Flashcards in Lab Practical 2 Deck (70):
fluid thioglycollate media
what does thioglycollate do
reacts with oxygen to remove it to create an anaerobic environment
What does red indicate in thyioglycollate media?
Resazurin is added to FTM, if it turns red/pink it indicates the presence of oxygen
a type of agar with methylene blue added.
Blue color indicates specimen has been in oxygenated environment. If placed in gaspak jar, it should turn clear if in anaerobic environment
creates an anaerobic environment - packets are put inside that get rid of oxygen
-Have SOD and catalase
-must have oxygen for metabolism (aerobic respiration) and growth
-Most fungi and protists, some bacteria (Bacillus, Pseudomonas)
-Usually have SOD and catalase
-“flexible” – can survive with or without oxygen
-Grow best with oxygen (aerobic respiration)
-E. coli, Staphylococcus and Saccharomyces (yeast)
-Only small amounts of SOD and catalase
-Need small concentrations of oxygen (2-10%) for aerobic respiration
-Large amounts of oxygen are inhibitory
-Found in mucous linings of hollow organs
-Usually lack both SOD and catalase
-Cannot grow if oxygen is present
-No aerobic respiration
-deep mud, lakes, oceans, inside animal bodies
- May have SOD, but not catalase
- Indifferent to oxygen
- Do not use oxygen – obligate fermenters
- Streptococcus pyogenes
degrates hydrogen peroxide into oxygen and water
converts superoxide ion to oxygen and hydrogen peroxide
breaks down hydrogen peroxide to water with the help of NAD+
inorganic compounds such as sulfate or nitrates replace oxygen as the terminal electron acceptor
results of growth patterns of FTM
Aerobic - grows only on top
Microaerophilic - towards top but not exposed to oxygen
Facultative - throughout media
Anaerobic - in the bottom of media
*Add lab 13
*study lab 13
-cold loving, - 5C to 15C
-Grow in polar and glacial regions
-“cold feeding,” 20C to 30C
-do not cause infections in humans
-responsible for spoiling of refrigerated and frozen food
-middle-loving, 25C to 45C
-optimum temperature for human pathogens is around 37C
- heat loving, 45oC to 70oC
-Found in natural hot springs, compost
-extreme heat loving, 70oC +
-usually Archaea, hydrothermal vents
Bacteria usually live in what kind of solution
cell wall gives protection from high osmotic pressure
what will bacteria do to maintain their environment
-Bacteria will maintain hypotonic environment outside cell
-Pump in K+ or produce extra amino acids
needs hypotonic environment
- up to 10% NaCl
- can tolerate moderate concentrations of salt
- Staphylococcus on skin
- (obligate halophiles)
- require a high level of NaCl
- marine microbes, extreme halophiles are Archaea living in salt lakes
grow in high sugar concentrations
Temperature Requirement Classifications (5)
1. Psychrophiles (cold-loving)
2. Mesophiles (middle-loving)
3. Thermophiles (heat-loving)
Classification using solute concentration (4)
2. Halophilis (obligate halophile)
Oxidation & Fermentation Reactions (4)
1. Sugar fermentation (Ferment Manitol, Glucose, Lactose)
2. MR-VP (Methyl Red - Vogues Proskauer) Test
3. Catalase Production Experiment
4. Citrate Utilization Experiment
Sugar fermentation (Ferment Manitol, Glucose, Lactose)
-Durham tube (upside down tube w/in a tube)
*Indicates the presence of gas that was produces as a result of fermentation (+/-)
-Each tube contains a phenol red pH indicator
**Tube begins red:
-NO color change= NO fermentation occurred
-Color changed to yellow= fermentation occurred
MR-VP (Methyl Red - Vogues Proskauer) Test
-two tubes (original & new w/ 1 ml of broth) & 2 solutions to add.
-Original tube: add methyl red indicator: Will turn red if pH dropped below 5 (red= mixed acid fermentation occurred)
-Tube w/ 1 ml: add VP solution A (15) & VP solution B (5): If turns PINK/RED = 2,3 butanedial and ethanol fermentation (instead of acid fermentation) occurred.
