Lab Practical Flashcards
Presence or absence of oxygen can be very _________ to the growth of bacteria
Important
Sodium thioglycate broth
Type of reducing media that is used to reduce the amount of oxygen concentration within the test tube
_____ is added to reduce the diffusion of oxygen to the rest of the media
Agar
Dye added to indicate where oxygen is present in the medium
resazurin
Turns pink in the presence of excess oxygen
Resazurin
Obligate Aerobe
1.Require oxygen. 2.See growth at the top of broth only
Obligate anaerobe
- Can’t tolerate oxygen
- Lack catalase
3.See growth at bottom of broth only
Majority of bacteria
Facultative anaerobes
Capable of living with or without oxygen
Facultative anaerobe
Growth is seen throughout medium but more at the surface
Facultative anaerobe
Cannot use oxygen but tolerate it well
Aerotolerant anaerobe
Most bacteria use fermentative metabolism
Aerotolerant anaerobe
Growth is seen throughout the medium, but more at the bottom
Aerotolerant anaerobe
Microaerophiles
Grow best in an atmosphere with increased carbon dioxide (5-10%)and lower concentrations of oxygen.
To culture microaerophiles on petri dishes, a ______ jar is needed
CO2
Generally, ____ kills microbes and _____ inhibits microbial growth
Heat
cold
How is heat sensitivity determined?
Heat sensitivity is genetically determined and partially reflected in the optimal growth ranges
Bacteria exhibit different tolerances to the application of_____
Heat
Examples of dry heating
1.Hot air ovens
2.incineration
Moist heat examples
- Pasteurization
2.boiling - autoclave
________ transfers heat energy to the microbial cell more efficiently
Moisture
Pasteurization maintains temperature at ____ for ______ mins
145 F for 30 mins
Pasteurization maintains temperature at _____ for __ ________ to kill microbes that are pathogenic or cause spoilage
160 F for 15 seconds
Boiling
212 F for 10 minutes kills vegetative bacterial cells but does not inactivate endospores
Most effective method of moist heat sterilization
Autoclave
What is autoclaving?
Use of steam under pressure
More pressure ________ the boiling point of water and produces steam with a higher temperature
Raises
What are the conditions for autoclaving?
15 PSI at 250F for 15 minutes
Sufficient to kill endospores and render materials sterile
Autoclaving
Thermal death time
The effectiveness of heat against a specific microbe
TDT
Length of time required to kill all bacteria in a liquid culture at a given temperature
TDP thermal death point
Temperature required to kill all bacteria in a liquid culture in 10 minutes (less common)
DRT decimal reduction time
Time in minutes in which 90% of a population of bacteria at a given temperature will be killed.
Antimicrobial agents
Control the growth of microbes
Disinfectants
Chemical agents used on inanimate objects to lower the level of microbes on their surface
Antiseptics
Chemicals used on living tissue to decrease number of microbes
Bactericidal agents
Those that result in bacterial death
-cidal
Lethal
Bacteriostatic agents
Those that cause temporary inhibition of growth
-static
Inhibiting
DRT Decimal reduction time
for the most common and persistent microbes identified at a healthcare facility should be determined
Limited to bacteriocidal compounds cannot be used for bacterostatic
Dilution test
The effectiveness of a disinfectant can be determined by the amount of resulting growth
Use-dilution test
Common and persistent microbes identified at healthcare facilities
1.Salmonella enterica
2.Staphylococcus aureus
3. Pseudomonas aeruginosa
Use dilution test
Rings are dipped into standardized cultures, removed, and dried.
