Lab Practical 2 Flashcards
Generation time
Amount of time from the formation of a cell until the cell divides.
Phases of bacterial enumeration
Lag, Log, Stationary, Death
Why is there a lag phase
Bacteria are adjusting to their new environment.
At what phase is it best to collect bacteria in lab?
Log or early stationary phase
What is enumeration?
The process of counting or calculating the number of cells in a population or sample. We used aerobic plate count.
What do you call the number of viable cells in a sample?
Colony forming units (CFU)
What is serial dilution?
Technique used to reduce the number of CFU in a sample.
Technique used in bacterial enumeration to evenly distribute a liquid sample on the surface of agar.
Spread plating
CFU per mL =
of colonies on a plate x ( 1/ dilution factor of the plate)
of colonies on a plate that can be used in bacterial enumeration calculations
30-300
What are exoenzymes?
Exoenzymes are secreted by bacteria into their environment. They can function as toxins, or to provide nutrients to the cell.
What is gelatinase?
A type of protease that catalyzes the hydrolysis of gelatin into its component amino acids. Proteases are a group of enzymes that break down proteins. Pathogens can use this to break down connective tissue in the process of invasion. This is a virulence factor.
What is a protease?
Proteases are a group of enzymes that break down proteins
Nutrient gelatin
Differential. Used to determine if an organism produces gelatins. Contains gelatin (collagen), peptone, and beef extract. Collagen is a protein.
What indicates that an organism produces gelatinse?
The solid gelatin will become liquid at room temperature. Must be at temperatures lower than 28 C, as it is always liquid at 28.
What is amylase?
An exoenzyme used to break down large molecules into their monomers so they can be taken in to the cell.
What does amylase do?
Catalyzes the hydrolyses of amylose (starch) into maltose and glucose. Often used in the brewing industry, and converts corn starch to high fructose corn syrup.
Starch agar
Differential. Detects hydrolysis of starch by a bacteria.
Nutrient agar that contains soluble starch. After incubation plate is covered with iodine to detect the presence of a zone of no cleaning.
What will an organism that can hydrolyze starch show?
A distinct halo (the zone of clearing), showing that the starch around the culture was digested.
Starch agar results: bacterial growth with a zone of cleaning
Organism produces the enxoenzyme amylase
Starch agar results: bacterial growth, no zone of clearing
Organism does not produce the exoenzyme amylase
Sulfide Indole Motility medium (SIM) detects…
Used to detect:
- sulfide production
- indole production
- motility
SIM medium characteristics
Differential. Contains sodium thiosulfate, ferrous ammonium sulfate, casein, and 3.5% agar
What is sodium thiosulfate recused to?
Hydrogen sulfide (H2S). This reacts with the iron in ferrous ammonium sulfate, forming ferrous sulfide.
How does ferrous sulfide appear on SIM?
As a black precipitate.
Black precipitate on SIM plate
Organism produces hydrogen sulfide
No black precipitate on SIM plate
Organism cannot produce hydrogen sulfide.
What is the amino acid tryptophan broken down into?
Indole
What is used to determine the presence of indole on a SIM plate?
Kovak’s reagent, which will turn red if indole is present.
Red Kovak’s reagent on SIM plate
Organism produces indole
Kovak’s reagent didn’t turn red on SIM plate
Organism does not produce indole
What is the purpose of the 3.5% agar in SIM?
It creates a medium that is solid enough to prevent the diffusion of non-motile bacteria.
How is motility indicated in SIM?
Growth or cloudiness outside of the single inoculation stab.
*obligate aerobes do not grow well in stabs, can only be used for facultative anaerobes or anaerobes.
What is catalase?
Enzyme found in bacteria and other organisms that catalyzes the conversion of peroxide anions into water and oxygen, protecting the organism from oxidative damage.
Catalase test
Differential. Determines if an organism produces the enzyme catalase.
Hydrogen peroxide added to sample on microscope slide. If bubbles form, catalase is produced.
No bubble formation on catalase test
Organism does not produce catalase
What does cytochrome oxidase do?