**always a + MR/- VP or -MR/+ VP result**
Catalase Production Experiment
-Uses nutrient agar plate
-->Results: Drop hydrogen peroxide on plate; if bubbling occurs = catalase positive
Citrate Utilization Experiment
-Uses Simmons agar slant (starts dark green) - double inoculate; stab with needle, smear slant
-->Positive= Change to blue
-->Negative= remains green
-refers to bacteria using enzymes to do catabolic chemical reactions.
1. Starch Hydrolysis
2. Urea Hydrolysis
3. Tryptophan Hydrolysis
-experiment determines if bacteria have enzyme called amylase that breaks down starch to sugar.
Starch Hydrolysis Results
Results--> Flood starch agar plate with Grams Iodine (IKI), IKI turns starch to a blue or dark color.
*Positive= “zone of clearing” (lighter coloration) around the colonies
-experiment determines if bacteria have enzyme called urease that breaks down urea into ammonia.
Urea Hydrolysis Results
Results--> Tubes contain phenol red pH indicators (nothing added to tube)
*Positive= broth turns red/purple (indicates presence of urea)
*Negative= NO color change
-experiment determines if bacteria have enzyme called tryptophanase that break down the amino acid tryptophan into indole and pyruvate.
Tryptophan Hydrolysis Results
Results--> Add “indole reagent” to tryptophan broth
*Positive= Indole is present (and tryptophan broken down), RED layer will form at top of tube.
Kligler’s Iron Agar
-experiment determines if the bacteria are capable of conducting acidic fermentation with glucose and lactose and if they produce hydrogen sulfide gas from amino acid cysteine.
Kligler’s Iron Agar Results
Results--> Possible color changes in tube:
*Bottom YELLOW, top REDDISH= glucose used during fermentation
*Entire tube YELLOW= glucose and lactose used during fermentation
*Tube turns BLACK= Amino acid cysteine into pyruvic acid, ammonia, and hydrogen sulfide
-used to identify unknown bacteria.
- the key always has two answers for each question asked, determines next question or test.
thermal death time
-time required to destroy a population of bacteria at a specific temperature
thermal death point
-temperature required to destroy a population of bacteria in 10 minutes
the area around the disk (antibiotic or cleanser) with no bacteria
zone of inhibition
- measured in mm
Oxygen Requirement Classifications (5)
1. Obligate aerobe
2. Facultate anaerobe
3. Obligate anaerobes
5. Aerotolerant (Aerotolerant Anaerobe, Obligate Fermenter)
no colony growth
101 - uncountable
why is UV light lethal bacteria
It causes mutations, such as thymine dimers
Dilution Method Experiment
- toothpicks soaked in bacteria where then soaked in a disinfectant for varying lengths of time and then rinsed and placed in nutrient broth.
filter paper disk method
a plate is inoculated and filter paper disks soaked in disinfectant are placed on the plate and incubated. The disinfectant with the greatest zone of inhibition is the most effective.
When performing plate counts what dilution is easiest to count
how to calculate cells/mL
(total # of cells counted in six med squares) x dilution factor x 1000
divided by 0.024
How to count squares
counts 6 squares, ignore cells touching upper and left borders of a box, but count the cells found on lower or right borders
Only count plates with how many colonies
30 - 300
If the cfus are >300, overcrowding on the plate could have inhibited some cells from growing. If cfus are <30, could involve a sampling error.
name of device used to count squares
Naubauer counting chamber
Types of direct counts
standard count, living organisms
living & dead cells
How are population counts conducted?
A sample can be diluted and cells in sample can be counted with a microscope and Petroff-Hauser chamber.
Why are population counts important?
To determine the number of bacteria in a sample.
Ex: A diagnosis of bladder infection depends on a certain threshold level of bacteria present in a urine sample.
What is a serial dilution?
Start with culture.
Add 1 mL of sample to 100 mL of sterile water = 1:100
Remove 1 mL of 1:100 sample and add to 100 mL of sterile water = 1:1000
Remove 1 mL of 1:1000 sample and add to 100 mL of sterile water = 1:10,000
Remove 1 mL of 1:10,000 sample and add to 100 mL of sterile water = 1:1,000,000
Why are serial dilutions necessary?
-Bacteria cells are diluted to an end point where a single cell divides giving rise to the visible colony on a plate.
-Multiply the number of colonies by the dilution factor to determine the number of bacteria in the original sample.