Rings are placed into a solution of disinfectant at the concentration recommended for 10 minutes at 68° F
Use-dilution test
Effectiveness of disinfectant can be determined by the amount of resulting growth
Use dilution test
Antibiotics
Substance produced by a microorganism that inhibits the growth of other microorganisms
Antimicrobial or chemotherapeutic drugs
Antimicrobial chemicals used internally whether natural (antibiotic) or synthetic
Disk diffusion method
1.Petri dish is innoculated with desired microbe
- Various antimicrobial agents are placed on the surface of the agar
- Antimicrobial agent diffuses
Zone of inhibition
No growth around the disk
1. Helps us see the diffusion rate of antimicrobial agent
- Growth rate of microbe
Kirby-Bauer test
Standardized disc diffusion method to minimize variance
Uses Mueller-Hinton agar
The bigger the zone of inhibition the more ______ the antibiotic is
Effective
Which bacteria produce 70% of all antibiotics
Streptomycetes
Earliest writers classified bacteria solely on their
Morphological characteristics
Biologic characters
Refers to information about the metabolism of bacteria
Exoenzymes
Break down large molecules outside a cell. Smaller molecules released by this reaction are taken into the cell and further degraded by endoenzymes
Endoenzymes
Degrade the products of exoenzymes
Majority of enzymes function on the inside of the cell
Endoenzymes
Enzymes that are released from the cell to catalyze reactions outside the cell
Exoenzymes
Mainly hydrolytic enzymes
Exoenzymes
What do exoenzymes do?
1.Leave the cell
2.break down large substrates
3.need the addition of water
4. transport smaller components into the cell
Example of exoenzyme
Amylase hydrolyzes the polysaccharide starch to glucose
Starch hydrolysis begins
Clear
Add iodine to see presence in substrate outside
Colony
O/F test
1.Oxidative vs fermentative
2. OF medium is semisolid nutrient agar deep
Low concentration of peptone is
Growth medium
Desired carbohydrate can be added
O/F test
Oxidative catabolism requires the presence of
O2
Fermentative catabolism does not require oxygen but may occur in its presence
Some gas may occur
Peptone are
Food
Bromothymol blue turns green to yellow in the presence of
Acids
Bromothymol blue turns green to yellow in the presence of acids
Catabolism of carbohydrates
Both tubes turn yellow
Carbohydrate catabolism= fermentation
Yellow at top half
Acids produced as intermediates in respiration
Yellow in open tube only
Aerobic conditions only
Dark blue
1.Alkaline conditions
2.peptone used but no carbohydrate
3.ammonia production
More specific
Fermentation
Carbohydrate fermentation
Many bacteria produce organic acids and carbon dioxide gases from carbohydrate fermentation
-glucose
MRVP broth
Used to distinguish organisms that produce large amounts of acid from glucose and organisms that produce neutral product Acetoin
Citrate agar
Used to test for ammonia production
Phenol red turns from red to yellow when
Acid is present
Carbohydrate fermentation test
1.Peptone is used for microbe growth
2. Each tube has the same microbe but different carbohydrates
Durham tube
Small inverted tube used to observe gas released by the microbe
This is not a test you want to leave longer than 24 hours because the color will change back to red
Carbohydrate fermentation test
Why can’t you leave the carbohydrate fermentation test for more than 24 hours
The color will change back to red because peptone is used after the carbohydrate is gone
MR test
Test for acid to neutral pH changes based on products produced by microbes
Red
pH is below 4.4
Orange red
pH is 4.4-6.0
Yellow
pH is above 6.0
MR VP
Methyl red voges proskauer
Is a glucose supplemented nutrient broth
MR VP
Voges proskauer test
Test for production of acetoin detection by the addition of reagent 1 (alpha napthol) and reagent 2 (KOH)
VP
Upper part is red
Acetoin present= positive test
VP Upper part is brown
No acetoin=negative test
Simmons citrate agar
Green=control and negative
Prussian blue=positive
Some bacteria ferment citrate which can be useful for identifying bacteria
Contain citrate lyase
When citric acid is in solution, it loses a proton to form
Citrate ion
Sole carbon source
Citrate lyase break down citrate to form
pyruvate which can be reduced in fermentation
Bacteria use ammonium for sources of nitrogen and produce
Ammonia
Bromothymol blue changes to blue when the medium is
Alkalized
Small chain of amino acids
Peptide
Large chain of amino acids
Polypeptide
Amino acids are used primarily in
Anabolic reactions
Bacteria can hydrolyze the peptides or Polypeptides to release amino acids
Use amino acids as energy sources when carbohydrates are unavailable
When is the nutrient gelatin test positive?