Enzyme responsible for reducing oxygen in the last step of aerobic respiration, forming H2O as an end product.
What does a blue result (after 20s) on a DrySlide indicate?
That an organism produces cytochrome oxidase
What is fermentation?
Organism oxidizes NADH back into NAD+, produces ATP, without oxygen.
Types of fermentation
Homolactic
Heterolactic
Mixed acid
2,3 butanediol
Phenol Red Carbohydrate Broth
Differential. Determines if a bacteria can ferment certain sugar, and if acids or gases are produced as end products.
Phenol Red broths (4)
Glucose
Lactose
Sucrose
Mannitol
What is phenol red?
A neutral pH indicator
Name of tube inside phenol red broth?
Durham tube
Phenol red test: broth is yellow, bubble forms
Organism can ferment whatever sugar is in the broth, and produces acid
Phenol red test: broth turns orange
Obligate aerobe converted the sugar into acid, but not using fermentation.
Man who did experiments on mice that led to understanding of bacterial transformation
Frederick Griffith
Scientists that continued Frederick Griffith’s work on bacterial transformation
Avery, McLeod, McCarty
What is transformation?
The uptake of naked DNA from the environment by a bacterial cell
What do you call the ability of a bacterial cell to be transformed (take in DNA from the environment)?
Competence
What are the steps in making E coli have artificial competency?
Being in a solution of CaCl2 (calcium chloride), subjected to heat shock.
What is the purpose of the heat shock step?
Forms transient pores in the cell walls, giving a path for the plasmid to enter through.
What is the purpose of the Calcium chloride?
The Ca ions bind to the bacterial cell wall, neutralizing it
pGLO plasmid
Contains ampicillin resistance and the production of the green fluorescent protein GFP
What promoter controls the transcription of the GFP gene?
The promoter for arabinose metabolism. There must be arabinose in the growth medium, so the ara promoter becomes active, so that GFP will be synthesized.
What gene causes the glowing?
GFP
What does beta-lactamase enzyme do?
Confers resistance to ampicillin. Only bacteria that have been transformed will be resistant to ampicillin.
What gene codes for beta-lactamase enzyme?
bla gene
What will E. coli do on plates with ampicillin.
E. coli that has not been transformed will not be able to grow on plates containing ampicillin
What is a promoter?
Region of DNA that initiates transcription of a piece of DNA
Final e- receptor in fermentation?
An inorganic material
Top of phenol red tube is red, bottom yellow?
Tube was left to incubate for too long.
End products of homolactic fermentation
Lactic acid
End products of heterolactic fermentation
lactic acid, ethanol, CO2
End products of mixed acid fermentation
lactic accidentally, succinc acid, acetic acid, formic acid, ethanol, hydrogen, CO2
End products of 2,3 butanediol fermentation
lactic acid, formic acid, ethanol, 2,3 butaneldiol, hydrogen, CO2
Antimicrobial definition
General term for a compound that negatively affects the growth of microbes.
Broad spectrum drugs target….
A common aspect of all bacterial cells.
Narrow spectrum drugs usually attack…
an aspect of cell physiology not found in all bacterial cells
Aminoglycosides
Streptomycin(S), kanamycin (K), neomycin (N). Prevent protein synthesis.
Bacitracin (B)
Prevents cell wall synthesis
Beta-Lactams
Penicillin (P), amoxicillin (AmC), prevents formation of peptidoglycan in the cell wall
Chloramphenicol (C)
Disrupts protein synthesis
Fluoroquinolones
Ciprofloxacin (CIP) prevents replication by binding to DNA gyrase
Macrolides
Erythromycin (E), disrupts protein synthesis
Tetracyclines
Tetracycline (TE), interfere with protein production
Sulfanomides/Trimethoprim
Sulfamethoxazole with trimethroprin (SxT) inhibits folate synthesis
Name of test with all the little dots on the tray
Kirby-Bauer Disk diffusion assay
UV light causes….
…the formation of thymine dimers in DNA molecules, which block DNA replication and transcription, killing bacteria.
