blue
Negative- remains green
Starch hydrolysis
Determines if bacteria have enzyme called amylase that breaks down starch to sugar
Results: flood starch agar plate with grams iodine (IKI), IKI turns starch to blue or a dark color
After 2 minutes if the zone is clearing (zone of clearing) turns to a lighter color around the colonies then it is positive
Urea hydrolysis
Determines if bacteria has the enzyme urease that breaks down urea into ammonia.
Results:
The tube contains phenol red pH indicators (nothing added)
*positive: broth turns red/purple, presence of urea
*negative: no color change
Tryptophan hydrolysis
Determines if bacteria have enzyme called tryptophanase that break down the amino acid tryptophan into insole and pyruvate
Add indole reagent to tryptophan broth
Results:
Positive: indole is present if red layer is at the top of the tube
Kilger’s iron agar
Experiment that determines if bacteria are capable of conducting acidic fermentation with glucose and lactose and if they produce sulfide gas from amino acid cysteine
Results for Kilger’s iron agar
- bottom: yellow, top: red then glucose was used during fermentation. If it is split apart then gas was produced
- Entire tube is yellow: glucose and lactose were both used during acidic fermentation . If the solid material is split then it gas was produced
- tube turns black: bacteria can degrade the amino acid cysteine into pyruvic acid, ammonia and hydrogen sulfide
Dichotomous key
The key always has two answers for each question asked, determines the next question or test
Why is UV light lethal to bacteria?
.
Is UV light harmful to humans?
.
Covered vs. uncovered Petri dishes
The card or the lid blocks the UV light which allows more bacteria to grow
What is thermal death time?
The time required to kill a suspension of cells or spores at a given temp
What is thermal death point?
Temp at which an organism is killed in 10 minutes or when the microorganisms start to die
Why is a control plate used?
Use the control to compare the results of the tests
What does “use dilution” refer to?
Moving away from the source
In this experiment, the drug moving away from the disk, and affecting the growth/death of bacteria
Is the “use dilution” or “paper disc” method more accurate?
The “use dilution” method is more accurate
What is the zone of inhibition?
The area around the disk with no bacteria
Which antibiotic is most effective? Against what bacteria? And how can you tell?
Iodine
Drugs used in the antibiotic evaluation experiment
Erythromycin: narrow spectrum, difficulty entering gram negative
Streptomycin: narrow spectrum, prevents formation of cell walls in gram positive cells
Sulfonamides: competitive inhibitor, must be used in high concentrations to be truly effective
Tetracycline: disrupts protein synthesis, very broad spectrum