If it stays liquid because it signifies the breakdown of proteins
Exoenzymes gelatinase hydrolyzes gelatin
It liquefies but does not solidify when cooled
Litmus milk
Bacteria can hydrolyzes the protein in milk called casein
Skim milk
Litmus indicator (blue/purple)
Cason hydrolysis peptonization
Turns clear blue
Lactose fermentation
Turns pink with acid
Excessive acid coagulation
Curd
Catabolism of litmus milk test
Alkaline reaction is purple
Reduce litmus
Turns white in lower part of two
Reduce litmus
Turns white in lower part of tube
Urea
Urea is a waste product of protein digestion in most vertebrates and is excreted in urine
Urease
Breaks down urea releasing ammonia
Urea agar contains
peptone, glucose, urea, and phenol red
Phenol red in urease test is originally
Orange
In urease test, yellow means a pH of
6.8
Urease test: Bright pink means a pH of
8.4
Once the amino acids are taken into a bacterial cell, various metabolic processes can occur using endoenzymes
- Use as carbon and energy source
- Have to remove the amino group
Removing the amino group is called
Deamination makes H2S and Indole
During deamination
Amino acid to ammonia, then released from the cell
Urease breaks down urea releasing
Ammonia
Deamination produces
Indole and H2S
Some bacteria have de-sulfhydrase enzymes
Remove hydrogen sulfide from the sulfur containing amino acids cysteine and methionine
De-sulfhydrase enzymes gas
Make rotten egg gas
H2S can also be produced from the reduction of inorganic compounds such as
thiosulfate
SIM Medium
Sulfide indole motility
Why do we use an inoculation needle in the sim medium?
To see if the bacteria grows out from where it was stabbed
Motility
The growth of black hydrogen sulfide is going to spread instead of being in a line
What is added to the Sim medium? What kind of salt?
Heavy metal salt such as iron, thiosulfate
Positive results shows a blackened area
Sulfide
Positive result shows red at top
Indole
Positive result shows black all over test tube
Motility
What does the hydrogen sulfide production test do?
Tests sulfide, indole and motility
Indole test
Useful diagnostic tool. Some bacteria deaminate amino acid tryptophan and produce indole, indigo
Uses MIO deep
Indole test
Which reagent is used for the indole test?
Kovac’s reagent
MIO stands for
Motility
Indole
Ornithine decarboxylase
Respiration
Living organisms obtain energy by removing electrons or oxidizing substrates
Example of respiration
Glycolysis; glucose is oxidized to pyruvic acid; The electron transport chain carries electrons; O2 is the final electron acceptor in aerobic respiration.
Cytochromes in the plasma membrane of bacterial cells carry electrons to
O2
Oxidase test
Used to determine the presence of cytochrome C
Four classes of cytochromes have been identified
Catalase test
During respiration, hydrogen combines with oxygen forming hydrogen peroxide.
Hydrogen peroxide is lethal to the cell
During respiration hydrogen combines with oxygen forming
Hydrogen peroxide
Nitrate reduction rest
Used to determine the ability to reduce nitrates
During anaerobic respiration, some bacteria reduce
1.Nitrate to nitrite
2.Nitrite to nitrous oxide
3.Nitrous oxide to nitrogen gas
Uses to reagents to detect nitrites
Reagent a & reagent b
Dimethyl-alpha-naphthylamine
Reagent A
Sulfanilic acid
Reagent B
What indicates a positive nitrate reduction test?
Red color-> nitrite is present= positive
Why should you check for gas first in the nitrate reduction test
Nitrogen has been reduced to nitrous, oxide (N2O) or nitrogen gas (N2)
A second test is used to further check if a negative is found
Zinc dust
Red color-> zinc will reduce nitrates to nitrites
Salt tolerance test
Tests the ability of an organisms ability to survive in high salt concentration
Percentage of salt for salt tolerance test
6.5% to 7.5%
What is a positive result of the salt tolerance test?
Cloudy is a positive result. Clear is a negative result
Which organisms are expected to grow in high salt concentrations?
Staphylococci enterococci, Aerococci
